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

Zebrafish Primordial Germ Cell Cultures Derived from vasa::RFP Transgenic Embryos

Although embryonic germ (EG) cell-mediated gene transfer has been successful in the mouse for more than a decade, this approach is limited in other species due to the difficulty of isolating the small numbers of progenitors of germ cell lineage (PGCs) from early-stage embryos and the lack of information on the in vitro culture requirements of the cells. In this study, methods were established for the culture of PGCs obtained from zebrafish embryos. Transgenic embryos that express the red fluorescent protein (RFP) under the control of the PGC-specific vasa promoter were used, making it possible to isolate pure populations of PGCs by fluorescence-activated cell sorting (FACS) and to optimize the culture conditions by counting the number of fluorescent PGC colonies produced in different media. Cultures initiated from 26-somite-stage embryos contained the highest percentage of PGCs that proliferated in vitro to generate colonies. The effect of growth factors, including Kit ligand a and b (Kitlga and Kitlgb) and stromal cell-derived factor 1a and 1b (Sdf-1a and Sdf-1b), on PGC proliferation was studied. Optimal in vitro growth and survival of the zebrafish PGCs was achieved when recombinant Kitlga and Sdf-1b were added to the culture medium through transfected feeder cells, resulting in a doubling of the number of PGC colonies. Results from RT-PCR and in situ hybridization analysis demonstrated that PGCs maintained in culture expressed the kita receptor, even though receptor expression was not detected in PGCs isolated by FACS directly from dissociated embryos. In optimal growth conditions, the PGCs continued to proliferate for at least 4 months in culture. The capacity to establish long-term PGC cultures from zebrafish will make it possible to conduct in vitro studies of germ cell differentiation and EG cell pluripotency in this model species and may be valuable for the development of a cell-mediated gene transfer approach