Anthony J. DiLeo

High Resolution Removal of Virus from Protein Solutions Using a Membrane of Unique Structure

We describe a new class of membrane that has the capability of removing particles such as viruses from solution with resolution and reproducibility superior to that of conventional membranes. This composite membrane is composed of a pre-formed microporous membrane plus a thin asymmetric, finely porous retentive layer that is quite different from conventional ultrafilters. The protein sieving characteristics of this membrane are nearly equivalent to, but slightly less than, that of conventional 100,000 Dalton cut-off ultrafiltration membranes. This membrane uniquely shows particle retention characteristics that increase monotonically from 3 to 8 logs as a function of particle diameter in the range of 28 to 93 nm. The performance of this membrane in both a single stage and a two stage system show that 4 to 6 log overall removal of virus particles in the size range 30 to 70 nm is possible with simultaneous high recovery of product protein. Clearance factors exceeding 6 logs are possible with viruses larger than 78 nm. In addition, the performance of process systems containing this membrane is predictable in accordance with the general membrane properties and equilibrium mass balance models. This membrane system is fully validatable and can be used in conjunction with other validated operations in a downstream process to reliably achieve an overall reduction of 12 logs of known or putative virus particles.

Deadend Microfiltration: Applications, Design, and Cost

The previous three chapters covered a general description of microfiltration and the theories of deadend and crossflow microfiltration. This chapter focuses on the practical aspects of microfiltration with special emphasis on deadend microfiltration using commercial membranes, important applications, design criteria, and cost estimates.

The effect of antiapoptosis genes on clarification performance

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 × 106 W/m3 and minimal damage to the cells.

A UNIQUE AND VALIDATABLE MEMBRANE-BASED SYSTEM CAPABLE OF HIGH RESOLUTION REMOVAL OF VIRUSES FROM PROTEIN SOLUTIONS

The increasing risk of viral contamination of therapeutic proteins derived from human or animal systems has led both producers of biologically based therapeutics and regulatory agencies to address issues of product safety that relate to to viral transmission. Millipore Corporation (Bedford, MA) has developed a unique and validatable system based on a new caliber of membrane filters successful in achieving reproducible high resolution removal of viruses from protein solutions. The structure and function of this novel membrane is discussed, as well as the experimental design and data employed in the development of a comprehensive mammalian virus validation package.