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.

[1]  W M Miller,et al.  Effects of elevated pCO(2) and/or osmolality on the growth and recombinant tPA production of CHO cells. , 1996, Biotechnology and bioengineering.

[2]  D. Liu,et al.  Deamidation: a source of microheterogeneity in pharmaceutical proteins. , 1992, Trends in biotechnology.

[3]  R. Schauer,et al.  Detection of CMP-N-acetylneuraminic acid hydroxylase activity in fractionated mouse liver. , 1989, The Biochemical journal.

[4]  Bernard Thorens,et al.  Chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion , 1986, Nature.

[5]  S. Wilson,et al.  Carbohydrate structures of human tissue plasminogen activator expressed in Chinese hamster ovary cells. , 1989, The Journal of biological chemistry.

[6]  W. Miller,et al.  Production of tPA in recombinant CHO cells under oxygen‐limited conditions , 1993, Biotechnology and bioengineering.

[7]  I. Goldstein,et al.  Binding properties of a mannose-specific lectin from the snowdrop (Galanthus nivalis) bulb. , 1988, The Journal of biological chemistry.

[8]  C. Goochee,et al.  Glycosidase Activities in Chinese Hamster Ovary Cell Lysate and Cell Culture Supernatant , 1993, Biotechnology progress.

[9]  I. Madshus,et al.  Regulation of intracellular pH in eukaryotic cells. , 1988, The Biochemical journal.

[10]  N Jenkins,et al.  Glycosylation of recombinant proteins: problems and prospects. , 1994, Enzyme and microbial technology.

[11]  R J Harris,et al.  Tissue plasminogen activator has an O-linked fucose attached to threonine-61 in the epidermal growth factor domain. , 1991, Biochemistry.

[12]  L. Chasin,et al.  Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[13]  D. Livingston,et al.  In Vivo Clearance of Tissue Plasminogen Activator: The Complex Role of Sites of Glycosylation and Level of Sialylation , 1993 .

[14]  R. Townsend,et al.  Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection. , 1988, Analytical biochemistry.

[15]  A. Billiau,et al.  Purification of Human Tissue-Type Plasminogen Activator in Centigram Quantities from Human Melanoma Cell Culture Fluid and Its Conditioning for Use In Vivo , 1982, Thrombosis and Haemostasis.

[16]  A. Gottschalk.  Glycoproteins: Their composition, structure and function , 1972 .

[17]  R. Dwek,et al.  Effects of N-glycosylation on in vitro activity of Bowes melanoma and human colon fibroblast derived tissue plasminogen activator. , 1989, Biochemistry.

[18]  C. Goochee,et al.  The effect of ammonia on the O‐linked glycosylation of granulocyte colony‐stimulating factor produced by chinese hamster ovary cells , 1995, Biotechnology and bioengineering.

[19]  C. Crowley,et al.  The Influence of Carbohydrate Structure on the Clearance of Recombinant Tissue-Type Plasminogen Activator , 1988, Thrombosis and Haemostasis.

[20]  D. Chow,et al.  Monosaccharide and oligosaccharide analysis of proteins transferred to polyvinylidene fluoride membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. , 1993, The Journal of biological chemistry.

[21]  J. Kamerling,et al.  Sialylated carbohydrate chains of recombinant human glycoproteins expressed in Chinese hamster ovary cells contain traces of N‐glycolylneuraminic acid , 1990, FEBS letters.

[22]  C. A. Berry,et al.  Regulation of cell pH by ambient bicarbonate, carbon dioxide tension, and pH in the rabbit proximal convoluted tubule. , 1988, The Journal of clinical investigation.

[23]  D. T. Berg,et al.  Signal and propeptide processing of human tissue plasminogen activator: activity of a pro-tPA derivative. , 1991, Biochemical and biophysical research communications.

[24]  A. Roos,et al.  Regulation of intracellular pH in human neutrophils , 1985, The Journal of general physiology.

[25]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[26]  S E Builder,et al.  Characterization studies of human tissue-type plasminogen activator produced by recombinant DNA technology. , 1986, Cold Spring Harbor symposia on quantitative biology.

[27]  B. Keyt,et al.  Identification of carbohydrate structures in glycoprotein peptide maps by the use of LC/MS with selected ion extraction with special reference to tissue plasminogen activator and a glycosylation variant produced by site directed mutagenesis. , 1993, Analytical chemistry.