GENERAL INFORMATION1655 GENERAL INFORMATION 1. AminoAcidAnalysis This test is harmonized with the European Pharmacopoeia and the U.S. Pharmacopeia. Amino acid analysis refers to the

16551655

GENERAL INFORMATION

1. Amino Acid AnalysisThis test is harmonized with the European Pharmacopoeia

and the U.S. Pharmacopeia.Amino acid analysis refers to the methodology used to de-

termine the amino acid composition or content of proteins,peptides, and other pharmaceutical preparations. Proteinsand peptides are macromolecules consisting of covalentlybonded amino acid residues organized as a linear polymer.The sequence of the amino acids in a protein or peptidedetermines the properties of the molecule. Proteins areconsidered large molecules that commonly exist as foldedstructures with a speciˆc conformation, while peptides aresmaller and may consist of only a few amino acids. Aminoacid analysis can be used to quantify protein and peptides, todetermine the identity of proteins or peptides based on theiramino acid composition, to support protein and peptidestructure analysis, to evaluate fragmentation strategies forpeptide mapping, and to detect atypical amino acids thatmight be present in a protein or peptide. It is necessary tohydrolyze a protein/peptide to its individual amino acidconstituents before amino acid analysis. Following pro-tein/peptide hydrolysis, the amino acid analysis procedurecan be the same as that practiced for free amino acids in otherpharmaceutical preparations. The amino acid constituents ofthe test sample are typically derivatized for analysis.

ApparatusMethods used for amino acid analysis are usually based on

a chromatographic separation of the amino acids present inthe test sample. Current techniques take advantage of the au-tomated chromatographic instrumentation designed for ana-lytical methodologies. An amino acid analysis instrument willtypically be a low-pressure or high-pressure liquid chromato-graph capable of generating mobile phase gradients thatseparate the amino acid analytes on a chromatographiccolumn. The instrument must have postcolumn derivatiza-tion capability, unless the sample is analyzed usingprecolumn derivatization. The detector is usually an ultrav-iolet-visible or ‰uorescence detector depending on thederivatization method used. A recording device (e.g.,integrator) is used for transforming the analog signal fromthe detector and for quantitation. It is preferred thatinstrumentation be dedicated particularly for amino acidanalysis.

General PrecautionsBackground contamination is always a concern for the

analyst in performing amino acid analysis. High purityreagents are necessary (e.g., low purity hydrochloric acid cancontribute to glycine contamination). Analytical reagents arechanged routinely every few weeks using only high-pressureliquid chromatography (HPLC) grade solvents. Potentialmicrobial contamination and foreign material that might bepresent in the solvents are reduced by ˆltering solvents before

use, keeping solvent reservoirs covered, and not placing ami-no acid analysis instrumentation in direct sunlight.

Laboratory practices can determine the quality of theamino acid analysis. Place the instrumentation in a low tra‹carea of the laboratory. Keep the laboratory clean. Clean andcalibrate pipets according to a maintenance schedule. Keeppipet tips in a covered box; the analysts may not handle pipettips with their hands. The analysts may wear powder-free la-tex or equivalent gloves. Limit the number of times a testsample vial is opened and closed because dust can contributeto elevated levels of glycine, serine, and alanine.

A well-maintained instrument is necessary for acceptableamino acid analysis results. If the instrument is used on aroutine basis, it is to be checked daily for leaks, detector andlamp stability, and the ability of the column to maintain reso-lution of the individual amino acids. Clean or replace allinstrument ˆlters and other maintenance items on a routineschedule.

Reference Standard MaterialAcceptable amino acid standards are commercially

available for amino acid analysis and typically consist of anaqueous mixture of amino acids. When determining aminoacid composition, protein or peptide standards are analyzedwith the test material as a control to demonstrate the integrityof the entire procedure. Highly puriêd bovine serumalbumin has been used as a protein standard for this purpose.

Calibration of InstrumentationCalibration of amino acid analysis instrumentation

typically involves analyzing the amino acid standard, whichconsists of a mixture of amino acids at a number of concen-trations, to determine the response factor and range ofanalysis for each amino acid. The concentration of each ami-no acid in the standard is known. In the calibration proce-dure, the analyst dilutes the amino acid standard to severaldiŠerent analyte levels within the expected linear range of theamino acid analysis technique. Then, replicates at each of thediŠerent analyte levels can be analyzed. Peak areas obtainedfor each amino acid are plotted versus the known concentra-tion for each of the amino acids in the standard dilution.These results will allow the analyst to determine the range ofamino acid concentrations where the peak area of a givenamino acid is an approximately linear function of the aminoacid concentration. It is important that the analyst preparethe samples for amino acid analysis so that they are withinthe analytical limits (e.g., linear working range) of the tech-nique employed in order to obtain accurate and repeatableresults.

Four to six amino acid standard levels are analyzed todetermine a response factor for each amino acid. Theresponse factor is calculated as the average peak area or peakheight per nmol of amino acid present in the standard. Acalibration ˆle consisting of the response factor for each ami-no acid is prepared and used to calculate the concentration ofeach amino acid present in the test sample. This calculationinvolves dividing the peak area corresponding to a given ami-

16561656 JP XVAmino Acid Analysis / General Information

no acid by the response factor for that amino acid to give thenmol of the amino acid. For routine analysis, a single-pointcalibration may be su‹cient; however, the calibration ˆle isupdated frequently and tested by the analysis of analyticalcontrols to ensure its integrity.

RepeatabilityConsistent high quality amino acid analysis results from an

analytical laboratory require attention to the repeatability ofthe assay. During analysis of the chromatographic separationof the amino acids or their derivatives, numerous peaks canbe observed on the chromatogram that correspond to theamino acids. The large number of peaks makes it necessary tohave an amino acid analysis system that can repeatedly iden-tify the peaks based on retention time and integrate the peakareas for quantitation. A typical repeatability evaluation in-volves preparing a standard amino acid solution and analyz-ing many replicates (i.e., six analyses or more) of the samestandard solution. The relative standard deviation (RSD) isdetermined for the retention time and integrated peak area ofeach amino acid. An evaluation of the repeatability is ex-panded to include multiple assays conducted over severaldays by diŠerent analysts. Multiple assays include the prepa-ration of standard dilutions from starting materials to deter-mine the variation due to sample handling. Often the aminoacid composition of a standard protein (e.g., bovine serumalbumin) is analyzed as part of the repeatability evaluation.By evaluating the replicate variation (i.e., RSD), the labora-tory can establish analytical limits to ensure that the analysesfrom the laboratory are under control. It is desirable to estab-lish the lowest practical variation limits to ensure the bestresults. Areas to focus on to lower the variability of the ami-no acid analysis include sample preparation, high back-ground spectral interference due to quality of reagentsand/or laboratory practices, instrument performance andmaintenance, data analysis and interpretation, and analystperformance and habits. All parameters involved are fully in-vestigated in the scope of the validation work.

Sample PreparationAccurate results from amino acid analysis require puriêd

protein and peptide samples. BuŠer components (e.g., salts,urea, detergents) can interfere with the amino acid analysisand are removed from the sample before analysis. Methodsthat utilize postcolumn derivatization of the amino acids aregenerally not aŠected by buŠer components to the extent seenwith precolumn derivatization methods. It is desirable tolimit the number of sample manipulations to reduce potentialbackground contamination, to improve analyte recovery,and to reduce labor. Common techniques used to removebuŠer components from protein samples include the follow-ing methods: (1) injecting the protein sample onto a reversed-phase HPLC system, eluting the protein with a volatile sol-vent containing a su‹cient organic component, and dryingthe sample in a vacuum centrifuge; (2) dialysis against a vola-tile buŠer or water; (3) centrifugal ultraˆltration for buŠerreplacement with a volatile buŠer or water; (4) precipitatingthe protein from the buŠer using an organic solvent (e.g.,acetone); and (5) gel ˆltration.

Internal StandardsIt is recommended that an internal standard be used to mo-

nitor physical and chemical losses and variations during ami-no acid analysis. An accurately known amount of internal

standard can be added to a protein solution prior to hydroly-sis. The recovery of the internal standard gives the generalrecovery of the amino acids of the protein solution. Free ami-no acids, however, do not behave in the same way as protein-bound amino acids during hydrolysis because their rates ofrelease or destruction are variable. Therefore, the use of aninternal standard to correct for losses during hydrolysis maygive unreliable results. It will be necessary to take this pointunder consideration when interpreting the results. Internalstandards can also be added to the mixture of amino acids af-ter hydrolysis to correct for diŠerences in sample applicationand changes in reagent stability and ‰ow rates. Ideally, an in-ternal standard is an unnaturally occurring primary aminoacid that is commercially available and inexpensive. It shouldalso be stable during hydrolysis, its response factor should belinear with concentration, and it needs to elute with a uniqueretention time without overlapping other amino acids. Com-monly used amino acid standards include norleucine,nitrotyrosine, and a-aminobutyric acid.

Protein HydrolysisHydrolysis of protein and peptide samples is necessary for

amino acid analysis of these molecules. The glassware usedfor hydrolysis must be very clean to avoid erroneous results.Glove powders and ˆngerprints on hydrolysis tubes maycause contamination. To clean glass hydrolysis tubes, boiltubes for 1 hour in 1 mol/L hydrochloric acid or soak tubesin concentrated nitric acid or in a mixture of concentratedhydrochloric acid and concentrated nitric acid (1:1). Cleanhydrolysis tubes are rinsed with high-purity water followedby a rinse with HPLC grade methanol, dried overnight in anoven, and stored covered until use. Alternatively, pyrolysis ofclean glassware at 5009C for 4 hours may also be used toeliminate contamination from hydrolysis tubes. Adequatedisposable laboratory material can also be used.

Acid hydrolysis is the most common method for hydrolyz-ing a protein sample before amino acid analysis. The acidhydrolysis technique can contribute to the variation of theanalysis due to complete or partial destruction of several ami-no acids. Tryptophan is destroyed; serine and threonine arepartially destroyed; methionine might undergo oxidation;and cysteine is typically recovered as cystine (but cystinerecovery is usually poor because of partial destruction orreduction to cysteine). Application of adequate vacuum(≦less than 200 mm of mercury or 26.7 Pa) or introduction ofan inert gas (argon) in the headspace of the reaction vesselcan reduce the level of oxidative destruction. In peptidebonds involving isoleucine and valine the amido bonds of Ile-Ile, Val-Val, Ile-Val, and Val-Ile are partially cleaved; andasparagine and glutamine are deamidated, resulting in aspar-tic acid and glutamic acid, respectively. The loss of trypto-phan, asparagine, and glutamine during an acid hydrolysislimits quantitation to 17 amino acids. Some of the hydrolysistechniques described are used to address these concerns.Some of the hydrolysis techniques described (i.e., Methods4-11) may cause modiˆcations to other amino acids. There-fore, the beneˆts of using a given hydrolysis technique areweighed against the concerns with the technique and are test-ed adequately before employing a method other than acidhydrolysis.

A time-course study (i.e., amino acid analysis at acidhydrolysis times of 24, 48, and 72 hours) is often employed toanalyze the starting concentration of amino acids that are

16571657JP XV General Information / Amino Acid Analysis

partially destroyed or slow to cleave. By plotting the observedconcentration of labile amino acids (i.e., serine and threo-nine) versus hydrolysis time, the line can be extrapolated tothe origin to determine the starting concentration of theseamino acids. Time-course hydrolysis studies are also usedwith amino acids that are slow to cleave (e.g., isoleucine andvaline). During the hydrolysis time course, the analyst willobserve a plateau in these residues. The level of this plateau istaken as the residue concentration. If the hydrolysis time istoo long, the residue concentration of the sample will beginto decrease, indicating destruction by the hydrolysis condi-tions.

An acceptable alternative to the time-course study is tosubject an amino acid calibration standard to the samehydrolysis conditions as the test sample. The amino acid infree form may not completely represent the rate of destruc-tion of labile amino acids within a peptide or protein duringthe hydrolysis. This is especially true for peptide bonds thatare slow to cleave (e.g., Ile-Val bonds). However, thistechnique will allow the analyst to account for some residuedestruction. Microwave acid hydrolysis has been used and israpid but requires special equipment as well as special precau-tions. The optimal conditions for microwave hydrolysis mustbe investigated for each individual protein/peptide sample.The microwave hydrolysis technique typically requires only afew minutes, but even a deviation of one minute may give in-adequate results (e.g., incomplete hydrolysis or destructionof labile amino acids). Complete proteolysis, using a mixtureof proteases, has been used but can be complicated, requiresthe proper controls, and is typically more applicable to pep-tides than proteins.

Note: During initial analyses of an unknown protein,experiments with various hydrolysis time and temperatureconditions are conducted to determine the optimal condi-tions.

Method 1Acid hydrolysis using hydrochloric acid containing phenol

is the most common procedure used for protein/peptidehydrolysis preceding amino acid analysis. The addition ofphenol to the reaction prevents the halogenation of tyrosine.

Hydrolysis Solution 6 mol/L hydrochloric acid contain-ing 0.1z to 1.0z of phenol.

Procedure—Liquid Phase Hydrolysis Place the protein or peptide

sample in a hydrolysis tube, and dry. [Note: The sample is d-ried so that water in the sample will not dilute the acid usedfor the hydrolysis.] Add 200 mL of Hydrolysis Solution per500 mg of lyophilized protein. Freeze the sample tube in a dryice-acetone bath, and ‰ame seal in vacuum. Samples are typi-cally hydrolyzed at 1109C for 24 hours in vacuum or inert at-mosphere to prevent oxidation. Longer hydrolysis times(e.g., 48 and 72 hours) are investigated if there is a concernthat the protein is not completely hydrolyzed.

Vapor Phase Hydrolysis This is one of the most commonacid hydrolysis procedures, and it is preferred for microanal-ysis when only small amounts of the sample are available.Contamination of the sample from the acid reagent is alsominimized by using vapor phase hydrolysis. Place vials con-taining the dried samples in a vessel that contains an ap-propriate amount of Hydrolysis Solution. The HydrolysisSolution does not come in contact with the test sample. App-ly an inert atmosphere or vacuum (≦ less than 200 mm of

mercury or 26.7 Pa) to the headspace of the vessel, and heatto about 1109C for a 24-hour hydrolysis time. Acid vaporhydrolyzes the dried sample. Any condensation of the acid inthe sample vials is minimized. After hydrolysis, dry the testsample in vacuum to remove any residual acid.

Method 2Tryptophan oxidation during hydrolysis is decreased by us-

ing mercaptoethanesulfonic acid (MESA) as the reducingacid.

Hydrolysis Solution 2.5 mol/L MESA solution.Vapor Phase Hydrolysis About 1 to 100 mg of the

protein/peptide under test is dried in a hydrolysis tube. Thehydrolysis tube is placed in a larger tube with about 200 mL ofthe Hydrolysis Solution. The larger tube is sealed in vacuum(about 50 mm of mercury or 6.7 Pa) to vaporize the Hydroly-sis Solution. The hydrolysis tube is heated to 1709C to 1859Cfor about 12.5 minutes. After hydrolysis, the hydrolysis tubeis dried in vacuum for 15 minutes to remove the residual acid.

Method 3Tryptophan oxidation during hydrolysis is prevented by

using thioglycolic acid (TGA) as the reducing acid.Hydrolysis Solution A solution containing 7 mol/L

hydrochloric acid, 10z of tri‰uoroacetic acid, 20z ofthioglycolic acid, and 1z of phenol.

Vapor Phase Hydrolysis About 10 to 50 mg of theprotein/peptide under test is dried in a sample tube. The sam-ple tube is placed in a larger tube with about 200 mL of theHydrolysis Solution. The larger tube is sealed in vacuum(about 50 mm of mercury or 6.7 Pa) to vaporize the TGA.The sample tube is heated to 1669C for about 15 to 30minutes. After hydrolysis, the sample tube is dried in vacuumfor 5 minutes to remove the residual acid. Recovery of tryp-tophan by this method may be dependent on the amount ofsample present.

Method 4Cysteine-cystine and methionine oxidation is performed

with performic acid before the protein hydrolysis.Oxidation Solution The performic acid is prepared fresh

by mixing formic acid and 30 percent hydrogen peroxide(9:1), and incubated at room temperature for 1 hour.

Procedure The protein/peptide sample is dissolved in20 mL of formic acid, and heated at 509C for 5 minutes; then100 mL of the Oxidation Solution is added. The oxidation isallowed to proceed for 10 to 30 minutes. In this reaction, cys-teine is converted to cysteic acid and methionine is convertedto methionine sulfone. The excess reagent is removed fromthe sample in a vacuum centrifuge. This technique may causemodiˆcations to tyrosine residues in the presence of halides.The oxidized protein can then be acid hydrolyzed usingMethod 1 or Method 2.

Method 5Cysteine-cystine oxidation is accomplished during the

liquid phase hydrolysis with sodium azide.Hydrolysis Solution 6 mol/L hydrochloric acid contain-

ing 0.2z of phenol, to which is added sodium azide to obtaina ˆnal concentration of 0.2z (w/v). The added phenol pre-vents halogenation of tyrosine.

Liquid Phase Hydrolysis The protein/peptide hydrolysisis conducted at about 1109C for 24 hours. During the hydrol-ysis, the cysteine-cystine present in the sample is converted tocysteic acid by the sodium azide present in the Hydrolysis So-


lution. This technique allows better tyrosine recovery thanMethod 4, but it is not quantitative for methionine. Methio-nine is converted to a mixture of the parent methionine andits two oxidative products, methionine sulfoxide and methio-nine sulfone.

Method 6Cysteine-cystine oxidation is accomplished with dimethyl

sulfoxide (DMSO).Hydrolysis Solution 6 mol/L hydrochloric acid contain-

ing 0.1z to 1.0z of phenol, to which DMSO is added toobtain a ˆnal concentration of 2z (v/v).

Vapor Phase Hydrolysis The protein/peptide hydrolysisis conducted at about 1109C for 24 hours. During the hydrol-ysis, the cysteine-cystine present in the sample is converted tocysteic acid by the DMSO present in the Hydrolysis Solution.As an approach to limit variability and compensate for par-tial destruction, it is recommended to evaluate the cysteicacid recovery from oxidative hydrolyses of standard proteinscontaining 1 to 8 mol of cysteine. The response factors fromprotein/peptide hydrolysates are typically about 30z lowerthan those for nonhydrolyzed cysteic acid standards. Becausehistidine, methionine, tyrosine, and tryptophan are also mo-diêd, a complete compositional analysis is not obtained withthis technique.

Method 7Cysteine-cystine reduction and alkylation is accomplished

by a vapor phase pyridylethylation reaction.Reducing Solution Transfer 83.3 mL of pyridine, 16.7 mL

of 4-vinylpyridine, 16.7 mL of tributylphosphine, and 83.3mL of water to a suitable container, and mix.

Procedure Add the protein/peptide (between 1 and 100mg) to a hydrolysis tube, and place in a larger tube. Transferthe Reducing Solution to the large tube, seal in vacuum(about 50 mm of mercury or 6.7 Pa), and incubate at about1009C for 5 minutes. Then remove the inner hydrolysis tube,and dry it in a vacuum desiccator for 15 minutes to removeresidual reagents. The pyridylethylated protein/peptide canthen be acid hydrolyzed using previously describedprocedures. The pyridylethylation reaction is performedsimultaneously with a protein standard sample containing 1to 8 mol of cysteine to improve accuracy in the pyridylethyl-cysteine recovery. Longer incubation times for thepyridylethylation reaction can cause modiˆcations to thea-amino terminal group and the e-amino group of lysine inthe protein.


by a liquid phase pyridylethylation reaction.Stock Solutions Prepare and ˆlter three solutions:

1 mol/L Tris hydrochloride (pH 8.5) containing 4 mmol/Ldisodium dihydrogen ethylendiamine tetraacetate (Stock So-lution A), 8 mol/L guanidine hydrochloride (Stock SolutionB), and 10z of 2-mercaptoethanol in water (Stock SolutionC).

Reducing Solution Prepare a mixture of Stock Solution Band Stock Solution A (3:1) to obtain a buŠered solutionof 6 mol/L guanidine hydrochloride in 0.25 mol/L Trishydrochloride.

Procedure Dissolve about 10 mg of the test sample in 50mL of the Reducing Solution, and add about 2.5 mL of StockSolution C. Store under nitrogen or argon for 2 hours at

room temperature in the dark. To achieve the pyridylethyla-tion reaction, add about 2 mL of 4-vinylpyridine to theprotein solution, and incubate for an additional 2 hours atroom temperature in the dark. The protein/peptide isdesalted by collecting the protein/peptide fraction from areversed-phase HPLC separation. The collected sample canbe dried in a vacuum centrifuge before acid hydrolysis.


by a liquid phase carboxymethylation reaction.Stock Solutions Prepare as directed for Method 8.Carboxymethylation Solution Prepare a solution con-

taining 100 mg of iodoacetamide per mL of ethanol (95).BuŠer Solution Use the Reducing Solution, prepared as

directed for Method 8.Procedure Dissolve the test sample in 50 mL of the BuŠer

Solution, and add about 2.5 mL of Stock Solution C. Storeunder nitrogen or argon for 2 hours at room temperature inthe dark. Add the Carboxymethylation Solution in a ratio 1.5fold per total theoretical content of thiols, and incubate foran additional 30 minutes at room temperature in the dark.[Note: If the thiol content of the protein is unknown, thenadd 5 mL of 100 mmol/L iodoacetamide for every 20 nmol ofprotein present.] The reaction is stopped by adding excess of2-mercaptoethanol. The protein/peptide is desalted by col-lecting the protein/peptide fraction from a reversed-phaseHPLC separation. The collected sample can be dried in avacuum centrifuge before acid hydrolysis. TheS-carboxyamidomethyl-cysteine formed will be converted toS-carboxymethylcysteine during acid hydrolysis.

Method 10Cysteine-cystine is reacted with dithiodiglycolic acid or

dithiodipropionic acid to produce a mixed disulˆde. [Note:The choice of dithiodiglycolic acid or dithiodipropionic aciddepends on the required resolution of the amino acid analysismethod.]

Reducing Solution A solution containing 10 mg ofdithiodiglycolic acid (or dithiodipropionic acid) per mL of0.2 mol/L sodium hydroxide.

Procedure Transfer about 20 mg of the test sample to ahydrolysis tube, and add 5 mL of the Reducing Solution. Add10 mL of isopropyl alcohol, and then remove all of the sampleliquid by vacuum centrifugation. The sample is then hydro-lyzed using Method 1. This method has the advantage thatother amino acid residues are not derivatized by side reac-tions, and the sample does not need to be desalted prior tohydrolysis.

Method 11Asparagine and glutamine are converted to aspartic acid

and glutamic acid, respectively, during acid hydrolysis.Asparagine and aspartic acid residues are added andrepresented by Asx, while glutamine and glutamic acidresidues are added and represented by Glx. Proteins/peptidescan be reacted with bis(1,1-tri‰uoroacetoxy)iodobenzene(BTI) to convert the asparagine and glutamine residues todiaminopropionic acid and diaminobutyric acid residues,respectively, upon acid hydrolysis. These conversions allowthe analyst to determine the asparagine and glutaminecontent of a protein/peptide in the presence of aspartic acidand glutamic acid residues.

Reducing Solutions Prepare and ˆlter three solutions: a


solution of 10 mmol/L tri‰uoroacetic acid (Solution A), asolution of 5 mol/L guanidine hydrochloride and 10 mmol/Ltri‰uoroacetic acid (Solution B), and a freshly preparedsolution of N,N-dimethylformamide containing 36 mg ofBTI per mL (Solution C).

Procedure In a clean hydrolysis tube, transfer about200 mg of the test sample, and add 2 mL of Solution A orSolution B and 2 mL of Solution C. Seal the hydrolysis tubein vacuum. Heat the sample at 609C for 4 hours in the dark.The sample is then dialyzed with water to remove the excessreagents. Extract the dialyzed sample three times with equalvolumes of n-butyl acetate, and then lyophilize. The proteincan then be acid hydrolyzed using previously described proce-dures. The a,b-diaminopropionic and a,g-diaminobutyricacid residues do not typically resolve from the lysine residuesupon ion-exchange chromatography based on amino acidanalysis. Therefore, when using ion-exchange as the mode ofamino acid separation, the asparagine and glutamine con-tents are the quantitative diŠerence in the aspartic acid andglutamic acid content assayed with underivatized and BTI-derivatized acid hydrolysis. [Note: The threonine, methio-nine, cysteine, tyrosine, and histidine assayed content can bealtered by BTI derivatization; a hydrolysis without BTI willhave to be performed if the analyst is interested in the compo-sition of these other amino acid residues of the protein/pep-tide.]

Methodologies of Amino Acid Analysis General PrinciplesMany amino acid analysis techniques exist, and the choice

of any one technique often depends on the sensitivityrequired from the assay. In general, about one-half of theamino acid analysis techniques employed rely on theseparation of the free amino acids by ion-exchangechromatography followed by postcolumn derivatization(e.g., with ninhydrin or o-phthalaldehyde). Postcolumndetection techniques can be used with samples that containsmall amounts of buŠer components, such as salts andurea, and generally require between 5 and 10 mg of proteinsample per analysis. The remaining amino acid techniquestypically involve precolumn derivatization of the free aminoacids (e.g., phenyl isothiocyanate; 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate or o-phthalaldehyde;(dimethylamino)azobenzenesulfonyl chloride; 9-‰uorenylmethylchloroformate; and, 7-‰uoro-4-nitrobenzo-2-oxa-1,3-diazole) followed by reversed-phase HPLC.Precolumn derivatization techniques are very sensitive andusually require between 0.5 and 1.0 mg of protein sample peranalysis but may be in‰uenced by buŠer salts in the samples.Precolumn derivatization techniques may also result inmultiple derivatives of a given amino acid, which complicatesthe result interpretation. Postcolumn derivatization tech-niques are generally in‰uenced less by performance variationof the assay than precolumn derivatization techniques.

The following Methods may be used for quantitativeamino acid analysis. Instruments and reagents for theseprocedures are available commercially. Furthermore, manymodiˆcations of these methodologies exist with diŠerentreagent preparations, reaction procedures, chromatographicsystems, etc. Speciˆc parameters may vary according to theexact equipment and procedure used. Many laboratories willutilize more than one amino acid analysis technique to exploitthe advantages oŠered by each. In each of these Methods, theanalog signal is visualized by means of a data acquisition sys-

tem, and the peak areas are integrated for quantiˆcation pur-poses.Method 1—Postcolumn Ninhydrin Detection GeneralPrinciple

Ion-exchange chromatography with postcolumn ninhydrindetection is one of the most common methods employed forquantitative amino acid analysis. As a rule, a Li-basedcation-exchange system is employed for the analysis of themore complex physiological samples, and the faster Na-basedcation-exchange system is used for the more simplistic aminoacid mixtures obtained with protein hydrolysates (typicallycontaining 17 amino acid components). Separation of theamino acids on an ion-exchange column is accomplishedthrough a combination of changes in pH and cation strength.A temperature gradient is often employed to enhance separa-tion.

When the amino acid reacts with ninhydrin, the reactanthas characteristic purple or yellow color. Amino acids, exceptimino acid, give a purple color, and show the maximum ab-sorption at 570 nm. The imino acids such as proline give ayellow color, and show the maximum absorption at 440 nm.The postcolumn reaction between ninhydrin and amino acideluted from column is monitored at 440 and 570 nm, and thechromatogram obtained is used for the determination of ami-no acid composition.

Detection limit is considered to be 10 pmol for most of theamino acid derivatives, but 50 pmol for proline. Responselinearity is obtained in the range of 20 to 500 pmol withcorrelation coe‹cients exceeding 0.999. To obtain good com-position data, samples larger than 1 mg before hydrolysis arebest suited for this amino acid analysis of protein/peptide.

Method 2—Postcolumn OPA Fluorometric DetectionGeneral Principle

o-Phthalaldehyde (OPA) reacts with primary amines in thepresence of thiol compound, to form highly ‰uorescent isoin-dole products. This reaction is utilized for the postcolumnderivatization in analysis of amino acids by ion-exchangechromatography. The rule of the separation is the same asMethod 1. Instruments and reagents for this form of aminoacid analysis are available commercially. Many modiˆcationsof this methodology exist.

Although OPA does not react with secondary amines(imino acids such as proline) to form ‰uorescent substances,the oxidation with sodium hypochlorite allows secondaryamines to react with OPA. The procedure employs a stronglyacidic cation-exchange column for separation of free aminoacids followed by postcolumn oxidation with sodiumhypochlorite and postcolumn derivatization using OPA andthiol compound such as N-acetyl-L-cysteine and 2-mercap-toethanol. The derivatization of primary amino acids are notnoticeably aŠected by the continuous supply of sodiumhypochlorite.

Separation of the amino acids on an ion-exchange columnis accomplished through a combination of changes in pH andcation strength. After postcolumn derivatization of elutedamino acids with OPA, the reactant passes through the‰uorometric detector. Fluorescence intensity of OPA-deriva-tized amino acids are monitored with an excitationwavelength of 348 nm and an emission wavelength of450 nm.

Detection limit is considered to be a few tens of picomolelevel for most of the amino acid derivatives. Response lineari-


ty is obtained in the range of a few picomole level to a fewtens of nanomole level. To obtain good compositional data,the starting with greater than 500 ng of sample before hydrol-ysis is best suited for the amino acid analysis ofprotein/peptide.

Method 3—Precolumn PITC Derivatization GeneralPrinciple

Phenylisothiocyanate (PITC) reacts with amino acids toform phenylthiocarbamyl (PTC) derivatives which can be de-tected with high sensitivity at 245 nm. Therefore, precolumnderivatization of amino acids with PITC followed by a rever-sed-phase HPLC separation with UV detection is used toanalyze the amino acid composition.

After the reagent is removed under vacuum, the deriva-tized amino acids can be stored dry and frozen for severalweeks with no signiˆcant degradation. If the solution for in-jection is kept cold, no noticeable loss in chromatographicresponse occurs after three days.

Separation of the PTC-amino acids on a reversed-phaseHPLC with ODS column is accomplished through a combi-nation of changes in concentrations of acetonitrile and buŠerionic strength. PTC-amino acids eluted from column aremonitored at 254 nm.

Detection limit is considered to be 1 pmol for most of theamino acid derivatives. Response linearity is obtained in therange of 20 to 500 pmol with correlation coe‹cients exceed-ing 0.999. To obtain good compositional data, samples largerthan 500 ng of protein/peptide before hydrolysis is best suit-ed for this amino acid analysis of proteins/peptides.

Method 4—Precolumn AQC Derivatization GeneralPrinciple

Precolumn derivatization of amino acids with 6-amino-quinolyl-N-hydroxysuccinimidyl carbamate (AQC) followedby reversed-phase HPLC separation with ‰uorometricdetection is used.

6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate(AQC) reacts with amino acids to form stable, ‰uorescent un-symmetric urea derivatives (AQC-amino acids) which are rea-dily amenable to analysis by reversed-phase HPLC. There-fore, precolumn derivatization of amino acids with AQC fol-lowed by reversed-phase HPLC separation is used to analyzethe amino acid composition.

Separation of the AQC-amino acids on ODS column isaccomplished through a combination of changes in concen-trations of acetonitrile and salt. Selective ‰uorescence detec-tion of the derivatives with excitation wavelength at 250 nmand emission wavelength at 395 nm allows for the directinjection of the reaction mixture with no signiˆcant interfer-ence from the only major ‰uorescent reagent by-product,6-aminoquinoline. Excess reagent is rapidly hydrolyzed (t1/2

º15 seconds) to yield 6-aminoquinoline, N-hydroxysuc-cinimide and carbon dioxide, and after 1 minute no furtherderivatization can take place.

Peak areas for AQC-amino acids are essentially unchangedfor at least 1 week at room temperature, and the derivativeshave more than su‹cient stability to allow for overnight au-tomated chromatographic analysis.

Detection limit is considered to be ranging from ca. 40 to320 fmol for each amino acid, except for Cys. Detection limitfor Cys is approximately 800 fmol. Response linearity is ob-tained in the range of 2.5 to 200 mmol/L with correlationcoe‹cients exceeding 0.999. Good compositional data could

be obtained from the analysis of derivatized protein hydroly-sates containing as little as 30 ng of protein/peptide.Method 5—Precolumn OPA Derivatization GeneralPrinciple

Precolumn derivatization of amino acids with o-phthalal-dehyde (OPA) followed by reversed-phase HPLC separationwith ‰uorometric detection is used. This technique does notdetect amino acids that exist as secondary amines (e.g.,proline).

o-Phthalaldehyde (OPA) in conjunction with a thiolreagent reacts with primary amine groups to form highly‰uorescent isoindole products. 2-Mercaptoethanol or 3-mer-captopropionic acid can be used as the thiol. OPA itself doesnot ‰uoresce and consequently produces no interferingpeaks. In addition, its solubility and stability in aqueoussolution, along with the rapid kinetics for the reaction, makeit amenable to automated derivatization and analysis usingan autosampler to mix the sample with the reagent. However,lack of reactivity with secondary amino acids has beenpredominant drawback. This method does not detect aminoacids that exist as secondary amines (e.g., proline). To com-pensate for this drawback, this technique may be combinedwith another technique described in Method 7 or Method 8.

Precolumn derivatization of amino acids with OPA isfollowed by a reversed-phase HPLC separation. Because ofthe instability of the OPA-amino acid derivative, HPLCseparation and analysis are performed immediately followingderivatization. The liquid chromatograph is equipped with a‰uorometric detector for the detection of derivatized aminoacids. Fluorescence intensity of OPA-derivatized amino acidsis monitored with an excitation wavelength of 348 nm and anemission wavelength of 450 nm.

Detection limits as low as 50 fmol via ‰uorescence havebeen reported, although the practical limit of analysisremains at 1 pmol.

Method 6—Precolumn DABS-Cl Derivatization GeneralPrinciple

Precolumn derivatization of amino acids with(dimethylamino)azobenzenesulfonyl chloride (DABS-Cl) fol-lowed by reversed-phase HPLC separation with visible lightdetection is used.

(Dimethylamino)azobenzenesulfonyl chloride (DABS-Cl)is a chromophoric reagent employed for the labeling ofamino acids. Amino acids labeled with DABS-Cl (DABS-amino acids) are highly stable and show the maximumabsorption at 436 nm.

DABS-amino acids, all 19 naturally occurring amino acidsderivatives, can be separated on an ODS column of areversed-phase HPLC by employing gradient systems consist-ing of acetonitrile and aqueous buŠer mixture. SeparatedDABS-amino acids eluted from column are detected at436 nm in the visible region.

This Method can analyze the imino acids such as prolinetogether with the amino acids at the same degree of sensitivi-ty, DABS-Cl derivatization method permits the simultaneousquantiˆcation of tryptophan residues by previous hydrolysisof the protein/peptide with sulfonic acids such as mercap-toethanesulfonic acid, p-toluenesulfonic acid or methanesul-fonic acid described under Method 2 in ``Protein Hydroly-sis''. The other acid-labile residues, asparagine and gluta-mine, can also be analysed by previous conversion into di-aminopropionic acid and diaminobutyric acid, respectively,


by treatment of protein/peptide with BTI described underMethod 11 in ``Protein Hydrolysis''.

The non-proteinogenic amino acid, norleucine cannot beused as internal standard in this method, as this compound iseluted in a chromatographic region crowded with peaks ofprimary amino acids. Nitrotyrosine can be used as an internalstandard, because it is eluted in a clean region.

Detection limit of DABS-amino acid is about 1 pmol. Aslittle as 2 to 5 pmol of an individual DABS-amino acid can bequantitatively analysed with reliability, and only 10 to 30 ngof the dabsylated protein hydrolysate is required for eachanalysis.

Method 7—Precolumn FMOC-Cl Derivatization GeneralPrinciple

Precolumn derivatization of amino acids with 9-‰uorenylmethyl chloroformate (FMOC-Cl) followed byreversed-phase HPLC separation with ‰uorometric detectionis used.

9-Fluorenylmethyl chloroformate (FMOC-Cl) reacts withboth primary and secondary amino acids to form highly‰uorescent products. The reaction of FMOC-Cl with aminoacid proceeds under mild conditions in aqueous solution andis completed in 30 seconds. The derivatives are stable, onlythe histidine derivative showing any breakdown. AlthoughFMOC-Cl is ‰uorescent itself, the reagent excess and‰uorescent side-products can be eliminated without loss ofFMOC-amino acids.

FMOC-amino acids are separated by a reversed-phaseHPLC using ODS column. The separation is carried out bygradient elution varied linearly from a mixture of acetonitrilemethanol and acetic acid buŠer (10:40:50) to a mixture ofacetonitrile and acetic acid buŠer (50:50), and 20 amino acidderivatives are separated in 20 minutes. Each derivative elut-ed from column is monitored by a ‰uorometric detector set atan excitation wavelength of 260 nm and an emissionwavelength of 313 nm.

The detection limit is in the low fmol range. A linearityrange of 0.1 to 50 mmol/L is obtained for most of the aminoacids.

Method 8—Precolumn NBD-F Derivatization GeneralPrinciple

Precolumn derivatization of amino acids with 7-‰uoro-4-nitrobenzo-2-oxa-1.3-diazole (NBD-F) followed by reversed-phase HPLC separation with ‰uorometric detection is used.

7-‰uoro-4-nitrobenzo-2-oxa-1.3-diazole (NBD-F) reactswith both primary and secondary amino acids to form highly‰uorescent products. Amino acids are derivatized with NBD-F by heating to 609C for 5 minutes.

NBD-amino acid derivatives are separated on an ODScolumn of a reversed-phase HPLC by employing gradientelution system consisting of acetonitrile and aqueous buŠermixture, and 17 amino acid derivatives are separated in 35minutes. e-Aminocaproic acid can be used as an internalstandard, because it is eluted in a clean chromatographicregion. Each derivative eluted from column is monitored by a‰uorometric detector set at an excitation wavelength of480 nm and an emission wavelength of 530 nm.

The sensitivity of this method is almost the same asfor precolumn OPA derivatization method (Method 5),excluding proline to which OPA is not reactive, and might beadvantageous for NBD-F against OPA. The detection limitfor each amino acid is about 10 fmol. Proˆle analysis was

achieved for about 1.5 mg of protein hydrolysates in the ˆnalprecolumn labeling reaction mixture for HPLC.Data Calculation and Analysis

When determining the amino acid content of a pro-tein/peptide hydrolysate, it should be noted that the acidhydrolysis step destroys tryptophan and cysteine. Serine andthreonine are partially destroyed by acid hydrolysis, whileisoleucine and valine residues may be only partially cleaved.Methionine can undergo oxidation during acid hydrolysis,and some amino acids (e.g., glycine and serine) are commoncontaminants. Application of adequate vacuum (≦0.0267kPa) or introduction of inert gas (argon) in the headspace ofthe reaction vessel during vapor phase hydrolysis can reducethe level of oxidative destruction. Therefore, the quantitativeresults obtained for cysteine, tryptophan, threonine, isoleu-cine, valine, methionine, glycine, and serine from a pro-tein/peptide hydrolysate may be variable and may warrantfurther investigation and consideration.

CalculationsAmino Acid Mole Percent This is the number of speciˆc

amino acid residues per 100 residues in a protein. This resultmay be useful for evaluating amino acid analysis data whenthe molecular weight of the protein under investigation isunknown. This information can be used to corroborate theidentity of a protein/peptide and has other applications.Carefully identify and integrate the peaks obtained as direct-ed for each Procedure. Calculate the mole percent for eachamino acid present in the test sample by the formula:100rU/r,in which rU is the peak response, in nmol, of the amino acidunder test; and r is the sum of peak responses, in nmol, for allamino acids present in the test sample. Comparison of themole percent of the amino acids under test to data fromknown proteins can help establish or corroborate the identityof the sample protein.

Unknown Protein Samples This data analysis techniquecan be used to estimate the protein concentration of anunknown protein sample using the amino acid analysis data.Calculate the mass, in mg, of each recovered amino acid bythe formula:mMW/1000,in which m is the recovered quantity, in nmol, of the aminoacid under test; and MW is the average molecular weight forthat amino acid, corrected for the weight of the watermolecule that was eliminated during peptide bond formation.The sum of the masses of the recovered amino acids will givean estimate of the total mass of the protein analyzed after ap-propriate correction for partially and completely destroyedamino acids. If the molecular weight of the unknown proteinis available (i.e., by SDS-PAGE analysis or mass spec-troscopy), the amino acid composition of the unknown pro-tein can be predicted. Calculate the number of residues ofeach amino acid by the formula:m/(1000M/MWT),in which m is the recovered quantity, in nmol, of the aminoacid under test; M is the total mass, in mg, of the protein; andMWT is the molecular weight of the unknown protein.

Known Protein Samples This data analysis technique canbe used to investigate the amino acid composition and pro-tein concentration of a protein sample of known molecularweight and amino acid composition using the amino acidanalysis data. When the composition of the protein being

16621662 JP XVAristolochic Acid / General Information

analyzed is known, one can exploit the fact that some aminoacids are recovered well, while other amino acid recoveriesmay be compromised because of complete or partial destruc-tion (e.g., tryptophan, cysteine, threonine, serine, methio-nine), incomplete bond cleavage (i.e., for isoleucine andvaline) and free amino acid contamination (i.e., by glycineand serine).

Because those amino acids that are recovered bestrepresent the protein, these amino acids are chosen toquantify the amount of protein. Well-recovered amino acidsare, typically, aspartate-asparagine, glutamate-glutamine,alanine, leucine, phenylalanine, lysine, and arginine. This listcan be modiêd based on experience with one's own analysissystem. Divide the quantity, in nmol, of each of the well-recovered amino acids by the expected number of residues forthat amino acid to obtain the protein content based on eachwell-recovered amino acid. Average the protein contentresults calculated. The protein content determined for eachof the well-recovered amino acids should be evenly distribut-ed about the mean. Discard protein content values for thoseamino acids that have an unacceptable deviation from themean. Typically »greater than 5z variation from the meanis considered unacceptable. Recalculate the mean proteincontent from the remaining values to obtain the protein con-tent of the sample. Divide the content of each amino acid bythe calculated mean protein content to determine the aminoacid composition of the sample by analysis.

Calculate the relative compositional error, in percentage,by the formula:

100m/mS,in which m is the experimentally determined quantity, innmol per amino acid residue, of the amino acid under test;and mS is the known residue value for that amino acid. Theaverage relative compositional error is the average of theabsolute values of the relative compositional errors of theindividual amino acids, typically excluding tryptophan andcysteine from this calculation. The average relative composi-tional error can provide important information on the stabil-ity of analysis run over time. The agreement in the aminoacid composition between the protein sample and the knowncomposition can be used to corroborate the identity and puri-ty of the protein in the sample.

2. Aristolochic AcidAristolochic acid, which occurs in plants of Aristolochia-

ceae, is suspected to cause renal damage. It is also reported tobe oncogenic (see References).

Aristolochic acid toxicity will not be a problem if crudedrugs of the origin and parts designated in the JP are used,but there may be diŠerences in crude drug nomenclature be-tween diŠerent countries, and it is known that crude drugpreparations not meeting the speciˆcations of the JP are cir-culating in some countries. Consequently, when crude drugsor their preparations are used, it is important that the materi-als should not include any plant containing aristolochic acid.

Since Supplement I to JP14, the test for aristolochic acid Iwas added to the Purity under Asiasarum Root, which con-sists of the rhizome and root. Because the aerial part of theplant may contain aristolochic acid and may have beenimproperly contaminated in Asiasarum Root.

It is considered that Akebia Stem, Sinomenium Stem andSaussurea Root do not contain aristolochic acid, unlessplants of origin other than that designated in the JP are used.However, contamination of aristolochic acid might occur, asmentioned above. In this case, the test described in the Purityunder Asiasarum Root is useful for checking the presence ofaristolochic acid.References:

Drug & Medical Device Safety Information (No.161) (July,2000).

New England Journal of Medicine (June 8, 2000).Mutation Research 515, 63–72 (2002).

3. Basic Requirements for ViralSafety of BiotechnologicalWBiological Products listed in

Japanese Pharmacopoeia

IntroductionThe primary role of speciˆcation of biotechnologicalWbio-

logical products listed in Japanese Pharmacopoeia (JP) is notonly for securing quality control or consistency of the qualitybut also for assuring their e‹cacy and safety. In the mean-time, the requirements to assure quality and safety of drugshave come to be quite strict recently, and a rigid attitudeaddressing safety assurance is expected for biotechnologicalWbiological products. The key points for quality and safety as-surance of biotechnologicalWbiological products are selectionand appropriate evaluation of source material, appropriateevaluation of manufacturing process and maintenance ofmanufacturing consistency, and control of speciˆc physicalproperties of the products. Now, how to assure quality andsafety of such drugs within a scope of JP has come to bequestioned. This General Information describes what sorts ofapproaches are available to overcome these issues.

It is desired that quality and safety assurance of JP listedproducts are achieved by state-of-the-art methods and con-cepts which re‰ect progress of science and accumulation ofexperiences. This General Information challenges to show thehighest level of current scientiˆc speculation. It is expectedthat this information will contribute to promotion of scien-tiˆc understanding of quality and safety assurance of notonly JP listed products but also the other biotechnologicalWbiological products and to promotion of active discussion ofeach O‹cial Monograph in JP.

1. Fundamental measures to ensure viral safety of JP listedbiotechnologicalWbiological products

The biotechnologicalWbiological product JP includes theproducts derived from living tissue and body ‰uid (urine,blood, etc.) of mammals, etc. Protein drugs derived from celllines of human or animal origin (e.g., recombinant DNAdrug, cell culture drug) are also included. The fundamentalmeasures required for comprehensive viral safety of JP listedbiotechnologicalWbiological products are as follows: 1) ac-quaintance of possible virus contamination (source of con-tamination); 2) careful examination of eligibility of rawmaterials and their sources, e.g. humanWanimal, andthorough analysis and screening of the sample chosen as asubstrate for drug production (e.g., pooled body ‰uid, cell

1663

Table 1. Items described in General Informationfor Viral Safety Assurance of JP listedBiotechnologicalWBiological Product

I. Introduction1. Fundamental measures to ensure viral safety of JP listed

biotechnologicalWbiological products2. Safety assurance measures described in the O‹cial

Monograph and this General Information3. Items and contents described in this General Informa-

tionII. General Matters

1. Purpose2. Background3. Unknown risk on the measures taken for ensuring viral

safety4. Applicable range5. Possible viral contamination to a JP listed biotechno-

logicalWbiological product (source of virus contamina-tion)

6. Basis for ensuring viral safety7. Limit of virus test8. Roles of viral clearance studies

III. Raw materialWsubstrate for drug production1. Issues relating to animal species and its region as a

source of raw materialWsubstrate for drug productionand countermeasures to be taken thereto

2. Qualiˆcation evaluation test on human or animal as asource of raw materialWsubstrate for drug production

IV. Points of concern with respect to manufacturing and vi-rus testing

1. Virus test conducted in advance of puriˆcation process2. Virus test as an acceptance test of an intermediate

material, etc.3. Virus test on a ˆnal product

V. Process evaluation on viral clearance1. Rationale, objective and general items to be concerned

with respect to viral clearance process evaluation2. Selection of virus3. Design of viral clearance studies4. Interpretation of viral clearance studies

1) Evaluation of viral clearance factor2) Calculation of viral clearance index3) Interpretation of results and items to be concerned at

evaluationVI. Statistics

1. Statistical considerations for assessing virus assays2. Reproducibility and conˆdence limit of viral clearance

studiesVII. Re-evaluation of viral clearanceVIII. Measurement for viral clearance studies

1. Measurement of virus infective titer2. Testing by nucleic-acid ampliˆcation test (NAT)

IX. Reporting and preservationX. Others

1663JP XV General Information / Basic Requirements for Viral

bank, etc.) to determine any virus contamination and deter-mination of type and nature of the virus, if contaminated; 3)evaluation to determine virus titer and virus-like particleshazardous to human, if exists; 4) selection of productionrelated material (e.g., reagent, immune antibody column)free from infectious or pathogenic virus; 5) performance ofvirus free test at an appropriate stage of manufacturing in-cluding the ˆnal product, if necessary; 6) adoption of eŠec-tive viral clearance method in the manufacturing process toremoveWinactivate virus. Combined method sometimesachieves higher level of clearance; 7) development of adeliberate viral clearance scheme; 8) performance of the testto evaluate viral removal and inactivation. It is consideredthat the stepwise and supplemental adoption of the saidmeasures will contribute to ensure viral safety and its im-provement.

2. Safety assurance measures described in the O‹cialMonograph and this General Information

As mentioned in above 1, this General Informationdescribes, in package, points to be concerned with and con-crete information on the measures taken for viral safety of JPlisted products. Except where any speciˆc caution is providedin O‹cial Monograph of a product in question, O‹cialMonograph provides in general that `Àny raw material, sub-strate for drug production and production related materialused for production of drug should be derived from healthyanimals and should be shown to be free of latent virus whichis infectious or pathogenic to human'', ``Cell line and culturemethod well evaluated in aspects of appropriateness and ra-tionality on viral safety are used for production, and thepresence of infectious or pathogenic latent virus to human inprocess related materials derived from living organismsshould be denied''. and ``biotechnologicalWbiological drugshould be produced through a manufacturing process whichis capable of removing infectious or pathogenic virus'', etc.,to raise awareness on viral safety and on necessity to conducttest and process evaluation for viral safety.

3. Items and contents described in this General InformationAs for viral safety of protein drug derived from cell line of

human or animal origin, there is a Notice in Japan entitled``Viral safety evaluation of biotechnology products derivedfrom cell lines of human or animal origin'' (Iyakushin No.329 issued on February 22, 2000 by Director, Evaluation andLicensing Division, Pharmaceutical and Medical SafetyBureau, Ministry of Health and Welfare) to re‰ect the inter-nationally harmonized ICH Guideline, and as for blood plas-ma protein fraction preparations, there is a document enti-tled ``Guideline for ensuring viral safety of blood plasmaprotein fraction preparations''. This General Informationfor ensuring viral safety of JP listed biotechnologicalWbiolog-ical products has been written, referencing the contents ofthose guidelines, to cover general points and their details tobe concerned for ensuring viral safety of not only JP listedbiotechnologicalWbiological products but also all productswhich would be listed in JP in future, i.e., biologicalproducts derived from living tissue and body ‰uid, such asurine, and protein drugs derived from cell line of human oranimal origin (Table 1).3.1 Purpose

The purpose of this document is to propose the compre-hensive concepts of the measures to be taken for ensuringviral safety of biotechnologicalWbiological products derived

from living tissue or body ‰uid of mammals, etc. and of pro-tein drugs derived from cell lines of human or animal origin.That is to say, this document describes the measures and thepoints of concern on the items, such as consideration ofthe source of virus contamination; appropriate evaluationon eligibility at selecting the raw material and on qualiˆcation

16641664 JP XVBasic Requirements for Viral / General Information

of its source, e.g. human or animal; virus test, and itsanalysis and evaluation at a stage of cell substrate for drugproduction; appropriate evaluation to choose productrelated materials derived from living organisms (e.g. reagent,immune antibody column, etc.); conduct of necessary vi-rus test on the product at an appropriate stage of manufac-turing; development of viral clearance test scheme; per-formance and evaluation of viral clearance test. This docu-ment is also purposed to comprehensively describe in detailsthat supplemental and combining adoption of the said meas-ures will contribute to secure viral safety and its improve-ment.3.2 Background

One of the most important issues to be cautioned for safetyof a biological product, which is directly derived from humanor animal, or of a protein drug, which is derived from cellline of human or animal origin (recombinant DNA derivedproduct, cell culture derived product, etc.), is risk of viruscontamination. Virus contamination may cause serious situa-tion at clinical use once it occurs. Virus contamination maybe from a raw material or from a cell substrate for drugproduction, or may be from an adventitious factor in-troduced to the manufacturing process.

JP listed biological drugs or protein drugs derived fromcell line have achieved drastic contribution to the medicalsociety, and to date, there has not been any evidence of anysafety problem on them caused by virus. But, social require-ment of health hazard prevention is strong, and it is now veryimportant to prevent accidental incidence, taking securitymeasures carefully supported by scientiˆc rationality. It isalways great concern among the persons involved that underwhat sort of viewpoint and to what extent we have to pursuefor ensuring viral safety of a biotechnologicalWbiologicalproduct. Before discussing these issues, two fundamentalpoints have to be reconˆrmed. One is that; we have to con-sider scientiˆc, medical, and social proˆles a drug has. Inother words, ``Medicine is a social asset which is utilized inmedical practice paying attention to the risk and beneˆt fromthe standpoints of science and society''. It is the destine andthe mission of the medicalWpharmaceutical society to realizeprompt and stable supply of such a social asset, drug, amongthe medical work front to bring gospel to the patients.

The other is that; issue of viral safety is independent fromsafety of the components of a drug per se (narrow sense ofsafety). It is important to consider that this is the matter ofgeneral safety of drug (broad sense of safety). In case of adrug which has been used for a long time in the medicalfront, such as a JP listed product, its broad sense of safety isconsidered to have been established epidemiologically, andits usage past records have a great meaning. However, diŠer-ent from safety of drug per se (its components), taking intoaccount any possibility of virus contamination, we have tosay that only the results accumulated can not always assureviral safety of a drug used in future. Accordingly, the basisfor securing broad sense of viral safety of JP listedbiotechnologicalWbiological products is to pay every attentionto the measures to take for prevention, while evaluating theaccumulated results.

Adopting strict regulations and conducting tests at maxi-mum level to the extent theoretically considered may be theways oŠ assuring safety, but applying such way generally,without su‹cient scientiˆc review of the ways and evaluationof usage results, causes excessive requirement of regulation

and test not having scientiˆc rationality. As the results, eŠec-tive and prompt supply of an important drug, already havingenough accumulation of experiences, to the medical workfront will be hampered, and the drug, a social asset, may notto be utilized eŠectively. Medicine is a sword used in medicalêld having double-edge named eŠectiveness and safety.EŠectiveness and safety factors have to be derived as thefruits of leading edge of science, and relatively evaluated on abalance sheet of usefulness. Usefulness evaluation should notbe unbalanced in a way that too much emphasis is placed onsafety concern without back-up of appropriate scientiˆc ra-tionality. A drug can play an important role as a social assetonly when well balanced appropriate scientiˆc usefulnessevaluation in addition to social concern of the age are given.In other words, drug is a common asset utilized by society formedication as a fruit of science of the age, and the key pointof its utilization lies on a balance of risk and beneˆt producedfrom scientiˆc and social evaluation. So, those factors haveto be taken into account when target and pursuance levels forensuring viral safety of a JP listed biotechnologicalWbiologi-cal product are reviewed.

And, in general, the risk and beneˆt of drugs should beconsidered with the relative comparison to alternative drugsor medical treatment. The usefulness of certain drug shouldbe reviewed ˆnally after the competitive assessment on therisk and beneˆt on the alternative drugs, relevant drugs andWor alternative medical treatment.

Under such background, the purpose of this article is todescribe the scientiˆc and rational measures to be taken forensuring viral safety of JP listed biotechnologicalWbiologicalproducts. Giving scientiˆc and rational measures mean that;appropriate and eŠective measures, elaborated from the cur-rent scientiˆc level, are given to the issues assumable underthe current scientiˆc knowledge. In other words, possiblecontaminant virus is assumed to have the natures of genus,morph, particle size, physicalWchemical properties, etc. whichare within the range of knowledge of existing virology, and isthose assumed to exist in human and animal, tissue and body‰uid, which are the source of biotechnologicalWbiologicalproduct, reagent, material, additives, etc. Accordingly, viralclearance studies using a detection method which target thoseviruses have to be designed.3.3 Unknown risk on the measures taken for ensuring viralsafety

There are known and unknown risks.It is easy to determine a test method and an evaluation

standard on the known risk, which exists in the drug per se(pharmaceutical component) or inevitably exists due to aquality threshold, and quantiˆcation of such risk is possible.In other words, it is easy to evaluate the known risk on abalance sheet in relation to the beneˆt, and we can say thatvaluation even in this respect has been established to some ex-tent.

On the other hand, as for the unknown risk which is inevit-able for ensuring viral safety, the subject of the risk can notbe deˆned and quantitative concept is hard to introduce, and,therefore, taking a counter measure and evaluating its eŠectare not so easy. Therefore, this is the subject to be challengedcalling upon wisdom of the related parties among the societyof drug.

Talking about the unknown risk, there are view points thatsay `Ìt is risky because it is unknown.'' and ``What are theunknowns, and how do we cope with them in ensuring safe-


ty?''.The view of `Ìt is risky because it is unknown.'' is already

nothing but a sort of evaluation result, and directly connectsto a ˆnal decision if it can be used as a drug. Such evaluationWdecision has to be made based upon a rational, scientiˆc orsocial judgment.

For example, in the case that `Ìn a manufacturing processof drug, virus, virus-like particle or retrovirus was detected,but its identiˆcation could not be conˆrmed, and, therefore,its risk can not be denied.'', the evaluation of `Ìt is risky be-cause it is unknown.'' is scientiˆcally rational and reasona-ble. On the other hand, however, if we reach a decision of `Ìtis risky because it is unknown.'' due to the reason that `Ìn amanufacturing process of drug, virus, virus-like particle orretrovirus was not detected, but there is a `concern' thatsomething unknown may exist.'', it can not be said that suchevaluation is based upon a rational, scientiˆc or social judg-ment. It goes without saying that the utmost care has to betaken for viral safety, but the substance of `concern' has tobe at least clearly explainable. Otherwise, the `concern' mayresult in causing contradiction in the meaningful mission toutilize a social asset, drug, in medical practice.

From scientiˆc view point, we should not be narrow mind-ed by saying `ìt is risky'' because ``there is a `concern' thatsomething unknown may exists'', but challenge to clarify thesubject of ``What is unknown, and how to cope with it forensuring safety'' using wisdom. What is important at thetime is to deˆne ``what is unknown'' based upon currentscientiˆc knowledge. Only through this way, is it possible forus to elaborate the measures for ensuring safety.

