References Materials for Chemical Analysis || Reference Materials for“Life” Analysis

Reference Materials for Chemical Analysis Certification, Availability, and Proper Usage

Edited by Markus Stoeppler, Wayne R. WOK PeterJjenks

0 Wiley-VCH Verlag GmbH, 2001

154 I

5

Reference Materials for “Life” Analysis Edited by PeterJ Jenks

So far we have examined the reference material and calibration needs of the conven- tional analytical chemist. But as we move into the zIst Century, new technologies are driving the quality concepts established over the past 30 years by analytical chemists into other analytical disciplines, including microbiology and molecular biology. Indeed, the next major challenge may be the development of reference materials, or the provision of a form of reference material, that gives to the molecular biologist the same ability to control and standardize their determinations and quantification as a chemical reference material gives the analytical chemist.

There have been a number of attempts to achieve this objective, but so far the challenge has not been fully met. This Chapter will examine some of the conven- tional approaches and then go on to consider how recent developments in the use of the Polymerase Chain Reaction (PCR) with DNA for the identification of species and individual organisms by DNA analysis, sometimes known as “DNA Fingerprinting”, have identified a yet unrealized need for a new dimension of certified reference materials.

We will also review the development of chemical metrology in the pharmaceutical industry, an industry that is ever more closely related to molecular biology. The phar- macists first developed quality protocols and reference materials more than 40 years ago, quite independently of the activities of I S 0 and the REMCO Technical Commit- tee. Only in the closing years of the 20th Century have these two important groups begun to realize that they share common objectives.

5.1 Standard Reference Materials for Microbiological Assays

Jane Tang and Shung-ChangJong

When the American Type Culture Collection (ATCC) was founded in 1925, one of its chief roles was to be a source of standards. In this context we mean standard organisms, rather than standard materials or chemicals known as Reference Materi- als or Certified Reference Materials and issued in the USA by the National Bureau of Standards (NBS). The National Bureau of Standards evolved into NIST and the abbreviation SRM became a trade mark of NIST.

I 155 5. I Standard Reference Materials fo r Microbiological Assays

ATCC biological standards were known as Type Strains (TS), but as they are used in the same ways as, and fill many of the requirements for, RMs we have described them in this article as biological RMs.

In the 1920’s microbiological “type strains” were acquired to support taxonomical studies. Since then many organizations have used ATCC as a repository for other types of biological RMs, especially for use in official assays and standardized test procedures in public health, microbiology and clinical determinations. The range of biological RMs held by ATCC includes bacteria, viruses, fungi, animal and human cell lines, protozoa and, as we move into the 21st Century, also includes genetically modified organisms (GMOs).

Unlike chemicals, microorganisms are living materials, so growth conditions, the physiological state of the cells, and their metabolic activities will all influence the results of tests in which they may be used.

The reproducibility of a determination is a critical element of a standard method, so they are carefully written to ensure that they can be followed accurately by any qualified laboratory. The designated test biological RMs must not only be reproducible in their reactions to the performance tests, but they must also respond in a pre- dicable manner. In order to guarantee that the same strain of microorganisms could always be available, many of these biological RMs have been deposited with the ATCC and other culture collections around the world.

5.1.1 Standards for Official Assays and Tests

Around the world there are hundreds of official methods from various government and professional sources that specify ATCC microorganisms as biological RMs. The following six examples serve as examples of many organizations around the world that specify ATCC microbial cultures as biological RMs:

ASTM, The American Society for Testing and Materials, is a scientific and technical organization that develops standards for a wide variety of materials and products. It is the worlds largest source of voluntary consensus standards. AOAC, The Association of Official Analytical Chemists International, describes methods for analyzing a wide range of materials. These methods are often adopted by other organizations and government regulatory agencies as standards. NCCLS, National Committee for Clinical Laboratory Standards provides a medium for communication among professionals in healthcare, government, and industry in developing guidelines for clinical laboratory practices. USDOD, The United States Department of Defense describes testing procedures for any product or material for use in the military. USFDA, The United States Food and Drug Administration details its standard methods in the Code of Federal Regulations, Title 21.

156 5 Reference Materials far “L$e”Analysis

USPC, The United States Pharmacopeia is a compendium of information on drug products. The standards set forth in the USP are recognized in both federal and state law.

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Other organizations whose standard procedures specify ATCC biological RMs include:

(I) American Association of Cereal Chemists (AACC) (2) American Association of Textile Chemists and Colorists (3) American Wood-Preservers’ Association (4) British Pharmacopoeia (BP) (5) British Standards Institution (BSI) (6) International Organization for Standardization (I SO)

(7) Radio Technical Commission for Aeronautics (8) Technical Association of the Pulp and Paper Industry (9) United States General Services Administration

For a comprehensive list of quality control and reference strains refer to ATCC QC and Reference Strains, 1st edition, 1997.

5.1.2 Quality Management of Biological RMs

Effective management of microbiology collections requires that the cultures meet current and future international standard requirements of biotechnology industries. The knowledge and competence of the scientists performing cell culture work obviously impact the quality of the cultures.

For biological RMs, quality control is the total accumulation of all means and activities by which desirable properties of each microbial culture are precisely and consistently maintained throughout production, storage, shipping, and customer use. To confirm such quality, ATCC have developed a quality system that uses certain principles from International Standard Organization (ISO) standards for quality systems and Good Laboratory Practices (GLPs) and the pharmaceutical industry’s Good Manufacturing Practices (GMP). All of these principles require written standard operating procedure (SOP) manuals, the most important documents for standardization and organization of all observations, tests, and functions in the collections.

ATCC issues “Certificates of Analysis” with cultures. Although ATCC Certificate of Analysis do not meet the specific requirements of IS0 Guide 31, they do show that the biological RM culture has been authenticated and/or specific characteristics have been verified. Each ATCC Certificate of Analysis is lot-specific and includes expiration dates, the specific seeds used for propagation, and selected biochemical and morphological characteristics that are indicative of the culture. ATCC also aims to provide a Product Sheet for each microbial culture, with instructions for propagation, special features of the organism, and any unusual observations or properties.

Microbial cultures at ATCC are normally preserved by lyophilization (freeze-drying) and/or storage over liquid nitrogen at -180°C. A seed stock system is used to maintain the distribution stock.

I 157 5.1 Standard Reference Materials for Microbiological Assays

The source ofall biological RMs i s “new accessions”, i.e. a new organism. Once one i s

received, it i s grown in fresh medium and a set of seed stock vials are made along with a distribution batch of vials. Quality control is performed on the seed material and the distribution batch. When the first distribution batch is exhausted, another new lot i s

made by propagating only from the seed material. The seed stock i s always the closest material to the original deposit available for propagation and verification.

The seed stock system allows the minimization of frequent subculturing and passages of microorganisms. With repeated culturing there are risks of contamination and mutation, which will alter the characteristics of the microorganisms. T h i s is especially crucial for maintaining biological RMs, thus every effort i s made to minimize transfers and passages of cultures. Users of biological RMs need to pay particular attention to the risk of change in the identity of the organism caused by subculturing, mutation or contamination. A recent personal communication from Masters and coworkers on human cell line cross-Contamination showed that HeLa cell lines used by a number of workers for work published in the literature were not, when checked by DNA analysis, what the authors thought they were.

To ensure microbial strains are viable and pure a suite of morphological, biochemical, and cytochemical tests are used to confirm characteristics specific to their taxons. A number of commercially available rapid identification kits are also employed for some common genera. In addition to these taxon specific tests, many of the cultures are tested for their fatty acid methyl ester (FAME) profiles using the commercial MIDI system. The FAME profiles can be compared to the MIDI database for species identification/confirmation purposes. The Biolog system, which yields a metabolic fingerprint of an organism, i s another alternative for rapid identification.

In recent years molecular taxonomic tests have been introduced. There are a number of molecular methods available. However, due to the volume and variety of organisms it i s important that any molecular method meet the following criteria:

(I) Be cost effective (2) Be reproducible (3 ) Provide data that can be converted to a database accessible format (4) Be robust enough to work with a variety of different organisms (5) Be rapid and simple to perfom

Based on these criteria DNA fingerprinting has started to be used as part of the quality control procedure for Biological RMs. Comparison of DNA profiles generated from the seed material and the distribution batches assure no alteration occurred during the propagation and preservation processes. Due to the diverse nature of the organisms no single system can work well with all strains. Therefore development to investigate other PCR-fingerprinting methods as possible molecular taxonomic characterization of microbial cultures i s ongoing at ATCC.

The goal i s to achieve a polyphasic taxonomic approach for authentication and quality control of microbial strains. Each morphological, physiological, cytochemical, and molecular method has limitations. Combining and comparing the results enable us to obtain a more accurate and complete picture ofthe microorganism’s identity.

158 5 Reference Materialsfor “L@”Analysis

5.2

Certified Microbiological Culture Materials

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Although the work of ATCC and others has done much to ensure the reproducibility and even demonstrate some traceability of microbiological reference materials the development of microbiological Certified Reference Materials (CRMs), certified for number of viable life forms is seen as important for control analyses of water and food. Somewhat of a “holy grail” the development of such CRMs has long been ham- pered by the unstable concentration and insufficient homogeneity of viable organisms in the materials.

