CHO CELL LINE SELECTION FOR DEVELOPMENT OF STABLE CELL LINE PRODUCING RECOMBINANT HUMAN FOLLICLE-STIMULATING HORMONE

DOI: https://doi.org/10.29296/25877313-2020-03-01
Issue: 
3
Year: 
2020

V.S. Monakhova Biologist, FGUP «State Research Institute of Highly Pure Biopreparations» of the Federal Biomedical Agency (Saint-Petersburg) E-mail: v.s.monakhova@hpb.spb.ru N.V. Pigareva Ph.D. (Med.), Senior Biologist, FGUP «State Research Institute of Highly Pure Biopreparations» of the Federal Biomedical Agency (Saint-Petersburg) A.S. Simbirtsev Corresponding member of RAS, Dr.Sc. (Med.), Professor, Scientific Supervisor, FGUP «State Research Institute of Highly Pure Biopreparations» of the Federal Biomedical Agency (Saint-Petersburg)

Follicle-stimulating hormone (FSH) is one of the most important hormones of reproductive system and it is widely used as a treatment in an assisted reproductive technology. Choice of productive cell line for recombinant human FSH (rhFSH) protein production is the major issue of biotechnological process. Study objective. The aim of the study was to generate stable cell lines for rhFSH production using different parental CHO cell lines and compare their characteristics. Material and methods. Two CHO suspension parental cell lines were used for stable cell line generation – CHO-K1 and CHO-DXB 11 adapted to sus-pension conditions. Cell count and viability were analyzed by trypan blue exclusion method. RhFSH concentration was measured by ELISA, protein iden-tity was confirmed by western blot analysis. Results. Four clones generated from CHO-DXB 11 cell line were selected after 3 steps – pool selection, clone selection and methotrexate amplification. Productivity during 6 days of cultivation for the most productive clone CHO-FSH 11/24 was 7,1±0,6 mg/l. Three clones were chosen after transfection and cell cloning of CHO-K1 cells transfected with plasmids containing of FSH α- and β-chains sequences. The highest productivity was detected for CHO-FSH91 clone and it was 10,8±0,9 mg/l. Molecular weight of rhFSH was identical for rhFSH purified from cell culture of optimal clones and com-mercial preparation Gonal-F (Merck Serono).

Keywords: 
CHO-FSH91 clone which was generated from CHO-K1 cell line was chosen for the further experiments

References: 
  1. Latash W.D., Karande V.C., Klipstein S. The development of recombinant follicle stimulating hormone (rFSH) for use in the treatment of infertility: new solutions to old challenges. Reviews in Gynaecological Practice. 2004; 4:203-210.
  2. Keene J.L., Matzuk M.M. et al. Expression of biologically active human follitropin in chinese hamster ovary cells. The Journal of Biological Chemistry. 1989; 264(9):4769-4775.
  3. Olijve W., Boer W. et al. Molecular biology and biochemistry of human recombinant follicle stimulating hormone (Puregon®). Molecular Human Reproduction. 1996; 2(5):371-382.
  4. NA K.H., Seo K.S., Kim W.B., Kim S.-Ch., Lee S.-H., Lee K.S. Purification and Characterization of Recombinant Human Follicle Stimulating Hormone Produced by Chinese Hamster Ovary Cells. J. Microbiol. Biotechnol. 2005; 15(2):395-402.
  5. Kim D.J., Seok S.-H., Baek M.-W., Lee H.-Y., Juhn J.-H., Lee S. et al. Highly expressed recombinant human follicle-stimulating hormone from Chinese hamster ovary cells grown in serum-free medium and its effect on induction of folliculogenesis and ovulation. 2010; 93(8):2652-2660.
  6. Patent № 2615447 (RF). Sinteticheskaja DNK, kodirujuschaja interlejkin-7 cheloveka, soderzhaschij ee eks-pressionnyj vektor (varianty), shtamm-produtsent inter-lejkina-7 cheloveka i sposob poluchenija interlejkina-7 cheloveka. Ischenko A.M., Kudling T.V., Petrov A.V., Pigareva N.V., Protasov E.A., Simbirtsev A.S. 2017. (Patent № 2615447 (RF). Sinteticheskaya DNK, ko-diruyushchaya interlejkin-7 cheloveka, soderzhashchij ee eks-pressionnyj vektor (varianty), shtamm-producent inter-lejkina-7 cheloveka i sposob polucheniya interlejkina-7 cheloveka. Ishchenko A.M., Kudling T.V., Petrov A.V., Pigareva N.V., Protasov E.A., Simbircev A.S. 2017).
  7. Patent № 2616273 (RF). Sinteticheskaja DNK, kodiru-juschaja antimjullerov gormon cheloveka, soderzhaschij ee ekspressionnyj vektor pTVK4pu/MISOPT i shtamm kletok jaichnikov kitajskogo homjachka CHO-MIS – produtsent rekombinantnogo antimjullerovogo gormona cheloveka. Petrov A.V., Karasev M.M., Kudling T.V., Pigareva N.V., Sergeeva V.E., Trofimov A.V., Ischenko A.M., GorbunovA.A.,Vahrushev A.V., Simbirtsev A.S. 2017. (Sinteticheskaya DNK, kodiruyushchaya antimyullerov gormon cheloveka, soderzha-shchij ee ekspressionnyj vektor pTVK4pu/MISOPT i shtamm kletok yaichnikov kitajskogo homyachka CHO-MIS – producent rekombinantnogo antimyullerovogo gormona cheloveka. Patent № 2616273 (RF). Petrov A.V., Karasev M.M., Kudling T.V., Pigareva N.V., Sergeeva V.E., Trofimov A.V., Ishchenko A.M., Gorbunov A.A., Vahrushev A.V., Simbircev A.S. 2017).
  8. Betts Z., Dickson A.J. Ubiquitous Chromatin Opening Elements (UCOEs) effect on transgene position and expression stability in CHO cells following methotrexate (MTX) amplification. Biotechnol J. 2016; 11(4):554-641.