Characterisation of Cell Lines with Engineered Golgi Organisation

N-linked glycosylation is an important modulator of protein structure and function, relevant for regulating cellular characteristics and properties of recombinantly expressed glycoprotein biopharmaceuticals, including monoclonal antibodies (mAbs). Biopharmaceutical glycosylation is tightly regulated, and glycan engineering is attractive to the biopharmaceutical industry. With no molecular template, glycosylation is controlled, inter alia, by localisation of enzymes within the Golgi apparatus. This requires tightly regulated membrane trafficking, involving specific vesicle tethering interactions, which are coordinated by the heterooctameric conserved oligomeric Golgi (COG) complex. COG mutations alter glycosylation, exemplified in congenital disorders of glycosylation, and in COG-mutant cell lines. Different COG subunit mutations cause unique glycosylation changes, highlighting the potential of targeting COG for generating glycan-engineered biotherapeutics.
The Ungar group previously identified mutations in COG subunits that altered specific interactions with Rabs, which are small GTPases with regulatory roles in membrane trafficking. A Cog4L36P mutation was identified with impaired Rab30 binding. In this work, a CHO-K1 cell line expressing Cog4L36P was engineered, characterised, and tested as a host for production of the mAb, Herceptin. The mutation was also engineered into an ex-production host CHO cell line from GlaxoSmithKline for testing Herceptin production in cells optimised for high-level mAb production. The goal was to dissect the function of the Cog4-Rab30 interaction in controlling glycosylation and how this affects cell properties and recombinant biotherapeutic expression. Results show that Cog4L36P cells have altered whole-cell glycosylation, impaired adhesion, slower proliferation and altered metabolic activity. Glycan profiling of WT, ΔCog4 and Cog4L36P cells cultured in monolayer and suspension also revealed cell line specific glycosylation changes in response to suspension adaptation. Cog4L36P did not affect Herceptin production in ex-production CHO cells. However, suspension-adapted Cog4L36P CHO-K1 cells have enhanced Herceptin production with similar glycoforms to Herceptin produced by WT cells, demonstrating the potential of the Cog4L36P mutation for aiding mAb production.