Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield
Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield
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DOI：
10.1038/sj.gt.3300938
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发表时间：
1999-06-01
期刊：
影响因子：
5.1
通讯作者：
Muzyczka, N
中科院分区：
文献类型：
--
作者：
Zolotukhin, S;Byrne, BJ;Muzyczka, N
基于氯化铯超离心的传统rAAV纯化方法经常产生质量不稳定的载体制备，并导致颗粒感染性的显著丧失。我们在这里报告了几种新的纯化策略，包括使用非离子碘二醇梯度，然后通过传统或高效液相色谱柱进行离子交换或肝素亲和层析。这些方法可以从粗裂解物中回收50%以上的rAAV，并且通常可以产生纯度超过99%的载体。更重要的是，新的纯化程序始终生产出颗粒与传染性比小于100的rAAV库存，这明显优于传统方法。新方案将感染性rAAV的总产量提高了至少10倍，并允许在1个工作日内完全纯化rAAV。其中一些方法也适用于大规模生产。
Conventional methods for rAAV purification that are based on cesium chloride ultracentrifugation have often produced vector preparations of variable quality and resulted in significant loss of particle infectivity. We report here several novel purification strategies that involve the use of non-ionic iodixanol gradients followed by ion exchange or heparin affinity chromatography by either conventional or HPLC columns. These methods result in more than 50% recovery of rAAV from a crude lysate and routinely produce vector that is more than 99% pure. More importantly, the new purification procedures consistently produce rAAV stocks with particle-to-infectivity ratios of less than 100, which is significantly better than conventional methods. The new protocol increases the overall yield of infectious rAAV by at least 10-fold and allows for the complete purification of rAAV in 1 working day. Several of these methods should also be useful for large-scale production.