This Section describes one of the first successful attempts, by a multi national project team funded by the European Commission’s DG XII, to produce and certify a microbiological CRM that is both fit for purpose and meets the requirements of the I S 0 Guides. The first results are two bacterial strains, Enterococcus fueciurn (CRM 506) and Sdmoneha typhimuriurn (CRM 507) (Janning et al. 1995). Both are available as part of the EU “ B C R range of CRMs.

The authors demonstrated that the main problems could be overcome by stabilization of the target microorganisms by spray-drying them in a preservation medium based on sterile evaporated milk. The resulting highly contaminated milk powder had, depending on the test strain and the contamination level of the milk suspension, a final contamination level of 104-107 colony-forming particles per gram (cfp/ 8). After a period of stabilization while stored at a temperature of +5’C or -2o”C, the contaminated milk powder was mixed with sterile milk powder to achieve the desired contamination level of the final material. Then, gelatin capsules were filled with 0.2-0.3 g of the mixed powder. The capsules stored at -20°C. Fig. 5.1 shows the most important steps of the production process.

Cultivation of bacterial strain

Suspension in evaporated milk

Spray drying

U

U

Highly contaminated milk powder U

Mixing with sterile milk powder U

Filling of gelatin capsules U

Storage at-20°C Fig. 5.1

a microbiological RM. Flow diagram: Production of

I 159 5.2 Certified Microbiological Culture Materials

The homogeneity determination of the bacteria in the materials is performed by viable count followed by statistical evaluation of the counts of sub-samples from the same capsule solution and of total counts of different capsules of one batch. An example for the homogeneity determination for a batch of capsules containing Enterococcusfaecium is also presented in (Janning et al. 1995).

After the performance of collaborative studies organized by the RIVM (National Institute of Public Health and Environmental Protection, Bilthoven, The Nether- lands) and SM&T certification studies were performed by a group of eleven or twelve qualified European laboratories. These examined simultaneously the materials using standardized methods following a very strict analytical protocol. Table 5.1 below shows data for CRM 506 (Mooijman et al. 1999).

The development of these CRMs has shown that it is possible to produce CRMs for public health microbiology and that whilst such a CRM can largely meet the I S 0 Guides requirements it does not completely satisfy all the requirements of CRMs. It has also demonstrated that their manufacture and certification places considerable challenges before the producers.

I S 0 7899/2 (1984) KFAa 76 71-81

Tab. 5.1

(WR63) in CRM 506 from artificially contaminated milk powder (Mooijman et al. 1999) Certified number concentration of colony forming particles of Enterococcus faeciurn

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160 5 Reference Materialsfor “Lif”Ana/ysis

5.3 Reference Materials for DNA Analysis Barbara C. Levin and DennisJ. Reeder

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Mention has already been made, in Section 5.1, of the use of DNA-based techniques for the control of microbiological DNA. In parallel to the work of ATCC the USA National Institute of Standards and Technology (NI ST) developed three Standard Reference Materials (SRM 2390, SRM 7,391 and SRM 2392) to provide quality assurance in the analysis of human DNA. Two of these SRMs, the DNA Profiling SRM 2390 and the PCR-based DNA Profiling SRM 2391, are intended for use in forensic and paternity identifications, for instructional law enforcement, or non-clinical research purposes. They are not intended for any human/animal clinical diagnostic use. The third NIST SRM - 2392 - is for standardization and quality control when sequencing the entire or any segment of human mitochondria1 DNA (16 569 base pairs) for forensic identification, disease diagnosis or mutation detection.

5.3.1 SRM 2390

The first human DNA SRM developed by NIST was designed in the late 1980’s to standardize Restriction Fragment Length Polymorphism (RFLP) procedures which at that time were very new developments in the application of DNA to forensic analysis.

SRM 2390 uses the HaeIII restriction enzyme to examine highly polymorphic human DNA loci containing variable numbers of tandem repeats (VNTR) (Reeder 1991). These sequences resemble mini-satellites in that they are com- posed of repeats of a core sequence and different individuals have different numbers of repeats. However, unlike mini-satellites, there is usually only one VNTR of a given type in an individual. Therefore, VNTR analysis gives patterns that are simpler and easier to analyze than DNA fingerprints, but several different probes must be used to be convincing. The use of RFLP depends on the presence of a polymorphism which can be as small as a single nucleotide change or as large as the tandem repeat segment in a particular DNA sequence. In this case, the HueIII restriction cleavage sites flank the tandem repeat sequences and will produce different length DNA fragments depending on the number of tandem repeats in an individual. In 1991, the Federal Bureau of Investigation (FBI), the Royal Canadian Mounted Police and many US State and local laboratories were using Hue111 in their RFLP procedures for forensic and paternity testing (Budowle 1988; Eisenberg et al. 1991). The restriction enzyme HueIII was chosen since it is an extremely hardy enzyme, recognizes a four-base sequence (GGCC), cuts approximately every 256 bases, produces smaller DNA fragments, and has better resolution of the fragment size variants than other enzymes, e.g. PstI, HinfI, A h 1 (Budowle 1988). In addition, DNA methylation does not interfere with HaeIII efficiency to recognize or cleave its restriction site.

I 5.3 Reference Materialsfor DNA Analysis

SRM 2390 provides quality control for the following procedures:

(I) extracting the DNA from the cell pellet ( 2 ) quantifying the extracted DNA (3) determining the restriction enzyme cutting efficiency (4) separating the DNA electrophoretically (5) blotting the separated DNA onto a nylon membrane using an alkaline trans-

fer solution (6) hybridizing the sample DNA to radioactively labelled DNA probes (7) exposing the membrane to X-ray film (8) imaging the developed autoradiogram with a computerized imaging system

to determine band sizes

The human DNA in this SRM comes from a female (K562) cell line and a male (TAW) source. The cells and DNA are provided as:

(I) a cell pellet containing approximately 3 x 10' cells (2) extracted genomic DNA (3) HaeIII-digested DNA

In addition, the SRM contains a DNA molecular size standard for sizing the allele fragments; a set of quantitative DNA standards in concentrations of 6-250 ng/6 pL; and a visualization marker set which produces twelve bands ranging from 594 to 35 937 base pairs and which is used to assess the DNA separation on the electrophoretic gel.

Twenty-nine laboratories, including NET, participated in an inter-laboratory evaluation of SRM 2390. All laboratories received the above components plus a set of four bloodstains [DzS44 (YNH24) was the target probe (Nakamura et al. 1987)], instructions, and a questionnaire. All laboratories used the basic FBI protocol (Budowle and Baechtel 1990) or modifications thereof for RFLP-sizing. The participants were asked to provide calculated band sizes for locus DzS44 and any other probes that they were willing to examine. Variations were noted among the participating laboratories in the type of agarose, sources of chemicals and probes, methods for labelling probes and the electrophoretic and imaging equipment.

Thirty-four sets of data on DzS44 were sent to NIST. Nineteen laboratories also reported results on D&jg (PHjo), 17 tested D1oS28, 16 laboratories examined D1S7 (MSI), and 12 provided results on D17S7g (VI). The certified values (number of DNA base pairs) for SRM 2390 are shown on the NIST SRM 2390 Certificate.

5.3.2 SRM 2391

The second NET human DNA SRM is a PCR-based DNA Profiling Standard. The PCR was first described by Saiki et al. (1985,1989). Since then it has developed into a highly versatile and widely used detection, identification, manipulation and analysis tool in molecular biology, including DNA profiling. In brief, two short synthetic oligonucleotides, or primers, are used to define an intervening DNA sequence

162 5 Reference Materials for “L@”Ana/ysis

which is then amplified in vitro using a thermo stable DNA polymerase, DNA pre- cursors, suitable reaction buffer and a thermal cycler. Correct amplification requires a specific interaction of the primers with their target sequence. The specificity of the reaction is generally assessed by gel electrophoresis or hybridization which deter- mines whether the size or internal sequence is consistent with correct amplification.

Because of the complexity of the complete DNA profiling procedure, including the PCR stage, NIST developed and certified a specific SRM for use in procedures based on those of the FBI (PCR Typing Protocols, FBI Laboratory, Quantico, VA, 121 12/94). This procedure employs the genetic locus DIS8o (pMCTIr8) (Nakamura et al. 1988) and specific primers (Kasai et al. 1990). The procedures covered by the use of SRM 2391 are:

I

(I) extraction of DNA from cells spotted on special filter paper (2) amplification of the DNA from the DIS8o locus (Kasai et al. 1990) (3) separation of the amplified alleles by electrophoresis (4) silver staining the gel or fluorescent dye detection plus image analysis

This SRM contains human cells from two cell culture lines from which DNA can be extracted, genomic DNA from those two cell lines plus eight individuals, PCR- amplified DNA from the two cell lines plus four of the eight individuals, a DIS8o allelic ladder for characterization of amplified DNA, and a DNA size marker to assure proper electrophoretic separations.

Prior to certification, various components of this SRM were tested by 20 laboratories. The 1995 certified values for D1S80 alleles, derived from pooled results from analyses performed at two laboratories plus NIST are shown in the certificate supplied with the SRM.

