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 in vitro and in vivo 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.

中文翻译：

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TPP与DGUC结合使用，是大规模纯化AAV载体的经济通用方法。

通常通过密度梯度超速离心（DGUC）或柱色谱法纯化腺伴随病毒（AAV）载体。尽管DGUC方法可以有效地将空病毒与完整病毒颗粒分离，但是由于每个离心管的体积有限，DGUC方法在大规模AAV纯化中的应用受到了阻碍。或者，柱层析是血清型依赖性的，昂贵的和复杂的，其共同纯化空的和完整的病毒颗粒。在这项研究中，我们描述了使用三相分配（TPP）与DGUC结合以纯化大量AAV载体的经济且通用的方法。首先，TPP用于去除细胞裂解液中多达90％的细胞杂质，同时将AAV载体浓缩至其原始裂解液体积的〜10％。第二，使用两轮DGUC分离空病毒和完整病毒颗粒，同时去除残留的细胞杂质。根据AAV血清型的产量，该组合过程将超速离心的能力提高了5到10倍。多种AAV血清型，例如AAV2，AAV5，AAV6，AAV9和AAVDJ已通过此过程成功纯化。体外和体内研究均表明，TPP对AAV的感染性没有有害影响。作为概念证明，我们进行了3至25 L的Sf9培养体积的几次纯化操作。在贝克曼库尔特超速离心机中，仅用两个SW28离心管，我们就能从3 L细胞培养体积中纯化出超过3e + 15的AAV病毒基因组（vg）。我们的数据表明，这种TPP-DGUC工艺是经济的，