Glycosylation of CHO-derived recombinant tPA produced under elevated pCO2.
Carbon dioxide is a metabolic byproduct of mammalian cell metabolism that can accumulate in poorly ventilated cultures. A buildup of CO2 at constant pH will be accompanied by an increase in medium osmolality. We have examined the glycosylation of tissue plasminogen activator (tPA) produced under serum-free conditions by recombinant Chinese hamster ovary (CHO) cells (MT2-1-8 cell line) in response to elevated pCO2 at constant or elevated osmolality. The proportion of sialic acids comprising N-glycolylneuraminic acid decreased from 2.3-4.0% under 36 mmHg pCO2 to 1.5-2.2% under 250 mmHg pCO2. No changes were observed in the total sialic acid content, the content of other monosaccharides, the relative amounts of type I and type II tPAs, the distribution of surface charges, or the proportion of high-mannose oligosaccharides-even though these conditions have previously been shown to inhibit the specific growth rate of MT2-1-8 cells by 30-40% and the specific tPA production rate by as much as 40%. These results suggest robust glycosylation of tPA by CHO cells.
Kimura
R
R
Chemical Engineering Department, Northwestern University, Evanston, Illinois 60208-3120, USA.
Miller
W M
WM
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells / Q000378:metabolism*; D002245:Carbon Dioxide / Q000008:administration & dosage*; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008358:Mannose / Q000032:analysis; D009005:Monosaccharides / Q000032:analysis; D019158:N-Acetylneuraminic Acid / Q000032:analysis; D009438:Neuraminic Acids / Q000032:analysis; D009994:Osmolar Concentration; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*
D009005:Monosaccharides; D009438:Neuraminic Acids; D011994:Recombinant Proteins; C032592:N-glycolylneuraminic acid; D002245:Carbon Dioxide; D010959:Tissue Plasminogen Activator; D019158:N-Acetylneuraminic Acid; D008358:Mannose