Cell culture production of a bioactive recombinant equine chorionic gonadotropin (reCG, FOLI-REC®): replacement of animals as a source of PMSG

VILLARRAZA, CARLOS JAVIER; ANTUÑA, SEBASTIÁN; TARDIVO, MARÍA BELÉN; RODRÍGUEZ, MARÍA CELESTE; FONTANA, DIEGO; BÓ, GABRIEL; CATTANEO, LUCIANO; CEAGLIO, NATALIA; PRIETO, CLAUDIO

Lisboa

Congreso; 27th ESACT Meeting; 2022

Background and novelty: Equine Chorionic Gonadotropin (eCG) is a glycoprotein hormone used to improve reproductive performance in different domestic species. Until recently, the only product available on the market consisted of partially purified eCG preparations from blood of pregnant mares (PMSG). This not only constitutes a health risk due to possible plasma contaminants, but also raises some bioethical issues. For these reasons, the development of a recombinant eCG (reCG, FOLI-REC®) represents a powerful strategy to substitute PMSG. The aim of this work was to develop a suitable manufacturing process for the production of a biologically active reCG expressed in suspension CHO-K1 (sCHO-K1) cells cultured in serum-free medium.Experimental approach: Cell lines were generated through lentiviral transgenesis of sCHO-K1 cells, and then cloned in 96-well plates using the limiting dilution method. The highest reCG producing clone was cultured in a one-liter bioreactor in perfusion mode. reCG was purified from supernatants using a CaptoBlue-Sepharose FF resin. Physicochemical characterization of purified reCG was evaluated following SDS-PAGE, western-blot and isoelectric focusing (IEF). The biopotency of purified reCG and of PMSG was evaluated in female rats using the Steelman and Pohley (SP) bioassay. The efficacy of different doses of reCG at the end of a timed artificial insemination (TAI) protocol on pregnancy rates (P/AI) was evaluated in anestrous Bos indicus x Bos taurus crossbred suckled cows.Results and discussion: The use of lentiviruses as a gene delivery system allowed the development of highly producer sCHO-K1 cell lines and clones, achieving a productivity around 1.8x105 IU of reCG per day in a perfusion bioreactor. reCG presented a smaller apparent molecular mass and a lower number of glycoforms compared to PMSG, which would indicate a greater homogeneity of the recombinant hormone compared to the natural variant. The specific biological activity of reCG was 4900 ± 500 IU eCG/mg total protein. Pregnancy rates to IA were 45.0% and 42.9% for cows receiving 140 IU of reCG and 400 IU PMSG, respectively. Thus, reCG not only demonstrated biological activity in cattle, but also a dose of 140 IU of reCG exerted a biological effect comparable to 400 IU of PMSG, which could indicate a higher bioactivity of the recombinant hormone. The results obtained show that the developed strategy represents an attractive option for the production of reCG and constitutes an auspicious alternative for the replacement of animals as a source of PMSG.