He Xiaoping Z, Powers Thomas W, Huang Sisi, Liu Zhenjiu, Shi Heliang, Orlet John D, Mo Jim J, Srinivasan Saipraveen, Jacobs Steven, Zhang Kun, Runnels Herbert A, Anderson Melissa M, Lerch Thomas F
Pfizer Inc., Analytical Research and Development, Chesterfield, MO, USA.
Mol Ther Methods Clin Dev. 2023 Mar 10;29:133-144. doi: 10.1016/j.omtm.2023.03.002. eCollection 2023 Jun 8.
Adeno-associated virus (AAV) gene therapy vectors, which contain a DNA transgene packaged into a protein capsid, have shown tremendous therapeutic potential in recent years. Methods traditionally used in quality control labs, such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), do not provide a complete understanding of capsid viral protein (VP) charge heterogeneity. In the present study, we developed simple, one-step sample preparation and charge-based VP separation using imaged capillary isoelectric focusing (icIEF) for monitoring AAV products. The robustness of the method was confirmed through a design of experiments (DoE) exercise. An orthogonal reverse-phase (RP) HPLC method coupled with mass spectrometry was developed to separate and identify charge species. Additionally, capsid point mutants demonstrate the capability of the method to resolve deamidation at a single site on the viral proteins. Finally, case studies using two different AAV serotype vectors establish the icIEF method as stability indicating and demonstrate that increases in acidic species measured by icIEF correlate with increased deamidation, which, we show, results in decreased transduction efficiency. The addition of a rapid and robust icIEF method to the AAV capsid analytical toolkit enables development and consistent manufacturing of well-characterized gene therapy products.
腺相关病毒（AAV）基因治疗载体将包装在蛋白质衣壳中的DNA转基因包含在内，近年来已显示出巨大的治疗潜力。传统上质量控制实验室使用的方法，如高效液相色谱（HPLC）和毛细管电泳（CE），无法全面了解衣壳病毒蛋白（VP）的电荷异质性。在本研究中，我们开发了一种简单的一步样品制备方法，并使用成像毛细管等电聚焦（icIEF）进行基于电荷的VP分离，以监测AAV产品。通过实验设计（DoE）验证了该方法的稳健性。开发了一种与质谱联用的正交反相（RP）HPLC方法，用于分离和鉴定电荷种类。此外，衣壳点突变体证明了该方法解析病毒蛋白单个位点脱酰胺化的能力。最后，使用两种不同AAV血清型载体的案例研究将icIEF方法确立为稳定性指示方法，并证明icIEF测量的酸性物种增加与脱酰胺化增加相关，我们发现这会导致转导效率降低。在AAV衣壳分析工具包中添加快速且稳健的icIEF方法，能够开发并一致地生产特征明确的基因治疗产品。
