0581-0586 [1055] Biotechnology-Derived Articles—Peptide Mapping

8/15/2019 0581-0586 [1055] Biotechnology-Derived Articles—Peptide Mapping

1/6

USP 35 General Information / 1055 Biotechnology-Derived Articles 581

desired protein product, to evaluate the stability of the ex-Points To Considerpression construct of cells used for recombinant DNA prod-ucts, to evaluate the consistency of the overall process, and1. Samples can be applied to any area on the gel, butto assess product stability, as well as to ensure the identityto protect the proteins from extreme pH environ-of the protein product or to detect the presence of proteinments, samples should not be applied close to either variant. The validation scheme presented differentiates be-electrode. During method development, the analysttween qualification of the method at an early stage in thecan try applying the protein in three positions on theregulatory process, the Investigational New Drug (IND)gel (e.g., middle and both ends); the pattern of alevel, and full validation in support of New Drug Applicationprotein applied at opposite ends of the gel may not (NDA), Product License Application (PLA), or Marketing Au-be identical.thorization Application (MAA). The validation concepts de-2. A phenomenon known as cathodic drift, where thescribed are consistent with the general information chapter pH gradient decays over time, may occur if a gel isValidation of Compendial Procedures 1225 and with the In- focused too long. Although not well understood,ternational Conference on Harmonization (ICH) documentelectroendoosmosis and absorption of carbon dioxideAnalytical Methods Validation .may be factors that lead to cathodic drift. Cathodic

drift is observed as focused protein migrating off thecathode end of the gel. Immobilized pH gradients THE PEPTIDE MAPmay be used to address this problem.

3. Efficient cooling (approximately 4 °) of the bed that Peptide mapping is not a general method, but involvesthe gel lies on during focusing is important. High developing specific maps for each unique protein. Although field strengths used during isoelectric focusing can the technology is evolving rapidly, there are certain meth-lead to overheating and affect the quality of the fo- ods that are generally accepted. Variations of these methodscused gel. will be indicated, when appropriate, in specific monographs. A peptide map may be viewed as a fingerprint of a pro-

tein and is the end product of several chemical processesthat provide a comprehensive understanding of the proteinbeing analyzed. Four major steps are necessary for the de-velopment of the procedure: isolation and purification of theprotein, if the protein is part of a formulation; selective1055 BIOTECHNOLOGY- cleavage of the peptide bonds; chromatographic separationof the peptides; and analysis and identification of the pep-DERIVED ARTICLES—PEPTIDE tides. A test sample is digested and assayed in parallel witha Reference Standard or Reference Material. Complete cleav-MAPPING age of peptide bonds is more likely to occur when enzymessuch as endoproteases (e.g., trypsin) are used instead of chemical cleavage reagents. A map should contain enoughpeptides to be meaningful. On the other hand, if there areThis chapter provides guidance and procedures used for too many fragments, the map might lose its specificity be-characterization of biotechnology-derived articles by peptidecause many proteins will then have the same profiles.mapping. This chapter is harmonized with the correspond-

ing chapter in JP and EP . Portions of the chapter that arenot harmonized with the other two pharmacopeias are

Isolation and Purificationmarked by the symbol . Other characterization tests, alsoharmonized, are shown in Biotechnology-Derived Articles— Isolation and purification are necessary for analysis of bulkAmino Acid Analysis 1052 , Biotechnology-Derived Articles— drugs or dosage forms containing interfering excipients andCapillary Electrophoresis 1053 , Biotechnology-Derived Arti- carrier proteins and, when required, will be specified in thecles—Isoelectric Focusing 1054 , Biotechnology-Derived Arti- monograph. Quantitative recovery of protein from the dos-cles—Polyacrylamide Gel Electrophoresis 1056 , and Biotech- age form should be validated.nology-Derived Articles—Total Protein Assay 1057 .

