New Mass Spectrometry Methods to Characterise Virus Based Drug Products

表征病毒药物产品的新质谱方法

• 批准号: BB/X002403/1
• 负责人: Perdita Barran
• 金额: $ 192.62万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX002403%2F1
• 关键词: New; Mass; Spectrometry; Methods; Characterise

AAV vectors have emerged at the forefront of gene therapy due to their lack of pathogenicity, relatively low immunogenicity and persistent gene expression in different tissue types. The analytics to support AAV production are very limited and to answer this challenge, this project will construct a new charge detection mass spectrometer - CDMS - that will significantly advance analytical capabilities for the manufacture and testing of AAV therapies.Gentle electrospray ionisation coupled to mass spectrometry so called native mass spectrometry, provides a facile way of delivering large multimeric protein particles to the gas phase for analysis. Whilst ESI based mass spectrometry has proved hugely beneficial for biological and biomedical science, in particular in the study of large intact protein assemblies, still some assemblies are so massive that they cannot be resolved using conventional MS methods.There has been considerable academic and commercial effort to development native mass spectrometry to investigate protein complexes and other assemblies including viruses, with masses into the mDa range. Despite extraordinary efforts and innovation in these areas, there remain challenges associated with the transmission and mass analysis of such large objects. The main issue is that the peaks in the m/z spectrum broaden and shift due to mass heterogeneity, either intrinsic or due to adduct formation whilst retaining the interactions that keep the assemblies intact.Charge detection mass spectrometry (CDMS) bypasses the need to resolve charge states. CDMS is a single particle technique, where the m/z and z of individual ions are measured concurrently, thereby allowing direct determination of the mass of each ion. CDMS can analyse heterogeneous mixtures of protein complexes and other large assemblies that are intractable by conventional MS methods. CDMS is highly simple and works by measuring the charge on a particle that passes through a chamber, along with the time it takes the particle to traverse the chamber. This charge will be some integer, z, multiplied by the fundamental unit of charge (e). The time it takes the particle to traverse the tube will be related to the m/z of the particle by Newton's equation of motion and these two measurements can be combined to provide M, the mass of the particle.To date three academic groups in the world have developed CDMS (two in the USA (Martin Jarrold and Evan Williams) and one in France (Philippe Dugourd), we are not aware of any commercial CDMS instrument that has been developed to date. The highest resolution commercial mass spectrometer is unable to provide sufficient mass resolution in measurements on AAVs of this nature and also has very low throughput in the study of such massive particles due to the method of mass analysis and difficulties in transmitting these ions through conventional ion optics.In this project we propose to build, install and test a new CDMS instrument with a novel ESI source for transmission of MDa ions and a different geometry to any previously reported, which will have better charge accuracy and hence higher throughput. We will use it to determine the DNA content of recombinant adenoassociated viral (AAV) vectors. We propose to compare the capabilities of this instrument with that of commercially available mass spectrometry platforms and other methods. We will also build a stage to allow subsequent characterisation via microscopy.We will develop standard operating procedures for the instrument using, large assembled protein standards (ferritin, Groel and IgEs) and subsequently develop its use for AAVs. The potential to study other large assemblies will also be explored especially heavily glycosylated protein assemblies. We will use the instrument to measure how many of a given AAV product have filled capsids versus empty and we will compare the data to the efficacy of the drug in animal models.

AAV载体因其缺乏致病性、相对较低的免疫原性以及在不同组织类型中持续的基因表达而成为基因治疗的前沿。支持 AAV 生产的分析非常有限，为了应对这一挑战，该项目将构建一种新型电荷检测质谱仪 - CDMS - 它将显着提高 AAV 疗法的制造和测试的分析能力。温和的电喷雾电离与质谱法（称为天然质谱法）相结合，提供了一种将大的多聚蛋白颗粒输送到气相进行分析的简便方法。虽然基于 ESI 的质谱法已被证明对生物和生物医学科学非常有益，特别是在大型完整蛋白质组装体的研究中，但仍然有一些组装体太大，无法使用传统 MS 方法进行解析。学术界和商业界已经做出了大量努力来开发天然质谱法，以研究蛋白质复合物和其他组装体（包括病毒），其质量达到 mDa 范围。尽管在这些领域付出了巨大的努力和创新，但此类大型物体的传输和质量分析仍然存在挑战。主要问题是，由于质量异质性（本质上的或由于加合物的形成），m/z 谱中的峰会变宽和移动，同时保留保持组件完整的相互作用。电荷检测质谱 (CDMS) 无需解析电荷态。 CDMS 是一种单粒子技术，同时测量单个离子的 m/z 和 z，从而可以直接测定每个离子的质量。 CDMS 可以分析传统 MS 方法难以处理的蛋白质复合物和其他大型组装体的异质混合物。 CDMS 非常简单，其工作原理是测量穿过腔室的粒子上的电荷以及粒子穿过腔室所需的时间。该电荷将是某个整数 z 乘以电荷的基本单位 (e)。粒子穿过管子所需的时间将通过牛顿运动方程与粒子的 m/z 相关联，这两个测量结果可以结合起来提供粒子的质量 M。 迄今为止，世界上三个学术团体已开发出 CDMS（两个在美国（Martin Jarrold 和 Evan Williams），一个在法国（Philippe Dugourd），我们不知道迄今为止已开发出任何商业 CDMS 仪器。最高分辨率的商业质量 光谱仪无法在这种性质的 AAV 测量中提供足够的质量分辨率，并且由于质量分析方法和通过传统离子光学传输这些离子的困难，在研究此类大颗粒时也具有非常低的通量。在这个项目中，我们建议建造、安装和测试一种新的 CDMS 仪器，该仪器具有用于传输 MDa 离子的新型 ESI 源和与之前报道的任何几何形状不同的几何形状，这将具有更好的电荷精度，因此更高 吞吐量。我们将用它来测定重组腺相关病毒 (AAV) 载体的 DNA 含量。我们建议将该仪器的功能与市售质谱平台和其他方法进行比较。我们还将建立一个平台，以便通过显微镜进行后续表征。我们将使用大型组装蛋白质标准品（铁蛋白、Groel 和 IgE）为仪器开发标准操作程序 并随后开发其在 AAV 中的用途。还将探索研究其他大型组装体的潜力，特别是高度糖基化的蛋白质组装体。我们将使用该仪器来测量给定 AAV 产品中填充衣壳的数量与空衣壳的数量，并将数据与药物在动物模型中的功效进行比较。