Akeprathumchai Saengchai, Han Binbing, Wickramasinghe S Ranil, Carlson Jonathan O, Czermak Peter, Preibeta Katrin
Department of Chemical Engineering, Colorado State University, Fort Collins, Colorado 80523-1370, USA.
Biotechnol Bioeng. 2004 Dec 30;88(7):880-9. doi: 10.1002/bit.20312.
Clearance of murine leukemia virus from CHO cell suspensions by flocculation and microfiltration was investigated. Murine leukemia virus is a retrovirus that is recommended by the U.S. Food and Drug Administration for validating clearance of retrovirus-like particles. Due to biosafety considerations, an amphotropic murine leukemia virus vector (A-MLV) that is incapable of self-replication was used. Further, A-MLV is incapable of infecting CHO cells, thus ensuring that infection of the CHO cells in the feed did not result in a reduced virus titer in the permeate. The virus vector contains the gene for the enhanced green fluorescent protein (EGFP) to facilitate assaying for infectious virus particles. The virus particles are 80-130 nm in size. The feed streams were flocculated using a cationic polyelectrolyte. Microfiltration was conducted using 0.1 and 0.65 microm pore size hollow fiber membranes. The level of virus clearance in the permeate was determined. For the 0.1 microm pore size membranes a 1,000-fold reduction in the virus titer in the permeate was observed for feed streams consisting of A-MLV, A-MLV plus flocculant, A-MLV plus CHO cells, and A-MLV plus flocculant and CHO cells. While the flocculant had little effect on the level of virus clearance in the permeate for 0.1 microm pore size membranes, it did lead to higher permeate fluxes for the CHO cell feed streams. Virus clearance experiments conducted with 0.65 microm pore size membranes indicate little clearance of A-MLV from the permeate in the absence of flocculant. However, in the presence of flocculant the level of virus clearance in the permeate was similar to that observed for 0.1 microm pore size membranes. The results obtained here indicate that significant clearance of A-MLV is possible during tangential flow microfiltration. Addition of a flocculant is essential if the membrane pore size is greater than the diameter of the virus particles. Flocculation of the feed stream leads to an increase in the permeate flux.
研究了通过絮凝和微滤从CHO细胞悬液中清除鼠白血病病毒的情况。鼠白血病病毒是一种逆转录病毒，美国食品药品监督管理局推荐其用于验证逆转录病毒样颗粒的清除效果。出于生物安全考虑，使用了一种不能自我复制的嗜性鼠白血病病毒载体（A-MLV）。此外，A-MLV不能感染CHO细胞，从而确保进料中的CHO细胞感染不会导致渗透液中病毒滴度降低。该病毒载体包含增强型绿色荧光蛋白（EGFP）基因，便于检测感染性病毒颗粒。病毒颗粒大小为80-130nm。使用阳离子聚电解质对进料流进行絮凝。使用孔径为0.1和0.65微米的中空纤维膜进行微滤。测定了渗透液中病毒的清除水平。对于孔径为0.1微米的膜，在由A-MLV、A-MLV加絮凝剂、A-MLV加CHO细胞以及A-MLV加絮凝剂和CHO细胞组成的进料流中，观察到渗透液中病毒滴度降低了1000倍。虽然絮凝剂对孔径为0.1微米的膜的渗透液中病毒清除水平影响不大，但它确实导致CHO细胞进料流的渗透通量更高。用孔径为0.65微米的膜进行的病毒清除实验表明，在没有絮凝剂的情况下，渗透液中A-MLV的清除很少。然而，在有絮凝剂的情况下，渗透液中病毒的清除水平与孔径为0.1微米的膜所观察到的相似。此处获得的结果表明，在切向流微滤过程中，A-MLV的显著清除是可能的。如果膜孔径大于病毒颗粒直径，则添加絮凝剂至关重要。进料流的絮凝会导致渗透通量增加。
