The “Push-to-Low” Approach for Optimization of High-Density Perfusion Cultures of Animal Cells

Abstract

High product titer is considered a strategic advantage of fed-batch over perfusion cultivationmode. The titer difference has been experimentally demonstrated and reported in the literature. However,the related theoretical aspects and strategies for optimization of perfusion processes with respect to theirfed-batch counterparts have not been thoroughly explored. The present paper introduces a unified frameworkfor comparison of fed-batch and perfusion cultures, and proposes directions for improvement of the latter.The comparison is based on the concept of “equivalent specific perfusion rate”, a variablethat conveniently bridges various cultivation modes. The analysis shows that development of economicallycompetitive perfusion processes for production of stable proteins depends on our ability to dramaticallyreduce the dilution rate while keeping high cell density, i.e., operating at low specific perfusion rates.Under these conditions, titer increases significantly, approaching the range of fed-batch titers. However,as dilution rate is decreased, a limit is reached below which performance declines due to poor growthand viability, specific productivity, or product instability. To overcome these limitations, a strategyreferred to as “push-to-low” optimization has been developed. This approach involves an iterativestepwise decrease of the specific perfusion rate, and is most suitable for production of stable proteinswhere increased residence time does not compromise apparent specific productivity or product quality. Thepush-to-low approach was successfully applied to the production of monoclonal antibody against tumor necrosisfactor (TNF). The experimental results followed closely the theoretical prediction, providing a multifoldincrease in titer. Despite the medium improvement, reduction of the specific growth rate along with increasedapoptosis was observed at low specific perfusion rates. This phenomenon could not be explained with limitationor inhibition by the known nutrients and metabolites. Even further improvement would be possible if thecause of apoptosis were understood.

In general, a strategic target in the optimization of perfusion processes should be the decreaseof the cell-specific perfusion rate to below 0.05 nL/cell/day, resulting in high, batch-like titers.The potential for high titer, combined with high volumetric productivity, stable performance over many months,and superior product/harvest quality, make perfusion processes an attractive alternative to fed-batch production,even in the case of stable proteins.