Understanding and Controlling Sialylation in a CHO Fc-Fusion Process

Abstract

A Chinese hamster ovary (CHO) bioprocess, where the product is a sialylated Fc-fusion protein, was operated at pilot and manufacturing scale and significant variation of sialylation level was observed. In order to more tightly control glycosylation profiles, we sought to identify the cause of variability. Untargeted metabolomics and transcriptomics methods were applied to select samples from the large scale runs. Lower sialylation was correlated with elevated mannose levels, a shift in glucose metabolism, and increased oxidative stress response. Using a 5-L scale model operated with a reduced dissolved oxygen set point, we were able to reproduce the phenotypic profiles observed at manufacturing scale including lower sialylation, higher lactate and lower ammonia levels. Targeted transcriptomics and metabolomics confirmed that reduced oxygen levels resulted in increased mannose levels, a shift towards glycolysis, and increased oxidative stress response similar to the manufacturing scale. Finally, we propose a biological mechanism linking large scale operation and sialylation variation. Oxidative stress results from gas transfer limitations at large scale and the presence of oxygen dead-zones inducing upregulation of glycolysis and mannose biosynthesis, and downregulation of hexosamine biosynthesis and acetyl-CoA formation. The lower flux through the hexosamine pathway and reduced intracellular pools of acetyl-CoA led to reduced formation of N-acetylglucosamine and N-acetylneuraminic acid, both key building blocks of N-glycan structures. This study reports for the first time a link between oxidative stress and mammalian protein sialyation. In this study, process, analytical, metabolomic, and transcriptomic data at manufacturing, pilot, and laboratory scales were taken together to develop a systems level understanding of the process and identify oxygen limitation as the root cause of glycosylation variability.

Increased MSX level improves biological productivity and production stability in multiple recombinant GS CHO cell lines

“…Cell culture samples were collected at designated days from a fed-batch culture. The cells were removed by centrifugation at 500 RCF for 5 min at 4 • C. The resulting supernatant was then analyzed for metabolites using LC-MS and NMR spectroscopy technology [53].…”

Improvements in protein production in mammalian cells from targeted metabolic engineering

“…For example, Majors et al [33] inhibited apoptosis by modifying GLUT1 and/or hexokinase expression, since a high glycolytic activity can slow the onset of apoptosis by improving energy efficiency [34]. Moreover, recent studies have altered glycosylation patterns by maintaining efficient glucose metabolism and avoiding accumulation of toxic byproducts, since central metabolism controls the supply of nucleotide sugar precursors [35, 36]. Finally, Mulukutla et al [32] highlighted several potential engineering targets within central metabolism (e.g.…”

Oxidative stress‐alleviating strategies to improve recombinant protein production in CHO cells

“…In large bioreactors, local dO 2 gradients can be formed (Xing, Kenty, Li, & Lee, 2009). This heterogeneity in oxygenation can result in localized hypoxic condition within the culture, and lead to an increase in the sensitivity of CHO cells to oxidative stress that is detrimental to cell growth, culture productivity and/or product quality (A. M. Lewis et al, 2016;Lin & Miller, 1992;Lin, Kimura, & Miller, 1993). On the other hand, hyperoxia can also be a problem in bioprocesses.…”