Selective Cleavage of Peptide BondsINTRODUCTIONThe selection of the approach used for the cleavage of Peptide mapping is an identity test for proteins, especially peptide bonds will depend on the protein under test. Thisthose obtained by rDNA technology. It involves the chemi- selection process involves determination of the type of cal or enzymatic treatment of a protein, resulting in the cleavage to be employed—enzymatic or chemical—and the formation of peptide fragments, followed by separation and type of cleavage agent within the chosen category. Severalidentification of the resultant fragments in a reproducible cleavage agents and their specificity are shown in Table 1 .manner. It is a powerful test that is capable of identifying

This list is not all-inclusive and will be expanded as other single amino acid changes resulting from events such as er- cleavage agents are identified.rors in the reading of complementary DNA (cDNA) se-quences or point mutations. Peptide mapping is a compara-tive procedure because the information obtained, compared P RETREATMENT OF S AMPLEto a Reference Standard or Reference Material similarlytreated, confirms the primary structure of the protein, is ca- Depending on the size or the configuration of the pro-pable of detecting whether alterations in structure have oc- tein, different approaches in the pretreatment of samplescurred, and demonstrates process consistency and genetic can be used. For monoclonal antibodies, the heavy andstability. Each protein presents unique characteristics that light chains will need to be separated before mapping. If must be well understood so that the scientific and analytical trypsin is used as a cleavage agent for proteins with a mo-approaches permit validated development of a peptide map lecular mass greater than 100,000 Da, lysine residues mustthat provides sufficient specificity. be protected by citraconylation or maleylation; otherwise,

This chapter provides detailed assistance in the application too many peptides will be generated.of peptide mapping and its validation to characterize the

ffici from y 1 2012opyri t c 2011 T e nited St tes rm copei on ention A ri ts reser ed

Accessed from 128 83 63 20 by nEwp0rt1 on Fri Dec 02 23:39:59 EST 2011


2/6

582 1055 Biotechnology-Derived Articles / General Information USP 35

Table 1. Examples of Cleavage Agents

Type Agent SpecificityEnzymatic Trypsin, EC 3.4.21.4 C -terminal side of Arg and Lys

Chymotrypsin, EC 3.4.21.1 C -terminal side of hydrophobic residues (e.g., Leu, Met, Ala, aromatics)

Pepsin, EC 3.4.23.1 and EC 3.4.23.2 Nonspecific digestLysyl endopeptidase (Lys-C endopeptidase), EC 3.4.21.50 C -terminal side of LysGlutamyl endopeptidase; (from S. aureus strain V8), EC 3.4.21. C -terminal side of Glu and Asp19

Peptidyl-Asp metalloendopeptidase (Asp-N endoproteinase), EC N -terminal side of Asp3.4.24.33

Clostripain, EC 3.4.22.8 C -terminal side of ArgChemical Cyanogen bromide C -terminal side of Met

2-Nitro-5-thiocyanobenzoic acid N -terminal side of Cyso -Iodosobenzoic acid C -terminal side of Trp and Tyr Dilute acid Asp and ProBNPS-skatole Trp

timal. As a general rule, the pH of the reaction milieuP RETREATMENT OF THE C LEAVAGE A GENTshould not alter the chemical integrity of the protein duringthe digestion and should not change during the course of Pretreatment of cleavage agents, especially enzymaticthe fragmentation reaction.agents, might be necessary for purification purposes to en-

sure reproducibility of the map. For example, trypsin used Temperature— A temperature between 25 ° and 37 ° isas a cleavage agent will have to be treated with tosyl- L- adequate for most digestions. The temperature used is in-phenylalanine chloromethyl ketone to inactivate chymotryp- tended to minimize chemical side reactions. The type of sin. Other methods, such as purification of trypsin by HPLC protein under test will dictate the temperature of the reac-or immobilization of enzyme on a gel support, have been tion milieu because some proteins are more susceptible tosuccessfully used when only a small amount of protein is denaturation as the temperature of the reaction increases.available. For example, digestion of recombinant bovine somatropin is

conducted at 4 ° because at higher temperatures it will pre-cipitate during digestion.P RETREATMENT OF THE P ROTEIN Time— If a sufficient amount of sample is available, atime course study is considered in order to determine theUnder certain conditions, it might be necessary to con- optimum time to obtain a reproducible map and avoid in-centrate the sample, or to separate the protein from added complete digestion. Time of digestion varies from 2 to 30substances and stabilizers used in the formulation of the hours. The reaction is stopped by the addition of an acidproduct if these interfere with the mapping procedure. that does not interfere with the tryptic map, or by freezing.Physical procedures used for pretreatment can include ultra-

