Abstract
Therapeutic antibodies are decorated with complex-type N-glycans that significantly affect their biodistribution and bioactivity. The N-glycan structures on antibodies are incompletely processed in wild-type CHO cells due to their limited glycosylation capacity. To improve N-glycan processing, glycosyltransferase genes have been traditionally overexpressed in CHO cells to engineer the cellular N-glycosylation pathway by using random integration, which is often associated with large clonal variations in gene expression levels. In order to minimize the clonal variations, we used recombinase-mediated-cassette-exchange (RMCE) technology to overexpress a panel of 42 human glycosyltransferase genes to screen their impact on antibody N-linked glycosylation. The bottlenecks in the N-glycosylation pathway were identified and then released by overexpressing single or multiple critical genes. Overexpressing B4GalT1 gene alone in the CHO cells produced antibodies with more than 80% galactosylated bi-antennary N-glycans. Combinatorial overexpression of B4GalT1 and ST6Gal1 produced antibodies containing more than 70% sialylated bi-antennary N-glycans. In addition, antibodies with various tri-antennary N-glycans were obtained for the first time by overexpressing MGAT5 alone or in combination with B4GalT1 and ST6Gal1. The various N-glycan structures and the method for producing them in this work provide opportunities to study the glycan structure-and-function and develop novel recombinant antibodies for addressing different therapeutic applications.
Highlights
The Fc region of IgGs possesses two identical N-glycans which are composed of a core heptasaccharide with three mannoses and two N-acetylglucosamine (GlcNAc)
Design of RMCE to screen for functional roles of human glycosyltransferase genes in N‐gly‐ cosylation of antibodies in Chinese Hamster Ovary (CHO) cells
In order to study the impact of overexpressing human glycosyltransferase genes on IgG glycosylation, we co-expressed the individual glycosylation gene together with the antibody gene in a CHO K1 master cell line (MCL) via RMCE-based targeted integration (Fig. 1A)
Summary
The Fc region of IgGs possesses two identical N-glycans which are composed of a core heptasaccharide with three mannoses and two N-acetylglucosamine (GlcNAc). Overexpression of other glycosyltransferase genes that regulate precursor b iosynthesis[23,24], nucleotide sugar transport[25] and branching e xtension[26] alone in CHO cells have been shown to increase sialylated N-glycan structures on erythropoietin (EPO) and IFNγ proteins. In this study we utilized a Flp/FRT-based RMCE system to first overexpress a panel of 42 human glycosyltransferase genes in CHO cells to screen their impact on the N-glycosylation of a model IgG antibody, rituximab. These genes are involved in the different steps of the entire core N-glycosylation pathway and have diverse functions, ranging from sugar precursor synthesis, glycan processing, branching, galactosylation to terminal capping (Table 1). We demonstrated that the enzyme expression level was critical in determining glycosylation outcomes and combinatorial engineering of multiple enzymes was required for obtaining highly complex N-glycan structures on mAbs
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