Development of technology for culturing a cell line producing a single-domain antibody fused with the Fc fragment of human IgG1

Objectives. To develop an effective technology for the cultivation of Chinese hamster ovary (CHO) cells stably producing GamP2C5 antibody which is a component I of the GamCoviMab candidate drug for emergency prevention and therapy of  infection caused by SARS-CoV-2 virus; to select optimal cultivation parameters and to scale this technology in production.
Methods. The study was performed on CHO GamP2C5 (clone 78) cell culture, producing a single-domain antibody fused to the Fc fragment of human IgG1 GamP2C5. Different culture media and supplements were used. Cells were cultured in Erlenmeyer flasks, Biostat^® RM 20 wave-mixed bioreactor, Ambr^® 250 mini bioreactors, STR 200 stirred-tank bioreactor.
Results. Using molecular-genetic and biotechnological methods, a stable clone producer of CHO GamP2C5 antibody, clone 78, was obtained. Then a technique was worked out for the cultivation of the obtained clone producer on different culture media. The most suitable cultivation regimes, culture media, and optimal supplements were selected. This technology was tested in laboratory conditions in a 10-L reactor, and then successfully scaled up for production at the MedGamal Branch of the Gamaleya National Research Center for Epidemiology and Microbiology.
Conclusions. This study demonstrates the fundamental feasibility of developing and scaling up a culture technology, in order to produce a drug based on a modified single-domain antibody with virus neutralizing activity against different strains of SARS-CoV-2 virus.

1. Sun J., Yang .D., Xie X., Li L., Zeng H.Z., Gong B., Xu J.Q., Wu J.H., Qu B.B., Song G.W. Clinical application of SARS-CoV-2 antibody detection and monoclonal antibody therapies against COVID-19. World J. Clin. Cases. 20236;11(10):2168–2180. https://doi.org/10.12998/wjcc.v11.i10.2168

2. Mitra S., Tomar P.S. Hybridoma technology; advancements, clinical significance, and future aspects. J. Genet. Eng. Biotechnol. 2021;19(1):159. https://doi.org/10.1186/s43141-021-00264-6

3. Lu R.M., Hwang Y.C., Liu I.J., Lee C.C., Tsai H.Z., Li H.J., Wu H.C. Development of therapeutic antibodies for the treatment f diseases. J. Biomed. Sci. 2020;27(1):1. https://doi.org/10.1186/s12929-019-0592-z

4. Quinteros D.A., Bermúdez J.M., Ravetti S., CidA., Allemandi D.A., Palma S.D. Therapeutic use of monoclonal antibodies: general aspects and challenges for drug delivery. Chapter 25. In: Nanostructures for Drug Delivery. Micro and Nano Technologies. 2017:807–833. https://doi.org/10.1016/B978-0-323-46143-6.00025-7

5. Garcia J., Hurwitz H.I., Sandler A.B., Miles D., Coleman R.L., Deurloo R., Chinot O.L. Bevacizumab (Avastin®) in cancer treatment: a review of 15 years of clinical experience and future outlook. Cancer Treat. Rev. 2020;86:102017. https://doi.org/10.1016/j.ctrv.2020.102017

6. Chisari C.G., Sgarlata E., Arena S., Toscan S., Luca M., Patti F. Rituximab for the treatment of multiple sclerosis: a review. J. Neurol. 2022;269(1):159–183. https://doi.org/10.1007/s00415-020-10362-z

7. Hemperly A., Vande Casteele N. Clinical Pharmacokinetics and Pharmacodynamics of Infliximab in the Treatment of Inflammatory Bowel Disease. Clin. Pharmacokinet. 2018;57(8):929–942. https://doi.org/10.1007/s40262-017-0627-0

8. Weinblatt M.E., Keystone E.C., Furst D.E., Kavanaugh A.F., Chartash E.K., Segurado O.G. Long term efficacy and safety of adalimumab plus methotrexate in patients with rheumatoid arthritis: ARMADA 4-year extended study. Ann. Rheum. Dis. 2006;65(6):753–759. https://doi.org/10.1136/ard.2005.044404

9. Salazar G., Zhang N., Fu T.M., An Z. Antibody therapies for the prevention and treatment of viral infections. NPJ Vaccines. 2017;2:19. https://doi.org/10.1038/s41541-017-0019-3

10. Pelegrin M., Naranjo-Gomez M., Piechaczyk M. Antiviral Monoclonal Antibodies: Can They Be More Than Simple Neutralizing Agents? Trends Microbiol. 2015;23(10): 653–665. https://doi.org/10.1016/j.tim.2015.07.005

11. Pantaleo G., Correia B., Fenwick C., Joo V.S., Perez L. Antibodies to combat viral infections: development strategies and progress. Nat. Rev. Drug Discov. 2022;21(9):676–696. https://doi.org/10.1038/s41573-022-00495-3

12. Miyazato Y., Yamamoto K., Nakaya Y., Morioka S., Takeuchi J.S., Takamatsu Y., Maeda K., Kimura M., Sugiura W., Mitsuya H., Yano M., Ohmagaria N. Successful use of casirivimab/imdevimab anti-spike monoclonal antibodies to enhance neutralizing antibodies in a woman on anti-CD20 treatment with refractory COVID-19. J. Infect. Chemother. 2022;28(7):991–994. https://doi.org/10.1016/j.jiac.2022.03.002

