Native Mass Spectrometry approaches for Electron and Photon mediated Top Down Sequencing of Biotherapeutics

在这个项目中，我们建议开发电子和光子介导的自上而下测序方法来分析重组生物疗法，包括单抗(MAbs)和腺相关病毒(AAV)载体。该项目与目前资助的BBSRC与Allergan和Barran的案例具有协同效应，但与之截然不同，并代表着他们战略关系的成熟。单抗是目前发展最快的一类治疗药物，目前有8/10的新药是以单抗或单抗为基础的，这些具有结构异质性的复杂生物分子在发现、开发和生产过程中分析和确定给定单抗产品的一致性是一个巨大的分析挑战。AAV载体因其缺乏致病性、免疫原性相对较低以及在不同组织类型中持续表达基因而成为基因治疗的前沿。Allergan在2016年收购RetroSense时，对将AAV载体用于眼部药物输送特别感兴趣，RetroSense是一家生物技术公司，正在开发一种改变游戏规则的基因疗法(AAV)，以恢复因视网膜色素变性(RP)和晚期干性年龄相关性黄斑变性(Advanced Dry-AMD)而失明的患者的视力。目前还没有FDA批准的治疗方法来改善或恢复这些视网膜退行性疾病患者的视力。支持AAV生产的分析也非常有限，希望通过这个项目，快速的自上而下测序将显著提高Allergan在生物治疗药物开发和制造方面的能力。学生将学习如何准备由阿莱根提供的样品和从其他来源获得的标准，使它们适合于直接进样质谱分析。然后，他们将学习本地的质谱学技术，将病毒和单抗完好无损地送入质谱仪，这样就可以确定它们的质量和化学计量比。使用离子迁移质谱仪，他们将能够确定分子的形状和大小。学生还将接受数据分析和数据整理方面的培训。除了UOM的培训外，学生还将在Allergan学习生物疗法的生产，以及如何改进生产以最大限度地提高产品产量和实现批次到批次的一致性。在Allergan，他们还将学习更多经典的样品分析生物物理方法，包括自下而上的质谱学方法和光谱方法。在这个最初的培训阶段之后，学生将开始这个项目的主要目标，用自上而下的测序方法来充分描述完整的生物治疗中的序列和相互作用。我们将在Q_Exactive Orbitrap质谱仪上安装一个新的电子陷阱，以便进行电子俘获解离(ECD)。这是一种非遍历的解离方法，它主要裂解多肽骨架以产生报告第三和第四折叠的独特片段，然后这些片段可以映射回天然结构。这些ECD测量将得到使用我们定制的离子迁移率质谱仪上的光子解离共价键的测量的补充。学生将开发高级数据分析来比较两种方法的数据。巴伦是开发质谱仪的国际专家。她的团队在这一领域拥有必要的专业知识。它属于BBSRC的“新工作方式”、“工业生物技术”和“生物科学促进健康”主题。

In this project we propose to develop electron and photon mediated top-down sequencing methods to analyse recombinant biological therapeutics including monoclonal antibodies (mAbs) and adeno-associated viral (AAV) vectors. This project has synergies with but is distinctive to the currently funded BBSRC case with Allergan and Barran, and represents maturation of their strategic relationship. mAbs are the fastest growing class of therapeutic and currently 8/10 of new drugs are mAbs or mAb based, these complex biomolecules with structural heterogeneity are complex to analyse and determining the consistency of a given mAb product in its discovery, development and production is a substantial analytical challenge. 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. Allergan is particularly interested in using AAV vectors for Ocular drug delivery as it purchased Retrosense in 2016 that is a biotechnology company that is developing a game-changing gene therapy (AAV) to restore vision in patients suffering from blindness due to retinitis pigmentosa (RP) and advanced dry age-related macular degeneration (advanced dry-AMD). There are currently no FDA approved therapies to improve or restore vision in patients with these retinal degenerative conditions. The analytics to support AAV production are also very limited and the hope is with this project that rapid top down sequencing will significantly advance Allergan's capabilities in the development and manufacture of biological therapeutics. The student will learn how to prepare the samples supplied by Allegan and standards obtained from other sources such that they are suitable for analysis by direct infusion mass spectrometry. They will then learn native mass spectrometry techniques to get the viruses and mAbs into the mass spectrometer intact such that their mass and stoichiometry can be determined. Using Ion mobility mass spectrometry they will then be able to determine the shape and size of the molecules. The student will also be trained in data analysis and in data curation. As well as this training at UoM the student will spend time at Allergan, learning about the production of the biological therapeutics and how production is improved to maximise the product yield and batch to batch consistency is achieved. At Allergan they will also learn more classical biophysical methods of sample analysis, including bottom up mass spectrometry methods and spectroscopic methods. After this initial training period the student will embark on the main aim of this project to fully characterise the sequence and interactions in the intact biotherapeutic with top down sequencing methods. We will install a new electron trap onto our Q_Exactive Orbitrap mass spectrometer such that electron capture dissociation (ECD) can be performed. This is a non-ergodic dissociation method, which primarily cleaves the polypetide backbone to produce distinctive fragments that report on the tertiary and quaternary fold which can then be mapped back to the native structure. These ECD measurement will be complemented by measurements taken using photons to dissociate covalent bonds on our customised ion mobility mass spectrometer. The student will develop advanced data analysis to compare data from both methods.Barran is an international expert in the development of mass spectrometry instrumentation. Her team have requisite expertise in this area. It falls in the BBSRC themes in 'new ways of working', industrial biotechnology' and 'bioscience for health.'