Once we chase up the substance of unknown risk for viralsafety without premises of ``what is unknown'', `ùnknown''will be an endless question because it theoretically remainsunresolved forever. If this kind of approach is taken, the is-sue and the measure can not be scientiˆcally connected toeach other, which will result in the excessive requirement ofregulation and of test to be conducted. Yet, it is unlikely thatthe measure which has no relation with science will be eŠec-tive to the subject of ``What is unknown is unknown.''

For example, ``what is unknown'' at the `èvaluation of apuriˆcation process which can completely clear up every virusthat contaminated in a manufacturing process'' should be thesubject of ``what sort of existing virus that contaminated isunknown'', not on the subject of ``what sort of virus that ex-ist in the world is unknown. In the former subject, thepremise of the study is based on all the knowledge on virusesincluding DNAWRNA-virus, virus withWwithout envelope,particle size, physicalWchemical properties, etc. The premiseis that the virus contaminated should be within range of exist-ing wisdom and knowledge of virus such as species, type, na-ture, etc., even though the virus that contaminated isunknown. Under such premise, when evaluation is made on apuriˆcation process to decide its capability of clearing a der-ived virus, which is within the range of existing wisdom andlearning, speciˆc viral clearance studies designed to combinea few model viruses with diŠerent natures, such as type ofnucleic acid, withWwithout envelope, particle size, physicalWchemical properties, etc., would be enough to simulate everysort of the virus already known, and will be a ``good measurefor ensuring safety''.

The issue of ``the sort of viruses that exist in the world isunknown'' may be a future study item, but it is not an ap-propriate subject for the viral clearance test. Further, even if

the subject of `ùnknown viruses, which have a particle sizesmaller than that of currently known viruses, may exists'' or`ùnknown viruses, which have special physicalWchemicalproperties that can not be matched to any of the currentlyknown viruses, may exists'' is set up as an armchair theory,any experimental work can not be pursued under the currentscientiˆc level, since such virus model is not available. Fur-ther, any viral clearance test performed by using the currentlyavailable methods and technologies will be meaningless ``forensuring safety'', since particle size or natures of such specu-lated virus are unknown. Likewise, any counter measures cannot be taken on the subject of `ùnknown virus, which cannot be detected by currently available screening method, mayexist'', and conducting any virus detection test at any stagewill be useless ``for ensuring safety''.

The requirement of regulations or tests excessively overscientiˆc rationality will raise human, economical and tem-poral burden to the pharmaceutical companies, and will ad-versely aŠect prompt, eŠective and economical supply of adrug to the medical front. As drug is a sort of social asset,which has to be scientiˆcally evaluated, how to assure max-imization of its safety by means of scientiˆcally rational ap-proach at minimum human, economical and time resources isimportant.

It is also important to reconˆrm that achievement of thoseissues is on the premise that appropriate measures are takenon the supply source of drugs. For example, in a case of `Ìn amanufacturing process of drug, virus, virus-like particle orretrovirus was not detected, but there is a `concern' thatsomething unknown may exist.'', appropriateness of the test,which resulted in the judgment that ``virus, virus-like particleor retrovirus was not detected in a process of drug produc-tion'', should be a prerequisite premise when judged byscience standard ate the time. If there is any question on thepremise, it is quite natural that the question of ``there is a`concern' that unknown something may exist.'' will be eŠec-tive.3.4 Applicable range

This General Information is on JP listed biologicalproducts, derived from living tissue or body ‰uid, and pro-tein drugs, derived from human or animal cell line, that inJapan. In the case of protein drugs derived from human oranimal cell line, the products developed and approved beforeenforcement of the Notice Iyakushin No. 329 entitled ``Viralsafety evaluation of biotechnology products derived from celllines of human or animal origin'' should have been treatedunder the Notice had there been one, and it is inevitable thatsome products approved after the Notice might not have beensu‹ciently treated. It is expected that such biodrug will besu‹ciently examined to meet such General Information be-fore being listed in JP. On the other hand, blood prepara-tions listed in the biological products standard and coveredby ``Guideline for securing safety of blood plasma proteinfraction preparations against virus'', are out of the scope ofthis General Information. Further, in case of a relatively low-er molecular biogenous substance, such as amino acid, sac-charide and glycerin, and of gelatin, which is even classiêdas infectious or pathogenic polymer, there are cases that viralcontamination can not be considered due to its manufactur-ing or puriˆcation process, and that potent viral inactivationWremoval procedure that can not be applied to protein, can beused, and, therefore, it is considered reasonable to omit suchsubstances from the subject for application. However, some


part of this General Information may be used as reference.Further, a comprehensive assurance measure for viral safetyis recommendable on a biotechnologicalWbiological productnot listed in JP using this document as a reference so long asit is similar to the biotechnologicalWbiological product JP.3.5 Possible viral contamination to a JP listed biotechno-logicalWbiological product (source of virus contamination)

Promoting awareness of virus contamination to a JP listedbiotechnologicalWbiological product (source of virus con-tamination) and citing countermeasure thereof are importantfor eradicating any possible virus contamination and raisingprobability of safety assurance. Many biotechnologicalWbio-logical products are produced from a ``substrate'' which isderived from human or animal tissue, body ‰uid, etc. as anoriginWraw material, and in puriˆcation or pharmaceuticalprocessing of such products column materials or additives,which are living organism origin, are occasionally used. Ac-cordingly, enough safety measures should be taken againstdiŠusion of the contaminant virus. Further, as mentioned inNotice Iyakushin No. 329, any protein drug derived from celllines of human or animal origin should be carefully examinedwith respect to the risk of virus contamination through thecell line, the cell substrate for drug production, and throughthe manufacturing process applied thereafter.

``Substrate for drug production'' is deˆned as a startingmaterial which is at a stage where it is deemed to be in a posi-tion to ensure qualityWsafety of an active substance. The``substrate for drug production'' is sometimes tissue, body‰uid, etc. of human or animal per se and pooled materialsuch as urine, and sometimes a material after some treat-ment. In many cases, it is considered rational that startingpoint of full-scale test, evaluation and control should be atthe stage of ``substrate for drug production''. The more strictlevels of test, evaluation and control achieved at the stage of``substrate for drug production'' can more rationalize evalu-ation and control of the raw material or individual level ofupper stream. On the contrary, strict evaluation and controlof the raw material or individual level at an upper streamstage can rationalize tests, evaluation or quality control at thestage of ``substrate for drug production''.

The measures taken for ensuring viral safety on abiotechnologicalWbiological product currently listed in JP canbe assumed from the provisions of manufacturing method,speciˆcation and test methods of each preparation. However,unitary principles or information with respect to the meas-ures to be taken for ensuring viral safety, totally reviewingthe entire process up to the ˆnal product rationally and com-prehensively, including source Wraw material Wsubstrate,puriˆcation process, etc. have not been clariêd. The mostimportant thing for ensuring viral safety is to take thoroughmeasures to eliminate the risk of virus contamination at anystage of source animal, raw material and substrate. Althoughnot the cases of a biotechnologicalWbiological product,known examples of a virus contamination from a raw materi-alWsubstrate for drug production in old times are Hepatitis AVirus (HAV) or Hepatitis C Virus (HCV) contamination inblood protein fraction preparations. It is also well knownthat Human Immunodeˆciency Virus (HIV) infection causedby blood plasma protein fraction preparations occurred in1980s. The aim of this General Information is to show con-crete guidelines for comprehensive viral safety assurance ofthe JP listed biotechnologicalWbiological products. Thepathogenic infectious viruses, currently known to con-

taminate to raw materials, etc. of drug and have to be cau-tioned, are HIV, HAV, Hepatitis B Virus (HBV), HCV, Hu-man T-Lymphotropic Virus (HTLV-IWII), Human Parvovi-rus B19, Cytomegalovirus (CMV), etc. BiotechnologicalWbio-logical products produced from raw materialWcell substratederived from tissue or body ‰uid of human or animal originalways have a risk of contamination of pathogenic or otherlatent virus. Therefore, safety measures should be thoroughlytaken. There is also the case that a material, other than thebiological component such as raw materialWsubstrate, causesvirus contamination. Using enzymatic or monoclonal an-tibody column or using albumin etc. as a stabilizer, is the ex-ample of the case, in which caution has to be taken on risk ofvirus contamination from the source animal or cell. Further,there is a possibility of contamination from environment orpersonnel in charge of production or at handling of theproduct. So, caution has to be taken on these respects as well.

In case of protein drugs derived from cell line of human oranimal origin, there may be cases where latent or persistentinfectious viruses (e.g., herpesvirus) or endogenousretroviruses exist in the cell. Further, adventitious virusesmay be introduced through the routes such as: 1) derivationof a cell line from an infected animal; 2) use of a virus todrive a cell line; 3) use of a contaminated biological reagent(e.g., animal serum components); 4) contamination duringcell handling. In the manufacturing process of drug, anadventitious virus may contaminate to the ˆnal productthrough the routes, such as 1) contamination through a rea-gent of living being origin, such as serum component, whichis used for culturing, etc.; 2) use of a virus for introduction ofa speciˆc gene expression to code an objective protein; 3)contamination through a reagent used for puriˆcation suchas monoclonal antibody a‹nity column; 4) contaminationthrough an additive used for formulation production; 5) con-tamination at handling of cell and culture media, etc. It isreported that monitoring of cell culture parameter may behelpful for early detection of an adventitious viral contami-nation.3.6 Basis for ensuring viral safety

Viral safety of a biotechnologicalWbiological product pro-duced from a raw materialWsubstrate, which derived from tis-sue, body ‰uid, cell line, etc. of human or animal origin, canbe achieved by supplemental and appropriate adoption of thefollowing plural methods.(1) Acquaintance of possible virus contamination (source

of contamination).(2) Careful examination of eligibility of the raw material

and its source, i.e., human or animal, thorough analysisand screening of the sample chosen as the substrate fordrug production to determine virus contamination andthrough examination of the type of virus and its nature,if contaminated.

(3) Evaluation to determine hazardous properties of the vi-rus or virus-like particle to human, if exists.

(4) Choosing a product related material of living organismorigin (e.g., reagent, immune anti-body column, etc.)which is free from infectious or pathogenic virus.

(5) Conduct virus free test at an appropriate stage ofmanufacturing including the ˆnal product, if necessary.

(6) Adoption of an eŠective method to removeWinactivatethe virus in the manufacturing process for viral clear-ance. Combined processes sometimes achieve higher lev-el of viral clearance.


(7) Develop a deliberate viral clearance scheme.(8) Conduct test and evaluation to conˆrm removalWinacti-

vation of the virus.Manufacturer is responsible for explaining rationality of

the way of approach adopted among the comprehensivestrategy for viral safety on each product and its manufactur-ing process. At the time, the approach described in thisGeneral Information shall be applicable as far as possible.3.7 Limit of virus test

Virus test has to be conducted to deˆne existence of virus,but it should be noted that virus test alone can not reach aconclusion of inexistence of virus nor su‹cient to secure safe-ty of the product. Examples of a virus not being detected areas follows: 1) Due to statistical reason, there is an inherentquantitative limit, such as detection sensitivity at lower con-centration depends upon the sample size. 2) Generally, everyvirus test has a detection limit, and any negative result of a vi-rus test can not completely deny existence of a virus. 3) A vi-rus test applied is not always appropriate in terms of speciˆci-ty or sensitivity for detection of a virus which exists in the tis-sue or body ‰uid of human or animal origin.

Virus testing method is improved as science and technologyprogress, and it is important to apply scientiˆcally the mostadvanced technology at the time of testing so that it can bepossible to raise the assurance level of virus detection. Itshould be noted, however, that the limit as mentioned abovecan not always be completely overcome. Further, risk of vi-rus contamination in a manufacturing process can not becompletely denied, and, therefore, it is necessary to elaboratethe countermeasure taken these eŠects into account.

Reliable assurance of viral free ˆnal product can not be ob-tained only by negative test results on the raw materialWsub-strate for drug production or on the product in general, it isalso necessary to demonstrate inactivationWremoval capabil-ity of the puriˆcation process.3.8 Roles of viral clearance studies

Under the premises as mentioned in the preceding clausethat there is a limit of a virus test, that there is a possibility ofexistence of latent virus in a raw materialWsubstrate for drugproduction and that there is a risk of entry of a non-en-dogenous virus in a manufacturing process, one of the im-portant measures for viral safety is how to remove or inacti-vate the virus, which exists in a raw material, etc. and can notbe detected, or the virus, which is contingently contaminatedin a manufacturing process. The purpose of viral clearancestudy is to experimentally evaluate the viral removalWinacti-vation capability of a step that mounted in a manufacturingprocess. So, it is necessary to conduct an experimental scalespike test using an appropriate virus that is selected by takingaccount the properties, such as particle size, shape, with orwithout envelope, type of nucleic acid (DNA type, RNAtype), heat and chemical treatment tolerance, etc., with anaim to determine removalWinactivation capability of the virusthat can not be detected in a raw material or contingentlycontaminated.

As mentioned above, the role of viral clearance study is tospeculate removalWinactivation capability of a processthrough a model test, and it contributes to give scientiˆc basisto assure that a biotechnologicalWbiological product of hu-man or animal origin has reached an acceptable level inaspect of viral safety.

At a viral clearance study, it is necessary to adopt an ap-propriate approach method which is deˆnitive and rational

and can assure viral safety of a ˆnal product, taking into con-sideration the source and the properties of the raw materialWsubstrate as well as the manufacturing process.

4. Raw materialWsubstrate for drug production4.1 Issues relating to animal species and its region as asource of raw materialWsubstrate for drug production andcountermeasures to be taken thereto

For manufacturing JP listed biotechnologicalWbiologicalproducts, which require measures for viral safety, a rawmaterialWsubstrate derived mainly from human, bovine,swine or equine is used, and it is obvious that such humanand animal has to be healthy nature. A wild animal should beavoided, and it is recommended to use animals derived froma colony controlled by an appropriate SPF (SpeciˆcPathogen-Free) condition and bred under a well deignedhygienic control, including appropriate control for preven-tion of microbial contamination and contamination monitor-ing system. If a meat standard for food is available, ananimal meeting this standard has to be used. The type of vi-rus to be concerned about depend on animal species, but itmay be possible to narrow down the virus for investigation bymeans of examining the hygiene control, applicability of ameat standard for food, etc. On the other hand, even with theanimals of the same species, a diŠerent approach may benecessary depending upon the region where the specimen fora raw materialWsubstrate is taken. For example, in case of ob-taining raw materialWsubstrate from blood or other speciˆcregion, it is necessary to be aware of the risk level, virus mul-tiplication risk, etc. which may speciˆcally exists dependingupon its region. Such approach may be diŠerent from thoseapplied to body waste such as urine, milk, etc. as a source ofraw materialWsubstrate. Further, caution has to be taken ontransmissible spongiform encephalopathy (TSE) when pitui-tary gland, etc. is used as a raw material. This report does notinclude detailed explanation on TSE, but recommendationsare to use raw material derived from 1) animals originated inthe countries (area) where incidence of TSE has not beenreported; 2) animals not infected by TSE; or 3) species ofanimal which has not been reported on TSE. It is recom-mended to discuss the matters concerned with TSE with theregulatory authority if there is any unclear point.

Followings are the raw materialWsubstrate used formanufacturing biotechnological Wbiological products inJapan.(1) Biological products derived from human

Blood plasma, placenta, urine, etc. derived from humanare used as the sources of raw material of biotechnologicalWbiological product. As for these raw materials, there are 2cases: 1) Appropriateness can be conˆrmed by interview orby examination of the individual who supplies each rawmaterial, and 2) Such su‹cient interview or examination ofthe individual can not be made due to type of raw material.In case that su‹cient examination of individual level is notpossible, it is necessary to perform test to deny virus contami-nation at an appropriate manufacturing stage, for example,the stage to decide it as a substrate for drug production.(2) Biological products derived from animal besides human

Insulin, gonadotropin, etc. are manufactured from bloodplasma or from various organs of bovine, swine and equine.(3) Protein drug derived from cell line of human or animalorigin

In the case of protein drugs derived from cell line of human


or animal origin, a cell line of human or animal is the rawmaterial per se, and the substrate for drug production is a cellbank prepared from cloned cell line (master cell bank orworking cell bank). Examination at cell bank level is consi-dered enough for viral safety qualiˆcation, but it goeswithout saying that the more appropriate and rational qualiˆ-cation evaluation test of cell bank can be realized when moreinformation is available on the virus of the source animal oron prehistory of driving the cell line, the base of cell bank.4.2 Qualiˆcation evaluation test on human or animal as asource of raw materialWsubstrate for drug production(1) Biological products derived from human

Body ‰uid etc. obtained from healthy human must be usedfor biological products production. Further, in case that in-terview or examination of the individual, who supplies theraw material, can be possible and is necessary, interview un-der an appropriate protocol and a serologic test well evaluat-ed in aspects of speciˆcity, sensitivity and accuracy have to beperformed, so that only the raw material, which is denied la-tent HBV, HCV and HIV, will be used. In addition to theabove, it is necessary to test for gene of HBV, HCV and HIVby a nucleic ampliˆcation test (NAT) well evaluated inaspects of speciˆcity, sensitivity and accuracy.

In case of the raw material (e.g., urine), which can not betested over the general medical examination of the individualwho supplies the material, or of the raw material which is ir-rational to conduct individual test, the pooled raw material,as the substrate for drug production, has to be conducted atleast to deny existence of HBV, HCV and HIV, using amethod well evaluated in aspects of speciˆcity, sensitivity andaccuracy, such as the antigen test or NAT.(2) Biological products derived from animal besides human

The animal used for manufacturing biological product hasto be under appropriate health control, and has to be con-ˆrmed of its health by various tests. Further, it is necessarythat the population, to which the animal belongs, has beenunder an appropriate breeding condition, and that no abnor-mal individual has been observed in the population. Further,it is necessary to demonstrate information or scientiˆc basiswhich can deny known causes infection or disease to human,or to deny such animal inherent latent virus by serologic testor by nucleic ampliˆcation test (NAT). The infectious virusthat is known to be common between human and animal, andknown to cause infection in each animal are tentatively listedin Table 2. It is necessary that the table is completed undercareful examination, and denial of all of them, by means oftests on individual animal, tissue, body ‰uid, etc. as a rawmaterial, or on pooled raw material (as a direct substrate fordrug production), is not always necessary. Table 2 can beused as reference information, in addition to the other infor-mation, such as; source of animal, health condition, healthand breeding control, conformity to the meat standard forfood, etc., to elaborate to which virus what kind of test has tobe performed, and for which virus it is not always necessaryto test for, etc. It is important to clarify and record the basisof choosing the virus and the test conducted thereof.

Table 2. Infectious viruses known to be commonbetween human and animal and known to

cause infection to each animal

bovine swine sheep goat equine

Cowpox virus w

Paravaccinia virus w w w w

Murray valley encephalitisvirus w w

Louping ill virus w w w w

Wesselsbron virus w

Foot-and-mouth diseasevirus w w

Japanese encephalitisvirus w

Vesicular stomatitis virus w

Bovine papular stomatitisvirus w

Orf virus w

Borna disease virus w w

Rabies virus w w w w w

In‰uenza virus w

Hepatitis E virus w

Encephalomyocarditisvirus w w

Rotavirus w

Eastern equineencephalitis virus w

Western equineencephalitis virus w

Venezuelan equineencephalitis virus w

Morbillivirus w

Hendra virus w

Nipah virus w

Transmissible gastroente-ritis virus w

Porcine respiratorycoronavirus w

Porcine epidemicdiarrhea virus w

Hemagglutinatingencephalomyelitis virus w

Porcine respiratory andreproductive syndrome virus w

Hog cholera virus w


Parain‰uenza virus Type 3 w

TelfanWTeschen diseasevirus w

Reovirus w

Endogenous retrovirus w

Porcine adenovirus w

Porcine circovirus w

Porcine parvovirus w

Porcine poxvirus w

Porcine cytomegalovirus w

Pseudorabies virus w

Russian spring-summerencephalitis virus w w

Rift Valley fever virus w w

Crimean-Congo hemor-rhagic fever virus(Nairovirus)

w w w

Torovirus w

(3) Protein drug derived from cell line of human or animalorigin

It is important to conduct thorough investigation on latentendogenous and non-endogenous virus contamination in amaster cell bank (MCB), which is the cell substrate for drugproduction, in accordance with the Notice Iyakushin No. 329entitled ``Viral safety evaluation of biotechnology productsderived from cell lines of human or animal origin''. Further,it is necessary to conduct an appropriate adventitious virustest (e.g., in vitro and in vivo test) and a latent endogenous vi-rus test on the cell at the limit of in vitro cell age (CAL) fordrug production. Each WCB as a starting cell substrate fordrug production should be tested for adventitious virus eitherby direct testing or by analysis of cells at the CAL, initiatedfrom the WCB. When appropriate non-endogenous virustests have been performed on the MCB and cells cultured upto or beyond the CAL have been derived from the WCB andused for testing for the presence of adventitious viruses, simi-lar tests need not be performed on the initial WCB.

5. Points of concern with respect to manufacturing andvirus testing

To ensure viral safety of a biological product derived fromtissue, body ‰uid etc. of human or animal origin, it is neces-sary to exclude any possibility of virus contamination from araw material, such as tissue and body ‰uid, or a substrate,paying attention to the source of virus contamination as men-tioned in above 3.5, and to adopt appropriate manufacturingconditions and technologies in addition to enhancement ofmanufacturing environment, so that virus contamination inthe course of process and handling and from operators, facil-ities and environment can be minimized.

In addition to the above, eŠective virus test and viral inac-tivationWremoval technology, which are re‰ected by rapidprogress of science, have to be introduced. Adoption of two

or more steps with diŠerent principles is recommended for vi-rus inactivationWremoval process. Further, it is important tominimize any possible virus derivation by using a reagent,which quality is equivalent to that of a drug. Examples of vi-rus inactivationWremoval processes are heating (It isreported that almost viruses are inactivated by heating at 55 –609C for 30 minutes with exceptions of hepatitis virus, etc.and that dry heating at 609C for 10 – 24 hours is eŠective incase of the products of blood or urine origin.), treatmentwith organic solventWsurfactant (SWD treatment), mem-brane ˆltration (15 – 50 nm), acid treatment, irradia-tion (g-irradiation, etc.), treatment with column chro-matograph (e.g. a‹nity chromatography, ion-exchange chro-matography), fractionation (e.g. organic solvent or am-monium sulfate fractionation), extraction.5.1 Virus test conducted in advance of puriˆcation process(1) Biological products derived from human

In many cases, samples for virus test before puriˆcationprocess are body ‰uid or tissue of individual collected as araw material, or its pooled material or extraction as a sub-strate. As mentioned in 4.2 (1), it is necessary to deny latentHBV, HCV and HIV by the test evaluated enough in aspectsof speciˆcity, sensitivity and accuracy. Even in a case that anon-puriêd bulk before puriˆcation process is producedfrom a substrate, it is not always necessary to conduct virustest again at the stage before puriˆcation, so long as thepresence of any latent virus can be denied at the stage of sub-strate by an appropriate virus test, with cases where the non-puriêd bulk is made from the substrate by adding any rea-gent etc. of living organisms origin are an exception.(2) Biological products derived from animal besides human

Similar to (1) above, samples for virus test before puriˆca-tion process are, in many cases, body ‰uid or tissue of in-dividual collected as a raw material, or its pooled material orextraction as a substrate. In these cases, it is necessary to havea data, which can deny latent virus of probable cause of hu-man infection or disease as mentioned in the above 4.2 (2), orto have a result of serologic test or nucleic ampliˆcation test(NAT) evaluated enough in aspects of speciˆcity, sensitivityand accuracy. The concept, which is applied to a case thatnon-puriêd bulk before puriˆcation process is producedfrom substrate, is the same as those provided in the above 4.2(1).(3) Protein drug derived from cell line of human or animalorigin

Generally, substrate in this case is cell bank, and the sam-ple for testing before puriˆcation process is a harvested cellafter cell culturing or unprocessed bulk which consists of sin-gle or pooled complex culture broth. The unprocessed bulkmay be sometimes culture broth without cell. Denial of latentvirus, which is determined by virus test at a MCB or WCBlevel, does not always deny latent virus in unprocessed bulkafter culturing. Further, it is noted that the viral test at theCAL is meaningful as a validation but can not guaranteedeˆnite assurance of latent virus denial, since the test isgenerally performed only once. In case of using a serum or acomponent of blood origin in a culture medium, deˆnitedenial of latent virus at the level of unprocessed bulk can notbe assured so long as the viral test has not been conducted oneach lot at the CAL, since lot renewal can be a variable factoron viral contamination.

A representative sample of the unprocessed bulk, removedfrom the production reactor prior to further processing,


represents one of the most suitable levels at which the pos-sibility of adventitious virus contamination can be deter-mined with a high probability of detection. Appropriate test-ing for viruses should be performed at the unprocessed bulklevel unless virus testing is made more sensitive by initial par-tial processing (e.g., unprocessed bulk may be toxic in testcell cultures, whereas partially processed bulk may not betoxic). In certain instances it may be more appropriate to testa mixture consisting of both intact and disrupted cells andtheir cell culture supernatants removed from the productionreactor prior to further processing.

In case of unprocessed bulk, it is required to conduct virustest on at least 3 lots obtained from pilot scale or commercialscale production. It is recommended that manufacturersdevelop programs for the ongoing assessment of adventitiousviruses in production batches. The scope, extent and fre-quency of virus testing on the unprocessed bulk should be de-termined by taking several points into consideration includ-ing the nature of the cell lines used to produce the desiredproducts, the results and extent of virus tests performed dur-ing the qualiˆcation of the cell lines, the cultivation method,raw material sources and results of viral clearance studies.Screening in vitro tests, using one or several cell lines, aregenerally employed to test unprocessed bulk. If appropriate,a NAT test or other suitable methods may be used.

Generally, harvest material in which adventitious virus hasbeen detected should not be used to manufacture theproduct. If any adventitious viruses are detected at this level,the process should be carefully checked to determine thecause of the contamination, and appropriate actions taken.5.2 Virus test as an acceptance test of an intermediatematerial, etc.

When a biological product is manufactured from tissue,body ‰uid etc. of human or animal origin, there are cases thatan intermediate material, partially processed as a raw materi-al or substrate by outside manufacturer, is purchased andused for manufacturing. In such case, if any test to meet thisGeneral Information has been conducted by such outsidemanufacturer, it is necessary for the manufacturer of the bio-logical product, who purchased the intermediate material, toexamine what sort of virus test has to be conducted as accep-tance tests, and to keep record on the basis of rationality in-cluding the details of the test conducted.

On the other hand, if no test to meet this General Informa-tion has been conducted by such outside manufacturer of theraw material, all necessary virus free test has to be conductedto meet this General Information on the intermediate materi-al regarding it as the direct substrate for drug production.5.3 Virus test on a ˆnal product

Virus tests to be conducted on a ˆnal product (or on aproduct to reach the ˆnal product) has to be deˆned undercomprehensive consideration of the type of raw material orsubstrate, the result of virus test conducted on raw materialWsubstrate, the result of evaluation on viral removalWinactiva-tion process, any possibility of virus contamination in themanufacturing process, etc. Comprehensive viral safety as-surance can only be achieved by appropriate selection of theraw materialWsubstrate, an appropriate virus test conductedon the raw materialWsubstrateWintermediate material, thevirus test conducted at an appropriate stage of manufactur-ing, an appropriate viral clearance test, etc. However, thereare cases of having speciˆc backgrounds, such as 1) use of theraw material derived from unspeciêd individual human, 2)

possible existence of virus at window period, 3) speciˆc detec-tion limit of virus test, etc. and in these cases, virus contami-nation to the ˆnal product may occur if there is any deˆciencyon the manufacturing process (e.g., damage of membraneˆlter) or any mix-up of the raw materials, etc. To avoid suchaccidental virus contamination, it may be recommended toconduct nucleic ampliˆcation test (NAT) on the ˆnal productfocusing on the most risky virus among those that may possi-bly to exist in the raw material.

6. Process evaluation on viral clearance6.1 Rationale, objective and general items to be concernedwith respect to viral clearance process evaluation

Evaluation of a viral inactivationWremoval process is im-portant for ensuring safety of a biological product derivedfrom tissue or body ‰uid of human or animal origin. Con-ducting evaluation on viral clearance is to assure, even tosome extent, elimination of the virus, which may exist in araw material, etc. or may be derived to the process due to un-expected situation. Viral clearance studies should be made bya carefully designed appropriate method, and has to be ra-tionally evaluated.

The objective of viral clearance studies is to assess processstep(s) that can be considered to be eŠective in inactivatingWremoving viruses and to estimate quantitatively the overalllevel of virus reduction obtained by the process. This shouldbe achieved by the deliberate addition (``spiking'') of sig-niˆcant amounts of a virus at diŠerent manufacturingWpuriˆ-cation steps and demonstrating its removal or inactivationduring the subsequent steps. It is not necessary to evaluate orcharacterize every step of a manufacturing process if ade-quate clearance is demonstrated by the use of fewer steps. Itshould be borne in mind that other steps in the process mayhave an indirect eŠect on the viral inactivationWremovalachieved. Manufacturers should explain and justify the ap-proach used in studies for evaluating viral clearance.

The reduction of virus infectivity may be achieved byremoval of virus particles or by inactivation of viral infectivi-ty. For each production step assessed, the possible mechan-ism of loss of viral infectivity should be described with regardto whether it is due to inactivation or removal. Forinactivation steps, the study should be planned in such a waythat samples are taken at diŠerent times and an inactivationcurve constructed.6.2 Selection of virus

To obtain broad range of information of viral inactivationWremoval, it is desirable that a model virus used for viralclearance studies should be chosen from the viruses withbroad range of characteristics in aspects of DNAWRNA, withor without envelope, particle size, signiˆcant resistance tophysicalWchemical treatment, etc. and it is necessary to com-bine about 3 model viruses to cover these characteristics.

At choice of a model virus, there are also the ways tochoose a virus closely related to or having the same character-istics of the virus known to exist in the raw material. In suchcase, it is in principle recommendable to choose a virus whichdemonstrates a higher resistance to inactivationWremovaltreatment if two or more candidate viruses are available forchoice. Further, a virus which can grow at a high titer isdesirable for choice, although this may not always be possi-ble. In addition to the above, choosing a virus, which willprovide eŠective and reliable assay result at each step, isnecessary, since sample condition to be tested at each step of

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Table 3. Example of viruses which have been used for viral clearance studies

Virus Family Genus Natural host Genome Env Size (nm) Shape Resistance

Vesicular Stomatitis Virus Rhabdo Vesiculovirus EquineBovine

RNA yes 70 × 150 Bullet Low

Parain‰uenza Virus Paramyxo Type 1,3Respirovirus

Type 2,4Rubulavirus

Various RNA yes 100 – 200＋ Pleo-Spher Low

MuLV Retro Type Concovirus

Mouse RNA yes 80 – 110 Spherical Low

Sindbis Virus Toga Alphavirus Human RNA yes 60 – 70 Spherical Low

BVDV Flavi Pestivirus Bovine RNA yes 50 – 70 Pleo-Spher Low

Pseudorabies Virus Herpes Varicellovirus Swine DNA yes 120 – 200 Spherical Med

Poliovirus Sabin Type 1 Picorna Enterovirus Human RNA no 25 – 30 Icosahedral Med

EncephalomyocardititisVirus

Picorna Cardiovirus Mouse RNA no 25 – 30 Icosahedral Med

Reovirus 3 Reo Orthoreovirus Various kind RNA no 60 – 80 Spherical Med

SV 40 Papova Polyomavirus Monkey DNA no 40 – 50 Icosahedral Very high

Parvovirus: canine, por-cine

Parvo Parvovirus CaninePorcine

DNA no 18 – 24 Icosahedral Very high


a production process may in‰uence the detection sensitivity.Consideration should also be given to health hazard whichmay pose to the personnel performing the clearance studies.

For the other items taken for consideration at choice ofvirus, the Notice, Iyakushin No. 329 can be used as a refer-ence. Examples of the virus which have been used for viralclearance studies are shown in Table 3 which was derivedfrom Iyakushin No. 329. However, the Notice, IyakushinNo. 329, is on viral safety of a product derived cell line of hu-man or animal origin, and a more appropriate model virushas to be chosen taking into account the originWraw materialof biological products.6.3 Design of viral clearance studies

The purpose of viral clearance studies is to quantitativelyevaluate removal or inactivation capability of a process, inwhich a virus is intentionally spiked to a speciˆc step of amanufacturing process.

Following are the precautions to be taken at planning viralclearance studies.

(1) Care should be taken in preparing the high-titer virusto avoid aggregation which may enhance physical removaland decrease inactivation thus distorting the correlation withactual production.

(2) Virus detection method gives great in‰uence to viralclearance factor. Accordingly, it is advisable to gain detec-tion sensitivity of the methods available in advance, and use amethod with a detection sensitivity as high as possible. Quan-titative infectivity assays should have adequate sensitivity andreproducibility in each manufacturing process, and should beperformed with su‹cient replicates to ensure adequatestatistical validity of the result. Quantitative assays not asso-ciated with infectivity may be used if justiêd. Appropriatevirus controls should be included in all infectivity assays to

ensure the sensitivity of the method. Also, the statistics ofsampling virus when at low concentrations (for example,number of virus is 1-1000WL) should be considered.

(3) Viral clearance studies are performed in a miniaturesize system that simulates the actual production process ofthe biotechnologicalWbiological product used by themanufacturer. It is inappropriate to introduce any virus intoa production facility because of GMP constraints. Therefore,viral clearance studies should be conducted in a separatelaboratory equipped for virological work and performed bystaŠ with virological expertise in conjunction with produc-tion personnel involved in designing and preparing a scaled-down version of the puriˆcation process. The viral clearancestudies should be performed under the basic concept of GLP.

(4) Each factor on a viral clearance study of a process,which is performed in miniature size, should re‰ect that ofactual manufacturing as far as possible, and its rationalityshould be clariêd. In case of chromatograph process, lengthof column bed, linear velocity, ratio of bed volume per veloc-ity (in other words, contact time), buŠer, type of columnpacking, pH, temperature, protein concentration, salt con-centration and concentration of the objective product are allcorrespondent to those of the actual production. Further,similarity of elution proˆle should be achieved. For the otherprocess, similar concept should be applied. If there is any fac-tor which can not re‰ect the actual production, its eŠect tothe result should be examined.

(5) It is desirable that two or more inactivationWremovalprocesses of diŠerent principles are selected and examined.

(6) As for the process which is expected to inactivateWre-move virus, each step should be evaluated in aspect of clear-ance capability, and carefully determined if it is the stage ofinactivation, removal or their combination for designing the


test. Generally, in viral clearance test, a virus is spiked in eachstep which is the object of the test, and after passing throughthe process in question, the reduction level of infectivity isevaluated. But, in some case, it is accepted that a high poten-tial virus is spiked at a step of the process, and virus concen-tration of each succeeding step is carefully monitored. Whenremoval of virus is made by separation or fractionation, it isdesirable to investigate how the virus is separated or fractio-nated (mass balance).

(7) For assessment of viral inactivation, unprocessedcrude material or intermediate material should be spiked withinfectious virus and the reduction factor calculated. It shouldbe recognized that virus inactivation is not a simple, ˆrst ord-er reaction and is usually more complex, with a fast ``phase1'' and a slow ``phase 2''. The study should, therefore, beplanned in such a way that samples are taken at diŠerenttimes and an inactivation curve constructed. It is recom-mended that studies for inactivation include at least one timepoint less than the minimum exposure time and greater thanzero, in addition to the minimum exposure time. Thereproducible clearance should be demonstrated in at least twoindependent studies. When there is a possibility that the virusis a human pathogen, it is very important that the eŠective in-activation process is designed and additional data are ob-tained. The initial virus load should be determined from thevirus which can be detected in the spiked starting material. Ifthis is not possible, the initial virus load may be calculatedfrom the titer of the spiking virus preparation. Where inacti-vation is too rapid to plot an inactivation curve using processconditions, appropriate controls should be performed todemonstrate that infectivity is indeed lost by inactivation.

(8) If antibody against virus exists in an unprocessedmaterial, caution should be taken at clearance studies, since itmay aŠect the behavior of virus at viral removal or inactiva-tion process.

(9) Virus spiked in unprocessed material should besu‹cient enough to evaluate viral removal or inactivationcapability of the process. However, the virus ``spike'' to beadded to the unprocessed material should be as small as pos-sible in comparison with the sample volume of the unproc-essed material so as not to cause characteristic change of thematerial by addition of the virus nor to cause behavioralchange of the protein in the material.

(10) It is desirable that the virus in the sample is subjectfor quantitative determination without applying ultracen-trifuge, dialysis, storage, etc. as far as possible. However,there may be a case that any handling before quantitativetest, such as remove procedure of inhibitor or toxic sub-stance, storage for a period to realize test at a time, etc., is in-evitable. If any manipulation, such as dilution, concentra-tion, ˆltration, dialysis, storage, etc., is applied for prepara-tion of the sample for testing, a parallel control test, whichpasses through a similar manipulation, should be conductedto assess infectivity variance at the manipulation.

(11) BuŠers and product (desired protein or other com-ponent contained therein) should be evaluated independentlyfor toxicity or interference in assays used to determine the vi-rus titer, as these components may adversely aŠect the indica-tor cells. If the solutions are toxic to the indicator cells, dilu-tion, adjustment of the pH, or dialysis of the buŠer contain-ing spiked virus might be necessary. If the product itself hasanti-viral activity, the clearance study may need to be per-formed without the product in a ``mock'' run, although

omitting the product or substituting a similar protein thatdoes not have anti-viral activity could aŠect the behaviour ofthe virus in some production steps.

(12) Many puriˆcation schemes use the same or similarbuŠers or columns, repetitively. The eŠects of this approachshould be taken into account when analyzing the data. TheeŠectiveness of virus elimination by a particular process mayvary with the stage in manufacture at which it is used.

(13) Overall reduction factors may be underestimatedwhere production conditions or buŠers are too cytotoxic orvirucidal and should be discussed on a case-by-case basis.Overall reduction factors may also be overestimated due toinherent limitations or inadequate design of viral clearancestudies.

(14) It has to be noted that clearance capability of viralremovalWinactivation process may vary depending upon thetype of virus. The viral removalWinactivation process, whichdisplays viral clearance by speciˆc principle or mechanism,may be quite eŠective to the virus, which meets such mechan-ism of action, but not eŠective to the other type of viruses.For example, SWD treatment is generally eŠective to the viruswith lipid membrane, but not eŠective to the virus not havingsuch membrane. Further, some virus is resistant to the gener-al heating process (55 – 609C, 30 minutes). When clearance isexpected for such virus, introduction of a further severe con-dition or process, which has diŠerent principle or mechan-ism, is necessary. Virus removal membrane ˆltration, whichis diŠerent from SWD or heat treatment in aspect of principle,is eŠective to a broad range of virus that can not pass throughthe membrane. A‹nity chromatography process, which spe-ciˆcally absorbs the objective protein, can thoroughly washout the materials other than the objective protein includingvirus etc. and is generally eŠective for viral removal. Separa-tionWfractionation of a virus from an objective protein issometimes very di‹cult, but there are not so rare that ion ex-change chromatography, ethanol fractionation, etc. is eŠec-tive for clearance of a virus which can not be su‹ciently inac-tivated or removed by the other process.

(15) EŠective clearance may be achieved by any of thefollowing: multiple inactivation steps, multiple complemen-tary separation steps, or combinations of inactivation andseparation steps. Separation methods may be dependent onthe extremely speciˆc physico-chemical properties of a viruswhich in‰uence its interaction with gel matrices and precipi-tation properties. However, despite these potential variables,eŠective removal can be obtained by a combination of com-plementary separation steps or combinations of inactivationand separation steps. Well designed separation steps, such aschromatographic procedures, ˆltration steps and extractions,can be also eŠective virus removal steps provided that theyare performed under appropriately controlled conditions.

(16) An eŠective virus removal step should givereproducible reduction of virus load shown by at least two in-dependent studies.

(17) Over time and after repeated use, the ability of chro-matography columns and other devices used in the puriˆca-tion scheme to clear virus may vary. Some estimate of the sta-bility of the viral clearance after several uses may providesupport for repeated use of such columns.

(18) The Notice, Iyakushin No. 329, would be used as areference when viral clearance studies on biological productsare designed.6.4 Interpretation of viral clearance studies


6.4.1 Evaluation on viral clearance factorViral clearance factor is a logarithm of reduction ratio of

viral amount (infectious titer) between each step applied forviral clearance of a manufacturing process. Total viral clear-ance factor throughout the process is sum of the viral clear-ance factor of each step appropriately evaluated.

Whether each and total viral clearance factor obtained areacceptable or should not be evaluated in aspects of everyvirus that can be realistically anticipated to derive into theraw material or the manufacturing process, and its rationalityshould be recorded.

In case that existence of any viral particle is recognized in asubstrate for drug production, e.g., a substrate of rodent ori-gin for biodrug production, it is important not only todemonstrate removal or inactivation of such virus, but alsoto demonstrate that the puriˆcation process has enoughcapability over the required level to assure safety of the ˆnalproduct at an appropriate level. The virus amount removedor inactivated in a manufacturing process should be com-pared with the virus amount assumed to exist in the substrateetc. used for manufacturing drug, and for this purpose, it isnecessary to obtain the virus amount in the raw materialsWsubstrate, etc. Such ˆgure can be obtained by measuring in-fectious titer or by the other method such as transmissionelectron microscope (TEM). For evaluation of overall proc-ess, a virus amount, far larger than that assumed to exist inthe amount of the raw materialsWsubstrate which is equiva-lent to single administration of the ˆnal product, has to be re-moved. It is quite rare that existence of virus can be assumedin a substrate for drug production, with the exception of thesubstrate of rodent origin, and such suspicious raw materialWsubstrate should not be used for manufacturing drug with aspecial exceptional case that the drug in question is not avail-able from the other process and is clinically indispensable,and that the information including infectious properties ofthe virus particle assumed to exist has been clariêd.6.4.2 Calculation of viral clearance index

Viral clearance factor, ``R'', for viral removalWinactivationprocess can be calculated by the following formula.

R＝log[(V1×T1)W(V2×T2)]

In whichR: Logarithm of reduction ratioV1: Sample volume of the unprocessed materialT1: Virus amount (titer) of the unprocessed materialV2: Sample volume of the processed materialT2: Virus amount (titer) of the processed material

At the calculation of viral clearance factor, it is recom-mendable to use the virus titer detected in the sample prepa-ration of the unprocessed material after addition of virus, notthe viral titer added to the sample preparation wherever pos-sible. If this is not possible, loaded virus amount is calculatedfrom virus titer of the solution used for spike.6.4.3 Interpretation of results and items to be concerned atevaluation

At the interpretation and the evaluation of the data oneŠectiveness of viral inactivationWremoval process, there arevarious factors to be comprehensively taken into account,such as appropriateness of the virus used for the test, design of the viral clearance studies, virus reduction ratioshown in logarithm, time dependence of inactivation, factorsWitems which give in‰uence to the inactivationW

removal process, sensitivity limit of virus assay method, possible eŠect of the inactivationWremoval process which isspeciˆc to certain class of viruses.

Additional items to be concerned at appropriate interpreta-tion and evaluation of the viral clearance data are as follows:(1) Behavior of virus used to the test

At interpretation of the vial clearance results, it is necessa-ry to recognize that clearance mechanism may diŠer depend-ing upon the virus used for the test. Virus used for a test isgenerally produced in tissue culture, but behavior of the virusprepared in the tissue culture may be diŠerent from that ofthe native virus. Examples are possible diŠerences of purityand degree of aggregation between the native and the cul-tured viruses. Further, change of surface properties of a vi-rus, e.g., addition of a sucrose chain which is ascribed tospeciˆc nature of a separation process, may give eŠect to theseparation. These matters should be also considered at inter-pretation of the results.(2) Design of test

Viral clearance test should have been designed taking intoaccount variation factors of the manufacturing process andscaling down, but there still remain some variance fromactual production scale. It is necessary to consider such vari-ance at the interpretation of the data and limitation of thetest.(3) Acceptability of viral reduction data

Total viral clearance factor is expressed as a sum oflogarithm of reduction ratio obtained at each step. The sum-mation of the reduction factor of multiple steps, particularlyof steps with little reduction (e.g., below 1 log10), may overes-timate viral removalWinactivation capability of the overallprocess. Therefore, virus titer of the order of 1 log10 or lesshas to be ignored unless justiêd. Further, viral clearance fac-tor achieved by repeated use of the same or similar methodshould be ignored for calculation unless justiêd.(4) Time dependence of inactivation

Inactivation of virus infectivity frequently shows biphasiccurve, which consists of a rapid initial phase and subsequentslow phase. It is possible that a virus not inactivated in a stepmay be more resistant to the subsequent step. For example, ifan inactivated virus forms coagulation, it may be resistant toany chemical treatment and heating.(5) Evaluation of viral reduction ratio shown logarithm

Viral clearance factor shown in logarithm of reductionratio of virus titer can demonstrate drastic reduction of resid-ual infectious virus, but there is a limit that infectious titercan never be reduced to zero. For example, reduction in in-fectivity of a preparation containing 8 log10 infectious unitper mL by a factor of 8 log10 leaves zero log10 per mL or oneinfectious unit per mL, taking into account the detectionlimit of assay.(6) Variable factor of manufacturing process

Minor variance of a variation factor of a manufacturingprocess, e.g., contact time of a spiked sample to a buŠer or acolumn, will sometimes give in‰uence to viral removal or in-activation eŠect. In such case, it may be necessary to inves-tigate to what extent such variance of the factor has given in-‰uence to the process concerned in aspect of viral inactiva-tion.(7) Existence of anti-viral antiserum

Anti-viral antiserum that exists in the sample preparationused for a test may aŠect sensitivity of distribution or inacti-vation of a virus, which may result in not only defusing the


virus titer but complicating interpretation of the test result.So, existence of anti-viral antiserum is one of the importantvariable factors.(8) Introduction of a new process for removalWinactivation

Viral clearance is an important factor for securing safety ofdrug. In case that an achievement level of infective clearanceof a process is considered insu‹cient, a process which ischaracterized by inactivationWremoval mechanism to meetthe purpose or an inactivationWremoval process which canmutually complement to the existence process has to be in-troduced.(9) Limit of viral clearance studies

Viral clearance studies are useful for contributing to the as-surance that an acceptable level of safety in the ˆnal productis achieved but do not by themselves establish safety.However, a number of factors in the design and execution ofviral clearance studies may lead to an incorrect estimate ofthe ability of the process to remove virus infectivity, as de-scribed above.

7. StatisticsThe viral clearance studies should include the use of

statistical analysis of the data to evaluate the results. Thestudy results should be statistically valid to support the con-clusions reached.7.1 Statistical considerations for assessing virus assays

Virus titrations suŠer the problems of variation commonto all biological assay systems. Assessment of the accuracy ofthe virus titrations and reduction factors derived from themand the validity of the assays should be performed to deˆnethe reliability of a study. The objective of statistical evalua-tion is to establish that the study has been carried out to anacceptable level of virological competence.Assay

1. Assay methods may be either quantal or quantitative.Both quantal and quantitative assays are amenable to statisti-cal evaluation.

2. Variation can arise within an assay as a result of dilu-tion errors, statistical eŠects and diŠerences within the assaysystem which are either unknown or di‹cult to control.These eŠects are likely to be greater when diŠerent assay runsare compared (between-assay variation) than when results wi-thin a single assay run are compared (within-assay variation).

3. The 95z conˆdence limits for results of within-assayvariation normally should be on the order of ±0.5 log10 ofthe mean. Within-assay variation can be assessed by standardtextbook methods. Between-assay variation can be moni-tored by the inclusion of a reference preparation, the estimateof whose potency should be within approximately 0.5 log10 ofthe mean estimate established in the laboratory for the assayto be acceptable. Assays with lower precision may be accepta-ble with appropriate justiˆcation.7.2 Reproducibility and conˆdence limit of viral clearancestudies

An eŠective virus inactivationWremoval step should givereproducible reduction of virus load shown by at least two in-dependent studies. The 95z conˆdence limits for the reduc-tion factor observed should be calculated wherever possiblein studies of viral clearance. If the 95z conˆdence limits forthe viral assays of the starting material are ±s, and for theviral assays of the material after the step are ±a, the 95zconˆdence limits for the reduction factor are ± s2＋a2.

8. Re-evaluation of viral clearanceWhenever signiˆcant changes in the production or puriˆca-

tion process are made, the eŠect of that change, both directand indirect, on viral clearance should be considered and thesystem re-evaluated as needed. Changes in process steps mayalso change the extent of viral clearance.

9. Measurement for viral clearance studies9.1 Measurement of virus infective titer

Assay methods may be either quantal or quantitative.Quantal methods include infectivity assays in animals or incultured cell infections dose (CCID) assays, in which theanimal or cell culture is scored as either infected or not. In-fectivity titers are then measured by the proportion ofanimals or culture infected. In quantitative methods, the in-fectivity measured varies continuously with the virus input.Quantitative methods include plaque assays where eachplaque counted corresponds to a single infectious unit. Bothquantal and quantitative assays are amenable to statisticalevaluation.9.2 Testing by nucleic-acid-ampliˆcation test (NAT)

NAT can detect individual or pooled raw materialWcell sub-strate or virus genome at a high sensitivity even in a stage thatserum test on each virus is negative. Further, it can detectHBV or HCV gene, which can not be measured in culture sys-tem. Window period can be drastically shortened at the teston HBV, HCV and HIV, and the method is expected to con-tribute as an eŠective measure for ensuring viral safety.However, depending upon a choice of primer, there may be acase that not all the subtype of objective virus can be detectedby this method, and, therefore, it is recommendable to evalu-ate, in advance, if subtype of a broad range can be detected.

NAT will be an eŠective evaluation method for virusremoval capability at viral clearance studies. However, incase of viral inactivation process, viral inactivation obtainedby this method may be underrated, since there is a case thatinactivated virus still shows positive on nucleic acid. Further,at introduction of NAT, cautions should be taken on ration-ality of detection sensitivity, choice of a standard which isused as run-control, quality assurance and maintenance of areagent used for primer, interpretation of positive and nega-tive results, etc.

10. Reporting and preservationAll the items relating to virus test and viral clearance stu-

dies should be reported and preserved.

11. OthersThe Notice, Iyakushin No. 329, should be used as a refer-

ence at virus test and viral clearance studies.

ConclusionAs mentioned at the Introduction, assurance of qualityW

safety etc. of JP listed drugs should be achieved by state-of-the-art methods and concepts re‰ecting the progress ofscience and accumulation of experiences.

The basis for ensuring viral safety of JP listed biotechno-logicalWbiological products is detailed in this General Infor-mation. What is discussed here is that an almost equal levelof measures are required for both development of new drugsand for existing products as well, which means that similarlevel of concerns should be paid on both existing and newproducts in aspect of viral safety. This document is intendedto introduce a basic concept that quality and safety assuranceof JP listed product should be based upon the most advanced

16751675JP XV General Information / Capillary Electrophoresis

methods and concepts. This document has been written tocover all conceivable cases, which can be applied to allbiotechnologicalWbiological products. Therefore, there maybe cases that it is not so rational to pursue virus tests and viralclearance studies in accordance with this document on eachproduct, which has been used for a long time without anysafety issue. So, it will be necessary to elaborate the most ra-tional ways under a case-by-case principle taking into dueconsideration source, origin, type, manufacturing process,characteristics, usages at clinical stage, accumulation of thepast usage record, etc. relating to such biotechnologicalWbio-logical products.

4. Capillary ElectrophoresisThis test is harmonized with the European Pharmacopoeia

and the U.S. Pharmacopeia.

General PrinciplesCapillary electrophoresis is a physical method of analysis

based on the migration, inside a capillary, of chargedanalytes dissolved in an electrolyte solution, under thein‰uence of a direct-current electric êld.

The migration velocity of an analyte under an electric êldof intensity E, is determined by the electrophoretic mobilityof the analyte and the electro-osmotic mobility of the buŠerinside the capillary. The electrophoretic mobility of a solute(mep) depends on the characteristics of the solute (electriccharge, molecular size and shape) and those of the buŠer inwhich the migration takes place (type and ionic strength ofthe electrolyte, pH, viscosity and additives). The elec-trophoretic velocity (nep) of a solute, assuming a sphericalshape, is given by the equation:

nep＝mepE＝Ø q6phr »Ø V

L »q: eŠective charge of the solute,h: viscosity of the electrolyte solution,r: Stoke's radius of the solute,V: applied voltage,L: total length of the capillary.

When an electric êld is applied through the capillary ˆlledwith buŠer, a ‰ow of solvent is generated inside the capillary,called electro-osmotic ‰ow. The velocity of the electro-os-motic ‰ow depends on the electro-osmotic mobility (meo)which in turn depends on the charge density on the capillaryinternal wall and the buŠer characteristics. The electro-os-motic velocity (neo) is given by the equation:

neo＝meoE＝Ø ezh »Ø V

L »e: dielectric constant of the buŠer,z: zeta potential of the capillary surface.

The velocity of the solute (n) is given by:

n＝nep＋neo

The electrophoretic mobility of the analyte and the electro-osmotic mobility may act in the same direction or in oppositedirections, depending on the charge of the solute. In normalcapillary electrophoresis, anions will migrate in the opposite

direction to the electro-osmotic ‰ow and their velocities willbe smaller than the electro-osmotic velocity. Cations will mi-grate in the same direction as the electro-osmotic ‰ow andtheir velocities will be greater than the electro-osmotic veloc-ity. Under conditions in which there is a fast electro-osmoticvelocity with respect to the electrophoretic velocity of the so-lutes, both cations and anions can be separated in the samerun.

The time (t) taken by the solute to migrate the distance (l)from the injection end of the capillary to the detection point(capillary eŠective length) is given by the expression:

t＝l

nep＋neo＝

l×L(mep＋meo)V

In general, uncoated fused-silica capillaries above pH 3have negative charge due to ionized silanol groups in theinner wall. Consequently, the electro-osmotic ‰ow is fromanode to cathode. The electro-osmotic ‰ow must remain con-stant from run to run if good reproducibility is to be obtainedin the migration velocity of the solutes. For some applica-tions, it may be necessary to reduce or suppress the electro-osmotic ‰ow by modifying the inner wall of the capillary orby changing the concentration, composition and/or pH ofthe buŠer solution.

After the introduction of the sample into the capillary,each analyte ion of the sample migrates within the back-ground electrolyte as an independent zone, according to itselectrophoretic mobility. Zone dispersion, that is the spread-ing of each solute band, results from diŠerent phenomena.Under ideal conditions the sole contribution to the solute-zone broadening is molecular diŠusion of the solute along thecapillary (longitudinal diŠusion). In this ideal case thee‹ciency of the zone, expressed as the number of theoreticalplates (N), is given by:

N＝(mep＋meo)×V×L

2×D×L

D: molecular diŠusion coe‹cient of the solute in thebuŠer.

In practice, other phenomena such as heat dissipation,sample adsorption onto the capillary wall, mismatchedconductivity between sample and buŠer, length of the injec-tion plug, detector cell size and unlevelled buŠer reservoirscan also signiˆcantly contribute to band dispersion.

Separation between two bands (expressed as the resolution,RS) can be obtained by modifying the electrophoretic mobili-ty of the analytes, the electro-osmotic mobility induced in thecapillary and by increasing the e‹ciency for the band of eachanalyte, according to the equation:

RS＝N(mepb－mepa)4( šmep＋meo)

mepa and mepb: electrophoretic mobilities of the two analytesseparated,

šmep: mean electrophoretic mobility of the two analytes

šmep＝12

(mepb＋mepa).

ApparatusAn apparatus for capillary electrophoresis is composed of:—a high-voltage, controllable direct-current power supply,—two buŠer reservoirs, held at the same level, containing

the prescribed anodic and cathodic solutions,

16761676 JP XVCapillary Electrophoresis / General Information

—two electrode assemblies (the cathode and the anode),immersed in the buŠer reservoirs and connected to thepower supply,

—a separation capillary (usually made of fused-silica)which, when used with some speciˆc types of detectors,has an optical viewing window aligned with the detector.The ends of the capillary are placed in the buŠer reser-voirs. The capillary is ˆlled with the solution prescribedin the monograph,

—a suitable injection system,—a detector able to monitor the amount of substances of

interest passing through a segment of the separationcapillary at a given time. It is usually based on absorp-tion spectrophotometry (UV and visible) or ‰uorometry,but conductimetric, amperometric or mass spectrometricdetection can be useful for speciˆc applications. Indirectdetection is an alternative method used to detect non-UV-absorbing and non-‰uorescent compounds,

—a thermostatic system able to maintain a constant tem-perature inside the capillary is recommended to obtain agood separation reproducibility,

—a recorder and a suitable integrator or a computer.The deˆnition of the injection process and its automation

are critical for precise quantitative analysis. Modes ofinjection include gravity, pressure or vacuum injection andelectrokinetic injection. The amount of each samplecomponent introduced electrokinetically depends on itselectrophoretic mobility, leading to possible discriminationusing this injection mode.

Use the capillary, the buŠer solutions, the preconditioningmethod, the sample solution and the migration conditionsprescribed in the monograph of the considered substance.The employed electrolytic solution is ˆltered to removeparticles and degassed to avoid bubble formation that couldinterfere with the detection system or interrupt the electricalcontact in the capillary during the separation run. A rigorousrinsing procedure should be developed for each analyticalmethod to achieve reproducible migration times of the so-lutes.

