Carbohydrate Structures of Human Tissue Plasminogen Activator Expressed in Chinese Hamster Ovary Cells

Spellman, Michael W.; Basa, Louisette J.; Leonard, C K; Chakel, John A.; O’Connor, John; Wilson, Sarah S.; Halbeek, Herman van

doi:10.1016/s0021-9258(18)71649-9

Cited by 202 publications

(64 citation statements)

References 59 publications

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“…The putative type I and type II peaks in the RP-HPLC assay were initially verified by mass spectrometry and carbohydrate composition analysis using previous results characterizing the oligosaccharide structures present on CHOproduced t-PA (Spellman et al, 1989). To confirm that these two peaks continued to reflect type I and type II for varying cell culture conditions, the following sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) methodology was used.…”

Section: Verification Of Type I/type II Peaksmentioning

confidence: 97%

Multiple cell culture factors can affect the glycosylation of Asn-184 in CHO-produced tissue-type plasminogen activator

Andersen¹,

Bridges²,

Gawlitzek³

et al. 2000

Biotechnol. Bioeng.

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Human tissue-type plasminogen activator (t-PA) contains a variably occupied glycosylation site at Asn-184 in naturally produced t-PA and in t-PA produced in recombinant Chinese hamster ovary (CHO) cells. The presence of an oligosaccharide at this site has previously been shown to reduce specific activity and fibrin binding. In this report, the site occupancy of t-PA is shown to increase gradually over the course of batch and fedbatch CHO cultures. Additional cell culture factors, including butyrate and temperature, are also shown to influence the degree of glycosylation. In each of these cases, conditions with decreased growth rate correlate with increased site occupancy. Investigations using quinidine and thymidine to manipulate the cell cycle distribution of cultures further support this correlation between site occupancy and growth state. Comparison of the cell cycle distribution across the range of cell culture factors investigated shows a consistent relationship between site occupancy and the fraction of cells in the G 0 /G 1 phase of the cell cycle. These results support a correlation between growth state and site occupancy, which fundamentally differs from site occupancy trends previously observed and illustrates the importance of the growth profile of CHO cultures in producing consistently glycosylated recombinant glycoproteins.

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Section: Verification Of Type I/type II Peaksmentioning

confidence: 97%

Multiple cell culture factors can affect the glycosylation of Asn-184 in CHO-produced tissue-type plasminogen activator

Andersen¹,

Bridges²,

Gawlitzek³

et al. 2000

Biotechnol. Bioeng.

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“…Reteplase is a glycoprotein with three potential N-glycosylation sites at Asn 12 , Asn 48 , and Asn 278 (corresponding to Asn 184 , Asn 218 and Asn 448 in tPA). In both tPA and rPA, glycosite Asn 218 (Asn 48 in rPA) is not glycosylated and Asn 184 (Asn 12 in rPA) is only 20–25% occupied [ 11 , 12 ].…”

Section: Resultsmentioning

confidence: 99%

“…tPA molecules that are glycosylated at position 184 are termed type I while type II molecules are not glycosylated at this position. Glycan analysis of CHO-derived Alteplase revealed that the potential Asn 218 glycosylation site is not occupied, Asn 117 carries exclusively high mannose oligosaccharides while Asn 184 and Asn 448 carry predominantly di-antennary complex glycans with different amounts of α2,3-terminal sialic acid and core α1,6-fucose, typical of CHO-derived glycoproteins [ 11 , 12 ]. tPA glycosylation was shown to affect its enzymatic activity [ 13 ] and its plasma clearance [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

Reteplase Fc-fusions produced in N. benthamiana are able to dissolve blood clots ex vivo

et al. 2021

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Thrombolytic and fibrinolytic therapies are effective treatments to dissolve blood clots in stroke therapy. Thrombolytic drugs activate plasminogen to its cleaved form plasmin, a proteolytic enzyme that breaks the crosslinks between fibrin molecules. The FDA-approved human tissue plasminogen activator Reteplase (rPA) is a non-glycosylated protein produced in E. coli. rPA is a deletion mutant of the wild-type Alteplase that benefits from an extended plasma half-life, reduced fibrin specificity and the ability to better penetrate into blood clots. Different methods have been proposed to improve the production of rPA. Here we show for the first time the transient expression in Nicotiana benthamiana of rPA fused to the immunoglobulin fragment crystallizable (Fc) domain on an IgG1, a strategy commonly used to improve the stability of therapeutic proteins. Despite our success on the expression and purification of dimeric rPA-Fc fusions, protein instability results in high amounts of Fc-derived degradation products. We hypothesize that the “Y”- shape of dimeric Fc fusions cause steric hindrance between protein domains and leads to physical instability. Indeed, mutations of critical residues in the Fc dimerization interface allowed the expression of fully stable rPA monomeric Fc-fusions. The ability of rPA-Fc to convert plasminogen into plasmin was demonstrated by plasminogen zymography and clot lysis assay shows that rPA-Fc is able to dissolve blood clots ex vivo. Finally, we addressed concerns with the plant-specific glycosylation by modulating rPA-Fc glycosylation towards serum-like structures including α2,6-sialylated and α1,6-core fucosylated N-glycans completely devoid of plant core fucose and xylose residues.

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“…The probe temperature was kept at 27 °C. Further experimental details have been described (Argade et al, 1988;Green et al, 1988;Spellman et al, 1989). Chemical shifts (5) are expressed in parts per million downfield from internal sodium 4,4-dimethyl-4-silapentane-l-sulfonate (DSS), measured by reference to internal acetone (5 2.225 in 2H20 at 27 °C) with an accuracy of 0.002 ppm.…”

Section: Methodsmentioning

confidence: 99%

Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast

Miller²,

Kubek³

et al. 1992

Biochemistry

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The N-glycans of purified recombinant middle surface protein (preS2+S) from hepatitis B virus, a candidate vaccine antigen expressed in a mnn9 mutant strain of Saccharomyces cerevisiae, have been characterized structurally. The glycans were released by N-glycanase treatment, isolated by size-exclusion chromatography on Sephadex G-50 and Bio-Gel P-4 columns, and analyzed by 500-MHz 1H NMR spectroscopy and fast atom bombardment mass spectrometry. The mixture of oligosaccharides was fractionated by HPLC, the major subfractions were isolated, and their carbohydrate compositions were determined by high-pH anion-exchange chromatography with pulsed amperometric detection. The combined results suggest that high-mannose oligosaccharides account for all the N-glycans released from preS2+S: structures include Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 isomers in the ratios of 3:6:1. Approximately 80% of the oligosaccharides contain the C2,C6-branched trimannosyl structural element typical of yeast high-mannose oligosaccharides but not usually found in high-mannose oligosaccharides in animal glycoproteins.

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Carbohydrate Structures of Human Tissue Plasminogen Activator Expressed in Chinese Hamster Ovary Cells

Cited by 202 publications

References 59 publications

Multiple cell culture factors can affect the glycosylation of Asn-184 in CHO-produced tissue-type plasminogen activator

Multiple cell culture factors can affect the glycosylation of Asn-184 in CHO-produced tissue-type plasminogen activator

Reteplase Fc-fusions produced in N. benthamiana are able to dissolve blood clots ex vivo

Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast

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