New Mass Spectrometry Methods to Characterise Virus Based Drug Products

AAV载体由于其致病性低、免疫原性相对较低以及基因在不同组织类型中的持续表达，已成为基因治疗的前沿。支持AAV生产的分析方法非常有限，为了应对这一挑战，该项目将构建一种新的电荷检测质谱仪CDMS，这将大大提高AAV治疗方法的制造和测试的分析能力。温和的电喷雾电离耦合到质谱，即所谓的天然质谱，提供了一种简便的方法，将大的多聚体蛋白质颗粒输送到气相进行分析。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合的研究中，但仍然有一些组合太大，无法使用传统的质谱方法进行解析。已经有相当多的学术和商业努力开发天然质谱法来研究蛋白质复合物和其他组件，包括病毒，质量在mDa范围内。尽管在这些领域做出了非凡的努力和创新，但在传输和大量分析这些大型物体方面仍然存在挑战。主要的问题是，由于质量的非均质性，m/z谱的峰变宽和移位，要么是由于内在的，要么是由于加合物的形成，同时保留了保持组装完整的相互作用。电荷检测质谱（CDMS）绕过了解析电荷状态的需要。CDMS是一种单粒子技术，其中同时测量单个离子的m/z和z，从而可以直接确定每个离子的质量。CDMS可以分析蛋白质复合物的异质混合物和其他难以用传统的质谱方法处理的大型组件。CDMS非常简单，它的工作原理是测量穿过一个腔室的粒子上的电荷，以及粒子穿过腔室所需的时间。这个电荷是一个整数，z，乘以电荷的基本单位e。通过牛顿运动方程，粒子穿过管道的时间与粒子的m/z有关，这两个测量可以结合起来，得到粒子的质量m。迄今为止，世界上有三个学术团体开发了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经开发了任何商业CDMS仪器。最高分辨率的商用质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，在研究这种大质量粒子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，该仪器带有一种新的ESI源，用于传输MDa离子，其几何形状与之前报道的任何仪器都不同，这将具有更好的电荷精度，从而提高通量。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能力与市售的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。我们将为使用大型组装蛋白标准（铁蛋白，Groel和IgEs）的仪器制定标准操作程序，并随后开发其在aav中的应用。研究其他大型组装体的潜力也将被探索，特别是重度糖基化的蛋白质组装体。我们将使用该仪器测量给定的AAV产品中有多少填充衣壳与空衣壳，并将数据与动物模型中的药物功效进行比较。

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.