Sciex, Brea, CA, United States.
AB Sciex, Beijing, China.
Curr Mol Med. 2020;20(10):814-820. doi: 10.2174/1566524020666200915105456.
Adeno-associated virus (AAV) is one of the most promising gene transfer vector types featuring long-term gene expression and low toxicity. The lack of pathogenicity and the availability of many serotypes augmented the applicability of AAV virions in gene therapy applications. The recombinant AAV capsid includes the therapeutic protein-coding transgene as well as a promoter to initiate translation and a poly A sequence portion for stabilization. Current AAV manufacturing technologies, however, cannot guarantee the generation of only full capsids, i.e., including the entire required genome. Partially filled and empty capsids are also part of the product, decreasing in this way the efficacy and safety upon clinical translation. Therefore, rapid, accurate and QC friendly analysis of the full and empty capsid ratio is of high importance during AAV vector manufacturing and release testing. In this paper, an automated capillary isoelectric focusing technique is introduced, readily applicable in the biopharmaceutical industry for fast and efficient determination of the full and empty capsid ratio. The method also reveals information about the proportion of partially filled capsids. For higher resolution (<0.1 pI unit), mixtures of wide and narrow range ampholytes were utilized. The isoelectric point and peak area percentage reproducibility (RSD) of the mixed ampholyte assay were as low as 1.67% and 2.45 %, respectively, requiring only 65 nL of sample volume per injection.
腺相关病毒（AAV）是最有前途的基因转移载体类型之一，具有长期的基因表达和低毒性。缺乏致病性和多种血清型的可用性增加了 AAV 病毒粒子在基因治疗应用中的适用性。重组 AAV 衣壳包括治疗蛋白编码转基因以及启动子以启动翻译和聚 A 序列部分以稳定。然而，当前的 AAV 制造技术不能保证仅产生完整的衣壳，即包括整个必需的基因组。部分填充和空衣壳也是产品的一部分，从而降低了临床转化时的功效和安全性。因此，在 AAV 载体制造和放行测试期间，快速、准确和符合 QC 要求的全空衣壳比分析非常重要。本文介绍了一种自动化毛细管等电聚焦技术，该技术在生物制药行业中易于应用，可快速高效地确定全空衣壳比。该方法还揭示了部分填充衣壳比例的信息。为了获得更高的分辨率（<0.1 pI 单位），使用了宽范围和窄范围两性电解质的混合物。混合两性电解质测定的等电点和峰面积百分比重现性（RSD）分别低至 1.67%和 2.45%，每次注射仅需 65 nL 样品体积。
