Historical Overview of Mycoplasma Testing for Production of Biologics

Disclaimer

“The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.”

Abstract

This review summarizes the evolution of mycoplasma testing methods as well as the historical development of requirements and recommendations for testing of vaccines and biologics produced in cell substrates. Although newly emerged molecular technologies provide a real possibility to expedite mycoplasma testing, the historical experience accumulated during evaluation and implementation of mycoplasma testing methods may be useful for the identification of challenges associated with the comparability between different methods, particularly when comparable tests rely on detection of different biological features of mycoplasmas. Even though mycoplasma contamination during production of biological products has been recognized for many years, a global consensus regarding the most appropriate testing methods for biological products has not yet been reached, as evidenced by the variations in the regulatory and compendial requirements and guidance recommendations.

Introduction

Mycoplasmas (trivial name for organisms of the class Mollicutes), the smallest free-living organisms, are frequent contaminants of mammalian cell cultures [1-4]. It is important for the safety and purity of vaccines and other biological cell-derived products to assure that the cell substrates and unprocessed bulk material used to manufacture these products are free of adventitious agents, including mycoplasmas [2, 3, 5-10].

Mycoplasmas represent a widely diverse group of organisms that includes more than 250 species. Their host range is broad and encompasses humans and other animals, including birds, reptiles, fish, and mammals as well as insects and plants [1]. The innate dependence of mycoplasmas on host cellular nutrients, as well as their broad distribution in nature, ability to pass through antibacterial filters, and generally covert growth in infected cells [1-4], makes them ideal agents for contamination of cell substrates. Generally, mycoplasma contamination represents a serious problem for biomedical research laboratories and facilities involved in development and manufacture of cell-derived biological and pharmaceutical products. Potentially, administration of mycoplasma-containing products could cause bacterial infections, especially in pediatric, geriatric, or immunocompromised patients. Despite precautionary measures and systematic mycoplasma monitoring, mycoplasma contamination is periodically detected in veterinary and human live virus vaccines or viral stocks produced by multiple manufacturers worldwide [11-14]. Other biologics (non-vaccine products, e.g., commercial diagnostic antigens) have also been reported to be contaminated with mycoplasmas [15, 16].

A majority of mycoplasmas that contaminate cell substrates and cell-derived biologics can be detected by incubation with specially formulated mycoplasma media [2, 3]. However, there are some fastidious species/isolates, e.g., M. hyorhinis, which can grow in cell cultures but not in the conventional mycoplasma media [2]. Del Giudice has shown that there are inhibitory factors in mycoplasma media components that can inhibit growth of some isolates of M. hyorhinis [17-19].

Mycoplasmas as Contaminants of Cell Cultures: Sources and Consequences

Mycoplasma contamination of cell cultures including the most frequently detected species and main sources of contamination were described previously [2, 3, 20]. Historically, 15% of US cultures screened for mycoplasma contamination have been infected with one or more mycoplasma species [2]. In other countries, surveys have reported the prevalence of contamination to vary from 10% to 80% [21]. To clarify, these ranges of infection refer to research cell cultures submitted for routine testing, not the percentage of cultures used for manufacture of biologics. A more recent survey suggests that the prevalence of mycoplasma contamination in samples submitted to commercial testing laboratories has declined (prevalence of contamination: 0.4 to 6.7% [22]) but mycoplasma contamination still remains a significant and costly problem for the biopharmaceutical industry [22]. Although approximately 20 species have been recovered from infected cell cultures, only a few species comprise 95% of the isolates, namely Acholeplasma laidlawii, Mycoplasma arginini, M. hyorhinis, M. orale, and M. fermentans, and in several cases M. hominis, M. salivarium, and M. pirum [2, 4, 20, 22].

For research laboratories, the most likely sources of mycoplasma infection of cell cultures are other previously contaminated cell cultures, laboratory equipment, media and sera used for cell cultivation [23], as well as personnel involved in cell maintenance [20]. Recently, contamination of plant-based media components by Acholeplasma laidlawii has been reported [24].

