Standardizing microbiological method to detect mycoplasma contamination in biological products: development of Acholeplasma laidlawii PG8 reference standard

INTRODUCTION. Mycoplasmas are one of the most challenging types of microbial contamination to detect in biological products (BP). Detecting micoplasmas in the BPs and standardizing microbiological control method necessitates highly sensitive culture media and pharmacopeial reference standards (RS) for test strains of various mycoplasmas.

AIM. This study aimed to develop and certify the new pharmacopeial reference standard Acholeplasma laidlawii PG8 for mycoplasma identification in the BPs using the microbiological method.

MATERIALS AND METHODS. A. laidlawii PG8 (NCPМ 930002, ATCC 23206, NCTC 10116) strain, Kagan's medium, and horse serum was used to develop the reference standard in compliance with the State Pharmacopoeia of the Russian Federation (SP RF). The certification included determination of strain titre by the most probable number method and quality tests for: Description, Vacuum, Reconstitution time, Description of dissolved sample, Sterility, Loss on drying, and Mass homogeneity (average mass and average deviation).

RESULTS. Development and specification of A. laidlawii PG8 reference standard was completed. Lyophilised samples of the master and working bank of A. laidlawii PG8 strain culture were prepared. It was established that the lyophilization process does not significantly affect strain viability. The stability of strain titers (10⁸ CFU/mL), cultural, and physicochemical properties of the samples was demonstrated for critical quality and reliability indicators during two years of storage at -20 to -30°C. Three batches of A. laidlawii PG8 RS were certified. The calculated titer values ranged from 10×10⁸ to 21×10⁸ CFU/mL. The pharmacopoeial reference standard is found to be comparable to the European Pharmacopoeia reference standard A. laidlawii BRP, batch 1 (titer 2.45×10⁶ CFU/mL). As a result of the conducted research, A. laidlawii PG8 RS was included in the Register and Collection of Reference Standards of the Russian Pharmacopoeia.

CONCLUSIONS. A new A. laidlawii PG8 reference standard has been developed and certified that can be used to evaluate the nutritive properties of culture media; determine the inhibitory effects; and serve as a positive control in BP and material tests for the presence of mycoplasmas using the microbiological method in accordance with SP RF requirements. Introducing the new RS will contribute to standardization and improve BP control, as well as harmonization of the Russian compendial requirements with the international standards.

1. David SA, Volokhov DV, Ye Z, Chizhikov V. Evaluation of Mycoplasma inactivation during production of biologics: egg-based viral vaccines as a model. Appl Environ Microbiol. 2010;76(9):2718–28. https://doi.org/10.1128/AEM.02776-09

2. Gladin DP, Kozlova NS, Edelstein IA, et al. Mycoplasmas. Biological properties (lecture). Russian Biomedical Research. 2023;8(4):103–15 (In Russ.). https://doi.org/10.56871/RBR.2023.58.16.012

3. Leonovich OA. Mycoplasma: Properties, detection and decontamination methods of cell cultures and viral strains (review). Applied Biochemistry and Microbiology. 2024;60:785–93 (In Russ.). https://doi.org/10.1134/S0003683824604943

4. Kalaivani M, Chaudhary P, Goyal M, et al. Current regulatory and pharmacopoeial status of mycoplasma testing in concern with vaccines safety for human use. J Basic Clin Pharma. 2021;12(S4):001.

5. Medvedeva ES, Mouzykantov AA, Kostenko VV, et al. Adaptation to antimicrobials and pathogenicity in mycoplasmas: Development of ciprofloxacin-resistance and evolution of virulence in Acholeplasma laidlawii. Dokl Biochem Biophys. 2021;501(1):444–8. https://doi.org/10.1134/S1607672921060028

6. Dumchenko NB, Sementsova AO, Nechaeva EA. Research of contamination with mycoplasma infections of cell FBSI SRC VB “Vector” of Rospotrebnadzor. PNRPU Bulletin. Chemical Tecnology and Biotechnology. 2023;(3):19–29 (In Russ.). EDN: JUIIMN

7. Rottem S, Kosower NS, Kornspan JD. Contamination of tissue cultures by mycoplasmas. In: Ceccherini-Nelli L, Matteoli B, eds. Biomedical Tissue Culture. 2012. https://doi.org/10.5772/51518

8. Milne C, Daas A. Establishment of European Pharmacopoeia mycoplasma reference strains. Pharmeuropa Bio. 2006;(1):57– 72. PMID: 17270132

9. Sukhanova SM, Berdnikova ZE, Tikhonova AS. Ways to improve quality control of culture medium used for mycoplasma detection. BIOpreparations. Prevention, Diagnosis, Treatment. 2019;19(3):161–8 (In Russ.). https://doi.org/10.30895/2221-996X-2019-19-3-161-168

10. Glantz SA. Primer of Biostatistics. 4th ed. New York: McGraw–Hill; 1994.

11. Fadeikina OV, Volkova RA. Elaboration of certification procedures for reference standards of biological drugs. Pharm Chem J. 2017;51(8):716–21. https://doi.org/10.1007/s11094-017-1680-6

12. Gegechkori VI, Shatilina AA, Shulga NA, et al. Biological reference materials: Topical issues of development and certification procedure. Measurement Standards. Reference Materials. 2023;19(3):21–9 (In Russ.). EDN: RKQAVM

13. Sukhanova SM, Berdnikova ZE, Fadeikina OV, et al. Stability analysis of a standard sample of the Mycoplasma arginini G230 used in testing for the presence of mycoplasmas of biological drugs. Biological Products. Prevention, Diagnosis, Treatment. 2022;22(3):331–46 (In Russ.). https://doi.org/10.30895/2221-996X-2022-22-3-331-346