∗doi:10.3969/j.issn.2095-1736.2023.04.107rAAV5阴离子交换层析工艺的开发许琳1,梁欢欢1,李红英2,韩旭2,戴柳冰2,马超2,朱涛1,2(1.天津科技大学生物工程学院,天津300457;2.康希诺生物股份公司,天津300000)摘要建立一种有效提高重组5型腺相关病毒(rAAV5)实心载体比率的阴离子交换层析的纯化方法。对亲和层析捕获的rAAV5样品,使用多种阴离子交换层析柱进行离子强度线性梯度洗脱,CIMmultusQA(CIMQ)层析柱能够有效分离不含目的基因组的病毒(病毒空颗粒)和含目的基因组的病毒(实心载体)。基于CIMQ层析柱,进行纯化缓冲液体系、紫外目标峰收集范围的优化。通过qPCR检测病毒基因组(Vg)含量计算回收率,高效液相色谱法(HPLC)或体积排阻色谱-多角度静态光散射技术(SEC-MALS)测定实心载体比率。使用Tris,Na2HPO4,pH8.0缓冲体系,洗脱时收集紫外吸收值UV260/UV280比值大于1的部分,收集的样品实心载体比率达到80.3%。成功建立一种简单方便的阴离子交换层析方法,能去除rAAV5样品中的病毒空颗粒,使实心载体比率达到基因治疗临床产品要求。关键词rAAV5;阴离子交换层析;实心载体比率;回收率;纯化中图分类号TQ425文献标识码B文章编号2095-1736(2023)04-0107-07DevelopmentofrAAV5anionexchangechromatographyprocessXULin1,LIANGHuanhuan1,LIHongying2,HANXu2,DAILiubing2,MAChao2,ZHUTao1,21.CollegeofBioengineeringTianjinUniversityofScienceandTechnologyTianjin300457China2.CansinoBiologicsInc.Tianjin300000ChinaAbstractApurificationmethodwasestablishedforeffectivelyincreasingtheproportionbetweenvectorcapsidsofrAAV5-aviralprod-uctcontainingthetherapeuticDNAsequenceandemptycapsidsviralvectorslackingthetherapeuticgene.FortherAAV5samplesobtainedfromaffinitychromatographyseveralanionexchangechromatographyandrelatedgradientelutionmethodsweretestedinwhichCIMmultusQACIMQcaneffectivelyremoveemptycapsidsfromvectorcapsids.FortheCIMQpurificationthebuffermatrixandthecollectionofrAAV5fractionswereoptimized.SubsequentlyqPCRwasusedtoquantifytherecoveryrateofthevirusgenomeVgcontentandhigh-performanceliquidchromatographyHPLCorsizeexclusionchromatography-multi-anglestaticlightscatteringSEC-MALSwasusedtodeterminethevectorcapsidsproportionofrAAV5samples.IntheCIMQpurificationprocesstheproportionofvectorca...