5.3.2.1 Recertification of SRM 2391 Since 1995, when SRM 2391 was issued, technologies, particularly the use of Short Tandem Repeat (STR) loci for human identity, have developed rapidly. The initial certificate for SRM 2391 had limited STR information on only four STR loci (tetra- plex system), HUMTHOI (THoI), HUMFI~AOI (FI~AoI), HUMVWFA~I (vWA) and HUMFESFPS (FES/FPS). The forensic community requested that SRM 2391 include STR information for all the DNA samples and that the STR information include the FBI’s CODIS (Combined DNA Index System) core loci. The CODIS core loci are HUMCSFIPO (CSFIPO), D3S1358, DgS818, D7S820, D8S1179, DqS317, DrGSgjg, DI8Sg1, DZISII, FGA, THoI, HUMTPOX (TPOX), and vWA. In addition, F13Ao1, HUMBFXIII ( F I ~ B ) , FES/FPS, HUMLIPOL (LPL), DrS80, DQAI, and Amelogenin were also analyzed. Therefore, a total of 20 loci were analyzed for the recertification of SRM 2391 and seven laboratories contributed data on specific genetic loci.

The STR data generated at NIST were based on amplifjmg 1.0 ng of the genomic DNA with fluorescent labelled primers. The PCR amplified products were analyzed by slab gel electrophoresis followed by imaging with a Molecular Dynamics FluorI- mager 595 or by capillary electrophoresis using a PE-ABI 310 Genetic Analyzer.

I 163 5.3 Reference Materials for DNA Analysis

When possible, both methods of analysis were used. The other laboratories used either a PE-ABI 377 sequencer, PE-ABI 310 Genetic Analyzer, or a Hitachi FMBIO imager.

Tables in the certificate provided with SRM 2391 provide the data for AmpliType@ HLA DQA1, AmpliType@ PM types including the Amelogenin typing information and information values for the 17 STR loci.

5.3 .3 SRM 2392

The third human DNA SRM developed by NIST was designed to meet the need for quality control when amplifying and sequencing human mitochondrial DNA (mtDNA).

Human mitochondrial DNA has been completely sequenced and found to be cir- cular double-stranded molecules containing 16 569 base pairs (Anderson et al. 1981). There i s very little wasted space in this genome, since there are no introns, no untranslated leaders or trailers in the encoded mRNA and few if any, bases between structural genes. The human mitochondrial genomes encode two rRNAs, 22 tRNAs and 13 proteins. Each human cell can have a few dozen to several thousand molecules of mtDNA (Bogenhagen and Clayton 1974; King and Attardi 1989). Sequence analysis of mtDNA is being used by the forensic community for human identification especially in those cases where genomic DNA is highly degraded or non-existent (Holland et al. 1993, 1995). Forensic analysis to distinguish between individuals is primarily based on the considerable sequence variation found in the two hypervariable regions (HVI, HV2) located in the non-coding displacement loop (D-loop). The medical community i s also using sequence analysis of mtDNA for diagnoses of diseases associated with specific mutations and deletions (Wallace 1992). A third area of research which is largely unexplored and which needs sequence analysis is the examination of the mutagenic effects of chemical and phy- sical agents on mtDNA (Grossman 1995; Ballinger et al. 1996).

5.3.3.1 DNA Source Most investigators examining human mtDNA have used the numbering system of Anderson et al. (1981) and have compared their findings to the sequence first described in 1981. However, the DNA sequenced by Anderson is not available for use as a positive control during actual experiments; whereas, NIST SRM 2392 is available. This SRM provides quality control when amplifying and sequencing human mtDNA (Levin et al. 1999). SRM 2392 includes DNA from two lymphoblastoid cell cultures (CHR and 9947A) and cloned DNA from the CHR HVI region which contains a C-stretch and i s difficult to sequence. The mtDNA sequence (but not the DNA) of a third human template GMo3798 is provided for comparison. All three DNA templates are from apparently normal individuals. Fifty-eight unique primer sets allow any area or the entire mtDNA (1G569 base pairs) to be amplified and sequenced. By using this number of sets of primers, about 58 overlapping fragments are generated, ensuring complete coverage of the whole genome while

164 5 Reference Materials for “Life”Analysis

sequencing. While none of the differences in these three templates correspond to published mutations associated with specific diseases (Wallace 1gg2), some of these differences did result in anima acid changes when compared with that published by Anderson et al. (1981). Table 5.2 shows the mtDNA differences compared to the Anderson sequence that were found at NISTwith all three templates. There were 13, g, and 4 differences in the non-coding regions of templates CHR, 9947.4, and GMo3798, respectively and 33, 23, and 19 differences in the coding regions of templates CHR, 9947A and GMo3798, respectively.

I

5.3.3.2 Three laboratories in addition to NIST participated in an inter-laboratory evaluation of the CHR template. All of the laboratories essentially followed the NIST protocol. Three of the four labs found essentially the same polymorphisms. Laboratory 4, who had less experience with sequencing mtDNA, did find differences that the other laboratories did not observe. The differences noted by Laboratory 4 confirm and emphasize the need for a standard reference material for sequencing mtDNA. Had Laboratory 4 had run NIST mtDNA SRM 2392 simultaneously with their unknown sample, they would have realized that they were finding an undue number of differences and could have reexamined their procedures to try to determine the reason for these differences.

Inter-Laboratory Evaluation of SRM 2392

5.3.4 Summary

These three NIST SRMs have a number of important quality control applications for forensic DNA profiling, medical diagnostics and mutation detection. The main applications are summarized below

SRM 2390 can be used in several different ways depending on quality assurance requirements. Components of the SRM are designed to provide assurance that each step of the RFLP protocol is functioning properly, but they can also be used for trouble shooting, for calibrating equipment, and for testing the efficacy ofnew lots ofreagents.

SRM 2391 is designed to provide quality assurance to laboratories that perform DNA profiling using PCR methods. This SRM can be used to verify that each step of the analysis system is operating correctly and within the proper limits.

SRM 2392 is designed to provide quality control when amplifying and sequencing any region or the entire 16 569 base pairs which comprise human mtDNA. It can also be used as a control when amplifying and sequencing any type of DNA. The two DNA templates (CHR and 9947A) included in the SRM have characteristic polymorphisms throughout the non-coding and coding regions of the DNA and therefore, can serve as positive controls during PCR amplification and sequencing. None of these polymorphisms correspond to any of the published base pair changes that have been correlated with specific diseases. Corroboration ofthe SRM results provides quality assurance that any unknown DNA is also being amplified and sequenced correctly.

Primer set

Ampli- Length Comparison with Anderson fied ofam-

region" plijed region

Amino Ander-

5.3 Reference Materials for DNA Analysis

Primer sets used for PCR amplification o f human mtDNA and differences with the Tab. 5.2 anderson sequence found in three templates at NlSTfor SRM 2392

1470 tart 39 G

C C A

G C(ins)

-

-

start 39

G C

-

-

-

G G

C(ins) C(ins) C(ins)

end 473

start 421

G end 846

-

start 55 -

- 73 93 195 204 207 214 263

309.1 309.2 315.1 C(ins)

end 436

start 429 A G

end 891

C (ins) end 454

start 415 A G

end 834

start 818 end 1146

start 938 end 1323

start 1295 G E

end 1654

2 361-921 561 709 750

G A

3 756-1425

4 873-1425

5 1234-1769

670

553

536

NONE start 778 start 778 end 1197 end 1278

start 931 start 928 end 1335 end 1377

start 1279 start 1275 G G A

end 1738 end 1741

-

NONE

1438 1719

A G

6 1587-2216 630 start 1632 start 1632 start 1649

end2106 end2106 end2031

start 1691 start 1686 start 1715

end2170 end2173 end2097

start 2036 start 2018 start 2069 end 2213 end 2217 end 2212



end 2920 end 2956 end 2915

A - -

A - -

G - -

1719* G

7 1657-2216 560 1719d G

8 1993-2216

9 2105-2660

10 2417-3006

224

556

590

NONE

NONE

2706 A

166 5 Reference Materialsfor “Li$e”Analysis

Tab. 5.2 (Continued) I

Primer 1 Ampli- fied

region‘

length of amplified region

11 2834-3557 724

12 2972-3557 586

13 32343557 324

14 3441-3940 500

15 36354162 528

16 3931-4728 798

17 41834728 546

18 4392-4982 591

19 4447-4982 536

20 4797-5553 757

21 4976-5553 578

22 5318-5882 565

23 5700-6262 563

Comparison with Anderson I

9947A GMo3798

Ander- son No.