Amount of Cleavage Agent— Although excessive filtration, column chromatography, and lyophilization. amounts of cleavage agent are used to accomplish a reason-Other pretreatments, such as the addition of chaotropicably rapid digestion time (i.e., 6 to 20 hours), the amountagents (e.g., urea) can be used to unfold the protein prior of cleavage agent is minimized to avoid its contribution toto mapping. To allow the enzyme to have full access tothe chromatographic map pattern. A protein-to-protease ra-cleavage sites and permit some unfolding of the protein, ittio between 20:1 and 200:1 is generally used. It is recom-is often necessary to reduce and alkylate the disulfide bondsmended that the cleavage agent be added in two or moreprior to digestion.stages to optimize cleavage. Nonetheless, the final reactionDigestion with trypsin can introduce ambiguities in thevolume remains small enough to facilitate the next step intryptic map as a result of side reactions occurring during thepeptide mapping—the separation step. To sort out digestiondigestion reaction, such as nonspecific cleavage, deamida-artifacts that might be interfering with the subsequent anal-tion, disulfide isomerization, oxidation of methionine resi-

ysis, a blank determination is performed using a digestiondues, or formation of pyroglutamic groups created from thecontrol with all the reagents except the test protein.deamidation of glutamine at the N -terminal side of a pep-

tide. Furthermore, peaks may be produced by autohydrolysis of trypsin. Their intensities depend on the ratio of tryp- Chromatographic Separationsin to protein. To avoid autohydrolysis, solutions of proteases may be prepared at a pH that is not optimal (e.g.,

Many techniques are used to separate peptides for map-at pH 5 for trypsin), which would mean that the enzyme ping. The selection of a technique depends on the proteinwould not become active until diluted with the digest being mapped. Techniques that have been successfully usedbuffer. for the separation of peptides are shown in Table 2 .

ESTABLISHMENT OF O PTIMAL D IGESTION C ONDITIONS Table 2. Techniques Used for the Separation of Peptides

Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC)Factors that affect the completeness and effectiveness of Ion-Exchange Chromatography (IEC)digestion of proteins are those that could affect any chemi-Hydrophobic Interaction Chromatography (HIC)cal or enzymatic reactions.Polyacrylamide Gel Electrophoresis (PAGE), nondenaturatingpH— The digestion mixture pH is empirically determined

to ensure the optimal performance of the given cleavage Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)agent. For example, when using cyanogen bromide as a Capillary Electrophoresis (CE)cleavage agent, a highly acidic environment (e.g., pH 2, Paper Chromatography—High Voltage (PCHV) formic acid) is necessary; however, when using trypsin as a

High-Voltage Paper Electrophoresis (HVPE)cleavage agent, a slightly alkaline environment (pH 8) is op-




3/6


In this section, a most widely used reverse-phase HPLC specimen chromatogram should be included with the USP(RP-HPLC) method is described as one of the procedures of Reference Standard or Reference Material for additionalchromatographic separation. comparison purposes. Other indicators may include visual

The purity of solvents and mobile phases is a critical fac- inspection of protein or peptide solubility, the absence of tor in HPLC separation. HPLC-grade solvents and water that intact protein, or measurement of responses of a digestion-are commercially available are recommended for RP-HPLC. dependent peptide. The critical system suitability parametersDissolved gases present a problem in gradient systems for peptide analysis will depend on the particular mode of where the solubility of the gas in a solvent may be less in a peptide separation and detection, and on the data analysismixture than in a single solvent. Vacuum degassing and agi- requirements.tation by sonication are often used as useful degassing pro- When peptide mapping is used as an identification test,cedures. When solid particles in the solvents are drawn into the system suitability requirements for the identified pep-the HPLC system, they can damage the sealing of pump tides cover selectivity and precision. In this case, as well asvalves or clog the top of the chromatographic column. Both when identification of variant proteins is done, the identifi-pre- and post-pump filtration are also recommended. cation of the primary structure of the peptide fragments in

the peptide map provides both a verification of the knownChromatographic Column— The selection of a chromat-primary structure and the identification of protein variantsographic column is empirically determined for each protein.by comparison with the peptide map of the USP ReferenceColumns with 100 ˚ A or 300 ˚ A pore size and silica supportStandard or Reference Material for the specified protein. Thecan give optimal separation. For smaller peptides, octylsi-use of a digested USP Reference Standard or Reference Ma-lane chemically bonded to totally porous silica particles 3 toterial for a given protein in the determination of peptide10 µm in diameter (L7) and of octadecylsilane chemicallyresolution is the method of choice. For an analysis of a vari-bonded to porous silica or ceramic microparticles 3 to 10ant protein, a characterized mixture of a variant and a Refer-µm in diameter (L1) column packings are more efficientence Standard can be used, especially if the variant peptidethan the butyl silane chemically bonded to totally porousis located in a less-resolved region of the map. The index of silica particles 5 to 10 µm in diameter (L26) packing.pattern consistency can be simply the number of major