The consequences of mycoplasma infection on a cell culture may vary from subtle to severe depending on the type of cells infected, the mycoplasma species infecting the cells, the mycoplasma burden (titer), and the duration of the infection [3]. In general, mycoplasma infection may dramatically change the metabolism of the infected cells (e.g., produce pH-altering metabolites), induce chromosomal aberrations, deplete media components, alter product yields (e.g., viral titers), and affect cytokine production and other functions of cells of the immune system [1, 3, 20, 25]. Mycoplasma infection may cause false negative or false positive results in reverse transcriptase (RT) assays [26]. Other observed effects include alteration of mutagenic assays [1] and lymphocyte and macrophage activation [1, 2, 27]. Steps for preventing mycoplasma contamination of cell cultures have been described previously [20].

In summary, mycoplasma contamination of cell substrates is a vital issue that could compromise both safety and efficacy. Current safety regulations require demonstrating the absence of any detectable levels of mycoplasma contamination in starting cell substrates intended for manufacture of biologics and at specified steps during product manufacture [5-10].

Mycoplasma Testing Considerations

Risk Assessment of the Product for Potential Mycoplasma Contamination

The biologics that need to be tested are those that are produced in cell substrates. Examples of such products include viral vaccines, monoclonal antibodies, immunologic modulators, cytokines, growth factors, cell therapy products, etc. In general, vaccines and recombinant products produced in bacteria or yeast have not been tested for mycoplasmas, primarily because those production organisms grow rapidly in media that would not be expected to support the replication of the slow-growing mycoplasmas. There is one caveat, however, that is based on the recent reports that peptones (e.g., tryptone soya products sterilized by filtration) used in microbiological and cell culture media can be contaminated with viable mycoplasmas, specifically, A. laidlawii [24, 28]. This observation makes it important to carefully evaluate all raw materials for potential mycoplasma contamination or to have procedures in place that will mitigate the risk of raw materials containing viable mycoplasma, such as pre-treatment of raw materials with ultraviolet radiation.

Stage of Manufacture where Mycoplasma Testing is Appropriate

Cell lines used for manufacturing human biological products should be tested for the presence of detectable microbial agents (including mycoplasmas) as prescribed in the US Code of Federal Regulations (CFR) for biological products [6]. In addition to master cell banks and working cell banks, any control cells and end of production cells, as well as the master and working virus seeds, should also be tested for mycoplasmas [5, 7-9]. For production lots, the testing should be performed at the stage where any mycoplasma adventitious agents would most likely be detected, i.e., unprocessed bulk (rather than in the processed bulk or final product). The test for Mycoplasma in 21 CFR 610.30 specifies that live virus vaccines should be tested prior to clarification or filtration and that inactivated virus vaccines should be tested prior to inactivation. The mycoplasma test results showing the absence of detectable mycoplasmas in the unprocessed bulk would typically be included in the lot release protocol for the vaccine.

Selection and Implementation of a Mycoplasma Testing Procedure: Evolution of Mycoplasma Testing Procedures

21 CFR 610.30 Test for Mycoplasma

Mycoplasma contamination of cell cultures was first reported in 1956 [29]. In the 1950’s and 1960’s many cell cultures and established cell lines were found to be contaminated with mycoplasma [2]. The development of cell cultures for production of viral vaccines raised concerns that mycoplasmal adventitious agents could be present in cell substrates used for vaccine preparation, and hence, could potentially cause deleterious effects in vaccine recipients [2]. To address this concern, in 1962 the United States Public Health Service (USPHS) established a mycoplasma test requirement for viral vaccines produced in cell cultures. Shortly after the Division of Biologics Standards, National Institutes of Health, transitioned to the Bureau of Biologics (BoB), Food and Drug Administration (FDA) in 1972, the mycoplasma test was codified in the regulations for food and drugs, in Chapter 21 of the Code of Federal Regulations (CFR), Subpart D  Mycoplasma, §610.30 Test for Mycoplasma (21 CFR 610.30) [5].

