title: The effect of antiapoptosis genes on clarification performance.
abstract: Optimal bioreactor harvest time is typically determined based on maximizing product titer without compromising product quality. We suggest that ease of downstream purification should also be considered during harvest. In this view, we studied the effect of antiapoptosis genes on downstream performance. Our hypothesis was that more robust cells would exhibit less cell lysis and thus generate lower levels of cell debris and host-cell contaminants. We focused on the clarification unit operation, measuring postclarification turbidity and host-cell protein (HCP) concentration as a function of bioreactor harvest time/cell viability. In order to mimic primary clarification using disk-stack centrifugation, a scale-down model consisting of a rotating disk (to simulate shear in the inlet feed zone of the centrifuge) and a swinging-bucket lab centrifuge was used. Our data suggest that in the absence of shear during primary clarification (typical of depth filters), a 20-50% reduction in HCP levels and 50-65% lower postcentrifugation turbidity was observed for cells with antiapoptosis genes compared to control cells. However, on exposing the cells to shear levels typical in a disk-stack centrifuge, the reduction in HCP was 10-15% while no difference in postcentrifugation turbidity was observed. The maximum benefit of antiapoptosis genes is, therefore, realized using clarification options that involve low shear, <1 × 10(6) W/m(3) and minimal damage to the cells.
authors.affiliation: Downstream Technologies, EMD Millipore, 80 Ashby Road, Bedford, MA, 01730.
authors.forename: Ajish S R
authors.initials: AS
authors.lastname: Potty
authors.forename: Alex
authors.initials: A
authors.lastname: Xenopoulos
authors.forename: Sonal
authors.initials: S
authors.lastname: Patel
authors.forename: Holly
authors.initials: H
authors.lastname: Prentice
authors.forename: Anthony
authors.initials: A
authors.lastname: Dileo
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D019149:Bioreactors; D016466:CHO Cells; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000235:genetics; D002498:Centrifugation / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D009391:Nephelometry and Turbidimetry / Q000379:methods*
keywords: apoptosis; disk-stack centrifugation; host-cell protein