Amino acid

change

3010 3106/3107

3106/3107d 3423

3423d

NONE

NONE

4135

NONE

4769

4769d

4985 5186

518Gd

NONE

NONE

G C

C G

G

T

A

A

G A

A

start 2861 start 2869 start 2888 - A

del del del end3350 end3373 end3243

start 2999 start 2999 start 3031 del del del

end 3422 end 3460 end 3425


end 3548 end 3545 end 3541


start 3667 start 3662 start 3725 end4126 end4061 end4044

start 3964 start 3968 start 3987 Try+

end 4399 end 4427 end 4436



end 4860 end 4935 end 4877


end4958 end4921 end4931


-

E T T Silent

T T T Silentd

- C - His

G G G Silent

G G G Silentd

A A A Silent G - - Silent

end5327 end5324 end5215


end5516 end5521 end5400



G - - Silentd

I 167 5.3 Reference Materialsfor DNA Analysis

Jemplate Jemplate Template Ander- son No. bp CHR 9947A CMo3798 I

Tab. 5.2 (Continued)

Amino acid

change

Primei

i 24

Ampli- Length

region" plifed 7 region

5999-6526

25 6242-6526

26 6426-7030

27 6744-7255

28 7075-7792

29 7215-7792

30 7645-8215

31 7901-8311

32 8164-8669

33 8539-9059

34 8903-9403

35 9309-9848

528

285

605

512

718

578

571

411

506

521

501

540


6221 6371

637Id

6791 6849"

6849d" 7028

NONE

7645

7861

NONE

8448 8503

8860

9315

9559

start 6043 start 6058 start 6047 T C - - Silent C T - Silent -

end6442 end6503 end6456


end6520 end6520 end6520


C T - - Silentd

- A G - Silent

end 6916 end 6930 end 6885 Ala"


A G(0.3A)h+: - - Thr+

A G(0.3A)h+c - - Thr-t C T - - Ala'k

end 7215 end 7221 end 7177 Silent



end 7722 end 7769 end 7706


end 8149 end 8155 end 8156



T - C - Silent

T - C - Silent

T - C - Met+ T C - - Thr

end 8646 end 8641 end 8637 Silent


end 9019 end 8999 end 8991 Ala


end 9380 end 9381 end 9370 Leu


end 9823 end 9827 end 9800 Pro

A G G G Thr+

T - C - Phe-t

G C C C A r g t

168 5 Reference Materiaisfor “Life”Ana1ysis


36

37

38

39

40

41

42

43

44

45

46

47

48

9449-9995

9 7 5 4 10275

10127- 10556

10386- 11166

10704- 11267

11001- 11600

11403- 11927

11760- 12189

11901- 12876

12357- 12876

12601- 13123

12793- 13343

13188- 13611

547

522

430

781

564

600

525

430

976

520

523

551

424

Amino Ander- sonNo. bp CHR 9947A CMo3798 change

Template Template Template acid I 9559d

NONE

NONE

NONE

NONE

11335

11719

11878

NONE

12612 12705

12705~~

NONE

13572

start 9476 start 9485 start 9479 G C C C A r g i

end 9964 end 9940 end 9911 Prod






end 11461 end 11517 end 11497


end 11795 end 11853 end 11855


end12159 end12164 end12163



T C C C Silent

G A - - Silent

T C - - Silent

A G - - Silent C T - - Silent

end12769 end12849 end12775


end 13102 end 13045 end 13024



end13587 end13593 end13590

C T - - Silentd

T - C - Silent

I 169 5.3 Reference Materials for DNA Analysis

Tab. 5.2 (Continued)

region

49

50

51

52

53

54

55

56

13518- 13935

13715- 14118

13899- 14388

14189- 14926

14470- 14996

14909- 15396

15260- 15774

15574- 16084

418

404

490

738

527

488

515

511


Amino Ander- son No.

13572d 13702 13708 13759

13966

1396Gd 14199 14272 14365

14272d 1436Sd 14368 14470 14766

14766d

15326

1532Gd 15 646

1564Gd

start 13541 start 13541 start 13571 T - C - Silentd

G A - - Arg G C C C Gly-

G - A - A l a i

end 13910 end 13921 end 13900 Thr Ala- Thr


end 14094 end 14110 end 14104 Ala


A G - - Thr+

A G - - T h r i G T T T Alad G C C C Pro+ G C C E Thr

end 14369 end 14374 end 14342 P h e i Leu

Silent

start 14216 start 14216 start 14240 G C C C Phe- G C C C Leud G C C c Silentd T C - - Phe+ T E C E Leu

end 14699 end 14806 end 14698 Silent I l e h Thr

T - C C Ile-

Thrd


end 14957 end 14972 end 14956


end 15380 end 15373 end 15359 Ala


A G G G Thr-t

A G G G Thr+

end 15754 end15950 end 15723 Silent


end 16056 end 16058 end 16030

C - - T Alad

C - - T Silentd

170 5 Reference Materials for “L@”Ana/ysis


Primer set

Ampli- Length Comparison with Anderson fied ofam-

region” plified region

Amino

9947A GMo3798 change Template Template acid Ander-

son No.

57 15971-

(HV1) 16451

58 16097-336

-21 16133-40 M13‘ cloned DNA

481 16183 16189 16311 16357

809 16183d 1618gd 16311d 16357d 16519

477 16183d 16189d 16193.1 16223 16278 1651gd

a) Numbers correspond to Anderson sequence

B) Base pair change came before the readable

E) Base pair change came after the readable

-) Base pair same as in Anderson sequence.

(Anderson et al. 1981)

sequence.

sequence.

h“) PossibIe heteroplasmic site. This heteroplasmy seen in the mtDNA from the first CHR cell culture line is not seen in the mtDNA from the second CHR cell culture line. It is DNA from the second CHR cell culture line which is supplied in NIST SRM 2392.

c) This primer is used for sequencing the cloned DNA of the HVI region.

d) Change also seen in previous primer set. Start)

end) Start of readable sequence.

End of readable sequence.

start 16014 start 16011 start 16004 A C - T C -

T E C -

T E C

-

-

-

end 16193 end 16430 end 16403

start 16125 start 16130 start 16151 A C -

T C - T E C -

T E C T E C C

- -

-

end16193 end59 end103

start 16131 A C ND ND T C

C T c T T C

C(ins)

end 40

I 171 5.4 Future Developments in Molecular Reference Materials

5.4 Future Developments in Molecular Reference Materials PeterJJenks and Vanessa Dekou

DNA analysis has become an invaluable tool having very many practical applications that aim to open new frontiers in science. The sequencing of the human genome will provide information that could be applied to the study of genetic disorders as well as complications affecting the behaviour of humans at molecular level.

Cloning genes has become an extensively used technique, with applications ranging from production of proteins (e.g. human growth protein) to production of improved new generation vaccines.

In the future it may be possible to treat genetic defects by transplanting cloned normal genes into patients whose own genes are damaged. Already the use of gene transfer techniques to produce so-called genetically modified organisms, or “GMOs” in agriculture is extensive and has become rather controversial.

Initial efforts by workers at the Institute for Reference Materials and Measure- ment (IRMM), Geel, Belgium, to produce certified reference materials for GMOs have demonstrated that the provision of suitable reference materials is not easy and that together with the development of suitable analytical methods there are many challenges to be solved ahead. The first two examples produced jointly by the IRMM in Belgium and Flulta Chernie AG in Switzerland were based on “Round-Up ReadyTM Soya and BT 17GTM Maize. The reference materials are needed to validate EU and Swiss regulations which permit non-GMO products to be contaminated by up to 1 % GMO material and still be accepted.

In each case the grains were supplied by the producers as “100 % GMO”. At the IRMM they were milled, heat-treated to inactivate enzymes found in the grain and then blended with non-GMO materials to produce a range of CRMs, from I % GMO to 5 % GMO. Certification was undertaken by 25 labs (soya) and 22 labs (maize) for the PCR qualitative screening method. Whilst the CRM prove completely suitable for the intended purpose later use in quantitative PCR analysis suggests that much more controlled and rigorous procedures may be needed if CRMs suitable for quantitative PCR are to be realized. It is believed that contamination of the exterior of the grains by both non-GMO or other GMO types, the breakdown of DNA in proces- sing, especially if site-specific and the consequences of the polyploidal nature of plant genomes must all be taken into consideration when developing matrix RMs certified for GMO (Pauwels and Schimmel, personal communication).

The detection of modified, or recombinant DNA (rDNA) in processed foods will pose even greater challenges. In the European Union, foods and food ingredients produced by means of, or containing more than I % of GMO material, have to be labelled declaring the fact. Detecting rDNA from GMO ingredients within processed foods will require the development of novel techniques, although some evidence exists to show that rDNA is reasonably stable and can be extracted in sufficient quantity to be amplified (Straub et al. 1999). Certainly Matrix CRMs will be needed, but so far no organization has done more than consider the need.

172 5 Reference Materials for “L@”Analysis

The efforts by NIST to produce a PCR SRM have already been described. Whilst SRMs 2390 and 2391 both meet the specific requirements of forensic DNA determination by a specific PCR-based method and. SRM 2392 can be used as a general control of PCR-based methods, but it is unrealistic to believe that similar reference materials will be developed for each application of PCR.

Even inside the controlled conditions of a research laboratory, analyzing clean and standardized test samples PCR procedures requires careful quality control, tak- ing into consideration differences in sample preparation, variation in pipetting, differences in reaction tube thickness, poor calibration or instability of the thermal cycler, and reagent quality.

As nucleic acid amplification techniques are applied to more diverse applications, as has been demonstrated by the experiences of the IRMM, a range of factors including DNA stability, external contamination, inhibition from other elements ofthe matrix and the inherent variability of biological matrices all conspire together to affect both the ability of the PCR to amplify a target and the specificity of the interaction of primer and target. Together these variables lead to false positives or negatives, over- or under-results, and generally make reliable quality control very difficult.

The use of internal standards for monitoring PCR analysis for certain food patho- gens has been developed. The internal standard is a DNA fragment flanked by the same primer recognition sites as the target sequence. It can be considered a mimic and can help control false positives or negatives but is no use monitoring the specificity of the PCR reaction. Indeed, as the mimic competes with the intended target it decreases assay sensitivity. As far back as 1989 Wang et al. demonstrated the use of mimics in the quantification of competitive PCR.