Based on the concept that different mycoplasma species might require different nutritional factors for their optimal growth, the requirements of 21 CFR 610.30 included a provision that two different media formulations be used in the test. The different media formulations were intended to allow for the growth of glucose-fermenter organisms such as A. laidlawii, M. pneumoniae or M. hyorhinis, as well as arginine-utilizing mycoplasma species such as M. arginini or M. orale. To allow different laboratories to work out the optimal conditions for mycoplasma detection for their process, the actual media formulations were not stipulated in the US regulations. The media formulation most frequently used for potential cell culture contaminants included mycoplasma broth base, supplemented with fresh yeast extract and animal serum [30-33]. The broth medium prescribed in 21 CFR 610.30 also included 0.05% agar, known as semi-solid medium [2]. To ensure the capability of the medium to support mycoplasma growth, two positive control strains of mycoplasma for each test were required. One of the positive control strains had to be M. pneumoniae, since that was the best known human pathogen [34] at the time the regulation was drafted and hence posed a theoretical safety concern for vaccine recipients.

Sample volumes included 2 mL of sample inoculated onto 5 agar plates of one medium and 5 agar plates of the second medium. One mL aliquots of the test sample were inoculated into each of 4 tubes of 10 mL of semi-solid broth medium. Subcultures of the broth tubes to the prescribed agar plates were to be performed at 3 and 14 days; all agar plates including the original plates and the subcultures were incubated for 14 days to allow mycoplasma colony growth to develop. Thus, the time required for completing the test is at least 28 days. Because diverse mycoplasma species might have different atmospheric requirements for growth, two atmospheric conditions, aerobic and anaerobic (5-10% CO2 in N2), were required for incubation of the test cultures.

Since this procedure is required as a regulation, this test is still necessary for each production lot of human vaccines licensed in the US, unless the FDA grants a waiver in accordance with 21 CFR 610.9(b) [35] for the use of an alternative mycoplasma testing method(s), for example as described in the EP [8].

Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals [1993 Cell Lines PTC]

In 1973, Hope Hopps at BoB/FDA reported the discovery of “non-cultivable” M. hyorhinis strains that were unable to grow in the typical broth and agar media used for mycoplasma cultivation, but could efficiently replicate when co-cultivated with cell cultures [36]. Thus, those fastidious strains could escape detection using the broth and agar culture procedures required in 21 CFR 610.30. As a result of these findings, the mycoplasma laboratory headed by Dr. M.F. Barile at FDA worked out conditions for an “indicator cell” assay to detect those strains of mycoplasma that do not grow using the broth and agar culture procedures [2]. Revised recommendations for mycoplasma testing, including the indicator cell culture assay, were published in Attachment 2 of the July 1993 Points to Consider (PTC) in the Characterization of Cell Lines Used to Produce Biologicals (abbreviated 1993 Cell Lines PTC, [10]). The major addition to the testing procedures in the 1993 Cell Lines PTC was the addition of the indicator cell culture assay able to detect fastidious organisms “non-cultivable” with the typical broth and agar procedures. In the indicator cell assay, cell cultures (e.g., Vero cells or equivalent) are prepared on cover slips and the biologic product or cell substrate test sample is added to the cell cultures and incubated for 3-5 days at 36±1ºC and 5% CO2. The cultures are fixed and then stained with a DNA-binding fluorochrome (e.g., a bisbenzimidazole stain such as Hoechst 33258) and examined by epi-fluorescence microscopy. Similar to the methods prescribed in 21 CFR 610.30, to allow different laboratories to work out the optimal conditions for mycoplasma detection for their process, specific media formulations were not prescribed. A protocol for the DNA staining procedure using the bisbenzimidazole fluorochrome stain can be found in the review by Barile and Rottem [2].

The broth and agar procedures described in the 1993 cell substrates PTC include inoculating 0.2 mL of the test sample onto 2 or more agar plates (1 medium formulation) and no less than 10 mL into 50 mL of broth medium, incubated at 36±1ºC. The broth culture is then sub-cultured (0.2 mL onto 2 or more agar plates) on days 3, 7, and 14. The agar plates are incubated anaerobically in 5-10% carbon dioxide in nitrogen and/or hydrogen atmosphere for no less than 14 days at 36±1ºC. Details of the procedures described in the 1993 cell substrates PTC can be found in the article by Olson and Barile [37].