Solvent— The most commonly used solvent is water with peptides detected. Peptide pattern consistency can be bestacetonitrile as the organic modifier to which less than 0.1% defined by the resolution of peptide peaks. Chromato-of trifluoroacetic acid is added. If necessary, add isopropyl graphic parameters—such as peak-to-peak resolution, maxi-alcohol or n-propyl alcohol to solubilize the digest compo- mum peak width, peak area, peak tailing factors, and col-nents, provided that the addition does not unduly increase umn efficiency—may be used to define peptide resolution.the viscosity of the components. Depending on the protein under test and the method of Mobile Phase— Buffered mobile phases containing phos- separation used, single peptide or multiple peptide resolu-

phate are used to provide some flexibility in the selection of tion requirements may be necessary.pH conditions, because shifts of pH in the 3.0 to 5.0 range The replicate analysis of the digest of the USP Referenceenhance the separation of peptides containing acidic resi- Standard or Reference Material for the protein under testdues (e.g., glutamic and aspartic acids). Sodium or potas- yields measures of precision and quantitative recovery. Re-sium phosphates, ammonium acetate, phosphoric acid, and covery of the identified peptides is generally ascertained bya pH between 2 and 7 (or higher for polymer-based sup- the use of internal or external peptide standards. The preci-ports) have also been used with acetonitrile gradients. Ace- sion is expressed as the relative standard deviation (RSD).tonitrile-containing trifluoroacetic acid is also used quite Differences in the recovery and precision of the identifiedoften. peptides are expected; therefore, the system suitability limits

Gradient Selection— Gradients can be linear, nonlinear, will have to be established for both the recovery and theor include step functions. A shallow gradient is recom- precision of the identified peptides. These limits are uniquemended in order to separate complex mixtures. Gradients for a given protein and will be specified in the individualare optimized to provide clear resolution of one or two monograph.peaks that will become “marker” peaks for the test. Visual comparison of the relative retention times, the peak

responses (the peak area or the peak height), the number of Isocratic Selection— Isocratic HPLC systems using a sin-peaks, and the overall elution pattern is completed initially.gle mobile phase are used on the basis of their convenienceIt is then complemented and supported by mathematicalof use and improved detector responses. Optimal composi-analysis of the peak response ratios and by the chromato-tion of a mobile phase to obtain clear resolution of eachgraphic profile of a 1:1 (v/v) mixture of sample and USPpeak is sometimes difficult to establish. Mobile phases for Reference Standard or Reference Material digest. If all peakswhich slight changes in component ratios or in pH signifi-in the sample digest and in the USP Reference Standard or cantly affect retention times of peaks in peptide mapsReference Material digest have the same relative retentionshould not be used in isocratic HPLC systems.times and peak response ratios, then the identity of theOther Parameters— Temperature control of the columnsample under test is confirmed.is usually necessary to achieve good reproducibility. The

If peaks that initially eluted with significantly different rela- flow rates for the mobile phases range from 0.1 to 2.0 mLtive retention times are then observed as single peaks in theper minute, and the detection of peptides is performed with1:1 mixture, the initial difference would be an indication of

a UV detector at 200 to 230 nm. Other methods of detec- system variability. However, if separate peaks are observedtion have been used (e.g., postcolumn derivatization), but in the 1:1 mixture, this would be evidence of the nonequiv-they are not as robust or as versatile as UV detection.alence of the peptides in each peak. If a peak in theSystem Suitability— The section System Suitability under 1:1 mixture is significantly broader than the correspondingChromatography 621 provides an experimental means for peak in the sample and USP Reference Standard or Refer-measuring the overall performance of the test method. The ence Material digest, it may indicate the presence of differ-acceptance criteria for system suitability depend on the ent peptides. The use of computer-aided pattern recognitionidentification of critical test parameters that affect data inter- software for the analysis of peptide mapping data has beenpretation and acceptance. These critical parameters are also proposed and applied, but issues related to the validation of criteria that monitor peptide digestion and peptide analysis. the computer software preclude its use in a compendial test An indicator that the desired digestion endpoint was in the near future. Other automated approaches have beenachieved is the comparison with a Reference Standard or used that employ mathematical formulas, models, and pat-Reference Material, which is treated exactly as the article tern recognition. Such approaches, for example the auto-under test. The use of a USP Reference Standard in parallel mated identification of compounds by IR spectroscopy andwith the protein under test is critical in the development the application of diode-array UV spectral analysis for identi-and establishment of system suitability limits. In addition, a fication of peptides, have been proposed. These methods




