McElroy Will, Huang Sisi, He Xiaoping, Zhou Cheng, Heger Christopher D, Powers Thomas W, Anderson Melissa M, Sloan Courtney, Lerch Thomas F
ProteinSimple, A Bio-Techne Brand, San Jose, California, USA.
Pfizer, Analytical Research and Development, Chesterfield, Missouri, USA.
Electrophoresis. 2025 Jan;46(1-2):22-33. doi: 10.1002/elps.202400201. Epub 2025 Jan 20.
A significant limitation of imaged capillary electric focusing (icIEF) is the inability to identify and characterize specific species in the electropherogram. This has led to the development of complementary ion-exchange chromatography (IEX)-based methods that are amenable to either fraction collection and subsequent characterization or online IEX coupled to mass spectrometry. To overcome this limitation while maintaining the use of icIEF, novel approaches, including an icIEF separation and fractionation technology (MauriceFlex, ProteinSimple), have been developed. This approach enables the fractionation of various icIEF peaks, which can then be characterized by mass spectrometry to confirm the identity of the separated charged species. Herein, the MauriceFlex technology was applied to adeno-associated viral (AAV) gene therapy products, which contain a DNA transgene packaged into a protein capsid and have shown tremendous therapeutic potential in recent years. Utilizing the MauriceFlex system, we developed an approach for the separation of charged species from AAV capsid viral proteins (VP) by icIEF and subsequent characterization by liquid chromatography and mass spectrometry (LC-MS). When applying the same sample preparation, charge profiles of AAV capsid proteins on the MauriceFlex instrument were demonstrated to be consistent with those from the original Maurice platform, the industrial gold standard. Optimization of the VP icIEF fractionation method required the development of a method for low concentration samples, optimization of mobilization conditions, enhancement of fraction recovery, and maintenance of protein stability post fractionation. Herein, we were able to successfully collect charge-separated VP fraction samples and subsequently analyze them by MS analysis. In addition, a workflow for AAV capsid protein characterization based on icIEF separation and fractionation coupled with downstream LC-MS has been established for the confirmation of VP identity and additional characterization of capsid protein heterogeneity.
成像毛细管电泳聚焦（icIEF）的一个显著局限性在于无法在电泳图中识别和表征特定物种。这促使了基于互补离子交换色谱（IEX）的方法的发展，这些方法适用于馏分收集及后续表征，或者与质谱联用的在线IEX。为了在保持使用icIEF的同时克服这一局限性，已经开发了包括icIEF分离和分馏技术（MauriceFlex，ProteinSimple）在内的新方法。这种方法能够对各种icIEF峰进行分馏，然后通过质谱对其进行表征，以确认分离出的带电物种的身份。在此，MauriceFlex技术被应用于腺相关病毒（AAV）基因治疗产品，该产品含有包装在蛋白质衣壳中的DNA转基因，近年来已显示出巨大的治疗潜力。利用MauriceFlex系统，我们开发了一种通过icIEF从AAV衣壳病毒蛋白（VP）中分离带电物种，并随后通过液相色谱和质谱（LC-MS）进行表征的方法。当采用相同的样品制备方法时，在MauriceFlex仪器上AAV衣壳蛋白的电荷分布被证明与原始Maurice平台（行业黄金标准）的电荷分布一致。VP icIEF分馏方法的优化需要开发一种针对低浓度样品的方法、优化迁移条件、提高馏分回收率以及在分馏后保持蛋白质稳定性。在此，我们能够成功收集电荷分离的VP馏分样品，并随后通过质谱分析对其进行分析。此外，基于icIEF分离和分馏并结合下游LC-MS的AAV衣壳蛋白表征工作流程已经建立，用于确认VP身份以及对衣壳蛋白异质性进行额外表征。