Unfortunately these and other existing quality control procedures do not answer all problems. There remains a clear need for development of PCR reference materials that will provide information both on quality and quantity levels. For quality the reference materials should be host-specific and PCR primers, for positive control, may correspond to host specific house keeping genes e.g. b-actin. For quantitative analysis, fluorescence dyes in specific primers might be used in order to measure accurately the amount of DNA present. Such practices, and other as yet un-realized procedures, will be needed to achieve reliable results in the quantification of DNA analysis.

I

5.5 Reference Substances and Spectra for Pharmaceutical Analysis

John H. McB. Miller, Agnbs Artiges, Ulrich Rose, Vincent Eglofand Emrnanuelle Charton

The aim of REMCO, The International Standards Organisation Committee on Ref- erence Materials, is to carry out and encourage a broad, international effort for the harmonisation, production, and application of CRMs. Unfortunately, the specifications of pharmaceutical analysis and the use of pharmacopoeia1 reference substances were not taken into account in the preparation of the ISO-REMCO guidelines. Chemical reference substances (CRS) required for the application of monographs for drug substances and pharmaceutical formulations are an integral part of

I 173 5.5 Reference Substances and Spectra for Pharmaceutical Analysis

these monographs, which are legally binding quality standards. Pharmacopoeias mostly follow the general philosophy of the ISO-REMCO guidelines. Differences have been recognized by ISO-REMCO with an amendment to the Introduction to I S 0 Guideline 34,znd Edition 2000:

‘‘Pharmacopoeia1 standards and substances are established and distributed by pharmacopoeial authorities following the general principles of this Guide. It should be noted, however, that a different approach is used by the pharmacopoeial authorities to give the user the information provided by certificates of analysis and expiration dates. Also, the uncertainty of their assigned values is not stated since it is negligible in relation to the defined limits of the method-specific assays of the pharmacopoeias for which they are used. ’’

5.5.1 Introduction

Reference substances (and spectra) form an integral part of the majority of monographs of the official compendia and as such their use is mandatory for the testing of pharmacopoeial substances and preparations. Reference substances for pharmaceutical analysis are available from a number of pharmacopoeias world-wide but the most extensively used are those of the European and United States Pharmacopoeias. However, in Europe some national pharmacopoeias may continue to publish addi- tional national monographs; in this case they publish the monographs of the Eur- opean Pharmacopoeia which require the use of European Pharmacopoeia chemical reference substances (Ph.Eur. CRSs), biological reference preparations (Ph. Eur. BRPs) or reference spectra, while national pharmacopoeial reference materials are required only for the substances or preparations which are not included in the Eur- opean Pharmacopoeia. The European Pharmacopoeia includes about 1500 monographs which describe the quality specifications for the corresponding substances or preparations. These norms are obligatory standards for these substances/preparations in all signatory countries of the “Convention on the Elaboration of the Eur- opean Pharmacopoeia” (European Treaty Series 1964) and are further enforced by Directives (EEC Directives 1975 ff.) of the European Union. This is also the situation in the United States of America where the USP is the legal instrument for the quality of pharmaceutical material (US Pharmacopoeia 1995). The International Phar- macopoeia chemical reference substances (IPCRSs) have no legal status except in those countries which have adopted the International Pharmacopoeia for the control of pharmaceutical substances, products and excipients within the framework of their national legislation.

It is essential to realize that reference materials must only be employed for the purpose(s) for which they are intended. The use of such materials for analytical methods other then those described in the monographs is unacceptable, except when the user carries out the necessary testing to validate the reference substance for a particular test.

Chemical and pharmaceutical manufacturers must establish their own internal reference materials, when no pharmacopoeial monograph exists, to satisfy the requirements of Good Manufacturing Practice (European Commission 1997).

174 5 Reference M a t e r d s for “1ife”Analysis

This Section will describe the procurement, characterization, production, packaging, storage, distribution, and repeat testing of pharmacopoeia1 reference materials and will to a large extent reflect the experiences and practices of the European Phar- macopoeia. A number of articles have previously been published describing the characterization and monitoring of chemical reference substances of the European Pharmacopoeia (Miller 1987, 1990, 1992; Rose 1996), chemical reference substances for microbiological assay (Sandrin et al. 1997) and biological reference preparations (The Biological Standardization Programme 1996; Charton et al. 1997).

I

5.5.2 Definitions and Guidelines

In 1975 the World Health Organization produced a guideline for the establishment, maintenance and distribution of chemical reference substances (WHO 1975). This document was intended to foster collaboration and harmonization of approval for the provision of reference substances by national authorities and organizations responsible for reference substances collections. This guideline was revised in 1982 (WHO 1982) and a further revision was completed more recently (WHO 1999) to take into account progress in pharmaceutical analysis. The latest guideline defines both primary chemical reference substance and secondary chemical reference substance as follows:

“A designated primary chemical reference substance is widely acknowledged as having appropriate qualities within a specified context, and whose value is accepted without reliance on comparison to another chemical substance.

“A secondary chemical reference substance is a substance whose characteristics are assigned and/or calibrated by comparison with a primary chemical reference substance. The extent of characterization and testing of a secondary chemical reference substance may be less extensive than for a primary chemical reference substance. This definition may apply to some substances termed Working Standards”.

This guideline covers all aspects of chemical reference substance production including procurement of candidate material, the evaluation of the material by various chemical and physico-chemical methods, the assignment of content for those substances to be employed in quantitative analysis, packaging, storage and distribution. However, a series of guidelines have been published by International Standardiza- tion Organization (ISO) comprising terms and definitions ( I S 0 Guide 30 1gg2), quality system required ( IS0 Guide 34 1996) and contents of certificates and labels ( I S 0 Guide 31 1981). A stand-alone guideline for accreditation of producers of reference materials is presently under elaboration ( I S 0 Document N 464 1998) by the ISO-REMCO group. IS0 defines a reference material as a “material or substance, one or more of whose properties are sufficiently homogeneous and well established to be used for the calibration of an apparatus, the assessment of a measurement method or for assigning values to materials” whilst a certified reference material (CRM) is a “reference material, accompanied by a certificate, one or more of whose property values are certified by a procedure which establishes its traceability to an


accurate realization of the unit in which the property values are expressed, and for which each certified value is accompanied by an uncertainty at a stated level of confidence”. The role of CRMs in chemical analysis is to provide a “measurement benchmarlts” that chemists can use to calculate or assess the accuracy of their analysis. When several laboratories can achieve the same analytical results for a given CRM, they demonstrate comparability of their measurement. The use of reference materials is encouraged whenever possible in the quality system guidelines of I S 0 ( I S 0 1990) and EN 45001 (CEN/CENELEC 1989) where it is stated that reference materials provide essential traceability in chemical measurements and are used to demonstrate the accuracy of results, calibrate equipment and methods, monitor laboratory performance, validate methods, and enable comparison of methods by use of transfer standards.

Guidance concerning the extent of testing required is also given in the “Technical Guide for the elaboration of monographs” (1996) of the European Pharmacopoeia. These guidelines are specifically addressed to the establishment of reference substances for pharmacopoeia1 use.

Thus, it can he seen that IS0 type reference materials or certijied reference materials are intended to he employed fo r a number of purposes using a variety of diferent analytical procedures, whilst pharmacopoeia1 reference substances/preparations are intended f o r a specijic purpose and are not to he used fo r methods or procedures which are not described i n the particular monograph.

5.5.3 Uses of Pharmacopoeia1 Reference Substances

Pharmacopoeia1 reference substances/preparations are an integral part of a monograph and employed for a variety of purposes within the monograph (Table 5 . 3 ) .

5.5.3.1

Reference substances can be used for confirmation of identity of the substance by, e.g. infrared spectrophotometry where the spectrum of the substance to be examined must be concordant with the spectrum of the CRS, or by thin layer chromatography where the migration and appearance of the spots of both the substance to be examined and the CRS are the same, or by liquid chromatography where the reten- tion time of both the substance to be examined and the CRS are the same.

Identification by peptide mapping requires the use of both a CRS and a reference chromatogram. The substance to be examined and the CRS are hydrolyzed with a pro- tease and the resulting digests are chromatographed. The profile of the chromatogram obtained with the substance to be examined must correspond to that of the chromatogram obtained with the CRS and must be similar to the reference chromatogram supplied with the CRS. In the monograph for insulin (Monograph 0276 1999). Figure 5.2, such a test allows the distinction between different insulin species.

Reference Substances Used for Identification

176 5 Reference Materials for “1ife”Analysis

Tab. 5.3 Use of reference products in the European Pharmacopoeia I

Code

I

2

3

3A 4 4‘4 5 5‘4

6 7

8

8A

9

I 0

I1

Uses

Identification by infrared spectrophotometry Identification by thin layer chromatography Identification by melting point

Identification by depolymerization Identification by liquid chromatography Identification by peptide mapping Identification by electrophoresis Identification by size-exclusion chromatography Identification by gas chromatography Identification by nuclear magnetic resonance spectrometry Identifkation by UV spectrophotometry or colorimetry Test for related substances by UV spectrophotometry or colorimetry Test for related substances by thin-layer chromatography Test for related substances by liquid chromatography Test for related substances by size-exclusion chromatography

Code

I2

13

I4

IS

15‘4 16 I7 I8

19 I9A

20

2oA

21

22

Uses

Test for related substances by electrophoresis Test for related substances by gas chromatography Assay by UV-spectrophotometiy or colorimetry Assay by liquid chromatography Assay by size-exclusion chromatography Assay by gas chromatography Microbiological assay Bioassay

Imrnunoassay Chromogenic substrate assay

Assay by volumetric titration

Assay by hydrolysis rate

General methods

Other purposes

5.5.3.2 Related substances are defined as known impurities which may be identified or uniden- tified (Technical Guide for the Elaboration of Monographs 1996). They include inter- mediates and by-products from a synthetically produced organic substance impurities, co-extracted from a natural product and degradation product ofthe substance.

