Abstract
Commercially available automated cell counters based on trypan blue dye-exclusion are widely used in industrial cell culture process development and manufacturing to increase throughput and eliminate inherent variability in subjective interpretation associated with manual hemocytometers. When using these cell counters, sample dilution is often necessary to stay within the assay measurement range; however, the effect of time and diluents on cell culture is not well understood. This report presents the adverse effect of phosphate buffered saline as a diluent on cell viability when used in combination with an automated cell counter. The reduced cell viability was attributed to shear stress introduced by the automated cell counter. Furthermore, length of time samples were incubated in phosphate buffered saline also contributed to the observed drop in cell viability. Finally, as erroneous viability measurements can severely impact process decisions and product quality, this report identifies several alternative diluents that can maintain cell culture viability over time in order to ensure accurate representation of cell culture conditions.
Highlights
Rapid, accurate and precise assessment of cell culture viability is critical to industrial cell culture process development and manufacturing of the monoclonal antibodybased therapeutic proteins
A discrepancy of viability was first observed when comparing experimental results obtained with an microscale bioreactor (MBR) system and those obtained with 3 L bioreactor systems
Cell culture samples from the 3 L bioreactors were only diluted with phosphate buffered saline (PBS) when the viable cell density (VCD) exceeded 1x107 cells/mL, and all samples from MBR were diluted with PBS
Summary
Accurate and precise assessment of cell culture viability (viability) is critical to industrial cell culture process development and manufacturing of the monoclonal antibody (mAb)based therapeutic proteins. Viability provides information on process performance and reproducibility, and a basis to calculate other important parameters such as viable cell density (VCD) [1]. Maintaining a desirable viability profile throughout the cell culture process has become progressively important to enhance protein production. The observed increases in mAb production in recent years is partly attributed to the increased understanding of cell engineering and process optimization to achieve and sustain high VCD throughout the culture duration [2,3]. The importance of cellular viability is beyond protein yield as it is a critical parameter for maintaining protein quality. Viability is often incorporated as part of the harvest criteria, due to the potential impact upon protein quality
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