The Effect of Cell Culture Parameters on Protein Glycosylation

Restelli, Veronica; Butler, Michael

doi:10.1007/0-306-47525-1_2

Cited by 16 publications

(9 citation statements)

References 84 publications

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“…It is evident that the influence of DO concentration on glycosylation of proteins may depend on the cell culture technique, glycoprotein studied, and host cell employed amongst other variables (Restelli and Butler, 2002). The effect of scale-up or process changes on the specific production or quality of a recombinant protein is difficult to predict and it is important to study the effect of those parameters on the glycosylation of the glycoprotein of interest and under the specific production conditions.…”

Section: Introductionmentioning

confidence: 99%

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: Introductionmentioning

confidence: 99%

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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“…For the production of a specific recombinant protein as a biotherapeutic it is essential to ensure that a consistent glycosylation profile is maintained between batches (Restelli and Butler 2002). Therefore, it is important to understand the culture parameters that can be controlled to enable strategies in the production bioprocess to ensure the consistency of biopharmaceutical production.…”

Section: Introductionmentioning

confidence: 99%

Optimisation of the Cellular Metabolism of Glycosylation for Recombinant Proteins Produced by Mammalian Cell Systems

2005

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Many biopharmaceuticals are now produced as secreted glycoproteins from mammalian cell culture. The glycosylation profile of these proteins is essential to ensure structural stability and biological and clinical activity. However, the ability to control the glycosylation is limited by our understanding of the parameters that affect the heterogeneity of added glycan structures. It is clear that the glycosylation process is affected by a number of factors including the 3-dimensional structure of the protein, the enzyme repertoire of the host cell, the transit time in the Golgi and the availability of intracellular sugar-nucleotide donors. From a process development perspective there are many culture parameters that can be controlled to enable a consistent glycosylation profile to emerge from each batch culture. A further, but more difficult goal is to control the culture conditions to enable the enrichment of specific glycoforms identified with desirable biological activities. The purpose of this paper is to discuss the cellular metabolism associated with protein glycosylation and review the attempts to manipulate, control or engineer this metabolism to allow the expression of human glycosylation profiles in producer lines such as genetically engineered Chinese hamster ovary (CHO) cells.

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“…171,172 Hence, it is essential to ensure that a consistent glycosylation profile is maintained between batches in the process of production. 6,173 However, culture conditions, such as temperature, pH, 174 DO, CO 2 , osmolarity, 175 time of culture, 176 as well as nutrient and metabolite concentrations, 174,175,177 have been shown to affect glycosylation. 6,178,179 Therefore, it is of extreme importance to have a further understanding of the parameters that control protein glycosylation in bioreactors, and to monitor product quality during each step of the optimization, to be able to ensure the quality and consistency needed for biopharmaceutical production.…”

Section: Parametersmentioning

confidence: 99%

Technological progresses in monoclonal antibody production systems

Rodrigues¹,

Costa²,

Henriques³

et al. 2009

Biotechnology Progress

101

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Monoclonal antibodies (mAbs) have become vitally important to modern medicine and are currently one of the major biopharmaceutical products in development. However, the high clinical dose requirements of mAbs demand a greater biomanufacturing capacity, leading to the development of new technologies for their large-scale production, with mammalian cell culture dominating the scenario. Although some companies have tried to meet these demands by creating bioreactors of increased capacity, the optimization of cell culture productivity in normal bioreactors appears as a better strategy. This review describes the main technological progresses made with this intent, presenting the advantages and limitations of each production system, as well as suggestions for improvements. New and upgraded bioreactors have emerged both for adherent and suspension cell culture, with disposable reactors attracting increased interest in the last years. Furthermore, the strategies and technologies used to control culture parameters are in constant evolution, aiming at the on-line multiparameter monitoring and considering now parameters not seen as relevant for process optimization in the past. All progresses being made have as primary goal the development of highly productive and economic mAb manufacturing processes that will allow the rapid introduction of the product in the biopharmaceutical market at more accessible prices.

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The Effect of Cell Culture Parameters on Protein Glycosylation

Cited by 16 publications

References 84 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

Optimisation of the Cellular Metabolism of Glycosylation for Recombinant Proteins Produced by Mammalian Cell Systems

Technological progresses in monoclonal antibody production systems

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