Virus clearance during microfiltration

Abstract

Biotechnology and biopharmaceutical industries use mammalian derived raw materials such as growth medium and genetically engineered vectors to produce recombinant proteins of prophylactic and therapeutic character. The demand of these compounds is increasing tremendously. The use of mammalian derived raw materials enhances the possibility of virus contamination in the final product. The Food and Drug administration (FDA) mandates that manufacturers demonstrate levels of virus removal in excess of calculated levels of contamination in the purification train. Consequently, there is a great need to validate virus clearance in existing unit operations. Microfiltration is usually used to remove cells, cell debris and small particulates from biomass suspensions. The filtrate flux is often relatively low since biomass suspensions are particularly difficult to filter due to their highly fouling nature and the fact that the cake formed is also highly compressible. Limited clearance of virus is expected during microfiltration since they are much smaller than the microfiltration membrane pores. Here, a method to improve filtrate flux and obtain virus clearance during microfiltration is proposed. In this project, a feed suspension containing CHO-DG44 cells and A-MLV pseudotypes is pretreated with cationic polyacrylamides prior to microfiltration. By flocculation, the average particle size is increased leading to filtrate flux enhancement. Further, while flocculation of biomass takes place, the capture of A-MLV pseudotypes into the floe network occurs. Therefore, significant removal of A-MLV from feed suspension may be attained during microfiltration. Cationic polyacrylamides with molecular weight of 4 - 8 x 106 and 40 % charge density were successfully used to flocculate CHO cell suspension. However, the combination of molecular weight and charge density may play a significant role on the level of virus clearance. The average particle size of the flocculated suspension is increased from 18 to ~120 μm. As a result, a considerable improvement of filtrate flux of flocculated CHO cell suspension is accomplished compared to that of unflocculated suspensions. Further, removal of A-MLV pseudotypes as high as 5 log titer reductions may be achieved by flocculating the suspension containing CHO cells and A-MLV prior to microfiltration.