Research Article: High-level expression of biologically active human follicle stimulating hormone in the Chinese hamster ovary cell line by a pair of tricistronic and monocistronic vectors

Date Published: July 5, 2019

Publisher: Public Library of Science

Author(s): Nadezhda A. Orlova, Sergey V. Kovnir, Yulia A. Khodak, Mikhail A. Polzikov, Victoria A. Nikitina, Konstantin G. Skryabin, Ivan I. Vorobiev, Paulo Lee Ho.

http://doi.org/10.1371/journal.pone.0219434

Abstract

Recombinant human follicle stimulating hormone (FSH), produced in Chinese hamster ovary (CHO) cells, is widely used for treatment of fertility disorders and is subject to biosimilars development. Cell lines with high specific productivities may simplify the FSH production process. Here, we used our previously established expression system based on vector p1.1 to create new cell lines secreting heterodimeric FSH protein. To this end, we linked open reading frames of both FSH subunits by the wild-type internal ribosome entry site from the encephalomyocarditis virus (EMCV IRES). Intact and double-negative for the dihydrofolate reductase CHO cells were stably transfected by the FSH-coding plasmids. Stably transfected intact cells showed higher level of the FSH secretion and were utilized for subsequent methotrexate-driven transgene amplification, which doubled their productivity. The excess of the free α-subunit was corrected by transfecting the cells by the additional p1.1-based plasmid encoding the β-subunit of the FSH. Clonal cell lines obtained secreted mostly the heterodimeric FSH and possessed specific productivities up to 12.3±1.7 pg/cell/day. Candidate clonal cell line C-P1.3-FSH-G4 maintained a constant specific productivity for at least 2 months of culturing without the section pressure. The resulting FSH protein conformed to the international pharmaceutical quality criteria as evidenced by the receptor binding kinetics, distribution pattern of hormone isoforms and biological activity. In conclusion, our expression system offers a simple and cost-effective approach to production of FSH.

Partial Text

Follicle stimulating hormone (FSH) is a heterodimeric glycoprotein produced by the pituitary gland that promotes growth and maturation of ovarian follicles in females and spermatogenesis in males [1]. Urinary-derived and recombinant forms of FSH are widely used in infertility treatment for assisted reproducing technologies [2]. Since both of the above have the same efficacy and safety profiles, the clinical choice of FSH source is usually defined by availability, convenience, and cost [2]. As patents on innovative recombinant FSH products approach expiration, there is an increasing global interest in developing methodology for biosimilar FSH generation [3].

The aim of this study was to develop a new recombinant FSH producing cell lines with high specific productivity and sufficient long-term genetic stability. We used previously designed specialized plasmid vectors, containing non-coding areas of the EEF1A1 gene and the fragment of the long terminal repeat from the Epstein-Barr virus. Open reading frames of two FSH subunits were connected by the wild-type EMCV IRES and linked to the mouse DHFR ORF area by the attenuated EMCV IRES, forming the tricistronic mRNAs. It was found that secretion of heterodimeric FSH is evident only in the case of α-chain ORF in the first cistron position, the opposite order of chain’s ORFs resulted in non-secreting stably transfected cell population. DHFR-positive CHO S cells, stably transfected by the p1.1-FSH-BIA, demonstrated much higher specific productivity, than DHFR-negative CHO DG44 cells. Excess of the free α-chain of FSH in the culture medium was compensated by the second transfection of the polyclonal cell population by the plasmid, coding only the β-chain of FSH. Several of the clonal cell lines derived from the doubly transfected cells secrete heterodimeric FSH in large quantities. Therefore, the combination of genome-amplifiable tricistronic plasmid vector and monocistronic vector with the DHFR-compatible selection marker is sufficient for generating cell lines secreting large quantities of heterodimeric glycoprotein hormones.

 

Source:

http://doi.org/10.1371/journal.pone.0219434