Joshi Pranav R H, Bernier Alice, Moço Pablo D, Schrag Joseph, Chahal Parminder S, Kamen Amine
Viral Vectors and Vaccine Bioprocessing Group, Department of Bioengineering, McGill University, Montreal, QC, Canada.
Human Health Therapeutics, National Research Council of Canada, Montreal, QC, Canada.
Mol Ther Methods Clin Dev. 2021 Mar 23;21:341-356. doi: 10.1016/j.omtm.2021.03.016. eCollection 2021 Jun 11.
Removal of empty capsids from adeno-associated virus (AAV) manufacturing lots remains a critical step in the downstream processing of AAV clinical-grade batches. Because of similar physico-chemical characteristics, the AAV capsid populations totally lacking or containing partial viral DNA are difficult to separate from the desired vector capsid populations. Based on minute differences in density, ultracentrifugation remains the most effective separation method and has been extensively used at small scale but has limitations associated with availabilities and operational complexities in large-scale processing. In this paper, we report a scalable, robust, and versatile anion-exchange chromatography (AEX) method for removing empty capsids and subsequent enrichment of vectors of AAV serotypes 5, 6, 8, and 9. On average, AEX resulted in about 9-fold enrichment of AAV5 in a single step containing 80% ± 5% genome-containing vector capsids, as verified and quantified by analytical ultracentrifugation. The optimized process was further validated using AAV6, AAV8, and AAV9, resulting in over 90% vector enrichment. The AEX process showed comparable results not only for vectors with different transgenes of different sizes but also for AEX runs under different geometries of chromatographic media. The herein-reported sulfate-salt-based AEX process can be adapted to different AAV serotypes by appropriately adjusting elution conditions to achieve enriched vector preparations.
从腺相关病毒（AAV）生产批次中去除空衣壳仍然是AAV临床级批次下游加工中的关键步骤。由于物理化学特性相似，完全缺乏或含有部分病毒DNA的AAV衣壳群体很难与所需的载体衣壳群体分离。基于密度的微小差异，超速离心仍然是最有效的分离方法，并且已在小规模中广泛使用，但在大规模加工中存在可用性和操作复杂性方面的限制。在本文中，我们报告了一种可扩展、稳健且通用的阴离子交换色谱（AEX）方法，用于去除空衣壳并随后富集AAV血清型5、6、8和9的载体。经分析超速离心验证和定量，平均而言，AEX在一步中使含有80%±5%含基因组载体衣壳的AAV5富集约9倍。使用AAV6、AAV8和AAV9对优化后的工艺进行了进一步验证，载体富集率超过90%。AEX工艺不仅对具有不同大小不同转基因的载体显示出可比的结果，而且对在不同几何形状的色谱介质下进行的AEX运行也显示出可比的结果。本文报道的基于硫酸盐的AEX工艺可以通过适当调整洗脱条件来适应不同的AAV血清型，以获得富集的载体制剂。
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