An additional change for the PTC method was the reduction of atmospheric incubation required from both anaerobic and aerobic conditions to only anaerobic incubation. The Mycoplasma Laboratory at FDA had evaluated the relative recovery of cell culture contaminants using anaerobic vs. aerobic conditions and found that increased recovery of cell culture contaminants using both aerobic and anaerobic incubation was minimal compared to anaerobic incubation alone, as also seen in other laboratories [38-40]. Also, incubation of the indicator cells in 95% air- 5% CO2 essentially provides an aerobic atmosphere for mycoplasmas that might be favored by aerobic conditions. Thus, a decrease in the number of broth and agar cultures required in the PTC assay was justified by the added redundancy of the indicator cell culture procedure and reduced somewhat the burden on industry of the broth and agar test procedures compared to the requirements of 21 CFR 610.30.

The broth and agar and indicator cell procedures were generally considered to be able to detect a few colony-forming units (CFU) per mL, based on experience in the Mycoplasma Laboratory at FDA. Note that in theory, the minimal unit detectable by growth of viable organisms is 1 CFU (per volume sampled), and if 5 CFUs are detected in a volume of 50 mL, the calculated sensitivity is 0.1 CFU/mL. The ability to detect mycoplasma is dependent on the quality of the media used for the cultures, and appropriate quality control of the mycoplasma broth and agar media components is critical. An evaluation for mycoplasma growth promotion needs to be performed on a lot-by-lot basis for the mycoplasma broth base, the serum, the purified agar, and yeast extract. For growth promotion evaluation, a screening test is performed with a variety of mycoplasma test organisms and various lots of the media components [2, 30]. If the test laboratory does not prepare the mycoplasma media in house and utilizes commercially prepared media, the test laboratory relies on the commercial supplier to perform this evaluation, and a certificate of analysis should be available describing this evaluation. Another potential concern is the acceptable shelf-life of the commercial media.

Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infections Disease Indications [2010 Cell Substrates Guidance]

The combination of the broth and agar culture assay and the indicator cell culture procedure described in the 1993 Cell Lines PTC is still considered the “gold standard” methodology for mycoplasma testing for biologics produced in cell substrates. The Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications, published in February 2010 [9], incorporated the main recommendations for mycoplasma testing described in the 1993 Cell Lines PTC. This guidance (abbreviated in this document as the 2010 Cell Substrate Guidance) was developed for evaluating cell substrates and raw materials used for the production of viral vaccines, but the guidance does not address all cell-derived biologics, which would still be covered by requirements described in the 1993 Cell Lines PTC.

There are, however, some specific recommendations in the final guidance that need to be emphasized. The 2010 Cell Substrate Guidance specifies that the methods described in the guidance are considered to be acceptable alternatives to the methods specified in 21 CFR 610.30, as long as the modification is FDA approved in accordance with 21 CFR 610.9(b) (Equivalent methods and processes) [35]. Hence, the vaccine manufacturer may utilize the alternative testing methods (for broth and agar, only a single medium formulation and only anaerobic incubation conditions, but includes the added indicator cell test) rather than the broth and agar tests required in 21 CFR 610.30. In addition, the Guidance contains a recommendation to perform the culture and DNA staining tests for Spiroplasma and related organisms. This testing could be recommended for situations when cell substrates or materials of insect origin are used during manufacture. The composition of the media and incubation conditions are not specified, which allows different laboratories to work out the most optimal conditions for Spiroplasma detection for their process.