1. Capillary Zone ElectrophoresisPrinciple

In capillary zone electrophoresis, analytes are separated ina capillary containing only buŠer without any anticonvectivemedium. With this technique, separation takes place becausethe diŠerent components of the sample migrate as discretebands with diŠerent velocities. The velocity of each banddepends on the electrophoretic mobility of the solute and theelectro-osmotic ‰ow in the capillary (see General Principles).Coated capillaries can be used to increase the separationcapacity of those substances adsorbing on fused-silicasurfaces.

Using this mode of capillary electrophoresis, the analysisof both small (Mrº2000) and large molecules (2000ºMr

º100,000) can be accomplished. Due to the high e‹ciencyachieved in free solution capillary electrophoresis, separationof molecules having only minute diŠerences in their charge-to-mass ratio can be eŠected. This separation mode also al-lows the separation of chiral compounds by addition of chiralselectors to the separation buŠer.Optimization

Optimization of the separation is a complex process whereseveral separation parameters can play a major role. The

main factors to be considered in the development of separa-tions are instrumental and electrolytic solution parameters.Instrumental parameters

Voltage: A Joule heating plot is useful in optimizing theapplied voltage and column temperature. Separation time isinversely proportional to applied voltage. However, anincrease in the voltage used can cause excessive heat produc-tion, giving rise to temperature and, as a result thereof,viscosity gradients in the buŠer inside the capillary. ThiseŠect causes band broadening and decreases resolution.

Polarity: Electrode polarity can be normal (anode at theinlet and cathode at the outlet) and the electro-osmotic ‰owwill move toward the cathode. If the electrode polarity isreversed, the electro-osmotic ‰ow is away from the outlet andonly charged analytes with electro-osmotic mobilities greaterthan the electro-osmotic ‰ow will pass to the outlet.

Temperature: The main eŠect of temperature is observedon buŠer viscosity and electrical conductivity, and thereforeon migration velocity. In some cases, an increase in capillarytemperature can cause a conformational change in proteins,modifying their migration time and the e‹ciency of the sepa-ration.

Capillary: The dimensions of the capillary (length and in-ternal diameter) contribute to analysis time, e‹ciency ofseparations and load capacity. Increasing both eŠectivelength and total length can decrease the electric êlds(working at constant voltage) which increases migration time.For a given buŠer and electric êld, heat dissipation, andhence sample band-broadening, depend on the internal di-ameter of the capillary. The latter also aŠects the detectionlimit, depending on the sample volume injected and thedetection system employed.

Since the adsorption of the sample components on thecapillary wall limits e‹ciency, methods to avoid theseinteractions should be considered in the development of aseparation method. In the speciˆc case of proteins, severalstrategies have been devised to avoid adsorption on the capil-lary wall. Some of these strategies (use of extreme pH and ad-sorption of positively charged buŠer additives) only requiremodiˆcation of the buŠer composition to prevent protein ad-sorption. In other strategies, the internal wall of the capillaryis coated with a polymer, covalently bonded to the silica, thatprevents interaction between the proteins and the negativelycharged silica surface. For this purpose, ready-to-use capilla-ries with coatings consisting of neutral-hydrophilic, cationicand anionic polymers are available.Electrolytic solution parameters

BuŠer type and concentration: Suitable buŠers forcapillary electrophoresis have an appropriate buŠer capacityin the pH range of choice and low mobility to minimizecurrent generation.

Matching buŠer-ion mobility to solute mobility, wheneverpossible, is important for minimizing band distortion. Thetype of sample solvent used is also important to achieveon-column sample focusing, which increases separatione‹ciency and improves detection.

An increase in buŠer concentration (for a given pH)decreases electro-osmotic ‰ow and solute velocity.

BuŠer pH: The pH of the buŠer can aŠect separation bymodifying the charge of the analyte or additives, and bychanging the electro-osmotic ‰ow. In protein and peptideseparation, changing the pH of the buŠer from above tobelow the isoelectric point (pI) changes the net charge of the


solute from negative to positive. An increase in the buŠer pHgenerally increases the electro-osmotic ‰ow.

Organic solvents: Organic modiêrs (methanol, acetoni-trile, etc.) may be added to the aqueous buŠer to increase thesolubility of the solute or other additives and/or to aŠect thedegree of ionization of the sample components. The additionof these organic modiêrs to the buŠer generally causes adecrease in the electro-osmotic ‰ow.

Additives for chiral separations: For the separation ofoptical isomers, a chiral selector is added to the separationbuŠer. The most commonly used chiral selectors arecyclodextrins, but crown ethers, polysaccharides and proteinsmay also be used. Since chiral recognition is governed by thediŠerent interactions between the chiral selector and each ofthe enantiomers, the resolution achieved for the chiral com-pounds depends largely on the type of chiral selector used. Inthis regard, for the development of a given separation it maybe useful to test cyclodextrins having a diŠerent cavity size(a-, b-, or g-cyclodextrin) or modiêd cyclodextrins with neu-tral (methyl, ethyl, hydroxyalkyl, etc.) or ionizable(aminomethyl, carboxymethyl, sulfobutyl ether, etc.) groups.When using modiêd cyclodextrins, batch-to-batch varia-tions in the degree of substitution of the cyclodextrins mustbe taken into account since it will in‰uence the selectivity.Other factors controlling the resolution in chiral separationsare concentration of chiral selector, composition and pH ofthe buŠer and temperature. The use of organic additives,such as methanol or urea can also modify the resolutionachieved.

2. Capillary Gel ElectrophoresisPrinciple

In capillary gel electrophoresis, separation takes placeinside a capillary ˆlled with a gel that acts as a molecularsieve. Molecules with similar charge-to-mass ratios areseparated according to molecular size since smaller moleculesmove more freely through the network of the gel and there-fore migrate faster than larger molecules. DiŠerent biologicalmacromolecules (for example, proteins and DNA frag-ments), which often have similar charge-to-mass ratios, canthus be separated according to their molecular mass by capil-lary gel electrophoresis.Characteristics of Gels

Two types of gels are used in capillary electrophoresis: per-manently coated gels and dynamically coated gels. Perma-nently coated gels, such as cross-linked polyacrylamide, areprepared inside the capillary by polymerization of themonomers. They are usually bonded to the fused-silica walland cannot be removed without destroying the capillary. Ifthe gels are used for protein analysis under reducing condi-tions, the separation buŠer usually contains sodium dodecylsulfate and the samples are denatured by heating a mixture ofsodium dodecyl sulfate and 2-mercaptoethanol ordithiothreitol before injection. When non-reducing condi-tions are used (for example, analysis of an intact antibody),2-mercaptoethanol and dithiothreitol are not used. Separa-tion in cross-linked gels can be optimized by modifying theseparation buŠer (as indicated in the free solution capillaryelectrophoresis section) and controlling the gel porosityduring the gel preparation. For cross-linked polyacrylamidegels, the porosity can be modiêd by changing the concentra-tion of acrylamide and/or the proportion of cross-linker. Asa rule, a decrease in the porosity of the gel leads to a decrease

in the mobility of the solutes. Due to the rigidity of these gels,only electrokinetic injection can be used.

Dynamically coated gels are hydrophilic polymers, such aslinear polyacrylamide, cellulose derivatives, dextran, etc.,which can be dissolved in aqueous separation buŠers givingrise to a separation medium that also acts as a molecularsieve. These separation media are easier to prepare thancross-linked polymers. They can be prepared in a vial andˆlled by pressure in a wall-coated capillary (with no electro-osmotic ‰ow). Replacing the gel before every injection gener-ally improves the separation reproducibility. The porosity ofthe gels can be increased by using polymers of higher molecu-lar mass (at a given polymer concentration) or by decreasingthe polymer concentration (for a given polymer molecularmass). A reduction in the gel porosity leads to a decrease inthe mobility of the solute for the same buŠer. Since the disso-lution of these polymers in the buŠer gives low viscosity solu-tions, both hydrodynamic and electrokinetic injection tech-niques can be used.

3. Capillary Isoelectric FocusingPrinciple

In isoelectric focusing, the molecules migrate under thein‰uence of the electric êld, so long as they are charged, in apH gradient generated by ampholytes having pI values in awide range (poly-aminocarboxylic acids), dissolved in theseparation buŠer.

The three basic steps of isoelectric focusing are loading,focusing and mobilization.

Loading step: Two methods may be employed:—loading in one step: the sample is mixed with ampholytes

and introduced into the capillary either by pressure orvacuum;

—sequential loading: a leading buŠer, then the ampho-lytes, then the sample mixed with ampholytes, again am-pholytes alone and ˆnally the terminating buŠer are in-troduced into the capillary. The volume of the samplemust be small enough not to modify the pH gradient.

Focusing step: When the voltage is applied, ampholytesmigrate toward the cathode or the anode, according to theirnet charge, thus creating a pH gradient from anode (lowerpH) to cathode (higher pH). During this step the componentsto be separated migrate until they reach a pH correspondingto their isoelectric point (pI) and the current drops to verylow values.

Mobilization step: If mobilization is required for detec-tion, use one of the following methods. Three methods areavailable:

—in the ˆrst method, mobilization is accomplished duringthe focusing step under the eŠect of the electro-osmotic‰ow; the electro-osmotic ‰ow must be small enough toallow the focusing of the components;

—in the second method, mobilization is accomplished byapplying positive pressure after the focusing step;

—in the third method, mobilization is achieved after thefocusing step by adding salts to the cathode reservoir orthe anode reservoir (depending on the direction chosenfor mobilization) in order to alter the pH in the capillarywhen the voltage is applied. As the pH is changed, theproteins and ampholytes are mobilized in the directionof the reservoir which contains the added salts and passthe detector.

The separation achieved, expressed as DpI, depends on the

16781678 JP XVCapillary Electrophoresis / General Information

pH gradient (dpH/dx), the number of ampholytes havingdiŠerent pI values, the molecular diŠusion coe‹cient (D), theintensity of the electric êld (E) and the variation of the elec-trophoretic mobility of the analyte with the pH (－dm/dpH):

DPI＝3D(dpH/dx)

E(－dm/dpH)

OptimizationThe main parameters to be considered in the development

of separations are:Voltage: Capillary isoelectric focusing utilises very high

electric êlds, 300 V/cm to 1000 V/cm in the focusing step.Capillary: The electro-osmotic ‰ow must be reduced or

suppressed depending on the mobilization strategy (seeabove). Coated capillaries tend to reduce the electro-osmotic‰ow.

Solutions: The anode buŠer reservoir is ˆlled with asolution with a pH lower than the pI of the most acidicampholyte and the cathode reservoir is ˆlled with a solutionwith a pH higher than the pI of the most basic ampholyte.Phosphoric acid for the anode and sodium hydroxide for thecathode are frequently used.

Addition of a polymer, such as methylcellulose, in theampholyte solution tends to suppress convective forces (ifany) and electro-osmotic ‰ow by increasing the viscosity.Commercial ampholytes are available covering many pHranges and may be mixed if necessary to obtain an expandedpH range. Broad pH ranges are used to estimate the isoelec-tric point whereas narrower ranges are employed to improveaccuracy. Calibration can be done by correlating migrationtime with isoelectric point for a series of protein markers.

During the focusing step precipitation of proteins at theirisoelectric point can be prevented, if necessary, using buŠeradditives such as glycerol, surfactants, urea or zwitterionicbuŠers. However, depending on the concentration, ureadenatures proteins.

4. Micellar Electrokinetic Chromatography (MEKC)Principle

In micellar electrokinetic chromatography, separationtakes place in an electrolyte solution which contains a sur-factant at a concentration above the critical micellar concen-tration (cmc). The solute molecules are distributed betweenthe aqueous buŠer and the pseudo-stationary phase com-posed of micelles, according to the partition coe‹cient of thesolute. The technique can therefore be considered as a hybridof electrophoresis and chromatography. It is a technique thatcan be used for the separation of both neutral and chargedsolutes, maintaining the e‹ciency, speed and instrumentalsuitability of capillary electrophoresis. One of the most wide-ly used surfactants in MEKC is the anionic surfactant sodiumdodecyl sulfate, although other surfactants, for examplecationic surfactants such as cetyltrimethylammonium salts,are also used.

The separation mechanism is as follows. At neutral andalkaline pH, a strong electro-osmotic ‰ow is generated andmoves the separation buŠer ions in the direction of thecathode. If sodium dodecyl sulfate is employed as the sur-factant, the electrophoretic migration of the anionic micelleis in the opposite direction, towards the anode. As a result,the overall micelle migration velocity is slowed down com-pared to the bulk ‰ow of the electrolytic solution. In the caseof neutral solutes, since the analyte can partition between the

micelle and the aqueous buŠer, and has no electrophoreticmobility, the analyte migration velocity will depend only onthe partition coe‹cient between the micelle and the aqueousbuŠer. In the electropherogram, the peaks corresponding toeach uncharged solute are always between that of the electro-osmotic ‰ow marker and that of the micelle (the time elapsedbetween these two peaks is called the separation window).For electrically charged solutes, the migration velocity de-pends on both the partition coe‹cient of the solute betweenthe micelle and the aqueous buŠer, and on the electrophoreticmobility of the solute in the absence of micelle.

Since the mechanism in MEKC of neutral and weaklyionized solutes is essentially chromatographic, migration ofthe solute and resolution can be rationalized in terms of theretention factor of the solute (k?), also referred to as massdistribution ratio (Dm), which is the ratio of the number ofmoles of solute in the micelle to those in the mobile phase.For a neutral compound, k? is given by:

k?＝tR－t0

t0Ø1－ tRtmc »

＝KVS

VM

tR: migration time of the solute,t0: analysis time of an unretained solute (determined by in-

jecting an electro-osmotic ‰ow marker which does notenter the micelle, for instance methanol),

tmc: micelle migration time (measured by injecting a micellemarker, such as Sudan III, which migrates while con-tinuously associated in the micelle),

K: partition coe‹cient of the solute,VS: volume of the micellar phase,VM: volume of the mobile phase.

Likewise, the resolution between two closely-migratingsolutes (RS) is given by:

RS＝N4

×a－1

a×

k?b

k?b＋1×

1－Ø t0tmc »

1＋Ø t0tmc »k?a

N: number of theoretical plates for one of the solutes,a: selectivity,k?a and k?b: retention factors for both solutes, respectively

(k?bÀk?a).

Similar, but not identical, equations give k? and RS valuesfor electrically charged solutes.Optimization

The main parameters to be considered in the developmentof separations by MEKC are instrumental and electrolytic so-lution parameters.Instrumental parameters

Voltage: Separation time is inversely proportional toapplied voltage. However, an increase in voltage can causeexcessive heat production that gives rise to temperaturegradients and viscosity gradients of the buŠer in the cross-section of the capillary. This eŠect can be signiˆcant withhigh conductivity buŠers such as those containing micelles.Poor heat dissipation causes band broadening and decreasesresolution.

Temperature: Variations in capillary temperature aŠectthe partition coe‹cient of the solute between the buŠer andthe micelles, the critical micellar concentration and the


viscosity of the buŠer. These parameters contribute to themigration time of the solutes. The use of a good cooling sys-tem improves the reproducibility of the migration time forthe solutes.

Capillary: As in free solution capillary electrophoresis,the dimensions of the capillary (length and internal diameter)contribute to analysis time and e‹ciency of separations. In-creasing both eŠective length and total length can decreasethe electric êlds (working at constant voltage), increasemigration time and improve the separation e‹ciency. The in-ternal diameter controls heat dissipation (for a given buŠerand electric êld) and consequently the sample band broaden-ing.Electrolytic solution parameters

Surfactant type and concentration: The type of sur-factant, in the same way as the stationary phase in chro-matography, aŠects the resolution since it modiês separa-tion selectivity. Also, the log k? of a neutral compoundincreases linearly with the concentration of surfactant in themobile phase. Since resolution in MEKC reaches a maximumwhen k? approaches the value of tm/t0, modifying the con-centration of surfactant in the mobile phase changes the reso-lution obtained.

BuŠer pH: Although pH does not modify the partitioncoe‹cient of non-ionized solutes, it can modify the electro-osmotic ‰ow in uncoated capillaries. A decrease in the buŠerpH decreases the electro-osmotic ‰ow and therefore increasesthe resolution of the neutral solutes in MEKC, resulting in alonger analysis time.

Organic solvents: To improve MEKC separation ofhydrophobic compounds, organic modiêrs (methanol,propanol, acetonitrile, etc.) can be added to the electrolyticsolution. The addition of these modiêrs usually decreasesmigration time and the selectivity of the separation. Since theaddition of organic modiêrs aŠects the critical micellar con-centration, a given surfactant concentration can be used onlywithin a certain percentage of organic modiêr before themicellization is inhibited or adversely aŠected, resulting inthe absence of micelles and, therefore, in the absence of par-tition. The dissociation of micelles in the presence of a highcontent of organic solvent does not always mean that theseparation will no longer be possible; in some cases thehydrophobic interaction between the ionic surfactantmonomer and the neutral solutes forms solvophobiccomplexes that can be separated electrophoretically.

Additives for chiral separations: For the separation ofenantiomers using MEKC, a chiral selector is included in themicellar system, either covalently bound to the surfactant oradded to the micellar separation electrolyte. Micelles thathave a moiety with chiral discrimination properties includesalts of N-dodecanoyl-L-amino acids, bile salts, etc. Chiralresolution can also be achieved using chiral discriminators,such as cyclodextrins, added to the electrolytic solutionswhich contain micellized achiral surfactants.

Other additives: Several strategies can be carried out tomodify selectivity, by adding chemicals to the buŠer. The ad-dition of several types of cyclodextrins to the buŠer can alsobe used to reduce the interaction of hydrophobic solutes withthe micelle, thus increasing the selectivity for this type ofcompound.

The addition of substances able to modify solute-micelleinteractions by adsorption on the latter, is used to improvethe selectivity of the separations in MEKC. These additives

may be a second surfactant (ionic or non-ionic) which givesrise to mixed micelles or metallic cations which dissolve in themicelle and form co-ordination complexes with the solutes.Quantiˆcation

Peak areas must be divided by the corresponding migrationtime to give the corrected area in order to:

—compensate for the shift in migration time from run torun, thus reducing the variation of the response,

—compensate for the diŠerent responses of sampleconstituents with diŠerent migration times.

Where an internal standard is used, verify that no peak ofthe substance to be examined is masked by that of theinternal standard.Calculations

From the values obtained, calculate the content of thecomponent or components being examined. Whenprescribed, the percentage content of one or more compo-nents of the sample to be examined is calculated by determin-ing the corrected area(s) of the peak(s) as a percentage of thetotal of the corrected areas of all peaks, excluding those dueto solvents or any added reagents (normalization procedure).The use of an automatic integration system (integrator ordata acquisition and processing system) is recommended.

System SuitabilityIn order to check the behavior of the capillary electropho-

resis system, system suitability parameters are used. Thechoice of these parameters depends on the mode of capillaryelectrophoresis used. They are: retention factor (k?) (only formicellar electrokinetic chromatography), apparent numberof theoretical plates (N), symmetry factor (AS) and resolution(RS). In previous sections, the theoretical expressions for Nand RS have been described, but more practical equationsthat allow these parameters to be calculated from the elec-tropherograms are given below.Apparent Number of Theoretical Plates

The apparent number of theoretical plates (N) may becalculated using the expression:

N＝5.54Ø tRwh »

2

tR: migration time or distance along the baseline from thepoint of injection to the perpendicular dropped fromthe maximum of the peak corresponding to the compo-nent,

wh: width of the peak at half-height.

ResolutionThe resolution (RS) between peaks of similar height of two

components may be calculated using the expression:

RS＝Ø 1.18(tR2－tR1)wh1＋wh2 »

tR2ÀtR1

tR1 and tR2: migration times or distances along the baselinefrom the point of injection to the perpendicu-lars dropped from the maxima of two adjacentpeaks,

wh1 and wh2: peak widths at half-height.

When appropriate, the resolution may be calculated bymeasuring the height of the valley (Hv) between two partlyresolved peaks in a standard preparation and the height ofthe smaller peak (Hp) and calculating the peak-to-valley ratio:

16801680 JP XVDecision of Limit for Bacterial / General Information

p/n＝ Hp

Hv

Symmetry FactorThe symmetry factor (AS) of a peak may be calculated

using the expression:

AS＝w0.05

2d

w0.05: width of the peak at one-twentieth of the peakheight,

d: distance between the perpendicular dropped from thepeak maximum and the leading edge of the peak at one-twentieth of the peak height.

Tests for area repeatability (standard deviation of areas orof the area/migration-time ratio) and for migration timerepeatability (standard deviation of migration time) areintroduced as suitability parameters. Migration timerepeatability provides a test for the suitability of the capillarywashing procedures. An alternative practice to avoid the lackof repeatability of the migration time is to use migration timerelative to an internal standard.

A test for the veriˆcation of the signal-to-noise ratio for astandard preparation (or the determination of the limit ofquantiˆcation) may also be useful for the determination ofrelated substances.Signal-to-noise Ratio

The detection limit and quantiˆcation limit correspond tosignal-to-noise ratios of 3 and 10 respectively. The signal-to-noise ratio (S/N) is calculated using the expression:

S/N＝2Hh

H: height of the peak corresponding to the componentconcerned, in the electropherogram obtained with theprescribed reference solution, measured from the maximumof the peak to the extrapolated baseline of the signal observedover a distance equal to twenty times the width at half-height,

h: range of the background in an electropherogram ob-tained after injection of a blank, observed over a distanceequal to twenty times the width at the half-height of the peakin the electropherogram obtained with the prescribed refer-ence solution and, if possible, situated equally around theplace where this peak would be found.

5. Decision of Limit for BacterialEndotoxins

The endotoxin limit for injections is to be decided as fol-lows:

Endotoxin limit＝KWM

where K is a minimum pyrogenic dose of endotoxin per kgbody mass (EUWkg), and M is equal to the maximum dose ofproduct per kg per hour.

M is expressed in mLWkg for products to be administeredby volume, in mgWkg or mEqWkg for products to beadministered by mass, and in UnitWkg for products to beadministered by biological units. Depending on the adminis-tration route, values for K are set as in the following table.

Intended route of administration K (EUWkg)

Intravenous 5.0

Intravenous, forradiopharmaceuticals 2.5

Intraspinal 0.2

Notes:1) For products to be administered by mass or by units,

the endotoxin limit should be decided based on the labeledamount of the principal drug.

2) Sixty kg should be used as the average body mass of anadult when calculating the maximum adult dose per kg.

3) The pediatric dose per kg body mass should be usedwhen this is higher than the adult dose.

4) The K values for the intravenous route are applicableto drugs to be administered by any route other than thoseshown in the table.

6. Disinfection and SterilizationMethods

Disinfection and Sterilization Methods are applied to killmicroorganisms in processing equipmentWutensils and areasused for drug manufacturing, as well as to perform microbio-logical tests speciêd in the monographs, and so diŠer from``Terminal Sterilization'' and ``Filtration Method'' describedin ``Terminal Sterilization and Sterilization Indicators''. Thekilling eŠect on microorganisms or the estimated level ofsterility assurance is greatly variable, so the conditions fordisinfection and sterilization treatment must be chosenappropriately for each application. Generally, the followingmethods are to be used singly or in combination afterappropriate optimization of operation procedures and condi-tions, in accordance with the kind and the degree of thecontaminating microorganisms and the nature of the item towhich the methods are applied.

The validation of sterilization in accordance with TerminalSterilization and Sterilization Indicators is required when themethods are applied to the manufacturing processes of drugproducts.

1. Disinfection methodsThese methods are used to reduce the number of living

microorganisms, but do not always remove or kill allmicroorganisms present. Generally, disinfection is classiêdinto chemical disinfection with the use of chemical drugs (dis-infectants) and physical disinfection with the use of moistheat, ultraviolet light, and other agents.

1-1. Chemical disinfectionMicroorganisms are killed with chemical drugs. The killing

eŠect and mechanisms of a chemical drug diŠer depending onthe type, applied concentration, action temperature, andaction time of the chemical drug used, the degree of contami-nation on the object to be disinfected, and the species andstate (e.g., vegetative bacteria or spore bacteria) of microor-ganisms.

16811681JP XV General Information / Disinfection and Sterilization

Therefore, in applying the method, full consideration isrequired of the sterility and permissible storage period of theprepared chemical drug, the possibility of resistance ofmicroorganisms at the site of application, and the eŠect ofresidual chemical drug on the product. In selecting a suitablechemical drug, the following items should be considered inrelation to the intended use.

1) The antimicrobial spectrum2) Action time for killing microorganisms3) Action durability4) EŠect of the presence of proteins5) In‰uence on the human body6) Solubility in water7) In‰uence on the object to be disinfected8) Odor9) Convenience of use

10) Easy disposability11) In‰uence on the environment at disposal12) Frequency of occurrence of resistance1-2. Physical disinfectionMicroorganisms are killed without a chemical drug.(i) Steam ‰ow methodMicroorganisms are killed by direct application of steam.

This method is used for a product which may be denatured bythe moist heat method. As a rule, the product is kept in‰owing steam at 1009C for 30 – 60 minutes.

(ii) Boiling methodMicroorganisms are killed by putting the object in boiling

water. This method is used for a product which may bedenatured by the moist heat method. As a rule, the product isput in boiling water for 15 minutes or more.

(iii) Intermittent methodMicroorganisms are killed by heating for 30 – 60 minutes

repeatedly, three to ˆve times, once a day in water at 80 –1009C or in steam. This method is used for a product whichmay be denatured by the moist heat method. There is anothermethod called the low temperature intermittent method withrepeated heating at 60 – 809C. During the intermissionperiods between heating or warming, a suitable temperaturefor the growth of microorganisms of 209C or higher, must bemaintained.

(iv) Ultraviolet methodAs a rule, microorganisms are killed by irradiation with

ultraviolet rays at a wavelength of around 254 nm. Thismethod is used for products which are resistant to ultravioletrays, such as smooth-surfaced articles, facilities, and equip-ment, or water and air. This method does not suŠer from theoccurrence of resistance, which is observed in chemical disin-fection, and shows a killing eŠect on bacteria, fungi, andviruses. It must be taken into consideration that direct ultrav-iolet irradiation of the human body can injure the eyes andskin.

2. Sterilization methods2-1. Heating methodsIn these methods, the heating time before the temperature

or pressure reaches the prescribed value diŠers according tothe properties of the product, the size of the container, andthe conditions. The duration of heating in conducting thesemethods is counted from the time when all the parts of theproduct have reached the prescribed temperature.

(i) Moist heat methodMicroorganisms are killed in saturated steam at a suitable

temperature and pressure. This method is generally used forheat-stable substances, such as glass, porcelain, metal, rub-ber, plastics, paper, and ˆber, as well as heat-stable liquids,such as water, culture media, reagents, test solutions, liquidsamples, etc. As a rule, one of the following conditions isused.

115 – 1189C for 30 minutes121 – 1249C for 15 minutes126 – 1299C for 10 minutes

(ii) Dry-heat methodMicroorganisms are killed in dry-heated air. This method

is generally used for heat-stable substances, such as glass,porcelain, and metal, as well as heat-stable products, such asmineral oils, fats and oils, powder samples, etc. This methodis generally conducted in the way of direct heating by gas orelectricity or circulating heated air. As a rule, one of the fol-lowing conditions is used.

160 – 1709C for 120 minutes170 – 1809C for 60 minutes180 – 1909C for 30 minutes

2-2. Irradiation methods(i) Radiation methodMicroorganisms are killed by gamma-rays emitted from a

radioisotope or electron beam and bremsstrahlung (X-ray)generated from an electron accelerator. This method is gener-ally used for radiation-resistant substances such as glass, por-celain, metal, rubber, plastics, ˆber, etc. The dose is decidedaccording to the material properties, and the degree of con-tamination of the product to be sterilized. Special considera-tion is necessary of the possibility of qualitative change of theproduct after the application of the method.

(ii) Microwave methodMicroorganisms are killed by the heat generated by direct

microwave irradiation. This method is generally used formicrowave-resistant products such as water, culture media,test solutions, etc. As a rule, microwave radiation with awavelength of around 2450±50 MHz is used.

2-3. Gas methodsMicroorganisms are killed by a sterilizing gas. Suitable

gases for killing microorganisms include ethylene oxide gas,formaldehyde gas, hydrogen peroxide gas, chlorine dioxidegas, etc. Temperature, humidity, the concentration of gas,and the exposure time diŠer in accordance with the species ofgas used. As sterilizing gases are generally toxic to humans,full consideration is required of the environmental controlfor the use of gases and the concentration of residual gas. Insome of the gas methods, it may be di‹cult to measure orestimate quantitatively the killing of microorganisms.

2-4. Filtration methodMicroorganisms are removed by ˆltration with a suitable

ˆltering device. This method is generally used for gas, water,or culture media and test solutions containing a substancethat is water-soluble and unstable to heat. As a rule, a ˆlterhaving a pore size of 0.22 mm or smaller is used for the sterili-zation. However, in this method, a ˆlter with a pore size of0.45 mm or smaller is permitted to be used.

16821682 JP XVGuideline for Residual Solvents, / General Information

7. Guideline for Residual Solvents,Residual Solvents Test, and Models

for the Test in Monographs1. Guideline for Residual Solvents

Refer to the Guideline for Residual Solvents in Phar-maceuticals (PABWELD Notiˆcation No.307; dated March30, 1998).

The acceptable limits of residual solvents recommended inthe Guideline were estimated to keep the safety of patients.The levels of residual solvents in pharmaceuticals should notexceed the limits, except for in a special case. Accordingly,pharmaceutical manufacturers should assure the quality oftheir products by performing the test with the productsaccording to the Residual Solvents Test, to keep the limitsrecommended in the Guideline.

2. Residual Solvents TestMethod—Perform the test as directed in the Residual Sol-

vents Test under the General Tests, Processes and Apparatus.International harmonization—The test may also be per-

formed according to the Residual solvents in the EP or theOrganic volatile impurities in the USP. Even in this case,Monographs should be described in the JP style.

3. Models for the Test in MonographsThe following are typical examples for the test in Mono-

graphs, but these do not necessarily imply that other suitableoperating conditions can not be used. It is important to pre-pare (draft) monographs according to the Guideline for thePreparation of the Japanese Pharmacopoeia.1) A model for test item, amounts of test sample and refer-ence standard (reference substance), preparation of the sam-ple solution and the standard solution, injection amount forgas chromatography, calculation formula, and preparationof the internal standard solution

Residual solvents (or name of the solvent)—Weigh ac-curately about 0.200 g of △△△ (name of the substance to betested), add exactly 5 mL of the internal standard solution todissolve, add water to make exactly 20 mL, and use this solu-tion as the sample solution. If necessary ˆlter or centrifuge.Separately, weigh exactly 0.10 g of ◯◯◯ reference sub-stance (name of the solvent), put in a vessel containing 50 mLof water, and add water to make exactly 100 mL. Pipet 5 mLof this solution, and add water to make exactly 100 mL. Pipet2 mL of this solution, add exactly 5 mL of the internal stan-dard solution and 20 mL of water, and use this solution as thestandard solution. Perform the test with 1 mL each of thesample solution and standard solution as directed (in thehead-space method) under Gas Chromatography accordingto the following conditions, and calculate the ratios, QT andQS, of the peak area of △△△ (name of the substance to betested) to that of the internal standard of each solution,respectively. The amount of △△△ should be not more than×× ppm.

Amount of ◯◯◯ (mg)＝amount of ◯◯◯ reference substance (mg)×

QT

QS

Internal standard solution—A solution of △△△ in ▽▽▽

(name of solvent) (1 in 1000).

2) Models for operating conditions for a head-space sampleinjection device

Operating conditions (1) for the head-space sample injectiondevice—

Equilibration temperature forinside vial

A constant tempera-ture of about 809C

Equilibration time for inside vial 60 minutesTransfer-line temperature A constant tempera-

ture of about 859CCarrier gas NitrogenPressurisation time 30 secondsInjection volume of sample 1.0 mL











3) Models for operating conditions and system suitabilityIn the operating conditions, generally, items required for

the test such as detector, column, column temperature, carri-er gas, ‰ow rate, and time span of measurement should bespeciêd, and in the system suitability, items such as test forrequired detectability, system performance, and systemrepeatability should be speciêd.

The following are several models for the operating condi-tions and the system suitability:Operating conditions—

Detector: Specify in the following manner: ``Hydrogen‰ame-ionization detector''.

Column: Specify the inside diameter, length, material ofcolumn, name of stationary phase liquid and thickness of sta-tionary phase in the following manner: ``Coat the inside wallof a fused silica tube, 0.3 mm in inside diameter and 30 m inlength, to 0.25 mm thickness with polyethylene glycol 20M.''Describe the inside diameter and length of the column, andthickness or particle size of the stationary phase based on thedata obtained for validation of the test method.

Column temperature: Specify in the following manner: `Àconstant temperature of about ×9C.'' or ``Maintain at 409Cfor 20 minutes, then increase to 2409C at 109C per minute,and keep at 2409C for 20 minutes.''

Carrier gas: Specify in the following manner: ``Helium''.Flow rate: Specify in the following manner: `Àdjust the

‰ow rate so that the retention time of ▲▲▲ is about ×

minutes.'' or ``35 cmWsecond''. Describe the ‰ow rate basedon the data obtained for validation of the test method.

Time span of measurement: About × times as long as the

16831683JP XV General Information / Guideline for Residual Solvents,

retention time of △△△ beginning after the air peak.System suitability—

Test for required detectability: Specify in the followingmanner: ``Measure exactly × mL of the standard solution,and add □□□ to make exactly × mL. Conˆrm that thepeak area of □□□ obtained from × mL of this solution isequivalent to × to ××z of that of □□□ obtained fromthe standard solution''. This item should be speciêd when,in Purity, the amount of an impurity is controlled by compar-ing the area of a speciˆc peak from the sample solution withthat of the peak of ×××× from the standard solution andthe system repeatability alone is not su‹cient to check thesystem suitability.

System performance: Specify in the following manner:``Dissolve × g of □□□ and × g of △△△ in × mL of ◯◯◯. When the procedure is run with × mL of this solution un-der the above operating conditions, □□□ and △△△ areeluted in this order with the resolution between these peaksbeing not less than ×, and the number of theoretical platesand the symmetry factor of the peak of □□□ are not lessthan ×× steps and not more than ×××, respectively.''This item should be speciêd in all of the test methods.Generally, the order of elution and the resolution, and in caseof need (such as when the peak is asymmetrical) the symmet-ry factor, should be speciêd. The order of elution and theresolution may be replaced by the resolution and the numberof theoretical plates. When no suitable reference substance isavailable to check separation, the number of theoreticalplates and the symmetry factor of the peak of the substanceto be tested may be speciêd.

System repeatability: Specify in the following manner:``When the test is repeated × times with × mL of the solutionfor the system suitability test under the above operating con-ditions, the relative standard deviation of the peak areas of△△△ is not more than ×z.'' The system repeatabilityshould be speciêd in any case, except in a qualitative test.

Examples of operating conditions and system suitabilityTest conditions (1)Operating conditions—

Detector: Hydrogen ‰ame-ionization detectorColumn: Coat the inside wall of a fused silica tube, 0.53

mm in inside diameter and 30 m in length, to 3 mm thicknesswith 6z cyanopropylphenyl-methyl silicon polymer for gaschromatography. Use a guard column if necessary.

Column temperature: Maintain at 409C for 20 minutes,then increase to 2409C at 109C per minute if necessary, andkeep at 2409C for 20 minutes.

Injection port temperature: A constant temperature ofabout 1409C

Detector temperature: A constant temperature of about2509C

Carrier gas: HeliumFlow rate: 35 cmWsecondSplit ratio: 1:5

System suitability—System performance: When the procedure is run with the

standard solution under the above operating conditions, theresolution between the peaks is not less than 1.0. (Note: Inthe case that the number of substances to be tested is two ormore.)

System repeatability: When the test is repeated 3 times withthe standard solution under the above operating conditions,

the relative standard deviation of the peak areas of the sub-stance to be tested is not more than 15z.

Test conditions (2)Operating conditions—


mm in inside diameter and 30 m in length, to 5 mm thicknesswith 5z phenyl-methyl silicon polymer for gas chro-matography. If necessary use a guard column prepared bycoating the inside wall of a fused silica tube, 0.53 mm in in-side diameter and 5 m in length, to 5 mm thickness with 5zphenyl-methyl silicon polymer for gas chromatography.

Column temperature: Maintain at 359C for 5 minutes,then increase to 1759C at 89C per minute, further increase to2609C at 359C per minute if necessary, and keep at 2609C for16 minutes.



Carrier gas: HeliumFlow rate: 35 cmWsecondSplit ratio: Splitless



System repeatability: When the test is repeated 3 times withthe standard solution under the above operating conditions,the relative standard deviation of the peak areas of the sub-stance to be tested is not more than 15z.

Test conditions (3)Operating conditions—


mm in inside diameter and 30 m in length, to 0.25 mm thick-ness with polyethylene glycol 20M for gas chromatography.Use a guard column if necessary.

Column temperature: Maintain at 509C for 20 minutes,then increase to 1659C at 69C per minute if necessary, andkeep at 1659C for 20 minutes.



Carrier gas: HeliumFlow rate: 35 cmWsecondSplit ratio: 1:5



System repeatability: When the test is repeated 3 times withthe standard solution under the above operating conditions,the relative standard deviation of the peak areas of the sub-stance to be tested is not more than 15z.

1684

Oct. 2004 (Rev. 2)

Harmonized items JP 15 Remarks

Residue on IgnitionWSulphated AshTest

(Introduction)

2.44 Residue on Ignition Test

(Introduction) JP's particular description: Explana-tion of the description in mongraph,etc.

Procedure Procedure

Nov. 2003


Specific Surface Area 3.02 Specific Surface Area

(Introduction) (Introduction) JP's particular description: Explana-tion on this test.

Multi-point measurement Multi-point measurement

Single-point measurement Single-point measurement

Sample preparation Sample preparationOutgassingAdsorbateQuantity of sample

Method 1: The dynamic flow method Method 1: The dynamic flow method

Method 2: The volumetric method Method 2: The volumetric method

Reference materilals Reference materilals

Figure 1 Schematic diagram of thedynamic flow method apparatus

Fig. 3.02-1 Schematic diagram of thedynamic flow method apparatus

Figure 2 Schematic diagram of thevolumetric method apparatus

Fig. 3.02-2 Schematic diagram of thevolumetric method apparatus

1684 JP XVInternational Harmonization / General Information

8. International HarmonizationImplemented in the Japanese

Pharmacopoeia Fifteenth EditionItems for which harmonization has been agreed among the

European Pharmacopoeia, the United States Pharmacopeiaand the Japanese Pharmacopoeia are implemented in theJapanese Pharmacopoeia Fifteenth Edition (JP 15). They are

shown in the tables below.The column headed Harmonized items shows the har-

monized items written in the Pharmacopoeial HarmonizationAgreement Document, and the column headed JP 15 showsthe items as they appear in JP 15. In the Remarks column,notes on any diŠerences between JP 15 and the agreement areshown as occasion demands.

The date on which the agreement has been signed is shownon the top pf each table. In the case where the harmonized i-tems have been revised, this is indicated in parenthesis.

1685

June 2004


3.04 Particle Size Determination

(Introduction) (Introduction) JP's particular description: Explana-tion on this test.

Optical microscopy Method 1. Optical microscopy JP's particular description: Explana-tion on this test.

Apparatus Apparatus

Adjustment Adjustment

Illumination Illumination

Visual characterization Visual characterization JP's particular description: Explana-tion on the method of particle sizemeasurement.

Photographic characterization Photographic characterization

Preparation of the mount Preparation of the mount

Crystallinity characterization Crystallinity characterization

Limit test of particle size bymicroscopy

Limit test of particle size bymicroscopy

Particle size characterization Particle size characterization

Particle shape characterization Particle shape characterization

General observations General observations

Figure 1 Commonly used measure-ments of particle size

Fig. 3.04-1 Commonly used measure-ments of particle size

Figure 2 Commonly used descriptionsof particle shape

Fig. 3.04-2 Commonly used descrip-tions of particle shape

Analytical sieving Method 2. Analytical sieving method JP's particular description: Explana-tion on this method.

Principles of analytical sieving Principles of analytical sieving

Test sieves Test sieves

Test specimen Test specimen

Agitation methods Agitation methods

Endpoint determination Endpoint determination

Sieving methods Sieving methods

1) Methanical agitationdry sieving method

Methanical agitationDry sieving method

JP's particular description: How totreat the fine powder adherent on backsurface of the sieve.

2) Air entrainment methodsair jet and sonic sifter sieving

Air entrainment methodsAir jet and sonic shifter sieving

Table 1 Size of standard sieve series inrange of interest

Table 3.04-1 Size of standard sieveseries in range of interest

Interpretation Interpretation

1685JP XV General Information / Isoelectric Focusing

1686

Jan. 2000


Bacterial Endotoxins Test 4.01 Bacterial Endotoxins Test

(Introduction) (Introduction)

Apparatus Apparatus

Preparation of standard endotoxinstock solution

Preparation of standard endotoxinstock solution

JP's particular description: Endotoxin10000 Reference Standard and En-dotoxin 100 Reference Standard.

Preparation of standard endotoxin so-lution

Preparation of standard endotoxin so-lution

Preparation of sample solutions Preparation of sample solutions JP's particular description: Additionof the preparation of sample solutionsfor containers, and deletion of that formedical devices.

Determination of maximum valid di-lution

Determination of maximum valid di-lution

JP's particular description: Additionof the concentration unit of sample so-lutions in the case where the endotoxinlimit per equivalent is specified.

Gel-clot technique Gel-clot technique

(1) Preparatory testing (1) Preparatory testing

(i) Test for confirmation of labeledlysate sensitivity

(i) Test for confirmation of labeledlysate sensitivity

(ii) Test for interfering factors (ii) Test for interfering factors

(2) Limit test (2) Limit test

(i) Procedure (i) Procedure

(ii) Interpretation (ii) Interpretation

(3) Assay (3) Assay


(ii) Calculation and interpretation (ii) Calculation and interpretation

Photometric techniques Photometric techniques

(1) Turbidimetric techniques (1) Turbidimetric techniques

(2) Chromogenic technique (2) Chromogenic technique

(3) Preparatory testing (3) Preparatory testing

(i) Assurance of criteria for thestandard curve

(i) Assurance of criteria for thestandard curve

JP's particular description: The test forassurance of criteria for the standardcurve must be carried out for each lotof lysate reagent.

(ii) Test for interfering factors (ii) Test for interfering factors JP's particular description:Two conditions which the test must

meet are specified.Explanation of the test method

where the interfering action is found.Explanation of the usual methods

for eliminating the interference.

(4) Assay (4) Assay


(ii) Calculation (ii) Calculation JP's particular description: Additionof the alternative requirement to whichsolution D must meet for valid test.

(iii) Interpretation (iii) Interpretation

1686 JP XVIsoelectric Focusing / General Information

1687

Regents, test solutions Deletion

Amebocyte lysate Deletion

Lysate TS Deletion

Water for bacterial endotoxins test(BET)

Deletion

Oct. 2002


Sterility 4.06 Sterility Test

(Introduction) (Introduction)

Precautions against microbial con-tamination

The same statement appears in the in-troduction.

Culture media and incubation temper-atures

Media and rinsing ‰uids

Fluid thioglycollate medium Fluid thioglycolate medium Note 1 – 5

Soya-bean casein digest medium Soybean-casein digest medium

Sterility Sterility of media

Growth promotion test of aerobes,anaerobes and fungi

Growth promotion test Note 6

Validation test Validation test

Membrane ˆltration Membrane ˆltration

Direct inoculation Direct inoculation

Test for sterility of the product to beexamined

Test for sterility of the products to beexamined

Membrane ˆltration Membrane ˆltration Note 7

Aqueous solutions a) Liquid medicines

Soluble solids b) Solid medicines

Oils and oily solutions c) Oils and oily solutions

Ointments and creams d) Ointments and creams

Direct inoculation of the culture medi-um

Direct inoculation of the culture medi-um

Oily liquids a) Oily liquids

Ointments and creams b) Ointments and creams

Catgut and other surgical sutures forveterinary use

The items not included in JP.

Observation and interpretation ofresults

Cultivation and observation, Observa-tion and interpretation of results

Note 8 – 9

Application of the test to parenteralpreparations, ophthalmic and othernon-injectable preparations requiredto comply with the test for sterility

A part of this is directed in the GeneralTest.

Table 2.6.1.-1. Strains of the testmicro-organisms suitable for use inthe Growth Promotion Test and theValidation Test

Table 4.06-1. Microorganisms forgrowth promotion test and the vali-dation test

Table 2.6.1.-2. Minimum quantityto be used for each medium

Table 4.06-3. Minimum quantity tobe used for each medium


1688

Table 2.6.1.-3. Minimum number ofitems to be tested

Table 4.06-2. Number of items to betaken from the lot

Note 10 – 11

Note: 1) Non-pharmaceutical media: Not to be used.2) Water content of agar: Not being speciêd.3) EŠective period of media: Unnecessary to be validated.4) EŠective period of media stored in hermetic containers: Usable for maximum one year.5) Medium for sterility test of the products containing a mercurial preservative: Speciêd.6) Periodic growth promotion test for a ready-made powder medium: Speciêd.7) Amount of the rinsing ‰uid each time in the membrane-ˆltration method: 100 mL per ˆlter.8) Transferring amount from turbid medium to fresh medium: A suitable amount.9) Requirements for the retesting in the case when the evidence of microbial growth is found: Speciêd.

10) The table of 'Number of items to be taken from the lot': Speciêd as a part of the General Test.11) Number of large-volume products (more than 100 mL) to be taken from the lot: Maximum 10 containers.

Feb. 2004


Uniformity of Dosage Units 6.02 Uniformity of Dosage Units

(Introduction) (Introduction) JP's particular description:Additional explanation on Liquids.Additional explanation for the partnot containing drug substance.

Content uniformity Content uniformity

Solid dosage forms Solid dosage forms

Liquid dosage forms Liquid dosage forms

Calculation of acceptance value Calculation of acceptance value

Mass variation Mass variation JP's particular description: Assumingthat the concentration of drug sub-stance is uniform in each lot.

Uncoated or film-coated tablets Uncoated or film-coated tablets

Hard capsules Hard capsules

Soft capsules Soft capsules

Solid dosage forms other than tabletsand capsules

Solid dosage forms other than tabletsand capsules

Liquid dosage forms Liquid dosage forms The phrase `ìn conditions of normaluse. If necessary, compute the equiva-lent volume after determining the den-sity.'' is deleted.

Calculation of acceptance value Calculation of acceptance value

Criteria Criteria

Solid and liquid dosage forms Solid and liquid dosage formsTable 1Application of content uniformity(CU) and mas variation (MV) test fordosage forms

Table 6.02-1Application of content uniformity(CU) and mas variation (MV) test fordosage forms

JP's particular description:Addition of ``(divided forms, lyophi-lized forms)'' and ``(true solution)''.

Table 2 Table 6.02-2 The phrases `àt time of manufacture''and ``For purposes of this Phar-macopoeia'' are deleted


1689

June 2004 (Rev.1)


Test for Extractable Volume of Paren-teral Preparations

6.05 Test for Extractable Volume ofParenteral Preparations

(Introduction) (Introduction) JP's particular description: Explana-tion of this Test

Single-dose containers (1) Single-dose containers

Multi-dose containers (2) Multi-dose containers

Cartridges and prefilled syringes (3) Cartridges and prefilled syringes

Parenteral infusions (4) Parenteral infusions

June 2004 (Rev.1)


Particulate Matter in Injectables 6.07 Insoluble Particulate Matter Testfor Injections

(Introduction) (Introduction) The phrase `òther than gas bubbles'' isdeleted.

Method 1. Method 1.

Light obscuration particle count test Light obscuration particle count test JP's particular description: Descriptionof evaluation frequency.

Apparatus

Calibration JP's particular description

Manual methods JP's particular description

Electronic methods JP's particular description

Automated methods JP's particular description

Sample volume accuracy JP's particular description

Sample flow rate JP's particular description

Sensor JP's particular description

Particle resolution JP's particular description

Particle counting accuracy JP's particular description

Threshold accuracy JP's particular description

Reagents JP's particular description

General precautions General precautions

Method Method

Evaluation Evaluation JP's particular description: `èqual toor more than 100 mL''.

Method 2. Method 2.

Microscopic particle count test Microscopic particle count test

Apparatus

General precautions General precautions

Method Method

Evaluation Evaluation JP's particular description: `èqual toor more than 100 mL''.

1. Circular diameter graticule Fig. 6.07-1 Circular diameter graticule


1690

June 2004


Disintegration 6.09 Disintegration JP's particular description: This test isapplied also to Granules and Pills.

Apparatus Apparatus

Basket-rack assembly Basket-rack assembly JP's particular description: The ap-paratus may be varied somewhatprovided the specifications.

Disk Disk

Auxiliary tube JP's particular description

Procedure Procedure JP's particular description: Making useof water also as the test fluid.Setting each intervals of the immersion.Making a definition of complete disin-tegration.Setting a procedure for Granules.

(1) Immediate-release preparation JP's particular description:Setting a test for Granules and Pills.

(2) Enteric coated preparations JP's particular description:Setting a test for enteric coated prepa-rations.

Figure 1 Disintegration apparatus Fig. 6.09-1 Disintegration apparatus

Fig. 6.09-2 Auxiliary tube JP's particular description.

June 2004


Dissolution 6.10 Dissolution Test JP's particular description: The testalso aims at preventing significantbioinequivalence.

Apparatus Apparatus

Apparatus 1 (Basket apparatus) Apparatus for Basket Method(Apparatus 1)

Apparatus 2 (Paddle apparatus) Apparatus for Paddle Method(Apparatus 2)

JP's particular description: The sinkeris allowed to use in case only when spe-cified in the monograph.

Apparatus 3 (Reciprocating cylinder)

Apparatus 4 (Flow-through cell) Apparatus for Flow-Through CellMethod (Apparatus 3)

JP's particular description: A pumpwithout the pulsation may also be used.

Procedure Procedure

Apparatus 1 or 2 Basket Method or Paddle Method

Immediate-release dosage forms Immediate-release dosage forms

Procedure Procedure

Dissolution medium Dissolution medium

Time Time

Extended-release dosage forms Extended-release dosage forms

Procedure Procedure


Time Time


1691

Delayed-release dosage forms Delayed-release dosage forms

Procedure Procedure In the Harmonized text: Alternativeusage of Method A or B.

Method A

Method B

Dissolution medium JP's particular description.

Time Time JP's particular description: Time isspecified each for the 1st and 2ndfluids.

Apparatus 3 not specified

Immediate-release dosage forms

Procedure

Dissolution medium

Time

Extended-release dosage forms

Procedure

Dissolution medium

Time

Delayed-release dosage forms

Procedure

Time

Apparatus 4 Flow-Through Cell Method

Immediate-release dosage forms Immediate-release dosage forms

Procedure Procedure


Time Time

Extended-release dosage forms Extended-release dosage forms

Procedure Procedure


Time Time

Delayed-release dosage forms

Procedure

Time

Interpretation Interpretation JP's particular description: Follow In-terpretation 1 when the value Q is spe-cified in the individual monograph,otherwise follow Interpretation 2.

Immediate-release dosage forms Immediate-release dosage forms JP's particular description:Setting Interpretation 2.

Interpretation 1

Interpretation 2

Extended-release dosage forms Extended-release dosage forms JP's particular description:Setting Interpretation 2.

Interpretation 1

Interpretation 2

Delayed-release dosage forms Delayed-release dosage forms Non-harmonized items: Different dis-solution medium. Deletion of dishar-monized part on the value Q.


1692

JP's particular description: Setting In-terpretation 2.

Interpretation 1 The value Q is specified in theindividual monograph.

Interpretation 2

Acceptance Table 1 Acceptance Table 6.10-1




Figure 1 Apparatus 1. Basket stirringelement

Fig. 6.10-1 Apparatus 1. Basket stir-ring element

Figure 2 Paddle stirring element Fig. 6.10-2 Paddle stirring element

Figure 2 a Alternative sinker Fig. 6.10-2 a Alternative sinker

Figure 3 Apparatus not specified

Figure 4 Apparatus 4(top) large cell tablets and capsules(bottom) tablet holder for the largecell

Fig. 6.10-3 Apparatus 3(top) large cell tablets and capsules(bottom) tablet holder for the largecell

Figure 5 Apparatus 4(top) small cell tablets and capsules(bottom) tablet holder for the smallcell

Fig. 6.10-4 Apparatus 3(top) small cell tablets and capsules(bottom) tablet holder for the smallcell

Sep. 2002 (Rev. 1)


Ethanol Ethanol

Identification A not specified as Identification Setting Specific gravity as specification.

Identification B Identification

Relative density Specific gravity Setting Specific gravity at 159C.

Appearance Purity (1) Clarity and color of solu-tion

Acidity or alkalinity Purity (2) Acid or alkali

Volatile impurities Purity (3) Volatile impurities

Absorbance Purity (4) Other impurities

Residue onevaporation Purity (5) Residue on evaporation

Storage Containers and storage

Sep. 2002 (Rev. 1)


Ethanol, Anhydrous Anhydrous Ethanol

Definition limits of content

Identification A not specified as Identification Setting Specific gravity as specification.

Identification B Identification

Relative density Specific gravity Setting Specific gravity at 159C.


1693

Appearance Purity (1) Clarity and color of solu-tion


Volatile impurities Purity (3) Volatile impurities

Absorbance Purity (4) Other impurities

Residue onevaporation Purity (5) Residue on evaporation

Storage Containers and storage

Nov. 2003 (Rev. 2)


Sodium Chloride Sodium Chloride

Deˆnition limits of the content

Identiˆcation A Identiˆcation (1)

Identiˆcation B Identiˆcation (2)

Acidity or alkalinity Purity (2) Acidity or alkalinity

Sulphates Purity (3) Sulfates

Phosphates Purity (4) Phosphates

Bromides Purity (5) Bromides

Iodides Purity (6) Iodides

Ferrocyanides Purity (7) Ferrocyanides

Iron Purity (9) Iron

Barium Purity (10) Barium

Magnesium and alkaline-earth metals Purity (11) Magnesium and alkaline-earth materials

Aluminium not speciêd

Nitrites not speciêd

Potassium not speciêd

Loss on drying Loss on drying

Assay Assay

July 2003 (Rev. 1)


Carboxymethylcellulose Calcium Carmellose Calcium

Deˆnition origin



Identiˆcation C Identiˆcation (3)

Identiˆcation D Identiˆcation (4)

Alkalinity Purity (1) Alkali


Residue on ignition Residue on ignition

Limit of chloride Purity (2) Chloride

Limit of sulfate Purity (3) Sulfate


1694

Nov. 2003 (Rev. 1)


Citric Acid, Anhydrous Anhydrous Citric Acid


Appearance of solution Purity (1) Clarity and color of solu-tion


Oxalic acid Purity (3) Oxalate

Readily carbonisable substances Purity (5) Readily carbonizable sub-stances


Water Water

Sulphated ash Residue on ignition

Assay Assay

Nov. 2003 (Rev. 1)


Citric Acid Monohydrate Citric Acid Hydrate




Oxalic acid Purity (3) Oxalate



Water Water


Assay Assay

Oct. 2001


Croscarmellose Sodium Definition

Croscarmellose Sodium origin

Identification A

B

C

Identification (1)

Identification (2)

Identification (3)

pH pH

Settling volume Precipitation test

Degree of substitution Degree of substitution

Loss on drying Residue on ignition

Loss on drying Residue on ignition

Packaging and storage Containers and storate


1695

Oct. 2001


Wheat Starch Wheat Starch

Deˆnition origin




pH pH


Total protein not speciêd

Oxidising substances Purity (2) Oxidizing substances

Sulphur dioxide Purity (3) Sulfur dioxide



Feb. 2003


Saccharin Saccharin


Limit of benzoate and salicylate Purity (3) Benzoate and salicylate




Assay Assay

Packaging and storage Containers and storage

Feb. 2004 (Rev. 1)


Saccharin Sodium Saccharin Sodium Hydrate


Identification A, B Identification (2)


Limit of benzoate and salicylate Purity (4) Benzoate and salicylate


Water Water

Assay Assay


1696

Oct. 2001


Cellacefate Cellulose Acetate Phthalate

Deˆnition content of the acetyl and carboxyben-zoyl groups

Identiˆcation Identiˆcation

Viscosity Viscosity

Limit of free acid Purity (2) Free acids

Water Water


Phthalyl content Assay (1) Carboxybenzoyl group

Content of acetyl Assay (2) Acetyl group

Packaging and storage Containers and storage

May 2005 (Rev. 1)


Microcrystalline Cellulose Microcrystalline Cellulose

Definition origin

Identification A Identification (1)

Identification B Identification (3)

pH pH

Water-soluble substances Purity (2) Water- soluble substances

Ether-soluble substances Purity(3) Diethyl ether-soluble sub-stances

Conductivity Conductivity



Bulk density Bulk density

May 2005 (Rev. 1)


Powdered Cellulose Powdered Cellulose

Definition origin


Identification B Identification (3)

pH pH

Water-soluble substances Purity (2) Water-soluble substances

Ether-soluble substances Purity (3) Diethyl ether- soluble sub-stances




1697

Feb. 2004 (Rev. 1)


Corn Starch Corn Starch

Deˆnition origin




pH pH



Limit of iron Purity (1) Iron

Limit of oxidizing substances Purity (2) Oxidizing substances

Sulfur dioxide determination Purity (3) Sulfur dioxide

Feb. 2003 (Rev. 1)


Anhydrous Lactose Anhydrous Lactose

Definition origin

Labeling labeling of relative quantities of a andb lactose

Identification Identification

Specific rotation Optical rotation

Clarity and color of solution Purity (1) Clarity and color of solu-tion

Acidity or alkalinity Purity (2) Acid and alkali

Protein and light-absorbing impurities Purity (4) Proteins and light absorb-ing substances


Water Water


Content of alpha and beta anomers Isomer ratio

Sep. 2002


Lactose Monohydrate Lactose Hydrate

Definition origin

Identification Identification

Specific rotation Specific rotation

Clarity and color of solution Purity (1) Clarity and color of solu-tion

Acidity or alkalinity Purity (2) Acid and alkali

Protein and light-absorbing impurities Purity (4) Protein and light absorbingsubstances

Water Water



1698

Feb. 2004


Ethyl Parahydroxybenzoate Ethyl Parahydroxybenzoate




Acidity Purity (2) Acid

Related substances Purity (4) Related substances System suitability is not specified.