4/6


have limitations due to inadequate resolutions, co-elution of THE USE OF PEPTIDE MAPPING FOR GENETIC fragments, or absolute peak response differences between STABILITY EVALUATIONUSP Reference Standard or Reference Material and sample fragments. A validated peptide map can be used to assess the integ-The numerical comparison of the retention times and rity of the predicted primary sequence of a protein productpeak areas or peak heights can be done for a selected group (i.e., its genetic stability). It can also be used to determineof relevant peaks that have been correctly identified in the lot-to-lot consistency of the biotechnology-derived productpeptide maps. Peak areas can be calculated using one peak

process. Furthermore, the performance of the protein ex-showing relatively small variation as an internal reference, pression of the production system is best assessed by pep-keeping in mind that peak area integration is sensitive to tide mapping of the expressed protein. Peptide maps of baseline variation and is likely to introduce error into the protein produced at various times of the protein expressionanalysis. Alternatively, the percentage of each peptide peak process, including a point well beyond the normal proteinheight relative to the sum of all peak heights can be calcu- expression time, compared with those of a USP Referencelated for the sample under test. The percentage is then Standard or Reference Material, will evaluate the genetic sta-compared to that of the corresponding peak of the USP bility of the expression system as a function of time.Reference Standard or Reference Material. The possibility of Variant protein sequences can arise from a genetic varia-autohydrolysis of trypsin is monitored by producing a blank tion at the DNA level (point mutation) or as an error in thepeptide map, that is, the peptide map obtained when a translation process. A validated peptide map is the best ap-blank solution is treated with trypsin. proach to the detection of protein variants. However, theThe minimum requirement for the qualification of peptide limitations of the peptide mapping itself must be taken intomapping is an approved test procedure that includes system consideration. The detection of a structured variant is possi-suitability as a test control. In general, early in the regula- ble only if the corresponding peptide variant is easily iso-tory process, qualification of peptide mapping for a protein lated and characterized. To establish genetic stability will re-is sufficient. As the regulatory approval process for the pro- quire the use of a battery of biochemical methods, providedtein progresses, additional qualifications of the test can in-that the variants have properties different from those of theclude a partial validation of the analytical procedure to pro- “normal” protein.vide assurance that the method will perform as intended in

the development of a peptide map for the specified protein.VALIDATION

Analysis and Identification of Peptides

This section gives guidance on the use of peptide map- Critical Factorsping during development in support of regulatoryapplications. Validation of peptide mapping requires that a protocol be

The use of a peptide map as a qualitative tool does not designed, outlining in detail the experiment to be con-require the complete characterization of the individual pep- ducted and the criteria for acceptance of the map. Criteriatide peaks. However, validation of peptide mapping in sup- for acceptance of mapping include detection limit, specific-port of regulatory applications requires rigorous characteri- ity, linearity, range, accuracy, precision, and reagent stabil-zation of each of the individual peaks in the peptide map. ity. Reproducibility of the peptide map is a critical elementMethods to characterize peaks range from N -terminal se- in the utilization of such a map as an identity test and for quencing of each peak followed by amino acid analysis to confirming genetic stability. Those technical aspects of pep-the use of mass spectroscopy (MS). tide mapping that influence the reproducibility of the mapFor characterization purposes, when N -terminal sequenc- will be discussed.ing and amino acid analysis are used, the analytical separa- The setting of limits, with respect to quantification (peaktion is scaled up. Because scale-up might affect the resolu- area or height) and identification (retention times) for thetion of peptide peaks, it is necessary, using empirical data, selected group of relevant peaks is based on empirical ob-to ensure that there is no loss of resolution due to scale-up. servations. These limits detect significant differences be-Eluates corresponding to specific peptide peaks are col- tween the sample and USP Reference Standard or Referencelected, vacuum-concentrated, and chromatographed again, Material within a series of analyses.if necessary. Amino acid analysis of fragments may be lim- Another critical issue is the recovery of peptides and itsited by the peptide size. If the N -terminus is blocked, it may impact on peak area determination and reproducibility andneed to be cleared before sequencing. C -terminal sequenc- on the establishment of acceptance criteria. The recoverying of proteins in combination with carboxypeptidase diges- criteria address all aspects of test methodology, from diges-tion and matrix-assisted laser desorption ionization–time-of- tion to chromatographic conditions. Determination of pep-

