The capture of recombinant antibodies from cell culture broth is the first critical step of downstream processing. We were able to develop a precipitation‐based method for the capture and purification of monoclonal antibodies based on divalent cations, namely ZnCl2. Traditional precipitation processes have to deal with high dilution factors especially for resolubilization and higher viscosity due to the use of PEG as precipitation or co‐precipitation agent. By the use of the crosslinking nature of divalent cations without the use of PEG, we kept viscosity from the supernatant and resolubilization dilution factors very low. This is especially beneficial for the solid–liquid separation for the harvest and wash of the precipitate in continuous mode. For this harvest and wash, we used tangential flow filtration that benefits a lot from low viscosity solutions, which minimizes the membrane fouling. With this precipitation based on ZnCl2, we were able to implement a very lean and efficient process. We demonstrated precipitation studies with three different antibodies, Adalimumab, Trastuzumab, and Denosumab, and a continuous capture case study using tangential flow filtration for precipitate recovery. In this study, we achieved yields of 70%.
Cysteine is considered an essential amino acid in the cultivation of Chinese hamster ovary (CHO) cells. An optimized cysteine supply during fed‐batch cultivation supports the protein production capacity of recombinant CHO cell lines. However, we observed that CHO production cell lines seeded at low cell densities in chemically defined media enriched with cysteine greater than 2.5 mm resulted in markedly reduced cell growth during passaging, hampering seed train performance and scale‐up. To investigate the underlying mechanism, seeding cell densities and initial cysteine concentrations ranging from low to high cysteine concentrations were varied followed by an analysis of cell culture performance. Additionally, cell cycle analysis, intracellular quantification of reactive oxygen species (ROS) as well as transcriptomic analyses by next‐generation sequencing were carried out. Our results demonstrate that CHO cells seeded at low cell densities at high initial cysteine concentrations encountered increased oxidative stress leading to a p21‐mediated cell cycle arrest in the G1/S phase. The resulting oxidative stress caused redox imbalance in the endoplasmic reticulum and activation of the unfolded protein response as well as the major antioxidant nuclear factor‐like 2 response pathways. Potential signature genes related to oxidative stress and the inhibition of the pentose phosphate pathway were identified in the study. Finally, the study presents that seeding cells at a higher concentration counteract oxidative stress in cysteine‐enriched cell culture media.
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