Abstract
In order to meet desired drug product quality targets, the glycosylation profile of biotherapeutics such as monoclonal antibodies (mAbs) must be maintained consistently during manufacturing. Achieving consistent glycan distribution profiles requires identifying factors that influence glycosylation, and manipulating them appropriately via well-designed control strategies. Now, the cell culture media supplement, MnCl2, is known to alter the glycosylation profile in mAbs generally, but its effect, particularly when introduced at different stages during cell growth, has yet to be investigated and quantified. In this study, we evaluate the effect of time-dependent addition of MnCl2 on the glycan profile quantitatively, using factorial design experiments. Our results show that MnCl2 addition during the lag and exponential phases affects the glycan profile significantly more than stationary phase supplementation does. Also, using a novel computational technique, we identify various combinations of glycan species that are affected by this dynamic media supplementation scheme, and quantify the effects mathematically. Our experiments demonstrate the importance of taking into consideration the time of addition of these trace supplements, not just their concentrations, and our computational analysis provides insight into what supplements to add, when, and how much, in order to induce desired changes.
Highlights
The global market for pharmaceuticals is predicted to grow to $1.5 trillion by 2021, with biologics such as monoclonal antibodies, hormones, and therapeutic enzymes accounting for nearly20% of the market share, based on current projections
Unlike the other quality attributes of monoclonal antibodies (mAbs), such studies have been limited to the introduction of media supplements cases where EDTA addition on day 0 (D0) or day 3 (D3) resulted in low titers, adding EDTA on day 6 (D6) along with MnCl2 exclusively at the start of the culture, and the results, when quantitative, have yielded only isolated supplementation on D0, D3 or D6, resulted in end of run (EOR) titers higher than the titer value single factor relationships
Most media supplementation studies do not take into consideration the impact of introducing media supplements at different stages of cell growth
Summary
The global market for pharmaceuticals is predicted to grow to $1.5 trillion by 2021, with biologics such as monoclonal antibodies (mAbs), hormones, and therapeutic enzymes accounting for nearly20% of the market share, based on current projections. With US sales rising from $8.29 billion in 2005 to $24.6 billion in 2012 [1,2]—coupled with the increase in regulatory agencies approvals of mAb treatments for different indications ranging from cancer to rheumatoid arthritis and Crohn’s disease [3,4], the growing market for mAbs has resulted in active development of these biological products. While several different mammalian cell expression systems can be used to synthesize mAb therapeutics, over half of all currently approved mAbs are produced in Chinese Hamster. CHO cells are the preferred host for a large number of recombinant mAb therapeutics because of the ease of adapting them to suspension growth, and the availability of powerful gene amplification systems to improve specific productivity [5]. More importantly, the post-translational modification machinery in CHO cells produces human-like structures in mAbs, ensuring biocompatibility. One important post-translational modification in mAbs is N-glycosylation, a process by which an oligosaccharyltransferase complex in the endoplasmic reticulum adds a sugar substrate (glycan) to the
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