flight (MALDI-TOF) MS can also be used for characterization tide recovery includes quantitative amino acid analysis, spikepurposes. addition, radiolabeling, and UV summation. An overall re-

The use of MS for characterization of peptide fragments is covery of about 80% is considered satisfactory. Recovery of by direct infusion of isolated peptides or by the use of on- individual peptides is more problematic and is handled on aline LC-MS for structure analysis. In general, it includes case-by-case basis. The critical factors considered in the vali-electrospray and MALDI-TOF analyzers as well as fast atom dation of a peptide map are as follows.bombardment (FAB). Tandem MS has also been used to se- Written Test Procedures— These procedures include aquence a modified protein and to determine the type of detailed description of the analytical method in which re-amino acid modification that has occurred. The comparison agents, equipment, sample preparation, method of analysis,of mass spectra of the digests before and after reduction and analysis of the data are defined.provides a method to assign the disulfide bonds to the vari-

Validation Protocol— A protocol is prepared that con-ous sulfhydryl-containing peptides.tains a procedure for test validation.If regions of the primary structure are not clearly demon-

Acceptance Criteria— The criteria can be minimal at thestrated by the peptide map, it might be necessary to de-early stages, but need to be better defined as validationvelop a secondary peptide map. The goal of a validatedstudies progress.method of characterization of a protein through peptide

mapping is to reconcile and account for at least 95% of the Reporting of Results— Results from the validation studytheoretical composition of the protein structure. are documented with respect to the analytical parameters

listed in the validation protocol.




5/6


Revalidation of the Test Procedure— If the method used ROBUSTNESSrequires alteration that could affect the analytical parameter previously assessed in the validation of the procedure, the Factors such as composition of the Mobile Phase , proteasetest procedure must be revalidated. Significant changes in quality or chemical reagent purity, column variation andthe processing of the article, in laboratories performing the age, and digest stability are likely to affect the overall per-analysis, in formulation of the bulk or the finished products, formance of the test and its reproducibility. Tolerances for and in any other significant parameter will require revalida- each of the key parameters are evaluated and baseline limitstion of the methods. established in case the test is used for routine lot release

purposes.Mobile Phase— The composition of the Mobile Phase isRequirements

optimized to obtain the maximum resolution of peptidesthroughout the elution profile. A balance between optimalresolution and overall reproducibility is desired. A lower pHmight improve peak separation but might shorten the life of P RECISIONthe column, resulting in lack of reproducibility. Peptidemaps at a pH above and below the pH of the procedure areIntratest Precision— This is a measure of the reproduc-compared to the peptide map obtained at the pH of theibility of peptide mapping. The two critical steps in peptideprocedure and checked for significant differences; they aremapping are fragmentation (i.e., digestion) and separationalso reviewed with respect to the acceptance criteria estab-of peptides. An acceptable precision occurs where the abso-lished in the validation protocol.lute retention times and the relative peak areas are constant

Protease Quality or Chemical Reagent Purity— A sam- from run to run, and the average variation in retention timeple of the USP Reference Standard or Reference Material for is small relative to that of a selected internal reference peak.the protein under test is prepared and digested with differ-The reproducibility of the map can be enhanced if a tem-ent lots of cleavage agent. The chromatograms for each di-perature-controlled column oven is used, if an extensivegest are compared in terms of peak areas, shape, and num-equilibration of the system is performed prior to the start of ber. The same procedure can be applied to other criticalthe test, if a blank (control digest mixture without protein)chemicals or pretreatment procedures used during sampleis run first to minimize “first run effects,” and if a USP Refer-preparation, such as reducing and carboxymethylationence Standard or a Reference Material digest is interspersedreagents.periodically with test samples to evaluate chromatographic

drift. Column Considerations— Column-to-column variability,The criteria for validation of the fragmentation step are even within a single lot, can affect the performance of the

similar to those described below for separation of peptides, column in the development of peptide maps. Column sizebut they are met for consecutive tests of a series of sepa- may also lead to significant differences. A USP Referencerately prepared digests of the protein under test. Standard or Reference Material of the protein under test is