The monographs of the European Pharmacopoeia describe a so-called test for related substances which is intended to control the level of impurities. Reference substances may be used in this test in order to ensure the system suitability and/or to control the content of related substances.

Reference Substances Used for Related Substance Tests

(I) System Suitability Tests These chromatographic performance tests are camed out in order to ensure that all impurities to be controlled are well separated from the substance to be examined (HPLC, GC andTLC). For this reason, preferably such reference substances are chosen which elute close to the main compound (HPLC, GC) or which have a similar Rfivalue (TLC) but can still be separated. These may be structurally related compounds which shall be separated with a minimum requirement for the resolution using the chromatographic system described, e.g. such as in the monograph for desmopressin (Monograph 0712 1999), Figure 5.3.

I 177 5.5 Reference Substances and Spectrafor Pharmaceutical Analysis

200~

150~

100

5 0 ~

1400

1200

1000

2 800 E 8 > E

600

400

200

0

1 Oxytocin 17.36

16.76

1 ‘L

I11 I1 I

Porcine Insulin h

I11 I1 1:; Human Insulin

5 10 15 20 25 30 35 40 45 50 Minutes

Fig. 5.2 Chromatogram ofthe enzymatic digests of different types o f insulin employed to demonstrate the suitability ofthe digestion procedure and the chromatographic system.

desmopressin required to confirm the suitability ofthe chromatographic system in the desmopressin monograph.

178 5 Reference Materialsfor “Life”Ana1ysis

A different approach is the use ofby-products or degradation products which has the advantage that the impurity itself, which must be controlled, is used to ensure the chromatographic performance and can simultaneously serve as external standard in the purity test. Occasionally, structural or optical isomers are used for these purposes (Rose 1996) such as diltiazem impurity A (“trans-diltiazem”) in the monograph for diltiazem hydrochloride (Monograph 1004 1997). Another approach to carry out a system suitability test in liquid chromatographic tests for related substances is the use of “performance test? mixtures or spiked materials. These mixtures are employed to adjust the chromatographic conditions required for the performance of the test by comparison to a representative chromatogram and may serve to identify the impurities on a given test sample. A characteristic example is atenolol for column validation CRS which is supplied together with a typical chromatogram depicted in Fig- ure 5.4 (Monograph 0703 1997).

I

(2) Purity Control The employment of reference substances in the related substances test as external standards for the determination of the content of impurities is often used. The impurities themselves may be described for this purpose but also a

W 1.4€+04

1.2E+04

1 .OE+04

8.OE+03

6.OE+03

4.OE+03

2.OE+03

O.OE+OO k

6 I

AJ

SPECIMEN CHROMATOGRAM

5.00 10.00 15.00 20.00 25.00

Minutes

Fig. 5.4 which is supplied with the CRS. A similar chromatogram must be obtained to assure the suitability ofthe chromatographic system. (Column:4.6x 150mm Nucleosil C-l8[5pm]: Mobile phase: 1.Og sodium octane-sulphonate, 0.4 g tetrabutyl ammonium hydroxide, 2.72 g potassium dihydrogen phosphate in 800 ml water [pH 3.01, 20 ml tetrahydrofuran and 180 ml methanol, flow rate: 1 .O rnl/min and detection wavelength: 226 nm).

Chromatogram ofatenolol for column validation CRS


Fig. 5.5 Chromatogram ofchlor- prothixene hydrochloride CRS which contains 2.7% ofthe E-isomer. (Column: 4 x 120 mm hypersil BDS [3 p]. Mobile phase: 6.0 g potassium dihydrogen phosphate, 2.9 g sodium lauryl sulphate, 9.0 g tetrabutylammonium bromide 550 ml water, 50 ml methanol and400 ml aceto- nitrile, flow rate: 1.5 ml/min and detection wavelength: 254 nm.)

dilution of the test solution can be used provided that the impurity and the monograph substance exhibit a similar detector response. In case of reduced availability of an impurity a possible approach is to pre- pare a “spiked sample”, i.e. a known amount of impurity is added to the CRS and may serve in a system suitability test as well as for the control of the level of this impurity. An example is given in the monograph for chlorprothixene hydrochloride (Monograph 0815 1999) where the content of the E-isomer is controlled to a level of not more than z per cent, Figure 5.5.

5.5.3.3 Pharmacopoeia1 reference substances are increasingly employed as assay standards since assay of content is determined more and more frequently by separation techniques.

In such a case, based on the results of a collaborative study, a content is assigned to the corresponding reference substance which is method specific, i.e. it is only to be used with the method described in the monograph. The establishment of these substances is described in more detail later in the Chapter.

Reference Substances Used for Assay

180 5 Reference Materials for “Life”Analysis

5.5.3.4 It is policy of the Commission of the European Pharmacopoeia to minimize the use of reference substances/preparations since the production, maintenance and distribution of chemical reference substances is a costly and time-consuming undertak- ing. Therefore the decision to establish a reference substance should not be taken lightly and consideration should be given to other approaches which could be adopted to avoid the use of reference substances. A number of strategies can be employed to reduce the need for reference substances.

Minimizing the Use of Reference Substances I

When the monograph requires the identification of a substance by infrared spectrophotometry, comparison can be made to a reference spectrum. When the test for related substances is a limit test, the peaks of the impurities in the chromatogram of the test solution can be compared to the peak of the test substance in the chromatogram of a dilution of the test solution at the limiting concentration. The approach is valid provided that the response factors of the impurities and the test substance are equivalent using the detector conditions described, otherwise correction factors need to be applied.

When there is a system suitability criterion for the resolution of a critical pair it is preferable to describe reagent grade material which is available in commerce rather than to establish a reference substance. Alternatively, it may be possible to describe a degradation procedure of the substance so that a particular impurity profile is obtained which can serve as a chromatographic performance check. This approach has been described (Rose 1998) and applied to a number of monographs of the Eur- opean Pharmacopoeia, e.g. in the revised monograph for spiramycin (Monograph 0293 1999). This monograph describes a test for related substances by liquid chromatography for the control of impurities and degradation products. Spiramycin is a mixture of three components: spiramycin I, 11 and I11 of which spiramycin I is the most important. One of the impurities to be controlled by the monograph and which may be obtained by acid hydrolysis is neospiramycin I. In the conditions described, neospiramycin I elutes close to spiramycin I and can therefore be used in the system suitability test. Thus, to avoid the use of a chemical reference substance, spiramycin is hydrolyzed as follows: a solution of spiramycin (I mg/ml) in the mobile phase (pH 2.2) is heated for 30 min at G O T . By this procedure peaks of similar heights of spiramycin 1 and neospiramycin I are obtained (Figure 5.6) which can be used for the identification of the impurity and also serves for the calculation of the resolution.

Non-specific absolute assay methods, e.g. volumetric titration, can be applied to avoid the establishment of a reference substance. This is only appropriate, however, when the monograph describes a separation test for related substances. This approach is certainly valid for the determination of the content of pharmaceutical raw materials but less acceptable for the assay of content of pharmaceutical preparations where the employment of specific assay methods is recommended (ICH Guideline 1994) to take account of decomposition of the active ingredient during the shelf life of the product and to avoid possible interference from excipients.

I 18’ 5.5 Reference Substances and Spectra for Pharmaceutical Analysis

Minutes

Fig. 5.6 from the impurity neospiramycin I produced by acid-hydrolysis.

Liquid chromatographic separation of spiramycin I

5.5.4 Procurement of Candidate Reference Substances

A candidate reference substance is selected from the normal production batches of a manufacturer and is not further usually purified. Candidate reference substances/ preparations should be supplied with:

a certificate of analysis including test methods, test results with complete identification by appropriate physico-chemical methods, e.g. nuclear magnetic resonance spectroscopy, infrared spectrophotometry, mass spectroscopy etc. stability data of the substance with an indication of the storage conditions to be employed information as to its hygroscopicity and its solid-state properties, e.g. amor- phous, crystalline, polymorphic form etc. a material safety data sheet a list of the potential impurities which may be present, if a monograph substance

The quantity requested should be sufficient to allow for all the pre-testing required and the preparation of a stock ofvials or ampoules which will last for several years.

5.5.5 Requirements for Candidate Reference Substances

Except in rare cases, the quality of the CRS to be established must comply with the requirements of the monograph. The purity of the candidate reference substances

182 5 Reference Materials for "L@"Ana/ysis

depends on the purpose for which it is required in the monograph. In most cases a purity of better than 99.0 % (on an anhydrous and solvent-free basis) is required, but some substances, e.g. antibiotics, and naturally occurring substances may be employed with higher levels of impurity. Where the CRS is intended for a non-specific assay the purity requirement may also be less (ultraviolet and visible spectrophotometric methods). Impurities with physico-chemical characteristics similar to the main component will not impair the use of the chemical reference substance whereas traces of impurities with significantly different properties will render the substances unsuitable for use in a non-specific assay. The response of impurities under the assay conditions should always be assessed.

