腺相关病毒(AAV)已成为体内治疗基因递送的主要平台,并在治疗和预防人类疾病方面具有巨大潜力。这类治疗方法的快速发展,加上它们内在的结构复杂性,对能够有效监测产品质量以确保安全性和有效性的分析方法提出了很高的要求,以及支持制造和工艺优化。重要的是,空的和部分填充的AAV衣壳亚群的存在和相对丰度是主要关注的问题,因为这些代表了AAV制造中最常见的产品相关杂质,并且对治疗潜力有直接影响。出于这个原因,衣壳内容,或者空衣壳和部分衣壳与包装有全长治疗基因组的衣壳的比例,已被监管机构确定为关键质量属性(CQA),必须仔细控制以满足临床规范。建立的用于定量衣壳含量比率的分析方法通常需要较长的周转时间,低吞吐量,和高样本需求,不适合狭窄的时间线和有限的样本可用性典型的过程开发。在这项研究中,我们提出了一个集成的在线天然质谱平台,旨在最大限度地减少样品处理,并最大限度地提高通量和鲁棒性,以快速和灵敏地定量AAV衣壳含量比。该平台用于AAV分析的主要优点包括能够在低流量条件下进行在线缓冲液交换,以最小的样品稀释保持样品稳定性,以及通过掺杂剂改性的去溶剂化气实现在线电荷减少的能力。通过利用后者,增强空信号的频谱分辨率,局部,和完整的AAV衣壳在m/z域中完成,以促进改进的光谱解释和定量,其与用于衣壳含量比测定的工业标准分析超速离心(AUC)方法良好相关。这种方法的实用性在几个应用中得到了进一步证明,包括快速和普遍筛选不同的AAV血清型,评估过程中样品的衣壳含量,以及在受到热应力条件下衣壳稳定性的监测。
Adeno-associated viruses (AAVs) have emerged as a leading platform for in vivo therapeutic gene delivery and offer tremendous potential in the treatment and prevention of human disease. The fast-paced development of this growing class of therapeutics, coupled with their intrinsic structural complexity, places a high demand on analytical methods capable of efficiently monitoring product quality to ensure safety and efficacy, as well as to support manufacturing and process optimization. Importantly, the presence and relative abundance of both empty and partially filled AAV
capsid subpopulations are of principal concern, as these represent the most common product-related impurities in AAV manufacturing and have a direct impact on therapeutic potential. For this reason, the
capsid content, or ratio of empty and partial capsids to those packaged with the full-length therapeutic genome, has been identified by regulatory agencies as a critical quality attribute (CQA) that must be carefully controlled to meet clinical specifications. Established analytical methods for the quantitation of capsid content ratios often suffer from long turnaround times, low throughput, and high sample demands that are not well-suited to the narrow timelines and limited sample availability typical of process development. In this study, we present an integrated online native mass spectrometry platform that aims to minimize sample handling and maximize throughput and robustness for rapid and sensitive quantitation of AAV
capsid content ratios. The primary advantages of this platform for AAV analysis include the ability to perform online buffer exchange under low flow conditions to maintain sample stability with minimal sample dilution, as well as the ability to achieve online charge reduction via dopant-modified desolvation gas. By exploiting the latter, enhanced spectral resolution of signals arising from empty, partial, and full AAV capsids was accomplished in the m/z domain to facilitate improved spectral interpretation and quantitation that correlated well with the industry standard analytical ultracentrifugation (AUC) method for capsid content ratio determination. The utility of this approach was further demonstrated in several applications, including the rapid and universal screening of different AAV serotypes, evaluation of
capsid content for in-process samples, and the monitoring of
capsid stability when subjected to thermal stress conditions.