Chemistry Department, Indiana University, 800 E Kirkwood Avenue, Bloomington, Indiana 47405, United States.
Analytical Research and Development, Pfizer Inc., 875 Chesterfield Pkwy. West, Chesterfield, Missouri 63017, United States.
Anal Chem. 2023 Mar 7;95(9):4310-4316. doi: 10.1021/acs.analchem.2c04234. Epub 2023 Feb 22.
Adeno-associated virus (AAV) is a widely used gene therapy vector. The intact packaged genome is a critical quality attribute and necessary for an effective therapeutic. In this work, charge detection mass spectrometry (CDMS) was used to measure the molecular weight (MW) distribution for the genome of interest (GOI) extracted from recombinant AAV (rAAV) vectors. The measured MWs were compared to sequence masses for a range of rAAV vectors with different GOIs, serotypes, and production methods (Sf9 and HEK293 cell lines). In most cases, the measured MWs were slightly larger than the sequence masses, a result attributed to counterions. However, in a few cases, the measured MWs were significantly smaller than the sequence masses. In these cases, genome truncation is the only reasonable explanation for the discrepancy. These results suggest that direct analysis of the extracted GOI by CDMS provides a rapid and powerful tool to evaluate genome integrity in gene therapy products.
腺相关病毒（AAV）是一种广泛使用的基因治疗载体。完整的包装基因组是一个关键的质量属性，也是有效治疗所必需的。在这项工作中，采用电荷检测质谱（CDMS）测量了从重组腺相关病毒（rAAV）载体中提取的目的基因组（GOI）的分子量（MW）分布。将测量的 MW 与具有不同 GOI、血清型和生产方法（Sf9 和 HEK293 细胞系）的一系列 rAAV 载体的序列质量进行了比较。在大多数情况下，测量的 MW 略大于序列质量，这归因于反离子。然而，在少数情况下，测量的 MW 明显小于序列质量。在这些情况下，基因组截断是差异的唯一合理解释。这些结果表明，通过 CDMS 对提取的 GOI 进行直接分析为评估基因治疗产品中的基因组完整性提供了一种快速而强大的工具。