The criteria for validation of the separation of peptides digested and the digest is chromatographed on differentstep include the following: lots of column from a single manufacturer. The maps are

1. The average standard deviation of the absolute reten- then evaluated in terms of the overall elution profile, reten-tion times of all major peaks for a set of consecutive tion times, selectivity resolution, and recovery. To evaluatetests of the same digest does not exceed a specified the overall lifetime of the column in terms of robustness,acceptance criterion. perform a peptide mapping test on different columns and

2. The average standard deviation of absolute peak area vary significantly the number of injections (e.g., from 10 for all fully resolved major peaks does not exceed a injections to 250 injections). The resulting maps can then bespecified percentage. compared for significant differences in peak broadening,

peak area, and overall resolution. As a column ages, an in-Intertest Precision— This is a measure of the reproduc-crease in back pressure might be observed that might affectibility of the peptide mapping when the test is performedthe peptide maps.on different days, by different analysts, in different laborato-

A sensible precaution in the use of peptide mapping col-ries, with reagents or enzymes from different suppliers or umns is to select alternative columns in case the originaldifferent lots from the same supplier, with different instru-columns become unavailable or are discontinued. Perform aments, on columns of different makes or columns of thepeptide mapping test using equivalent columns from differ-same make from different lots, and on individual columns of ent manufacturers, and examine the maps. Differences inthe same make from the same lot. Although it would beparticle shape and size, pore size and volume, carbon load,desirable, from a scientific perspective, to validate all of and end-capping can lead to significant differences in reten-these variables in terms of their impacts on precision, ation times, elution profile selectivity, resolution, and recov-practical approach is to validate the test using those vari-ery. Slight modifications in the gradient profile may be re-ables most likely to be encountered under operational con-quired to achieve equivalency of mapping when usingditions. Additional variables can be included when needed.columns from different manufacturers. [ NOTE —TheThe experimental design allows the analyst to make com-equivalency between instrumentation used for the validationparisons using peak retention times and areas that are ex-of the test and for routine quality control testing should bepressed relative to a highly reproducible internal reference considered. It might be preferable to use the same HPLCpeak within the same chromatogram. The relative peak areasystem for all applications. Otherwise, equivalency of theis expressed as the ratio of the peak area to that of thesystems is determined, which may require some changes ininternal reference peak. The relative retention time can bethe chromatographic test conditions.]expressed as the difference between the absolute retention

time and that of the reference peak. The use of relative Digest Stability— The length of time a digest can bevalues eliminates the need to make separate corrections for kept before it is chromatographed, as well as the conditionsdifferences due to injector-to-injector volumes, units of under which the digest is stored before chromatography, ismeasure for peak areas, column dimensions, and instrument assessed. Several aliquots from a single digest are stored atdead volumes. The variability in the retention times and different storage conditions and chromatographed. Thesepeak areas for the Intertest Precision experiments is expected maps are then evaluated for significant differences.to be slightly higher than the variability observed for In-tratest Precision .




6/6


mixture migrate at different speeds during electrophoresisREPRODUCIBILITYand thus are separated into discrete fractions. Electropho-retic separations can be conducted in systems without sup-Determination of various parameters indicated above is re-port phases (e.g., free solution separation in capillary elec-peated using the same USP Reference Standard or Referencetrophoresis) and in stabilizing media, such as thin-layer Material and test sample in at least two different laborato-plates, films, or gels.ries by two analysts equipped with similar HPLC systems.

The generated peptide maps are evaluated for significantdifferences.

CHARACTERISTICS OF POLYACRYLAMIDEGELS

The sieving properties of polyacrylamide gels are estab-lished by the three-dimensional network of fibers and poresthat is formed as the bifunctional bisacrylamide cross-linksadjacent to polyacrylamide chains. Polymerization is cata-1056 BIOTECHNOLOGY- lyzed by a free-radical-generating system composed of am-monium persulfate and N,N,N ′ ,N ′ -tetramethylethylenedi-DERIVED ARTICLES— amine (TEMED).

