The glycosylation profile of therapeutic monoclonal antibodies (mAbs) is one of the critical quality attributes (CQAs) that must be closely monitored throughout its development. The most common types of glycans expressed on mAbs contain N-acetylglucosamine (GlcNAc), mannose, galactose, fucose and sialic acid modifications, and their abundances and patterns factor into the molecule’s lifetime and therapeutic effect. Mimicking the glycosylation profile of an originator biologic is key for the development of biosimilars. As recently reviewed by Yoon and coworkers, there are a growing set of modeling tools and metabolic analysis that are focused on optimizing the control of glycosylation profiles from changes in cell genotypes and culture conditions. Such culture conditions like media additives, cell density, temperature, ammonia concentration, dissolved oxygen concentrations and nucleotide sugar precursors have all been explored as tools to adjust the glycosylation profile of the protein.
In “Combined effects of glycosylation precursors and lactate on the glycoprofile of IgG produced by CHO cells,” the team compares traditional glucose fed cultures with pairs of alternative carbon sources when the initial glucose levels are depleted and the culture shifts to lactate consumption as a carbon source. A CHO-K1 line was grown in fed-batch mode in shake flasks with glucose added at the start of culture. On day 5 of the process, the control flask was given 20 mM glucose and the other six flasks were given 20 mM total (10 mM each) of two different sugars: mannose and fructose, mannose and galactose, mannose and fucose, fructose and galactose, fructose and fucose, and galactose and fucose. For the 8 days the experiment was conducted, intra- and extracellular metabolites, titer, and N-glycan profiling were collected.
The glucose-only fed culture produced approximately 10-20% more IgG (by day 8) and had minimal variation in viable cell density and viability compared to all the other six alternative sugar cultures. There was little difference in IgG concentrations between the six-alternative sugar-treated flasks, although there were significant variations in cell viabilities and densities likely due to carbon source depletion. Only the glucose fed cultures had a net increase in the specific lactate production-to-consumption rate. All other cultures exhibited negative rates indicating that the cultures were net consumers of lactate with the three mannose-fed cultures having lower net rates than the fructose-fucose, fructose-galactose, and galactose-fucose conditions. This supported the notion that in glucose-deficient cultures, lactate could be an adequate carbon-source substrate used in conjunction with non-glucose sugars.
Cell specific consumption of each non-glucose sugar decreased from day 6 through day 8 as the total concentration of each non-glucose sugar decreased over time. The lone exception was fucose consumption in the mannose-fucose and fructose-fucose cultures where the fucose consumption was more than an order-of-magnitude lower than all other sugar molecules. Intracellular nucleotide sugar concentrations were correlated to the non-glucose sugar blends in the cultures. The three cultures fed fucose had a substantially higher intracellular concentration of GDP-Fuc compared to fucose deficient cultures. The mannose-galactose culture had the highest intracellular concentrations of both GDP-Man and UDP-Gal, and variations in the levels of CMP-Neu5Ac were nearly identical to GDP-Man and UDP-Gal. The three non-glucose fed cultures produced the highest mannose glycosylation profiles, and the three galactose containing cultures produced the highest G1 and G2 level glycans. The lowest galactose G1 and G2 level cultures were those fed mannose-fructose and fructose-fucose. Overall, there was not a significant difference in the afucosylation levels indicating that most of the glycans were fucosylated. Additionally, there were no sialyated glycans detected, and there was no correlation between the ammonium concentration and the levels of galactosylation that were observed.
Understanding how non-glucose sugar blends affect both cell culture growth and a biologic’s glycosylation profile contributes to the toolbox of cell media additives and nutrients that may provide alternative strategies for producing the desired profile. In the work featured here, the authors utilized a single cell line to investigate the roles of non-glucose sugars fed to cultures after glucose depletion. The study outlined some essential characteristics of how some blends of sugars may influence the glycosylation of the expressed mAb and how it may affect the intracellular levels of nucleotide sugars, which influence the metabolic pathways that lead to the observed modifications. This approach may prove useful in biosimilar projects, where regulators require close correlation to the innovator biologic, but intellectual property restrictions require alternative approaches to obtain it.
By: Glenn A. Harris, Director of Integrated Life Sciences Platforms