A Master Cell Bank (MCB) banking troubleshooting case study: Challenges and process improvements with comprehensive root cause analysis

Mammalian cells, especially Chinese hamster ovary (CHO) cells, are routinely used in the biopharmaceutical industry for production of recombinant therapeutic proteins. Master Cell banking is one of the key step during drug development, which ensures preservation of cells at low temperatures for an extended period of time for GMP drug substance manufacturing. CHO cells can show significant variation in growth characteristics during cell line development. This variation necessitates the need for a robust Master Cell Bank (MCB) manufacturing process to ensure consistent MCB thaw and growth. Numerous efforts have been done to understand the cryopreservation mechanism as well as techniques to improve the robustness of banking processes. However, failure of MCB releasing still happens across the industry. A case study will be presented highlighting experiments carried out to identify root cause of MCB thaw and expansion variability. In this study, the health of the cells was examined using an Apoptosis assay and Transmission Electron Microscopy (TEM) analysis to gain a better understanding of the cell bank health. Process improvements that included further passaging of the cell line for improved robustness of the MCB manufacturing process will be discussed

Improved Titer in Late-Stage Mammalian Cell Culture Manufacturing by Re-Cloning

Improving productivity to reduce the cost of biologics manufacturing and ensure that therapeutics can reach more patients remains a major challenge faced by the biopharmaceutical industry. Chinese hamster ovary (CHO) cell lines are commonly prepared for biomanufacturing by single cell cloning post-transfection and recovery, followed by lead clone screening, generation of a research cell bank (RCB), cell culture process development, and manufacturing of a master cell bank (MCB) to be used in early phase clinical manufacturing. In this study, it was found that an additional round of cloning and clone selection from an established monoclonal RCB or MCB (i.e., re-cloning) significantly improved titer for multiple late phase monoclonal antibody upstream processes. Quality attributes remained comparable between the processes using the parental clones and the re-clones. For two CHO cells expressing different antibodies, the re-clone performance was successfully scaled up at 500-L or at 2000-L bioreactor scales, demonstrating for the first time that the re-clone is suitable for late phase and commercial manufacturing processes for improvement of titer while maintaining comparable product quality to the early phase process

PCSK9 is not involved in the degradation of LDL receptors and BACE1 in the adult mouse brain

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that regulates hepatic low-density lipoprotein receptor (LDLR) levels in humans. PCSK9 has also been shown to regulate the levels of additional membrane-bound proteins in vitro, including the very low-density lipoprotein receptor (VLDLR), apolipoprotein E receptor 2 (ApoER2) and the β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1), which are all highly expressed in the CNS and have been implicated in Alzheimer's disease. To better understand the role of PCSK9 in regulating these additional target proteins in vivo, their steady-state levels were measured in the brain of wild-type, PCSK9-deficient, and human PCSK9 overexpressing transgenic mice. We found that while PCSK9 directly bound to recombinant LDLR, VLDLR, and apoER2 protein in vitro, changes in PCSK9 expression did not alter the level of these receptors in the mouse brain. In addition, we found no evidence that PCSK9 regulates BACE1 levels or APP processing in the mouse brain. In conclusion, our results suggest that while PCSK9 plays an important role in regulating circulating LDL cholesterol levels by reducing the number of hepatic LDLRs, it does not appear to modulate the levels of LDLR and other membrane-bound proteins in the adult mouse brain