Incorporation of 15N from ammonium into the N-linked oligosaccharides of an immunoadhesin glycoprotein expressed in Chinese hamster ovary cells

Gawlitzek, Martin; Papac, Damon I.; Sliwkowski, Mary B.; Ryll, Thomas

doi:10.1093/glycob/9.2.125

Cited by 34 publications

(29 citation statements)

References 38 publications

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“…In fact, glucosamine supplementation appeared to decrease t-PA site occupancy, which is, perhaps, surprising given that glucosamine has been shown previously to increase specific branching glycosylation reactions in recombinant protein production in BHK cells, apparently by increasing UDP-GNAc pools (Gawlitzek et al, 1998). In another study, elevated UDP-GNAc pools, resulting from ammonium addition, failed to increase glycosylation branching for an immunoadhesin glycoprotein expressed in CHO cells (Gawlitzek et al, 1999). In another model in which variable-site occupancy was evaluated, Nyberg et al (1999) showed a correlation between increased UDP-GNAc levels and increased site occupancy for interferon-␥.…”

Section: Discussionmentioning

confidence: 89%

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: Discussionmentioning

confidence: 89%

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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“…These and other studies demonstrated that galactose or uridine supplementation can lead to increased levels of intracellular UDP-Gal, likely leading to increased intracellular biosynthesis of galactosylated antibodies. This increase could compensate for reduced endogenous precursor synthesis over the course of culture, reduced transport, reduced galactosyltransferase expression or activity, or for increased sequestration of galactose into N-acetylgalactosamine or of uridine into UDP-GlcNAc from increased levels of ammonia (Cayli et al, 1999;Chen and Harcum, 2006;Clark et al, 2005;Gawlitzek et al, 1999;Grammatikos et al, 1998;Pels Rijcken et al, 1995;Ryll et al, 1994;Valley et al, 1999;Wong et al, 2010a,b;Yang and Butler, 2002). In one case, the addition of uridine alone led to increased intracellular pools of UDP-Gal and increased antibody galactosylation (Ryll, 2003 US Patent Application 20030211573).…”

Section: Options For More Specifically Impacting Galactosylationmentioning

confidence: 99%

Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose

Gramer

Eckblad

Donahue

et al. 2011

Biotech & Bioengineering

160

182

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Through process transfer and optimization for increased antibody production to 3 g/L for a GS-CHO cell line, an undesirable drop in antibody Fc galactosylation was observed. Uridine (U), manganese chloride (M), and galactose (G), constituents involved in the intracellular galactosylation process, were evaluated in 2-L bioreactors for their potential to specifically increase antibody galactosylation. These components were placed in the feed medium at proportionally increasing concentrations from 0 to 20 × UMG, where a 1× concentration of U was 1 mM, a 1× concentration of M was 0.002 mM, and a 1× concentration of G was 5 mM. Antibody galactosylation increased rapidly from 3% at 0× UMG up to 21% at 8× UMG and then more slowly to 23% at 20× UMG. The increase was primarily due to a shift from G0F to G1F, with minimal impact on other glycoforms or product quality attributes. Cell culture performance was largely not impacted by addition of up to 20× UMG except for suppression of glucose consumption and lactate production at 16 and 20× UMG and a slight drop in antibody concentration at 20× UMG. Higher accumulation of free galactose in the medium was observed at 8× UMG and above, coincident with achieving the plateau of maximal galactosylation. A concentration of 4× UMG resulted in achieving the target of 18% galactosylation at 2-L scale, a result that was reproduced in a 1,000-L run. Follow-up studies to evaluate the addition of each component individually up to 12× concentration revealed that the effect was synergistic; the combination of all three components gave a higher level of galactosylation than addition of the each effect independently. The approach was found generally useful since a second cell line responded similarly, with an increase in galactosylation from 5% to 29% from 0 to 8× UMG and no further increase or impact on culture performance up to 12× UMG. These results demonstrate a useful approach to provide exact and specific control of antibody galactosylation through manipulation of the concentrations of uridine, manganese chloride, and galactose in the cell culture medium.

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“…These methods can be divided into several general approaches involving the use of labeling techniques. General labeling methods, such as the use of 2 H 2 O, 18 O, 15 N or 13 C to label the whole organism, were used to show de novo synthesis, as indicated by an increase in the density of the resulting proteins (Gonzalez-Prieto et al, 1995;Gawlitzek et al, 1999;Yao et al, 2001;Goshe and Smith, 2003;Cargile et al, 2004;Roessner-Tunali et al, 2004;Busch et al, 2006;Belloto et al, 2007;Schaff et al, 2008;Zhao et al, 2009). Specific isotopically-labeled (radioactive or stable isotope) amino acids have been used to label cellular proteins, with labeling rates reflecting de novo synthesis, and the rate of loss monitoring proteolysis (Cooke et al, 1979;Thompson et al, 1989;Ong et al, 2002;Doherty et al, 2005;Krü ger et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Measuring the turnover rates of Arabidopsis proteins using deuterium oxide: an auxin signaling case study

et al. 2010

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SUMMARYRapid environmental responses in plants rely on endogenous signaling mechanisms, which in many cases are mediated by changes in protein turnover rates. It is therefore necessary to develop methods for measuring protein dynamics that monitor large sets of plant proteins to begin to apply a systems biology approach to the study of plant behavior. The use of stable isotope labeling strategies that are adaptable to proteomic methods is particularly attractive for this purpose. Here, we explore one example of such methods that is particularly suitable for plants at the seedling stage, where measurement of amino acid and protein turnover rates is accomplished using a heavy water labeling strategy. The method is backed by microarray evaluation to define its feasibility for specific experimental approaches, and the CULLIN-ASSOCIATED AND NEDDYLATION DISSOCIATED 1 (CAND1) and TRANSPORT INHIBITOR RESPONSE 1 (TIR1) proteins are used to illustrate the potential utility in understanding hormonal signaling regulation. These studies provide insight not only into the potential utility of the method, but also address possible areas of concern regarding the use of heavy water labeling during plant growth. These considerations suggest a prescription for specific experimental designs that minimize interference resulting from the induction of treatment-specific gene expression in the results obtained.

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Incorporation of 15N from ammonium into the N-linked oligosaccharides of an immunoadhesin glycoprotein expressed in Chinese hamster ovary cells

Cited by 34 publications

References 38 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

Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose

Measuring the turnover rates of Arabidopsis proteins using deuterium oxide: an auxin signaling case study

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