Protein glycosylation is post-translational modification (PTM) which is important for pharmacokinetics and immunogenicity of recombinant glycoprotein therapeutics. As a result of variations in monosaccharide composition, glycosidic linkages and glycan branching, glycosylation introduces considerable complexity and heterogeneity to therapeutics. The host cell line used to produce the glycoprotein has a strong influence on the glycosylation because different host systems may express varying repertoire of glycosylation enzymes and transporters that contributes to specificity and heterogeneity in glycosylation profiles. In this review, we discuss the types of host cell lines currently used for recombinant therapeutic production, their glycosylation potential and the resultant impact on glycoprotein properties. In addition, we compare the reported glycosylation profiles of four recombinant glycoproteins: immunoglobulin G (IgG), coagulation factor VII (FVII), erythropoietin (EPO) and alpha-1 antitrypsin (A1AT) produced in different mammalian cells to establish the influence of mammalian host cell lines on glycosylation.

To identify polymorphisms associated with variability of iron overload severity in HFE-associated hemochromatosis, we performed exome sequencing of DNA from 35 male HFE C282Y homozygotes with either markedly increased iron stores (n=22; cases) or with normal or mildly increased iron stores (n=13; controls). The 35 participants, residents of the U.S., Canada, and Australia, reported no or light alcohol consumption. Sequencing data included 82,068 single nucleotide variants, and 10,337 genes were tested for a difference between cases and controls. A variant in the GNPAT gene showed the most significant association with severe iron overload (p = 3×10−6, p=0.033 by the likelihood ratio test after correction for multiple comparisons). Sixteen of 22 participants with severe iron overload had GNPAT polymorphism p.D519G (rs11558492) (15 heterozygotes, one homozygote). No control participant had this polymorphism. To examine functional consequences of GNPAT deficiency, we performed siRNA-based knockdown of GNPAT in the human liver-derived cell line HepG2/C3A. This knockdown resulted in a >17-fold decrease in expression of the mRNA encoding the iron regulatory hormone hepcidin. Conclusion: GNPAT p.D519G is associated with a high-iron phenotype in HFE C282Y homozygotes and may participate in hepcidin regulation.

Mammalian host cell lines are the preferred expression systems for the manufacture of complex therapeutics and recombinant proteins. However, the most utilized mammalian host systems, namely Chinese hamster ovary (CHO), Sp2/0 and NS0 mouse myeloma cells, can produce glycoproteins with non-human glycans that may potentially illicit immunogenic responses. Hence, we developed a fully human expression system based on HEK293 cells for the stable and high titer production of recombinant proteins by first knocking out GLUL (encoding glutamine synthetase) using CRISPR-Cas9 system. Expression vectors using human GLUL as selection marker were then generated, with recombinant human erythropoietin (EPO) as our model protein. Selection was performed using methionine sulfoximine (MSX) to select for high EPO expression cells. EPO production of up to 92700 U/mL of EPO as analyzed by ELISA or 696 mg/L by densitometry was demonstrated in a 2 L stirred-tank fed batch bioreactor. Mass spectrometry analysis revealed that N-glycosylation of the produced EPO was similar to endogenous human proteins and non-human glycan epitopes were not detected. Collectively, our results highlight the use of a human cellular expression system for the high titer and xenogeneic-free production of EPO and possibly other complex recombinant proteins.