The "push-to-low" approach for optimization of high-density perfusion cultures of animal cells.

High product titer is considered a strategic advantage of fed-batch over perfusion cultivation mode. 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 their fed-batch counterparts have not been thoroughly explored. The present paper introduces a unified framework for 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 variable that conveniently bridges various cultivation modes. The analysis shows that development of economically competitive perfusion processes for production of stable proteins depends on our ability to dramatically reduce 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 growth and viability, specific productivity, or product instability. To overcome these limitations, a strategy referred to as "push-to-low" optimization has been developed. This approach involves an iterative stepwise decrease of the specific perfusion rate, and is most suitable for production of stable proteins where increased residence time does not compromise apparent specific productivity or product quality. The push-to-low approach was successfully applied to the production of monoclonal antibody against tumor necrosis factor (TNF). The experimental results followed closely the theoretical prediction, providing a multifold increase in titer. Despite the medium improvement, reduction of the specific growth rate along with increased apoptosis was observed at low specific perfusion rates. This phenomenon could not be explained with limitation or inhibition by the known nutrients and metabolites. Even further improvement would be possible if the cause of apoptosis were understood. In general, a strategic target in the optimization of perfusion processes should be the decrease of 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.