School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva, Switzerland.
Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva, Switzerland.
Int J Mol Sci. 2022 Oct 15;23(20):12332. doi: 10.3390/ijms232012332.
Gene therapy is opening unprecedented opportunities for novel therapeutic approaches. Based on the concept of rescuing function mutations by co-expressing the correct gene to allow biological functions to be restored, it requires the use of viral vectors to ensure the proper delivery of therapeutic genes. In this context, recombinant adeno-associated viruses (rAAV) are the most widely used vectors. Their biomanufacturing process requires the insertion of the therapeutic gene into the rAAV (full capsids). However, a percentage of rAAV that do not contain the desired gene (empty capsids), as well as partly filled capsids, might also be produced, potentially impacting the efficiency of the therapy. Therefore, the determination of the rAAV capsids' full/empty ratio needs to be monitored to ensure consistent product quality and efficacy. Anion-exchange chromatography (AEX) can serve this need. In this contribution, thorough AEX method development, including a mobile phase, a stationary phase and gradient conditions, has highlighted its potential in supporting gene therapy. Taking advantage of the fact that viral capsids follow an "on/off" retention behavior, the application of a step gradient approach to the rAAV serotype 8 (rAAV8) allowed the unprecedented separation of rAAV8 full/empty capsids, with a resolution gain of 3.7 as compared to the resolution obtained with a fully optimized linear gradient. Finally, the developed analytical approach allowed a precise and accurate baseline separation and quantification of full and empty rAAV8 capsids, with the potential to be applied as a high-throughput quality control (QC) method.
基因治疗为新型治疗方法开辟了前所未有的机会。基于通过共表达正确基因来挽救功能突变以恢复生物功能的概念，它需要使用病毒载体来确保治疗基因的正确递送至。在这种情况下，重组腺相关病毒（rAAV）是最广泛使用的载体。它们的生物制造过程需要将治疗基因插入 rAAV（完整衣壳）中。然而，可能也会产生不包含所需基因的 rAAV（空衣壳）和部分填充的衣壳，这可能会影响治疗的效率。因此，需要监测 rAAV 衣壳的满/空比，以确保一致的产品质量和疗效。阴离子交换色谱（AEX）可以满足这一需求。在本研究中，深入的 AEX 方法开发，包括流动相、固定相和梯度条件，突出了其在支持基因治疗方面的潜力。利用病毒衣壳遵循“开/关”保留行为的事实，应用阶跃梯度方法对 rAAV 血清型 8（rAAV8）进行处理，能够前所未有地分离 rAAV8 满/空衣壳，与使用完全优化的线性梯度获得的分辨率相比，分辨率提高了 3.7。最后，所开发的分析方法能够精确、准确地基线分离和定量满和空 rAAV8 衣壳，有望作为高通量质量控制（QC）方法应用。
