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.

由于缺乏致病性，相对较低的免疫原性和持续的基因表达，AAV载体已经在基因治疗的最前沿出现。支持AAV生产的分析技术非常有限，要应对这一挑战，该项目将构建一个新的电荷检测质谱仪-CDMS-将显着提高AAV疗法的制造和测试的分析能力。Gentle电喷雾电离电离偶联，耦合到所谓的天然质谱范围的质谱量，可为大型分析量构成大型蛋白质。虽然事实证明，基于ESI的质谱法对生物学和生物医学科学非常有益，特别是在大型完整蛋白质组件的研究中，但仍然有些组件是如此巨大，以至于无法使用常规MS方法来解决它们。在包括蛋白质组合和其他Masses和其他Masses and Moses and Moses和其他群落中，使用常规的MS方法进行了相当大的学术和商业努力。尽管在这些领域做出了巨大的努力和创新，但仍然存在与此类大物体的传输和质量分析有关的挑战。主要问题是，M/z光谱中的峰宽大并因质量异质性而引起的，无论是内在的还是加合物的形成，同时保留了保持组件完整的相互作用。充电检测质谱法（CDMS）绕过需要解决电荷状态的需求。 CDMS是一种单个粒子技术，其中同时测量单个离子的M/z和z，从而直接确定每个离子的质量。 CDM可以分析蛋白质复合物和其他大型组合的异质混合物，这些混合物通过常规MS方法棘手。 CDM非常简单，可以通过测量通过腔室的粒子上的电荷以及粒子穿越腔室的时间来起作用。该费用将是一些整数，z，乘以基本电荷单位（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吞吐量。我们将使用它来确定重组腺体相关病毒（AAV）向量的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。我们还将建立一个阶段，允许通过显微镜进行后续表征。我们将使用大型组装蛋白标准品（铁蛋白，凹槽和IGES）为仪器制定标准操作程序，然后随后开发其用于AAV的使用。研究其他大型组件的潜力也将被探索，特别是重度糖基化的蛋白质组件。我们将使用该仪器来测量多少个给定的AAV产品填充了衣壳与空的胶囊，我们将将数据与动物模型中药物的功效进行比较。