The effect of dissolved oxygen (DO) concentration on the glycosylation of recombinant protein produced by the insect cell–baculovirus expression system

Zhang, Fuming; Saarinen, Mark A.; Itle, Laura J.; Lang, Sybil C.; Murhammer, David W.; Linhardt, Robert J.

doi:10.1002/bit.10131

Cited by 24 publications

(19 citation statements)

References 23 publications

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“…Extreme oxidative or anaerobic conditions, however, appear to interfere with the core-fucosylation of the glycan structures. Similar results were found by Zhang et al (2002), who studied the effect of DO on the glycosylation of secreted alkaline phosphatase (SEAP) produced by insect cells. They found that the fraction of the most highly processed N-glycans (Man3F) were decreased at both 10% and 190% of air saturation compared to 50% or air saturation.…”

Section: Discussionsupporting

confidence: 84%

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

Restelli

Wang

Huzel

et al. 2006

Biotech & Bioengineering

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Human recombinant erythropoietin (rHuEPO) was produced from Chinese hamster ovary (CHO) cells transfected with the human EPO gene. The cells were grown in batch cultures in controlled bioreactors in which the set-points for dissolved oxygen varied between 3% and 200%. The cell-specific growth rate and final cell yield was significantly lower under hyperoxic conditions (200% DO). However, there was no significant difference in growth rates at other oxygen levels compared to control cultures run under a normoxic condition (50% DO). The specific productivity of EPO was significantly lower at a DO set-point of 3% and 200% but maintained a consistently high value between 10% to 100% DO. The EPO produced under all conditions as analyzed by two-dimensional electrophoresis showed a molecular weight range of 33 to 37 kDa and a low isoelectric point range of 3.5 to 5.0. This corresponds to a highly glycosylated and sialylated protein with a profile showing at least seven distinct isoforms. The glycan pattern of isolated samples of EPO was analyzed by weak anion exchange (WAX) HPLC and by normal-phase HPLC incorporating sequential digestion with exoglycosidase arrays. Assigned structures were confirmed by mass spectrometry (MALDI-MS). The most prominent glycan structures were core fucosylated tetranntenary with variable sialylation. However, significant biantennary, triantennary, and non-fucosylated glycans were also identified. Detailed analysis of these glycan structures produced under variable dissolved oxygen levels did not show consistently significant variations except for the ratio of fucosylated to non-fucosylated isoforms. Maximum core fucosylation (80%) was observed at 50% and 100% DO, whereas higher or lower DO levels resulted in reduced fucosylation. This observation of lower fucosylation at high or low DO levels is consistent with previous data reported for glycoprotein production in insect cells.

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Section: Discussionsupporting

confidence: 84%

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

Restelli

Wang

Huzel

et al. 2006

Biotech & Bioengineering

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“…Sialyltransferase activity, sialic acid content, and specific productivity of a recombinant glycoprotein, human follicle stimulating hormone, in CHO cells all increased as DO increased (43). In a well-controlled bioreactor, the glycan processing of the recombinant human secreted alkaline phosphatase in insect cell-baculovirus system was inhibited at both low and high dissolved oxygen concentrations (44). In contrast, another study indicated that the N-glycosylation of an interleukin-2 variant from BHK cells did not changed under low oxygen concentration (45).…”

Section: Process Optimizationmentioning

confidence: 94%

Fermentation Process Development of Carbohydrate-Based Therapeutics

Shu

2003

ACS Symposium Series

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This chapter focuses on the latest advances in the development of fermentation processes in carbohydrate-based therapeutics. Although fermentation has been the major route in producing bioactive compounds historically, the complexity of carbohydrates and the current remarkable accomplishments of glycobiology have forced a multidiscipline approach to the development of carbohydrate-based therapeutics. Current remarkable achievements of glycotechnology have been demonstrated in metabolic engineering, process optimization, analytical chemistry, monitoring techniques, enzymatic/ chemical synthesis, and separation techniques, etc. By intentionally incorporating these techniques into fermentation processes, numerous novel approaches have been generated to produce carbohydrate-based therapeutics with higher qualities in terms of authenticity and efficacy, and better yields. Besides, many novel bioactive compounds or drug candidates can be obtained by fermentation in a relatively economic approach.

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“…The inability of insect cells to perform complex glycosylation is apparently due to the absence or insufficient amounts of the terminal glycosyltransferases that are required to convert N-linked side chains to complex forms (Jarvis et al 1998). Strict control of growth and infection parameters, especially dissolved oxygen also play a major role in glycosylation (Zhang et al 2002). The baculovirus/insect cell system is seen as a promising expression system for obtaining authentic post-translational modification compared to the mammalian and prokaryotic systems.…”

Section: Improving Receptor Expression and Purification For Large Scamentioning

confidence: 99%

New tools for G-protein coupled receptor (GPCR) drug discovery: combination of baculoviral expression system and solid state NMR

Ratnala

2006

Biotechnol Lett

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Biotechnology using molecular biology, biochemistry, biophysics, and computational approaches provides an alternative approach for classical pharmacological screening to look at ligand-receptor interactions and receptor specificity, which should support the design of selective drugs based on detailed structural principles. This review addresses specific approaches to study function, structure and relevance of a major pharmaceutical target, namely the G-Protein Coupled Receptors (GPCRs). The main aim of this review has been to exploit and combine GPCR over-expression in a baculoviral expression system with solid-state MAS NMR (ssNMR) approaches for the elucidation of electronic structures of the coordinating ligands/drugs and their modes of interactions with the GPCRs. This review summarizes the approaches, possible future experiments and developments using the above combination of tools for GPCR drug discovery.

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The effect of dissolved oxygen (DO) concentration on the glycosylation of recombinant protein produced by the insect cell–baculovirus expression system

Cited by 24 publications

References 23 publications

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

Fermentation Process Development of Carbohydrate-Based Therapeutics

New tools for G-protein coupled receptor (GPCR) drug discovery: combination of baculoviral expression system and solid state NMR

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