New Mass Spectrometry Methods to Characterise Virus Based Drug Products

AAV载体由于其缺乏致病性、相对低的免疫原性和在不同组织类型中的持续基因表达而出现在基因治疗的最前沿。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的电荷检测质谱仪- CDMS -，这将大大提高AAV治疗的生产和测试的分析能力。温和的电喷雾电离耦合质谱法，称为原生质谱法，提供了一种将大的多聚体蛋白质颗粒输送到气相中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是如此巨大，以至于它们无法使用常规MS方法进行解析。尽管在这些领域做出了非凡的努力和创新，但仍然存在与这种大型物体的传输和质量分析相关的挑战。主要问题是m/z谱中的峰由于质量不均匀性而变宽和移动，无论是固有的还是由于加合物形成，同时保留保持组装体完整的相互作用。CDMS是一种单粒子技术，其中同时测量单个离子的m/z和z，从而允许直接确定每个离子的质量。CDMS可以分析蛋白质复合物和其他大型组装体的异质混合物，这些混合物是传统MS方法难以处理的。CDMS非常简单，其工作原理是测量穿过腔室的粒子上的电荷，沿着粒子穿过腔室所需的时间。这个电荷将是某个整数z乘以基本电荷单位（e）。根据牛顿运动方程，粒子穿过管道所需的时间与粒子的m/z有关，这两个测量值可以结合起来提供粒子的质量M。迄今为止，世界上有三个学术团体开发了CDMS（两个在美国（马丁·贾罗德和埃文威廉姆斯），一个在法国（Philippe Dugourt），我们不知道迄今为止已经开发出任何商业CDMS仪器。最高分辨率的商用质谱仪无法在这种性质的AAV上的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，在研究这种大质量颗粒时具有非常低的通量。在本项目中，我们建议建立，安装并测试一种新的CDMS仪器，该仪器具有用于传输MDa离子的新型ESI源和与先前报道的任何仪器不同的几何结构，这将具有更好的电荷准确性，因此具有更高的通量。我们将使用它来确定重组腺相关病毒（AAV）载体的DNA含量。我们建议将该仪器的能力与市售质谱平台和其他方法进行比较。我们还将建立一个阶段，使随后的表征通过显微镜。我们将开发标准操作程序的仪器使用，大组装蛋白质标准（铁蛋白，Groel和IgE），并随后开发其用于AAV。研究其他大型组装体的潜力也将被探索，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有多少给定的AAV产品具有填充的衣壳与空衣壳，并且我们将比较数据与药物在动物模型中的功效。

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.