Modeling the Effect of Ammonia on the Glycosylation Pattern of Monoclonal Antibodies Produced by Chinese Hamster Ovary Cells
Date
2012-05
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Publisher
University of Delaware
Abstract
Monoclonal antibodies (mAbs) are therapeutic recombinant proteins which are typically produced industrially by Chinese Hamster Ovary Cells (CHO). In order to approve mAbs for therapeutic purposes, regulatory agencies require manufacturers to assure the bioactivity, purity, and potency of these proteins to consumers. Regulatory agencies now encourage manufacturers to develop strategies to control quality attributes online. One of the most important factors affecting the quality and bioactivity of mAbs is glycosylation – the addition of a carbohydrate chain to a protein. Glycosylation is a post-translational modification which exhibits heterogeneity that arises from variations in site occupancy and structure of attached glycans. As a result of such heterogeneity, glycosylation presents a challenge to manufacturers of mAbs yet an on-line control strategy does not exist for glycosylation. While a number of culture conditions have been found to influence mAb glycoform distribution, in order to develop effective control schemes, it is important to understand the mechanisms underlying how these culture conditions affect glycosylation. One such culture condition is ammonia levels in culture media. Ammonia has been found to adversely affect cell growth and viability. It is also known to induce heterogeneity in glycosylation by affecting the extent of terminal sialylation, the transfer of a sialic acid residue to a glycoprotein. A few mechanisms have been proposed to describe how ammonia affects sialylation: one involves the reduction of CMP-sialic acid transporter (CMP-SAT) expression level by ammonia; another, the inhibition of CMP-SAT by an increase in UDP-GlcNAc in the presence of ammonia; and still another, the increase in the trans-Golgi pH causing a decrease in sialylation enzymes. The aim of this study is to develop a model based on these mechanisms in order to understand at a fundamental level the effects of ammonia on glycosylation. The model developed in this study can be used in conjunction with the Krambeck and Betenbaugh (2005) model to determine the ammonia concentration required in the cell culture to achieve the desired sialic acid content on the mAb. The model approach can also be employed for other culture conditions that affect glycosylation. This model work will aid in the development of a method for on-line quality control of the glycosylation pattern of mAbs.
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Keywords
monoclonal antibodies, Chinese Hamster Ovary Cells, mAbs, glycosylation, ammonia