As the acrylamide concentration of a gel increases, its ef-POLYACRYLAMIDE GEL fective pore size decreases. The effective pore size of a gel isoperationally defined by its sieving properties, that is, by theELECTROPHORESIS resistance it imparts to the migration of macromolecules.There are limits to the acrylamide concentrations that canbe used. At high acrylamide concentrations, gels break

This chapter provides guidance and procedures used for much more easily and are difficult to handle. As the porecharacterization of biotechnology-derived articles by polya- size of a gel decreases, the migration rate of a proteincrylamide gel electrophoresis. Portions of the chapter that through the gel decreases. By adjusting the pore size of aare not harmonized with the other two pharmacopeias are gel, through manipulating the acrylamide concentration, themarked by the symbol . This chapter is harmonized with resolution of the method can be optimized for a given pro-the corresponding chapter in JP and EP . Other characteriza- tein product. Thus, a given gel is physically characterized bytion tests, also harmonized, are shown in Biotechnology- its respective composition of acrylamide and bisacrylamide.Derived Articles—Amino Acid Analysis 1052 , Biotechnology- In addition to the composition of the gel, the state of theDerived Articles—Capillary Electrophoresis 1053 , Biotechnol- protein is an important component of electrophoretic mobil-ogy-Derived Articles—Isoelectric Focusing 1054 , Biotechnol- ity. In the case of proteins, electrophoretic mobility is de-ogy-Derived Articles—Peptide Mapping 1055 , and Biotechnol- pendent on the pK value of the charged groups and theogy-Derived Articles—Total Protein Assay 1057 . size of the molecule. It is influenced by the type, the con-

centration, and the pH of the buffer; by the temperatureand the field strength; and by the nature of the supportINTRODUCTION material.

Denaturing Polyacrylamide Gel ElectrophoresisScope This method cited is limited to the analysis of monomeric

Polyacrylamide gel electrophoresis (PAGE) is used for the polypeptides with a mass range of 14,000 to 100,000 Da. Itqualitative characterization of proteins in biological prepara- is possible to extend the mass range by various techniquestions, for control of purity and for quantitative (e.g., gradient gels or particular buffer systems), but thosedeterminations. techniques are not discussed in this chapter.

Denaturing PAGE using sodium dodecyl sulfate (SDS)(SDS-PAGE) is the most common mode of electrophoresisPurpose used in assessing the pharmaceutical quality of proteinproducts and will be the focus of the example method. Typ- Analytical gel electrophoresis is an appropriate method ically, analytical electrophoresis of proteins is carried out inwith which to identify and to assess the homogeneity of polyacrylamide gels under conditions that ensure dissocia-proteins in drug substances. The method is routinely used tion of the proteins into their individual polypeptide for the estimation of protein subunit molecular masses and subunits and that minimize aggregation. Most commonly, for the determination of the subunit compositions of puri- the strongly anionic detergent SDS is used in combination fied proteins. with heat to dissociate the proteins before they are loadedReady-to-use gels and reagents are widely available on the on the gel. The denatured polypeptides bind to SDS, be-

market and can be used instead of those described in this come negatively charged, and exhibit a consistent charge-chapter, provided that they give equivalent results and that to-mass ratio regardless of protein type. Because thethey meet the validity requirements given below under Vali- amount of SDS bound is almost always proportional to thedation of the Test . molecular mass of the polypeptide and is independent of itssequence, SDS-polypeptide complexes migrate throughpolyacrylamide gels with mobilities dependent on the size of General Principle of Electrophoresisthe polypeptide.

The electrophoretic mobilities of the resultantUnder the influence of an electrical field, charged particlesdetergent–polypeptide complexes all assume the same func-migrate in the direction of the electrode bearing the oppo-tional relationship to their molecular masses. Migration of site polarity. In gel electrophoresis, the movements of theSDS complexes is toward the anode in a predictable man-particles are retarded by interactions with the surroundingner, with low molecular mass complexes migrating faster gel matrix, which acts as a molecular sieve. The opposingthan larger ones. The molecular mass of a protein can there-interactions of the electrical force and molecular sieving re-

fore be estimated from its relative mobility in calibratedsult in differential migration rates according to the sizes,SDS-PAGE and the occurrence of a single band in such a gelshapes, and charges of particles. Because of their differentis a criterion of purity.physicochemical properties, different macromolecules of a



Documents

0581-0586 [1055] Biotechnology-Derived Articles—Peptide Mapping