Assay Assay

Feb. 2004


Butyl Parahydroxybenzoate Butyl Parahydroxybenzoate


Identification A Identification





Assay Assay

Feb. 2004


Propyl Parahydroxybenzoate Propyl Parahydroxybenzoate







Assay Assay

Feb. 2004


Methyl Parahydroxybenzoate Methyl Parahydroxybenzoate





1699




Assay Assay

Oct. 2001


Potato Starch Potato Starch

Deˆnition origin




pH pH


Oxidising substances Purity (2) Oxidizing substances

Sulphur dioxide Purity (3) Sulfur dioxide



Nov. 2003


Hydroxypropyl Methylcellulose Hypromellose

Definition limits of content of methoxy groupand hydroxypropoxy group

Labeling labeling of viscosity

Identification (1) Identification (1)





Viscosity Viscosity

Method 1 Method I

Method 2 Method II

pH pH

Heavy metals Purity Heavy metals



Assay Assay


1700

July, 2000


Benzyl Alcohol Benzyl Alcohol


Identiˆcation Identiˆcation


Refractive index Refractive index


Benzaldehyde and other relatedsubstances

Purity (3) Benzaldehyde and otherrelated substances

Peroxide value Purity (4) Peroxide value

Residue on evaporation Purity (5) Residue on evaporation

Assay Assay

Nov. 2003


Methylcellulose Methylcellulose

Definition limits of content of methoxy group

Labeling labeling of viscosity






Viscosity Viscosity

Method 1 Method I

Method 2 Method II

pH pH

Heavy metals Purity Heavy metals



Assay Assay

Sep. 2002


General Information

Amino Acid Analysis Amino Acid Analysis

Apparatus Apparatus

General Precautions General Precautions

Reference Standard Material Reference Standard Material

Calibration of Instrumentation Calibration of Instrumentation

Repeatability Repeatability

Sample Preparation Sample Preparation

Internal Standards Internal Standards


1701

Protein Hydrolysis Protein Hydrolysis

Method 1 Method 1

Hydrolysis Solution Hydrolysis Solution

Procedure Procedure

Method 2 Method 2


Vapor Phase Hydrolysis Vapor Phase Hydrolysis

Method 3 Method 3



Method 4 Method 4

Oxidation Solution Oxidation Solution

Procedure Procedure

Method 5 Method 5


Liquid Phase Hydrolysis Liquid Phase Hydrolysis

Method 6 Method 6



Method 7 Method 7

Reducing Solution Reducing Solution

Procedure Procedure

Method 8 Method 8

Stock Solutions Stock Solutions


Procedure Procedure

Method 9 Method 9

Stock Solutions Stock Solutions

Carboxymethylation Solution Carboxymethylation Solution

BuŠer Solution BuŠer Solution

Procedure Procedure

Method 10 Method 10


Procedure Procedure

Method 11 Method 11

Reducing Solutions Reducing Solutions

Procedure Procedure

Methodologies of amino acid analysisgeneral principles

Methodologies of Amino Acid Analy-sis General Principles

Method 1-Postcolumn ninhydrindetection general principle

Method 1-Postcolumn NinhydrinDetection General Principle

Method 2-Postcolumn OPA ‰uoro-metric detection general principle

Method 2-Postcolumn OPA Fluoro-metric Detection General Principle

Method 3-Precolumn PITC derivati-zation general principle

Method 3-Precolumn PITC Derivati-zation General Principle

Method 4-Precolumn AQC derivati-zation general principle

Method 4-Precolumn AQC Derivati-zation General Principle


1702

Method 5-Precolumn OPA derivati-zation general principle

Method 5-Precolumn OPA Derivati-zation General Principle

Method 6-Precolumn DABS-Clderivatization general principle

Method 6-Precolumn DABS-ClDerivatization General Principle

Method 7-Precolumn FMOC-Clderivatization general principle

Method 7-Precolumn FMOC-ClDerivatization General Principle

Method 8-Precolumn NBD-Fderivatization general principle

Method 8-Precolumn NBD-FDerivatization General Principle

Data calculation and analysis Data Calculation and Analysis

Calculations Calculations

Amino acid mole percent Amino Acid Mole Percent

Unknown protein samples Unknown Protein Samples

Known protein samples Known Protein Samples

Oct. 1999


Sodium Dodecyl Sulphate Poly-acrylamide Gel Electrophoresis(SDS-PAGE)

General Information

SDS-Polyacrylamide Gel Electrophore-sis

Characteristics of polyacrylamide gels 1. Characteristics of PolyacrylamideGels

Denaturing polyacrylamide gel elec-trophoresis

Reducing conditions

Non-reducing conditions

2. Polyacrylamide Gel Electrophore-sis under Denaturing Conditions

1) Reducing conditions

2) Non-reducing conditions

Characteristics of discontinuousbuŠer system gel electrophoresis

3. Characteristics of DiscontinuousBuŠer System Gel Electrophoresis

Preparing vertical discontinuousbuŠer SDS polyacrylamide gels

Assembling of the gel moulding cas-sette

Preparation of the gel

4. Preparing Vertical DiscontinuousBuŠer SDS-Polyacrylamide Gels

1) Assembling of the gel mouldingcassette

2) Preparation of the gel

Mounting the gel in the electrophore-sis apparatus and electrophoreticseparation

3) Mounting the gel in the electro-phoresis apparatus and electro-phoretic separation

Detection of protein in gels

Coomassie staining

Silver staining

5. Detection of Proteins in Gels

1) Coomassie staining

2) Silver staining

Drying of stained SDS polyacrylamidegels

6. Drying of Stained SDS-Polyacrylamide Gels

Molecular-mass determination 7. Molecular-Mass Determination

Validation of the test 8. Suitability of the Test

Quantiˆcation of impurities 9. Quantiˆcation of Impurities

Reagents, test solutions Test Solutions

Blocking solution Blocking TS

Coomassie staining solution Coomassie staining TS

Destaining solution Destaining TS

Developer solution Developer TS


1703

Fixing solution Fixing TS

Silver nitrate reagent Silver nitrate TS for silver staining

Trichloroacetic acid reagent Trichloroacetic acid TS for ˆxingTable 1 － Preparation of resolvinggel

Table 2 － Preparation of stacking gel

Table 1. Preparation of resolving gel

Table 2. Preparation of stacking gel

Sep. 2002


General Information

Capillary Electrophoresis Capillary Electrophoresis

Apparatus Apparatus

Capillary zone electrophoresis

Optimisation

Instrumental parameters

Voltage

Polarity

Temperature

Capillary

Electrolytic solution parameters

BuŠer type and concentration

BuŠer pH

Organic solvents

Additives for chiral separations

1. Capillary Zone Electrophoresis

Optimization


Voltage

Polarity

Temperature

Capillary


BuŠer type and concentration

BuŠer pH

Organic solvents


Capillary gel electrophoresis

Characteristics of gels

2. Capillary Gel Electrophoresis

Characteristics of Gels

Capillary isoelectric focusing

Loading step

loading in one step

sequential loading

Focusing step

Mobilisation step

Optimisation

Voltage

Capillary

Solutions

3. Capillary Isoelectric Focusing

Loading step

loading in one step

sequential loading

Focusing step

Mobilization step

Optimization

Voltage

Capillary

Solutions

Micellar electrokinetic chro-matography (MEKC)

Optimisation


Voltage

Temperature

Capillary


4. Micellar Electrokinetic Chro-matography (MEKC)

Optimization


Voltage

Temperature

Capillary


Surfactant type and concentration

BuŠer pH

Surfactant type and concentration

BuŠer pH


1704

Organic solvents


Other additives

Organic solvents


Other additives

Quantiˆcation

Calculations

Quantiˆcation

Calculations

System Suitability

Apparent number of theoreticalplates

Resolution

Symmetry factor

Signal-to-noise ratio

System Suitability

Apparent Number of TheoreticalPlates

Resolution

Symmetry Factor

Signal-to-noise Ratio

Feb. 2004


General InformationTablet Friability Tablet Friability Test

Sep. 2002


General Information

Total Protein Assay Total Protein Assay

Method 1

Standard Solution

Test Solution

Procedure

Light-scattering

Calculations

Method 1

Standard Solution

Test Solution

Procedure

Light-Scattering

Calculations

Method 2

Standard solutions

Test solution

Blank

Reagents and solutions

Copper sulfate reagent

SDS Solution

Sodium Hydroxide Solution

Alkaline copper reagent

Diluted Folin-Ciocalteu's PhenolRagent

Procedure

Calculations

Interfering substances

Sodium deoxycholate reagent

Trichloroacetic acid reagent

Procedure

Method 2

Standard Solutions

Test Solution

Blank

Reagents and Solutions

Copper Sulfate Reagent

5z SDS TS

Sodium Hydroxide Solution

Alkaline Copper Reagent

Diluted Folin's TS

Procedure

Calculations

Interfering Substances

Sodium Deoxycholate Reagent

Trichloroacetic Acid Reagent

Procedure

Explanatory footnote `Èxample: theMinimum Requirements for BiologicalProducts and individual monograph ofJP'' is added.

Method 3

Standard solutions

Method 3

Standard Solutions


1705

Test solution

Blank

Test Solution

Blank

Coomassie reagent Coomassie Reagent

Procedure

Calculations

Procedure

Calculations

Method 4

Standard solutions

Test solution

Blank

Reagents

BCA Reagent

Copper sulfate reagent

Copper-BCA reagent

Procedure

Calculations

Method 4

Standard Solutions

Test Solution

Blank


BCA Reagent

Copper Sulfate Reagent

Copper-BCA Reagent

Procedure

Calculations

Method 5

Standard solutions

Test solution

Blank

Biuret reagent

Procedure

Calculations

Interfering substances

Comments

Method 5

Standard Solutions

Test Solution

Blank

Biuret Reagent

Procedure

Calculations

Interfering Substances

Comments

Method 6

Standard solutions

Test solution

Blank

Reagents

Borate buŠer

Stock OPA reagent

OPA reagent

Procedure

Calculations

Method 6

Standard Solutions

Test Solution

Blank


Borate BuŠer

Stock OPA Reagent

OPA Reagent

Procedure

Calculations

Method 7

Procedure A

Procedure B

Calculations

Method 7

Procedure A

Procedure B

Calculations

Sep. 2002


General Information

Isoelectric Focusing Isoelectric Focusing

Theoretical aspects Theoretical Aspects

Practical aspects Practical Aspects


1706

Apparatus Apparatus

Isoelectric Focusing in polyacrylamidegels: detailed procedure

Preparation of the gels

1) 7.5 per cent polyacrylamide gel

2) Preparation of the mould

Isoelectric Focusing in Poly-acrylamide Gels: Detailed Procedure

Preparation of the Gels

7.5z Polyacrylamide gel

Preparation of the mould

Method Method

Variations to the detailed procedure(subject to validation)

Variations to the Detailed Procedure(Subject to Validation)

Validation of iso-electric focusingprocedures

Validation of Iso-Electric FocusingProcedures

Speciêd variation to the generalmethod

Speciêd Variations to the GeneralMethod

Point to consider Points to Consider

Figure-Mould Figure. Mould

Reagents

Fixing solution for isoelectric focus-ing in polyacrylamide gel


Fixing solution for isoelectric focus-ing in polyacrylamide gel

Coomassie staining TS

Destaining solution

Coomassie staining TS and destainingsolution are speciêd.

June 2004


General InformationPowder Flow Powder Flow

Angel of repose 1. Angel of repose

Basic methods for angel of repose 1.1 Basic methods for angel of re-pose

Variations in angel of reposemethods

1.2 Variations in angel of reposemethods

Angel of repose general scale offlowability

1.3 Angel of repose general scale offlowability

Experimental considerations for an-gle of repose

1.4 Experimental considerations forangle of repose

Recommended procedure for angleof repose

1.5 Recommended procedure for an-gle of repose

Compressibility index and Hausnerratio

2. Compressibility index and Hausnerratio

Basic methods for compressibility in-dex and Hausner ratio

2.1 Basic methods for compressibili-ty index and Hausner ratio

Experimental considerations for thecompressibility index and Hausnerratio

2.2 Experimental considerations forthe compressibility index andHausner ratio

Recommended procedure for com-pressibility index and Hausner ra-tio

2.3 Recommended procedure forcompressibility index and Hausnerratio

Flow through an orifice 3. Flow through an orifice

Basic methods for flow through anorifice

3.1 Basic methods for flow throughan orifice


1707

Variations in methods for flowthrough an orifice

3.2 Variations in methods for flowthrough an orifice

General scale of flowability for flowthrough an orifice

3.3 General scale of flowability forflow through an orifice

Experimental considerations forflow through an orifice

3.4 Experimental considerations forflow through an orifice

Recommended procedure for flowthrough an orifice

3.5 Recommended procedure forflow through an orifice

Shear cell methods 4. Shear cell methods

Basic methods for shear cell 4.1 Basic methods for shear cell

Recommendation for shear cell 4.2 Recommendation for shear cell

Table 1 Flow properties and cor-responding angle repose

Table 1 Flow properties and cor-responding angle repose

Table 2 Scale of flowability Table 2 Scale of flowability

Sep. 2002


General Information

Peptide Mapping Peptide Mapping

Purpose and scope Purpose and Scope

The peptide map The Peptide Map

Isolation and puriˆcation Isolation and Puriˆcation

Selective cleavage of peptide bonds Selective Cleavage of Peptide Bonds

Pretreatment of sample Pretreatment of Sample

Pretreatment of the cleavage agent Pretreatment of the Cleavage Agent

Pretreatment of the protein Pretreatment of the Protein

Establishment of optimal digestionconditions

pH

Temperature

Time

Amount of cleavage agent

Establishment of Optimal DigestionConditions

pH

Temperature

Time

Amount of Cleavage Agent

Chromatographic separation

Chromatographic column

solvent

Chromatographic Separation

Chromatographic Column

Solvent

Mobile phase

Gradient selection

Isocratic selection

Other parameters

Validation

Mobile Phase

Gradient Selection

Isocratic Selection

Other Parameters

Validation

Analysis and identiˆcation of peptides Analysis and Identiˆcation of Pep-tides

Table 1. Examples of cleavage agents.

Table 2. Techniques used for the sepa-ration of peptides.

Table 1. Examples of Cleavage Agents

Table 2. Techniques Used for theSeparation of Peptides


1708

Figure. Mould


9. Isoelectric FocusingThis test is harmonized with the European Pharmacopoeia

and the U.S. Pharmacopeia. The parts of the text that are notharmonized are marked with symbols ( ).

General PrinciplesIsoelectric focusing (IEF) is a method of electrophoresis

that separates proteins according to their isoelectric point.Separation is carried out in a slab of polyacrylamide oragarose gel that contains a mixture of amphoteric electrolytes(ampholytes). When subjected to an electric êld, the ampho-lytes migrate in the gel to create a pH gradient. In some casesgels containing an immobilized pH gradient, prepared by in-corporating weak acids and bases to speciˆc regions of the gelnetwork during the preparation of the gel, are used. Whenthe applied proteins reach the gel fraction that has a pH thatis the same as their isoelectric point (pI), their charge is neu-tralized and migration ceases. Gradients can be made overvarious ranges of pH, according to the mixture of ampho-lytes chosen.

Theoretical AspectsWhen a protein is at the position of its isoelectric point, it

has no net charge and cannot be moved in a gel matrix by theelectric êld. It may, however, move from that position bydiŠusion. The pH gradient forces a protein to remain in itsisoelectric point position, thus concentrating it; this concen-trating eŠect is called ``focusing''. Increasing the appliedvoltage or reducing the sample load result in improvedseparation of bands. The applied voltage is limited by theheat generated, which must be dissipated. The use of thin gelsand an e‹cient cooling plate controlled by a thermostatic cir-culator prevents the burning of the gel whilst allowing sharpfocusing. The separation is estimated by determining theminimum pI diŠerence (DpI), which is necessary to separate 2neighboring bands:

DpI＝3D(dpH/dx)

E(－dm/dpH)

D: DiŠusion coe‹cient of the proteindpH/dx: pH gradientE: Intensity of the electric êld, in volts per centimeter－dm/dpH: Variation of the solute mobility with the pH in

the region close to the pI

Since D and －dm/dpH for a given protein cannot bealtered, the separation can be improved by using a narrowerpH range and by increasing the intensity of the electric êld.Resolution between protein bands on an IEF gel preparedwith carrier ampholytes can be quite good. Improvements inresolution may be achieved by using immobilized pHgradients where the buŠering species, which are analogous tocarrier ampholytes, are copolymerized within the gel matrix.Proteins exhibiting pIs diŠering by as little as 0.02 pH unitsmay be resolved using a gel prepared with carrier ampholyteswhile immobilized pH gradients can resolve proteins diŠeringby approximately 0.001 pH units.

Practical AspectsSpecial attention must be paid to sample characteristics

and/or preparation. Having salt in the sample can be prob-

lematic and it is best to prepare the sample, if possible, indeionized water or 2 per cent ampholytes, using dialysis or gelˆltration if necessary.

The time required for completion of focusing in thin-layerpolyacrylamide gels is determined by placing a coloredprotein (e.g. hemoglobin) at diŠerent positions on the gel sur-face and by applying the electric êld: the steady state isreached when all applications give an identical band pattern.In some protocols the completion of the focusing is indicatedby the time elapsed after the sample application.

The IEF gel can be used as an identity test when themigration pattern on the gel is compared to a suitablestandard preparation and IEF calibration proteins, the IEFgel can be used as a limit test when the density of a band onIEF is compared subjectively with the density of bandsappearing in a standard preparation, or it can be used as aquantitative test when the density is measured using adensitometer or similar instrumentation to determine therelative concentration of protein in the bands subject tovalidation.

ApparatusAn apparatus for IEF consists of:—a controllable generator for constant potential, current

and power. Potentials of 2500 V have been used and areconsidered optimal under a given set of operatingconditions. Supply of up to 30 W of constant power isrecommended,

—a rigid plastic IEF chamber that contains a cooled plate,of suitable material, to support the gel,

—a plastic cover with platinum electrodes that are connect-ed to the gel by means of paper wicks of suitable width,length and thickness, impregnated with solutions of a-nodic and cathodic electrolytes.

Isoelectric Focusing in Polyacrylamide Gels: DetailedProcedure

The following method is a detailed description of an IEFprocedure in thick polyacrylamide slab gels, which is used un-less otherwise stated in the monograph.

Preparation of the GelsMould The mould (see Figure) is composed of a glass

plate (A) on which a polyester ˆlm (B) is placed to facilitatehandling of the gel, one or more spacers (C), a second glassplate (D) and clamps to hold the structure together.

7.5z Polyacrylamide gel Dissolve 29.1 g of acrylamideand 0.9 g of N,N?-methylenebisacrylamide in 100 mL ofwater. To 2.5 volumes of this solution, add the mixture ofampholytes speciêd in the monograph and dilute to 10


volumes with water. Mix carefully and degas the solution.Preparation of the mould Place the polyester ˆlm on the

lower glass plate, apply the spacer, place the second glassplate and ˆt the clamps. Place 7.5z polyacrylamide gelprepared before use on a magnetic stirrer, and add 0.25volumes of a solution of ammonium persulfate (1 in 10) and0.25 volumes of N,N,N?,N?-tetramethylethylenediamine.Immediately ˆll the space between the glass plates of themould with the solution.

MethodDismantle the mould and, making use of the polyester ˆlm,

transfer the gel onto the cooled support, wetted with a fewmillilitres of a suitable liquid, taking care to avoid formingair bubbles. Prepare the test solutions and reference solutionsas speciêd in the monograph. Place strips of paper for sam-ple application, about 10 mm×5 mm in size, on the gel andimpregnate each with the prescribed amount of the test andreference solutions. Also apply the prescribed quantity of asolution of proteins with known isoelectric points as pH mar-kers to calibrate the gel. In some protocols the gel has pre-cast slots where a solution of the sample is applied instead ofusing impregnated paper strips. Cut 2 strips of paper to thelength of the gel and impregnate them with the electrolyte so-lutions: acid for the anode and alkaline for the cathode. Thecompositions of the anode and cathode solutions are given inthe monograph. Apply these paper wicks to each side of thegel several millimetres from the edge. Fit the cover so that theelectrodes are in contact with the wicks (respecting the anodicand cathodic poles). Proceed with the isoelectric focusing byapplying the electrical parameters described in the mono-graph. Switch oŠ the current when the migration of the mix-ture of standard proteins has stabilized. Using forceps, re-move the sample application strips and the 2 electrode wicks.Immerse the gel in ˆxing solution for isoelectric focusing inpolyacrylamide gel. Incubate with gentle shaking at roomtemperature for 30 minutes. Drain oŠ the solution and add200 mL of destaining solution. Incubate with shaking for 1hour. Drain the gel, add coomassie staining TS. Incubate for30 minutes. Destain the gel by passive diŠusion with destain-ing solution until the bands are well visualized against a clearbackground. Locate the position and intensity of the bands inthe electropherogram as prescribed in the monograph.

Variations to the Detailed Procedure (Subject to Validation)Where reference to the general method on isoelectric focus-

ing is made, variations in methodology or procedure may bemade subject to validation. These include:—the use of commercially available pre-cast gels and of com-

mercial staining and destaining kits,—the use of immobilized pH gradients,—the use of rod gels,—the use of gel cassettes of diŠerent dimensions, including

ultra-thin (0.2 mm) gels,—variations in the sample application procedure, including

diŠerent sample volumes or the use of sample applicationmasks or wicks other than paper,

—the use of alternate running conditions, including varia-tions in the electric êld depending on gel dimensions andequipment, and the use of ˆxed migration times ratherthan subjective interpretation of band stability,

—the inclusion of a pre-focusing step,—the use of automated instrumentation,—the use of agarose gels.

Validation of Iso-Electric Focusing ProceduresWhere alternative methods to the detailed procedure are

employed they must be validated. The following criteria maybe used to validate the separation:—formation of a stable pH gradient of desired characteris-

tics, assessed for example using colored pH markers ofknown isoelectric points,

—comparison with the electropherogram provided with thechemical reference substance for the preparation to beexamined,

—any other validation criteria as prescribed in the mono-graph.

Speciêd Variations to the General MethodVariations to the general method required for the analysis

of speciˆc substances may be speciêd in detail in mono-graphs. These include:—the addition of urea in the gel (3 mol/L concentration is

often satisfactory to keep protein in solution but up to 8mol/L can be used): some proteins precipitate at their isoe-lectric point. In this case, urea is included in the gel formu-lation to keep the protein in solution. If urea is used, onlyfresh solutions should be used to prevent carbamylation ofthe protein,

—the use of alternative staining methods,—the use of gel additives such as non-ionic detergents (e.g.

octylglucoside) or zwitterionic detergents (e.g., CHAPS orCHAPSO), and the addition of ampholyte to the sample,to prevent proteins from aggregating or precipitating.

Points to ConsiderSamples can be applied to any area on the gel, but to pro-

tect the proteins from extreme pH environments samplesshould not be applied close to either electrode. Duringmethod development the analyst can try applying the proteinin 3 positions on the gel (i.e. middle and both ends); thepattern of a protein applied at opposite ends of the gel maynot be identical.

A phenomenon known as cathodic drift, where the pHgradient decays over time, may occur if a gel is focused toolong. Although not well understood, electroendoosmosis andabsorption of carbon dioxide may be factors that lead tocathodic drift. Cathodic drift is observed as focused proteinmigrating oŠ the cathode end of the gel. Immobilized pHgradients may be used to address this problem.

E‹cient cooling (approximately 49C) of the bed that thegel lies on during focusing is important. High êld strengthsused during isoelectric focusing can lead to overheating andaŠect the quality of the focused gel.

Reagents and Solutions—Fixing solution for isoelectric focusing in polyacrylamide

gel Dissolve 35 g of 5-sulfosalicylic acid dihydrate and 100 gof trichloroacetic acid in water to make 1000 mL.

Coomassie staining TS Dissolve 125 mg of coomassiebrilliant blue R-250 in 100 mL of a mixture of water,methanol and acetic acid (100) (5:4:1), and ˆlter.

Destaining solution A mixture of water, methanol andacetic acid (100) (5:4:1).

17101710 JP XVLaser DiŠraction Measurement / General Information

10. Laser DiŠraction Measurementof Particle Size

Particle size is one of the important factors concerning thepowder characteristics of solid-state drugs and pharmaceuti-cal excipients, and therefore prompt and highly accurate de-termination method is necessary. The laser diŠractionmethod is considered as a potent candidate, but due to thelimit of kind of particles and the range of particle diameterthat can be measured, the optical microscopy method shouldbe sometimes applied in parallel. In the pharmaceutical êld,the reliable and reproducible particle size determinationmethod is required for quality control of the composition ofthe formulation, especially, of the pharmaceutical excipientsthat are di‹cult to apply the analytical sieving method due tothe particle diameter of not larger than 100 mm.

In the Japanese Pharmacopoeia, the optical microscopy(Method 1) for the particles ranging from approximately 1mm to 100 mm and the analytical sieving method (Method 2)for the particles of not smaller than 75 mm are listed as thepowder particle size determination.

This document describes the laser diŠraction technique forparticle size determination as the method for analyzing theparticles having the diameter ranging from approximately 1mm to 1000 mm by utilizing the diŠraction phenomenonagainst the laser beam (Fraunhofer diŠraction). There aremethods utilizing the principles such as the Mie scatteringother than the Fraunhofer diŠraction, which can be also usedfor particle size determination of the particles in sub-micronrange (not larger than 1 mm).

1. Principle of laser diŠraction techniqueThe laser diŠraction technique is based upon the beam

diŠraction phenomenon (Fraunhofer diŠraction), in whichparticle size-dependent intensity patterns exhibit when thepowder particles are exposed to the parallel rays, and ac-curate and reproducible particle size distribution image canbe obtained by mathematical analysis of the diŠraction pat-tern. In other words, this apparatus is designed to detect thebeams diŠracted to each angle by the particles when the laserbeam is radiated to the powder sample dispersed in a liquid orgas, in a way that the beam passes over the specimen and, thediŠraction pattern is obtained and recorded for data analysis.For the analysis of data, the particles are qualiêd as spheri-cal form. In this technique, at every particle diameter intervalthat is provided in the apparatus, distribution of the sphericalequivalent diameter in the standard volume is calculated byinverse operation from the optical model to attain the highestcoherence to the diŠraction intensity pattern.

For the particles in sub-micron region, it is not easy to ob-tain an accurate particle size distribution due to di‹culty indistinction of the particle diameter-dependent scattering pat-tern because of weakness of the forward light scattering in-tensity, and it is generally considered that the FraunhoferdiŠraction can not be applied for the evaluation of particlediameter in the sub-micron range. Therefore, for the particlesin sub-micron region, the method that applies the Mie scat-tering theory is introduced here. The particle diameter ismeasured by the analysis of the scattered lights toward backand side that are easy to catch the signals of transmitted and

refracted light. However, since the scattering patterns of thenon-spherical particles vary depending on the particle shape,the correct particle diameter can not be obtained even if theaccurate physical properties (refraction index or transmit-tance) of the dispersing medium are given. Therefore, theMie scattering theory can be eŠectively applied if the particlesare nearly spherical, and for the other cases, evaluation of thedata is in general di‹cult due to strongly be aŠected by thephysical properties or the particle concentration.

2. ApparatusA typical set-up for a laser diŠraction instrument is given

in Figure 1. As the light source, the laser beam of a singlewavelength is generally used. A beam expander is equipped asthe laser beam processing unit H for irradiation to the parti-cle ensemble dispersed. When the sample (particle ensembleF) passes through the laser beam, a part of the laser beam isscattered by the particles, and the diŠraction images given bythe scattered and transmitted beams are transcribed by thedetector J placed at the focal length of the Fourier lens D.

A representative sample is dispersed in a liquid or gas, andthe recirculating system consisting of the optical measure-ment cell, a dispersion bath (usually equipped with the stirrerand ultrasonic elements), a pump and tubing are commonlymost used.(1) Calibration of apparatus

According to the description in ISO standard 13320-1(1999): Particle size analysis-Laser diŠraction methods-Part1: General principles, apparatus should be calibrated by us-ing the plural standard particles for particle size measurementhaving a known particle size distribution and over at least onedecade of size.(2) Particle size determination in sub-micron region

In order to extend the measuring range of particle diameterto sub-micron region, apparatuses based on the Mie scatter-ing theory have been developed and used. This type of instru-ment is designed so that the scattered lights toward back andside are detected and analyzed, but there are large variationsin the analytical results depending on the type of instrument.However, this type of apparatus can produce reproducibledata even in a sub-micron region, if the apparatus used andthe sample tested are restricted under the speciˆc conditionsin which the input number for data analysis, e.g., physicalproperties, are ˆxed for the apparatus.(3) Particle size determination of emulsion

In case of particle size determination of emulsion, themethod utilizing dynamic light scattering or static light scat-tering is generally used. Since the emulsion particles transmitthe light, the laser diŠraction method is not very appropriateto apply. However, it is possible to evaluate emulsion particlediameter when the apparatus has been appropriately calibrat-ed using the standard particles for particle size measurement.

3. MeasurementIn case of the laser diŠraction method, the particle concen-

tration aŠects the accuracy of measurement. Therefore, forthe sample having the size near the upper measuring limit ofparticle diameter, the particle concentration should be highenough to attain the static analysis. On the contrary, for thesample having smaller particle diameter, it is necessary foraccurate measurement to adjust the particle concentrationlower to some extent to prevent the multiple scattering.(1) Conditions of measurement･Location of the apparatus: It is important that the appara-

1711

Fig. 1 Construction of the laser diffraction apparatus

1711JP XV General Information / Laser DiŠraction Measurement

tus is not aŠected by electrical noise and mechanical vibra-tions. It is also desirable that the apparatus is located underthe environment where there is no variation of temperatureand humidity to the extent that aŠects the measurement andis no exposure of direct sun light.･Blank measurement: After selection of proper alignment ofthe optical part of the instrument, a blank measurement per-formed in a pure dispersing medium using the same methodas that for measurement of the sample.･Preliminary observation of the sample: Prior to the meas-urement, the powder sample should be observed in advanceby visual/microscopic inspection to conˆrm the approxi-mate particle diameter range and particle shape.･Establishment of the measurement conditions: The time formeasurement, the reading time of detector and the numberof repetition should be experimentally decided in accor-dance with required measurement accuracy.･Measurement of the sample: The sample is measured underthe established measurement conditions. Data analysis isperformed based on the pulse of dispersed particles from thediŠerence of diŠraction intensity between the sample andthe blank.

(2) Preparation of the sampleThe dry powder sample can be dispersed in a liquid or gas,

and the appropriate method is selected depending on the pur-pose of measurement. There are two types of dispersionmethod, wet type using a liquid as the dispersion medium anddry type using a gas as the dispersion medium. It is advisablethat the powder sample for measurement is divided to an ap-propriate apparent volume using a sample splitting tech-nique.

The result of the particle size distribution much varies de-pending on the conditions of preparation of the sample.Therefore, the procedures and conditions of sample prepara-tion should be deˆned in advance.(3) Sampling

The amount of the sample necessary for the laser diŠrac-tion for powder particle size measurement has deceased dueto technical advancement of the apparatus, but the samplefor the measurement should represent the whole sample.Therefore, how to sample a small amount for actual meas-urement from a sample of large volume is an important prob-lem. It is general that a large amount of the sample isdivided/reduced to reach the amount necessary for measure-

ment, and the divider or the cone and quartering method, etc.are available for this purpose. The cone and quarteringmethod is used for reduction of a small amount of samplewith poor ‰owability, and is a simple method without usingany divider.

For sampling of the powder particles dispersed in a liquid,the liquid is ˆrst stirred uniformly and mixed, and it shouldbe conˆrmed that no precipitate remains at the bottom of thevessel. Then, the amount necessary for measurement isquickly sampled using a pipette, etc. In case of the samplethat contains lager particles, it is easy to cause precipitates,and they are precipitated at the bottom after once stoppingagitation. Therefore, the sampling of the suspension shouldbe made under agitation.(4) Pretreatment of the sample for measurement

If the sample contains the particles or agglomerates overthe upper limit of measurable particle diameter range of thelaser diŠraction instrument, they should be removed by siev-ing in advance, and the mass and percent of such particles oragglomerates removed should be recorded.(5) Liquids used as the dispersion medium

A variety of liquids is available for the dispersion of thepowder samples, and at selection of the dispersion liquids,the followings should be taken into account:—be compatible with the materials used in the instrument

(O-ring, tubing, etc.)—not dissolve or alter the size of particular materials,—favour easy and stable dispersion of the particulate materi-

als,—have suitable viscosity in order to enable recirculation,—not be hazardous to health and should meet safety require-

ments.

A low-foaming surfactant and dispersant may be used tofacilitate the wetting of the particles, and to stabilize the dis-persion. A preliminary check on the dispersion quality can bemade by visual/microscopic inspection of the suspension.(6) Dispersion of the agglomerated particles by ultrasonica-tion

For the powder sample containing ˆne particles, intensephysical power should be given to disperse the agglomeratedparticles. The ultrasonication is generally used for this pur-pose. In this case, since the ultrasonic output, irradiationtime and the volumes of both the suspension and the vessel

17121712 JP XVMedia Fill Test / General Information

used signiˆcantly aŠect the intensiêd dispersion eŠect, theoptimum irradiation conditions should be established takingaccount of these factors. In case that an ultrasonic bath isused for dispersion, kinetic condition of the dispersant anddispersion eŠect may change depending on the altitude of itssurface. Therefore, it is necessary to conˆrm the resonancepoint where the ultrasonic wave is intensely irradiated. Fur-ther, in case of stronger ultrasonic output or longer irradia-tion time, the particles for measurement may be destructed.Therefore, the change of the particle size distribution de-pending on the variation of the irradiation conditions shouldbe checked to determine the appropriate ultrasonic outputand irradiation time.(7) Dispersion gases as the dispersion medium

An appropriate dry type dispersion apparatus using com-pressed gas is used for dispersion of the powder sample in agas. Any substance that may aŠect the measurement, such asoil, water and particulate matter, should not be contaminatedin the dispersion medium gas, and such substances should beremoved, if necessary, by a ˆlter, etc. In case of the dry dis-persion method, it is advisable to measure all the samplesreduced. A larger amount of sample diminishes the statisticalerror at a rough and large particle size area even if the particlesize distribution is broad. It is also important that the parti-cles are dispersed uniformly, It can be conˆrmed by compar-ing the results obtained by dry and wet dispersion methods. Itis desirable that the equivalent results are obtained by dry andwet measurements.

4. Data analysisVarious softwares have been developed to analyze the data

utilizing some mathematical methods, and the particle di-ameter and particle size distribution can be calculated fromthe data to meet its object. As for the accuracy of measure-ment, the reproducibility required for each apparatus is at-tained when measured on the independent triplicate sam-plings of the same batch.(1) Calculation of the particle size distribution

The instrument manufacturers may use diŠerent al-gorithms for calculation of the particle size distribution fromthe intensity of diŠraction (or scattering) beams obtained bythe laser diŠraction instrument, but the principle of estimat-ing particle diameter from the intensity of light diŠractionobtained by the detector under the diŠraction (or scattering)theory is fundamentally the same. Since this method is basedupon the optical model that assumes spherical particle, thesphere equivalent diameter is obtained for the non-sphericalparticles. The laser diŠraction method can not distinguish be-tween the diŠraction of a single particle and that of the ag-glomerate or of the primary particle cluster that constitutesan aggregates or agglomerates.(2) Weaving of the results

For the samples having particle size distributions, the me-dian diameter or the modal diameter are used as the charac-teristic diameters, and the particle size distribution is ex-pressed either as the cumulative size distribution or the fre-quency size distribution. The cumulative size distribution isexpressed as a quantitative proportion of the particles smallerthan the speciêd particle diameter among the whole sample.The analytical sieving method is generally used for the parti-cle size distribution determination, and this cumulative distri-bution is represented as the cumulative undersize distribu-tion. On the other hand, the frequency size distribution is ex-

pressed as the frequency that is shared by the particles thatbelong to the speciêd particle diameter fraction among thewhole sample.･Mathematical expression of the particle size distribution

The particle size distribution is expressed by a function ofeither the normal distribution or the lognormal distribution.The lognormal distribution is characterized by distributionpattern that splays out toward large particle size range, and isgenerally applied for the samples having a large amount oflarger particles and a small amount of smaller particles.

5. Experimental considerations on the measurementAccurate particle diameter measurement can not be expect-

ed for the laser diŠraction method in case of the samples hav-ing wide range of particle size distribution. Therefore, thesetting the measurement range is essential for the measure-ment of the particle diameter by the laser diŠraction method,because the scattering intensity is much in‰uenced by the low-er limit diameter (1 mm) and upper limit diameter (1000 mm)of sample particles. It is desirable in this method that themeasurement diameter range is conˆrmed by interpolationusing the standard sample having a known particle size distri-bution considering the accuracy of measurement and the sen-sitivity of instrument used. For the particles of not largerthan 10 mm, it is di‹cult to obtain accurate particle diameter,because the diŠerence of diŠraction pulse detected by the de-tector is small. Particularly, for the particles in sub-micronregion, there is almost no diŠerence of the scattered light for-ward, and it is hard to distinguish them. Further, in additionto the error caused by recognizing the non-spherical particlesas spherical particles, decrease of the scattering intensity,which is much bigger than that estimated from the reductionof geometrical particle diameter, occurs as the particle di-ameter decreases, and securing the accuracy of measurementcomes to be di‹cult. Therefore, the lower limit for measure-ment of the particle diameter by the laser diŠraction tech-nique is approximately 1 mm, similar to that by the opticalmicroscopic method. In case of the laser diŠraction tech-nique, since the scattering intensity is related to the volumeconcentration of the particles, and the maximum measure-ment range of particle diameter should be decided taking ac-count of the relative variation of the scattering intensity. Aparticle of 10 times larger in diameter has a volume of 1000times larger. This means that in case of 1 mm for lower limitmeasurement of particle diameter, the particles of 1000 mmhave a volume of 1 billion times larger, and consequently thesample necessary for measurement is 10 billion times larger,similar to the case of volume. Therefore, approximately 1000mm is appropriate for the maximum particle diameter formeasurement.

Example of the standard reference particlesUse the standard reference particles for SAP 10-03, the

Speciˆcation of Association of Powder Process Industry andEngineering, Japan. Either of MBP1-10 or MBP10-100 isused to meet the particle size measurement range (1 – 10 mmor 10 – 100 mm).

11. Media Fill TestThe media ˆll test (MFT) is one of the processing valida-

tions employed to evaluate the propriety of the aseptic

1713

Table 2. Upper 95z confidence limit of a Poisson variablefor numbers of contaminated units

Observed numbers of contaminatedunits (k)

Upper 95z confidencelimit (U)

0 2.99571 4.74392 6.29583 7.75374 9.15375 10.51306 11.84247 13.14818 14.43469 15.7052

10 16.9622

Using the 95z confidence limit (U) in Table 2, the contaminationrate (P) of observed numbers of contaminated units (k) per filledunits (n) can be calculated as P ＝ UWn (equation 1). For example:

If 5,000 units were filled and two contaminated units were ob-served, n ＝ 5,000 and U ＝ 6.30 (k ＝ 2) are substituted in the equa-tion 1; P ＝ 6.30W5,000 ＝ 0.0013. The upper 95z confidence limitfor the contamination rate would be 0.13z.

Table 3. Initial performance qualification: Media fills

Productionlot size

Numbers ofmedia fill runs

Alert level andaction required

Action level andaction required

º 500

A minimum of10 media fillruns using themaximum lotsize of theproduct

One contaminat-ed unit in anyrun. Investigatecause.

Two contaminat-ed units in singlerun, or one eachin two runs.Investigatecause and repeatinitial qualifica-tion media fills.

500 – 2,999

A minimum of 3media fill runsusing the maxi-mum lot size ofthe product

The same asabove

The same asabove

≧ 3,000

A minimum of 3media fill runsusing at least3,000 units

When any of themedia fill runsexceeds the alertlevel shown inTable 1, takethe action setout in 2.1.1.

When any of themedia fill runsexceeds the ac-tion level shownin Table 1, takethe action setout in 2.1.1

Table 4. Periodic performance requalification: Media fills

Productionlot size

Numbers ofmedia fill runs

Alert level andaction required

Action level andaction required

º 500

A minimum of 3media fill runsusing the maxi-mum lot size ofthe product

One contaminat-ed unit in anyrun. Investigatecause and repeatinitial qualifica-tion media fillruns.

500 – 2,999

One media fillrun using themaximum lotsize of theproduct

One contaminat-ed unit. Inves-tigate cause andrepeat initialqualification me-dia fill runs.

≧ 3,000

One media fillrun usingat least 3,000units

When the mediafill run exceedsthe alert levelshown in Table1, take the ac-tion set out in 2.1. 1.

When the mediafill run exceedsthe action levelshown in Table1, take the ac-tion set out in 2.1. 1

Table 1. Alert and action levels for large numbers ofmedia filled units

Number ofunits*1

Number of contaminated units

Acceptance levels Alert levels Action levels

3,000 0 not applicable ≧ 14,750 0 1 ≧ 26,300 0 1 – 2 ≧ 37,760 0 1 – 3 ≧ 49,160 0 1 – 4 ≧ 5

10,520 1 2 – 5 ≧ 611,850 1 2 – 6 ≧ 713,150 1 – 2 3 – 7 ≧ 814,440 1 – 2 3 – 8 ≧ 915,710 1 – 3 4 – 9 ≧1016,970 1 – 3 4 – 10 ≧11

*1 It is not necessary to relate the number of units with lot size ofactual products.

1713JP XV General Information / Media Fill Test

processing of pharmaceutical products using sterile media,etc. instead of actual products. Therefore, media ˆll testsshould be conducted with the manipulations normally per-formed in actual processing, e.g. ˆlling and closing opera-tion, operating environment, processing operation, numberof personnel involved, etc., and conducted under processingconditions that include ``worst case'' conditions. Refer toGMP (1), WHOWGMP for pharmaceutical products (2), andISO 13408 (3), etc. for necessary information to conduct thistest.

1. Frequency of media ˆlls1.1 Initial performance qualiˆcationInitial performance qualiˆcation should be conducted for

each new facility, item of equipment, ˆlling line, and contain-er design (except for multiple sizes of the same container de-sign), etc. For production batch sizes exceeding 3,000 units, aminimum of three media ˆll runs should be conducted onseparate days. For production batch sizes of less than 3,000units, see Table 3.

1.2 Periodic performance requaliˆcation1) Conduct media ˆll requaliˆcations periodically on

each working shift for the ˆlling line. Employees working inthe aseptic processing area should be trained for asepticprocessing operations and take part in media ˆlls.

2) When ˆlling lines have not been used for over sixmonths, conduct appropriate numbers of media ˆll runs inthe same way as for the initial performance qualiˆcationprior to resumption of use of the ˆlling lines.

3) In cases of facility and equipment modiˆcation (inter-changing parts may not require requaliˆcation), changes inpersonnel working in critical aseptic processing (e.g. newcrews), anomalies in environmental testing results, or a

17141714 JP XVMedia Fill Test / General Information

product sterility test showing contaminated products, con-duct appropriate numbers of media ˆll runs in the same wayas for the initial performance qualiˆcation prior to the sched-uled media ˆlls.

2. Acceptance criteria of media ˆllsA large number of media ˆlled units is required to detect

0.1z contamination rate. The alert level is more than 0.05zbut less than 0.1z, and the action level is more than 0.1zcontamination rate at the upper 95z conˆdence level.Table 1 shows alert and action levels for media ˆlled units.Table 2 is to be used for the calculation of contamination rateof contaminated units found in media ˆlled units. The con-tamination rate of 0.05z in media ˆlls is the minimum ac-ceptable level, and manufacturers should make eŠorts toachieve lower contamination rate than this. Table 3 shows thealert and action levels for initial performance qualiˆcation ofan aseptic processing line, and actions required for each level.Table 4 shows the alert and action levels for requaliˆcation ofan aseptic processing line, and actions required for each level.The alert and action levels for production batch sizes of lessthan 3,000 units take semiautomatic or manual operationinto consideration.

2.1 Actions required for each level2.1.1 Initial performance qualiˆcation1) When the result of the media ˆll run is less than the

alert level, the media ˆll run meets the requirement of theMFT.

2) When the results of any media ˆll run done with atleast three replicate runs reach the alert or the action level, aninvestigation regarding the cause is required, and initialqualiˆcation media ˆlls are to be repeated. When the result ofeach media ˆll run is less than the alert level, the initialqualiˆcation media ˆlls meet the requirement of the MFT.

2.1.2 Requaliˆcation1) When the result of the media ˆll run is less than the

alert level, the media ˆll run meets the requirement of theMFT.

2) When the result of the media ˆll run exceeds the alertlevel, an investigation regarding the cause is required, andone more media ˆll run is to be done. If the result is less thanthe alert level, the media ˆll run meets the requirement of theMFT.

3) When the result of the media ˆll run exceeds the actionlevel, a prompt review of all appropriate records relating toaseptic production between the current media ˆll and the lastsuccessful one, and an investigation regarding the cause mustbe conducted simultaneously. If necessary, appropriate ac-tion to sequester stored andWor distributed products shouldbe taken. After investigation regarding the cause, repeatthree serial media ˆll runs. If the results are less than the alertlevel, the media ˆll runs meet the requirement of the MFT.

2.2 Parameters which aŠect sterilityWhen media ˆll alert and action levels are exceeded, an in-

vestigation should be conducted regarding the cause, takinginto consideration the following points:

1) Microbial environmental monitoring data2) Particulate monitoring data3) Personnel monitoring data (microbial monitoring data

on gloves, gowns, etc. at the end of work)4) Sterilization cycles for media, commodities, equip-

ment, etc.5) Calibration of sterilization equipment

6) Storage conditions of sterile commodities7) HEPA ˆlter evaluation (airborne particulate levels,

DOP test, velocity measurements, etc.)8) Pre and post ˆlter integrity test data (including ˆlter

housing assembly)9) Room air ‰ow patterns and pressures

10) Unusual events that occurred during the media ˆll run11) Characterization of contaminants12) Hygienic control and training programs13) Gowning procedures and training programs14) Aseptic processing technique and training programs15) Operator's health status (especially coughing, sneez-

ing, etc., due to respiratory diseases)16) Other factors that aŠect sterility

3. Data guidance for media ˆllsEach media ˆll run should be fully documented and the

following information recorded:1) Data and time of media ˆll2) Identiˆcation of ˆlling room and ˆlling line used3) ContainerWclosure type and size4) Volume ˆlled per container5) Filling speed6) Filter lot and catalogue number7) Type of media ˆlled8) Number of units ˆlled9) Number of units not incubated and reason

10) Number of units incubated11) Number of units positive12) Incubation time and temperature13) Procedures used to simulate any step of a normal

production ˆll (e.g., mock lyophilization or substitution ofvial headspace gas)14) Microbiological monitoring data obtained during the

media ˆll set-up and run15) List of personnel who took part in the media ˆll16) Growth promotion results of the media (in case of

powder ˆll, an antimicrobial activity test for the powder isnecessary)17) Characterization of the microorganisms from any

positive units18) Review

4. Media ˆll proceduresMethods to validate aseptic processing of liquid, powder

and freeze-dried products are described. Basically, it is possi-ble to apply media ˆll procedures for liquid products to otherdosage forms and container conˆgurations.

4.1 Media selection and growth promotionSoybean-casein digest medium or other suitable media are

used. As growth promotion testing microorganisms, strainslisted in the Sterility Test and, if necessary, one to tworepresentative microorganisms which are frequently isolatedin environmental monitoring should be used. The media in-oculated with 10 to 100 viable microorganisms of each strainshould show obvious growth when incubated at the predeter-mined temperature for 5 days.

4.2 Sterile medium preparationThe medium is sterilized according to the pre-validated

method.4.3 Incubation and inspection of media ˆlled unitsLeaking or damaged media ˆll evaluation units should be

removed and recorded prior to incubation of media ˆlledunits. Incubate at 20 – 259C for 1 week, and then at 30 –

17151715JP XV General Information / Microbial Attributes

359C for 1 week (or at 30 – 359C for 1 week, and then at 20 –259C for 1 week), or at 30 – 359C for 2 weeks. Observe themedia ˆlled units for growth of microorganisms at least oncebetween the third day and seventh day and on the last day ofthe test period, twice in total. Microorganisms present in con-taminated units should be characterized.

A. Liquid productsMedia ˆll procedureMedia ˆll should include normal facilityWequipment opera-

tions and clean-up routines. Containers, closures, parts ofthe ˆlling machine, trays, etc. are washed and sterilized ac-cording to the standard operating procedures. Media ˆllsshould be conducted under processing conditions that include``worst case'' conditions, e.g., correction of line stoppage,repair or replacement of ˆlling needlesWtubes, replacement ofon-line ˆlters, permitted interventions, duration and size ofrun, number of personnel involved, etc.

A predetermined volume of medium is ˆlled into sterilizedcontainers at a predetermined ˆlling speed and the containersare sealed. The media are contacted with all product contactsurfaces in the containers by an appropriate method, andthen incubated at the predetermined temperature.

B. Powder productsB.1 Powder selection and antimicrobial activity testActual products or placebo powder are used. In general,

lactose monahydrate, D-mannitol, polyethylene glycol 6,000,carboxymethyl cellulose salts or media powder, etc. are usedas placebo powders. Prior to employing any of the powders,evaluate whether the powder has antimicrobial activity. Me-dia powders are dissolved in water and other powders in liq-uid medium, and the solutions are inoculated with 10 to 100viable microorganisms of each kind, shown in 4.1, for thegrowth promotion test. If obvious growth appears in themedium incubated at the predetermined temperature for 5days, the powder has no antimicrobial activity and is availa-ble for the media ˆll test.

B.2 Sterilization of powdersDry powders are bagged in suitable containers (e.g. double

heat-sealed polyethylene bags), and are subjected to radiationsterilization.

B.3 Sterility of ˆlling powdersThe powders must pass the Sterility Test. However, if the

sterilization is fully validated, sterility testing of the powderscan be omitted.

B.4 Media ˆll proceduresChose a suitable procedure from among the following

procedures.1) Fill sterilized liquid media into containers by suitable

methods, and then ˆll actual products or sterilized placebopowder with the powder ˆlling machine. If sterilized powdermedia are used as a placebo powder, ˆll sterilized water in-stead of sterilized liquid media.

2) Distribute liquid media into containers, and then steri-lize them in an autoclave. Remove the containers to the ˆllingarea, and then ˆll actual products or sterilized placebo pow-der into the containers with the powder ˆlling machine.

3) Fill actual products or sterilized placebo powder intocontainers with the powder ˆlling machine, and then ˆll steri-lized liquid media into the containers by appropriatemethods. If sterilized powder media are used as a placebopowder, ˆll sterilized water instead of sterilized liquid media.

C. Lyophilized productsIn the case of lyophilized products, it may be impossible to

conduct a media ˆll run in the same way as used for actualprocessing of lyophilized products. The process of freezingand lyophilization of the solution may kill contaminant or-ganisms and change the characteristics of the media too. Theuse of inert gas as a blanket gas may inhibit the growth ofaerobic bacteria and fungi. Therefore, in general, the actualfreezing and lyophilization process should be avoided and airused as the blanket gas.

Media ˆll proceduresUse the following method or other methods considered to

be equivalent to these methods.1) After ˆlling of the media into containers by the ˆlling

machine, cap the containers loosely and collect them in pre-sterilized trays.

2) After placing the trays in the lyophilizer, close thechamber door, and conduct lyophilization according to theprocedures for production operation. Hold them withoutfreezing under weak vacuum for the predetermined time.

3) After the vacuum process, break the vacuum, and sealthe stoppers.

4) Contact the media with all product contact surfaces inthe containers by appropriate methods, and then cultivatethem at the predetermined temperature.

References1) Good manufacturing practices for pharmaceutical

products (WHO-GMP, 1992)2) ISO 13408-1 (Aseptic processing of health care

products: Generals)

12. Microbial Attributes ofNonsterile Pharmaceutical

ProductsThe presence of microbial contaminants in nonsterile phar-

maceutical products can reduce or even inactivate the ther-apeutic activity of the product and has the potential to aŠectadversely the health of patients. Manufacturers, therefore,should ensure as low as possible a contamination level for ˆn-ished dosage forms, raw materials and packaging compo-nents to maintain appropriate quality, safety and e‹cacy ofnonsterile pharmaceutical products. This chapter providesguidelines for acceptable limits of viable microorganisms(bacteria and fungi) existing in raw materials and nonsterilepharmaceutical products. Testing methods for the countingof total viable microorganisms and methods for the detectionand identiˆcation of speciêd microorganisms (Escherichiacoli, Salmonella, Pseudomonas aeruginosa, Staphylococcusaureus, etc.) are given under the ``Microbial Limit Test''.When these tests are carried out, a microbial control programmust be established as an important part of the qualitymanagement system of the product. Personnel responsiblefor conducting the tests should have specialized training inmicrobiology and in the interpretation of the testing results.

1. Deˆnitions1.1 Nonsterile pharmaceutical products: Nonsterile drugs

shown in monographs of the JP and nonsterile productsincluding intermediate products and ˆnished dosage

17161716 JP XVMicrobial Attributes / General Information

forms.1.2 Raw materials: All materials, including raw ingredients

and excipients, used for the preparation of drugs, exceptfor water and gases.

1.3 Bioburden: Number and type of viable microorganismsexisting in nonsterile pharmaceutical products.

1.4 Action levels: Established bioburden levels that requireimmediate follow-up and corrective action if they are ex-ceeded.

1.5 Alert levels: Established bioburden levels that give earlywarning of a potential drift from normal bioburden level,but which are not necessary grounds for deˆnitive correc-tive action, though they may require follow-up investiga-tion.

1.6 Quality management system: The procedures, opera-tion methods and organizational structure of a manufac-turer (including responsibilities, authorities and relation-ships between these) needed to implement quality manage-ment.

2. ScopeIn general, the tests for total viable aerobic count and for

the detection of speciêd microorganisms are not applied toantibiotic or bacteriostatic drugs. However, the tests shouldbe done for microorganisms that are not aŠected by the drug.The test for total viable aerobic count is not applied to drugscontaining viable microorganisms as an active ingredient.

3. Sampling plan and frequency of testing3.1 Sampling methods

Microbial contaminants are usually not uniformly dis-tributed throughout the batches of non-sterile pharmaceuti-cal products or raw materials. A biased sampling plan, there-fore, cannot be used to estimate the real bioburden in theproduct. A sampling plan which can properly re‰ect the sta-tus of the product batch should be established on the basis ofthe bioburden data obtained by retrospective validation andWor concurrent validation. In general, a mixture of samplesrandomly taken from at least diŠerent three portions, almostthe same amount for each portion, is used for the tests of theproduct. When the sampling is di‹cult in a contamination-controlled environment, special care is required during sam-pling to avoid introducing microbial contamination into theproduct or aŠecting the nature of the product bioburden. If itis conˆrmed that the product bioburden is stable for a certainperiod, as in the case of nonaqueous or dried products, it isnot necessary to do the tests for total viable aerobic countsand for the detection of speciêd microorganisms, immedi-ately after the sampling.3.2 Testing frequency

The frequency of the tests should be established on the ba-sis of a variety of factors unless otherwise speciêd. Thesefactors include:a) Dosage forms of non-sterile pharmaceutical products

(dosage directions);b) Manufacturing processes;c) Manufacturing frequency;d) Characteristics of raw materials (natural raw material,

synthetic compound, etc.);e) Batch sizes;f) Variations in bioburden estimates (changes in batches,

seasonal variations, etc.);g) Changes aŠecting the product bioburden (changes in

manufacturing process, supplier of raw materials, lot

number of raw materials, etc.);h) Others.

In general, the tests may be performed at a high frequencyduring the initial production of a drug to get information onthe microbiological attributes of the product or raw materialsused for the production. However, this frequency may bereduced as bioburden data are accumulated throughretrospective validation andWor concurrent validation. Forexample, the tests may be performed at a frequency based ontime (e.g., weekly, monthly or seasonally), or on alternatebatches.

4. Microbial control programWhen the ``Microbial Limit Test'' is applied to a nonsterile

pharmaceutical product, the methods for the recovery, culti-vation and estimation of the bioburden from the productmust be validated and a ``Microbial control program'' cover-ing the items listed below must be prepared.a) Subject pharmaceutical name (product name);b) Frequency of sampling and testing;c) Sampling methods (including responsible person, quanti-

ty, environment, etc. for sampling);d) Transfer methods of the samples to the testing area (in-

cluding storage condition until the tests);e) Treatment of the samples (recovery methods of microbi-

al contaminants);f) Enumeration of viable microorganisms (including testing

quantity, culture media, growth-supporting test of themedia, culturing methods, etc.);

g) Detection of speciêd microorganisms (including testingquantity, culture media, growth-supporting test of themedia, culturing methods, etc.);

h) Estimation of the number of and characterization ofmicrobial contaminants;

i) Establishment of ``Microbial contamination limits'' (in-cluding alert level and action level);

j) Actions to be taken when the levels exceed ``Microbialcontamination limits'';

k) Persons responsible for the testing and evaluation, etc.;l) Other necessary items

5. Microbial contamination limits for nonsterile phar-maceutical products

By establishing ``Microbial contamination limits'', it ispossible to evaluate at the initial processing stage of theproduct whether the microbiological quality of the rawmaterials is adequate or not. Furthermore, it is then possibleto implement appropriate corrective action as needed tomaintain or improve the microbiological quality of theproduct. The target limits of microbial levels for raw materi-als (systhetic compounds and minerals) are shown in Table 1.

