Large-scale purification of human tissue-type plasminogen activator using monoclonal antibodies

Einarsson, Monica; Brandt, Johnny; Kaplan, Lennart

doi:10.1016/0167-4838(85)90123-2

Cited by 62 publications

(32 citation statements)

References 16 publications

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“…A difference in carbohydrate structure affecting biological activity in an analogous manner as for antithrombin has been shown for tissue plasminogen activator, expressed in CHO cells (Spellman et al, 1989). A fully glycosylated form of this enzyme having three oligosaccharide side chains thus had only about two-thirds of the activity of a form lacking one of these chains (Einarsson et al, 1985).…”

Section: Discussionmentioning

confidence: 93%

Decreased affinity of recombinant antithrombin for heparin due to increased glycosylation

Björk

Ylinenjärvi

Olson

et al. 1992

Biochemical Journal

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Recombinant antithrombin produced by baby hamster kidney (BHK) or Chinese hamster ovary (CHO) cells was separated into two fractions, containing comparable amounts of protein, by affinity chromatography on matrix-linked heparin. Fluorescence titrations showed that the more tightly binding fraction had a heparin affinity similar to that of plasma antithrombin (Kd 20 nM), whereas the affinity of the more weakly binding fraction was nearly 10-fold lower (Kd 175 nM). Analyses of the heparin-catalysed rate of inhibition of thrombin further showed that the fractions differed only in their affinity for heparin and not in the intrinsic rate constant of either the uncatalysed or the heparin-catalysed inactivation of thrombin. The recombinant antithrombin fraction with lower heparin affinity migrated more slowly than both the fraction with higher affinity and plasma antithrombin in SDS/PAGE under reducing conditions, consistent with a slightly higher apparent relative molecular mass. This apparent size difference was abolished by the enzymic removal of the carbohydrate side chains from the proteins. Such removal also increased the heparin affinity of the weakly binding fraction, so that it eluted from matrix-linked heparin at a similar position to the deglycosylated tightly binding fraction or plasma antithrombin. Analyses of N-linked carbohydrate side chains showed that the weakly binding fraction from CHO cells had a higher proportion of tetra-antennary and a lower proportion of biantennary oligosaccharides than the tightly binding fraction. We conclude that the recombinant antithrombin produced by the two cell lines is heterogeneously glycosylated and that the increased carbohydrate content of a large proporti6n of the molecules results in a substantial decrease in the affinity of these molecules for heparin. These findings are of particular relevance for studies aimed at characterizing the heparin-binding site of recombinant antithrombin by site-directed mutagenesis.

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Section: Discussionmentioning

confidence: 93%

Decreased affinity of recombinant antithrombin for heparin due to increased glycosylation

Björk

Ylinenjärvi

Olson

et al. 1992

Biochemical Journal

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“…In the case of tissuetype plasminogen activator (t-PA), variable site occupancy is commonly observed at Asn-184, but not at the other two glycosylation sites (Asn-117 and Asn-448), leading to the secretion of a mixture of material with either two N-linked oligosaccharides (type II) or three N-linked oligosaccharides (type I). Type I t-PA has been observed to display reduced clot lysis activity and reduced fibrin binding compared with type II t-PA (Einarsson et al, 1985;. The presence of an oligosaccharide at Asn-184 has also been shown to affect plasma clearance rates in rabbit studies (Beebe and Aronson, 1988;Cole et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

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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“…The glycosylation of t-PA was shown to affect its enzymatic activity [60–62] and the plasma clearance of the molecule [63–65]. Glycosylation was also shown to be necessary for the activity [66–68] and plasma clearance of erythropoietin (reviewed by Takeuchi [21]).…”

Section: An Historical Perspectivementioning

confidence: 99%

Carbohydrate analysis throughout the development of a protein therapeutic

Higgins¹

2009

Glycoconj J

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This review discusses the challenges involved in the characterization of the glycosylation of therapeutic glycoproteins. The focus is on methods that are most commonly used in regulatory filings and lot release testing of therapeutic glycoproteins. The different types of assays for carbohydrate analysis are reviewed, including the distinction between assays appropriate for lot release or better suited to testing during early drug development or in-depth characterization of the glycosylation. Characteristics of the glycoprotein and production process that should be considered when determining the amount of testing, the number of different methods to employ and when the testing should be performed during development of protein therapeutics is also discussed.

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Large-scale purification of human tissue-type plasminogen activator using monoclonal antibodies

Cited by 62 publications

References 16 publications

Decreased affinity of recombinant antithrombin for heparin due to increased glycosylation

Decreased affinity of recombinant antithrombin for heparin due to increased glycosylation

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

Carbohydrate analysis throughout the development of a protein therapeutic

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