Background and Novelty
Key Words: Monoclonal antibody glycosylation, stable & transient gene expression, mild hypothermia, mathematical modelling, flux balance analysis
With positive outcomes from medical treatments, monoclonal antibodies (mAbs) are the best-selling biologics in the pharmaceutical market to date. The medical value and safety of these molecules have been sometimes reported to be impacted by the carbohydrate structures that are attached to the N-linked glycosylation site on the constant region of the mAb, such as rituximab in which the glycan pattern on its Fc-region determines the function of rituximab in eliciting immune response against diseases such as leukemias and autoimmune disorders that are characterised by abnormal production of dysfunctional B cells . It has also been reported that different bioprocess conditions during recombinant mAb production directly impact glycan compositions [2, 3], although the mechanism behind this change is not fully understood. This lack of understanding limits process design and optimisation efforts. Herein, we examine the effect of mild hypothermic conditions. Mild hypothermia during cell culture (e.g. temperature shift from 36.5°C to 32°C) is commonly employed to increase the specific recombinant product productivity (qP) [4, 5]. Cells are commonly cultured at physiological temperature during the start of the production stage bioreactor step until desired viable cell density is obtained; the temperature is then lowered to mild hypothermic levels (ranges from 28°C to 34°C), where the lower rates of cell growth are typically compensated for by an the increase in qP. In this study, we investigate experimentally the effect of mild hypothermia (32°C) on mAb N-linked glycosylation, using flux balance analysis and mathematical modelling to identify resulting differences in cell metabolism. A mathematical model that mechanistically and quantitatively has been constructed to describe four different elements: 1. CHO cell behaviour and metabolism; 2. mAb synthesis; 3. Nucleotide sugar donor (NSD) metabolism; and 4. mAb Fc N-linked glycosylation profiles, before and after the induction of mild hypothermia. We believe that this is the first quantitative model that relates mild hypothermia to the four elements mentioned above. As the model aids understanding of the way bioprocess conditions affect product quality, it also provides a platform for bioprocess design, control and optimisation in industry and helps the implementation of the Quality by Design principles.