Intensified perfusion processes for biopharmaceutical manufacturing have gained significant attention over the last few years as a means to significantly improve productivity over conventional fed batch processes, but also pose some additional challenges that have to be addressed. In order to achieve process intensification, alternating tangential flow (ATF) devices are frequently used for cell retention, but determining the actual death rate due to cell lysis in these high-density processes is far from straightforward. While a generic measure of &gt;90% cell viability is typically used as an acceptable level of culture healthiness, lysis of cells causes the release of various degrading agents, such as proteases, sialidases, reducing agents, and others, and keeping viability as high as possible is mostly preferred. We monitored several CHO bioprocesses, intensified and non-intensified, for the production of monoclonal antibodies, Fc fusion proteins, and enzymes with a focus on cell viability. Trypan Blue-based viabilities, which is the industry standard, and viabilities estimated from the activity of lactate dehydrogenase (LDH) released from dead cells, were compared for the same processes run at various cell densities for the production of the afore-mentioned therapeutic modalities. Our results indicate that degradation of LDH in the bioreactor was more significant in high-density processes. In all cases observed, the instantaneous viabilities given by Trypan Blue methods are much higher than those obtained from LDH measurements, and the difference in some cases is up to 10-15%. The additional shear due to higher sparging rates and ATF recirculation can plausibly result in significantly higher lysis levels. Our results suggesting, that dead cell counts estimated through LDH may be more accurate than those from Trypan Blue methods, are in good agreement with previously reported literature. Our measurements from intensified processes demonstrate that that accuracy is even more pronounced at higher cell densities, and LDH assays may prove to be more suitable for death rate determination for those processes. Here we present in detail the steps of how to achieve the desired level of accuracy in death rate and how the analysis translates to cell culture health evaluation. We will show that a routine LDH analysis is complimentary to Trypan Blue cell counts and gives more information about shear damage in a given intensified process.

Increased CHO cell lysis in intensified high-density perfusion processes with alternating tangential filtration (ATF) elucidated by a lactate dehydrogenase (LDH) activity assay

2019 Spring National Meeting

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