Therapeutic monoclonal antibodies (mAbs) derived from mammalian cell cultures are currently one of the best-selling and fastest-growing product classes within the biopharmaceutical market. As glycoproteins, mAbs undergo a series of post-translational modifications and degradations, leading to the generation of charge heterogeneity, which has been shown to affect the in vitro and in vivo properties of mAbs. However, mAb charge heterogeneity is still poorly controlled since the lack of the understanding of this critical quality attribution. Actually, charge heterogeneity is generated both inside and outside of cells, consisting of both intracellular and extracellular process. This work aims to get insights into the generation of mAb charge heterogeneity and its relationship with cell culture process by cell-free assays and kinetics model.<br/>Firstly, cell-free assays were performed to simulate and investigate the extracellular process. It is found that the extracellular process is a pseudo first-order reaction and the apparent activation energy (Eapp) value estimated from the Arrhenius equation suggests that the extracellular acidic variant generation might be mainly attributed to asparagine deamidation. Process parameters were investigated as their potential roles in the extracellular process and temperature and media composition showed remarkable effects. A higher temperature led to a higher acidic variant generation rate and a higher C-terminal lysine processing rate. Besides, a kinetics model was established to get a deep understanding of the mAb charge variant generation during cell culture process, especially the intracellular process. The rates of both intracellular process and extracellular process were calculated and it is found that the extracellular process plays a dominant role in modulating the final acidic variant level. Based on the above conclusions, the effects of culture temperature on CHO cell cultures, especially mAb charge distribution were further investigated. As expected, lowering culture temperature significantly decreased the acidic variant content while increased the basic variant content. This work helps to better understand and control charge heterogeneity in mAb manufacturing.

Understanding the generation of monoclonal antibody charge heterogeneity in CHO cell cultures

253rd National Meeting (2017)

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