Khaparde Ashish, Patra Riya, Roy Shomereeta, Babu G R Sharath, Ghosh Arkasubhra
GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore 560099, India.
Manipal Academy of Higher Education, Manipal 576104, India.
ACS Omega. 2025 May 31;10(22):22657-22670. doi: 10.1021/acsomega.4c10900. eCollection 2025 Jun 10.
Adeno-associated viruses (AAVs) have emerged as a promising tool for gene therapy due to their excellent safety profile and efficient transduction in multiple target tissues. Currently generated AAV yields at lab scale are in the range of 10-10 vgc/L or between 10 and 10 vg/cell. Maximizing yields would significantly impact the overall production cost and accessibility. However, existing challenges to improving the overall yield from upstream and downstream processes persist. Using an adherent cell manufacturing process, we compared two different purification approaches to optimize the recovery from cell lysate (CsCl ultracentrifugation) and media supernatant (affinity column chromatography). We achieved combined purified yields of 2.25 × 10 for AAV6 (1.00 × 10 (44.44%) from ultracentrifugation and 1.25 × 10 (55.56%) from affinity chromatography), 1.11 × 10 for AAV8 (2.37 × 10 (21.39%) from ultracentrifugation and 8.70 × 10 (78.61%) from affinity chromatography), and 7.32 × 10 for AAV9 (1.10 × 10 (15.03%) from ultracentrifugation and 6.22 × 10 (84.97%) from affinity chromatography), with AAV9 showing a ∼1.5-fold increase compared to previous reports. Interestingly, we found each serotype to elute at different pH values which may influence yields. The chromatography approach obtained higher percentage recoveries for AAV6 (88.35 ± 12.87%), AAV8 (96.67 ± 0.60%), and AAV9 (93.54 ± 1.08%) from the media supernatant and also demonstrated significantly higher transduction rates. Thus, we demonstrate the potential for further expanding the AAV production capacity while also improving the final product quality.
腺相关病毒（AAV）因其出色的安全性和在多种靶组织中的高效转导能力，已成为一种很有前景的基因治疗工具。目前实验室规模生产的AAV产量在10-10 vgc/L范围内，或在10至10 vg/细胞之间。最大化产量将显著影响总体生产成本和可及性。然而，在上游和下游工艺中提高总体产量仍存在现有挑战。使用贴壁细胞制造工艺，我们比较了两种不同的纯化方法，以优化从细胞裂解物（氯化铯超速离心）和培养基上清液（亲和柱色谱）中的回收率。我们实现了AAV6的组合纯化产量为2.25×10（超速离心法得到1.00×10（44.44%），亲和色谱法得到1.25×10（55.56%）），AAV8为1.11×10（超速离心法得到2.37×10（21.39%），亲和色谱法得到8.70×10（78.61%）），AAV9为7.32×10（超速离心法得到1.10×10（15.03%），亲和色谱法得到6.22×10（84.97%）），其中AAV9与先前报道相比显示出约1.5倍的增长。有趣的是，我们发现每种血清型在不同的pH值下洗脱，这可能会影响产量。色谱法从培养基上清液中获得的AAV6（88.35±12.87%）、AAV8（96.67±0.60%）和AAV9（93.54±1.08%）回收率更高，并且还显示出显著更高的转导率。因此，我们证明了进一步扩大AAV生产能力同时提高最终产品质量的潜力。