In general, synthetic compounds have low bioburden levelsdue to the high temperatures, organic solvents, etc., used intheir manufacturing processes. Raw materials originatedfrom plants and animals in general have higher bioburdensthan synthetic compounds.

The microbial quality of the city water or puriêd waterused in the processing of active ingredients or nonsterilepharmaceuticals may have a direct eŠect on the quality of theˆnished dosage form. This means it is necessary to keep thelevel of microbial contaminants in the water as low as possi-ble.

The microbial contamination limits for nonsterile ˆnisheddosage forms are shown in Table 2. These microbial limits

17171717JP XV General Information / Microbiological Evaluation

are based primarily on the type of dosage form, water activi-ty, and so on. For oral liquids and pharmaceutical productshaving a high water activity, in general, low microbial con-tamination limits are given. In this guideline, Escherichiacoli, Pseudomonas aeruginosa, Staphylococcus aureus, andCandida albicans are shown as speciêd microorganisms, butit is also necessary to test certain pharmaceutical products forother microorganisms (for example, certain species of Clos-tridia, Pseudomonas, Burkholderia, Aspergillus, and En-terobacter species) that may have the potential to present amicrobial risk to patients. The selection of the speciêdmicroorganisms was based on following criteria; indicatorfor poor hygienic practices, pathogenic potential for route ofadministration, and survival proˆle of the microorganismand recoverability in the product. Due to the inherent preci-sion limitations of the enumeration methods, a value exceed-ing the target limit by not more than 2 times.

6. Microbial contamination limits for herbal drugsTarget limits of microbial contamination for herbal drugs

and herbal drug containing preparations are shown in Table3. Category 1 indicates herbal drugs and their preparations towhich boiling water is added before use, and category 2 indi-cates other herbal drugs and their preparations. In this guide-line, enterobacteria and other gram-negative bacteria, Es-cherichia coli, Salmonella, and Staphylococcus aureus arementioned as specidied microorganisms, but other microor-ganisms such as certain species of Bacillus cereus, Clostridia,Pseudomonas, Burkholderia, Asperigillus and Enterobacterspecies are also necessary to be tested depending on the originof the herbal drug raw materials or the preparation methodof the preparations.

Table 1. Microbial enumeration limits for raw materials

MicroorganismsTarget limit(cfuWg orcfuWmL)

Total aerobic microbial count (TAMC) ≦1000

Total combined yeastsWmolds count(TYMC)

≦100

Table 2. Microbial enumeration limits fornonsterile ˆnished dosage forms

Route ofadministration

TAMC(cfuWg orcfuWmL)

TYMC(cfuWg orcfuWmL)

Examples ofobjectionable

microorganisms

Inhalation(liquid)

≦20 ≦20 StaphylococcusaureusPseudomonasaeruginosa

Inhalation(powder)

≦100 ≦50 StaphylococcusaureusPseudomonasaeruginosa

Nasal ≦100 ≦50 Staphylococcusaureus

Pseudomonasaeruginosa

Varginal ≦100 ≦50 EscherichiacoliStaphylococcusaureusCandida albicans

Otic or Topical(includingtransdermalpatches)

≦100* ≦50* PseudomonasaeruginosaStaphylococcusaureus

Rectal ≦1000 ≦100 Not speciêd

Oral (solid) ≦1000 ≦100 Escherichia coli

Oral (liquid) ≦100 ≦50 Escherichia coli

* For transdermal patches, the limits are expressed as cfu pertransdermal patch.

Table 3. Microbial enumeration limits forherbal drugs and their preparations

MicroorganismsCategory 1

(cfuWg or cfuWmL)Category 2

(cfuWg or cfuWmL)

Aerobic bacteria 107 105

Molds and yeasts 104 103

Enterobacteriaand other gram-negative bacteria

* 103

Escherichia coli 102 not detectedSalmonella not detected not detectedStaphylococcusaureus * *

* The limits are not speciêd.

13. Microbiological Evaluation ofProcessing Areas for

Sterile Pharmaceutical ProductsThis chapter describes the methods for the control and

evaluation of microbial contamination in areas used for theprocessing of sterile pharmaceutical products. Such process-ing areas are classiêd into critical areas and clean areasaccording to the required levels of air-cleanliness. A criticalarea is a deˆned space in which the airborne particulate andmicroorganism levels are controlled to meet grade A. Thecleanliness requirements for such a space extend to the sur-faces of the facilities and equipment which form or are locat-ed within the space, as well as to the supplied raw materials,chemicals, water, etc. Environmental conditions, such astemperature, humidity, and air pressure, are also controlledin this space when required. A clean area is a controlled spacesuch that the levels of contaminants (particulates andmicroorganisms) in air, gases and liquids are maintainedwithin speciêd limits, which are less stringent than those ofgrade A. When sterile pharmaceutical products are manufac-

1718

Table 1. Air-cleanliness requirements for processing of sterile pharmaceutical products

Air cleanliness Maximum number of airborne particulates per m3

Grade*1at rest

≧0.5 mm

in operation

≧0.5 mm

A (Laminar-airflow zone) 3,530 3,530B (Non laminar-airflow zone) 3,530 353,000C 353,000 3,530,000D 3,530,000 —*2

*1 The maximum permitted number of particles in the `ìn operation'' condition corresponds to the standards described under USP <1116> asfollows.

Grade A: Class 100 (M3.5); Grade B: Class 10,000 (M5.5); Grade C: Class 100,000 (M6.5);Grade D: no corresponding standard.

*2 The limit for this area will depend on the nature of the operation carried out there.

Table 2. Suggested frequency of environmental monitoring

Processing area Frequency of monitoring

Critical area (Grade A) Each shiftClean area adjacent to critical area (Grade B) Each shiftOther clean areas (Grade C, D)

Potential productWcontainer contact areas Twice a weekNon-productWcontainer contact areas Once a week

Table 3. Media and culture conditions

Microorganismsto be detected Media*1 Culture conditions

AerobesSoybean-casein digest agar (or fluid) mediumBrain-heart infusion agar (or fluid) mediumNutrient agar (or fluid) medium

30 – 359C*2

More than 5 days*3

Yeast and fungi

Soybean-casein digest agar (or fluid) mediumSabouraud dextrose agar (or fluid) mediumPotato-dextrose agar (or fluid) mediumGlucose peptone agar (or fluid) medium

20 – 259C*2

More than 5 days*3

Anaerobes*4

Soybean-casein digest agar mediumFluid cooked meat mediumReinforced clostridial agar (or fluid) mediumThioglycolate medium I (or thioglycolate agar

medium) for sterility test

30 – 359CMore than 5 days*3

*1 If necessary, antibiotics may be added to media in an appropriate concentration (see Microbial Limit Test). If the existence of disinfectantsthat may interfere with the test on the surface of the specimen is suspected, add a substance to inactivate them.

*2 When soybean-casein digest agar medium is used for the detection of aerobes, yeast and fungi, incubation at 25 to 309C for more than 5days is acceptable.

*3 If a reliable count is obtained in a shorter incubation time than 5 days, this may be adopted.*4 Generally, anaerobes are not targets for the monitoring. For the detection of anaerobes, agar medium is incubated in an appropriate

anaerobic jar.

Table 4. Recommended limits for environmental microorganisms*1

GradeAirborne

microorganisms*2

(CFUWm3)Minimum air sample

(m3)

CFU on a surface

instrumentsWfacilities gloves

(CFUW24–30 cm2)*3

A º1 0.5 º1 º1B 10 0.5 5 5C 100 0.2 25 —D 200 0.2 50 —

*1 Maximum acceptable average numbers of microorganisms under each condition.*2 These values are by using a slit sampler or equivalent.*3 Viable microbe cell number per contact plate (5.4–6.2 cm in diameter). When swabbing is used in sampling, the number of microorganisms

is calculated per 25 cm2. For gloves, usually, put their all fingers on the plate.

1718 JP XVMicrobiological Evaluation / General Information

17191719JP XV General Information / Microbiological Evaluation

tured, the environment, facilitiesWequipment, and personnelshould be routinely monitored to ensure appropriatemicrobiological control in the processing areas. The detectionof microorganisms should be performed under normal opera-tional conditions, using an appropriate sampling device,according to an environmental control program establishedpreviously. The sampling, cultivation, counting, and evalua-tion methods for airborne microorganisms, as well as thosefound on surfaces, should also be chosen appropriately,depending on the monitoring purpose, monitoring items, andmicroorganisms being detected. Sampling devices, measure-ment methods, media, culture conditions, frequency ofmonitoring, and recommended limits for environmentalmicroorganisms shown in this chapter are for informationonly, and are not requirements.

1. DeˆnitionsFor the purposes of this chapter, the following deˆnitions

apply.1) Processing areas: Areas in which actions such as culti-

vation, extractionWpuriˆcation, weighing of raw materials,washing and drying of containers and stoppers, preparationof solutions, ˆlling, sealing and packaging are performed, in-cluding the gowning area.

2) Action levels: Established microbial levels (and type ofmicroorganisms, if appropriate) that require immediate fol-low-up and corrective action if they are exceeded.

3) Alert levels: Established microbial levels (and type ofmicroorganisms if appropriate) that give early warning of apotential drift from normal operating conditions, but whichare not necessarily grounds for deˆnitive corrective action,though they may require follow-up investigation.

4) Contaminants: Particulates and microorganisms caus-ing contamination by adhering to surfaces or by being incor-porated into materials.

5) Cleanliness: A quantity which indicates the conditionof cleanliness of a monitored item, expressed as mass or num-ber of contaminants contained in a certain volume or area.

6) Contamination control: The planning, establishmentof systems and implementation activities performed in orderto maintain the required cleanliness of a speciêd space orsurface.

7) Shift: Scheduled period of work or production, usual-ly less than 12 hours in length, during which operations areconducted by a single deˆned group of workers.

8) Characterization of contaminants: Procedures forclassifying contaminants so that they can be diŠerentiated. Inroutine control, classiˆcation to the genus level is su‹cient;as required, identiˆcation to the species level is performed.

2. Air-cleanliness of processing areas for sterile pharmaceu-tical products

Airborne particulates in areas used for the processing ofpharmaceutical products may act physically as a source of in-soluble particles in the products, and biologically as a carrierof microorganisms. So, it is necessary to control strictly thenumber of particles in the air. The air-cleanliness criteria areshown in Table 1.2.1 Terminally sterilized products

Solutions should generally be prepared in a grade C en-vironment. Solution preparation may be permitted in a gradeD environment if additional measures are taken to minimizecontamination. For parenterals, ˆlling should be done in agrade A workstation in a grade B or C environment. The re-

quirements during the preparation and ˆlling of other sterileproducts are generally similar to those for parenterals.2.2 Sterile products prepared aseptically after ˆltration

The handling of starting materials and the preparation ofsolutions should be done in a grade C environment. These ac-tivities may be permitted in a grade D environment if addi-tional measures are taken to minimize contamination, such asthe use of closed vessels prior to ˆltration. After sterile ˆltra-tion, the product must be handled and ˆlled into containersunder grade A aseptic conditions.2.3 Sterile products prepared aseptically from sterile start-ing materials

The handling of starting materials and all further process-ing should be done under grade A conditions.

3. Microbiological environmental monitoring programEnvironmental monitoring is especially important in steril-

ity assurance for sterile pharmaceutical products that aremanufactured by aseptic processing. The major purpose ofenvironmental monitoring is to predict potential deteriora-tion of the processing environment before it occurs, and toproduce high-quality, sterile pharmaceutical products underappropriate contamination control.3.1 Monitoring of environmental microorganisms

a) An environmental control program document is pre-pared for each area used for the processing of sterile phar-maceutical products. The procedures in the document in-clude: 1) items to be monitored, 2) type of microorganisms tobe monitored, 3) frequency of monitoring, 4) methods ofmonitoring, 5) alert and action levels, and 6) actions to betaken when speciêd levels are exceeded.

b) The aseptic processing areas and other processingareas maintained under controlled conditions are monitoredon a routine basis. The critical processing areas where sterileproducts are in contact with environmental air are monitoredduring every operational shift. The items to be monitored in-clude the air, ‰oor, walls, equipment surfaces, and the gownsand gloves of the personnel. Table 2 shows suggested fre-quencies of the environmental monitoring.

c) The sampling devices used for monitoring environ-mental microorganisms, as well as the methods and culturemedia, should be suitable to detect microorganisms that maybe present (aerobic bacteria, anaerobic bacteria, molds,yeast, etc.). The cultivation conditions, such as incubationtemperature and time, are selected to be appropriate for thespeciˆc growth requirements of microorganisms to be detect-ed. Table 3 shows the culture media and cultivation condi-tions that are generally used in testing for environmentalmicroorganisms.

d) The number of microorganisms in the samples is esti-mated by using the Membrane Filtration, Pour Plating,Spread Plating, or Serial Dilution (Most Probable Number)Methods described in the Microbial Limit Test.

e) Table 4 shows recommended limits for environmentalmicroorganisms. The alert and action levels may be adjustedif necessary after su‹cient data have been accumulated. Themost important point in environmental monitoring is to con-ˆrm that an acceptable value of each monitoring item ismaintained consistently.

f ) Microorganisms isolated are characterized if necessa-ry. In addition, analysis of hourly or daily variation of air-borne particulate numbers will provide data to assist in thecontrol of the cleanliness of processing areas.

17201720 JP XVMicrobiological Evaluation / General Information

3.2. Evaluation of environmental monitoring dataa) The data from the environmental monitoring are eval-

uated on a routine basis for each area and location. Thesource of any discrepancy should be investigated immediatelyand the investigation should be documented in a report. Af-ter corrective action has been taken, follow-up monitoringshould be done to demonstrate that the aŠected area is onceagain within speciˆcation.

b) The report is reviewed and approved by personnelresponsible for quality control and distributed to all key per-sonnel associated with the aseptic processing operation.

4. Sampling devices and measuring methodologyVarious types of sampling devices and measurement

methods are available for the sampling and measurement ofmicroorganisms in the air and on surfaces, and appropriatesamplers and measuring methodology are selected accordingto the purpose of monitoring and the items to be monitored.4.1 Evaluation of airborne microorganisms

a) Settle platesPetri dishes of a speciêd diameter containing a suitable

culture medium are placed at the measurement location andthe cover is removed there. The plates are exposed for a giventime and the microorganisms deposited from the air onto theagar surface are enumerated after incubation. This method isnot eŠective for quantitative monitoring of total airbornemicroorganisms because it does not detect microorganismsthat do not settle onto the surface of the culture media, andthe settling velocity of aggregates of microorganisms is aŠect-ed by air currents and disturbances in air‰ow. Although theresults obtained by the settle plate method are only qualita-tive or semi-quantitative, this method is suitable for long-term evaluation of possible contamination of products ordevices by airborne microorganisms.

b) Active microbial sampling methods1) Measuring methodsMethods in which a ˆxed volume of air is aspirated include

ˆltration-type sampling devices and impact-type samplingdevices. With the ˆlter-type sampling devices the desiredvolume of air can be collected by appropriately changing theair intake rate or the ˆlter size. However, care must be takento ensure that sterility is maintained while the ˆlter is placedin and removed from the holder. When air sampling devicesare used in critical areas, care must be taken to avoid distur-bance of the air‰ow around the products. There are two typesof ˆlters; wet-type used gelatin ˆlters and dry-type used mem-brane ˆlters. With the dry-type ˆlters, static electricity eŠectscan make it impossible to collect quantitatively microorgan-isms on the ˆlter. When an impact-type sampling device isused, the following points are important: 1) The speed atwhich the collected air strikes the culture medium surfacemust be su‹cient to capture the microorganisms, but mustnot have an adverse eŠect on the collected microorganisms.2) A su‹cient volume of air must be sampled so that even ex-tremely low levels of microbiological contaminants are de-tected, but the procedure must not cause a signiˆcant changein the physical or chemical properties of the culture medium.3) When the device is used in critical areas, care must be exer-cised to ensure that the processing of the sterile pharmaceuti-cal products is not adversely aŠected by the air disturbance.

2) Sampling devicesThe most commonly used samplers are as follows: Slit

sampler, Andersen sampler, pinhole sampler, centrifugal

sampler and ˆltration-type sampler. Each sampler hasspeciˆc characteristics. The slit sampler is a device to trapmicroorganisms in a known volume of air passed through astandardized slit. The air is impacted on a slowly revolvingPetri dish containing a nutrient agar. The rotation rate of thePetri dish and the distance from the slit to the agar surfaceare adjustable and it is possible to estimate the number ofmicroorganisms in the air passed through the device for aperiod of up to 1 hr. The Andersen sampler consists of a per-forated cover and several pieces of Petri dishes containing anutrient agar, and a known volume of air passed through theperforated cover impacts on the agar medium in the Petri dis-hes. The sampler is suitable for the determination of the dis-tribution of size ranges of microorganism particulates in theair. The pinhole sampler resembles the slit of the slit sampler,but has pinholes in place of the slit. A known volume of airpassed through several pinholes impacts on agar medium in aslowly revolving Petri dish. The centrifugal sampler consistsof a propeller that pulls a known volume of air into the deviceand then propels the air outward to impact on a tangentiallyplaced nutrient agar strip. The sampler is portable and can beused anywhere, but the sampling volume of air is limited.

See above 1) on the characteristics of the ˆltration-typesampler.4.2 Measurement methods for microorganisms on surfaces

a) Contact platesUse a contact plate with an appropriate contact surface.

The culture medium surface should be brought into contactwith the sampling site for several seconds by applyinguniform pressure without circular or linear movement. Aftercontact and removal, the plates are covered and, as soon aspossible, incubated using appropriate culture conditions. Af-ter a contact plate has been used, the site to which the platewas applied must be wiped aseptically to remove any adher-ent culture medium.

b) SwabsA piece of sterilized gauze, absorbent cotton, cotton swab,

or other suitable material premoistened with an appropriaterinse ‰uid is stroked in closely parallel sweeps or slowly rotat-ed over the deˆned sampling area. After sampling, the swabis agitated in a speciêd amount of an appropriate sterilizedrinse ‰uid, and the rinse ‰uid is assayed for viable organisms.

5. Test methods for collection performance of a samplingdevice for airborne microorganisms

The testing of the collection performance of samplingdevices for airborne microorganisms is performed in accor-dance with JIS K 3836 (Testing methods for collectione‹ciency of airborne microbe samplers) or ISO 14698 – 1(Cleanrooms and associated controlled environments.Biocontamination control. General principles).

6. Growth-promotion test of media and conˆrmation of an-timicrobial substances

This test and conˆrmation are performed according to`ÈŠectiveness of culture media and conˆrmation of an-timicrobial substances'' in the Microbial Limit Test.

Media

Brain-heart infusion agar medium or Fluid brain-heart infu-sion medium

Bovine brain extract powder*1

17211721JP XV General Information / Mycoplasma Testing

An amount equivalent to 200 g of calf brainBovine heart extract powder*2

An amount equivalent to 250 g of the materialPeptone 10.0 gGlucose 2.0 gSodium chloride 5.0 gDisodium hydrogenphosphate

dodecahydrate 2.5 gAgar 15.0 gWater 1,000 mL

Sterilize by heating in an autoclave at 1219C for 15 to 20min. pH after sterilization: 7.2 – 7.6.

Fluid cooked meat mediumBovine heart extract powder*2

An amount equivalent to 450 g of the materialPeptone 20.0 gGlucose 2.0 gSodium chloride 5.0 gWater 1,000 mL


Glucose peptone agar medium or Fluid glucose peptonemedium

See Microbial Limit Test. Antibiotic is added if necessary.

Nutrient agar medium or Fluid nutrient mediumMeat extract 3.0 gPeptone 5.0 gAgar 15.0 gWater 1,000 mL


Potato-dextrose agar medium or Fluid potato-dextrose medi-um


Reinforced clostridial agar medium or Fluid reinforced clos-tridial medium

Meat extract 10.0 gPeptone 10.0 gYeast extract 3.0 gSoluble starch 1.0 gGlucose 5.0 gL-Cystein hydrochloride monohydrate 0.5 gSodium chloride 5.0 gSodium acetate trihydrate 3.0 gAgar 15.0 gWater 1,000 mL

For ‰uid medium, add 0.5 g of agar. Sterilize by heatingin an autoclave at 1219C for 15 to 20 min. pH aftersterilization: 6.7 – 6.9.

Sabouraud dextrose agar medium or Fluid sabouraud dex-trose medium


Soybean-casein digest agar medium or Fluid soybean-caseindigest medium

See Microbial Limit Test.

Thioglycolate agar medium or thioglycolate medium I forsterility test

See Sterility Test. The agar concentration of Thioglycolateagar medium is about 1.5z.

*1 Bovine brain extract powder Dried extract of bovinefresh brain. A yellow-brown powder having a characteristicodor.

Loss on drying: not more than 5z.*2 Bovine heart extract powder Dried extract of bovinefresh heart. A yellow-brown powder having a characteristicodor.

Loss on drying: not more than 5z.

Rinsing Liquids

BuŠered sodium chloride-peptone solution (pH 7.0)See Microbial Limit Test.

LP liquidCasein peptone 1.0 gSoybean lecithin 0.7 gPolysorbate 80 1.0 – 20.0 gWater 1,000 mL

Sterilize by heating in an autoclave at 1219C for 15 to 20min. pH after sterilization: 7.2.

Phosphate buŠered solution (pH 7.2)See Microbial Limit Test.

Ringer's solution, 1W4 concentrationSodium chloride 2.25 gPotassium chloride 0.105 gCalcium chloride dihydrate 0.16 gWater 1,000 mL


Thiosulfate-Ringer's solutionSodium thiosulfate pentahydrate 0.8 gRinger's solution, 1W4 concentration 1,000 mL


14. Mycoplasma Testing forCell Substrates used for the

Production of BiotechnologicalWBiological Products

This document describes the currently available methodsof mycoplasma testing that should be performed for cell sub-strates that are used in the manufacture of biotechnologicalWbiological products.

Methods suggested for detection of mycoplasma are, A.culture method, B. indicator cell culture method, and C.polymerase chain reaction (PCR) method.

Mycoplasma testing should be performed on the mastercell bank (MCB) and the working cell bank (WCB), as well ason the cell cultures used during the manufacturing process of

17221722 JP XVMycoplasma Testing / General Information

the product. For the assessment of these cells, mycoplasmatesting should be performed using both methods A and B.Method B, however, does not detect only DNA derived frommycoplasma. Therefore, if a positive result is obtained onlyfrom method B, method C can be used to determine whethermycoplasma is actually present. When method C is used, it isnecessary to demonstrate the rationale for determining anegative result. In such a case, the sensitivity and speciˆcityof the method, the appropriateness of the sample prepara-tion, and the suitability of the selection of the test method,including selection of reagents, reaction conditions andprimers should be taken into account.

Prior to mycoplasma testing, the sample should be testedto detect the presence of any factors inhibiting the growth ofmycoplasma. If such growth-inhibiting factors are detectedthey should be neutralized or eliminated by an appropriatemethod, such as centrifugation or cell passage.

If the test will be performed within 24 hours of obtainingthe sample, the sample should be stored at a temperature be-tween 29C and 89C. If more than 24 hours will elapse beforethe test is performed, the sample should be stored at －609Cor lower.

If mycoplasma is detected, additional testing to identifythe species may be helpful in determining the source of con-tamination.

A. Culture Method1. Culture Medium

Both agar plates and broth are used. Each lot of agar andbroth medium should be free of antibiotics except for penicil-lin. Refer to the Minimum Requirements for BiologicalProducts regarding selection of the culture media. Other cul-ture media may be used if they fulˆll the requirementsdescribed in the following section 2.2. Suitability of Culture Medium

Each lot of medium should be examined for mycoplasmagrowth-promoting properties. To demonstrate the capacityof the media to detect known mycoplasma, each test shouldinclude control cultures of at least two known species orstrains of mycoplasma, one of which should be a dextrosefermenter (i.e., M. pneumoniae strain FH or equivalent spe-cies or strains) and one of which should be an argininehydrolyser (i.e., M. orale CH 19299 or equivalent species orstrains). The mycoplasma strains used for the positive controltests should be obtained from an o‹cial or suitably accredit-ed agency, and handled appropriately. Inoculate the culturemedium with no more than 100 colony-forming units (CFU).3. Culture and Observation

1) Inoculate no less than 0.2 mL of test sample (cell sus-pension) in evenly distributed amounts over the surface ofeach of four or more agar plates. After the surfaces of the in-oculated plates are dried, one half of the plates should be in-cubated under aerobic conditions in an atmosphere of aircontaining 5 to 10 percent carbon dioxide and adequate hu-midity, and the other half under anaerobic conditions in anatmosphere of nitrogen containing 5 to 10 percent carbon di-oxide and adequate humidity at 36±19C for no less than 14days.

2) Inoculate no less than 10 mL of the test sample (cellsuspension) into each of two vessels containing 100 mL ofbroth medium. One vessel should be incubated under aerobicconditions, and the other under anaerobic conditions.

If the culture medium for the sample cells contains any

growth-inhibiting factors, such as antibiotics, these factorsmust be removed. A method such as centrifugation is recom-mended for this purpose.

3) Subculture 0.2 mL of broth culture from each vesselon the 3rd, 7th, and 14th days of incubation onto two or moreagar plates. The plates inoculated with aerobic or anaerobicbroth cultures should be incubated aerobically or anaerobi-cally, respectively at 36±19C for no less than 14 days.

4) Examination of all plates for mycoplasma coloniesshould be done microscopically on the 7th and 14th day at 100times magniˆcation or greater.

B. Indicator Cell Culture MethodUsing Vero cell culture substrate, pretest the suitability of

the method using an inoculum of no more than 100 CFU ofM. hyorhinis DBS 1050 or M. orale CH 19299.

An equivalent indicator cell substrate and suitablemycoplasma strains may be acceptable if data demonstrate atleast equal sensitivity for the detection of known mycoplasmacontaminants. The mycoplasma strains should be obtainedfrom an o‹cial or suitably accredited agency, and handledappropriately. The cell substrate used should be obtainedfrom a qualiêd cell bank and certiêd to be mycoplasmafree. The acquired cells should be carefully cultured andpropagated, and su‹cient volumes of seed stock should beprepared with the proper precautions to avoid mycoplasmacontamination. The stock should be tested for mycoplasmacontamination using at least one of the methods described inthis document, then frozen for storage. For each test a newcontainer from the stock should be thawed and used within 6passages.

Indicator cell cultures should be grown on cover slips sub-merged in culture dishes or equivalent containers for one day.Inoculate no less than 1 mL of the test sample (cell culture su-pernatant) into two or more of the culture dishes.

The test should include a negative (non-infected) controland two positive mycoplasma controls, such as M. hyorhinisDBS 1050 or M. orale CH 19299. Use an inoculum of nomore than 100 CFU for the positive controls.

Incubate the cell cultures for 3 to 6 days at 36±19C in anatmosphere of air containing 5 percent carbon dioxide.

Examine the cell cultures after ˆxation for the presence ofmycoplasma by epi‰uorescence microscopy (400 to 600 timesmagniˆcation or greater) using a DNA-binding‰uorochrome, such as bisbenzimidazole or an equivalentstain. Compare the microscopical appearance of the test cul-tures with that of the negative and positive controls.Procedure

1) Aseptically place a sterilized glass cover slip into eachcell culture dish (35 mm diameter).

2) Prepare Vero cell suspension in Eagle's minimum es-sential medium containing 10 percent bovine calf serum at aconcentration of 1×104 cells per 1 mL. The bovine calfserum should be tested and conˆrmed to be free frommycoplasma prior to use.

3) Inoculate aliquots of 2 mL of the Vero cell suspensioninto each culture dish. Ensure that the cover slips are com-pletely submerged, and not ‰oating on the surface of the cul-ture medium. Incubate the cultures at 36±19C in an at-mosphere of air containing 5 percent carbon dioxide for oneday, so that the cells are attached to the glass cover slip.

4) Replace 2 mL of the culture medium with fresh medi-um, then add 0.5 mL of the test sample (cell culture super-

17231723JP XV General Information / Mycoplasma Testing

natant) to each of two or more culture dishes. Perform thesame procedure for the positive (2 types of mycoplasma, suchas M. hyorhinis and M. orale) and negative controls.

5) Incubate the cultures for 3 to 6 days at 36±19C in anatmosphere of air containing 5 percent carbon dioxide.

6) Remove the culture medium from the culture dishes,and add 2 mL of a mixture of acetic acid (100) and methanol(1:3) (ˆxative) to each dish; then, allow them to stand for 5minutes.

7) Remove the ˆxative from each dish, then add the sameamount of ˆxative again, and leave the dishes to stand for 10minutes.

8) Remove the ˆxative and then completely air-dry all thedishes.

9) Add 2 mL of bisbenzamide ‰uorochrome staining so-lution to each culture dish. Cover the dishes and let themstand at room temperature for 30 minutes.

10) Aspirate the staining solution and rinse each dishwith 2 mL of distilled water 3 times. Take out the glass coverslips and dry them.

11) Mount each cover slip with a drop of a mounting‰uid. Blot oŠ surplus mounting ‰uid from the edges of thecover slips.

12) Examine by epi‰uorescence microscopy at 400 to 600times magniˆcation or greater.

13) Compare the microscopic appearance of the test sam-ple with that of the negative and positive controls.

14) The test result is judged to be positive if there aremore than 5 cells per 1000 (0.5z) that have minute ‰uores-cent spots that appear to surround, but are outside, the cellnucleus.

C. Polymerase Chain Reaction (PCR) Detection MethodThe PCR method is a highly speciˆc method that enables

the detection of trace amounts of mycoplasma DNA, and hascome to be widely used in recent years as a means of detectingmycoplasma contamination. However, the sensitivity andspeciˆcity depend on the procedure employed, and a positiveresult from PCR does not always indicate the presence of via-ble mycoplasma.

The PCR method is based on amplifying DNA extractedfrom the cell culture with speciˆc primers so that the presenceof the target DNA is detected. A two-step PCR (nested PCR)is recommended in order to increase sensitivity and speciˆci-ty. The tests should include both a positive control (such asM. hyorhinis of 100 CFU or less) and a negative control.

Mycoplasma DNA from the sample of cells or cell culturesis ampliêd using primers which should be able to amplifysome common conserved mycoplasma DNA sequence. Theampliˆcation should be performed using an appropriate heat-resistant DNA polymerase, and suitable conditions. The am-pliêd DNA can be identiêd after agarose gel electrophore-sis, followed by ethidium bromide staining and UV irradia-tion of the gel.

For this method, it is important to use primers that arespeciˆc to mycoplasma by choosing base sequences that arewell-conserved for a wide range of mycoplasma species, forexample, the spacer region between the 16S-23S ribosomegenes.

It is recommended that a two-step PCR using nestedprimers should be performed to increase the sensitivity andspeciˆcity, if the one-step PCR is negative.

The primers to be selected for the second stage of a two-

step PCR are nested primers from the inner portion of thesequence. The outer and inner primers should have proveneŠectiveness and speciˆcity as described in publications or bevalidated experimentally.

It is possible to increase the accuracy of the detection ofmycoplasma DNA by performing PCR tests after cultivationof mycoplasma that may be present in samples using Verocells.

The following is an example of a two-step PCR procedure.The reagents and reaction conditions in this example are notexclusive. If the suitability of other reagents and conditions isveriêd, they may be used. If another procedure is used, theprocedure should be justiêd and documented in detail, andthe information provided should include the sensitivity andspeciˆcity of the method.Example Procedure1. Preparation of template

1) Place 600 mL of the test cell suspension (if necessary,subcultured with Vero cells) in a tube and dissolve the cellswith 0.1z SDS or an equivalent. Add an equal volume (600mL) of TE (10 mmolWL tris-hydrochloric acid (pH 8.0), 1mmolWL EDTA) buŠer-saturated phenol, and mix.

2) Centrifuge at 15,000 min－1 for 5 minutes at room tem-perature.

3) Transfer 400 mL of the supernatant to another tube,and add 10 mL of 3 molWL sodium acetate.

4) Add 1 mL (2.5 volumes) of ethanol (95) and stirthoroughly. Ice the mixture for 15 minutes, then centrifuge at15,000 min－1 for 10 minutes at 49C.

5) Discard the supernatant and rinse the precipitate onceor twice with 200 to 300 mL of 80z ethanol. Remove the rinsesolution using a pipette. Centrifuge at 15,000 min－1 for 10minutes at 49C, then remove the supernatant thoroughly anddry up the precipitate.

6) Dissolve the precipitate in 40 mL of distilled water.2. Perform the same procedure for the positive and nega-tive controls3. First stage of a two-step PCR

1) Make a mixture of the heat-resistant DNA poly-merase, dNTP solution, outer primer, and reaction buŠer so-lution (including Mg ions), and place 90 mL in each tube.

2) Add 10 mL of the template prepared as above to eachtube containing the ˆrst stage PCR solution (90 mL).

3) Perform the DNA ampliˆcation by repeating 30 cyclesof denaturation at 949C for 30 seconds, annealing at an ap-propriate temperature for the primer (559C for the primer inthis example), and elongation at 729C for 2 minutes. Adddrops of mineral oil or suitable equivalent as needed duringthe reaction to prevent evaporation.4. Second stage of a two-step PCR

1) Make a mixture of the heat-resistant DNA poly-merase, dNTP solution, inner primer, and reaction buŠersolution (including Mg ions), and place 99 mL in each tube.

2) Add 1 mL of the ˆrst stage PCR product from eachtube to a tube containing the second stage PCR solution (99mL).

3) Perform the DNA ampliˆcation by repeating 30 cyclesof denaturation at 949C for 30 seconds, annealing at an ap-propriate temperature for the primer (559C for the primer inthis example), and elongation at 729C for 2 minutes. Adddrops of mineral oil or suitable equivalent as needed duringthe reaction to prevent evaporation.5. Agarose gel electrophoresis

17241724 JP XVPeptide Mapping / General Information

1) Mix 10 mL of each of the ˆrst stage and second stagePCR products with 2 mL of an appropriate dye as a migrationmarker, and perform 1z agarose gel electrophoresis.

2) Stain the gel with ethidium bromide and take a photo-graph under UV irradiation.

3) The test is judged to be positive if a DNA band is de-tected.

[An Example of Primer]For mycoplasma detectionOuter primerF15?-ACACCATGGGAG(CWT)TGGTAAT-3?R15?-CTTC(AWT)TCGACTT(CWT)CAGACCCAAGG-

CAT-3?Inner primerF25?-GTG(GWC)GG(AWC)TGGATCACCTCCT-3?R25?-GCATCCACCA(AWT)A(AWT)AC(CWT)CTT-3?

( ) indicates a mixture.

[PCR reaction solution][First stage] [Second stage]

dNTP solution (each 1.25 mol) 16 mL 16 mLPrimer (10 pmolWmL) F1 2 mL F2 2 mLPrimer (10 pmolWmL) R1 2 mL R2 2 mLHeat-resistant DNA poly-

merase (1 UWmL) 2 mL 2 mL

Reaction buŠer solution 68 mL 77 mL25 mmolWL magnesium chlo-

ride hexahydrate 8 mL 8 mL

10-fold buŠer solution* 10 mL 10 mLSterile distilled water 50 mL 59 mL

*Composition of 10-fold buŠer solution2-amino-2-hydroxymethyl-1,3-

propanediol-hydrochloric acid(pH 8.4) 100 mmolWL

Potassium chloride 500 mmolWLMagnesium chloride hexahydrate 20 mmolWLGelatin 0.1 gWL

[Method of cultivating mycoplasma within Vero cells]1) Use at least two cell culture dishes for each of the test

sample, positive control and negative control.2) Into each cell culture dish (diameter 35 mm), inoculate

2 mL of the Vero cell suspension (1×104 cells per 1 mL) inEagle's minimum essential medium containing 10 percent bo-vine calf serum (tested in advance using the PCR method toverify that it does not contain any detectable mycoplasmaDNA). Incubate the cultures at 36±19C in an atmosphere ofair containing 5 percent carbon dioxide for one day.

3) Replace the culture media with fresh media, and add0.5 mL of the test sample (cell culture supernatant) to each oftwo or more Vero cell culture dishes. Perform the sameprocedure for the positive (such as 100 CFU or less M.hyorhinis) and negative controls.

4) Incubate the Vero cell culture dishes for the test sam-ple, positive and negative controls for 3 to 6 days at 36±19Cin an atmosphere of air containing 5 percent carbon dioxide.

15. Peptide MappingThis test is harmonized with the European Pharmacopoeia

and the U.S. Pharmacopeia.

Purpose and ScopePeptide mapping is an identity test for proteins, especially

those obtained by r-DNA technology. It involves the chemi-cal or enzymatic treatment of a protein resulting in the for-mation of peptide fragments followed by separation andidentiˆcation of the fragments in a reproducible manner. It isa powerful test that is capable of identifying single aminoacid changes resulting from events such as errors in thereading of complementary DNA (cDNA) sequences or pointmutations. Peptide mapping is a comparative procedurebecause the information obtained, compared to a referencestandard or reference material similarly treated, conˆrms theprimary structure of the protein, is capable of detectingwhether alterations in structure have occurred, and demon-strates process consistency and genetic stability. Each proteinpresents unique characteristics which must be wellunderstood so that the scientiˆc and analytical approachespermit validated development of a peptide map that providessu‹cient speciˆcity.

This chapter provides detailed assistance in the applicationof peptide mapping and its validation to characterize thedesired protein product, to evaluate the stability of the ex-pression construct of cells used for recombinant DNAproducts and to evaluate the consistency of the overall proc-ess, to assess product stability as well as to ensure the identityof the protein product, or to detect the presence of proteinvariant.

The Peptide MapPeptide mapping is not a general method, but involves

developing speciˆc maps for each unique protein. Althoughthe technology is evolving rapidly, there are certain methodsthat are generally accepted. Variations of these methods willbe indicated, when appropriate, in speciˆc monographs.

A peptide map may be viewed as a ˆngerprint of a proteinand is the end product of several chemical processes that pro-vide a comprehensive understanding of the protein being ana-lyzed. Four major steps are necessary for the development ofthe procedure: isolation and puriˆcation of the protein, if theprotein is part of a formulation; selective cleavage of the pep-tide bonds; chromatographic separation of the peptides; andanalysis and identiˆcation of the peptides. A test sample isdigested and assayed in parallel with a reference standard or areference material. Complete cleavage of peptide bonds ismore likely to occur when enzymes such as endoproteases(e.g., trypsin) are used, instead of chemical cleavage rea-gents. A map should contain enough peptides to be meaning-ful. On the other hand, if there are too many fragments, themap might lose its speciˆcity because many proteins will thenhave the same proˆles.

Isolation and PuriˆcationIsolation and puriˆcation are necessary for analysis of bulk

drugs or dosage forms containing interfering excipients andcarrier proteins and, when required, will be speciêd in themonograph. Quantitative recovery of protein from thedosage form should be validated.

Selective Cleavage of Peptide BondsThe selection of the approach used for the cleavage of pep-

tide bonds will depend on the protein under test. This selec-tion process involves determination of the type of cleavage tobe employed —enzymatic or chemical— and the type of

17251725JP XV General Information / Peptide Mapping

cleavage agent within the chosen category. Several cleavageagents and their speciˆcity are shown in Table 1. This list isnot all-inclusive and will be expanded as other cleavageagents are identiêd.

Table 1. Examples of Cleavage Agents

Type Agent Speciˆcity

Enzymatic Trypsin (EC 3.4.21.4) C-terminal side ofArg and Lys

Chymotrypsin(EC 3.4.21.1)

C-terminal side ofhydrophobicresidues (e.g.,Leu, Met, Ala,aromatics)

Pepsin (EC 3.4.23.1 & 2) Nonspeciˆc digestLysyl endopeptidase(Lys-C Endopeptidase)(EC 3.4.21.50)

C-terminal side ofLys

Glutamyl endopeptidase(from S. aureus strain V8)(EC 3.4.21.19)

C-terminal side ofGlu and Asp

Peptidyl-Asp metalloendopeptidase(Endoproteinase Asp-N)(EC 3.24.33)

N-terminal side ofAsp

Clostripain (EC 3.4.22.8) C-terminal side ofArg

Chemical Cyanogen bromide C-terminal side ofMet

2-Nitro-5-thio-cyanobenzoicacid N-terminal side of

Cyso-Iodosobenzoic acid C-terminal side of

Trp and TyrDilute acid Asp and ProBNPS-skatole Trp

Pretreatment of Sample Depending on the size or theconˆguration of the protein, diŠerent approaches in thepretreatment of samples can be used. For monoclonalantibodies the heavy and light chains will need to be separat-ed before mapping. If trypsin is used as a cleavage agent forproteins with a molecular mass greater than 100,000 Da,lysine residues must be protected by citraconylation or maley-lation; otherwise, many peptides will be generated.

Pretreatment of the Cleavage Agent Pretreatment ofcleavage agents —especially enzymatic agents— might benecessary for puriˆcation purposes to ensure reproducibilityof the map. For example, trypsin used as a cleavage agent willhave to be treated with tosyl-L-phenylalanine chloromethylketone to inactivate chymotrypsin. Other methods, such aspuriˆcation of trypsin by HPLC or immobilization of en-zyme on a gel support, have been successfully used when onlya small amount of protein is available.

Pretreatment of the Protein Under certain conditions, itmight be necessary to concentrate the sample or to separatethe protein from added substances and stabilizers used in for-mulation of the product, if these interfere with the mappingprocedure. Physical procedures used for pretreatment can in-clude ultraˆltration, column chromatography, and lyophili-

zation. Other pretreatments, such as the addition ofchaotropic agents (e.g., urea) can be used to unfold the pro-tein prior to mapping. To allow the enzyme to have full ac-cess to cleavage sites and permit some unfolding of the pro-tein, it is often necessary to reduce and alkylate the disulˆdebonds prior to digestion.

Digestion with trypsin can introduce ambiguities in thetryptic map due to side reactions occurring during thedigestion reaction, such as nonspeciˆc cleavage, deamida-tion, disulˆde isomerization, oxidation of methionineresidues, or formation of pyroglutamic groups created fromthe deamidation of glutamine at the N-terminal side of a pep-tide. Furthermore, peaks may be produced by autohydrolysisof trypsin. Their intensities depend on the ratio of trypsin toprotein. To avoid autohydrolysis, solutions of proteases maybe prepared at a pH that is not optimal (e.g., at pH 5 fortrypsin), which would mean that the enzyme would notbecome active until diluted with the digest buŠer.

Establishment of Optimal Digestion Conditions Factorsthat aŠect the completeness and eŠectiveness of digestion ofproteins are those that could aŠect any chemical or enzymaticreactions.

pH: The pH of the digestion mixture is empirically deter-mined to ensure the optimization of the performance of thegiven cleavage agent. For example, when using cyanogenbromide as a cleavage agent, a highly acidic environment(e.g., pH 2, formic acid) is necessary; however, when usingtrypsin as a cleavage agent, a slightly alkaline environment(pH 8) is optimal. As a general rule, the pH of the reactionmilieu should not alter the chemical integrity of the proteinduring the digestion and should not change during the courseof the fragmentation reaction.

Temperature: A temperature between 259C and 379C isadequate for most digestions. The temperature used isintended to minimize chemical side reactions. The type ofprotein under test will dictate the temperature of the reactionmilieu, because some proteins are more susceptible to denatu-ration as the temperature of the reaction increases. For exam-ple, digestion of recombinant bovine somatropin is conduct-ed at 49C, because at higher temperatures it will precipitateduring digestion.

Time: If su‹cient sample is available, a time coursestudy is considered in order to determine the optimum time toobtain a reproducible map and avoid incomplete digestion.Time of digestion varies from 2 to 30 hours. The reaction isstopped by the addition of an acid which does not interfere inthe tryptic map or by freezing.

Amount of Cleavage Agent: Although excessive amountsof cleavage agent are used to accomplish a reasonably rapiddigestion time (i.e., 6 to 20 hours), the amount of cleavage a-gent is minimized to avoid its contribution to the chromato-graphic map pattern. A protein to protease ratio between20:1 and 200:1 is generally used. It is recommended that thecleavage agent can be added in two or more stages to op-timize cleavage. Nonetheless, the ˆnal reaction volumeremains small enough to facilitate the next step in peptidemapping —the separation step. To sort out digestion artifactsthat might be interfering with the subsequent analysis, ablank determination is performed, using a digestion controlwith all the reagents, except the test protein.

Chromatographic SeparationMany techniques are used to separate peptides for

17261726 JP XVPeptide Mapping / General Information

mapping. The selection of a technique depends on the proteinbeing mapped. Techniques that have been successfully usedfor separation of peptides are shown in Table 2. In this sec-tion, a most widely used reverse-phase High PerformanceLiquid Chromatographic (RP-HPLC) method is described asone of the procedures of chromatographic separation.

Table 2. Techniques Used for the Separation of Peptides

Reverse-Phase High Performance Liquid Chromatography(RP-HPLC)

Ion-Exchange Chromatography (IEC)Hydrophobic Interaction Chromatography (HIC)Polyacrylamide Gel Electrophoresis (PAGE), nondenaturat-

ingSDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)Capillary Electrophoresis (CE)Paper Chromatography-High Voltage (PCHV)High-Voltage Paper Electrophoresis (HVPE)

The purity of solvents and mobile phases is a critical factorin HPLC separation. HPLC-grade solvents and water thatare commercially available, are recommended for RP-HPLC. Dissolved gases present a problem in gradientsystems where the solubility of the gas in a solvent may be lessin a mixture than in a single solvent. Vacuum degassing andagitation by sonication are often used as useful degassingprocedures. When solid particles in the solvents are drawninto the HPLC system, they can damage the sealing of pumpvalves or clog the top of the chromatographic column. Bothpre- and post-pump ˆltration is also recommended.

Chromatographic Column The selection of a chromato-graphic column is empirically determined for each protein.Columns with 100 Å or 300 Å pore size with silica supportcan give optimal separation. For smaller peptides, octylsilanechemically bonded to totally porous silica articles, 3 to 10 mmin diameter (L7) and octadecylsilane chemically bonded toporous silica or ceramic micro-particles, 3 to 10 mm in di-ameter (L1) column packings are more e‹cient than the butylsilane chemically bonded to totally porous silica particles, 5to 10 mm in diameter (L26) packing.

Solvent The most commonly used solvent is water withacetonitrile as the organic modiêr to which less than 0.1ztri‰uoroacetic acid is added. If necessary, add isopropylalcohol or n-propyl alcohol to solubilize the digest compo-nents, provided that the addition does not unduly increasethe viscosity of the components.

Mobile Phase BuŠered mobile phases containing phos-phate are used to provide some ‰exibility in the selection ofpH conditions, since shifts of pH in the 3.0 to 5.0 rangeenhance the separation of peptides containing acidic residues(e.g., glutamic and aspartic acids). Sodium or potassiumphosphates, ammonium acetate, phosphoric acid, and a pHbetween 2 and 7 (or higher for polymer-based supports) havealso been used with acetonitrile gradients. Acetonitrile con-taining tri‰uoroacetic acid is used quite often.

Gradient Selection Gradients can be linear, nonlinear, orinclude step functions. A shallow gradient is recommended inorder to separate complex mixtures. Gradients are optimizedto provide clear resolution of one or two peaks that willbecome ``marker'' peaks for the test.

Isocratic Selection Isocratic HPLC systems using a single

mobile phase are used on the basis of their convenience of useand improved detector responses. Optimal composition of amobile phase to obtain clear resolution of each peak is some-times di‹cult to establish. Mobile phases for which slightchanges in component ratios or in pH signiˆcantly aŠectretention times of peaks in peptide maps should not be usedin isocratic HPLC systems.

Other Parameters Temperature control of the column isusually necessary to achieve good reproducibility. The ‰owrates for the mobile phases range from 0.1 to 2.0 mL perminute, and the detection of peptides is performed with a UVdetector at 200 to 230 nm. Other methods of detection havebeen used (e.g., postcolumn derivatization), but they are notas robust or versatile as UV detection.

Validation This section provides an experimentalmeans for measuring the overall performance of the testmethod. The acceptance criteria for system suitability dependon the identiˆcation of critical test parameters that aŠect datainterpretation and acceptance. These critical parameters arealso criteria that monitor peptide digestion and peptide anal-ysis. An indicator that the desired digestion endpoint wasachieved is by the comparison with a Reference Standard,which is treated exactly as the article under test. The use of areference standard or reference material in parallel with theprotein under test is critical in the development and establish-ment of system suitability limits. In addition a specimenchromatogram should be included with the Reference Stan-dard or Reference Material for additional comparison pur-poses. Other indicators may include visual inspection of pro-tein or peptide solubility, the absence of intact protein, ormeasurement of responses of a digestion-dependent peptide.The critical system suitability parameters for peptide analysiswill depend on the particular mode of peptide separation anddetection and on the data analysis requirements.

When peptide mapping is used as an identiˆcation test, thesystem suitability requirements for the identiêd peptidescovers selectivity and precision. In this case, as well as whenidentiˆcation of variant protein is done, the identiˆcation ofthe primary structure of the peptide fragments in the peptidemap provides both a veriˆcation of the known primary struc-ture and the identiˆcation of protein variants by comparisonwith the peptide map of the reference standard/referencematerial for the speciêd protein. The use of a digested refer-ence standard or reference material for a given protein in thedetermination of peptide resolution is the method of choice.For an analysis of a variant protein, a characterized mixtureof a variant and a reference standard or reference materialcan be used, especially if the variant peptide is located in aless-resolved region of the map. The index of pattern con-sistency can be simply the number of major peptides detect-ed. Peptide pattern consistency can be best deˆned by theresolution of peptide peaks. Chromatographic parameters—such as peak-to-peak resolution, maximum peak width,peak area, peak tailing factors, and column e‹ciency— maybe used to deˆne peptide resolution. Depending on the pro-tein under test and the method of separation used, single pep-tide or multiple peptide resolution requirements may benecessary.

The replicate analysis of the digest of the referencestandard or reference material for the protein under testyields measures of precision and quantitative recovery.Recovery of the identiêd peptides is generally ascertained bythe use of internal or external peptide standards. The

17271727JP XV General Information / pH Test for Gastrointestinal

precision is expressed as the relative standard deviation(RSD). DiŠerences in the recovery and precision of theidentiêd peptides are expected; therefore, the systemsuitability limits will have to be established for both therecovery and the precision of the identiêd peptides. Theselimits are unique for a given protein and will be speciêd inthe individual monograph.

Visual comparison of the relative retention times, the peakresponses (the peak area or the peak height), the number ofpeaks, and the overall elution pattern is completed initially. Itis then complemented and supported by mathematical analy-sis of the peak response ratios and by the chromatographicproˆle of a 1:1 (v/v) mixture of sample and reference stan-dard or reference material digest. If all peaks in the sampledigest and in the reference standard or reference materialdigest have the same relative retention times and peaksresponse ratios, then the identity of the sample under test isconˆrmed.

If peaks that initially eluted with signiˆcantly diŠerent rela-tive retention times are then observed as single peaks in the1:1 mixture, the initial diŠerence would be an indication ofsystem variability. However, if separate peaks are observedin the 1:1 mixture, this would be evidence of the nonequiva-lence of the peptides in each peak. If a peak in the 1:1 mixtureis signiˆcantly broader than the corresponding peak in thesample and reference standard or reference material digest, itmay indicate the presence of diŠerent peptides. The use ofcomputer-aided pattern recognition software for the analysisof peptide mapping data has been proposed and applied, butissues related to the validation of the computer softwarepreclude its use in a compendial test in the near future. Otherautomated approaches have been used that employ mathe-matical formulas, models, and pattern recognition. Such ap-proaches are, for example, the automated identiˆcation ofcompounds by IR spectroscopy and the application of diode-array UV spectral analysis for identiˆcation of peptides.These methods have limitations due to inadequate resolu-tions, co-elution of fragments, or absolute peak responsediŠerences between reference standard or reference materialand sample fragments.

The numerical comparison of the retention times and peakareas or peak heights can be done for a selected group ofrelevant peaks that have been correctly identiêd in thepeptide maps. Peak areas can be calculated using one peakshowing relatively small variation as an internal reference,keeping in mind that peak area integration is sensitive tobaseline variation and likely to introduce error in theanalysis. Alternatively, the percentage of each peptide peakheight relative to the sum of all peak heights can be calculatedfor the sample under test. The percentage is then compared tothat of the corresponding peak of the reference stan-dard/reference material. The possibility of auto-hydrolysisof trypsin is monitored by producing a blank peptide map,that is, the peptide map obtained when a blank solution istreated with trypsin.

The minimum requirement for the qualiˆcation of peptidemapping is an approved test procedure that includes systemsuitability as a test control. In general, early in the regulatoryprocess, qualiˆcation of peptide mapping for a protein issu‹cient. As the regulatory approval process for the proteinprogresses, additional qualiˆcations of the test can include apartial validation of the analytical procedure to provideassurance that the method will perform as intended in the de-

velopment of a peptide map for the speciêd protein.

Analysis and Identiˆcation of PeptidesThis section gives guidance on the use of peptide mapping

during development in support of regulatory applications.The use of a peptide map as a qualitative tool does not

require the complete characterization of the individualpeptide peaks. However, validation of peptide mapping insupport of regulatory applications requires rigorous cha-racterization of each of the individual peaks in the peptidemap. Methods to characterize peaks range from N-terminalsequencing of each peak followed by amino acid analysis tothe use of mass spectroscopy (MS).

For characterization purposes, when N-terminal sequenc-ing and amino acids analysis are used, the analytical separa-tion is scaled up. Since scale-up might aŠect the resolution ofpeptide peaks, it is necessary, using empirical data, to assurethat there is no loss of resolution due to scale-up. Eluates cor-responding to speciˆc peptide peaks are collected, vacuum-concentrated, and chromatographed again, if necessary.Amino acid analysis of fragments may be limited by the pep-tide size. If the N-terminus is blocked, it may need to becleared before sequencing. C-terminal sequencing of proteinsin combination with carboxypeptidase and MALDITOF-MScan also be used for characterization purposes.

The use of MS for characterization of peptide fragments isby direct infusion of isolated peptides or by the use of on-lineLC-MS for structure analysis. In general, it includes elec-trospray and matrix-assisted laser desorption ionization cou-pled to time-of-‰ight analyzer (MALDITOF) as well as fastatom bombardment (FAB). Tandem MS has also been usedto sequence a modiêd protein and to determine the type ofamino acid modiˆcation that has occurred. The comparisonof mass spectra of the digests before and after reduction pro-vides a method to assign the disulˆde bonds to the varioussulfhydryl-containing peptides.

If regions of the primary structure are not clearly demon-strated by the peptide map, it might be necessary to develop asecondary peptide map. The goal of a validated method ofcharacterization of a protein through peptide mapping is toreconcile and account for at least 95z of the theoretical com-position of the protein structure.

16. pH Test for GastrointestinalMedicine

In this test, medicine for the stomach and bowels, which issaid to control stomach acid, is stirred in a ˆxed amount ofthe 0.1 molWL hydrochloric acid for a ˆxed duration, and thepH value of this solution is obtained. The pH value of astomach medicine will be based on the dose and the dosage ofthe medicine (when the dosage varies, a minimum dosage isused) and expressed in the pH value obtained from the testperformed by the following procedure.

Preparation of SampleSolid medicine which conforms to the general regulations

for medicine (the powdered medicine section) can be used asa sample. When the medicine is in separate packages, thecontent of 20 or more packages is accurately weighed to cal-culate the average mass for one dose and mixed evenly to

17281728 JP XVPlastic Containers / General Information

make a sample. For granules and similar types in separatepackages, among the solid medicine which does not conformto the general regulations for medicine (the powdered medi-cine section), the content of 20 or more packages is accuratelyweighed to calculate the average mass for one dose and isthen powdered to make sample. For granules and similartypes not in separate packages, among solid medicine whichdoes not conform to the general regulations for medicine (thepowdered medicine section), 20 doses or more are powderedto make a sample. For capsules and tablets, 20 doses or moreare weighed accurately to calculate the average mass for onedose or average mass and then powdered to make a sample.

Liquid medicine is generously mixed to make a sample.

ProcedurePut 50 mL of the 0.1 molWL hydrochloric acid with the

molarity coe‹cient adjusted to 1.000, or equivalent 0.1 molWL hydrochloric acid with its volume accurately measured in a100-mL beaker. Stir this solution with a magnetic stirrer anda magnetic stirrer rotator (35 mm length, 8 mm diameter) atthe speed of about 300 revolutions per minute. While stirring,add the accurately weighed one-dose sample. After 10minutes, measure the pH value of the solution using the pHDetermination. The solution temperature should be main-tained at 37±29C throughout this operation.

17. Plastic Containers forPharmaceutical Products

Various kinds of plastics are used in the manufacture ofcontainers for pharmaceutical products. Such plastics shouldnot alter the e‹cacy, safety or stability of the pharmaceuticalproducts. In selecting a suitable plastic container, it is desira-ble to have full information on the manufacturing processesof the plastic container including the substances added. Sinceeach plastic has speciˆc properties and a wide variety of phar-maceutical products may be stored in containers made fromit, the compatibility of plastic containers with pharmaceuticalproducts should be judged for each combination of containerand the speciˆc pharmaceutical product to be containedtherein. This judgement should be carried out by verifyingthat a type sample of the container for the pharmaceuticalpreparation fulˆlls the essential requirements, i.e., the designspeciˆcations, according to experiments andWor scientiˆcdocumentation, etc. In addition, the compatibility must beensured based upon an appropriate quality assurance system.

Furthermore, in introducing a plastic container, it isdesirable that proper disposal after use is taken into consider-ation.

