Understanding and Controlling Sialylation in a CHO Fc-Fusion Process.
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.
Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, United States of America.
Amanda M
AM
Lewis
Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, United States of America.
William D
WD
Croughan
Research and Development, Bristol-Myers Squibb Company, Princeton, NJ, United States of America.
Nelly
N
Aranibar
Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, United States of America.
Alison G
AG
Lee
Research and Development, Bristol-Myers Squibb Company, Princeton, NJ, United States of America.
Bethanne
B
Warrack
Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, United States of America.
Nicholas R
NR
Abu-Absi
Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, United States of America.
Rutva
R
Patel
Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, United States of America.
Barry
B
Drew
Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, United States of America.
Michael C
MC
Borys
Research and Development, Bristol-Myers Squibb Company, Princeton, NJ, United States of America.
Michael D
MD
Reily
Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, United States of America.
Zheng Jian
ZJ
Li
D000105:Acetyl Coenzyme A / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D020869:Gene Expression Profiling; D005947:Glucose / Q000378:metabolism; D006019:Glycolysis / Q000235:genetics; D006031:Glycosylation; D008358:Mannose / Q000235:genetics / Q000378:metabolism; D055432:Metabolomics; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D018384:Oxidative Stress / Q000235:genetics*; D010100:Oxygen / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism*; D059467:Transcriptome / Q000235:genetics*
D012794:Sialic Acids; D000105:Acetyl Coenzyme A; D019158:N-Acetylneuraminic Acid; D005947:Glucose; D008358:Mannose; D010100:Oxygen