The 2010 Cell Substrate Guidance does allow for using polymerase chain reaction (PCR)-based assays (or other nucleic acid amplification technique [NAT] or alternative assays) if the alternative assay is shown to be comparable to the agar and broth media and indicator cell assays. Of note, the term used was “comparable to” the agar and broth media and indicator cell assays, rather than the term “of equivalent sensitivity,” which likely reflects a recognition that the methods are based on different readouts and detect different features of potentially contaminating mycoplasmas. Also, this guidance accepts the concept that acceptable assays might combine culture and alternative methods (e.g., using PCR to detect mycoplasma growth in the broth used for enrichment, in place of subculture to agar), and the document recognizes that PCR testing might be necessary. For example, for viral vaccines, there is the potential that the virus can interfere with the indicator cell assay due to cytopathic effects on the indicator cells. Two approaches have been recommended to address this situation: (i) neutralize the virus with specific antibodies, or (ii) utilize an indicator cell that is non-permissive for growth of the vaccine virus. However, there may be circumstances when these approaches are not sufficient, and other testing methods such as PCR may be required.

European Pharmacopoeia, 2.6.7 Mycoplasmas [EP 2.6.7]

The latest publication of the general chapter EP 2.6.7 Mycoplasmas was published in Supplement 6.1 and implemented in July 2007. EP 2.6.7 prescribes the test for mycoplasma for a master cell bank, a working cell bank, a virus seed lot or for control cells, using the culture (broth and agar) method and indicator cell culture method. For a virus harvest or a bulk vaccine or final lot the culture method alone is recommended and the indicator cell method may be used for screening of media. This approach differs somewhat from the 1993 Cell Lines PTC and the 2010 Cell Substrate Guidance, both of which recommend that the indicator cell culture procedure, as well as the broth and agar culture procedures, be performed for all test articles evaluated. EP 2.6.7 also states that NAT methods may be used as an alternative to either method after suitable validation [41, 42]. The latest revision shortened the test by 7 days, i.e., the agar plates from the 21 day subculture may be read at 7 days, so that the total test time is 28 days, vs. 35 days required in a previous version [43].

The media utilized should be shown to have satisfactory growth promotion for the mycoplasma strains listed in the compendium, i.e., A. laidlawii, M. gallisepticum, M. hyorhinis, M. orale, M. pneumoniae and M. synoviae. These organisms should be used as positive controls, depending on the product to be tested. The suitable strains are available from National Collection of Type Cultures (NCTC), Collection de l’Institut Pasteur (CIP), American Type Culture Collection (ATCC), and the European Directorate for the Quality of Medicines and Healthcare (EDQM) [44]. Incubation conditions for the broth and agar cultures are 35-38ºC and microaerophilic conditions (5-10% CO2 in N2) for the solid media. An evaluation for inhibitory substances in the test product should also be performed. The test product (10 mL) is inoculated into 100 mL of liquid (broth) medium and the broth cultures are sub-cultured onto agar plates at 2-4, 6-8, 13-15, and 19-21 days of incubation. The agar plates are incubated for not less than 14 days, except the subcultures at 20-21 days are incubated for only 7 days. EP 2.6.7 provides a listing of recommended media [8].

A procedure for the indicator cell culture method is also provided, which is similar to the indicator cell culture method described in the 1993 Cell Lines PTC, although a subculture of the cells inoculated with the test product is additionally recommended in EP 2.6.7. The implementation of this additional step may increase the sensitivity of mycoplasma contamination detection using the indicator cell culture method. EP 2.6.7 also provides for the use of NAT procedures, e.g., in case the conventional culture tests cannot be applicable for the test sample or when a rapid method is needed. Guidelines for the validation of NATs are also provided, for information. To allow use of NAT as an alternative method to the broth and agar procedures, the NAT method should detect 10 CFU/ml, and the NAT method should detect 100 CFU/ml as an alternative to the indicator cell culture method. However, these numbers do not represent the limit of detection for the culture procedures, and a comparability study comparing the respective detection limits for the alternative method and the culture procedures should be conducted and the obtained results used to select the most appropriate test.