Substances (impurities) which are not the subject of a monograph are usually synthesized or supplied by the manufacturer. In this case a purity in excess of 90.0 % is required provided that the reference substance is employed in a limit test. If the reference substance (impurity) is employed in a quantitative test then the purity requirement is normally better than 99.0 %. When it is less, then an assigned content must be established.

I

5.5.6 Evaluation

Although pharmacopoeia1 reference substanceslpreparations are employed for specific tests and assays in the monographs of the pharmacopoeia, the candidate substances are tested against a wide variety of analytical methods, including all the tests prescribed in the monograph. However, the extent of testing and the number of laboratories involved depends on the use of the reference substances. The following testing programme is carried out by the European Pharmacopoeia applying a quality assurance system based on EN 45001.

5.5.6.1 The batch must comply to the requirements of the monograph. However, the most important control is the application of the test@) for which the substance is intended. It is usual for at least one laboratory (usually the Ph. Eur. laboratory) to apply all tests of the monograph. The structure of the substance must be clearly identified by comparison of the IR spectrum to spectra published in the literature and interpreta- tion of the NMR and mass spectral data.

Reference Substances Used for Identification

5.5.6.2 As previously described three types of reference substances may be used in this test:

Reference Substances Used for Related Substance Tests

(I) structurally related compounds (2) by-products or degradation products (3) performance test mixtures or spiked materials

The substances described under (I) and (2) are characterized by the following physico-chemical and spectrophotometric techniques:

5.5 Reference Substances and Spectra for Pharmaceutical Analysis

structural identification by 'H-NMR, MS and IR resolution test as described in the monograph purity tests by LC (usually by external standard), TLC (semi-quantitative or densitometric), GC (peak area normalization) or DSC water determination by Karl Fischer or by coulometry, occasionally TGA

Although not used for assay purposes, the purity of the CRS should be at least go %. Howevei-, when the substance is used in a quantitative test and the purity is not greater than gg % a content will be assigned.

For the mixtures described under (3) it is sufficient to determine the chromatographic profile of the CRS and to demonstrate that all impurities are well separated according to the monograph description. When the spiked sample is also used in the purity control, then the content of the impurity in the CRS material must be determined by appropriate chromatographic methods and a value assigned to the material.

5.5.6.3

Physico-Chemical Assay Standards Where the CRS is to be employed as an assay standard, the extent of testing is very much greater. A number of ways for the assignment of content values of reference materials have been described ( I S 0 Guide 34 1996):

Reference Substances Used for Assay

single definitive method by a single Organization two or more reference methods by one Organization number of methods of known and acceptable accuracy and precision by a net- work of qualified Organizations method specific approach (inter-laboratory study) giving only a method specific assessed property value

The laboratory of the European Pharmacopoeia applies the method specific approach (inter-laboratory study) as has been previously described (Technical Guide for the Elaboration of Monographs 1996).

Several collaborating laboratories (usually five participating laboratories) test the proposed substance using a variety of techniques. The relative reactivity or relative absorbance of the impurities present in a substance must be checked when a non- specific assay method is employed, e.g. by colorimetry or ultraviolet spectrophotometry. It is particularly important to quantify the impurities when a selective assay is employed. In such a case, it is best to examine the proposed substance by as many methods as practicable, including, where possible, absolute methods. For acidic and basic substances, titration with alkali or acid is simple but other reactions which are known to be stoichiometric may be used. Phase solubility analysis and differential scanning calorimetry may also be employed in certain cases.

The European Pharmacopoeia prepares a protocol which must be strictly followed by the participants of the collaborative trial to assign the content. The protocol usually requires:

184 5 Reference Materialsfor "Life"Ana1ysis

the determination ofwater (or loss on drying) the estimation of the impurities using the separation technique described in the monograph the estimation of residual solvents by head space gas chromatography when a test for loss on drying is not prescribed the determination of the content of the substance by an absolute method (usually volumetric titration) may be included to ensure that significant levels of inorganic impurities are not present. This is a confirmatory determination and the result is not used in the calculation of the assigned value

I

Summation of the results of the determinations of water, organic solvents, mineral impurities and the organic components amounts to roo %. The results of methods employed to analyze the substance other than these given above are not used for the calibration of the assigned value but are reported to support the results obtained by the defining methods. For most reference substances intended as assay standards the assigned content is normally expressed "as is" so that it is essential (when establishing the CRS) to determine the content ofwater and residual solvents for a non-specific assay and also, for a selective assay, to determine the content of impurities.

The European Pharmacopoeia Commission has adopted the policy that the value assigned to a reference substance as a result of an inter-laboratory trial should have an uncertainty not greater than a predetermined value. The following formula (Equation 5.1) may be used to calculate the estimated approximate uncertainty

where: n = the number of participating laboratories oi2 = variance for the estimation of impurities ow2 = variance for the determination of water 0s' = variance for the determination of residual solvents t = studentst

Assuming an analytical error of +2 % which is based on the analysis of the results of a number of proficiency tests and collaborative trials (Daas and Miller 1998), a m a - imum uncertainty of 0.24% would imply a 0.1% probability of rejecting a good result. Given that the uncertainty of a content to be assigned is below a predetermined value, the results of the collaborative trial are acceptable; otherwise it is recommended to repeat the trial in whole or in part.

An example for the application of this calculation is given in Table 5.4 which shows the results of a collaborative trial for the establishment of ciprofloxacin hydrochloride CRS 2. The uncertainty was calculated to be 0.11 %.

In the specific case of the European Pharmacopoeia reference substances, it is considered that the recommendation in the IS0 guidelines, to give the uncertainty value with the assigned value on the label of the CRS, is not relevant because of the following reasons:

I 185 5.5 Reference Substances and Spectrafor Pharmaceutical Analysis

Tab. 5.4 Uncertainty ofthe assigned value of ciprofloxacin HCI CRS z

fmpurities Ph) Laboratory Related substances Wafer

1 2 3 4 5 6 7 Mean

SD (0) Variance (0’)

0.08 0.04 0.07 0.05 0.04 0.05 0.03 0.05 (n= 7) 0.018 0.0003

5.88 5.94 6.07 5.77 6.08 6.07 5.96 6.0 (n= 7) 0.116 0.014

The content of residual solvents is negligible.

Approximate Uncertainty= JT x 2.447 = 0.11

If the risk of rejecting a good result is considered to be acceptable, there is no justification of giving uncertainty values with the assigned value. The I S 0 guide is particularly concerned with the establishment of reference materials which contain the analyte as a small, or even trace, quantity in a complex matrix. These reference materials serve as measurement bench- marks when applying an appropriate analytical procedure for the determination of an analyte in the sample. The value attributed to the reference material is usually the mean of results obtained from a variety of methods and laboratories. Thus, the value attributed to the substance may have a high degree of uncertainty. A particular reference material is subjected to the same procedure as the test samples so that greater confidence can be given to the results of the test samples provided that the value found for the reference material falls within the given uncertainty. A pharmacopoeia1 reference substance is intended for the determination of the main component of a substance or for the active ingredient of a pharmaceutical formulation which is usually present at a high proportion of the total. The reference substance is to be used as a primary standard in a specific method validated as prescribed in the ICH Guideline “Validation of Analyti- cal Procedure: Methodology” (Technical Guide for the Elaboration of Mono- graphs 1996; ICH Guideline 1gg7), the reproducibility of which is known. This is taken into account when the limits of acceptance (tolerance) for the substance or product are fxed (Daas and Miller 1997,1998).

Microbiological Assay Standards

The potencies of some antibiotics described in the European Pharmacopoeia are determined by microbiological assay (Microbiological Assay of Antibiotics 2.7.2

186 5 Reference Materials for “1fe”Analysis

1997). The procedure employed for the establishment of the chemical reference substances used in these assays has been previously published (Sandrin et al. 1997). The CRSs for the microbiological assays of antibiotics are first submitted to the chemical tests of the monograph. If the results are satisfactory, a collaborative microbiological assay is carried out, using the International Standard as calibrator. Thus, these reference substances are considered to be secondary reference substances since they are calibrated against existing standards. Potency is expressed in Interna- tional Units. If an International Standard does not exist, European Pharmacopoeia Units are used.

Due to the inherent variability of these assays either by agar-plate diffusion measurement or turbidimetry measurement, the fiducial limits are calculated according

I

1 3 0 0

1 2 0 0

1 1 0 0

1 0 0 0

9 0 0

8 0 0

7 0 0 1 2 4 5 6 7

Fig. 5.7 Results (IU/mg) of an interlaboratory study to determi- nate the potency oftylosin CRS 1 by microbiological assay (diffusion method).


1200"

" o o - J

1000'.

g o o - .

8 0 0 ' .

7 0 0 '

i300r

!ill

A l l -

1 3 4

Fig. 5.8 the potency of tylosin CRS 1 by microbiological assay (turbidimetric method): individual estimates per assay (empty dots indicate rejected assays).

Results (IU/rng) of an interlaboratory study to establish

to thc statistical procedures given in the pharmacopoeia (Statistical analysis of results of biological assays and tests 5.3 1997). The variability obtained for these assays is shown for tylosin CRS (Figures 5.7 and 5.8 for the difision assay and the turbidimetric assay respectively). The decision tree employed for the statistical treat- ment of the results is shown in Figure 5.9. In this case, the two assay methods were considered equivalent and the fiducial limits were calculated from all valid data. Tylosin was assigned a potency of 1035 IU/mg with confidence limits of roz8 IU/mg to 1044 IU/mg (corresponding to * 0.8 % of the assigned value).

