How does mild hypothermia affect monoclonal antibody glycosylation

The application of mild hypothermic conditions to cell culture is a routine industrial practice used to improve recombinant protein production. However, a thorough understanding of the regulation of dynamic cellular processes at lower temperatures is necessary to enhance bioprocess design and optimization. In this study, we investigated the impact of mild hypothermia on protein glycosylation. Chinese hamster ovary (CHO) cells expressing a monoclonal antibody (mAb) were cultured at 36.5°C and with a temperature shift to 32°C during late exponential/early stationary phase. Experimental results showed higher cell viability with decreased metabolic rates. The specific antibody productivity increased by 25% at 32°C and was accompanied by a reduction in intracellular nucleotide sugar donor (NSD) concentrations and a decreased proportion of the more processed glycan structures on the mAb constant region. To better understand CHO cell metabolism at 32°C, flux balance analysis (FBA) was carried out and constrained with exometabolite data from stationary phase of cultures with or without a temperature shift. Estimated fluxomes suggested reduced fluxes of carbon species towards nucleotide and NSD synthesis and more energy was used for product formation. Expression of the glycosyltransferases that are responsible for N-linked glycan branching and elongation were significantly lower at 32°C. As a result of mild hypothermia, mAb glycosylation was shown to be affected by both NSD availability and glycosyltransferase expression. The combined experimental/FBA approach generated insight as to how product glycosylation can be impacted by changes in culture temperature. Better feeding strategies can be developed based on the understanding of the metabolic flux distribution. Biotechnol. Bioeng. 2015;112: 1165–1176. © 2014 Wiley Periodicals, Inc.