Production of therapeutic proteins comparable to references is a crucial issue for biopharmaceutical industry to develop biosimilars or biobetters. Among all the critical quality attributes, the N-glycosylation is closely related to protein stability, solubility and especially in vivo efficacy. However, it is quite difficult to control glycosylation in the manufacturing process due to the lack of thorough understanding of the intracellular process. In this work, a nucleotide precursor was supplemented to CHO cell culture media to increase high mannose glycan levels of two monoclonal antibodies (mAbs) and one fusion protein, which could be used as a tool to obtain a desired range of glycosylation of therapeutic proteins, and providing more knowledge of post translational modification as well. As a nucleotide precursor, uridine is commonly used as a media component to increase the level of galactosylation, especially combined with manganese and galactose to improve complement-dependent cytotoxicity (CDC). However, we found it could also lead to elevated levels of high mannose glycans and consequently increased antibody dependent cellular cytotoxicity (ADCC) for mAbs as expected. With careful titration, cell growth and product titer were found unaffected. Incubation with culture supernatant demonstrated that intracellular process was the main cause of this glycosylation change. Then, glycan distribution analysis was conducted to reveal that glycan biosynthesis process was relatively trapped in the early stage. Therefore, two key glycosyltransferases, N-acetylglucosaminyltransferase I (GnT-I) and α-1,6-fucosyltransferase (FUT8), were measured but no significant difference was found. Moreover, as building-blocks of glycan biosynthesis, nucleotide sugars were quite sufficient in the cultures supplemented with uridine. To further investigate the causal link between uridine addtion and increased levels of high mannose glycans, untargeted metabolite profiling was performed by LC-MS/MS to provide wider scope of this phenomena, which revealed that intracellular redox balance might be a key factor. Although a clear correlation is still under investigation, our findings provide a useful tool to specifically modulate protein glycosylation and give more information on the intracellular glycan biosynthesis for developers to achieve desired product quality.

Tuning high mannose glycan levels of therapeutic proteins via CHO cell culture media supplementation

253rd National Meeting (2017)

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