United States Pharmacopoeia Chapter <63> Mycoplasma Tests [USP <63>]

The United States Pharmacopoeia (USP) recently published a general chapter on mycoplasma testing, which includes a description of the broth and agar culture method and the indicator cell culture method [7]. The chapter introduction states that validated nucleic acid amplification (NAT) or an enzymatic activity-based method may also be used to detect mycoplasma provided such a method is shown to be comparable to both the broth and agar culture and indicator cell procedures. However, USP <63> does not describe specific alternative NAT methods or the requirements for their validation.

The chapter indicates that mycoplasma testing is a necessary quality control requirement for pure biotechnological products and allied materials and that the broth and agar media and indicator cell culture procedures may be used to detect mycoplasma contamination of test articles, tissues and/or cell cultures used to produce test articles, digest broth, or any other material in which mycoplasma contamination is suspected. Based on the uses listed for the positive controls, the USP chapter could also be applied to vaccines and/or cell-derived materials/cultures for human and veterinary use, non-avian veterinary vaccines or cell cultures, vaccines or cell banks for human use, materials derived from avian material used during production, and vaccine or cell banks intended for use in poultry.

The broth and agar media procedures parallel the requirements of EP 2.6.7 and do not differ to a great extent from those described in the 1993 Cell Lines PTC and in the 2010 Cell Substrate Guidance. A test for the nutritive properties for each batch of medium is included as well as a test for inhibitory substances in the articles to be tested (which has not been a requirement for the 1993 Cell Lines PTC or the 2010 Cell Substrate Guidance). The positive controls include a dextrose fermenter and an arginine hydrolyser, and the species appropriate for each type of product to be tested include A. laidlawii, M. gallisepticum, M. hyorhinis, M. orale, M. pneumoniae (or M. fermentans), and M. synoviae. A Spiroplasma control is needed only for testing insect cell lines. As for EP 2.6.7 and the 2010 Cell Substrate Guidance, the positive cultures should have undergone a limited number of subcultures, i.e., not more than 15 passages from isolation [8, 9, 44]. Incubation conditions are at 36±1ºC and in microaerophilic conditions.

A detailed description of the differences between USP, EP 2.6.7, and the 1993 PTC document can be found in the article by Nims and Meyers [45].

Parenteral Drug Association Technical Report No. 50 Alternative Methods for Mycoplasma Testing [PDA Technical Report No. 50]

Also published in 2010 is the Parenteral Drug Association Technical Report No. 50 Alternative Methods for Mycoplasma Testing [28], abbreviated in this review as PDA Technical Report No. 50. This monograph provides a comprehensive discussion of the alternative methods that could potentially have utility to replace the classical broth and agar and indicator cell methods. The report also includes a summary of the existing assays and compendial methods, and tends to emphasize the limitations of the current broth and agar and indicator cell methods, in particular the time required, i.e., ≥28 days, to complete the incubation and inspection of the subculture agar plates. The report also includes in Appendix 1 a comparison of the methods described in regulations, compendia, and guidance documents, i.e., 9 CFR 113.28 for veterinary products, 21 CFR 610.30 for human viral vaccines, FDA 1993 Cell Lines PTC, EP 2.6.7, and the Japanese Pharmacopoeia XV.

Additional Articles and References Related to Issues of Mycoplasma Testing

In addition to the compendial and regulatory requirements discussed above, there are several other sources regarding mycoplasma contamination of cell culture that might be of interest and use to the reader. Selected papers resulting from the Third PDA Workshop on Mycoplasmas, held in Berlin (Germany) on March 24-26th, 2009, were published in a special section of Biologicals (Elsevier) [46]. The section was introduced in an editorial by Barbara Potts [47] and includes a summary of recent surveillance of mycoplasma contamination by a commercial testing laboratory [22].