Essential Requirements in Designing Plastic Containers forPharmaceutical Products

The plastic material for the container should be of highquality. Therefore, recycled plastic materials, which are ofunknown constitution, must not be used.

The leachables or migrants from the container should notalter the e‹cacy or stability of the pharmaceutical productscontained therein. In addition, the possible toxic hazards ofthe leachables or migrants should not exceed a given level.Furthermore, the amounts of leachable or migratable chemi-cal substances, such as monomers and additives, from thecontainers to the pharmaceutical products contained therein

must be su‹ciently small from the viewpoint of safety.The container should have a certain level of physical

properties such as hardness, ‰exibility, shock resistance, ten-sile strength, tear strength, bending strength, heat resistanceand the like, in accordance with the intended usage.

The quality of the pharmaceutical products contained inthe container must not deteriorate during storage. For exam-ple, in the case of pharmaceutical products which are unsta-ble to light, the container should provide a su‹cient level oflight shielding. In the case of pharmaceutical products whichare easily oxidized, the container material should not allowthe permeation of oxygen. In the case of aqueous pharmaceu-tical products and pharmaceutical products that must be keptdry, the container material should not allow the permeationof water vapor. In addition, care should be taken that thecontainer is impermeable to the solvent in the case of solventsother than water. The concentration of the pharmaceuticalsmust not be decreased by more than a certain level due to theabsorption of the pharmaceuticals on the surface of the con-tainer, the migration of the pharmaceuticals into the inside ofthe material of the container, or the loss of pharmaceuticalsthrough the container. Also, the pharmaceutical productscontained therein must not be degraded by an interactionwith the material of the container.

The container should not be deformed, should not de-teriorate and should not be degraded by the pharmaceuticalproducts contained therein. Unacceptable loss of function ofthe container should not result from possible high tempera-ture or low temperature or cycles thereof encountered duringstorage or transportation.

The container should be of a required level of transparen-cy, when it is necessary to examine foreign insoluble matterandWor turbidity of the pharmaceutical products by visual ob-servation.

In the case of pharmaceutical products which must be steri-lized, it is required to satisfy the above-mentioned essentialrequirements of the container after the sterilization if there isa possibility that the quality of the container may changeafter the sterilization. There should not be any residue orgeneration of new toxic substances of more than certain risklevel after the sterilization. In addition, the container shouldnot have any inappropriate structure andWor material thatmight result in any bacterial contamination of the phar-maceutical products contained therein during storage andtransportation after sterilization.

Toxicity Evaluation of Container at Design PhaseFor design veriˆcation, the toxicity of the container should

be evaluated. For the toxicity evaluation, it is desirable toselect appropriate test methods and criteria for the evalua-tion, and to clarify the rationales for the selection. The testsshould be conducted using samples of the whole or a part ofthe prototype container. If the container consists of pluralparts of diŠerent materials, each part should be testedseparately. Such materials as laminates, composites, and likeare regarded as a single material. To test containers made ofsuch materials, it is recommended to expose the inner surfaceof the container, which contacts the pharmaceutical productscontained therein, to the extraction media used in the tests asfar as possible.

The tests required for the toxicity evaluation of the con-tainer are diŠerent depending upon the tissue to which thepharmaceutical products contained therein are to be applied.

17291729JP XV General Information / Powder Flow

The following tests are required for containers for1) preparations contacting blood:

Acute toxicity test, cytotoxicity test, sensitization testand hemolysis test

2) preparations contacting skin or mucous membranes:Cytotoxicity test and sensitization test

3) liquid orally administered preparations:Cytotoxicity test

It is recommended to conduct the tests in accordance withthe latest versions of the standard test methods on medicaldevices and materials published in Japan and other countries.

Those standard test methods are listed for information:(A) Selection of Tests・Guidelines for Basic Biological Tests of Medical Devices

and Materials (PAB Notiˆcation, YAKU-KI NO.99, June 27,1995), Principles and selection of tests・ISO 10993-1: Biological evaluation of medical

devices—Evaluation and testing(B) Acute Toxicity Test・ASTM F750-82: Standard practice for evaluating material

extracts by systemic injection in the mice・BS5736: Part 3 Method of test for systemic toxicity; as-

sessment of acute toxicity of extracts from medical devices・USP 24 <88> Biological reactivity tests, in vivo(C) Cytotoxicity Test・Guidelines for Basic Biological Tests of Medical Devices

and Materials, I. Cytotoxicity Test 10. Cytotoxicity test usingextract of medical device or material・ISO 10993-5: Biological evaluation of medical

devices—Tests for cytotoxicity : in vitro methods・USP 24 <87> Biological reactivity tests, in vitro(D) Hemolysis Test・Guidelines for Basic Biological Tests of Medical Devices

and Materials, VII. Hemolysis Test・ISO 10993-4: Biological evaluation of medical

devices—Selection of tests for interaction with blood. AnnexD・ASTM F756-82: Standard practice for assessment of

hemolytic properties of materials(E) Sensitization Test・Guidelines for Basic Biological Tests of Medical Devices

and Materials, II. Sensitization Test・ISO 10993-10: Biological evaluation of medical

devices—Tests for irritation and sensitization

Test Results to be Recorded per Production UnitAt the line production phase, it is required to establish the

values of acceptable limits on at least the test items men-tioned below and to record the test results of each productionunit of plastic containers for pharmaceutical products. In ad-dition, it is desirable to clarify the rationales for setting thevalues of limits. However, these requirements should not beapplied to orally administered preparations except liquidones.

1) Combustion Tests: Residue of ignition, heavy metals.If necessary, the amounts of the speciêd metals (lead, cad-mium, etc.)

2) Extraction Tests: pH, ultraviolet absorption spectra,potassium permanganate-reducing substances, foaming,non-volatile residue

3) Cytotoxicity Test4) Any other necessary tests for the speciˆc container for

aqueous infusions.

18. Powder FlowThis test is harmonized with the European Pharmacopoeia


Four commonly reported methods for testing powder ‰oware (1) angle of repose, (2) compressibility index of Hausnerratio, (3) ‰ow rate through an oriˆce, and (4) shear cell. Inaddition, numerous variations of each of these basic methodsare available.

In general, any method of measuring powder ‰ow shouldbe practical, useful, reproducible, sensitive, and yieldmeaningful results. It bears repeating that no one simplepowder ‰ow method will adequately or completely character-ize the wide range of ‰ow properties. An appropriate strategymay well be the use of multiple standardized test methods tocharacterize the various aspects of powder ‰ow as needed bythe pharmaceutical scientist.

1. Angle of ReposeThe angle of repose is the constant, three-dimensional an-

gle (relative to the horizontal base) assumed by a cone-likepile of material formed by any of several diŠerent methods.

The angle of repose has been used in several branches ofscience to characterize the ‰ow properties of solids. Angle ofrepose is a characteristic related to interparticulate friction,or resistance to movement between particles. Angle of reposetest results are reported to be very dependent upon themethod used. Experimental di‹culties arise due to segrega-tion of material and consolidation or aeration of the powderas the cone is formed.

1.1 Basic Methods for Angle of ReposeA variety of angle of repose test methods are reported in

the literature. The most common methods for determiningthe static angle of repose can be classiêd based on two im-portant experimental variables:

(1) The height of the ``funnel'' through which the powderpasses may be ˆxed relative to the base, or the height may bevaried as the pile forms.

(2) The base upon which the pile forms may be of ˆxeddiameter or the diameter of the powder cone may be allowedto vary as the pile forms.

1.2 Variations in Angle of Repose MethodsIn addition to the above methods, variations of them have

been used to some extent.･Drained angle of repose. This is determined by allowing anexcess quantity of material positioned above a ˆxed di-ameter base to ``drain'' from the container. Formation of acone of powder on the ˆxed diameter base allows determina-tion of the drained angle of repose.

･Dynamic angle of repose. This is determined by ˆlling acylinder (with a clear, ‰at cover on one end) and rotating itat a speciêd speed. The dynamic angle of repose is the an-gle (relative to the horizontal) formed by the ‰owing pow-der. The internal angle of kinetic friction is deˆned by theplane separating those particles sliding down the top layer ofthe powder and those particles that are rotating with thedrum (with roughened surface).1.3 Angle of Repose General Scale of Flowability

17301730 JP XVPowder Flow / General Information

While there is some variation in the qualitative descriptionof powder ‰ow using the angle of repose, much of the phar-maceutical literature appears to be consistent with the classiˆ-cation by Carr1, which is shown in Table 1. There are exam-ples of formulations with an angle of repose in the range of40 to 50 degrees that manufactured satisfactorily. When theangle of repose exceeds 50 degrees, the ‰ow is rarely accepta-ble for manufacturing purposes.

Table 1 Flow Properties and CorrespondingAngles of Repose1)

Flow Property Angle of Repose (degrees)

Excellent 25 – 30Good 31 – 35Fair 36 – 40

Passable 41 – 45Poor 46 – 55

Very poor 56 – 65Very, very poor À66

1.4 Experimental Considerations for Angle of ReposeAngle of repose is not an intrinsic property of the powder,

that is to say, it is very much dependent upon the methodused to form the cone of powder. On this subject, the existingliterature raises these important considerations:･The peak of the cone of powder can be distorted by the im-pact of powder from above. By carefully building the pow-der cone, the distortion caused by impact can be minimized.･The nature of the base upon which the powder cone isformed in‰uences the angle of repose. It is recommendedthat the powder cone be formed on a ``common base'',which can be achieved by forming the cone of powder on alayer of powder. This can be done by using a base of ˆxeddiameter with a protruding outer edge to retain a layer ofpowder upon which the cone is formed.

1.5 Recommended Procedure for Angle of ReposeForm the angle of repose on a ˆxed base with a retaining

lip to retain a layer of powder on the base. The base shouldbe free of vibration. Vary the height of the funnel to carefullybuild up a symmetrical cone of powder. Care should be takento prevent vibration as the funnel is moved. The funnelheight should be maintained approximately 2 – 4 cm from thetop of the powder pile as it is being formed in order tominimize the impact of falling powder on the tip of the cone.If a symmetrical cone of powder cannot be successfully orreproducibly prepared, this method is not appropriate. De-termine the angle of repose by measuring the height of thecone of powder and calculating the angle of repose, a, fromthe following equation:tan a＝height/0.5 base

2. Compressibility Index and Hausner RatioIn recent years the compressibility index and the closely

related Hausner ratio have become the simple, fast and popu-lar methods of predicting powder ‰ow characteristics. Thecompressibility index bas been proposed as an indirect meas-ure of bulk density, size and shape surface area, moisturecontent, and cohesiveness of materials because all of thesecan in‰uence the observed compressibility index. The com-pressibility index and the Hausner ratio are determined bymeasuring both the bulk volume and tapped volume of a

powder.

2.1 Basic Methods for Compressibility Index and Haus-ner Ratio

While there are some variations in the method of determin-ing the compressibility index and Hausner ratio, the basicprocedure is to measure (1) the unsettled apparent volume,Vo, and (2) the ˆnal tapped volume, Vf, of the powder aftertapping the material until no further volume changes occur(refer to the General Test Method, 3.01 Determination ofBulk and Tapped Densities). The compressibility index andthe Hausner ratio are calculated as follows:

Compressibility Index＝100×｛(Vo－Vf)/Vo｝

Hausner Ratio＝Vo/Vf

Alternatively, the compressibility index and Hausner ratiomay be calculated using measured values for bulk density(rB) and tapped density (rT) as follows:

Compressibility Index＝100×｛(rT－rB) /rT｝

Hausner Ratio＝rT/rB

In a variation of these methods, the rate of consolidation issometimes measured rather than, or in addition to, thechange in volume that occurs on tapping. For the compres-sibility index and the Hausner ratio, the generally acceptedscale of ‰owability is given in Table 2.

Table 2. Scale of Flowability1)

CompressibilityIndex (z) Flow Character Hausner Ratio

≦10 Excellent 1.00 – 1.1111 – 15 Good 1.12 – 1.1816 – 20 Fair 1.19 – 1.2521 – 25 Passable 1.26 – 1.3426 – 31 Poor 1.35 – 1.4532 – 37 Very poor 1.46 – 1.59»38 Very, very poor À1.60

2.2 Experimental Considerations for the CompressibilityIndex and Hausner Ratio

Compressibility index and Hausner ratio are not intrinsicproperties of the powder, that is to say, they are dependentupon the methodology used. The existing literature pointsout several important considerations aŠecting the determina-tion of the (1) unsettled apparent volume, Vo, (2) the ˆnaltapped volume, Vf, (3) the bulk density, rB, and (4) thetapped density, rT:･The diameter of the cylinder used･The number of times the powder is tapped to achieve thetapped density

･The mass of material used in the test･Rotation of the sample during tapping

2.3 Recommended Procedure for Compressibility Indexand Hausner Ratio

Use a 250-mL volumetric cylinder with a test sample weightof 100 grams. Smaller weights and volumes may be used, butweight of the test sample and volume of the cylinder usedshould be described with the results. An average of three de-terminations is recommended.

17311731JP XV General Information / Powder Flow

3. Flow through an OriˆceThe ‰ow rate of a material depends upon many factors,

some of which are particle-related and some related to theprocess. Monitoring the rate of ‰ow of material through anoriˆce has been proposed as a better measure of powder‰owability. Of particular signiˆcance is the utility ofmonitoring ‰ow continuously since pulsating ‰ow patternshave been observed even for free ‰owing materials. Changesin ‰ow rate as the container empties can also be observed.Empirical equations relating ‰ow rate to the diameter of theopening, particle size, and particle density have been deter-mined. However, determining the ‰ow rate through an oriˆceis useful only with free-‰owing materials.

The ‰ow rate through an oriˆce is generally measured asthe mass per time ‰owing from any of a number of types ofcontainers (cylinders, funnels, hoppers). Measurement of the‰ow rate can be in discrete increments or continuous.

3.1 Basic Methods for Flow through an OriˆceThere are a variety of methods described in the literature.

The most common for determining the ‰ow rate through anoriˆce can be classiêd based on three important experimen-tal variables:

(1) The type of container used to contain the powder.Common containers are cylinders, funnels and hoppers fromproduction equipment.

(2) The size and shape of the oriˆce used. The oriˆce di-ameter and shape are critical factors in determining powder‰ow rate.

(3) The method of measuring powder ‰ow rate. Flow ratecan be measured continuously using an electronic balanceand with some sort of recording device (strip chart recorder,computer). It can also be measured in discrete samples (forexample, the time it takes for 100 grams of powder to passthrough the oriˆce to the nearest tenth of a second or theamount of powder passing through the oriˆce in 10 secondsto the nearest tenth of a gram).

3.2 Variations in Methods for Flow through an OriˆceEither mass ‰ow rate or volume ‰ow rate can be deter-

mined. Mass ‰ow rate is the easier of the methods, but itbiases the results in favor of high-density materials. Since dieˆll is volumetric, determining volume ‰ow rate may bepreferable.

3.3 General Scale of Flowability for Flow through anOriˆce

No general scale is available because ‰ow rate is criticallydependent on the method used to measure it.

3.4 Experimental Considerations for Flow through anOriˆce

Flow rate through an oriˆce is not an intrinsic property ofthe powder. It is very much dependent upon the methodologyused. The existing literature points out several important con-siderations aŠecting these methods:･The diameter and shape of the oriˆce･The type of container material (metal, glass, plastic)･The diameter and height of the powder bed.

3.5 Recommended Procedure for Flow through anOriˆce

Flow rate through an oriˆce can be used only for materialsthat have some capacity to ‰ow. It is not useful for cohesivematerials. Provided that the height of the powder bed (the

`head' of powder) is much greater than the diameter of theoriˆce, the ‰ow rate is virtually independent of the powderhead. Use a cylinder as the container because the cylindermaterial should have little eŠect on ‰ow. Powder ‰ow rateoften increases when the height of the powder column is lessthan two times the diameter of the column. The oriˆce shouldbe circular and the cylinder should be free of vibration.General guidelines for dimensions of the cylinder are as fol-lows:･Diameter of opening À6 times the diameter of the particles･Diameter of the cylinder À2 times the diameter of the open-ingUse of a hopper as the container may be appropriate and

representative of ‰ow in a production situation. It is not ad-visable to use a funnel, particularly one with a stem, because‰ow rate will be determined by the size and length of the stemas well as the friction between the stem and the powder. Atruncated cone may be appropriate, but ‰ow will be in-‰uenced by the powder—wall friction coe‹cient, thus, selec-tion of an appropriate construction material is important.

For the opening in the cylinder, use a ‰at-faced bottomplate with the option to vary oriˆce diameter to provide maxi-mum ‰exibility and better ensure a powder-over-powder ‰owpattern. Rate measurement can be either discrete or continu-ous. Continuous measurement using an electronic balancecan more eŠectively detect momentary ‰ow rate variations.

4. Shear Cell MethodsA variety of powder shear testers and methods that permit

more thorough and precisely deˆned assessment of powder‰ow properties have been developed. Shear cell methodologyhas been used extensively in the study of pharmaceuticalmaterials. From these methods, a wide variety of parameterscan be obtained, including the yield loci representing theshear stress-shear strain relationship, the angle of internalfriction, the unconˆned yield strength, the tensile strength,and a variety of derived parameters such as the ‰ow factorand other ‰owability indices. Because of the ability to moreprecisely control experimental parameters, ‰ow propertiescan also be determined as a function of consolidation load,time, and other environmental conditions.

4.1 Basic Methods for Shear CellOne type of shear cell is the cylindrical shear cell which is

split horizontally, forming a shear plane between the lowerstationary base and the upper movable portion of the shearcell ring. After powder bed consolidation in the shear cell (us-ing a well-deˆned procedure), the force necessary to shear thepowder bed by moving the upper ring is determined. Annularshear cell designs oŠer some advantages over the cylindricalshear cell design, including the need for less material. A dis-advantage, however, is that because of its design, the powderbed is not sheared as uniformly because material on the out-side of the annulus is sheared more than material in the innerregion. A third type of shear cell (plate-type) consists of athin sandwich of powder between a lower stationary roughsurface and an upper rough surface that is moveable.

All of the shear cell methods have their advantages and dis-advantages. A signiˆcant advantage of shear cell methodolo-gy in general is a greater degree of experimental control. Themethodology generally is rather time-consuming and requiressigniˆcant amounts of material and a well-trained operator.

4.2 Recommendations for Shear Cell

17321732 JP XVPreservatives-EŠectiveness / General Information

Because of the diversity of available equipment and ex-perimental procedures, no speciˆc recommendations regard-ing methodology are presented in this chapter. It is recom-mended that the results of powder ‰ow characterization usingshear cell methodology include a complete description of eq-uipment and methodology used.

References1) Carr R.L.: Evaluating ‰ow properties of solids. Chem.

Eng., 72: 163–168(1965).

19. Preservatives-EŠectivenessTests

The purpose of the Preservatives-EŠectiveness Tests is toassess microbiologically the preservative e‹cacy, either dueto the action of product components themselves or any addedpreservative(s), for multi-dose containers. The e‹cacy of thepreservatives is assessed by direct inoculation and mixing ofthe test strains in the product, and titration of survival of thetest strains with time.

Preservatives must not be used solely to comply with GMPfor drugs or to reduce viable aerobic counts. In addition,preservatives themselves are toxic substances. Therefore,preservatives must not be added to products in amountswhich might jeopardize the safety of human beings, and con-sideration must be given to minimizing the amounts ofpreservative used. These tests are commonly used to verifythat products maintain their preservative eŠectiveness at thedesign phase of formulation or in the case of periodicmonitoring. Although these tests are not performed for lotrelease testing, the e‹cacy of the preservative present in theproduct packaged in the ˆnal containers should be veriêdthroughout the entire dating period.

1. Products and their CategoriesThe products have been divided into two categories for

these tests. Category I products are those made with aqueousbases or vehicles, and Category II products are those madewith nonaqueous bases or vehicles. Oil-in-water emulsionsare considered Category I products, and water-in-oil emul-sions Category II products. Category I is further divided intothree subtypes depending on the dosage forms.

Category IA: Injections and other sterile parenteralsCategory IB: Nonsterile parenteralsCategory IC: Oral products made with aqueous bases (in-

cluding syrup products to be dissolved or suspended beforeuse)

Category II: All the dosage forms listed under Category Imade with nonaqueous bases or vehicles.

2. Test Microorganisms and Culture MediaThe following strains or those considered to be equivalent

are used as the test microorganisms.Escherichia coli ATCC 8739, NBRC 3972Pseudomonas aeruginosa ATCC 9027, NBRC 13275Staphylococcus aureus ATCC 6538, NBRC 13276Candida albicans ATCC 10231, NBRC 1594, JCM 2085Aspergillus niger ATCC 16404, NBRC 9455These test microorganisms are representative of those that

might be found in the environment in which the product ismanufactured, used or stored, and they are also recognized

as opportunistic pathogens. In addition to these strains desig-nated as test microorganisms, it is further recommended touse strains that might contaminate the product and grow onor in it, depending on its characteristics. For the test microor-ganisms received from coordinated collections of microor-ganisms, one passage is deˆned as the transfer of microor-ganisms from an established culture to fresh medium, andmicroorganisms subjected to not more than ˆve passagesshould be used for the tests. Single-strain challenges ratherthan mixed cultures should be used. The test strains can beharvested by growth on solid agar or liquid media.

Cultures on agar plate media: Inoculate each of the ˆve teststrains on the surface of agar plates or agar slants. Forgrowth of bacteria, use Soybean-Casein Digest Agar Medi-um, and for yeasts and moulds, use Sabouraud Agar,Glucose-Peptone Agar or Potato Dextrose Agar Medium. In-cubate bacterial cultures at 309C to 359C for 18 to 24 hours,the culture of C. albicans at 209C to 259C for 40 to 48 hoursand the culture of A. niger at 209C to 259C for one week oruntil good sporulation is obtained. Harvest these culturedcells aseptically using a platinum loop, etc. Suspend the col-lected cells in sterile physiological saline or in 0.1z peptonewater and adjust the viable cell count to about 108 microor-ganisms per mL. In the case of A. niger, suspend the culturedcells in sterile physiological saline or 0.1z peptone watercontaining 0.05 wWvz of polysorbate 80 and adjust the sporecount to about 108 per mL. Use these suspensions as the inoc-ula.

Liquid cultures: After culturing each of the four strains ex-cept for A. niger in a suitable medium, remove the mediumby centrifugation. Wash the cells in sterile physiological sa-line or 0.1z peptone water and resuspend them in the samesolution with the viable cell or spore count of the inoculumadjusted to about 108 per mL.

When strains other than the ˆve listed above are cultured,select a culture medium suitable for growth of the strain con-cerned. The cell suspension may also be prepared by amethod suitable for that strain. Use the inoculum suspen-sions within 24 hours after they have been prepared from thecultivations on agar plate media or in liquid media. Store theinoculum suspensions in a refrigerator if it is not possible toinoculate them into the test specimens within 2 hours. Titratethe viable cell count of the inocula immediately before use,and then calculate the theoretical viable cell count per mL (g)of the product present just after inoculation.

3. Test Procedure3.1 Category I products

Inject each of the cell suspensions aseptically into ˆve con-tainers containing the product and mix uniformly. When it isdi‹cult to inject the cell suspension into the container asepti-cally or the volume of the product in each container is toosmall to be tested, transfer aseptically a su‹cient volume ofthe product into each of alternative sterile containers, andmix the inoculum. When the product is not sterile, incubateadditional containers containing the uninoculated product ascontrols and calculate their viable cell counts (the viablecounts of bacteria and those of yeasts and moulds). A sterilesyringe, spatula or glass rod may be used to mix the cell sus-pension uniformly in the product. The volume of the suspen-sion mixed in the product must not exceed 1W100 of thevolume of the product. Generally, the cell suspension is in-oculated and mixed so that the concentration of viable cells is

17331733JP XV General Information / Preservatives-EŠectiveness

105 to 106 cells per mL or per gram of the product. Incubatethese inoculated containers at 209C to 259C with protectionfrom light, and calculate the viable cell count of 1 mL or 1 gof the product taken at 0, 14 and 28 days subsequent to in-oculation. Record any marked changes (e.g., changes in coloror the development of a bad odor) when observed in the mix-ed samples during this time. Such changes should be consi-dered when assessing the preservative e‹cacy of the productconcerned. Express sequential changes in the viable counts aspercentages, with the count at the start of the test taken as100. Titration of the viable cell counts is based, in principle,on the Pour Plate Methods in ``Microbial Limit Tests''. Inthis case, conˆrm whether any antimicrobial substance ispresent in the test specimen. If a conˆrmed antimicrobialsubstance needs to be eliminated, incorporate an eŠective in-activator of the substance in the buŠer solution or liquidmedium to be used for dilution of the test specimen, as wellas in the agar plate count medium. However, it is necessary toconˆrm that the inactivator has no eŠect on the growth of themicroorganisms. When the occurrence of the preservative orthe product itself aŠects titration of the viable cell count andthere is no suitable inactivator available, calculate the viablecell counts by the Membrane Filtration Method in ``Microbi-al Limit Tests''.3.2 Category II products

The procedures are the same as those described for Catego-ry I products, but special procedures and considerations arerequired for both uniform dispersion of the test microorgan-ism in the product and titration of viable cell counts in thesamples.

For semisolid ointment bases, heat the sample to 459C to509C until it becomes oily, add the cell suspension and dis-perse the inoculum uniformly with a sterile glass rod or spat-ula. Surfactants may also be added to achieve uniform dis-persion, but it is necessary to conˆrm that the surfactantadded has no eŠect on survival or growth of the test microor-ganisms and that it does not potentiate the preservative e‹ca-cy of the product. For titration of the viable cell count, a sur-factant or emulsiêr may be added to disperse the productuniformly in the buŠer solution or liquid medium. Sorbitanmonooleate, polysorbate 80 or lecithin may be added to im-prove miscibility between the liquid medium and semisolidointments or oils in which test microorganisms were inoculat-ed. These agents serve to inactivate or neutralize many of themost commonly used preservatives.

4. InterpretationInterpret the preservative e‹cacy of the product according

to Table 1. When the results described in Table 1 are ob-tained, the product examined is considered to be eŠectivelypreserved. There is a strong possibility of massive microbialcontamination having occurred when microorganisms otherthan the inoculated ones are found in the sterile product to beexamined, and caution is required in the test procedures andWor the control of the manufacturing process of the product.When the contamination level in a nonsterile product to beexamined exceeds the microbial enumeration limit speciêdin ``Microbial Attributes of Nonsterile PharmaceuticalProducts'' in General Information, caution is also requiredin the test procedures andWor the control of the manufactur-ing process of the product.

Table 1. Interpretation criteria by product category

Productcategory Microorganisms

Interpretation criteria

After 14 days After 28 days

Category IA

Bacteria 0.1z of inocu-lum count orless

Same or lessthan level after14 days

YeastsWmoulds Same or lessthan inoculumcount

Same or lessthan inoculumcount

Category IB

Bacteria 1z of inoculumcount or less




Category IC

Bacteria 10z of inocu-lum count orless




Category II

Bacteria Same or lessthan inoculumcount




5. Culture MediaCulture media and buŠer solution used for Preservatives-

EŠectiveness Tests are described below. Other media may beused if they have similar nutritive ingredients and selectiveand growth-promoting properties for the microorganisms tobe tested.

Soybean Casein Digest Agar MediumCasein peptone 15.0 gSoybean peptone 5.0 gSodium chloride 5.0 gAgar 15.0 gWater 1000 mL

Mix all of the components and sterilize at 1219C for 15 –20 minutes in an autoclave. pH after sterilization: 7.1 – 7.3.

Sabouraud Glucose Agar MediumPeptone (animal tissue and casein) 10.0 gGlucose 40.0 gAgar 15.0 gWater 1000 mL


Glucose Peptone (GP) Agar MediumGlucose 20.0 gYeast extract 2.0 gMagnesium sulfate heptahydrate 0.5 g

17341734 JP XVQualiˆcation of Animals / General Information

Peptone 5.0 gMonobasic potassium phosphate 1.0 gAgar 15.0 gWater 1000 mL


Potato Dextrose Agar MediumPotato extract 4.0 gGlucose 20.0 gAgar 15.0 gWater 1000 mL


0.1z Peptone WaterPeptone 1.0 gSodium chloride 8.0 gWater 1000 mL


20. Qualiˆcation of Animals asOrigin of Animal-derived MedicinalProducts provided in the General

Notices of JapanesePharmacopoeia and

Other Standards

IntroductionThe O‹cial Gazette issued on March 29, 2002 announced

that General Notices of the Japanese Pharmacopoeia andother standards were amended to add a provision that``When a drug product or a drug substance which is used tomanufacture a drug product, is manufactured from a rawmaterial of animal origin, the animal in question should be inprinciple a healthy subject, if not otherwise provided.''.

The Notice Iyaku-hatsu No. 0329001, which was issued onthe same date, provided that ``Healthy subject herein pro-vided is the animal which does not cause any disease or anyinfection to human being at an appropriate production proc-ess and use of the drug product, and as for the oral or exter-nal drug for example, the animal, as its raw material ofanimal origin, should be conˆrmed at this stage to meet theFood Standard. It has to be noted that this standard ofhealthy subject has to be revised timely taking into accountthe up-to-date information with respect to the amphixenosisinfections common between human beings and animals.''.

This General Information describes safety assuranceagainst infection of drugs, which are manufactured from rawmaterials of animal origin, to follow up the Notice as men-tioned above.

1. Basic conceptWhen drugs derived from raw materials of animal origin

including human are used, it is important to take into ac-count any possibility that communicable disease agents suchas virus may cause infectious disease or any possible hazardsto patients. In such case, it goes without saying that the pri-

mary subject that has to be considered is the absence of anyinfectious agents such as virus in the raw materials of animalorigin including human as the source of the drug. More im-portant points are whether the drugs derived from such rawmaterials are free of such infectious agents and whether thereis any possibility of transmission of infectious agents whenthe drugs are administered to patient. The eligibility ofanimals including human, as the source of raw materials ofdrugs, in other words ``the subject which is free from any dis-ease or transmission of infectious agents that is infectious tohuman being at an appropriate production process and use ofthe drug product'' is that ``The drug should be entirely freefrom any risk of infections by means of whole procedureswhich include evaluation of appropriateness of the animalsincluding human as the source of their raw materials, estab-lishment of appropriate production processes and their ap-propriate control, and strict adherence to the clinical indica-tions of the ˆnal product.''

2. Animals including human as the source of raw materialsof drugs

What is the most clear and appropriate preventive meas-ures against infection to human being due to administrationof drugs which are derived from animals including human isto assure the absence of any infectious agents such as virus inits raw materials or an appropriate critical raw material byeach of the followings: (1) the use of raw materials of healthyanimal origin, which are proved to be free from communica-ble disease agents to human, or (2) the use of appropriatecritical raw materials (e.g., cell substrate, blood plasma,pooled urine after some treatments) for drug production,which are proved to be free from communicable diseaseagents after certain appropriate processing on raw materialsof animal origin.

As for raw materials of drugs of human origin, cell, tissue,blood, placenta, urine, etc. are used. Whenever it is su‹cientand possible each donor, as the origin of such raw materials,should be asked his (her) health condition and undergoes his(her) medical examination at this stage, so that the ap-propriateness as a donor can be conˆrmed from the stan-dpoint of safety concerning communicable disease agentssuch as virus.

For example, ``Basic concept on handling and use of adrug product, etc. which is derived from cellWtissue''(Attachment 1 of the Notice Iyaku-Hatsu No. 1314 dated De-cember 26, 2000) and ``Guidance for quality and safety assur-ance of a drug product, etc. which is derived from human cellWtissue (Attachment 2 of the Notice Iyaku-Hatsu No. 1314dated December 26, 2000)'' issued by the Director-General ofthe Medicinal Safety Bureau, Ministry of Health and Wel-fare, states that since the cellWtissue supplied by a humandonor comes to be applied to patients without processingthrough any su‹cient inactivation or removal of communica-ble disease agents, the selection and qualiˆcation criteria onsuch donor has to be established. These criteria are to becomposed with the respect to the check items on the casehistory and the physical conditions as well as the test items onthe various transmission of infectious agents through cellWtis-sue, and that the appropriateness of these criteria has to beclariêd. Hepatitis Type-B (HBV), Hepatitis Type-C (HCV),Human Immune Deˆciency Viral infections (HIV), AdultT-Cell Leukemia and Parvovirus B19 Infections should bedenied through the interview to the donor and the tests (sero-

17351735JP XV General Information / Qualiˆcation of Animals

logic test, nucleic-acid ampliˆcation test, etc.). Further, ifnecessary, Cytomegalovirus infection and EB Virus infectionshould be denied by tests. `Ìnfections caused by bacteriasuch as Treponema pallidum, Chlamydia, Gonococci, Tuber-cule bacillus, etc.'', ``septicemia and its suspicious case'',``vicious tumor'', ``serious metabolic or endocrine-relateddisorders'', ``collagenosis and haematological disorder'',``hepatic disease'' and ``dementia (transmissible spongiformencephalopathies and its suspicious case)'' should be checkedon the case history or by the interview, etc. and the ex-perience of being transfused orWand transplanted should bechecked to conˆrm eligibility as a donor. The most appropri-ate check items and test methods then available are to beused, which need to be reconsidered at appropriate timingtaking into account the updated knowledge and the progressof the science and the technologies. At screening of a donor,reexaminations has to be made at appropriate timing usingthe eligible check items and the test methods taking into ac-count the window period (Initial period after infection, inwhich antibody against bacteria, fungi or virus is not detect-ed.)

In the case of plasma derivatives produced from the donat-ed blood in Japan, the donor should be checked by means ofself-assessed report about health conditions, and a serologiccheck and a nucleic acid ampliˆcation test (NAT) on minipooled plasma should be performed at the stage of donatedblood. Further, the plasma material (i.e., critical raw materi-al ) for fractionation should be stored 4 months in minimumso that the arrangement could be taken based on the informa-tion available after collection of the blood and the blood in-fusion to exclude the possibility of using any critical rawmaterial which might cause infection to patients.

On the other hand, as for the materials such as urine whichare taken from the unspeciêd number of the donors andcome to be critical raw materials for drug production aftersome treatments, it is unrealistic and not practical to conductthe tests of virus infection, etc. on the individual donor. Con-sequently, appropriate tests such as virus test has to be per-formed on such critical raw materials for drug production.

In the case of the animals besides human, the wild onesshould be excluded. Only the animals, which are raised underwell sanitarily controlled conditions taken to prevent bacteri-al contamination or under the eŠective bacterial pollutionmonitoring systems, have to be used, and it is recommendedthat the animals from a colony appropriately controlled un-der speciˆc pathogen-free (SPF) environment are to be usedas far as possible. Further, for the animals regulated underthe Food Standard, only the animals that met this standardshould be used. It should be conˆrmed by appropriate teststhat the animals were free from pathogen, if necessary.

The concrete measures to avoid transmittance or spread ofinfectivity of prion, which is considered to be the pathogen oftransmissible spongiform encephalopathies (TSEs), as far aspossible are the followings: avoidance of use of animals,which are raised in the areas where high incidence or high riskof TSEs (Scrapie in sheep and goat, bovine spongiform en-cephalopathies (BSE) in cattle, chronic wasting disease(CWD) in deer, new type of Creutzfeldt-Jacob-Disease (CJD)in human, etc.) is reported, and humans, who have stayedlong time (more than 6 months) in such areas, as raw materi-als or related substances of drugs; avoidance of use of anysubstances that are derived from the individual infected withscrapie, BSE, CJD, etc.; avoidance of using a material

derived from organ, tissue and cell, etc. of high risk of TSEs;and taking appropriate measures basing on the informa-tion collected, which includes incidence of TSEs, the resultsof epidemiological investigation and the experimentalresearch on prion, and incidence of tardive infection ondonors after collecting raw materials, etc.

3. Human or animal cells which are used as critical rawmaterials for drug production

Cell substrates derived from humans or animals are usedfor drug production. In such case, it is desirable that thehumans or the animals, which are the origins of the cell sub-strates, are healthy subjects. However, it is considered practi-cal that viral safety of the drugs derived from the cell sub-strates are evaluated on the cells, which are so called criticalraw materials for production of such drugs. In such case, thesafety should be conˆrmed through the test and analysis onestablished cell bank thoroughly with respect to virus etc., asfar as possible. The items and the methods of the tests thathave been followed in this case are described in detail in theNotice of Japanese version on the internationally acceptedICH Guideline entitled ``Viral safety evaluation ofbiotechnology products derived from cell lines of human oranimal origin'' (Iyakushin No. 329 issued on February 22,2000 by Director, Evaluation and Licensing Division, Phar-maceutical and Medical Safety Bureau, Ministry of Healthand Welfare). In the meantime, it is important how to handlethe cell in case that any virus has been detected under the celllevel tests. This Notice describes how to cope with this situa-tion as follows: `Ìt is recognised that some cell lines used forthe manufacture of product will contain endogenousretroviruses, other viruses or viral sequences. In such circum-stances, the action plan recommended for manufacturer isdescribed in Section V (Rationale and action plan for viralclearance studies and virus tests on puriêd bulk) of the No-tice. The acceptability of cell lines containing viruses otherthan endogenous retroviruses will be considered on an in-dividual basis by the regulatory authorities, by taking into ac-count a riskWbeneˆt analysis based on the beneˆt of theproduct and its intended clinical use, the nature of the con-taminating viruses, their potential for infecting humans orfor causing disease in humans, the puriˆcation process forthe product (e.g., viral clearance evaluation data), and the ex-tent of the virus tests conducted on the puriêd bulk.'' Forexample, it is well known that Type A-, R- and C-endogenousparticles like retrovirus are observed in the cells of the ro-dents used most often for drug production. It is also knownthat they are not infectious to human and is not dangerous,and CHO cells are generally used for drug production. Theestablished cell lines (e.g., NAMALWA Cell, BALL-1 Cell,etc.) derived from cancer patients are sometimes used, butthrough the thorough virus tests, etc., their safety are con-ˆrmed. The established cell lines are assumed to be safer thanthe primary cultured cells which are hard to conduct thethorough virus test.

4. Establishment and control of appropriate productionprocess and adherence to the clinical indication of ˆnalproduct for safety assurance

Safety assurance against potential infections at only thelevel of animals that are source of raw materials of drugs islimited. Further, ``health of animal'' can not be deˆnedunivocally, and the various factors have to be taken into ac-count. The ˆnal goal of this subject is to protect human from

17361736 JP XVQuality Control of Water / General Information

any infectious disease caused by drugs. Achieving this goal,the establishment and control of appropriate productionprocesses of each drug and the adherence to the clinical indi-cations of the ˆnal product are important.

As mentioned above, the rodent cells used most often forthe production of the drugs are known to have endogenousretrovirus sometimes. The reason why such cells can be usedfor the production of the drugs is that multiple measures areapplied for safety in the puriˆcation stages which include ap-propriate inactivation or removal processes. There are casesin which the production procedure involves intentional use ofa virus or a microorganism. In this case, relevant measurescapable of removing or inactivating of such virus or microor-ganism are appropriately incorporated in the puriˆcationprocess, so that the risk of infection to human can be fullydenied and its safety can be assured when it is used as a drug.Further, even in the case that it is di‹cult to clarify the risk ofcontamination of the infectious agents or that the raw materi-al are contaminated by viruses etc., the raw material in ques-tion may be used for the production of drugs so long as ap-propriate inactivation or removal processes are introduced,their eŠectiveness can be conˆrmed and the safety can be as-sured by appropriate control of the manufacturing processesunder GMP, etc.

5. ConclusionThe qualiˆcation of animals including human, as the

source of raw materials of drugs, in other words ``the subjectwhich does not cause any infectious diseases to human beingat an appropriate production process and use of the drugproduct'' is that ``the drug has to be entirely free from anyrisk of infections by means of whole procedures which in-clude evaluation of appropriateness of the animal includinghuman as the source of their raw materials, establishment ofappropriate production processes and their appropriate con-trol, and strict adherence to the clinical indication of the ˆnalproduct.''

To cope with this subject, the advanced scientiˆc measures,which actually re‰ect the updated knowledge and progress ofthe science and the technology about infectious diseases inhuman and infection of animal origin, have to be taken intoaccount timely.

21. Quality Control of Water forPharmaceutical Use

Water used for producing pharmaceutical products, clean-ing pharmaceutical containers and equipment, and the like isreferred to as ``Pharmaceutical Water.'' To consistently as-sure the quality of pharmaceutical water, it is important toverify through appropriate validation that water of requiredquality is supplied, and to maintain that quality by routinecontrol.

Types of Pharmaceutical Water

WaterThe speciˆcations for Water are speciêd in the corre-

sponding monograph of the Japanese Pharmacopoeia. It isrequired for Water to meet the quality standards for drinkingwater provided by the Japanese Water Supply Law and anadditional requirement for ammonium of ``no greater than

0.05 mg/L.'' In preparing Water at various facilities fromsource water such as well water or industrial water, it is need-ed to assure compliance with speciˆcations set forth forWater in the Japanese Pharmacopoeia. Furthermore, whenWater is used after storage for a long period of time, microbi-al proliferation should be prevented.

Water is used as source water for ``Puriêd Water'' and``Water for Injection.'' It also is used in the production of in-termediates of active pharmaceutical ingredients (APIs) andin pre-washing the equipments used for pharmaceutical pur-poses.

Puriêd WaterThe speciˆcations of Puriêd Water are included in `Ò‹-

cial Monographs'' of the Japanese Pharmacopoeia. PuriêdWater is prepared from Water by distillation, ion-exchangetreatment, reverse osmosis (RO), ultraˆltration (UF) capableof removing substances having molecular weights of 6,000and above, or a combination of these methods. Because Puri-êd Water contains no components capable of inhibitingmicrobial growth, appropriate microbiological control isneeded. Particularly in the case of ion-exchange treatment,reverse osmosis or ultraˆltration, the appropriate treatmentfor microbial growth inhibition or periodical sterilizationshould be conducted.

Puriêd Water which has been sterilized or treated withchemical agents for the purpose of microbial growth inhibi-tion or maintaining the endotoxin level within an appropriatecontrol range should be appropriately controlled in order tomaintain the quality in compliance with standards set forthfor the individual purposes of use. Puriêd Water that hasbeen sterilized is referred to as ``Sterilized Puriêd Water.''

Water for InjectionThe speciˆcations of ``Water for Injection'' are described

in `Ò‹cial Monographs'' of the Japanese Pharmacopoeia.Water for Injection is prepared from Water or PuriêdWater by distillation, or from Puriêd Water by reverse os-mosis (RO), ultraˆltration (UF) capable of removing sub-stances having molecular weights of 6,000 and above, or acombination of RO and UF. The quality of processing waterby RO and/or UF should be maintained consistently at thesame level as that prepared by distillation, by the validationthrough long-term operation of the process and elaborateroutine control. Because Water for Injection contains nocomponents that inhibit microbial growth, stringent controlis needed for microorganisms and endotoxins. The standardfor endotoxins requires a level lower than 0.25 EU/mL.

Selection of Pharmaceutical WaterDepending on the purpose of use, pharmaceutical water

can be selected from the above-described group of phar-maceutical waters that allow us to assure the quality of the ˆ-nal product without causing any trouble during the produc-tion process. Table 1 shows some example criteria for suchselection (in the case of charge-in water for the preparation ofdrug products).

For the production of sterile drug products, for which con-tamination by microorganisms or endotoxins is not permit-ted, Water for Injection should be used. For ophthalmics andeye ointments, Water for Injection or Puriêd Water shouldbe used. For the production of non-sterile drug products,water of a quality higher than that of Puriêd Water shouldbe used, however for a non-sterile drug product that requires

1737

Table 1. Criteria for Selecting Pharmaceutical Water (Charge-In Water)

ClassiˆcationClass of

PharmaceuticalWater

Application Remarks

DrugProduct

Water forInjection

Injections, Ophthalmics, Eye Ointments

Puriêd Water

Ophthalmics, Eye Ointment

For ophthalmics and eye ointments forwhich precautions should be taken againstmicrobial contamination, puriêd waterthat is subjected to microbiological con-trol by treatment with sterilization,ultraˆltration, or the like, should be used.

Aerosols, Liquids and Solutions, Extracts,Elixirs, Capsules, Granules, Pills, Suspen-sions and Emulsions, Suppositories,Powders, Spirits, Tablets, Syrups, Infu-sions and Decoctions, Plasters, Tinctures,Troches, Ointments, Cataplasms, Aro-matic Waters, Liniments, Lemonades,Fluidextracts, Lotions, and Pharmaceuti-cal preparations of percutaneous absorp-tion type

For liquids, solutions, ointments, suspen-sions, emulsions, aerosols, and the like forwhich precautions should be taken againstmicrobial contamination, select and usepuriêd water subjected to appropriatemicrobiological control.

ActivePharmaceutical

Ingredient(API)

Water forInjection

Sterile APIs, and APIs rendered sterile inthe formulation process

Puriêd WaterAPIs, APIs rendered sterile in the formu-lation process, and API intermediates

In the production of APIs that arerendered sterile in the formulation processand have no subsequent processes capableof removing endotoxins, Puriêd Waterthat is controlled to maintain endotoxinsat a low level should be used.

Water API Intermediates

1737JP XV General Information / Quality Control of Water

care against microbiological contamination, such as liquids,ointments, suspensions, emulsions, suppositories, and aer-osols, water should have a quality appropriately controlledfrom microbiological viewpoints, considering the possibleimpacts of preservatives formulated in the dosage form.Water for pre-washing containers or equipment surfaces thatcome in direct contact with the drug should be of a qualityhigher than that of Water. Water for ˆnal rinsing shouldhave the same quality as that of charge-in water.

For the production of non-sterile drug products, the quali-ty of water should be higher than that of Puriêd Water;however for the production of liquids, ointments, suspen-sions, emulsions, suppositories, and aerosols for which careshould be taken against microbial contamination, the qualityof water should be controlled appropriately from microbio-logical viewpoints in consideration of the possible eŠects offormulated preservatives. Water for pre-washing containersor equipment surfaces that come in direct contact with theproduct should be of a quality not less than that of Water.The water for ˆnal rinsing should be the same quality as thatof charge-in water.

In selecting pharmaceutical water for Active Pharmaceuti-cal Ingredient (API), the characteristics and formulation

process of the drug products for which the API is used shouldbe considered, so that the quality of the ˆnal drug products ismaintained at an appropriate level. Water used in the produc-tion of sterile API or to clean pharmaceutical containers orequipment surfaces that come in direct contact with theproduct should have the quality controlled chemically andmicrobiologically at the level of Water or higher, even if thewater is to be used at an earlier stage of a synthetic or extrac-tion process. In the ˆnal puriˆcation process, water shouldhave a quality equal to Puriêd Water or higher. Water usedfor the ˆnal rinsing of containers or equipment surfaces thatcome in direct contact with the product should have the samequality as that of the charge-in water. Pharmaceutical waterused in sterile API should be Water for Injection. Similarly,in the case of APIs for drug products, where endotoxin con-trol is required and there are no subsequent processes capableof removing endotoxins, Water for Injetion or PuriêdWater, for which endotoxins are controlled to an appropriatelevel, should be used.Quality Control of Pharmaceutical Water

3.1 OutlineRoutine control of pharmaceutical water is conducted on


the premise that the production of water of a required qualityhas been thoroughly veriêd by validation studies at an earli-er stage of the system that produces the pharmaceutical water(pharmaceutical water system). If these requirements aremet, the following control methods are applicable.

For routine control, the control of electrical conductivityand Total Organic Carbon (TOC) is markedly useful. Itemsto be controlled regularly should be determined according tothe intended use of the pharmaceutical water. In addition tothe above, control should be made for several chemical sub-stances, viable counts, endotoxins, insoluble foreign parti-cles, and the like. The measurement frequency should be de-termined in consideration of the stability of the water qualityin question.

The following shows microbiological control items andcontrol items for electrical conductivity and Total OrganicCarbon (TOC), for which special attention should be paid.Similar considerations should be made of the other controlitems so that the water quality meets the speciˆcations ofpharmaceutical water.

3.2 SamplingMonitoring should be conducted at a su‹cient enough fre-quency to ensure that the pharmaceutical water system is incontrol and that water of acceptable quality is continuouslyproduced. Sampling should be made at representative loca-tions in the production and supply systems of the pharmaceu-tical water, with particular care that the collected samplere‰ects the pharmaceutical water system. Normally, point-of-use sites are appropriate for the sampling locations. Samplingfrequency is established on the basis of data from validationstudies of the pharmaceutical water system. In making a sam-pling plan for pharmaceutical water system, it is important tocover all important points of the system by thoroughly con-sidering the quality characteristics required for the water tobe sampled. Particularly in microbiological control, attentionshould be paid to the diŠerence in conditions among sam-pling locations.

3.3 Alert and Action LevelsIn a pharmaceutical water system, monitoring is done for

microorganisms and other quality attributes that assure waterof acceptable quality is produced during operation within thedesigned speciˆcations. Monitoring data thus obtained iscompared against an alert level, action level, other controllevels, and allowable limits for pharmaceutical waters. Thisimplies the alert level and action level are used for processcontrol, rather than for judging the acceptance.

Deˆnition of Alert LevelAn alert level indicates that, when exceeded, a process may

have drifted from its normal operating condition. Alert levelsconstitute a warning and exceeding them does not necessarilyrequire a corrective action. Alert levels are generally estab-lished either at a mean＋2s on the basis of past trend ana-lyses or at a level of 70z (50z for viable counts) of the ac-tion criteria, whichever is smaller.

Deˆnition of Action LevelAn action level indicates that, when exceeded, a process

has drifted from its normal operating range. Exceeding an ac-tion level indicates that corrective action must be taken to br-ing the process back within its normal operating range.

Alert and action levels should be established within processand product speciˆcation tolerances and based on integrated

considerations for technology and product quality. Conse-quently, exceeding an alert or action level does not necessari-ly indicate that the product quality has been compromised.

3.4 Microbiological MonitoringThe main purpose of a microbiological monitoring pro-

gram for pharmaceutical water system is to predict anymicrobiological quality deterioration of the produced waterand to prevent any adverse eŠects it may have on productquality．Consequently, detecting all of the microorganismspresent may not be necessary; however it is required to adopta monitoring technique capable of detecting a wide range ofmicroorganisms, including slow growing microorganisms.

The following indicates culture-based microbiologicalmonitoring methods for pharmaceutical water systems. Inadopting a rapid detection system for microorganisms, it isneeded to conˆrm in advance that the obtained microbialcounts are equivalent to or above the data obtained throughincubation.

3.4.1 Media and Incubation ConditionsThere are many mesophilic bacteria of heterotrophic type

that are adaptable to poor nutrient water environments. Inmany pharmaceutical water systems, heterotrophic bacteriamay form bio-ˆlms and cause water quality deterioration. It,therefore, is useful to monitor the water quality by use ofR2A Agar Medium, which is excellent for growing bacteriaof oligotrophic type. On the other hand, in routine microbialmonitoring, an approach that identiês the trend inmicrobiological quality change is widely employed; a stan-dard agar plate is used for counting the total number of via-ble microorganisms capable of proliferating at 30 – 359C in acomparatively short period of time.

Table 2 shows examples of measurement methods, mini-mum sample sizes, media, and incubation periods for es-timating viable counts.

3.4.2 Media Growth Promotion TestIn the media growth promotion test with R2A Agar Medi-

um, use the strains listed below or other strains considered tobe equivalent to these strains. Prior to the media growth pro-motion test, inoculate these strains into sterile puriêd waterand incubate at 20 – 259C for 3 days.

Methylobacterium extorquens: NBRC 15911Pseudomonas ‰uorescens: NBRC 15842, ATCC 17386, or

the likeDilute the ‰uid containing the starved strain in puriêd

water with sterile puriêd water to prepare a ‰uid containingabout 50 – 200 cfu per mL. When pipeting 1 ml of the dilutedsolution onto the R2A agar medium to be used for incubatingat 20 – 259C or 30 – 359C for 4 – 7 days, a su‹cient numberof recovered colonies of the microorganism must be observedin comparison with the inoculated cfus.

In the media growth promotion test with standard agarmedium, use the strains listed below or other strains consi-dered to be equivalent to these strains. When pipeting 1 mLof the solution containing the microorganism for incubatingat 30 – 359C for 48 hours, a su‹cient number of recoveredcolonies of the microorganism must be observed in compari-son with the inoculated counts.

Staphylococcus aureus: ATCC 6538, NCIMB 9518, CIP4.83 or NBRC 13276

Pseudomonas aeruginosa: ATCC 9027, NCIMB 8626, CIP82.118 or NBRC 13275

1739

Table 2. Methods for Assessment of Viable Counts in harmaceutical Water

Method Pharmaceutical Water

Water Puriêd Water in Bulk Water for Injection in Bulk

MeasurementMethod

Pour Plate Method orMembrane Filtration

Pour Plate Method or MembraneFiltration

Membrane Filtration

MinimumSample Size

1.0 mL 1.0 mL 100 mL

Media Standard Agar MediumR2A Agar Medium, StandardAgar Medium

R2A Agar Medium, StandardAgar Medium

IncubationPeriod

Standard Agar Medium:48 – 72 hours (or longer)

R2A Agar Medium: 4 – 7 days(or longer)Standard Agar Medium: 48 – 72hours (or longer)

R2A Agar Medium: 4 – 7 days(or longer)Standard Agar Medium: 48 – 72hours (or longer)

IncubationTemperature

Standard Agar Medium:30 – 359C

R2A Agar Medium: 20 – 259C or30 – 359CStandard Agar Medium: 30 – 359C

R2A Agar Medium: 20 – 259C or30 – 359CStandard Agar Medium: 30 – 359C

1739JP XV General Information / Quality Control of Water

Colon bacillus (Escherichia coli): ATCC 8739, NCIMB8545, CIP 53.126 or NBRC 3972

3.4.3 Action Levels for Microorganisms in Pharmaceuti-cal Water System

The following action levels are considered appropriate andgenerally applicable to pharmaceutical water systems. Actionlevels for Puriêd Water and Water for Injection should bethose obtained using R2A Agar Medium.

Action Levels for viable counts in various pharmaceuticalwaters

Water: 100 cfu/mL (according to the criteria in the WaterSupply Law)

Puriêd Water: 100 cfu/mLWater for Injection: 10 cfu/100 mL(cfu: colony-forming units)

When actual counts exceed such action levels in validationstudies or routine control studies, it is necessary to character-ize the isolated microorganisms and to sterilize or disinfectthe aŠected system.

3.5 Physicochemical MonitoringPhysicochemical monitoring of a pharmaceutical water

system is generally performed using electrical conductivityand Total Organic Carbon (TOC) as indicators. Monitoringby electrical conductivity can predict probable quantities ofthe total inorganic salts present. Monitoring by TOC can esti-mate the total quantity of the organic compounds present.Basically, the tests for electrical conductivity and total organ-ic carbon speciêd in ``General Tests'' of the Japanese Phar-macopoeia are applied as these physicochemical monitoringmethods. However, tests for monitoring are diŠerent in pro-ˆle from those described in ``Monographs'' of the JapanesePharmacopoeia. The following describes the particulars forcompleting the part that cannot be worked out with the in-dividual descriptions in ``General Tests'' of the Phar-macopoeia alone. When monitoring is done at a production

facility using electrical conductivity or TOC as an indicator,appropriate alert and action levels, as well as countermeas-ures against contingency should be established for each indi-cator.

3.5.1 Monitoring with an Indicator of Electrical Conduc-tivity

Measurement of electrical conductivity for monitoring isusually conducted continuously with an in-line meter havinga ‰ow-through type or pipe-insertion type cell. Alternatively,batch testing may be done using a dip type cell with samplestaken at point-of-use sites or other appropriate locations ofthe pharmaceutical water system. The following guidelinesfor the operation control of a pharmaceutical water systemaddress how to interpret the results of electrical conductivitytests, and how to approve the continuation of operation, de-pending on whether measurements are made at the standardtemperature (209C) or at temperatures outside the standard.

(1) When monitoring is done at the standard temperature(209C)

Tests for electrical conductivity of the Japanese Phar-macopoeia commonly require the tester to measure at thestandard temperature (209C), however measurement at atemperature within a range of 20±59C may be acceptable oncondition that the correct equation is used. When monitoringelectrical conductivity at the standard temperature of Puri-êd Water and Water for Injection, the recommended allow-able (action level) conductivity is as follows.

･Action Level 1.0 mS/cm (209C)The above allowable conductivity is established on the sup-

position of in-line monitoring, so this standard level may bechanged in batch testing.

(2) When monitoring is done at temperatures other thanthe standard temperature

When the quality of water is monitored by the measure-ment of electrical conductivity at a temperature other than


the standard temperature, the following three-stage approachis applied. The method described below complies with thedescription in ``General Chapter <645> WATER CONDUC-TIVITY'' of the United States Pharmacopoeia (USP28,2005), which speciês the establishment of allowable levels ofelectrical conductivity individually corresponding to diŠerentmeasurement temperatures and pH levels of specimens meas-ured by the three-stage approach (Stage 1 to Stage 3).

Stage 11. When a non-temperature-compensated conductivity

instrument is used, measure the temperature of the waterspecimen, in addition to the conductivity.

2. Using Table 3, ˆnd the conductivity value corre-sponding to the measured temperature or a temperature listedin Table 3 just below the measured temperature that can beused to calculate the allowable electrical conductivity at themeasured temperature.

3. If the measured conductivity is not greater than the al-lowable conductivity value, the water specimen is judged aspassing the electrical conductivity test. If the conductivityvalue exceeds the allowable conductivity value, proceed withStage 2.

Table 3 Stage 1 Allowable Electrical Conductivity Meas-ured at DiŠerent Temperatures*

Temperature(9C)

AllowableConductivity

(mS/cm)

Temperature(9C)

AllowableConductivity

(mS/cm)

0 0.65 0.8 55 2.1

10 0.9 60 2.215 1.0 65 2.420 1.1 70 2.525 1.3 75 2.730 1.4 80 2.735 1.5 85 2.740 1.7 90 2.745 1.8 95 2.950 1.9 100 3.1

*Applied to the conductivity value measured with a non-tem-perature-compensated conductivity instrument.

