aav及其抗体复合物电荷检测质谱分析的在线缓冲交换平台

IF 2.7 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS
Chen Du, , , Victoria C. Cotham*, , , Garima Thakur, , , Sheldon Mink, , , Andrew D. Tustian, , , Sven Møller-Tank, , , Shunhai Wang*, , and , Ning Li, 
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引用次数: 0

摘要

腺相关病毒(aav)是基因治疗的主要载体,在对抗多种人类疾病方面显示出巨大的潜力。为了增强特异性和减少剂量要求,抗体重靶向aav已成为一种有前途的策略,可将载体重定向到新的受体,从而提高疗效和安全性。然而,由于衣壳蛋白组装、基因组完整性和抗体修饰的变化所引起的异质性,aav和aav -抗体复合物的有效表征变得复杂,这需要超越传统方法的高分辨率技术。电荷检测质谱(CDMS)是一种通过直接测量离子电荷来有效表征复杂AAV样品的新兴技术。该技术通常使用基于静态注入的纳米电喷雾电离,采集时间长,这是高度手动的,并且受到不稳定的喷雾和样品不稳定性的影响。为了解决CDMS静态注射的局限性,我们开发了一种新的自动化平台:基于粒径排除色谱的流动注射-CDMS (SEC-FI-CDMS)。该平台通过双流量设置简化和自动化样品导入,实现高流量模式下的快速在线脱盐和低流量模式下稳定、长时间的输注,用于基于orbitrap的CDMS分析。它能准确测定AAV基因组包装比例和质量,区分衣壳组装和有效载荷变化,并解决AAV-抗体复合物上的抗体修饰问题。这种全面的表征支持AAV的制造和开发,为更有效的下一代基因治疗铺平了道路。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
An Online Buffer Exchange Platform for Charge Detection Mass Spectrometry Analysis of AAVs and AAV-Antibody Complexes

Adeno-associated viruses (AAVs) are leading vectors in gene therapy that have demonstrated great potential in combating a wide range of human diseases. To enhance specificity and reduce dosing requirements, antibody-retargeted AAVs have emerged as a promising strategy to redirect vectors to novel receptors, thereby achieving improved efficacy and safety. However, effective characterization of AAVs and AAV-antibody complexes is complicated by heterogeneities that arise from variations in capsid protein assembly, genome integrity, and antibody decorations, demanding high-resolution techniques beyond traditional methods. Charge detection mass spectrometry (CDMS) is an emerging technique that effectively characterizes complex AAV samples by directly measuring ion charge. This technique is typically performed using static infusion-based nanoelectrospray ionization with long acquisition times, which is highly manual and subject to unstable spray and sample instability. To address limitations of static infusion in CDMS, we have developed a novel automated platform: size-exclusion chromatography-based flow injection coupled with CDMS (SEC-FI-CDMS). This platform streamlines and automates sample introduction with a dual-flow setup, enabling fast online desalting in high-flow mode and stable, prolonged infusion in low-flow mode for Orbitrap-based CDMS analysis. It accurately determines AAV genome packaging ratios and masses, differentiates capsid assembly and payload variations, and resolves antibody decoration on AAV-antibody complexes. This comprehensive characterization supports AAV manufacturing and development, paving the way for more effective next generation gene therapies.

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来源期刊
CiteScore
5.50
自引率
9.40%
发文量
257
审稿时长
1 months
期刊介绍: The Journal of the American Society for Mass Spectrometry presents research papers covering all aspects of mass spectrometry, incorporating coverage of fields of scientific inquiry in which mass spectrometry can play a role. Comprehensive in scope, the journal publishes papers on both fundamentals and applications of mass spectrometry. Fundamental subjects include instrumentation principles, design, and demonstration, structures and chemical properties of gas-phase ions, studies of thermodynamic properties, ion spectroscopy, chemical kinetics, mechanisms of ionization, theories of ion fragmentation, cluster ions, and potential energy surfaces. In addition to full papers, the journal offers Communications, Application Notes, and Accounts and Perspectives
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