Metabolic Effects on Recombinant Interferon-γ Glycosylation in Continuous Culture of Chinese Hamster Ovary Cells

Nyberg, Gregg; Balcarcel, R. Robert; Follstad, Brian D.; Stephanopoulos, Gregory; Wang, Daniel I. C.

doi:10.1147/rd.445.0770

Cited by 8 publications

(16 citation statements)

References 53 publications

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“…It has been proposed that under glucose-limited condition, all available carbon is preferentially used for energy production, causing reduced nucleotide biosynthesis (Nyberg et al, 1999;Rearick et al, 1981). Exposure of cells to four or more hours to a glucose-depleted media can have impacted the cell's ability to produce precursors for RNA synthesis, nucleoside triphosphates such as UTP become depleted.…”

Section: Discussionmentioning

confidence: 99%

“…These effects depend on several cellular factors: (1) enzyme repertoire and localization (Ferrara et al, 2006;Meynial-Salles and Combes, 1996); (2) competition between different enzymes for one substrate (Umaña and Bailey, 1997); (3) transit time of the glycoproteins through the Golgi apparatus (Hooker et al, 1999;Nabi and Dennis, 1998); (4) levels of nucleotide sugar donors (Nyberg et al, 1999;Valley et al, 1999); and last but not the least (5) competition between different glycosylation sites on the protein for the same pool of enzymes (Schachter et al, 1983).…”

Section: Heterogeneity In N-glycosylation Pathwaymentioning

confidence: 99%

“…Culture conditions such as accumulation of ammonia, dissolved oxygen level, nutrient contents, pH level and temperature, may have great effects on the distribution of glycan structures (microheterogeneity and macroheterogeneity) found on the final recombinant protein products. Those factors may cause one or more mechanisms in the following: (1) the depletion of the cellular energy state (Rearick et al, 1981;Valley et al, 1999); (2) disruption of the local ER and Golgi environment, such as pH (Andersen and Goochee, 1995) or redox potential (Restelli et al, 2006); (3) alteration of glycosyltransferase activities (Chen and Harcum, 2006;Chotigeat et al, 1994); and (4) changes in the Nglycosylation precursor pool, such as nucleotides, nucleotide sugars (Kochanowski et al, 2008;Nyberg et al, 1999) and lipid precursors (Stoll and Krag, 1988). …”

Section: Culture Parameters That May Affect Glycosylationmentioning

confidence: 99%

“…It is known that limited glucose levels during protein production can result in the accumulation of truncated N-glycosylation precursors, with only 15 min turn-over time (Rearick et al, 1981). Low glucose concentration has been proven to cause low Nglycosylation site-occupancy in IFN-γ (Hayter et al, 1992;Nyberg et al, 1999).…”

Section: Objectives Of the Present Researchmentioning

confidence: 99%

“…Different mechanisms have been found to be potentially responsible for N-glycosylation site occupancy, including the availability of dolichol phosphate (Crick and Waechter, 1994;Rosenwald et al, 1990) and nucleotide sugars (Nyberg et al, 1999), activity of oligosaccharyl transferase (OST) (Hendrickson and Imperiali, 1995;Kaufman et al, 1994), and variable glycosylation site accessibility due to the competition with protein folding (Allen et al 1995;Holst et al, 1996;Imperiali, 1997;Shelikoff et al, 1996). Limitations in glucose and glutamine have been found to affect IFN-γ site-occupancy in CHO cultures (Hayter et al, 1992;Nyberg et al, 1999). In 2000, Andersen's group found that the site-occupancy of recombinant human t-PA was elevated gradually over the course of batch and fed-batch CHO cultures (Andersen et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

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The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

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Section: Discussionmentioning

confidence: 99%

Section: Heterogeneity In N-glycosylation Pathwaymentioning

confidence: 99%

Section: Culture Parameters That May Affect Glycosylationmentioning

confidence: 99%

Section: Objectives Of the Present Researchmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

Liu

Spearman

Doering

et al. 2014

Journal of Biotechnology

100

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Manganese increases high mannose glycoform on monoclonal antibody expressed in CHO when glucose is absent or limiting: Implications for use of alternate sugars

Surve

Gadgil

2014

Biotechnology Progress

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Alternate sugars such as galactose and fructose are metabolized at a slower rate than glucose and result in lower accumulation of lactate. While low lactate accumulation is desirable, we report that complete substitution of glucose with these sugars results in an increase in M5 high mannose glycans. Surprisingly, this increase is much higher when the culture is supplemented with manganese: for example, when cells are cultured with galactose, M5 high mannose glycan content increased from 5% at 1 nM Mn(2+) in the basal medium to 32% with 16 µM Mn(2+) supplementation. When galactose is supplemented with glucose maintained at low concentrations, a small reduction in high mannose glycans is seen. In control cultures with glucose, the high mannose content was however <2% in this range of Mn(2+) concentration. By varying Mn(2+) and glucose supplementation levels, with or without galactose, we systematically demonstrate that Mn(2+) concentration and glucose availability, together, significantly affect the high mannose glycan content. To our knowledge, this is the first report that shows that the effect of Mn(2+) on high mannose glycan content depends on glucose availability. At each Mn(2+) supplementation level evaluated, galactosylation percentages were highest for cultures where galactose was supplemented with glucose at non-limiting concentration.

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Effect of surfactant pluronic F‐68 on CHO cell growth, metabolism, production, and glycosylation of human recombinant IFN‐γ in mild operating conditions

Clincke

Guédon

Yen

et al. 2010

Biotechnology Progress

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The control of glycosylation to satisfy regulatory requirements and quality consistency of recombinant proteins produced by different processes has become an important issue. With two N-glycosylation sites, γ-interferon (IFN-γ) can be seen as a prototype of a recombinant therapeutic glycoprotein for this purpose. The effect of the nonionic surfactant Pluronic F-68 (PF-68) on cell growth and death was investigated, as well as production and glycosylation of recombinant IFN-γ produced by a CHO cell line that was maintained in a rich protein-free medium in the absence or presence of low agitation. Under these conditions, a dose-dependent effect of PF-68 (0-0.1%) was shown not only to significantly enhance growth but also to reduce cell lysis. Interestingly, supplementing the culture medium with PF-68 led to increased IFN-γ production as a result of both higher cell densities and a higher specific production rate of IFN-γ. If cells were grown with agitation, lack of PF-68 in the culture medium decreased the fraction of the fully glycosylated IFN-γ glycoform (2N) from 80% to 65-70% during the initial period. This effect appeared to be due to a lag phase in cell growth observed during this period. Finally, a global kinetic study of CHO cell metabolism indicated higher efficiency in the utilization of the two major carbon substrates when cultures were supplemented with PF-68. Therefore, these results highlight the importance of understanding how media surfactant can affect cell growth as well as cell death and the product quality of a recombinant glycoprotein expressed in CHO cell cultures.

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Metabolic Effects on Recombinant Interferon-γ Glycosylation in Continuous Culture of Chinese Hamster Ovary Cells

Cited by 8 publications

References 53 publications

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

Manganese increases high mannose glycoform on monoclonal antibody expressed in CHO when glucose is absent or limiting: Implications for use of alternate sugars

Effect of surfactant pluronic F‐68 on CHO cell growth, metabolism, production, and glycosylation of human recombinant IFN‐γ in mild operating conditions

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