Wu Di, Zhao Xiaonan, Jimenez Diego Antonio, Piszczek Grzegorz
Biophysics Core Facility, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Laboratory of Cell and Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Cells. 2023 Sep 13;12(18):2264. doi: 10.3390/cells12182264.
Over the past decade, adeno-associated viruses (AAVs) have attained significant prominence in gene therapy and genome editing applications, necessitating the development of robust and precise methodologies to ensure the quality and purity of AAV products. Existing AAV characterization techniques have proven effective for the analysis of pure and homogeneous AAV samples. However, there is still a demand for a rapid and low-sample-consumption method suitable for the characterization of lower purity or heterogeneous AAV samples commonly encountered in AAV products. Addressing this challenge, we propose the SEC-MP method, which combines size exclusion chromatography (SEC) with mass photometry (MP). In this novel approach, SEC effectively separates monomeric AAV particles from impurities, while the UV detector determines the virus particle concentration. MP complements this process by estimating the fraction of fully packaged AAVs in the total population of AAV particles. This combined methodology enables accurate determination of the titer of effective, fully packaged AAVs in samples containing aggregates, incorrectly packaged AAVs with incomplete genomes, protein or DNA fragments, and other impurities. Our experimental results demonstrate that SEC-MP provides valuable guidance for sample quality control and subsequent applications in the field of AAV research.
在过去十年中，腺相关病毒（AAV）在基因治疗和基因组编辑应用中已占据显著地位，因此需要开发强大而精确的方法来确保AAV产品的质量和纯度。现有的AAV表征技术已被证明对分析纯的和均一的AAV样品有效。然而，对于适用于表征AAV产品中常见的较低纯度或异质AAV样品的快速且低样品消耗的方法仍有需求。为应对这一挑战，我们提出了SEC-MP方法，该方法将尺寸排阻色谱（SEC）与质量光度法（MP）相结合。在这种新方法中，SEC有效地将单体AAV颗粒与杂质分离，而紫外检测器可测定病毒颗粒浓度。MP通过估计完全包装的AAV在AAV颗粒总数中的比例来补充这一过程。这种组合方法能够准确测定含有聚集体、基因组不完整的错误包装AAV、蛋白质或DNA片段以及其他杂质的样品中有效、完全包装的AAV的滴度。我们的实验结果表明，SEC-MP为AAV研究领域的样品质量控制及后续应用提供了有价值的指导。
