A two-step procedure for the design of perfusion bioreactors

Abstract

A two-step procedure for the design and optimization of mammalian cell perfusion cultures for therapeutic protein production, for a given expression system, is developed. The considered operating variables include the cell specific perfusion rate (CSPR), the perfusion rate (P) and the viable cell density (VCD). The procedure is articulated in two steps, each including a reactor operation through which different steady states are explored sequentially. In the first one, a suitable minimum value of the CSPR is found. This can be achieved through two alternative approaches. These include keeping the perfusion rate or the VCD constant, while changing the other so as to decrease the CSPR. Cellular growth and metabolite in- and output rates decrease when minimizing the CSPR resulting in a lower bleed rate and a better process performance. In the second step, steady states at constant CSPR but increasing VCD values are investigated, by properly adjusting the perfusion rate. In this case, the cellular activity remains constant, while the harvest yield and volumetric productivity further increase with higher perfusion rates. The product quality (charge variants, N-linked glycosylation) investigated during both steps of the procedure shows only minor changes among the different steady states. If product quality properties obtained at this point of the procedure are not satisfactory, they have to be fine-tuned in an additional step. As reported previously, the addition of supplements in the feed stream can be applied to induce significant changes in product quality.