Qu Weihong, Wang Mingxi, Wu Yaqing, Lv Yinghui, Wang Qizhao, Xu Ruian
School of Medicine and Institute of Molecular Medicine, Huaqiao University, Quanzhou, Fujian 361021, China; College of Medicine and Life Science, Jiujiang University, Jiujiang, Jiangxi 332000, China.
School of Medicine and Institute of Molecular Medicine, Huaqiao University, Quanzhou, Fujian 361021, China; Engineering Research Centre of Molecular Medicine, Ministry of Education, Xiamen, Fujian 361021, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 May 15;990:15-22. doi: 10.1016/j.jchromb.2015.03.003. Epub 2015 Mar 19.
Column chromatography has been widely used as a scalable purification strategy for recombinant adeno-associated virus (rAAV) vectors. The rAAV1, 2, 4, 5, 6, 8 and 9 serotypes could be separated using affinity resins, ion exchange resins or other types of resins. Apatite resin has displayed outstanding performance in protein purification in the past 10 years, and ceramic hydroxyapatite (CHT) chromatography resin with a polyethylene glycol (PEG) modulation has recently been used for rAAV1 and rAAV9 vectors. This study reports the use of CHT chromatography modulated by calcium ions instead of PEG for rAAV9 purification. Calcium-ion-containing buffers effectively improve the inclusion of CHT as a capture resin, the resin-binding capacity and the yield. The optimum calcium ion concentration is 30ppm, and the optimum pH is 7.0. A frontal analysis indicated that the binding capacity of CHT at 2ml/min reaches 65.1mg total protein per ml of resin. A previously developed purification strategy consists of CHT followed by ANX anion exchange chromatography. The vector yield of this approach is approximately 70%, and a software analysis indicated a vector purity exceeding 98%. The residual host cell (HEK293) protein contents are 24.75±2.32ng and 67.21±2.10ng, and the Benzonase residue contents are 1.55±0.10pg and 1.95±0.16ng per 10(13) vector genome copies (G.C.) separated by CHT/ANX and CsCl. In addition, CHT/ANX yields 798.44±50.10pg of plasmid DNA and 2.17±0.11ng of HEK293 DNA, while CsCl purification yields 840.27±76.14pg of plasmid DNA and 2.43±0.19 of HEK293 DNA. The two methods produce vectors with similar in vitro and in vivo potencies. The results indicated that the CHT/ANX method is suitable for the scalable purification of the rAAV9 vector.
柱色谱法已被广泛用作重组腺相关病毒（rAAV）载体的一种可扩展纯化策略。rAAV1、2、4、5、6、8和9血清型可以使用亲和树脂、离子交换树脂或其他类型的树脂进行分离。在过去10年中，磷灰石树脂在蛋白质纯化方面表现出卓越性能，最近一种带有聚乙二醇（PEG）调节的陶瓷羟基磷灰石（CHT）色谱树脂已被用于rAAV1和rAAV9载体。本研究报告了使用钙离子而非PEG调节的CHT色谱法用于rAAV9的纯化。含钙离子缓冲液有效提高了CHT作为捕获树脂的包容性、树脂结合能力及产量。最佳钙离子浓度为30ppm，最佳pH值为7.0。前沿分析表明，流速为2ml/min时CHT的结合能力达到每毫升树脂65.1mg总蛋白。先前开发的纯化策略包括先使用CHT，然后进行ANX阴离子交换色谱法。该方法的载体产量约为70%，软件分析表明载体纯度超过98%。每10¹³个通过CHT/ANX和CsCl分离的载体基因组拷贝（G.C.）中，残留宿主细胞（HEK293）蛋白含量分别为24.75±2.32ng和67.21±2.10ng，Benzonase残留量分别为1.55±0.10pg和1.95±0.16ng。此外，CHT/ANX产生798.44±50.10pg质粒DNA和2.17±0.11ng HEK293 DNA，而CsCl纯化产生840.27±76.14pg质粒DNA和2.43±0.19ng HEK293 DNA。这两种方法产生的载体在体外和体内效力相似。结果表明，CHT/ANX方法适用于rAAV9载体的可扩展纯化。