Stage 21. Transfer a su‹cient amount of water specimen to a

suitable container, and stir the specimen. Adjust the tempera-ture (25±19C), and while vigorously agitating the specimen,measure the conductivity periodically. If the change in con-ductivity is not greater than 0.1 mS/cm, due to the uptake ofatmospheric carbon dioxide, interpret the observed value asthe conductivity of the specimen (in equilibrium with the at-mosphere).

2. If the observed conductivity value at 259C (with thespecimen in equilibrium with the atmosphere) is not greaterthan 2.1 mS/cm, the specimen is judged as passing the con-ductivity test. If the observed value is greater than the level,proceed with Stage 3.

Stage 31. While maintaining the sample at 25±19C, perform the

conductivity measurement as explained in Step 1 of Stage 2.Add 0.3 mL of saturated potassium chloride solution to 100

mL of the water specimen, and determine the pH to thenearest 0.1 pH.

2. Use Table 4 to ˆnd the allowable conductivity valuecorresponding to the observed pH. If the observed conductiv-ity is lower than the allowable level, the water specimen isjudged as passing the electrical conductivity test. If the ob-served valued is greater than the allowable level, or if the pHof the specimen is outside of a range of pH 5.0 – 7.0, thewater specimen is judged as unacceptable.

Table 4 Third Step Allowable Electrical Conductivity atDiŠerent pHs*

pHAllowable

Conductivity(mS/cm)

pHAllowable

Conductivity(mS/cm)

5.0 4.75.1 4.1 6.1 2.45.2 3.6 6.2 2.55.3 3.3 6.3 2.45.4 3.0 6.4 2.35.5 2.8 6.5 2.25.6 2.6 6.6 2.15.7 2.5 6.7 2.65.8 2.4 6.8 3.15.9 2.4 6.9 3.86.0 2.4 7.0 4.6

*Applied only to a sample in equilibrium with the air at259C.

3.5.2 Monitoring with an Indicator of Total OrganicCarbon (TOC)

Drinking Water Standards (Prescribed under the Article 4of the Japanese Water Supply Law) require that TOC shouldbe ``not greater than 5 ppm''. However it is preferable for in-dividual facilities to conduct TOC monitoring on Water withalert and action levels separately determined for water-quali-ty control by TOC monitoring.

The Japanese Pharmacopoeia speciês the Test for TotalOrganic Carbon, and usually, TOC measurement is conduct-ed using an apparatus in compliance with the speciˆcations,however if the apparatus conforms to the apparatus suitabil-ity test requirements described in ``General Chapter <643>TOTAL ORGANIC CARBON'' of the United States Phar-macopeia (USP28, 2005), otherwise described in ``Methodsof Analysis 2.2.44. TOTAL ORGANIC CARBON INWATER FOR PHARMACEUTICAL USE'' of the Europe-an Pharmacopoeia (EP 5.0, 2005), the apparatus may be usedfor monitoring a pharmaceutical water supplying system,provided that sufficiently pure water not contaminated withionic organic substances or organic substances containingnitrogen, sulfur or chlorine atoms is used as source water forthe system.

A TOC apparatus, characterized by calculating the amountof organic carbon from the diŠerence in conductivity beforeand after the decomposition of organic substances withoutseparating carbon dioxide from the sample solution, may bein‰uenced negatively or positively, when applied to a samplesolution containing ionic organic substances or organic sub-stances comprised of nitrogen, sulfur, or halogens such aschlorine and the like; therefore, the apparatus should beselected appropriately depending on the purity of phar-

17411741JP XV General Information / Rapid Identiˆcation

maceutical water to be measured and on the contaminationrisk in the case of apparatus failure.

3.6 Storage of Water for InjectionIn storing Water for Injection, the water should be heated

during circulation or other action to inhibit microbialproliferation. The maximum storage time allowed shouldalso be appropriately established on the basis of validationstudies, with considerations given to risks of contaminationand quality deterioration.

22. Rapid Identiˆcation ofMicroorganisms Based on

Molecular Biological MethodThis chapter describes the methods for the identiˆcation or

estimation of microorganisms (bacteria and fungi), found inin-process control tests or lot release tests of pharmaceuticalproducts, at the species or genus level based on their DNA se-quence homology. The identiˆcation of isolates found in thesterility test or aseptic processing can be helpful for inves-tigating the causes of contamination. Furthermore, informa-tion on microorganisms found in raw materials used forpharmaceutical products, processing areas of pharmaceuticalproducts, and so on is useful in designing measures to controlthe microbiological quality of drugs. For the identiˆcation ofmicroorganisms, phenotypic analysis is widely used, based onmorphological, physiological, and biochemical features andanalysis of components. Commercial kits based on diŠer-ences in phenotype patterns have been used for the identiˆca-tion of microorganisms, but are not always applicable tomicroorganisms found in raw materials used for pharmaceu-tical products and in processing areas of pharmaceuticalproducts. In general, the identiˆcation of microorganismsbased on phenotypic analysis needs special knowledge andjudgment is often subjective. It is considered that the evolu-tionary history of microorganisms (bacteria and fungi) ismemorized in their ribosomal RNAs (rRNAs), so that sys-tematic classiˆcation and identiˆcation of microorganisms inrecent years have been based on the analysis of these se-quences. This chapter presents a rapid method to identify orestimate microorganisms based on partial sequences of diver-gent regions of the 16S rRNA gene for bacteria and of the in-ternal transcribed spacer 1 (ITS1) region located between 18SrRNA and 5.8S rRNA for fungi, followed by comparison ofthe sequences with those in the database. Methods describedin this chapter do not take the place of usual other methodsfor the identiˆcation, and can be modiêd based on the ex-aminer's experience, and on the available equipment ormaterials. Other gene regions besides those mentioned in thischapter can be used if appropriate.Apparatuses(1) DNA sequencer

Various types of sequencers used a gel board or capillarycan be used.(2) DNA ampliêr

To amplify target DNA and label ampliêd (PCR)products with sequencing reagents.

ProceduresThe following procedures are described as an example.

1. Preparation of template DNAIt is important to use a pure cultivated bacterium or fungus

for identiˆcation. In the case of colony samples, colonies arepicked up with a sterilized toothpick (in the case of fungi, asmall fragment of colony sample is picked up), and suspend-ed in 0.3 mL of DNA releasing solution in a 1.5 mL cen-trifuge tube. In the case of culture ‰uid, a 0.5 mL portion of‰uid is put in a 1.5 mL centrifuge tube and centrifuged at10,000 rpm for 10 min. After removal of the supernatant, thepellet is suspended in 0.3 mL of DNA releasing solution, andthen heated at 1009C for 10 min. In general, PCR can be runfor bacteria and yeasts heated in DNA releasing solution. Forfungi, DNA extraction after treatment with a mixer or ultra-sonic generator may be necessary before PCR.2. PCR

Add 2 mL of template DNA in PCR reaction solution. Use10F/800R primers for bacteria and ITS1F/ITS1R primers forfungi, and then perform 30 ampliˆcation cycles at 949C for30 sec, 559C for 60 sec, and 729C for 60 sec. DNA fragmentsare ampliêd about 800 bp in the case of bacteria and about150 – 470 bp depending on the strain in the case of fungi. In-clude a negative control (water instead of the test solution) inthe PCR.3. Conˆrmation of PCR products

Mix 5 mL of PCR product with 1 mL of loading buŠersolution, place it in a 1.5 w/vz agarose gel well, and carryout electrophoresis with TAE buŠer solution (1-fold concen-tration). Carry out the electrophoresis together withappropriate DNA size markers. After the electrophoresis, ob-serve PCR products on a trans-illuminator (312 nm) and con-ˆrm the presence of a single band of the targeted size. If mul-tiple bands are observed, cut the targeted band out of the gel,and extract DNA by using appropriate commercial DNA ex-traction kit.4. Puriˆcation of PCR products

Remove unincorporated PCR primers and deoxynucleo-side triphosphates (dNTP) from PCR products by usingappropriate puriˆcation methods.5. Quantiˆcation of puriêd DNA

When puriêd DNA is measured by spectrophotometer,calculate 1 OD260 nm as 50 mg/mL.6. Labeling of PCR products with sequencing reagents

Use an appropriate ‰uorescence-labeled sequencingreagent suitable for the available DNA sequencer or itsprogram and label the PCR products according to theinstructions provided with the reagent.7. Puriˆcation of sequencing reagent-labeled PCR products

Transfer the product in 75 mL of diluted ethanol (7 in 10)into a 1.5 mL centrifuge tube, keep in an ice bath for 20 min,and centrifuge at 15,000 rpm for 20 min. After removal ofsupernatant, add 250 mL of diluted ethanol (7 in 10) to theprecipitate and centrifuge at 15,000 rpm for 5 min. Removethe supernatant and dry the precipitate.8. DNA homology analysis

Place sequencing reagent-labeled PCR products in theDNA sequencer and read the nucleotide sequences of thePCR products. Compare the partial nucleotide sequence withthose in the BLAST database.

JudgmentIf sequencing data show over 90z identity with a sequence

in the database, in general, judgment may be made as fol-lows.

17421742 JP XVSDS-Polyacrylamide Gel / General Information

1. In the case of bacteria, compare about 300 nucleotidesbetween positions 50 to 350 in the product obtained withthe 10F primer, with the BLAST database. Higherranked species are judged as identiêd species or closelyrelated species.

2. In the case of fungi, compare sequencing data for theproduct obtained with the ITS1F primer, with theBLAST database. Higher ranked species are judged asidentiêd species or closely related species.

Reagents, Test Solutions(1) 0.5 mol/L Disodium dihydrogen ethylenediaminetetraacetate TS

Dissolve 18.6 g of disodium dihydrogen ethylenediaminetetraacetate dihydrate in water to make 100 mL.(2) 1 mol/L Tris buŠer solution, pH 8.0

Dissolve 24.2 g of 2-amino-2-hydroxymethyl-1,3-propanediol in a suitable amount of water, adjust the pH to8.0 with 0.2 mol/L hydrochloric acid TS, and add water tomake 200 mL.(3) TE buŠer solution

Mix 1.0 mL of 1 mol/L tris buŠer solution, pH 8.0 and 0.2mL of 0.5 mol/L disodium dihydrogen ethylenediaminetetraacetate TS, and add water to make 100 mL.(4) DNA releasing solution

Divide TE buŠer solution containing Triton X-100 (1 in100) into small amounts and store frozen until use.(5) PCR reaction solution

10-fold buŠer solution* 5 mLdNTP mixture** 4 mL10 mmol/L Sense primer 1 mL10 mmol/L Anti-sense primer 1 mLHeat-resistant DNA polymerase (1 U/mL) 1 mLWater 36 mL

* Being composed of 100 mmol/L 2-amino-2-hydrox-ymethyl-1,3-propanediol hydrochloride, pH 8.4, 500mmol/L potassium chloride, 20 mmol/L magnesium chlo-ride and 0.1 g/L gelatin.** A solution containing 2.5 mmol/L each of dGTP (sodi-um 2?-deoxyguanosine 5?-triphosphate), dATP (sodium 2?-deoxyadenosine 5?-triphosphate), dCTP (sodium 2?-deox-ycytidine 5?-triphosphate) and dTTP (sodium 2?-deox-ythymidine 5?-triphosphate). Adequate products containingthese components as described above may be used.(6) Sequencing reagent

There are many kinds of sequencing methods, such as thedye-primer method for labeling of primer, the dye-terminatormethod for labeling of dNTP terminator and so on. Use anappropriate sequencing reagent kit for the apparatus andprogram to be used.(7) 50-Fold concentrated TAE buŠer solution

Dissolve 242 g of 2-amino-2-hydroxymethyl-1,3-propanediol in 57.1 mL of acetic acid (100) and 100 mL of0.5 mol/L disodium dihydrogen ethylenediamine tetraacetateTS, and add water to make 1000 mL.(8) 1-Fold concentrated TAE buŠer solution

Diluted 50-fold concentrated TAE buŠer solution (1 in 50)prepared before use is referred to as 1-fold concentrated TAEbuŠer solution.(9) Agarose gel

Mix 1.5 g of agarose, 2.0 mL of 50-fold concentrated TAEbuŠer solution, 10 mL of a solution of ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide) (1 in

100) and 100 mL of water. After dissolving the materials byheating, cool the solution to about 609C, and prepare gels.(10) Loading buŠer solution (6-fold concentrated)

Dissolve 0.25 g of bromophenol blue, 0.25 g of xylenecyanol FF and 1.63 g of disodium dihydrogen ethylenedia-mine tetraacetate dihydrate in 50 mL of water, and add30 mL of glycerol and water to make 100 mL.(11) PCR primers

For Primer

Bacteria 10F800R

5?-GTTTGATCCTGGCTCA-3?5?-TACCAGGGTATCTAATCC-3?

Fungi ITS1FITS1R

5?-GTAACAAGGT(T/C)TCCGT-3?5?-CGTTCTTCATCGATG-3?

23. SDS-Polyacrylamide GelElectrophoresis

This test is harmonized with the European Pharmacopoeiaand the U.S. Pharmacopeia.

The SDS-Polyacrylamide Gel Electrophoresis is used forthe characterization of proteins in biotechnological and bio-logical products and for control of purity and quantitativedeterminations.

This technique is a suitable analytical method with whichto identify and to assess the homogeneity of proteins inbiotechnological and biological products. The method is alsoroutinely used for the estimation of protein subunit molecu-lar masses and for determining the subunit compositions ofpuriêd proteins.

Ready-to-use gels and reagents are widely available on themarket and can be used instead of those described in this text,provided that they give equivalent results and that they meetthe validity requirements given below under Validation of theTest.

1. Characteristics of Polyacrylamide GelsThe sieving properties of polyacrylamide gels are aŠorded

by the three-dimensional network of ˆbers and pores which isformed as the bifunctional bisacrylamide cross-links adjacentpolyacrylamide chains. Polymerization is catalyzed by a freeradical-generating system composed of ammoniumpersulfate and N,N,N?,N?-tetramethylethylenediamine(TEMED).

As the acrylamide concentration of a gel increases, itseŠective pore size decreases. The eŠective pore size of a gel isoperationally deˆned by its sieving properties; that is, by theresistance it imparts to the migration of macromolecules.There are limits on the acrylamide concentration that can beused. At high acrylamide concentrations, gels break muchmore easily and are di‹cult to handle. As the pore size of agel decreases, the migration rate of a protein through the geldecreases. By adjusting the pore size of a gel, throughmanipulating the acrylamide concentration, the resolution ofthe method can be optimized for a given protein product.Thus, the physical characteristics of a given gel are deter-mined by the relative concentrations of acrylamide and bi-sacrylamide, used in its preparation.

In addition to the composition of the gel, the state of the

17431743JP XV General Information / SDS-Polyacrylamide Gel

protein is an important determinant of the electrophoreticmobility. In the case of proteins, the electrophoretic mobilityis dependent on the pK values of the charged groups and thesize of the molecule. It is also in‰uenced by the type, concen-tration and pH of the buŠer, the temperature and the êldstrength, as well as by the nature of the support material.

2. Polyacrylamide Gel Electrophoresis under DenaturingConditions

The method cited as an example is limited to the analysis ofmonomeric polypeptides with a mass range of 14,000 to100,000 daltons. It is possible to extend this mass range byvarious techniques (e.g., by using gradient gels, particularbuŠer systems, etc.), but those techniques are not discussedin this chapter.

Analysis by electrophoresis on sodium dodecyl sulfate(SDS) polyacrylamide gel (SDS-Polyacrylamide Gel Elec-trophoresis) under denaturing conditions is the most com-mon mode of electrophoresis used in assessing the quality ofproteins in biotechnological and biological products, and willbe the focus of the example described here. Typically, analyt-ical electrophoresis of proteins is carried out in poly-acrylamide gels under conditions that ensure dissociation ofthe proteins into their individual polypeptide subunits andthat minimize aggregation. Most commonly, the strongly an-ionic detergent SDS is used in combination with heat to dis-sociate the proteins before they are loaded on the gel. Thedenatured polypeptides bind to SDS, become negativelycharged and exhibit a consistent charge-to-mass ratio regard-less of protein type. Because the amount of SDS bound isalmost always proportional to the molecular mass of thepolypeptide and is independent of its amino acid sequence,SDS-polypeptide complexes migrate through polyacrylamidegels with mobilities that are dependent on the size of the poly-peptides.

The electrophoretic mobilities of the resultant SDS-polypeptide complexes all assume the same functionalrelationship to their molecular masses. Migration of SDS-complexes occurs toward the anode in a predictable manner,with low-molecular-mass complexes migrating faster thanlarger ones. The molecular mass of a protein can therefore beestimated from its relative mobility calibrated in SDS-Polyacrylamide Gel Electrophoresis and the occurrence of asingle band in such a gel is a criterion of purity.

However, modiˆcations to the polypeptide backbone, suchas N- or O-1inked glycosylation, have a signiˆcant impact onthe apparent molecular mass of a protein, since SDS does notbind to a carbohydrate moiety in a manner similar to a poly-peptide. Thus, a consistent charge-to-mass ratio is not main-tained. The apparent molecular masses of proteins that haveundergone post-translational modiˆcations do not trulyre‰ect the masses of the polypeptides.1) Reducing conditions

Polypeptide subunits and three-dimensional structure ofproteins are often ˆxed, at least in part, by the presence ofdisulˆde bonds. A goal of SDS-Polyacrylamide Gel Elec-trophoresis under reducing conditions is to disrupt this struc-ture by reducing the disulˆde bonds. Complete denaturationand dissociation of proteins by treatment with 2-mercap-toethanol or dithiothreitol (DTT) will result in unfolding ofthe polypeptide backbone and subsequent complexation withSDS. Under these conditions, the molecular masses of thepolypeptide subunits can be calculated by interpolation in the

presence of suitable molecular-mass standards.2) Non-reducing conditions

For some analyses, complete dissociation of the protein ofinterest into subunit peptides is not desirable. In the absenceof treatment with reducing agents such as 2-mercaptoethanolor DTT, disulˆde covalent bonds remain intact, preservingthe oligomeric form of the protein. Oligomeric SDS-proteincomplexes migrate more slowly than their SDS-polypeptidesubunits. In addition, non-reduced proteins may not be com-pletely saturated with SDS and, hence, may not bind the de-tergent in the expected mass ratio. This makes molecular-mass determinations of these molecules by SDS-Polyacrylamide Gel Electrophoresis less straightforward thananalyses of fully denatured polypeptides, since it is necessarythat both standards and unknown proteins be in similar con-ˆgurations for valid comparisons. However, the staining of asingle band in such a gel is a criterion of purity.

3. Characteristics of Discontinuous BuŠer System Gel Elec-trophoresis

The most widely used electrophoretic method for the anal-ysis of complex mixtures of proteins involves the use of a dis-continuous buŠer system consisting of two contiguous, butdistinct gels: a resolving or separating (lower) gel and a stack-ing (upper) gel. The two gels are cast with diŠerent porosities,pH, and ionic strengths. In addition, diŠerent mobile ions areused in the gel and electrode buŠers. The buŠer discontinuityacts to concentrate large-volume samples in the stacking gel,resulting in improved resolution. When power is applied, avoltage drop develops across the sample solution whichdrives the proteins into the stacking gel. Glycinate ions fromthe electrode buŠer follow the proteins into the stacking gel.A moving boundary region is rapidly formed with the highlymobile chloride ions in the front and the relatively slowglycinate ions in the rear. A localized high-voltage gradientforms between the leading and trailing ion fronts, causing theSDS-protein complex to form into a very thin zone (called thestack) and to migrate between the chloride and glycinatephases. Regardless of the height of the applied sample solu-tion in the wells, all SDS-protein complexes condense withinbroad limits and enter the resolving gel as a well-deˆned, thinzone of high protein density. The large-pore stacking gel doesnot retard the migration of most proteins and serves mainlyas an anticonvective medium. At the interface of the stackingand resolving gels, the proteins experience a sharp increase inretardation due to the smaller pore size of the resolving gel.Once the proteins are in the resolving gel, their mobility con-tinues to be slowed down by the molecular sieving eŠect ofthe matrix. The glycinate ions overtake the proteins, whichthen move in a space of uniform pH formed by thetris(hydroxymethyl)aminomethane and glycine. Molecularsieving causes the SDS-polypeptide complexes to separate onthe basis of their molecular masses.

4. Preparing Vertical Discontinuous BuŠer SDS-Polyacrylamide Gels1) Assembling of the gel moulding cassette

Clean the two glass plates (size: e.g. 10 cm × 8 cm), thesample comb made of polytetra‰uoroethylene, the twospacers and the silicone rubber tubing (diameter, e.g. 0.6 mm× 35 cm) with mild detergent and rinse extensively withwater. Dry all the items with a paper towel or tissue. Lubri-cate the spacers and the silicone rubber tubing with non-sili-cone grease. Apply the spacers along each of the two short


sides of the glass plate 2 mm away from the edges and 2 mmaway from the long side corresponding to the bottom of thegel. Begin to lay the silicone rubber tubing on the glass plateby using one spacer as a guide. Carefully twist the siliconerubber tubing at the bottom of the spacer and follow the longside of the glass plate. While holding the silicone rubber tub-ing with one ˆnger along the long side again twist the tubingand lay it on the second short side of the glass plate, using thespacer as a guide. Place the second glass plate in perfectalignment and hold the mould together by hand pressure.Apply two clamps on each of the two short sides of themould. Carefully apply four clamps on the longer side of thegel mould, thus forming the bottom of the gel mould. Verifythat the silicone rubber tubing is running along the edge ofthe glass plates and has not been extruded while placing theclamps.2) Preparation of the gel

In a discontinuous buŠer SDS polyacrylamide gel, it isrecommended to pour the resolving gel, let the gel set, andthen pour the stacking gel, since the compositions of the twogels in acrylamide-bisacrylamide, buŠer and pH are diŠerent.Preparation of the resolving gel: In a conical ‰ask, preparethe appropriate volume of solution containing the desiredconcentration of acrylamide for the resolving gel, using thevalues given in Table 1. Mix the components in the ordershown. Where appropriate, before adding the ammoniumpersulfate solution and the tetramethylethylenediamine(TEMED), ˆlter the solution if necessary under vacuumthrough a cellulose acetate membrane (pore size: 0.45 mm);keep the solution under vacuum by swirling the ˆltration unituntil no more bubbles are formed in the solution. Add ap-propriate amounts of ammonium persulfate solution andTEMED as indicated in Table 1, swirl and pour immediatelyinto the gap between the two glass plates of the mould. Leavesu‹cient space for the stacking gel (the length of the teeth ofthe sample comb plus 1 cm). Using a pipette, carefully over-lay the solution with water-saturated isobutanol. Leave thegel in a vertical position at room temperature to allow poly-merization to occur.Preparation of the stacking gel: After polymerization iscomplete (about 30 minutes), pour oŠ the isobutanol andwash the top of the gel several times with water to remove theisobutanol overlay and any unpolymerized acrylamide. Drainas much ‰uid as possible from the top of the gel, and then re-move any remaining water with the edge of a paper towel.

In a conical ‰ask, prepare the appropriate volume of solu-tion containing the desired concentration of acrylamide,using the values given in Table 2. Mix the components in theorder shown. Where appropriate, before adding the ammoni-um persulfate solution and the TEMED, ˆlter the solution ifnecessary under vacuum through a cellulose acetate mem-brane (pore size: 0.45 mm); keep the solution under vacuumby swirling the ˆltration unit until no more bubbles areformed in the solution. Add appropriate amounts of ammo-nium persulfate solution and TEMED as indicated inTable 2, swirl and pour immediately into the gap between thetwo glass plates of the mould directly onto the surface of thepolymerized resolving gel. Immediately insert a clean samplecomb into the stacking gel solution, taking care to avoid trap-ping air bubbles. Add more stacking gel solution to ˆll com-pletely the spaces of the sample comb. Leave the gel in a ver-tical position and allow to polymerize at room temperature.3) Mounting the gel in the electrophoresis apparatus and

electrophoretic separationAfter polymerization is complete (about 30 minutes),

remove the sample comb carefully. Rinse the wells immedi-ately with water or with the running buŠer for SDS-Polyacrylamide Gel Electrophoresis to remove any un-polymerized acrylamide. If necessary, straighten the teeth ofthe sample comb of the stacking gel with a blunt hypodermicneedle attached to a syringe. Remove the clamps on one shortside, carefully pull out the silicone rubber tubing and replacethe clamps. Proceed similarly on the other short side. Re-move the silicone rubber tubing from the bottom part of thegel. Mount the gel in the electrophoresis apparatus. Add theelectrophoresis buŠers to the top and bottom reservoirs. Re-move any bubbles that become trapped at the bottom of thegel between the glass plates. This is best done with a benthypodermic needle attached to a syringe. Never pre-run thegel before loading solutions, such as samples, since this willdestroy the discontinuity of the buŠer systems. Before load-ing solutions, such as samples, carefully rinse the stacking gelwells with the running buŠer for SDS-Polyacrylamide GelElectrophoresis. Prepare the test and reference solutions inthe recommended sample buŠer and treat as speciêd in theindividual monograph. Apply the appropriate volume ofeach solution to the stacking gel wells. Start the electrophore-sis using suitable operating conditions for the electrophoresisequipment to be used. There are commercially available gelsof diŠerent surface area and thickness that are appropriatefor various types of electrophoresis equipment. Electropho-resis running time and currentWvoltage may need to be altereddepending on the type of apparatus used, in order to achieveoptimum separation. Check that the dye front is moving intothe resolving gel. When the dye is reaching the bottom of thegel, stop the electrophoresis. Remove the gel assembly fromthe apparatus and separate the glass plates. Remove thespacers, cut oŠ and discard the stacking gel and immediatelyproceed with staining.

5. Detection of Proteins in GelsCoomassie staining is the most common protein staining

method, with a detection level of the order of 1 mg to 10 mg ofprotein per band. Silver staining is the most sensitive methodfor staining proteins in gels and a band containing l0 ng to100 ng can be detected. All of the steps in gel staining aredone at room temperature with gentle shaking in any con-venient container. Gloves must be worn when staining gels,since ˆngerprints will stain.1) Coomassie staining

Immerse the gel in a large excess of Coomassie staining TSand allow to stand for at least 1 hour. Remove the stainingsolution.

Destain the gel with a large excess of destaining TS.Change the destaining solution several times, until the stainedprotein bands are clearly distinguishable on a clear back-ground. The more thoroughly the gel is destained, the smalleris the amount of protein that can be detected by the method.Destaining can be speeded up by including 2 to 3 g of anion-exchange resin or a small sponge in the destaining TS.

NOTE: the acid-alcohol solutions used in this proceduredo not completely ˆx proteins in the gel. This can lead to loss-es of some low-molecular-mass proteins during the stainingand destaining of the gel. Permanent ˆxation is obtainable byallowing the gel to stand in trichloroacetic acid TS for ˆxingfor 1 hour before it is immersed in Coomassie staining TS.

1745

Table 1. Preparation of resolving gel

Solution componentsComponent volumes (mL) per gel mould volume of

5 mL 10 mL 15 mL 20 mL 25 mL 30 mL 40 mL 50 mL

6z AcrylamideWater 2.6 5.3 7.9 10.6 13.2 15.9 21.2 26.5Acrylamide solution(1) 1.0 2.0 3.0 4.0 5.0 6.0 8.0 10.01.5 molWL Tris solution (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5100 gWL SDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5100 gWL APS(4) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5TEMED(5) 0.004 0.008 0.012 0.016 0.02 0.024 0.032 0.04






(1) Acrylamide solution: 30z acrylamideWbisacrylamide (29:1) solution(2) 1.5 molWL Tris solution (pH 8.8): 1.5 molWL tris-hydrochloride buŠer solution, pH 8.8(3) 100 gWL SDS: 100 gWL solution of sodium dodecyl sulfate(4) 100 gWL APS: 100 gWL solution of ammonium persulfate. Ammonium persulfate provides the free radicals that drive

polymerization of acrylamide and bisacrylamide. Since ammonium persulfate solution decomposes slowly, fresh solu-tions must be prepared before use.

(5) TEMED: N,N,N?,N?-tetramethylethylenediamine

1745JP XV General Information / SDS-Polyacrylamide Gel

1746

Table 2. Preparation of stacking gel

Solution componentsComponent volumes (mL) per gel mould volume of

1 mL 2 mL 3 mL 4 mL 5 mL 6 mL 8 mL 10 mL

Water 0.68 1.4 2.1 2.7 3.4 4.1 5.5 6.8Acrylamide solution(1) 0.17 0.33 0.5 0.67 0.83 1.0 1.3 1.71.0 molWL Tris solution (pH 6.8)(2) 0.13 0.25 0.38 0.5 0.63 0.75 1.0 1.25100 gWL SDS(3) 0.01 0.02 0.03 0.04 0.05 0.06 0.08 0.1100 gWL APS(4) 0.01 0.02 0.03 0.04 0.05 0.06 0.08 0.1TEMED(5) 0.001 0.002 0.003 0.004 0.005 0.006 0.008 0.01

(1) Acrylamide solution: 30z acrylamideWbisacrylamide (29:1) solution(2) 1.0 molWL Tris solution (pH 6.8): 1 molWL tris-hydrochloride buŠer solution, pH 6.8(3) 100 gWL SDS: 100 gWL solution of sodium dodecyl sulfate(4) 100 gWL APS: 100 gWL solution of ammonium persulfate. Ammonium persulfate provides the free radicals that drive

polymerization of acrylamide and bisacrylamide. Since ammonium persulfate solution decomposes slowly, fresh solu-tions must be prepared before use.

(5) TEMED: N,N,N?,N?-tetramethylethylenediamine


2) Silver stainingImmerse the gel in a large excess of ˆxing TS and allow to

stand for 1 hour. Remove the ˆxing solution, add fresh ˆxingsolution and incubate either for at least 1 hour or overnight,if convenient. Discard the ˆxing solution and wash the gel inwater for 1 hour. Soak the gel for 15 minutes in a 1 volzglutaraldehyde solution. Wash the gel twice for 15 minutes inwater. Soak the gel in fresh silver nitrate TS for silver stainingfor 15 minutes, in darkness. Wash the gel three times for 5minutes in water. Immerse the gel for about 1 minute in de-veloper TS until satisfactory staining has been obtained. Stopthe development by incubation in blocking TS for 15minutes. Rinse the gel with water.

6. Drying of Stained SDS-Polyacrylamide GelsDepending on the staining method used, gels are pretreated

in a slightly diŠerent way. For Coomassie staining, after thedestaining step, allow the gel to stand in a diluted solution ofconcentrated glycerin (1 in 10) for at least 2 hours (overnightincubation is possible). For silver staining, add to the ˆnalrinsing a step of 5 minutes in a diluted solution of concentrat-ed glycerin (1 in 50).

Immerse two sheets of porous cellulose ˆlm in water andincubate for 5 to 10 minutes. Place one of the sheets on adrying frame. Carefully lift the gel and place it on the cellu-lose ˆlm. Remove any trapped air bubbles and pour 2 to 3mL of water around the edges of the gel. Place the secondsheet on top and remove any trapped air bubbles. Completethe assembly of the drying frame. Place in an oven or leave atroom temperature until dry.

7. Molecular-Mass DeterminationMolecular masses of proteins are determined by compari-

son of their mobilities with those of several marker proteinsof known molecular mass. Mixtures of proteins with pre-cisely known molecular masses blended for uniform stainingare commercially available for calibrating gels. They are ob-tainable in various molecular mass ranges. Concentratedstock solutions of proteins of known molecular mass arediluted in the appropriate sample buŠer and loaded on thesame gel as the protein sample to be studied.

Immediately after the gel has been run, the position of thebromophenol blue tracking dye is marked to identify the

leading edge of the electrophoretic ion front. This can bedone by cutting notches in the edges of the gel or by insertinga needle soaked in India ink into the gel at the dye front.After staining, measure the migration distances of each pro-tein band (markers and unknowns) from the top of theresolving gel. Divide the migration distance of each proteinby the distance traveled by the tracking dye. The normalizedmigration distances so obtained are called the relative mobili-ties of the proteins (relative to the dye front) and convention-ally denoted as Rf. Construct a plot of the logarithm of therelative molecular masses (Mr) of the protein standards as afunction of the Rf values. Note that the graphs are slightlysigmoid. Unknown molecular masses can be estimated bylinear regression analysis or interpolation from the curves oflog Mr against Rf as long as the values obtained for theunknown samples are positioned along the linear part of thegraph.

8. Suitability of the Test (Validation)The test is not valid unless the front end of the molecular

mass marker migrates 80z of the migrating distance of thedye, and over the required separation range (e.g., the rangecovering the product and its dimer or the product and itsrelated impurities) the separation obtained for the relevantprotein bands shows a linear relationship between thelogarithm of the molecular mass and the Rf as described in 7.Additional requirements with respect to the solution undertest may be speciêd in individual monographs.

9. Quantiˆcation of ImpuritiesWhere the impurity limit is speciêd in the individual

monograph, a reference solution corresponding to that levelof impurity should be prepared by diluting the test solution.For example, where the limit is 5z, a reference solutionwould be a 1:20 dilution of the test solution. No impurity(any band other than the main band) in the electrophoreto-gram obtained with the test solution may be more intensethan the main band obtained with the reference solution.

Under validated conditions, impurities may be quantiêdby normalization to the main band, using an integrating den-sitometer. In this case, the responses must be validated forlinearity.

17471747JP XV General Information / Solid and Particle Densities

Test solutions:Coomassie staining TS Dissolve 125 mg of coomassie

brilliant blue R-250 in 100 mL of a mixture of water,methanol and acetic acid (100) (5:4:1), and ˆlter.

Developer TS Dissolve 2 g of citric acid monohydrate inwater to make 100 mL. To 2.5 mL of this solution add 0.27mL of formaldehyde solution and water to make 500 mL.

Fixing TS To 250 mL of methanol add 0.27 mL of for-maldehyde solution and water to make 500 mL.

Silver nitrate TS for silver staining To 40 mL of sodiumhydroxide TS add 3 mL of ammonia solution (28), then adddropwise 8 mL of a solution of silver nitrate (1 in 5) whilestirring, and add water to make 200 mL.

Destaining TS A mixture of water, methanol and aceticacid (100) (5:4:1).

Blocking TS To 10 mL of acetic acid (100) add water tomake 100 mL.

Trichloroacetic acid TS for ˆxing Dissolve 10 g oftrichloroacetic acid in a mixture of water and methanol (5:4)to make 100 mL.

24. Solid and Particle DensitiesDensity of a solid or a powder as a state of aggregation has

diŠerent deˆnitions depending on the way of including of theinterparticulate and intraparticulate voids that exist betweenthe particles or inside the powder. DiŠerent ˆgures are ob-tained in each case, and there are diŠerent practical mean-ings. Generally, there are three levels of deˆnitions of thesolid or powder density.

(1) Crystal density: It is assumed that the system ishomogeneous with no intraparticulate void. Crystal density isalso called true density.

(2) Particle density: The sealed pores or the experimental-ly non-accessible open pores is also included as a part of thevolumes of the solid or the powder.

(3) Bulk density: The interparticulate void formed in thepowder bed is also included as a part of the volumes of thesolid or the powder. Bulk density is also called apparentdensity. Generally, the powder densities at loose packing andat tapping are deˆned as the bulk density and the tapped den-sity, respectively.

Generally, the densities of liquid or gas are aŠected only bytemperature and pressure, but the solid or powder density isaŠected by the state of aggregation of the molecules or theparticles. Therefore, the solid or powder densities naturallyvary depending on crystal structure or crystallinity of the sub-stance concerned, and also varies depending on the methodof preparation or handling if the sample is amorphous formor partially amorphous. Consequently, even in a case thattwo solids or powders are chemically identical, it may be pos-sible that the diŠerent ˆgures of density are obtained if theircrystal structures are diŠerent. As the solid or powder parti-cle densities are important physical properties for the pow-dered pharmaceutical drugs or the powdered raw materials ofdrugs, the Japanese Pharmacopoeia speciês each density de-termination as ``Powder Particle Density Determination'' forthe particle density and as ``Determination of Bulk andTapped Densities'' for the bulk density.

The solid or powder densities are expressed in mass perunit volume (kg/m3), and generally expressed in g/cm3 (1

g/cm3＝1000 kg/m3).

Crystal DensityThe crystal density of a substance is the average mass per

unit volume, exclusive of all voids that are not a fundamentalpart of the molecular packing arrangement. It is an intrinsicproperty concerning the speciˆc crystal structure of the sub-stance, and is not aŠected by the method of determination.The crystal density can be determined either by calculation orby simple measurement.A. The calculated crystal density is obtained using:

1) For example, the crystallographic data (volume andcomposition of the unit cell) obtained by indexingthe perfect crystal X-ray diŠraction data from singlecrystal or the powder X-ray diŠraction data.

2) Molecular mass of the substance.B. The measured crystal density is obtained as the mass to

volume ratio after measuring the single crystal mass andvolume.

Particle DensityThe particle density takes account both the crystal density

and the intraparticulate porosity (sealed and/or experimen-tally non-accessible open pores) as a part of the particlevolume. The particle density depends on the value of thevolume determined, and the volume in turn depends on themethod of measurement. Particle density can be determinedeither by gas displacement pycnometry or mercury porosi-metry, but the Japanese Pharmacopoeia speciês thepycnometry as the ``Powder Particle Density Determina-tion''.A. The pycnometric density is obtained by assuming that

the volume of the gas displaced, which is measured withthe gas displacement pycnometer, is equivalent to thatof a known mass of the powder. In pycnometric densitymeasurements, any volume with the open pores accessi-ble to the gas is not included as a part of volume of thepowder, but the sealed pores or pores inaccessible to thegas is included as a part of the volume of the powder.Due to the high diŠusivity of helium which can penetrateto most open pores, it is recommendable as the measure-ment gas of particle density. Therefore, the pycnometricparticle density of a ˆnely milled powder is generally notvery diŠerent from the crystal density. Hence, the parti-cle density by this method is the best estimate of the truedensity of an amorphous or partially crystalline sample,and can be widely used for manufacturing control of theprocessed pharmaceutical powder samples.

B. The mercury porosimetric density is also called granulardensity. This method also includes the sealed pores as apart of the volumes of the solid or the powder, butexcludes the volume only from the open pores largerthan some size limit. This pore size limit or minimalaccess diameter depends on the maximal mercuryintrusion pressure applied during the measurement andunder normal operating pressure, the mercury does notpenetrate the ˆnenest pores accessible to helium. Sincethis method is capable of measuring the density whichcorresponds to the pore size limit at each mercuryintrusion pressure, the various granular densities can beobtained from one sample.

Bulk Density and Tapped DensityThe bulk density of a powder includes the contribution of

17481748 JP XVSterility Assurance / General Information

interparticulate void volume as a part of the volume of thepowder. Therefore, the bulk density depends on both thepowder particle density and the space arrangement ofparticles in the power bed. Further, since the slightestdisturbance of the bed may result in variation of the space ar-rangement, it is often very di‹cult to determine the bulk den-sity with good reproducibility. Therefore, it is essential tospecify how the determination was made upon reporting thebulk density.

The Japanese Pharmacopoeia speciês ``Determination ofBulk and Tapped Densities''.A. The bulk density is determined by measuring the

apparent volume of a known mass of powder samplethat has been passed through a screen in a graduatedcylinder (constant mass method). Separately, the Phar-macopoeia speciês the method of determining bulkdensity by measuring the mass of powder in a vessel hav-ing a known volume (constant volume method).

B. The tapped density is obtained by mechanically tappinga measuring cylinder containing a powder sample. Afterdetermining the initial bulk volume, carry out tappingunder a ˆxed measurement condition (tapping rate anddrop height), and the measurement is carried out repeat-edly until the bulk volume variation obtained at consecu-tive two measurements is within an acceptable range(constant mass method). Separately, the Pharmacopoeiaspeciês the method of determining the tapped densityby measuring the mass of a ˆxed volume of the tappedpowder (constant volume method).

25. Sterility Assurance forTerminally Sterilized

Pharmaceutical ProductsAs indicated in the ``Terminal Sterilization and Steriliza-

tion Indicators'', the pharmaceuticals to which terminalsterilization can be applied, generally must be sterilized sothat a sterility assurance level of 10－6 or less is obtained. Thesterility assurance level of 10－6 or less can be proven by usinga sterilization process validation based on physical andmicrobiological methods, but cannot be proven by sterilitytests of the sterilized products. This chapter deals with thenecessary requirements for the appropriate management ofthe important control points of the sterilization process forthe parametric release of products, without performing steril-ity tests on products which have been subjected to terminalsterilization (in the case of radiation sterilization, called dosi-metric release). Parametric release is a method that can be ap-plied in cases where the sterilization system is clearly deˆned,important control points are clearly speciêd, and the sterili-zation system process can be validated by microbiologicalmethods using appropriate biological indicators.

1. DeˆnitionsThe deˆnitions of the terminology used in this chapter are

provided below.1.1 Terminal sterilization

A process whereby a product is sterilized in its ˆnal con-tainer or packaging, and which permits the measurement andevaluation of quantiâble microbial lethality.

1.2 ValidationA documented procedure for obtaining, recording and in-

terpreting the results needed to show that a process will con-sistently yield a product complying with predeterminedspeciˆcations.1.3 Periodic re-validation

Validation that is regularly performed to reconˆrm that aprocess is consistently yielding a product complying withpredetermined speciˆcations. It should conˆrm that variablesand the acceptable ranges are permissible to yield a productconsistently of the required quality.1.4 FacilityWequipment qualiˆcation

This is to provide evidence that the manufacturing facilitiesWequipment, measuring equipment, and manufacturing en-vironment control facilities, etc. have been properly selected,correctly installed, and are operated in conformity with thespeciˆcations at the time of installation and during opera-tion.1.5 Operation qualiˆcation

This is to provide evidence to conˆrm physically, chemical-ly and microbiologically that equipment, operated in accor-dance with its operational instructions, operates as speciêdand aŠords a product meeting the speciˆcations.1.6 Support system for sterilization process

This refers to the facilityWequipment that is associated withthe sterilization devices, such as the preconditioning and aer-ation for ethylene oxide sterilization, the steam supply equip-ment for moist heat sterilization, and the loading devices forradiation sterilization.1.7 Quality system

The procedures, resources and organizational structure ofa manufacturer (responsibilities, authorities and relation-ships between these) required to implement quality manage-ment.1.8 Change control system

A system designed to evaluate all of the changes that mayaŠect the quality of the pharmaceutical product, in order toensure that the process is continuously controlled.1.9 F0 value

Assume a value of 109C for the Z value deˆned as the num-ber of degrees of temperature required for a 10-fold changein the D value. The F0 value indicates the time (minutes) re-quired to give the equivalent lethality at Tb of the sterilizationheat obtained by integrating the lethality rate (L) over an en-tire heating cycle.

L＝log－1 T0－Tb

Z＝10

T0－Tb

Z

T0＝Temperature inside the chamber or inside the productto be sterilized

Tb＝Reference temperature (1219C)

F0＝ft1

t0Ldt

t1－t0＝Processing time (minutes)

1.10 Control deviceA general term for the devices and measurement equip-

ment, including the equipment for controlling, measuringand recording the physical parameters that can be measured(temperature, humidity, pressure, time, radiation dose, etc.).1.11 Parametric release

A release procedure based on an evaluation of the produc-

17491749JP XV General Information / Sterility Assurance

tion records and critical parameters of the sterilizationprocess (temperature, humidity, pressure, time, radiationdose, etc.) based on the results of validation, in lieu of releasebased on testing results of the ˆnal product.

2. Sterilization Validation2.1 Subject of the Implementation

A manufacturer of sterile pharmaceuticals (hereafter,``manufacturer'') must establish a quality system, implementproduct sterilization validation for the categories below as ageneral rule, and continuously control the sterilizationprocess based on the results of the sterilization validation.

a. Sterilization processb. Sterilization process support system

2.2 Documenting Sterilization Validation Procedure2.2.1 The manufacturer must prepare a ``Sterilization

Validation Procedure'' deˆning the items listed below regard-ing the procedures for managing the sterilization process.

a. Details related to the range of duties of the personsresponsible for the validation, as well as the extent of theirauthority

b. Details related to the implementation period for thesterilization validation

c. Details related to the creation, modiˆcation, and ap-proval of the sterilization validation plan documents

d. Details related to the reporting, evaluation, and ap-proval of the sterilization validation implementation results

e. Details related to the storage of documentation con-cerning the sterilization validation

f. Other required matters2.2.2 The sterilization validation procedure must list the

names of the enactors, the date of enactment, and when thereare revisions, must also list the revisers, date of revisions,revised sections and reasons for the revisions.

2.2.3 The manufacturer must properly store and main-tain the sterilization validation procedure after clarifying theprocedures related to alterations and deletions of the contentsof the sterilization validation procedure.2.3 Persons Responsible for the Validation

The manufacturer must assign persons to be responsiblefor the sterilization validation. The responsible parties mustperform each of the duties listed below according to thesterilization validation procedure.

2.3.1 For products that are to be produced according tothe sterilization validation procedure, a written sterilizationvalidation implementation plan must be prepared. The im-plementation plan will specify the following points based ona consideration of the implementation details of the steriliza-tion validation.

a. Subject pharmaceutical name (product name)b. Purpose of the applicable sterilization validationc. Expected resultsd. Veriˆcation methods (including inspection results and

evaluation methods)e. Period of veriˆcation implementationf. Names of persons performing the sterilization valida-

tion (persons-in-charge)g. Names of the persons who created the plan, creation

date, and in the event of revisions, the names of the revisers,date of the revisions, revised sections, and reasons for revi-sion.

h. Technical requirements for the applicable sterilizationvalidation

i. Other required matters for the implementation of theapplicable sterilization validation

2.3.2 The following sterilization validation is implement-ed according to the plan deˆning the items above.

a. When the manufacturing license and additional(modiˆcation) licenses for product production are obtained,implementation items for the sterilization validation to be ex-ecuted

1 Product qualiˆcation2 FacilityWequipment qualiˆcation

1) Installation qualiˆcation2) Operation qualiˆcation

3 Performance qualiˆcation1) Physical performance qualiˆcation2) Microbiological performance qualiˆcation

b. Sterilization validation to be executed until it is time torenew the manufacturing license

1 Re-validation when there are changes2 Periodic re-validation (The items implemented, etc.

must be determined based on a consideration ofrelevant factors such as the sterilization method.)

2.3.3 Evaluate the results of the sterilization validationand verify that sterility is assured.

2.3.4 Make a written report of the results of the steriliza-tion validation to the manufacturer's authorized person.

2.3.5 Perform the day-to-day management of the sterili-zation process.

3. Microorganism Control ProgramWhen parametric release is adopted, it is important to con-

trol the bioburden in the raw materials of the product, thecontainers and stoppers, and in the product before steriliza-tion. The bioburden is measured with a previously speciêdmethod and frequency, and when required, surveys of thecharacteristics of the isolated microorganisms are made to in-vestigate their resistance to the applicable sterilizationmethod. Refer to the ``Microbiological Evaluation of ProcessAreas for Sterile Pharmaceutical Products'' regarding themethod for evaluating the environmental microorganisms inthe processing areas of pharmaceutical products.

4. Sterilization IndicatorsBiological indicators (BI), chemical indicators (CI), and

dosimeters are among the means used to monitor a steriliza-tion process and as indices of sterility (refer to TerminalSterilization and Sterilization Indicators). When using sterili-zation indicators it is important to consider environmentaland human safety, and to take all necessary precautions. TheBI used for sterilization validation and daily process controlmust be deˆned in the speciˆcation, and recorded in writing.When BI are used for daily process control it must be veriêdthat the loading pattern on the form, product, or simulatedproduct has a resistance equal to or greater than that used forthe microbiological performance qualiˆcation.

5. Establishment of a Change Control SystemChanges which have a large eŠect on the sterile quality,

such as changes in sterilization equipment, loading pattern,and sterilization conditions, correspond to changes of theparametric release conditions for the relevant pharmaceuticalproduct. A change control system must be deˆned in thesterilization validation procedure; and when there arechanges in the causes of variation that have been previouslyspeciêd, there must be an investigation of the causes of vari-

17501750 JP XVSterility Assurance / General Information

ation and of acceptable conditions to verify that the phar-maceutical product is guaranteed always to conform to thequality standards. Furthermore, before modiˆcations aremade to a sterilization process that has been validated, it ismandatory to obtain approval for the implementation of themodiˆcations in question from the appropriate authorizedperson.

6. Release ProcedureA release procedure must be created to clarify the condi-

tions required for shipment based on parametric release ofterminally sterilized products. The following points must beevaluated and recorded when a product is released.

Depending on the sterilization method, some of these itemsmay be omitted or modiêd.

a) Batch recordb) Microorganism evaluation data of production en-

vironmentc) Bioburden data for the raw materials and product be-

fore sterilizationd) Data related to the sterilization indicatorse) Data on the maintenance management of the steriliza-

tion process and sterilization process support systemsf ) Data on the management of sterilization parametersg) Data on the calibrations of measurement equipmenth) Re-validation datai) Other

7. Critical Control PointsThe important control points for each sterilization method

are presented.7.1 Moist heat sterilization

Moist heat sterilization is a method for killing microorgan-isms in which saturated water vapor is generated or in-troduced into a sterilization chamber at the appropriate tem-perature and pressure, and the chamber is then heated for acertain period of time. It is roughly classiêd into saturatedvapor sterilization, in which the target microorganisms aredirectly exposed to the saturated vapor, and unsaturatedvapor sterilization, in which the ‰uid inside a container, suchas an ampule, is subjected to moist heat energy or highfre-quency energy from the outside.

7.1.1 Important control pointsA process control procedure must be created, specifying

the process parameters that aŠect the sterile quality of thepharmaceutical product, and the permissible range of varia-tion for each parameter. The important control points for themoist heat sterilization are indicated below.

a) Heating history (usually indicated by F0 value)b) Temperaturec) Pressured) Timee) Product loading formatWloading densityf ) Other necessary matters7.1.2 UtilitiesThe utilities and control devices required for moist heat

sterilization determine the quality and precision.a) Quality of the vapor usedb) Quality of the air introduced into the sterilization

chamber to restore pressure, etc.c) Quality of the water used for coolingd) Precision of the temperature control devicese) Precision of the pressure control devicesf ) Precision of the time control devices

g) Other7.2 Ethylene oxide gas sterilization

Ethylene oxide gas allows sterilization at low temperatures,so there is typically little injury to the substance being steri-lized; however, since the gas is toxic it must be handled withextreme caution. The sterilization process consists of precon-ditioning, a sterilization cycle, and aeration. The precon-ditioning is performed before the sterilization cycle to processthe product so that temperature and relative humidity in theroom or container are within the range in the speciˆcations.The sterilization cycle indicates the stage at which the actualsterilization is performed, and consists of removal of the air,conditioning (when used), injection of the sterilization gas,maintenance of the sterilization conditions, removal of thesterilization gas, and replacement of the air. The aeration isthe process of eliminating the residual ethylene oxide gasfrom the product, either inside the sterilization chamber or ina separate location.

7.2.1 Important control pointsThe important control points for the ethylene oxide gas

sterilization are indicated below.7.2.1.1 Preconditioning (when performed)a) Time, temperature, humidityb) Product loading patternWloading densityc) Sterilization loading temperature andWor humidityd) Time from the end of preconditioning until the start of

the sterilizatione) Other necessary matters7.2.1.2 Conditioninga) If pressure reduction is performed, the pressure

achieved and required timeb) Reduced pressure maintenance periodc) Time, temperature, pressure, humidityd) Sterilization loading temperature and humiditye) Other necessary matters7.2.1.3 Sterilization cyclea) Pressure increase, injection time, and ˆnal pressure

for the injection of the sterilization gasb) Concentration of the ethylene oxide gas (it is desirable

to analyze directly the gas concentration inside the steriliza-tion chamber, but the following alternatives are acceptable ifdirect analysis is di‹cult)

i) Mass of gas usedii) Volume of gas usediii) Conversion calculation using the initial low pressure

level and the gas injection pressurec) Temperature within the sterilization chamberd) Temperature of the loaded products to be sterilizede) EŠect time (exposure time)f ) Product loading patternWloading densityg) BI placement points and cultivation resultsh) Other necessary matters7.2.1.4 Aerationa) Time, temperatureb) Loaded sterilized substance temperaturec) Pressure variation in the sterilization chamber andWor

the aeration roomd) Rate of change of the air or other gases in the aeration

roome) Other necessary matters

7.2.2 UtilitiesThe utilities and control devices required for ethylene oxide

sterilization determine the quality and precision.

17511751JP XV General Information / Tablet Friability Test

a) Quality of the ethylene oxide gasb) Quality of the injected vapor or waterc) Quality of the replacement air after the completion of

sterilizationd) Quality of the BIe) Precision of the temperature control devicesf ) Precision of the pressure control devicesg) Precision of the humidity control devicesh) Precision of the time control devicesi) Other

7.3 Irradiation SterilizationIrradiation sterilization refers to methods of killing

microorganisms through exposure to ionizing radiation. Thetypes of ionizing radiation used are gamma-rays (g-rays)emitted from a radioisotope such as 60Co or 137Cs, or electronbeams and bremsstrahling (X-ray) generated from an elec-tron accelerator. In the case of g-rays, the cells are killed bysecondarily generated electrons, while in the case of the elec-tron beam, the cells are killed by the electrons generateddirectly from the electron accelerator. For this reason, theprocessing time for electron beam sterilization is generallyshorter than that for g-ray sterilization; but, since thepenetration of the g-rays is better than that of the electronbeam, there must be appropriate consideration of the densityand thickness of the substance being sterilized when choosingbetween these methods. For an irradiation sterilizationprocess, the control procedures primarily make use ofdosimeters and measure the absorbed dose in the substancebeing sterilized. This is called dosimetric release.

7.3.1 Important control pointsThe important control points for the irradiation steriliza-

tion are indicated below.7.3.1.1 g-ray radiationa) Irradiation time (timer setting or conveyor speed)b) Absorbed dosec) Product loading patternd) Other necessary matters7.3.1.2 Electron beam and X-ray radiationa) Electron beam characteristics (average electron beam

current, electron energy, scan width)b) Conveyor speedc) Absorbed dosed) Product loading patterne) Other necessary matters7.3.2 UtilitiesA traceable calibration, performed according to national

standards, must be performed for the radiation devices anddose measurement systems. This calibration must be per-formed as speciêd in a written plan in order to verify thatthe equipment is kept within the required range of accuracy.

7.3.2.1 Required calibration items for gamma-radiationequipment

a) Cycle time or conveyor speedb) Weighing devicec) Dose measurement systemd) Other7.3.2.2 Required calibration items for electron-beam and

X-ray radiation equipmenta) Electron beam characteristicsb) Conveyor speedc) Weighing deviced) Dose measurement systeme) Other

References1) Validation Standards, PAB Notiˆcation No.158,

Ministry of Health and Welfare 19952) Sterilization Validation Standards, PMSBWIGD Notiˆ-

cation No.1, Ministry of Health and Welfare 19973) Quality Assurance Standards for Medical Devices, PAB

Notiˆcation No.1128, Ministry of Health and Welfare1994

4) ISO 9000 series, International Standards for QualityAssurance

5) ISO 11134 Industrial moist heat sterilization6) ISO 11135 Ethylene oxide sterilization7) ISO 11137 Radiation sterilization8) ISO 11138 Biological indicators9) ISO 11140 Chemical indicators

10) ISO 11737-1 Microbiological Methods Part 1: Estima-tion of population of microorganisms on products

11) USP 〈1222〉 Terminally Sterilized PharmaceuticalProducts - Parametric Release

26. Tablet Friability TestThis test is harmonized with the European Pharmacopoeia

and the U.S. Pharmacopeia.The Tablet Friability Test is a method to determine the

friability of compressed uncoated tablets. The test procedurepresented in this chapter is generally applicable to most com-pressed tablets. Measurement of tablets friability supple-ments other physical strength measurement, such as tabletcrushing strength.

Use a drum, with an internal diameter between 283 and 291mm and a depth between 36 and 40 mm, of transparent syn-thetic polymer with polished internal surface, and not subjectto minimum static build-up (see ˆgure for a typical appara-tus). One side of the drum is removable. The tablets are tum-bled at each turn of the drum by a curved projection with aninside radius between 75.5 and 85.5 mm that extends fromthe middle of the drum to the outer wall. The outside di-ameter of the central shaft ring is between 24.5 and 25.5 mm.The drum is attached to the horizontal axis of a device thatrotates at 25±1 rpm. Thus, at each turn the tablets roll orslide and fall onto the drum wall or onto each other.

For tablets with a unit mass equal to or less than 650 mg,take a sample of whole tablets n corresponding as near aspossible to 6.5 g. For tablets with a unit mass of more than650 mg, take a sample of 10 whole tablets. The tablets shouldbe carefully dedusted prior to testing. Accurately weigh thetablet sample, and place the tablets in the drum. Rotate thedrum 100 times, and remove the tablets. Remove any loosedust from the tablets as before, and accurately weigh.

Generally, the test is run once. If obviously cracked,cleaved, or broken tablets are present in the tablet sampleafter tumbling, the sample fails the test. If the results aredi‹cult to interpret or if the weight loss is greater than thetargeted value, the test should be repeated twice and the meanof the three tests determined. A maximum mean weight lossfrom the three samples of not more than 1z is considered ac-ceptable for most products.

If tablet size or shape causes irregular tumbling, adjust thedrum base so that the base forms an angle of abut 109with

17521752 JP XVTerminal Sterilization / General Information

the bench top and the tablets no longer bind together whenlying next to each other, which prevents them from fallingfreely.

EŠervescent tablets and chewable tablets may have diŠer-ent speciˆcations as far as friability is concerned. In the caseof hygroscopic tablets, an appropriate humidity-controlledenvironment is required for testing.

Drums with dual scooping projections, or apparatus withmore than one drum, for the running of multiple samples atone time, are also permitted.

27. Terminal Sterilization andSterilization Indicators

Sterilization is a process whereby the killing or removal ofall forms of viable microorganisms in substances is accom-plished. It is achieved by terminal sterilization or a ˆltrationmethod. For substances to which terminal sterilization can beapplied, an appropriate sterilization method should be select-ed in accordance with the properties of the product, includingthe packaging, after full consideration of the advantages anddisadvantages of each sterilization method, from among theheat method, irradiation method and gas method. After in-stallation of the sterilizer (including design and developmentof the sterilization process), validation is required to conˆrmthat the sterilization process is properly performing itsdesigned function, under conditions of loading and unload-ing of the product, on the basis of su‹cient scientiˆc evi-dence. After the process has been validated and the steriliza-tion of the product commenced, the process must be con-trolled correctly, and qualiˆcation tests of the equipment andprocedures must be performed regularly. The bioburden perproduct, prior to terminal sterilization, must be evaluatedperiodically or on the basis of batches. Refer to the ISO stan-dard (ISO 11737-1) relevant to bioburden estimation. For asubstance to which terminal sterilization can be applied,generally use sterilization conditions such that a sterility as-surance level of less than 10－6 can be obtained. The proprietyof the sterilization should be judged by employing an ap-propriate sterilization process control, with the use of a suita-ble sterilization indicator, and if necessary, based on theresult of the sterility test. The ˆltration procedure is used forthe sterilization of a liquid product, to which terminal sterili-zation can not be applied. Concerning the disinfection andWor sterilization necessary for processing equipment and areas

of pharmaceutical products, and performing microbiologicaltests speciêd in the monographs, see Disinfection andSterilization Methods.

1. DeˆnitionsThe deˆnitions of the terms used in this text are as follows.Terminal sterilization: A process whereby a product is

sterilized in its ˆnal container or packaging, and which per-mits the measurement and evaluation of quantiâblemicrobial lethality.

Product: A generic term used to describe raw materials, in-termediate products, and ˆnished products, to be sterilized.

Bioburden: Numbers and types of viable microorganismsin a product to be sterilized.

Sterility assurance level (SAL): Probability of a viablemicroorganism being present in a product unit after exposureto the proper sterilization process, expressed as 10－n.

Integrity test: A non-destructive test which is used topredict the functional performance of a ˆlter instead of themicroorganism challenge test.

D value: The value which shows the exposure time (decimalreduction time) or absorbed dose (decimal reduction dose) re-quired to cause a 1-logarithm or 90z reduction in the popu-lation of test microorganisms under stated exposure condi-tions.

Sterilization indicator: Indicators used to monitor thesterilization process, or as an index of sterility, including bio-logical indicators (BI), chemical indicators (CI), dosimetersand the like.

2. Sterilization2-1. Heat MethodIn the heat method, microorganisms are killed by heating.(i) Moist heat methodMicroorganisms are killed in saturated steam under pres-

sure. In this method, factors which may aŠect the steriliza-tion include temperature, steam pressure and exposure time.Therefore, in routine sterilization process control, it is re-quired to monitor continuously the temperature, steam pres-sure and exposure time, and they should be included in thespeciˆcations of the sterilizer.

(ii) Dry-heat methodMicroorganisms are killed in dry heated air. This method is

usually conducted in a batch-type dry heat sterilizer or a tun-nel-type dry heat sterilizer. In this method, factors which mayaŠect the sterilization include temperature and exposuretime. Therefore, in routine sterilization process control, it isrequired to monitor continuously the temperature and ex-posure time, and they should be included in the speciˆcationsof the sterilizer.

2-2. Irradiation methodMicroorganisms are directly killed by ionizing radiation, or

by the heat generated by microwave radiation.(i) Radiation methodIonizing radiations which may be used are gamma (g) rays

emitted from a radioisotope such as cobalt 60, an electronbeam and bremsstrahlung (X rays) generated from an elec-tron accelerator. Although any procedure can be applied tothermally unstable products with no radioactivity residue, itis necessary to consider the possibility of material degrada-tion. Although a 25 kGy dose is traditionally used as a sterili-zation dose, there are some ways to calculate the dose as fol-lows: the bioburden of the substance to be sterilized is meas-ured and the sterilization dose is calculated based on the

17531753JP XV General Information / Terminal Sterilization

mean bioburden and the standard resistance distribution(Method 1 in ISO 11137), the dose is calculated from the frac-tion positive information from a sterility test in whichrepresentative product samples are exposed to a substerilizingdose (Method 2 in ISO 11137), or the dose is calculated basedon the bioburden and D value of the most resistant microor-ganisms (Log method) (see 5-3). In the case of the radiationsterilization procedure, factors which may aŠect the steriliza-tion include dose (absorbed dose) and exposure time. There-fore, in g ray sterilization process control, it is required to de-termine the dose (the absorbed dose) at appropriate intervalsand to monitor continuously the exposure time in terms ofthe operating parameters (the conveyer speed, the cycle time).The dose control mechanism should be included in thespeciˆcations of the sterilizer. In the case of electron beam orbremsstrahlung irradiation, it is required to monitor the ac-celeration voltage, the beam current and beam scanningwidth besides the above-mentioned items.

(ii) Microwave methodMicroorganisms are killed by the heat generated by micro-

wave radiation, usually at the frequency of 2450±50 MHz.This method is applied to liquids or water-rich products insealed containers. Since a glass or plastic container may bedestroyed or deformed due to the rise of the inner pressure,the containers must be certiêd to be able to withstand theheat and the inner pressure generated during microwavesterilization. Leakage of electromagnetic radiation must be ata su‹ciently low level to cause no harm to humans and no in-terference with radio communications and the like. In thismethod, factors which may aŠect the sterilization includetemperature, processing time and microwave output power.Therefore, in routine sterilization process control, it is re-quired to monitor continuously the temperature, time and themicrowave output power, and they should be included in thespeciˆcations of the sterilizer.

2-3. Gas methodEthylene oxide (EO) is widely used as a sterilization gas.

Since EO gas has an explosive nature, a 10 – 30z mixturewith carbon dioxide is commonly used. Also, as EO gas is astrong alkylating agent, it can not be applied to the productswhich are likely to react with or absorb it. Furthermore, be-cause EO gas is toxic, the residual concentration of EO gasand other secondarily generated toxic gases in products steri-lized with EO gas must be reduced to less than the safe levelsthereof by means of aeration and the like before the productis shipped. In this method, factors which may aŠect thesterilization include temperature, gas concentration (pres-sure), humidity and exposure time. Therefore, in routinesterilization process control, it is required to monitor con-tinuously the temperature, gas concentration (pressure), hu-midity and exposure time, and they should be included in thespeciˆcations of the sterilizer.

3. Filtration methodMicroorganisms are removed by using a sterilizing ˆlter

made of an appropriate material. However, this method isnot intended for microorganisms smaller than bacteria.Generally, a sterilizing ˆlter challenged with more than 107

microorganisms of a strain of Brevundimonas diminuta(ATCC 19146, NBRC 14213, JCM 2428), cultured under theappropriate conditions, per square centimeter of eŠectiveˆlter area should provide a sterile eŒuent. In this method,factors which may aŠect the sterilization include pressure,

‰ow rate, ˆlter unit characteristics and the like. In routineˆltration process control, it is required to perform integritytests of the sterilizing ˆlter after each ˆltration process (alsoprior to the ˆltration process, if necessary).

4. Sterilization Indicators4-1. Biological indicator (BI)A BI is prepared from speciˆc microorganisms resistant to

the speciêd sterilization process and is used to develop andWor validate a sterilization process. The dry type BI is classiêdinto two kinds. In one, bacterial spores are added to a carriersuch as ˆlter paper, glass or plastic and then the carriers aredried and packaged. In the other, bacterial spores are addedto representative units of the product to be sterilized or tosimulated products. Packaging materials of the BI shouldshow good heat penetration in dry heat sterilization and goodgas or steam penetration in ethylene oxide and moist heatsterilizations. It should be conˆrmed that any carrier does notaŠect the D value of the spores. In the case of a liquidproduct, the spores may be suspended in the same solution asthe product or in a solution showing an equivalent eŠect inthe sterilization of biological indicator. However, when thespores are suspended in liquid, it is necessary to ensure thatthe resistance characteristics of the spores are not aŠecteddue to germination.

Typical examples of biological indicator

SterilizationRepresentative

microorganisms* Strain name

Moist heatmethod

Geobacillusstearo-thermophilus

ATCC 7953, NBRC13737, JCM 9488ATCC 12980, NBRC12550, JCM 2501

Dry heatmethod

Bacillusatrophaeus ATCC 9372, NBRC 13721

Gas method Bacillusatrophaeus ATCC 9372, NBRC 13721

* In addition to these microorganisms, other microorgan-isms with the greatest resistance to the sterilization proce-dure concerned, found in the bioburden, can be used as thebiological indicator.

4-1-1. D value of BIMethods for determination of the D value include the sur-

vival curve method and the fraction negative method(Stumbo, Murphy & Cochran procedure, Limited Spearman-Karber procedure and the like). In using marketed BIs, it isusually unnecessary to determine the D value before use if theD value indicated on the label has been determined by a stan-dardized biological indicator evaluation resistometer (BIER)under strictly prescribed conditions in accordance with ISO11138-1. It is acceptable that the D value indicated on thelabel shows a scattering of not more than ±30 seconds.

4-1-2. Setting up procedure of BI(i) In the case of dry materialsA Dry type BI is placed at predetermined cold spots in the

product to be sterilized or a suitable product showing anequivalent eŠect in the sterilization. The BIs are usually pri-mary packaged in the same way as the product, including asecondary packaging, if applicable.

(ii) In the case of wet materialsSpores are suspended as the BI in the same solution as the

17541754 JP XVTest for Trace Amounts / General Information

product or in an appropriate similar solution, and should beplaced at cold spots in the sterilizer.

4-1-3. Culture conditions of BISoybean casein digest medium is generally used. General

culture conditions are at 55 – 609C for 7 days in the case ofG. stearothermophilus and at 30 – 359C for 7 days in the caseof B. atrophaeus.

4-2. Chemical indicator (CI)CI is an indicator which shows a color change of a sub-

stance applied to a paper slip, etc. as a result of physical andWor chemical change due to exposure to heat, gas or radiation.The CI can be classiêd into three types. The ˆrst is employedto identify whether or not sterilization has already been im-plemented, the second is employed to control the sterilizationprocess (for example, its color changes after sterilization fora su‹cient time), and the third is the Bowie & Dick type usedto evaluate the eŠectiveness of air removal during the pre-vacuum phase of the pre-vacuum sterilization cycle.

4-3. DosimeterIn the radiation (g-ray) method, the sterilization eŠect de-

pends on the absorbed radiation dose, so the sterilizationprocess control is mainly performed by measuring the dose.A dosimeter is installed at a position corresponding to theminimum dose region of an exposed container or a positionwhere the dose is in a known relation to that in the aboveregion. Measurement should be done for each radiationbatch. If there are many containers in the same batch,dosimeters should be employed so that more than onedosimeter is always installed at the eŠective radiation sectionof the irradiation chamber. It should be noted thatdosimeters may be aŠected by environmental conditions(temperature, humidity, ultraviolet light, time until reading,etc.) before and during irradiation. Practical dosimeters forg-ray and bremsstrahlung sterilization include thedyed polymethylmethacrylate dosimeter, clear poly-methylmethacrylate dosimeter, ceric-cerous sulfatedosimeter, alanine-EPR dosimeter and the like. A dosimeterfor gamma radiation can not generally be used for steriliza-tion process control with an electron beam of less than 3 MeVenergy. Dosimeters for electron beam sterilization include thecellulose acetate dosimeter, radiochromic ˆlm dosimeter andthe like. A practical dosimeter must be calibrated against anappropriate national or international standard dosimetry sys-tem.

5. Determination of sterilization conditions using microor-ganism as an indicator

Taking account of the characteristics upon the sterilizationconcerned, bioburden, etc. of a product to be sterilized,chose a suitable method from the followings and determinethe conditions.

5-1. Half-cycle methodIn this method, a sterilization time of twice as long as that

required to inactive all of 106 counts of BI placed in theproduct is used, regardless of bioburden count in the productbeing sterilized or the resistance of the objective microorgan-isms to the sterilization.

5-2. Overkill methodIn this method, a sterilization condition giving a sterility

assurance level of not more than 10－6 counts is used, regard-less of bioburden count in the product being sterilized or theresistance of the objective microorganisms to the steriliza-tion. Generally, a sterilization condition providing 12

logarithmic reduction (12D) of a known count of BI of morethan 1.0 D value is used.

5-3. Combination of BI and bioburdenGenerally, a count of mean bioburden added three times of

its standard deviation obtained by an extensive bioburden es-timation is considered as the maximum bioburden count, andthe sterilization time (or radiation dose) is calculated with thebioburden count based on an objective sterility assurance lev-el. When this procedure is used, it is required to determinethe resistance of the bioburden to the sterilization as well asthe bioburden count in the product being sterilized. If a moreresistant microorganism than the BI spore is found in the bio-burden estimation, it should be used as the BI.

Sterilization time (or radiation dose) ＝D×log N0

N

D: D value of the BIN: Sterility assurance levelN0: Maximum bioburden count in the product

5-4. Absolute bioburden methodThe sterilization conditions are determined by employing

the D value of the most resistant microorganism found in theproduct or environment by the resistant estimation and beingbased on the bioburden count in the product. Generally, acount of mean bioburden added three times of its standarddeviation obtained by an extensive bioburden estimation isemployed as the bioburden count. When this procedure isused, it is required to make frequent counting and resistancedetermination of microorganisms in daily bioburden estima-tion.

References1) ISO 11134 Industrial moist heat sterilization2) ISO 11135 Ethylene oxide sterilization3) ISO 11137 Radiation sterilization4) ISO 11138 Biological indicators5) ISO 11140 Chemical indicators6) ISO 11737 Microbiological methods

Part 1: Estimation of population of micro-organisms on products

28. Test for Trace Amounts ofAluminum in Trans Parenteral

Nutrition (TPN) SolutionsTrans parenteral nutrition solutions (TPNs) are nutrient

preparations for intravenous injection packed in a largevolume. Since toxic eŠects to the central nervous system,bone, etc. due to trace amounts of aluminum have recentlybeen reported in several countries, testing methods for traceamounts of aluminum contaminating TPNs are required forthe o‹cial standard. The following three analytical methodsare available: (1) High-Performance Liquid Chromatographyusing a ‰uorescence photometric detector (HPLC with‰uorescence detection), (2) Inductivity Coupled Plasma-Atomic Emission Spectrometry (ICP-AES method), (3) In-ductivity Coupled Plasma-Mass Spectrometry (ICP-MSmethod). Detection sensitivity by HPLC with ‰uorescencedetection is about 1 mg/L (ppb), while ICP-AES ˆtted withspecial apparatus and ICP-MS have higher sensitivity.

17551755JP XV General Information / Test for Trace Amounts

Since TPNs are nutrient preparations, they contain manynutrients such as sugars, amino acids, electrolytes, etc., invarious compositions. Thus, care is needed in the selection ofa suitable analytical method, because these coexisting compo-nents may aŠect the measurement of trace amounts of alumi-num.

In view of the general availability of HPLC apparatus, thepresent general information describes procedures for the de-termination of trace levels of aluminum in TPNs by means ofHPLC with a ‰uorescence photometric detector, using twokinds of ‰uorescent chelating agents, i.e., (1) Quinolinolcomplexing method, (2) Lumogallion complexing method.

(1) Quinolinol complexing methodAfter forming a complex of aluminum ion in the sample

solution with quinolinol, the assay for aluminum by HPLCˆtted with a ‰uorescence photometer is performed.

Preparation of sample solutionPipet 1 mL of the sample (TPNs) exactly, and after adding

10 mL of water for aluminum test, make up the sample solu-tion to 10 mL exactly by adding the mobile phase.

Preparation of a series of standard solutions for calibra-tion curve

Pipet 1 mL of water for aluminum test exactly, and afteradding 10 mL each of standard solutions of aluminum (1)??(5), make up the standard solutions for calibration curve to10 mL (Aluminum concentration: 0, 1.25, 2.5, 5.0, and 10.0ppb).

Standard testing methodPipet 0.1 mL each of the sample solution and standard so-

lutions, and perform the test by HPLC under the followingconditions. Calculate the aluminum cotent in the sample so-lution using a calibration curve method.Operating conditions—

Detector: Fluorescence photometer(excitation wavelength:380 nm, emission wavelength: 520 nm)

Column: A stainless steel column 4.6 mm in inside di-ameter and 15 cm in length, packed with phenylsilanized sili-ca gel for liquid chromatography (5 mm in particle diameter).

Column temperature: A constant temperature of about409C.

Mobile phase: A mixture of 8-quinolinol in acetonitrile (3in 100) and diluted 0.5 mol/L ammonium acetate TS (2 in 5)(1:1).

Flow rate: Adjust the ‰ow rate so that the retention time ofaluminum/8-quinolinol complex is about 9 minutes.System suitability—

The correlation coe‹cient of the calibration curve, whichis prepared using a series of standard solutions, is not lessthan 0.99.

Furthermore there is an alternative method, in which thechelating agent 8-quinolinol is not included in the mobilephase. In this method also, aluminum is detected as a com-plex with 8-quinolinol in the sample solution by using HPLCˆtted with ‰uorescence photometer. But it is necessary toform a more stable aluminum/8-quinolinol complex in thesample solution, because the chelating agent is not includedin the mobile phase. Further, since the analytical wavelengthfor the ‰uorescence detection is diŠerent from that in thestandard method, excitation WL: 370 nm, emission WL: 504nm, the detection sensitivity is diŠerent. Thus, it is appropri-ate to obtain the calibration curve between 0 – 25 ppb of alu-

minum. Other than the above- mentioned diŠerences, the sizeof column, column temperature, and the mobile phase arealso diŠerent from those used in the standard method, sosuitable analytical conditions should be established for per-forming precise and reproducible examinations of traceamounts of aluminum in the sample specimen.

(2) Lumogallion complexing methodAfter forming a complex of aluminum ion in the sample

specimen with the ‰uorescent reagent of lumogallion, the so-lution is examined by HPLC ˆtted with a ‰uorescence pho-tometer.

Preparation of sample solutionPipet 70 mL of the sample specimen (TPN) exactly, add

0.15 mL of lumogallion hydrochloric acid TS and 0.6 mL ofbuŠer solution for aluminum test, pH 7.2 exactly, then mixthe solution. After this solution has been allowed to stand for4 hours at 409C, it can be used for the measurement as a sam-ple solution.

Preparation of a series of standard solutions for calibrationcurve

Pipet 1 mL each of standard aluminum solutions (1) – (5)exactly, and add diluted nitric acid for aluminum test (1 in100) to make exactly 100 mL. Pipet 70 mL each of these solu-tions exactly, and add exactly 0.15 mL of lumogallionhydrochloric acid TS and exactly 0.6 mL of buŠer solutionfor aluminum test, pH 7.2 then allow to stand for 4 hours at409C to make a series of standard solutions for obtaining thecalibration curve (Aluminum: 0, 1.07, 2.13, 4.27, and 8.54ppb).

Standard examination methodTake 0.1 mL each of the sample solution and standard alu-

minum solutions for the calibration curve, and performHPLC analysis under the following conditions. Calculate thealuminum content in the sample solution by using a calibra-tion curve method.Operating conditions—

Detector: Fluorescence photometer(excitation wavelength505 nm, emission wavelength 574 nm)

Column: A stainless steel column 6.0 mm in inside di-ameter and 10 cm in length, packed with octylsilanized silicagel for liquid chromatography (5 mm in particle diameter).

Column temperature: A constant temperature of about409C.

Mobile phase: Take 100 mL of 2-propanol, and add adiluted 1 mol/L acetic acid-sodium acetate buŠer solution ofpH 5.0 (1 in 10) to make 1000 mL.

Flow rate: Adjust the ‰ow rate so that the retention time ofaluminum/lumogallion complex is about 5 minutes.System suitability—

The correlation coe‹cient of the calibration curve, whichis prepared using a series of standard solutions, is not lessthan 0.99.

The following points should be noted in connection withmeasuring trace amounts of aluminum in TPN preparations.1) Regarding water, solvents, reagents, vessels and other

tools used for the examination, select those not contami-nated with aluminum. Further, keep the testing environ-ment clean and free from dust in the testing room.

2) Before the measurement, it is necessary to conˆrm that thecharacteristic properties of the sample do not aŠect the for-mation of the complex.

17561756 JP XVTotal Protein Assay / General Information

3) Reference substances of river water for analysis of traceelements, distributed by the Japan Society for AnalyticalChemistry, contain certiêd amounts of aluminum: JSAC0301-1 and JSAC 0302 (a known amount of aluminum isartiˆcially added to JSAC 0301-1).

Standard Solutions, Reagents and Test SolutionsOther than the standard solutions, reagents and test solu-

tions speciêd in the Japanese Pharmacopoeia, those de-scribed below can be used in this test.

N,N-Bis(2-hydroxyethyl)-2-aminoethane sulfonic acid C6

H15NO5S White crystals or powder.

Hydrochloric acid for aluminum test Same as the reagentHydrochloric acid. Further, it contains not more than 1 ppbof aluminum.

Lumogallion [5-Chloro-2-hydroxy-3(2,4-dihydrox-yphenylazo)benzenesulfonic acid] C12H9ClN2O6S Red-brownto dark brown powder. Further, it contains not more than 1ppm of aluminum.

Lumogallion hydrochloric acid TS Dissolve 0.86 g of lu-mogallion in 300 mL of 2-propanol, and add 350 mL of dilut-ed Hydrochloric acid for aluminum test (9 in 50) and Waterfor aluminum test to make 1000 mL exactly.

Nitric acid for aluminum test Same as the reagent Nitricacid. Further, it contains not more than 1 ppb of aluminum.

pH buŠer solution for aluminum test, pH 7.2 Dissolve106.6 g of N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonicacid in 800 mL of Water for aluminum test, adjust the pH 7.2by using Tetramethylammonium hydroxide aqueous solu-tion, and add Water for aluminum test to make 1000 mL.

Standard aluminum solution Pipet a constant volumeeach of Water for aluminum test or the Standard aluminumstock solution, dilute and adjust the aluminum concentrationto 0, 1.25, 2.5, 5.0, and 10 ppm by using diluted Nitric acidfor aluminum test (1 in 100), to make Standard aluminum so-lutions (1) – (5).

Tetramethylammonium hydroxide TS (CH3)4NOHIt is a 25z aqueous solution, prepared for aluminum test.

Further, it contains not more than 1 ppb of aluminum.

Water for aluminum test Same as the monograph Puri-êd Water. Further, it contains not more than 1 ppb of alu-minum.

29. Total Protein AssayThis test is harminized with the European Pharmacopoeia


The following procedures are provided as illustrations ofthe determination of total protein content in pharmacopoeialpreparations. Other techniques, such as HPLC, are also ac-ceptable if total protein recovery is demonstrated. Many ofthe total protein assay methods described below can be per-formed successfully using kits from commercial sources.

Note: Where water is required, use distilled water.

Method 1Protein in solution absorbs UV light at a wavelength of 280

nm, due to the presence of aromatic amino acids, mainlytyrosine and tryptophan. This property is the basis of thismethod. Protein determination at 280 nm is mainly a func-tion of the tyrosine and tryptophan content of the protein. Ifthe buŠer used to dissolve the protein has a high absorbancerelative to that of water, there is an interfering substance inthe buŠer. This interference can be compensated for whenthe spectrophotometer is adjusted to zero buŠer absorbance.If the interference results in a large absorbance thatchallenges the limit of sensitivity of the spectrophotometer,the results may be compromised. Furthermore, at low con-centrations protein can be absorbed onto the cuvette, therebyreducing the content in solution. This can be prevented bypreparing samples at higher concentrations or by using a non-ionic detergent in the preparation.

Note: Keep the Test Solution, the Standard Solution, andthe buŠer at the same temperature during testing.Standard Solution Unless otherwise speciêd in theindividual monograph, prepare a solution of the referencestandard or reference material for the protein under test inthe same buŠer and at the same concentration as the TestSolution.Test Solution Dissolve a suitable quantity of the protein un-der test in the appropriate buŠer to obtain a solution having aconcentration of 0.2 to 2 mg per mL.Procedure Concomitantly determine the absorbances of theStandard Solution and the Test Solution in quartz cells at awavelength of 280 nm, with a suitable spectrophotometer,using the buŠer as the blank. To obtain accurate results, theresponse should be linear in the range of protein concentra-tions to be assayed.Light-Scattering The accuracy of the UV spectroscopicdetermination of protein can be decreased by the scatteringof light by the test specimen. If the proteins in solution existas particles comparable in size to the wavelength of the meas-uring light (250 to 300 nm), scattering of the light beamresults in an apparent increase in absorbance of the test speci-men. To calculate the absorbance at 280 nm due to light-scat-tering, determine the absorbances of the Test Solution atwavelengths of 320, 325, 330, 335, 340, 345, and 350 nm. Us-ing the linear regression method, plot the log of the observedabsorbance versus the log of the wavelength, and determinethe standard curve best ˆtting the plotted points. From thegraph so obtained, extrapolate the absorbance value due tolight-scattering at 280 nm. Subtract the absorbance fromlight-scattering from the total absorbance at 280 nm to ob-tain the absorbance value of the protein in solution. Filtra-tion with a ˆlter having a 0.2-mm porosity or clariˆcation bycentrifugation may be performed to reduce the eŠect of light-scattering, especially if the solution is noticeably turbid.Calculations Calculate the concentration, CU, of protein inthe test specimen by the formula:

CU＝CS (AU/AS),

in which CS is the concentration of the Standard Solution;and AU and AS are the corrected absorbances of the TestSolution and the Standard Solution, respectively.

Method 2This method, commonly referred to as the Lowry assay, is

based on the reduction by protein of the phosphomolybdic-

1757

1) Example: the Minimum Requirements for Biological Products and in-dividual monograph of JP.

2) Dye purity is important in the reagent preparation.

1757JP XV General Information / Total Protein Assay

tungstic mixed acid chromogen in the Folin-Ciocalteu'sphenol reagent, resulting in an absorbance maximum at750 nm. The Folin-Ciocalteu's phenol reagent (Folin's TS)reacts primarily with tyrosine residues in the protein, whichcan lead to variation in the response of the assay to diŠerentproteins. Because the method is sensitive to interferingsubstances, a procedure for precipitation of the protein fromthe test specimen may be used. Where separation of interfer-ing substances from the protein in the test specimen isnecessary, proceed as directed below for InterferingSubstances prior to preparation of the Test Solution. TheeŠect of interfering substances can be minimized by dilutionprovided the concentration of the protein under test remainssu‹cient for accurate measurement. Variations of the Lowrytest that are indicated in national regulatory documents1) canbe substituted for the method described below.Standard Solutions Unless otherwise speciêd in theindividual monograph, dissolve the reference standard orreference material for the protein under test in the buŠer usedto prepare the Test Solution. Dilute portions of this solutionwith the same buŠer to obtain not fewer than ˆve StandardSolutions having concentrations between 5 and 100 mg ofprotein per mL, the concentrations being evenly spaced.Test Solution Dissolve a suitable quantity of the protein un-der test in the appropriate buŠer to obtain a solution having aconcentration within the range of the concentrations of theStandard Solutions. An appropriate buŠer will produce a pHin the range of 10 to 10.5.Blank Use the buŠer used for the Test Solution and theStandard Solutions.Reagents and Solutions—

Copper Sulfate Reagent Dissolve 100 mg of copper (II)sulfate pentahydrate and 200 mg of sodium tartrate dihy-drate in water, dilute with water to 50 mL, and mix. Dissolve10 g of anhydrous sodium carbonate in water to a ˆnalvolume of 50 mL, and mix. Slowly pour the sodium car-bonate solution into the copper sulfate solution with mixing.Prepare this solution fresh daily.

5z SDS TS Dissolve 5 g of sodium dodecyl sulfate inwater, and dilute with water to 100 mL.

Alkaline Copper Reagent Prepare a mixture of 5z SDSTS, Copper Sulfate Reagent, and Sodium HydroxideSolution (4 in 125) (2:1:1). This reagent may be stored atroom temperature for up to 2 weeks.

Diluted Folin's TS Mix 10 mL of Folin's TS with 50 mLof water. Store in an amber bottle, at room temperature.Procedure To 1 mL of each Standard Solution, the Test So-lution, and the Blank, add 1 mL of Alkaline Copper Reagent,and mix. Allow to stand at room temperature for 10 minutes.Add 0.5 mL of the Diluted Folin's TS to each solution, andmix each tube immediately after the addition, and allow tostand at room temperature for 30 minutes. Determine the ab-sorbances of the solutions from the Standard Solutions andthe Test Solution at the wavelength of maximum absorbanceat 750 nm, with a suitable spectrophotometer, using the solu-tion from the Blank to set the instrument to zero.Calculations [Note: The relationship of absorbance toprotein concentration is nonlinear; however, if the standardcurve concentration range is su‹ciently small, it willapproach linearity.] Using the linear regression method, plot

the absorbances of the solutions from the Standard Solutionsversus the protein concentrations, and determine the stan-dard curve best ˆtting the plotted points. From the standardcurve so obtained and the absorbance of the Test Solution,determine the concentration of protein in the Test Solution.Interfering Substances In the following procedure, deoxy-cholate-trichloroacetic acid is added to a test specimen toremove interfering substances by precipitation of proteins be-fore testing. This technique also can be used to concentrateproteins from a dilute solution.

Sodium Deoxycholate Reagent Prepare a solution ofsodium deoxycholate in water having a concentration of150 mg in 100 mL.

Trichloroacetic Acid Reagent Prepare a solution oftrichloroacetic acid in water having a concentration of 72 g in100 mL.Procedure Add 0.1 mL of Sodium Deoxycholate Reagentto 1 mL of a solution of the protein under test. Mix on avortex mixer, and allow to stand at room temperature for 10minutes. Add 0.1 mL of Trichloroacetic Acid Reagent, andmix on a vortex mixer. Centrifuge at 3000×g for 30 minutes,decant the liquid, and remove any residual liquid with apipet. Redissolve the protein pellet in 1 mL of Alkaline Cop-per Reagent. Proceed as directed for Test Solution. [Note:Color development reaches a maximum in 20 to 30 minutesduring incubation at room temperature, after which there is agradual loss of color. Most interfering substances cause alower color yield; however, some detergents cause a slight in-crease in color. A high salt concentration may cause aprecipitate to form. Because diŠerent protein species maygive diŠerent color response intensities, the standard proteinand test protein should be the same.]

Method 3This method, commonly referred to as the Bradford assay,

is based on the absorption shift from 470 nm to 595 nm ob-served when the brilliant blue G-250 dye binds to protein.The Coomassie Brilliant Blue G-250 dye binds most readily toarginyl and lysyl residues in the protein, which can lead tovariation in the response of the assay to diŠerent proteins.Standard Solutions Unless otherwise speciêd in theindividual monograph, dissolve the reference standard or thereference material for the protein under test in the buŠer usedto prepare the Test Solution. Dilute portions of this solutionwith the same buŠer to obtain not fewer than ˆve StandardSolutions having concentrations between 100 mg and 1 mg ofprotein per mL, the concentrations being evenly spaced.Test Solution Dissolve a suitable quantity of the protein un-der test in the appropriate buŠer to obtain a solution having aconcentration within the range of the concentrations of theStandard Solutions.Blank Use the buŠer used to prepare the Test Solution andthe Standard Solutions.Coomassie Reagent Dissolve 100 mg of brilliant blue G-2502) in 50 mL of ethanol (95). [Note: Not all dyes have the samebrilliant blue G content, and diŠerent products may givediŠerent results.] Add 100 mL of phosphoric acid, dilute withwater to 1000 mL, and mix. Filter the solution through ˆlterpaper (Whatman No.1 or equivalent), and store the ˆlteredreagent in an amber bottle at room temperature. [Note: Slowprecipitation of the dye will occur during storage of the rea-

17581758 JP XVTotal Protein Assay / General Information

gent. Filter the reagent before use.]Procedure Add 5 mL of the Coomassie Reagent to 100 mLof each Standard Solution, the Test Solution, and the Blank,and mix by inversion. Avoid foaming, which will lead to poorreproducibility. Determine the absorbances of the solutionsfrom the Standard Solutions and the Test Solution at 595nm, with a suitable spectrophotometer, using the Blank to setthe instrument to zero.

[Note: Do not use quartz (silica) spectrophotometer cells:the dye binds to this material. Because diŠerent proteinspecies may give diŠerent color response intensities, thestandard protein and test protein should be the same.] Thereare relatively few interfering substances, but detergents andampholytes in the test specimen should be avoided. Highlyalkaline specimens may interfere with the acidic reagent.Calculations [Note: The relationship of absorbance toprotein concentration is nonlinear; however, if the standardcurve concentration range is su‹ciently small, it willapproach linearity.] Using the linear regression method, plotthe absorbances of the solutions from the Standard Solutionsversus the protein concentrations, and determine the stan-dard curve best ˆtting the plotted points. From the standardcurve so obtained and the absorbance of the Test Solution,determine the concentration of protein in the Test Solution.

Method 4This method, commonly referred to as the bicinchoninic

acid or BCA assay, is based on reduction of the cupric (Cu2＋)ion to cuprous (Cu＋) ion by protein. The bicinchoninic acidreagent is used to detect the cuprous ion. The method has fewinterfering substances. When interfering substances arepresent, their eŠect may be minimized by dilution, providedthat the concentration of the protein under test remainssu‹cient for accurate measurement.Standard Solutions Unless otherwise speciêd in theindividual monograph, dissolve the reference standard or thereference material for the protein under test in the buŠer usedto prepare the Test Solution. Dilute portions of this solutionwith the same buŠer to obtain not fewer than ˆve StandardSolutions having concentrations between 10 and 1200 mg ofprotein per mL, the concentrations being evenly spaced.Test Solution Dissolve a suitable quantity of the protein un-der test in the appropriate buŠer to obtain a solution having aconcentration within the range of the concentrations of theStandard Solutions.Blank Use the buŠer used to prepare the Test Solution andthe Standard Solutions.Reagents and Solutions—

BCA Reagent Dissolve about 10 g of bicinchoninic acid,20 g of sodium carbonate monohydrate, 1.6 g of sodiumtartrate dihydrate, 4 g of sodium hydroxide, and 9.5 g ofsodium hydrogen carbonate in water. Adjust, if necessary,with sodium hydroxide or sodium hydrogen carbonate to apH of 11.25. Dilute with water to 1000 mL, and mix.

Copper Sulfate Reagent Dissolve about 2 g of copper (II)sulfate pentahydrate in water to a ˆnal volume of 50 mL.

Copper-BCA Reagent Mix 1 mL of Copper SulfateReagent and 50 mL of BCA Reagent.Procedure Mix 0.1 mL of each Standard Solution, the TestSolution, and the Blank with 2 mL of the Copper-BCAReagent. Incubate the solutions at 379C for 30 minutes, notethe time, and allow to come to room temperature. Within 60minutes following the incubation time, determine the absor-

bances of the solutions from the Standard Solutions and theTest Solution in quartz cells at 562 nm, with a suitablespectrophotometer, using the Blank to set the instrument tozero. After the solutions are cooled to room temperature, thecolor intensity continues to increase gradually. If substancesthat will cause interference in the test are present, proceed asdirected for Interfering Substances under Method 2. BecausediŠerent protein species may give diŠerent color response in-tensities, the standard protein and test protein should be thesame.Calculations [Note: The relationship of absorbance to pro-tein concentration is nonlinear; however, if the standardcurve concentration range is su‹ciently small, it willapproach linearity.] Using the linear regression method, plotthe absorbances of the solutions from the Standard Solutionsversus the protein concentrations, and determine the stan-dard curve best ˆtting the plotted points. From the standardcurve so obtained and the absorbance of the Test Solution,determine the concentration of protein in the Test Solution.

Method 5This method, commonly referred to as the Biuret assay, is

based on the interaction of cupric (Cu2＋) ion with protein inan alkaline solution and the resultant development of absor-bance at 545 nm.Standard Solutions Unless otherwise speciêd in the in-dividual monograph, prepare a solution of Albumin Humanfor which the protein content has been previously determinedby nitrogen analysis (using the nitrogen-to-protein conver-sion factor of 6.25) or of the reference standard or referencematerial for the protein under test in sodium chloride solu-tion (9 in 1000). Dilute portions of this solution with sodiumchloride solution (9 in 1000) to obtain not fewer than threeStandard Solutions having concentrations between 0.5 and 10mg per mL, the concentrations being evenly spaced. [Note:Low responses may be observed if the sample under test hassigniˆcantly diŠerent level of proline than that of AlbuminHuman. A diŠerent standard protein may be employed insuch cases.]Test Solution Prepare a solution of the test protein insodium chloride solution (9 in 1000) having a concentrationwithin the range of the concentrations of the StandardSolutions.Blank Use sodium chloride solution (9 in 1000).Biuret Reagent Dissolve about 3.46 g of copper (II) sulfatepentahydrate in 10 mL of water, with heating if necessary,and allow to cool (Solution A). Dissolve about 34.6 g ofsodium citrate dihydrate and 20.0 g of anhydrous sodiumcarbonate in 80 mL of water, with heating if necessary, andallow to cool (Solution B). Mix Solutions A and B, and dilutewith water to 200 mL. This Biuret Reagent is stable at roomtemperature for 6 months. Do not use the reagent if it de-velops turbidity or contains any precipitate.Procedure To one volume of the Standard Solutions and asolution of the Test Solution add an equal volume of sodiumhydroxide solution (6 in 100), and mix. Immediately add avolume of Biuret Reagent equivalent to 0.4 volume of theTest Solution, and mix. Allow to stand at a temperaturebetween 159C and 259C for not less than 15 minutes. Within90 minutes after the addition of the Biuret Reagent, deter-mine the absorbances of the Standard Solutions and thesolution from the Test Solution at the wavelength of maxi-mum absorbance at 545 nm, with a suitable spectrophotome-

17591759JP XV General Information / Total Protein Assay

ter, using the Blank to set the instrument to zero. [Note: Anysolution that develops turbidity or a precipitate is not accept-able for calculation of protein concentration.]Calculations Using the least-squares linear regressionmethod, plot the absorbances of the Standard Solutionsversus the protein concentrations, and determine the stan-dard curve best ˆtting the plotted points, and calculate thecorrelation coe‹cient for the line. [Note: Within the givenrange of the standards, the relationship of absorbance to pro-tein concentration is approximately linear.] A suitable systemis one that yields a line having a correlation coe‹cient of notless than 0.99. From the standard curve and the absorbanceof the Test Solution, determine the concentration of proteinin the test specimen, making any necessary correction.Interfering Substances To minimize the eŠect of interferingsubstances, the protein can be precipitated from the initialtest specimen as follows. Add 0.1 volume of 50z trichloroa-cetic acid to 1 volume of a solution of the test specimen,withdraw the supernatant layer, and dissolve the precipitatein a small volume of 0.5 mol/L sodium hydroxide TS. Usethe solution so obtained to prepare the Test Solution.Comments This test shows minimal diŠerence betweenequivalent IgG and albumin samples. Addition of the sodiumhydroxide and the Biuret Reagent as a combined reagent, in-su‹cient mixing after the addition of the sodium hydroxide,or an extended time between the addition of the sodiumhydroxide solution and the addition of the Biuret Reagentwill give IgG samples a higher response than albumin sam-ples. The trichloroacetic acid method used to minimize theeŠects of interfering substances also can be used to determinethe protein content in test specimens at concentrations below500 mg per mL.

Method 6This ‰uorometric method is based on the derivatization of

the protein with o-phthalaldehyde (OPA), which reacts withthe primary amines of the protein (i.e., NH2-terminal aminoacid and the e-amino group of the lysine residues). Thesensitivity of the test can be increased by hydrolyzing the pro-tein before testing. Hydrolysis makes the a-amino group ofthe constituent amino acids of the protein available for reac-tion with the OPA reagent. The method requires very smallquantities of the protein.

Primary amines, such as tris(hydroxymethyl)amino-methane and amino acid buŠers, react with OPA and must beavoided or removed. Ammonia at high concentrations willreact with OPA as well. The ‰uorescence obtained when a-mine reacts with OPA can be unstable. The use of automatedprocedures to standardize this procedure may improve the ac-curacy and precision of the test.Standard Solutions Unless otherwise speciêd in theindividual monograph, dissolve the reference standard or thereference material for the protein under test in the buŠer usedto prepare the Test Solution. Dilute portions of this solutionwith the same buŠer to obtain not fewer than ˆve StandardSolutions having concentrations between 10 and 200 mg ofprotein per mL, the concentrations being evenly spaced.Test Solution Dissolve a suitable quantity of the protein un-der test in the appropriate buŠer to obtain a solution having aconcentration within the range of the concentrations of theStandard Solutions.Blank Use the buŠer used to prepare the Test Solution andthe Standard Solutions.

Reagents and Solutions—Borate BuŠer Dissolve about 61.83 g of boric acid in

water, and adjust with potassium hydroxide to a pH of 10.4.Dilute with water to 1000 mL, and mix.

Stock OPA Reagent Dissolve about 120 mg of o-phthalaldehyde in 1.5 mL of methanol, add 100 mL ofBorate BuŠer, and mix. Add 0.6 mL of polyoxyethylene (23)lauryl ether, and mix. This solution is stable at room temper-ature for at least 3 weeks.

OPA Reagent To 5 mL of Stock OPA Reagent add 15 mLof 2-mercaptoethanol. Prepare at least 30 minutes prior touse. This reagent is stable for one day.Procedure Adjust each of the Standard Solutions and theTest Solution to a pH between 8.0 and 10.5. Mix 10 mL of theTest Solution and each of the Standard Solutions with 100 mLof OPA Reagent, and allow to stand at room temperature for15 minutes. Add 3 mL of 0.5 mol/L sodium hydroxide TS,and mix. Using a suitable ‰uorometer, determine the ‰uores-cent intensities of solutions from the Standard Solutions andthe Test Solution at an excitation wavelength of 340 nm andan emission wavelength between 440 and 455 nm. [Note: The‰uorescence of an individual specimen is read only once be-cause irradiation decreases the ‰uorescent intensity.]Calculations The relationship of ‰uorescence to proteinconcentration is linear. Using the linear regression method,plot the ‰uorescent intensities of the solutions from the Stan-dard Solutions versus the protein concentrations, and deter-mine the standard curve best ˆtting the plotted points. Fromthe standard curve so obtained and the ‰uorescent intensityof the Test Solution, determine the concentration of proteinin the test specimen.

Method 7This method is based on nitrogen analysis as a means of

protein determination. Interference caused by the presence ofother nitrogen-containing substances in the test protein canaŠect the determination of protein by this method. Nitrogenanalysis techniques destroy the protein under test but are notlimited to protein presentation in an aqueous environment.Procedure A Determine the nitrogen content of the proteinunder test as directed elsewhere in the Pharmacopoeia. Com-mercial instrumentation is available for the Kjeldahl nitrogenassay.Procedure B Commercial instrumentation is available fornitrogen analysis. Most nitrogen analysis instruments usepyrolysis (i.e., combustion of the sample in oxygen attemperatures approaching 10009C), which produces nitricoxide (NO) and other oxides of nitrogen (NOx) from thenitrogen present in the test protein. Some instrumentsconvert the nitric oxides to nitrogen gas, which is quantiêdwith a thermal conductivity detector. Other instruments mixnitric oxide (NO) with ozone (O3) to produce excited nitrogendioxide (NO2

.), which emits light when it decays and can bequantiêd with a chemiluminescence detector. A proteinreference standard or reference material that is relatively pureand is similar in composition to the test proteins is used to op-timize the injection and pyrolysis parameters and to evaluateconsistency in the analysis.Calculations The protein concentration is calculated bydividing the nitrogen content of the sample by the knownnitrogen content of the protein. The known nitrogen contentof the protein can be determined from the chemical composi-tion of the protein or by comparison with the nitrogen

17601760 JP XVValidation of Analytical / General Information

content of the appropriate reference standard or referencematerial.

30. Validation of AnalyticalProcedures

The validation of an analytical procedure is the process ofconˆrming that the analytical procedure employed for a testof pharmaceutics is suitable for its intended use. In otherword, the validation of an analytical procedure requires us todemonstrate scientiˆcally that risks in decision by testingcaused by errors from analytical steps are acceptably small.The performance of an analytical procedure is established byvarious kinds of validation characteristics. The validity of aproposed analytical procedure can be shown by demonstrat-ing experimentally that the validation characteristics of theanalytical procedure satisfy the standards set up according tothe acceptable limits of testing.

When an analytical procedure is to be newly carried in theJapanese Pharmacopoeia, when a test carried in the JapanesePharmacopoeia is to be revised, and when the test carried inthe Japanese Pharmacopoeia is to be replaced with a new testaccording to regulations in general notices, analytical proce-dures employed for these tests should be validated accordingto this document.

Required data for analytical procedures to be carried in theJapanese Pharmacopoeia

(1) OutlineThis section should provide a brief explanation of the prin-

ciple of a proposed analytical procedure, identify the necessi-ty of the analytical procedure and its advantage comparedwith other procedures, and summarize the validation. Whenan analytical procedure is revised, the limitation of the cur-rent analytical procedure and the advantage oŠered by thenew analytical procedure should be described.

(2) Analytical procedureThis section should contain a complete description of the

analytical procedure to enable skilled persons to evaluate cor-rectly the analytical procedure and replicate it if necessary.Analytical procedures include all important operating proce-dures for performing analyses, the preparation of standardsamples, reagents and test solutions, precautions, proceduresto verify system suitability (e.g. the veriˆcation of theseparating performance of a chromatographic system), for-mulas to obtain results, the number of replications and soforth. Any instruments and apparatus that are not stated inthe Japanese Pharmacopoeia should be described in detail.The physical, chemical or biological characteristics of anynew reference standards should be clariêd and their testingmethods should be established.

(3) Data showing the validity of analytical proceduresThis section should provide complete data showing the

validity of the analytical procedures. This includes the ex-perimental designs to determine the validation characteris-tics, experimental data, calculation results and results ofhypothesis tests.

Validation characteristicsThe deˆnition of typical validation characteristics to be as-

sessed in validation of analytical procedures and examples ofassessing procedures are given below.

The terminology and deˆnitions of the validation charac-teristics may possibly vary depending upon the êlds to whichanalytical procedures are applied. The terminology and deˆ-nitions shown in this document are established for the pur-pose of the Japanese Pharmacopoeia. Typical methods forassessing the validation characteristics are shown in the itemof assessment. Various kinds of methods to determine thevalidation characteristics have been proposed and anymethods that are widely accepted will be accepted for thepresent purpose. However, since values of the validationcharacteristics may possibly depend upon methods of deter-mination, it is required to present the methods of determiningthe validation characteristics, the data and calculationmethods in su‹cient detail.

Although robustness is not listed as a validation charac-teristic, it should be considered during the development ofanalytical procedures. Studying the robustness may help toimprove analytical procedures and to establish appropriateanalytical conditions including precautions.

(1) AccuracyWTruenessDeˆnition: The accuracy is a measure of the bias of ob-

served values obtained by an analytical procedure. The ac-curacy is expressed as the diŠerence between the averagevalue obtained from a large series of observed values and thetrue value.

Assessment: The estimate of accuracy of an analyticalmethod is expressed as the diŠerence between the total meanof observed values obtained during investigation of thereproducibility and the true value. The theoretical value isused as the true value (e.g., in the case of titration methods,etc.). When there is no theoretical value or it is di‹cult to ob-tain a theoretical value even though it exists, a certiêd valueor a consensus value may be used as the true value. When ananalytical procedure for a drug product is considered, the ob-served value of the standard solution of the drug substancemay be used as the consensus value.

It may be inferred from speciˆcity data that an analyticalprocedure is unbiased.

The estimate of accuracy and a 95z conˆdence interval ofthe accuracy should be calculated using the standard errorbased on the reproducibility (intermediate precision). Itshould be conˆrmed that the conˆdence interval includes zeroor that the upper or lower conˆdence limits are within therange of the accuracy required of the analytical procedure.

(2) PrecisionDeˆnition: The precision is a measure of the closeness of

agreement between observed values obtained independentlyfrom multiple samplings of a homogenous sample and is ex-pressed as the variance, standard deviation or relative stan-dard deviation (coe‹cient of variation) of observed values.

The precision should be considered at three levels withdiŠerent repetition conditions; repeatability, intermediateprecision and reproducibility.

(i) RepeatabilityWIntra-assay precisionThe repeatability expresses the precision of observed values

obtained from multiple samplings of a homogenous sampleover a short time interval within a laboratory, by the sameanalyst, using the same apparatus and instruments, lots ofreagents and so forth (repeatability conditions).

(ii) Intermediate precisionThe intermediate precision expresses the precision of ob-

served values obtained from multiple samplings of ahomogenous sample by changing a part of or all of the oper-

17611761JP XV General Information / Validation of Analytical

ating conditions including analysts, experimental dates, ap-paratus and instruments and lots of reagents within a labora-tory (intermediate precision condition).

(iii) ReproducibilityThe reproducibility expresses the precision of observed

values obtained from multiple samplings of a homogenoussample in diŠerent laboratories (reproducibility condition).

Assessment: A su‹cient volume of a homogenous sampleshould be prepared before studying the precision. The solu-tion is assumed to be homogenous. When it is di‹cult to ob-tain a homogenous sample, the following samples may beused as homogenous samples; e.g., a large amount of drugproducts or mixture of drug substance and vehicles that arecrushed and mixed well until they can be assumed to behomogenous.

Suitable experimental designs such as one-way layout maybe employed when more than one level of precision is to beinvestigated simultaneously. A su‹cient number of repeti-tions, levels of operating conditions and laboratories shouldbe employed. Sources of variations aŠecting analytical resultsshould be evaluated as thoroughly as possible through thevalidation.

It is required to show the variance, standard deviation andrelative standard deviation (coe‹cient of variation) of eachlevel of precision. The 90z conˆdence interval of the vari-ance and corresponding intervals of the standard deviationand relative standard deviation should also be established.The validity of the proposed analytical procedure for its in-tended use may be conˆrmed by comparing obtained valueswith the required values of the analytical procedure. Whetherthe proposed analytical procedure is acceptable may normal-ly be decided based on the reproducibility.

(3) SpeciˆcityDeˆnition: The speciˆcity is the ability of an analytical

procedure to measure accurately an analyte in the presence ofcomponents that may be expected to be present in the samplematrix. The speciˆcity is a measure of discriminating ability.Lack of speciˆcity of an analytical procedure may be com-pensated by other supporting analytical procedures.

Assessment: It should be conˆrmed that the proposed ana-lytical procedure can identify an analyte or that it can ac-curately measure the amount or concentration of an analytein a sample. The method to conˆrm the speciˆcity dependsvery much upon the purpose of the analytical procedure. Forexample, the speciˆcity may be assessed by comparing analyt-ical results obtained from a sample containing the analyteonly with results obtained from samples containing ex-cipients, related substances or degradation products, and in-cluding or excluding the analyte. If reference standards ofimpurities are unavailable, samples that are expected to con-tain impurities or degradation products may be used (e.g.samples after accelerated or stress tests).

(4) Detection limitDeˆnition: The detection limit is the lowest amount or con-

centration of the analyte in a sample that is detectable, butnot necessarily quantiâble.

Assessment: The detection limit should be normally deter-mined so that producer's and consumer's risks are less than5z. The detection limit may be calculated using the standarddeviation of responses of blank samples or samples contain-ing an analyte close to the detection limit and the slope of thecalibration curve close to the detection limit. The followingequation is an example to determine the detection limit using

the standard deviation of responses of blank samples and theslope of the calibration curve.

DL＝3.3sWslope

DL: detection limits: the standard deviation of responses of blank

samplesslope: slope of the calibration curve

The noise level may be used as the standard deviation ofresponses of blank samples in chromatographic methods. Itshould be ensured that the detection limit of the analyticalprocedure is lower than the speciêd limit for testing.

(5) Quantitation limitDeˆnition: The quantitation limit is the lowest amount or

concentration of the analyte in a sample that can be deter-mined. The precision expressed as the relative standard devia-tion of samples containing an analyte at the quantitationlimit is usually 10z.

Assessment: The quantitation limit may be calculated us-ing the standard deviation of responses of blank samples orsamples containing an analyte close to the quantitation limitand the slope of the calibration curve close to the quantita-tion limit. The following equation is an example to determinethe quantitation limit using the standard deviation ofresponses of blank samples and the slope of the calibrationcurve.

QL＝10sWslope

QL: quantitation limits: the standard deviation of responses of blank

samplesslope: slope of the calibration curve

The noise level may be used as the standard deviation ofresponses of blank samples in chromatographic methods. Itshould be ensured that the quantitation limit of the analyticalprocedure is lower than the speciêd limit for testing.

(6) LinearityDeˆnition: The linearity is the ability of an analytical

procedure to elicit responses linearly related to the amount orconcentration of an analyte in samples. A well-deˆnedmathematical transformation may sometimes be necessary toobtain a linear relationship.

Assessment: Responses are obtained after analyzing sam-ples with various amounts or concentrations of an analyte ac-cording to described operating procedures. The linearity maybe evaluated in terms of the correlation coe‹cient, and theslope and y-intercept of the regression line. It may be alsohelpful for evaluating the linearity to plot residual errorsfrom the regression line against the amount or concentrationand to conˆrm that there is no particular tendency in thegraph. Samples with ˆve diŠerent amounts or concentrationsof an analyte should be usually investigated.

(7) RangeDeˆnition: The range for the validation of analytical

procedures is the interval between the lower and upper limitsof the amount or concentration of an analyte providingsu‹cient accuracy and precision. The range for the valida-tion of analytical procedures for an analytical procedure withlinearity is the interval between the lower and upper limitsproviding su‹cient accuracy, precision and linearity.

Assessment: When the range for the validation of analyti-cal procedures is investigated, 80 to 120z of speciêd limits

17621762 JP XVValidation of Analytical / General Information

of testing should be usually considered. The accuracy, preci-sion and linearity should be evaluated using samples contain-ing the lower and upper limits and in the middle of the range.

Categories of tests employing analytical proceduresTests covered with this document are roughly classiêd

into three categories shown below according to their pur-poses. The table lists the normally required validation charac-teristics to be evaluated in the validation of analytical proce-dures used in these tests. This list should be considered torepresent typical validation characteristics. A diŠerent ap-proach to validating analytical procedures should be consi-dered depending upon the characteristics of analytical proce-dures and their intended use.

Type I Identiˆcation. Tests for identifying major compo-nents in pharmaceuticals according to their characteristics.

Type II Impurity tests. Tests for determination of impur-ities in pharmaceuticals.

Type III Tests for assaying drug substances, active in-gredients, and major components in pharmaceuticals.(Additives such as stabilizing agents and preservatives are in-cluded in major components.) Tests for determining perfor-mance of pharmaceuticals, such as dissolution testing.

Table Lists of validation characteristics required to beevaluated in tests of each type

＼　＼　　＼

Type of test

Validationcharacteristics

Type I Type II

Quantitationtest

Limittest

Type III

AccuracyWTrueness －＋－＋

PrecisionRepeatability －＋－＋

Intermediate precision －－* －－*Reproducibility －＋* －＋*

Specificity** ＋＋＋＋

Detection limit －－＋－

Quantitation limit －＋－－

Linearity －＋－＋

Range －＋－＋

－ Usually need not to be evaluated.＋ Usually need to be evaluated.* Either intermediate precision or reproducibility should be

evaluated depending upon circumstances in which analyticalprocedures or tests are performed. The latter should be normallyevaluated in the validation of analytical procedures proposed tobe included in the Japanese Pharmacopoeia.

** The lack of the specificity of an analytical procedure may becompensated by other relevant analytical procedures

Terminology used in the validation of analytical proceduresAnalytical procedure: This document covers analytical

procedures applied to identiˆcation, and ones that providesresponses depending upon the amount or concentration ofanalytes in samples.

Laboratory: The laboratory means an experimental roomor facility where tests are performed. In this document diŠer-ent laboratories are expected to perform an analytical proce-dure using diŠerent analysts, diŠerent experimental appara-tus and instruments, diŠerent lots of reagents and so forth.

Number of replications: The number of replications is onethat is described in analytical procedures. An observed valueis often obtained by more than one measurement in order toachieve good precision of analytical procedures. Analytical

procedures including the number of replications should bevalidated. This is diŠerent from repetition in the validation ofanalytical procedures to obtain accuracy or precision.

Observed value: The value of a characteristic obtained asthe result of performing an analytical procedure.

Consumer's risk: This is the probability that products outof the speciˆcation of tests are decided to be accepted aftertesting. It is usually expressed as b, and is called the probabil-ity of type II error or the probability of false negative in im-purity tests.

Producer's risk: This is the probability that products satis-fying the speciˆcation of tests are decided to be rejected aftertesting. It is usually expressed as a, and is called the probabil-ity of type I error or the probability of false positive in im-purity tests.

Robustness: The robustness is a measure of the capacity toremain unaŠected by small but deliberate variations in ana-lytical conditions. The stability of observed values may bestudied by changing various analytical conditions within suit-able ranges including pH values of solutions, reaction tem-perature, reaction time or amount of reagents added. Whenobserved values are unstable, the analytical procedure shouldbe improved. Results of studying robustness may be re‰ectedin the developed analytical procedure as precautions or sig-niˆcant digits describing analytical conditions.

Test: Tests mean various tests described in general testsand o‹cial monographs in the Japanese Pharmacopoeia suchas impurity tests and assay. They includes sampling methods,speciˆcation limits and analytical procedures.

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