title: Glycosylation and antiproliferative activity of hyperglycosylated IFN-α2 potentiate HEK293 cells as biofactories.
abstract: Both CHO and HEK cells are interesting hosts for the production of biotherapeutics due to their ability to introduce post-translational modifications such as glycosylation. Even though oligosaccharide structures attached to proteins are conserved among eukaryotes, many differences have been found between therapeutic glycoproteins expressed in hamster and human derived cells. In this work, a hyperglycosylated IFN-α2b mutein (IFN4N) was produced in CHO and HEK cell lines and an extensive characterization of their properties was performed. IFN4NCHOexhibited a higher average molecular mass and more acidic isoforms compared to IFN4NHEK. In agreement with these results, a 2-times higher sialic acid content was found for IFN4NCHOin comparison with the HEK-derived protein. This result was in agreement with monosaccharide quantification and glycan's analysis using WAX chromatography and HILIC coupled to mass spectrometry; all methods supported the existence of highly sialylated and also branched structures for IFN4NCHOglycans, in contrast with smaller and truncated structures among IFN4NHEKglycans. Unexpectedly, those remarkable differences in the glycosylation pattern had not a considerable impact on the clearance rate of both molecules in rats. In fact, although IFN4NHEKreached maximum plasma concentration 3-times faster than IFN4NCHO, their elimination profile did not differ significantly. Also, despite the in vitro antiviral specific biological activity of both proteins was the same, IFN4NHEKwas more efficient as an antiproliferative agent in different tumor-derived cell lines. Accordingly, IFN4NHEKshowed a higher in vivo antitumor activity in animal models. Our results show the importance of an appropriate host selection to set up a bioprocess and potentiate the use of HEK293 cells for the production of a new hyperglycosylated protein-based pharmaceutical.
authors.affiliation: UNL, CONICET, FBCB, Cell Culture Laboratory, Ciudad Universitaria UNL.C.C. 242, (S3000ZAA) Santa Fe, Argentina.
authors.forename: Agustina
authors.initials: A
authors.lastname: Gugliotta
authors.affiliation: UNL, CONICET, FBCB, Cell Culture Laboratory, Ciudad Universitaria UNL.C.C. 242, (S3000ZAA) Santa Fe, Argentina.
authors.forename: Natalia
authors.initials: N
authors.lastname: Ceaglio
authors.affiliation: UNL, FBCB, Cell Culture Laboratory, Ciudad Universitaria UNL.C.C. 242, (S3000ZAA) Santa Fe, Argentina.
authors.forename: Brenda
authors.initials: B
authors.lastname: Raud
authors.affiliation: UNL, FBCB, Cell Culture Laboratory, Ciudad Universitaria UNL.C.C. 242, (S3000ZAA) Santa Fe, Argentina; Zelltek S.A., PTLC RN 168, (S3000ZAA) Santa Fe, Argentina.
authors.forename: Guillermina
authors.initials: G
authors.lastname: Forno
authors.affiliation: Zelltek S.A., PTLC RN 168, (S3000ZAA) Santa Fe, Argentina.
authors.forename: Laura
authors.initials: L
authors.lastname: Mauro
authors.affiliation: UNL, CONICET, FBCB, Cell Culture Laboratory, Ciudad Universitaria UNL.C.C. 242, (S3000ZAA) Santa Fe, Argentina.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Kratje
authors.affiliation: UNL, CONICET, FBCB, Cell Culture Laboratory, Ciudad Universitaria UNL.C.C. 242, (S3000ZAA) Santa Fe, Argentina. Electronic address: moggero@fbcb.unl.edu.ar.
authors.forename: Marcos
authors.initials: M
authors.lastname: Oggero
mesh_terms: D000818:Animals; D016466:CHO Cells; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects*; D002846:Chromatography, Affinity; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D016898:Interferon-alpha / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D016898:Interferon-alpha
keywords: Antitumoral activity; CHO cells; Glycoproteins; HEK293 cells; Hyperglycosylated IFN-α2b; N-glycosylation; Pharmacokinetics