(67a) Modifying IgG Glycan Profile Using a High-Throughput Multi-Gene Assembly Platform
Protein N-glycosylation is a post-translational modification that is crucial for protein folding, biological activity, half-life and stability. It is one of the most complex post-translational modifications in that it involves a complex network of enzymes that may act on identical substrates to generate many different glycosylation patterns. A drawback of protein expression in mammalian cells is the presence of heterogeneous populations of the same protein due to differences in protein N-glycosylation. The complex nature of glycosylation makes it a good target for control using genetic engineering approaches. While pipelines exist for the generation of multi-gene constructs they are limited in their utility. Utilizing the latest DNA synthesis and assembly technology we developed a platform for combinatorial assembly of multi-gene constructs for use in altering glycosylation of immunoglobulin G (IgG) produced in chinese hamster ovary cells (CHO). A kinetic based model of N-glycosylation was used to predict N-glycosylation genes responsible for generating targeted glycosylation profiles of IgG. Expression of selected glycosylation genes from multi-gene constructs led to altered glycan profiles of IgG demonstrating the efficacy of our platform and validating predictions of our model.