13. Jovčevska I., Muyldermans S. The Therapeutic Potential of Nanobodies. BioDrugs. 2019;34(1):11–26. https://doi.org/10.1007/s40259-019-00392-z

14. Esmagambetov I.B., Shcheblyakov D.V., Egorova D.A., Voronina O.L., Derkaev A.A., Voronina D.V., Popova O., Ryabova E.I., Shcherbinin D.N., Aksenova E.I., SemenovA.N., Kunda M.S., Ryzhova N.N., Zubkova O.V., Tukhvatulin A.I., Logunov D.Y., Naroditsky B.S., Borisevich S.V., Gintsburg A.L. Nanobodies Are Potential Therapeutic Agents for the Ebola Virus Infection. Acta Naturae. 2021;13(4): 53–63. https://doi.org/10.32607/actanaturae.11487 ]

15. Derkaev A.A., Ryabova E.I., Esmagambetov I.B., Shcheblyakov D.V., Godakova S.A., Vinogradova I.D., Noskov A.N., Logunov D.Y., Naroditsky B.S., Gintsburg A.L. rAAV expressing recombinant neutralizing antibody for the botulinum neurotoxin type A prophylaxis. Front. Microbiol. 2022;13:960937. https://doi.org/10.3389/fmicb.2022.960937

16. Voronina D.V., Shcheblyakov D.V., Favorskaya I.A., Esmagambetov I.B., Dzharullaeva A.S., Tukhvatulin A.I., Zubkova O.V., Popova O., Kan V.Y., Bandelyuk A.S., Shmarov M.M., Logunov D.Y., Naroditskiy B.S., Gintsburg A.L. Cross-Reactive Fc-Fused Single-Domain Antibodies to Hemagglutinin Stem Region Protect Mice from Group 1 Influenza a Virus Infection. Viruses. 2022;14(11):2485. https://doi.org/10.3390/v14112485

17. Panova E.A., Kleymenov D.A., Shcheblyakov D.V., Bykonia E.N., Mazunina E.P., Dzharullaeva A.S., Zolotar A.N., Derkaev A.A., Esmagambetov I.B., Sorokin I.I., Usachev E.V., Noskov A.N., Ivanov I.A., Zatsepin T.S., Dmitriev S.E., Gushchin V.A., Naroditsky B.S., Logunov D.Y., Gintsburg A.L. Single-domain antibody delivery using an mRNA platform protects against lethal doses of botulinum neurotoxin A. Front. Immunol. 2023;14:1098302. https://doi.org/10.3389/fimmu.2023.1098302

18. Godakova S.A., Noskov A.N., Vinogradova I.D., Ugriumova G.A., Solovyev A.I., Esmagambetov I.B.., Tukhvatulin AI., Logunov D.Y., Naroditsky B.S.., Shcheblyakov D.V., Gintsburg A.L. Camelid VHHs Fused to Human Fc Fragments Provide Long Term Protection Against Botulinum Neurotoxin A in Mice. Toxins (Basel). 2019;11(8):464. https://doi.org/10.3390/toxins11080464

19. Esmagambetov I.B., Ryabova E.I., Derkaev A.A., Shcheblyakov D.V., Dolzhikova I.V., Favorskaya I.A., Grousova D.M., Dovgiy M.A., Prokofiev V.V., GosudarevA.I., Byrikhina D.V., Zorkov I.D., Iliukhina A.A., Kovyrshina A.V., Shelkov A.Y., Naroditsky B.S., Logunov D.Y., Gintsburg A.L. rAAV expressing recombinant antibody for emergency prevention and long-term prophylaxis of COVID-19. Front. Immunol. 2023;14:1129245. https://doi.org/10.3389/fimmu.2023.1129245

20. Favorskaya I.A., Shcheblyakov D.V., Esmagambetov I.B., Dolzhikova I.V., Alekseeva I.A., Korobkova A.I., Voronina D.V., Ryabova E.I., Derkaev A.A., Kovyrshina A.V., et al. Single-Domain Antibodies Efficiently Neutralize SARS-CoV-2 Variants of Concern. Front. Immunol. 2022;13:822159. https://doi.org/10.3389/fimmu.2022.822159

21. Sedova E.S., Shcherbinin D.N., Bandelyuk A.S., Verkhovskaya L.V., Viskova N.Yu., Avdonina E.D., Prokofiev V.V., Ryabova E.I., Esmagambetov I.B., Pervoykina K.A., Bogacheva E.A., Lysenko A.A., Shmarov M.M. Method for obtaining recombinant antibodies produced by a cell line transduced with recombinant adenoviruses. Tonk. Khim. Tekhnol. = Fine Chem. Technol. 2023;18(1):48–64 (Russ., Eng.). https://doi.org/10.32362/2410-6593-2023-18-1-48-64 ]

22. Yao T., Asayama Y. Animal-cell culture media: History, characteristics, and current issues. Reprod. Med. Biol. 2017;16(2):99–117. https://doi.org/10.1002/rmb2.12024

23. Xu P., Clark C., Ryder T., Sparks C., Zhou J., Wang M., Russell R., Scott C. Characterization of TAP Ambr 250 disposable bioreactors, as a reliable scale-down model for biologics process development. Biotechnol. Prog. 2017;33(2):478–489. https://doi.org/10.1002/btpr.2417