Investigating the dynamics of CHO cell surface glycosylation as a robust predictor of therapeutic protein glycoform distribution

Monoclonal antibodies (mAbs) are currently the highest-selling class of pharmaceuticals due to their high effectiveness in treating many types of life-threatening diseases, including sever-al types of cancer. The global mAb market was worth over USD 188.18 Billion in 2021 and is projected to exhibit a compound annual growth rate (CAGR) of 11.30 % between 2022-2030 [3]. The market for mAbs corresponded to 65.6% of total biopharmaceutical sales in 2017 [4]. Importantly, mAbs are extremely costly drugs, with a median sales price of approximately $14,000 per gram of product and a median annual treatment price of $58,968 from 1997 to 2016 [5, 6]. On the other hand, the biopharmaceutical industry seeks to develop new products for other chronic indications and extend their use into the developing world. Therefore, there is an increasing requirement to substantially reduce the cost to the patient. A fundamental dif-ference between mAbs and ‘conventional’ drugs is that they are extremely complex molecules that are produced by living organisms (Chinese Hamster Ovary cells are the most commonly used) [7]. Due to the complexity of mAb molecules, ensuring their safety and quality is an extremely complex task. It is important to emphasise that mAb complexity contributes direct-ly to the high cost of these products. Glycosylation, as the most prominent post-translational modification (PTM), plays a critical role in modulating the function, stability, and structural integrity of mAbs [8]. Therefore, con-trolling glycosylation through modulating manufacturing conditions can result in mAbs with desirable glycosylation patterns and optimal therapeutic efficacy. A factor that has limited control strategy development is the low throughput of mAb glycan analysis [7]. The bulk of mammalian cells glycosylation occurs in the endoplasmic reticulum and the Golgi apparatus [9]. While cellular machinery glycosylates the secreted recombinant therapeutic product, it simultaneously glycosylates endogenous glycoproteins that are localised, mainly to the cell surface [10]. Because cell surface glycoproteins and the mAb product are simultane-ously processed by the same cellular machinery, the glycan distribution of cell surface proteins and mAbs should be correlated [11]. This work aimed to evaluate this correlation and explore whether cell surface glycosylation signatures can be used as a robust predictor of mAb glycosylation during cell culture. A dis-tinct advantage of using cell surface glycosylation as a predictor is that it can be monitored rapidly and easily. In all, this project proposes the first steps towards developing a robust mAb glycosylation monitoring and control strategy, a tool that addresses a pressing need of the biopharmaceutical industry.