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目的选择适用于重组人凝血因子Ⅷ(recombinant human coagulation factorⅧ,rh FⅧ)制备工艺中的纳米膜。方法将rh FⅧ收获液Ⅰ(含吐温80)和收获液Ⅱ(不含吐温80)经4种不同型号的滤器(Duropore~、Kleenpak~、Minisart~、Virosart~max)进行预过滤,检测样品活性回收率及蛋白回收率。用不同厂家的20 nm孔径纳米膜(Nano1~Nano7)对预过滤样品进行过滤,检测样品的活性回收率、蛋白回收率及过滤通量。以粒径约20 nm的细小病毒(minute virus of mice,MVM)作为指示病毒,对筛选出的纳米膜进行病毒去除验证。结果确定最佳预过滤膜为minisart~,收获液Ⅰ和收获液Ⅱ的活性收获率分别为87.9%和99.1%,蛋白回收率分别为97.3%和98.2%。确定收获液Ⅰ的最佳过滤纳米膜为Nano6,样品活性回收率为100%,蛋白回收率为92.6%,过滤通量为16.44 L/(m2·h);收获液Ⅱ的最佳纳米膜为Nano7,样品活性回收率为98%,蛋白回收率为86%,过滤通量为26.54 L/(m2·h)。Nano6和Nano7的病毒去除验证均合格。结论筛选的纳米膜可用于rh FⅧ的产业化生产工艺中。
Objective To select nanofilms suitable for the preparation of recombinant human coagulation factor VIII (rh FⅧ). Methods Four different types of filters (Duropore ~ , Kleenpak ~ , Minisart ~ , Virosart ~ max) were used for rhFVIII harvest Ⅰ (containing Tween 80) and harvest liquid Ⅱ Pre-filtration, detection of sample activity recovery and protein recovery. The pre-filtered samples were filtered with 20 nm-diameter nanofilms from different manufacturers (Nano1 ~ Nano7) to measure the activity recovery, protein recovery and filtration flux of the samples. Virus screening was performed on the screened nanofilms using virus-in mice (MVM) with a particle size of about 20 nm as the indicator virus. The results showed that the optimal prefiltration membrane was minisart ~ , the harvest rates of harvest liquid Ⅰ and harvest liquid Ⅱ were 87.9% and 99.1% respectively, and the recoveries of protein were 97.3% and 98.2% respectively. The optimum filtration nanofilm of harvest Ⅰ was 100%, the recovery rate of protein was 92.6% and the filtration flux was 16.44 L / (m2 · h). The best nanofilm of harvest Ⅱ was Nano7, the sample activity recovery was 98%, protein recovery was 86%, filtration flux was 26.54 L / (m2 · h). Nano6 and Nano7 virus removal validation were qualified. Conclusion The nanofilter screened can be used in the industrial production of rh FⅧ.