Yu Zhe, Zhou Siyun, Luo Ningguang, Ho Ching Yi, Chen Min, Chen Haifeng
Virovek, 22429 Hesperian Boulevard, Hayward, CA 94541, USA.
Mol Ther Methods Clin Dev. 2019 Nov 22;17:34-48. doi: 10.1016/j.omtm.2019.11.009. eCollection 2020 Jun 12.
Adeno-associated virus (AAV) vectors have been commonly purified through density gradient ultracentrifugation (DGUC) or column chromatography methods. Although the DGUC method can efficiently separate the empty from the full virus particles, its application in large-scale AAV purification is hindered due to its limitation in volume of each centrifuge tube. Alternatively, column chromatography is serotype-dependent, expensive, and complicated, which co-purifies both empty and full virus particles. In this study, we describe an economical and universal process using three-phase partitioning (TPP) combined with DGUC to purify large quantities of AAV vectors. First, TPP is used to remove up to 90% of the cellular impurities in the cell lysate and at the same time condense the AAV vectors into ∼10% of their original lysate volume. Second, two rounds of DGUC are employed to separate the empty from the full virus particles and at the same time remove the remaining cellular impurities. This combined process increases the capacity of ultracentrifugation by a factor of 5- to 10-fold depending on the yields of AAV serotypes. A variety of AAV serotypes such as AAV2, AAV5, AAV6, AAV9, and AAVDJ have been successfully purified with this process. Both and studies demonstrate that TPP has no detrimental impact on AAV infectivity. In a proof of concept, we performed several purification runs ranging from 3 to 25 L of Sf9 culture volume. We were able to purify more than 3e+15 viral genomes (vg) of AAV vectors from 3 L of cell culture volume with just two SW28 centrifuge tubes in a Beckman Coulter ultracentrifuge. Our data indicate that this TPP-DGUC process is economic, universal, and can be used to purify a large quantity of AAV vectors for clinical applications with just a few ultracentrifuges.
腺相关病毒（AAV）载体通常通过密度梯度超速离心（DGUC）或柱色谱法进行纯化。尽管DGUC方法能够有效地将空病毒颗粒与完整病毒颗粒分离，但由于每个离心管的体积限制，其在大规模AAV纯化中的应用受到阻碍。另外，柱色谱法依赖血清型，成本高且操作复杂，会同时共纯化空病毒颗粒和完整病毒颗粒。在本研究中，我们描述了一种经济且通用的方法，即使用三相分离（TPP）结合DGUC来纯化大量的AAV载体。首先，TPP用于去除细胞裂解物中高达90%的细胞杂质，同时将AAV载体浓缩至其原始裂解物体积的约10%。其次，采用两轮DGUC来分离空病毒颗粒与完整病毒颗粒，同时去除剩余的细胞杂质。根据AAV血清型的产量，这种联合方法可将超速离心的容量提高5至10倍。多种AAV血清型，如AAV2、AAV5、AAV6、AAV9和AAVDJ，已通过该方法成功纯化。 和 研究均表明TPP对AAV感染性没有不利影响。在一个概念验证中，我们进行了几次纯化实验，Sf9培养体积从3 L到25 L不等。在贝克曼库尔特超速离心机中仅使用两个SW28离心管，我们就能够从3 L细胞培养体积中纯化出超过3×10¹⁵个病毒基因组（vg）的AAV载体。我们的数据表明，这种TPP-DGUC方法经济、通用，仅使用几台超速离心机就可用于纯化大量用于临床应用的AAV载体。
