The effect of glucose depletion and media supplementation on the productivity and quality of monoclonal antibodies production

Abstract

N-linked glycosylation of the Fc domain is an important post-translational modification that can have a great impact on the therapeutic efficacy of monoclonal antibodies (Mabs). The purpose of this study was to evaluate the effect that different culture parameters have on N-glycans of Mabs. For instance, glucose depletion, observed in both low and high density cultures, was responsible for a decrease of up to 51% site-occupancy and galactosylation content (GI< 0.43) in EG2-hFc Mabs. This reduced glycosylation was shown to be a result of limited synthesis of glycosylation precursors (0.03-0.23 fmoles/cell) and a reduced cell energy state (AEC < 0.57). In an attempt to improve galactosylation in Mabs, NS0 cells were supplemented with different glycosylation precursors. The combination of MnCl2 and galactose was the most successful in increasing galactosylation by up to 33% in NS0-IgG1 Mabs. The latter was a result of an increase of up to 3.5-fold in UDP-Gal intracellular levels in NS0 cells. Interestingly, inducing an increase in sialylation was not as simple as providing NS0 cells with sialic acid precursors. In fact, peracetylated mannosamine (Ac4ManNAc) and peracetylated glucosamine (Ac4GlcNAc) alone or in combination with uridine or cytidine respectively failed to improve sialylation in IgG1 Mabs, a feeding strategy previously shown to be successful in other glycoproteins. Instead, a decrease in sialylation of 33-64% was observed in IgG1 Mabs. In addition, the initial ratio of Neu5Gc:Neu5Ac (8:2) was reduced in cultures containing: a) M+Urd and M+Urd+Gal- 1:1; b) Ac4GlcNAc (>50 μM- 4:1); c) Ac4ManNAc (>200 μM-1:1); chelated iron (>0.1 μM- 1:1). A reduction in Neu5Gc is particularly desired to avoid any immunogenic reactions in humans. Here, supplementation of low-IgG1 FBS serum successfully reduced the Neu5Gc-content (by up to 24%) and increased Neu5Ac-content (5X) in NS0-IgG1 Mabs. The results presented in this thesis show that glycosylation patterns were strongly dependent on the cell producers’ machinery, protein itself, nutrient availability and culture media supplements. Improving Mab’s N-glycosylation required more than just providing cells with glycosylation precursors, it also required addressing the effect that feeding strategies and culture parameters can have on the presence of immunogenic residues.