生物学杂志

生物学杂志 ›› 2023, Vol. 40 ›› Issue (4): 107-.doi: 10.3969/j.issn.2095-1736.2023.04.107

• 技术方法 • 上一篇    下一篇

rAAV5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. 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
  • 出版日期:2023-08-18 发布日期:2023-08-10
  • 通讯作者: 朱涛，博士，教授，研究方向为疫苗及其他生物制品的开发，E-mail:tao.zhu@cansinotech.com
  • 作者简介:许琳，硕士研究生，研究方向为基因治疗，E-mail:xulxlin@ 163.com
  • 基金资助:
    天津市自然科学基金项目(No.18JCQNJC09700)

Development of rAAV5 anion exchange chromatography process

XU Lin1, LIANG Huanhuan1, LI Hongying2, HAN Xu2, DAI Liubing2, MA Chao2, ZHU Tao1,2   

  1. 1. College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China;
    2. Cansino Biologics Inc., Tianjin 300000, China)
  • Online:2023-08-18 Published:2023-08-10

PDF (PC)

39

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，CIMmultus QA（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, pH 8.0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

Abstract: A purification method was established for effectively increasing the proportion between vector capsids of rAAV5-a viral product containing the therapeutic DNA sequence and empty capsids, viral vectors lacking the therapeutic gene. For the rAAV5 samples obtained from affinity chromatography, several anion exchange chromatography and related gradient elution methods were tested, in which CIMmultus QA (CIMQ) can effectively remove empty capsids from vector capsids. For the CIMQ purification, the buffer matrix and the collection of rAAV5 fractions were optimized. Subsequently, qPCR was used to quantify the recovery rate of the virus genome (Vg) content, and high-performance liquid chromatography (HPLC) or size exclusion chromatography-multi-angle static light scattering (SEC-MALS) was used to determine the vector capsids proportion of rAAV5 samples. In the CIMQ purification process, the proportion of vector capsids in the collected fraction (at UV absorbance UV260/UV280 greater than 1) eluted with Tris-Na2HPO4buffer at pH 8.0 reached 80.3%. A simple and convenient method using anion exchange chromatography was successfully established, which can effectively eliminate empty capsids of rAAV5 from vector capsids so that vector capsids proportion can meet the requirements of clinical application of gene therapy.

Key words: rAAV5, anion exchange chromatography, vector capsids proportion, recovery, purification

中图分类号: