Investigating the rules governing N-linked glycosylation of Biologics

Biologics, drugs produced in living organisms, such as monoclonal antibodies (mAbs) can treat various medical conditions, so improving their production and efficacy is important. Large scale mAb manufacturing involves expressing recombinant DNA in mammalian cells, for example Chinese hamster ovary (CHO) cells, which are cultured in bioreactors. mAb glycosylation is monitored for quality control, as glycans dictate mAb stability, functionality, and efficacy. Glycans are processed in the Golgi and glycosylation enzyme localisation within Golgi cisternae is determined by intra Golgi vesicle transport. The conserved oligomeric Golgi (COG) complex participates in retrograde trafficking of glycosylation enzymes within the Golgi facilitated through Rab interactions. Understanding the glycosylation machinery and how alterations influence glycosylation patterns aids in improving mAb production and efficacy. This thesis addresses the role mutations in COG complex subunit Cog4 play in mAb glycosylation. Cog4 interactions with Rabs and stability of Cog4 mutants were examined using a computationally generated Cog4 structure. Rab30 and Rab41 structures were computationally docked to the Cog4 structure, revealing a preferential binding to the Cog4 N-terminus. Stability assessment of Cog4 upon mutation predicted Cog4-L36P is unstable, suggesting L36 is indirectly involved in Rab30 binding. Whereas R34C, Q67R and F137S had negligible effect on Cog4 stability suggesting these positions are directly involved in Rab41 binding. To determine how Cog4 mutations affect mAb glycosylation CHO cell lines Cog4-WT, Cog4-KO, Cog4-L36P, Cog4-R729W and Cog4-L773R were transiently transfected with Herceptin. Glycan profiling of Herceptin products unveiled potential decreases in trans-Golgi to medial-Golgi glycosylation enzyme recycling in Cog4-KO and Cog4-L36P cell lines. Comparing the Herceptin glycan profiles of Cog4-L773R and Cog4-WT revealed an increase in galactosylation, suggesting increased recycling of galactosylation enzymes to the trans-Golgi. Furthering the understanding of Cog4 functions and how Cog4 mutants influence glycosylation can facilitate the development of CHO cell lines with specific mAb glycosylation.