Differences in the production of hyperglycosylated IFN alpha in CHO and HEK 293 cells

GUGLIOTTA, AGUSTINA; OGGERO EBERHARDT, MARCOS; ETCHEVERRIGARAY, MARINA; KRATJE, RICARDO; CEAGLIO, NATALIA

Lille

Congreso; 23rd Meeting of the European Society for Animal Cell Technology; 2013

ESACT

IFN4N is an IFN-alpha2b mutein developed in our laboratory using glycoengineering. This molecule contains 4 potential N-glycosylation sites, resulting in higher apparent molecular mass and longer plasmatic half- life compared to non glycosylated IFN-alpha used for clinical applications. CHO cells are widespread used for the large-scale production of therapeutic recombinant proteins and HEK 293 cells are an interesting system for the generation of recombinant cell lines because they are easy to transfect and, consequently, they allow the production of high levels of the protein of interest.In this work, lentiviral vectors containing the sequence of IFN4N were assembled and used for transduction of CHO and HEK 293 cells. The recombinant cell lines were subjected to a process of selective pressure using increasing concentrations of puromycin. After cloning, 6 clones were selected for the study of their growth, production parameters and characterization of the secreted IFN4N.The CHO and HEK IFN4N producing cell lines resistant to the highest concentration of puromycin showed an 8-fold and 15-fold increment in IFN4N specific productivity, respectively, compared to the parental line. HEK clones exhibited lower µ and maximum cell density than CHO clones, but higher IFN4N cumulative production was achieved. Significant differences in the glycosylation pattern of IFN4N produced in CHO (CHO-IFN4N) and HEK293 (HEK-IFN4N) were observed by isoelectric focusing followed by western blot. Specifically, IFN4N produced in CHO cells showed more acidic isoforms than the one produced in HEK. Furthermore, this result was consistent with a lower in vitro antiviral and antiproliferative specific biological activity evidenced by the CHO-IFN4N compared to the HEK-IFN4N.Considering that glycosylation affects protein stability, solubility, pharmacokinetics and immunogenicity, differences between CHO and HEK cells should be capitalized to select the proper system for the cytokine´s production.