188 5 Reference Materidsfor “Lfe”Ana/ysis I

I and statistics of validity I 1

c No

arations smal

Situation

// // I f I!!7 //

Fig. 5.9 laborative trials to establish the potencies o f antibiotics to be used as chemical reference substances for microbiological assay standards.

Decision tree used to accept or reject results from col-

Biological Reference Preparations Biological reference preparations are established for specific assay methods by collaborative trials following a strict protocol. The results are statistically evaluated and potencies are assigned (Statistical analysis of results of biological assays and tests 5.3 1997). Reports of such trials are regularly published in special issues of Pharmeur- opa (Phamzeuropa Bio, published by the Council of Europe). Since 1996, results of collaborative studies for the establishment of the following BRPs have been published in Pharmeuropa Bio: Low Molecular Mass Heparins, Factor VIII, Human Immunoglobulin Inactivated Poliomyelitis Vaccine, Live Measles Vaccine, Live


Rubella Vaccine, Live Mumps Vaccine, rDNA Hepatitis B Vaccine, Oral Poliovirus Vaccine, Erythropoietin, and Factor IX etcetera.

5.5.6.4 CRS as Calibrators Where the CRS is to be employed as a verification material in general methods published in the European Pharmacopoeia, then like a CRS used as an assay standard, the extent of testing is considerable. It is desirable that several collaborating laboratories test the proposed substances using a variety of techniques to ascertain that its purity as adequate. An appropriate number of collaborating laboratories will also participate, after the substance has been deemed suitable, in the study to establish a value by the measurement of the essential property of the substance using an appropriate instrument.

Examples of such substances are calcium oxalate CRS employed for the verification of the electrobalance in thermogravimetry and the solvents, trimethylpentane CRS, toluene CRS and methylnaphthalene CRS, employed for verification of the performance of refractometers.

5.5.7 Monitoring Programme

It is necessary to test the CRSs and the BRPs for stability during their storage, in order to ensure their continued fitness for use. A standardized testing procedure has been introduced which is designed to detect at an early stage any sign of decomposition using appropriate analytical techniques. These screening methods have also been employed during the establishment phase and these initial results served as the “base-line” values. The reference substance collection of the European Pharmacopoeia consists of more than 1300 items. Consequently, for regular screening, the methods must be rapid. They must also be sensitive so that the quantities used are small to avoid depletion of the stock. The monitoring programme includes:

determination of water, loss on drying or TGA (not necessary for substances in ampoules) thin-layer chromatography using high loadings of samples and silica as absor- bent liquid chromatography usually employing reverse-phase stationary phase as a complement to the thin-layer chromatography. Other stationary phases are also used as considered appropriate when appropriate, differential scanning calorimetry is employed to determine the purity any other specific tests for detecting impurities

The frequency of testing of a reference substances depends on the known or sus- pected stability and of its prescribed use. Reference substances used as assay standards are re-examined on a two-yearly basis whilst the other reference substances are

190 5 Reference Materialsfor “Life”Ana1ysis

tested every three to four years. Substances known to be unstable are monitored more frequently.

Any differences observed compared to the last examination will lead to a more extensive examination of the batch and, if considered necessary, a replacement batch is obtained and established.

Implementation of this procedure precludes the necessity of giving expiry dates for these substances. However, no studies are undertaken to test the stability ofthe substance in opened vials or ampoules nor the substances in solution. Users of reference substances should not store solutions of chemical reference substances or opened vials.

I

5.5.8 Packaging and Filling

All operations conform to the requirement of Good Manufacturing Practice (Eur- opean Commission 1997). The reference substances are distributed into suitable containers using appropriate filling conditions.

A quantity sufficient to carry out the required tests, generally in duplicate, are filled into the containers. Most substances for which there are no concerns for either toxicity or stability are filled by weighing the appropriate quantity into antibiotic vials in a horizontal laminar flow work station. These operations are carried out in a self-contained cubicle to avoid cross-contamination. The vials are then closed with butyl rubber stoppers and sealed with an aluminium crimp seal using an automatic crimping and labelling machine.

A substance which is either hygroscopic or easily oxidized is filled in a pressur- ized glove box (isolator) fitted with a gas purification system in which the atmo- spheric air is replaced by argon. Under these conditions the contents of water and oxygen are restricted to less than I ppm.

A substance which is highly toxic is filled in a depressurized glove box (isolator). Increasingly, lyophilization is employed for the preparation of chemical and par-

ticularly biological reference preparations. Substances which are expensive and available only in small quantities are prepared in solution and distributed by an automatic liquid filling machine into containers which are then placed in a freeze- drier. On completion of the lyophilization cycle the vacuum is broken by the introduction of an inert gas. If vials are employed they are automatically stoppered in the freeze-drying chamber. Reference substances prepared in this manner include antibiotic standards for microbiological assay, synthetic peptides, biologicals and expensive chemical substances in short supply. In such cases the content or potency of the substance is expressed as a quantity per vial and the whole quantity has to be dis- solved for further use. Instructions on the preparation of the solution used is the assay standard are either given in an information leaflet which accompanies the CRS or in the monograph itself. Thus, CRSs prepared in this manner are standards for which no weighing by the analyst is required.


5.5.9 Certificates of Analysis / Expiry Date / Catalogue

Since the CRSs and BRPs are officially certified by the European Pharmacopoeia Commission, which adopts the reports establishing their suitability for the intended use, it should be noted that neither certificates of analysis nor data which are not relevant to the use of the substances as defined by the Ph. Eur. monograph, are provided with the reference products or substances. Information required for the correct use of the chemical reference substance or biological reference preparation is provided. The label on the vials or ampoules gives:

0 the batch number

If used as an assay standard the following information is also fronted:

(I) the assigned content of the chemical entity or (2) the content in mg or ml of the chemical entity or (3) the assigned potency (for microbiological assays or biological tests)

the name of the organization the name of the substance both in English and in French (as written in the corresponding monograph)

An accompanying explanatory leaflet may also be provided, if necessary, to describe the preparation of the substance for use. In some uses, a chromatogram may also be provided. As mentioned before, no expiry date is indicated because the products and substances comply with the requirements of the corresponding monograph and are monitored regularly.

A catalogue is issued three times a year, after each session of the European Phar- macopoeia Commission, where new and replacement batches of chemical reference substances or biological reference preparations are adopted. This catalogue indicates for each substance or preparation:

the order code the name of the substance or preparation in English the batch number the assigned value or assigned content or assigned potency, if applicable the validity or status of the previous batch if a replacement batch has been adopted the chemical formula (for impurities) the sales unit the unit quantity the code indicating the presented use(s) of the substance or preparation the corresponding reference number of the monograph(s) where its use is prescribed

Both the catalogue and material Safety Data Sheets are available on request or may be obtained from the web site of the European Pharmacopoeia.

192 5 Reference Materials for “Lfe”Analysis

5.5.10

Storage and Distribution

1

Most of the reference substanceslpreparations are stored in cold rooms controlled at between 2°C and 8°C. However, a number of substances/preparations which are relatively unstable are stored at -20°C or, in a few cases, e.g. vaccines, at -80°C. The reference substanceslpreparations may be stored for years under these conditions and their fitness for use is continually monitored as described above. Thus, the status of each reference substance/preparation is indicated in the catalogue. It is recommended that purchasers only order a sufficient amount for immediate use since the stability of the contents of opened vials or ampoules cannot be guaranteed.

Reference substances are distributed world-wide. Special packaging is employed to minimize the risk of damage during transport. Substances which are normally stored at 4’C are dispatched by normal mail. However, products which are stored at low temperatures (-20°C or -80°C) are packed on ice or solid carbon dioxide respectively and dispatched by courier.

5.5.1 1 International Harmonization

Users of reference substances have expressed the wish that reference substances from one pharmacopoeia be employed for a test described in another pharmacopoeia. It is clear from the previous text that this is not always possible since the monograph requirements of the different pharmacopoeias are generally not identi- cal and therefore that the reference substanceslpreparations have not been established for the same purposes.

However, the establishment of a new endotoxin standard by the World Health Organization is a recent example of successful international collaboration between the World Health Organization, the United Stated Pharmacopoeia and the European Pharmacopoeia (Poole et al. 1997). Thus this standard is available from any of these organizations to be employed as a reference in the harmonized Livnulus Amoebocyte Lysate test.

Exchanges between pharmacopoeias are co-ordinated by the Pharmacopoeia1 Dis- cussion Group (PDG) (International Harmonisation 1995) and it is frequent that one pharmacopoeia participates in a collaborative study organized by another pharmacopoeia, or that several pharmacopoeias share the same batch of reference substance to be used in their respective monographs: nevertheless, in this case the reference substance can not be considered as harmonized. A new batch of erythromy- cin was shared between the United States Pharmacopoeia and the European Pharmacopoeia and was established in a common collaborative study both for the microbiological assay (used in the USP for formulations) and the liquid chromatographic assay (used in the Ph. Eur. and USP for bulk material).

It is to be hoped that in the future there may be greater harmonization between the pharmacopoeia monographs leading to a greater number of common reference substances or standards.

I 193 5. G References

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Documents

References Materials for Chemical Analysis || Reference Materials for“Life” Analysis