The International Organization for Mycoplasmology (IOM) includes a standing committee, the International Research Programme on Comparative Mycoplasmology (IRPCM), with the goal to advance and disseminate knowledge on all aspects of mycoplasmas. There are a number of teams associated with the IRPCM, and the General Diagnostics and Cell Culture Team summarizes recent published and unpublished findings of the team members in biennial reports. The team has also recently published a report, Prevention and Control of Mycoplasma Contamination in Cell Cultures, available on the IOM website [4]

Nims and Meyers [45] have compared the language of the requirements in USP <63> with EP 2.6.7 and with the 1993 Cell Lines PTC, but that review does not include 21 CFR 610.30 or the PDA Technical Report No. 50 in the discussion. While EP 2.6.7 provided a section (for information) on the validation of nucleic acid amplification techniques (NAT) for the detection of mycoplasmas, USP<63> states only that alternative methods must be suitably validated but does not address validation requirements for alternate methods. In a recent article, John Duguid [48] provides a comprehensive summary of the concerns and the “top ten” validation considerations for implementing a rapid mycoplasma test.

Volokhov et al. [49] have recently published a review on alternative methods. This review includes analysis of advantages and disadvantages of different mycoplasma testing methods including compendial methods and alternative approaches and is focused on the strategy suitable for adequate evaluation and comparison of conventional and alternative mycoplasma testing methods. The importance of properly prepared and characterized mycoplasma reference strains required for a side-by-side comparability study is emphasized. An older review of techniques and recommended laboratory practices for avoiding mycoplasma infection of cell cultures is still relevant [50].

Final Thoughts

Although mycoplasma contamination of biological products has been recognized for many years, there still is no global consensus regarding the most appropriate test methods for all products, as evidenced by the variations in the regulatory and compendial requirements and guidance recommendations. It may not be possible to put forth a “one size fits all” test procedure due to the diversity of the potential mycoplasma contaminants and differences in the products that need to be evaluated. What may be more relevant is a careful, risk-based consideration of the most appropriate mycoplasma detection method to be used for a specific product at a required stage of manufacture.

The emphasis of this review is based on the requirements for vaccines intended for human use, and especially when those humans are infants and children, it makes sense to use the most conservative and sensitive screening methods. For example, the time consuming broth and agar culture and indicator cell culture procedures may still be appropriate for evaluating master and working cell banks and viral seeds when the urgency for the test results is not high. On the other hand, use of an NAT or similar method to test for the most frequent contaminants in an unprocessed bulk provides an advantage where expeditious results can help avoid contamination of columns and other equipment used in downstream processing.

In summary, except for the testing specified in 21 CFR 610.30, the selection of the most appropriate mycoplasma testing method(s), which are detailed in USP<63>, 21 CFR 610.30, and the 2010 Cell Substrate Guidance [9], depends primarily on the specific biological product and its manufacture. In the end, scientific judgment needs to be applied to determine which products and which stage of manufacture should be tested for mycoplasma as well as the most appropriate method. These considerations will be reflected in the Biologics License Application for a specific biologic product.

Acknowledgements

We thank Drs. Laurie Graham and Maureen K. Davidson for critical review of this article.

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Author Biographies

Dr. Donna K.F. Chandler is currently an independent consultant for vaccine development. Previously, she was Deputy Director, Divison of Vaccines and Related Products Applications, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration (1997-2006). She was a reviewer at FDA from 1989 to 1997, and researched pathogenicity of mycoplasmas and food-borne organisms at FDA from 1978-1989.

Dr. Dmitriy Volokhov holds a Visiting Associate position in the Laboratory of Methods Development at the Center for Biologics Evaluation and Research (CBER), US FDA. Dr. Volokhov’s areas of expertise include Virology, Microbiology, Infectious Diseases, and Molecular Biology. He is the author of more than 20 publications on microbiology in scientific peer-reviewed journals.

Dr. Vladimir Chizhikov holds a Principal Investigator position in the Laboratory of Methods Development at the Center for Biologics Evaluation and Research (CBER), FDA. Dr. Chizhikov’s areas of expertise include Virology, Microbiology, and Molecular Biology. He is the author of more than 80 publications in scientific peer-reviewed journals.

This article was printed in the May/June 2011 issue of American Pharmaceutical Review - Volume 14, Issue 4. Copyright rests with the publisher. For more information about American Pharmaceutical Review and to read similar articles, visit www.americanpharmaceuticalreview.com  and subscribe for free.

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