RECOMBINANT HUMAN LUTEINIZING HORMONE FOR THE TREATMENT OF INFERTILITY: THE GENERATION OF PRODUCER CELL LINES

The aim of the study was to obtain the cell line secreting the maximum amounts of the human luteinizing hormone (LH) heterodimer without significant amounts of free LH alpha-subunit.

Materials and methods. A pair of original plasmids of the p1.1 family containing long non coding segments of the Chinese hamster translation elongation factor 1 alpha gene and a fragment of the concatemer of the Epstein-Barr virus long terminal repeat were used for the persistent expression of human LH subunit genes in Chinese hamster ovary cells. The open reading frame areas of the LH subunits genes were cloned into vectors with the dihydrofolate reductase (DHFR) or glutamine synthase (GS) genes, transfected into the cells, and amplified by methotrexate; following the final procedure of limiting dilution, clonal producer cells were obtained.

Results. For a pair of plasmids, where the LH alpha subunit gene was associated with GS and the beta subunit gene was associated with DHFR, the rate of LH production by the initial stably transfected cell population was 0.1 pg/cell/day and the doubling time was 37 hours, for the reverse pair of plasmids – 0.3 pg/cell/day and 48 hours, respectively. For the initial cell population and for their clonal lines, we used increasing concentrations of methotrexate to amplify the genetic cassettes in the cell genome. In the polyclonal population an increase in the cell productivity up to 2.2 μg/10⁶ cells was observed at a concentration of methotrexate 8 μM; for the clonal lines – a significant productivity increase up to 0.09 μg/10⁶ cells was achieved in only one of six cell lines. Conclusion. Cell lines secreting significant amounts of heterodimeric LH without a significant admixture of free subunit can be obtained using a pair of plasmid vectors encoding the LH subunit genes and the selection markers DHFR or GS. Co-transfection of the plasmids and their subsequent amplification in the genome of producer cells under the selective pressure of methotrexate results in a significant increase in the biosynthesis rate of both LH subunits. The present study provides a rationale for a potential use of these lines in the industrial production of human luteinizing hormone.

1. Munoz E., Bosch E., Fernandez I., Portela S., Ortiz G., Remohi J., Pellicer A. The role of LH in ovarian stimulation. Curr Pharm Biotechnol. 2012; 13 (3): 409-16.

2. Leao Rde B., Esteves S.C. Gonadotropin therapy in assisted reproduction: an evolutionary perspective from biologics to biotech. Clinics (Sao Paulo). 2014; 69 (4): 279-93.

3. Ata B., Seli E. Strategies for Controlled Ovarian Stimulation in the Setting of Ovarian Aging. Semin Reprod Med. 2015; 33 (6): 436-48.

4. Bleau N., Agdi M., Son W., Tan S., Dahan M.H. A Comparison of Outcomes from In Vitro Fertilization Cycles Stimulated with Follicle Stimulating Hormone Plus either Recombinant Luteinizing Hormone or Human Menopausal Gonadotropins in Subjects Treated with Long Gonadotropin Releasing Hormone Agonist Protocols. Int J Fertil Steril. 2017; 11 (2): 79-84.

5. Mullen M.P., Cooke D., Crow M. Structural and functional roles of FSH and LH as glycoproteins regulating reproduction in mammalian species. In: Gonadotropin [Ed. J. Vizcarra]. Chapter 8. InTech: International Publisher. 2013: 155-80.

6. Vorobiev I.I., Orlova N.A., Kovnir S.V., Khodak Yu.A. Plasmid for expression in CHO cells of recombinant human follicle stimulating hormone (FSH), a plasmid for expression in CHO cells of the beta subunit of recombinant human FSH, a CHO-producer of recombinant human FSH, and a method for producing said hormone [Plazmida dlya ekspressii v kletkah SNO rekombinantnogo follikulostimuliruyushchego gormona (FSG) cheloveka, plazmida dlya ekspressii v kletkah SNO beta-sub"edinicy rekombinantnogo FSG cheloveka, kletka SNO – producent rekombinantnogo FSG cheloveka i sposob polucheniya ukazannogo gormona]. Patent RF № 2560596. 2015; Byul. № 23. URL: http://www.findpatent.ru/patent/256/2560596.html (in Russian) [Accessed: 20.05.2017].

7. Orlova N.A., Kovnir S.V., Hodak J.A., Vorobiev I.I., Gabibov A.G., Skryabin K.G. Improved elongation factor-1 alpha-based vectors for stable high-level expression of heterologous proteins in Chinese hamster ovary cells. BMC Biotechnol. 2014; 14: 56.

8. Bochkov Y.A., Palmenberg A.C. Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location. Biotechniques. 2006; 41 (3): 283-284, 286, 288 passim.

9. Almo S.C., Love J.D. Better and faster: improvements and optimization for mammalian recombinant protein production. Curr Opin Struct Biol. 2014; 26: 39-43.

10. Jazayeri S.H., Amiri-Yekta A., Gourabi H., Abd Emami B., Halfinezhad Z., Abolghasemi S. et al. Comparative Assessment on the Expression Level of Recombinant Human Follicle-Stimulating Hormone (FSH) in Serum-Containing Versus Protein-Free Culture Media. Mol Biotechnol. 2017; 37 (4): 1-9. DOI: 10.1007/s12033-017-0037-4.

11. Nakamura Y., Gojobori T., Ikemura T. Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Res. 2000; 28 (1): 292.

12. Kovnir S.V., Orlova N.A., Khodak C. et al. Approaches to Controlled Co-Amplification of Genes for Production of Biopharmaceuticals: Study of the Insertion and Amplification Dynamics of Genetic Cassettes in the Genome of Chinese Hamster Ovary Cells during Co-Expression of Compatible Pair of Plasmids. Bull Exp Biol Med. 2017; 163 (2): 245-9.

13. Kovnir S.V., Orlova N.A., Vorobiev I.I. et al. Plasmid for expression of recombinant factor IX blood coagulability, CHO cell – producer of recombinant factor IX blood coagulability and the method of obtaining this factor [Plazmida dlya ekspressii rekombinantnogo faktora svyortyvaemosti krovi IX cheloveka, kletka SNO – producent rekombinantnogo faktora svyortyvaemosti krovi IX cheloveka i sposob polucheniya ukazannogo faktora]. Patent RF № 2585532. 2015; Byul. № 15. URL: https://edrid.ru/rid/216.015.4457.html (in Russian) [Accessed: 20.05.2017].

14. Urlaub G., Kas E., Carothers A.M., Chasin L.A. Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells. Cell. 1983; 33 (2): 405-12.