Characterisation of Membrane Processes in the Manufacture of Vectors for Cell and Gene Therapies

在英国，先进治疗药物的临床试验数量正在增加，其中许多已经过了I/II期，因此需要可扩展和强大的制造工艺[1]。在许多这些疗法的递送中，一个关键组成部分是能够将治疗性遗传物质携带到宿主细胞的载体。最近，在抗击传染病中使用病媒使制造用于先进疗法的这类关键部件的能力受到关注。这些载体的制造大致可细分为上游加工（生成载体）和下游加工（收获、澄清和纯化步骤）。很大比例的损失发生在纯化和配方步骤[2]。膜处理通常用于细胞和基因治疗载体的制备和制造：从澄清过程中的终端过滤到超滤/滤除中载体浓缩/配制过程中的切向流过滤，最后经过无菌过滤产生最终载体。这些膜操作对载体功能损失的系统研究还有待研究。目前，还没有缩小比例的模型来研究这些损失并开发优化的膜处理系统。最近的一项研究开发了一个超缩小模型来研究抗体治疗药物[3]膜加工所需的最佳参数。将采用类似的超缩小方法来了解使用膜过滤器处理载体。这个博士项目的总体目标是研究膜材料类型、细胞培养基或缓冲液对这些膜操作的影响，并探索稳健有效处理载体的实验设计空间。将研究的载体的例子是慢病毒载体和细胞外囊泡，特别是外泌体。拟议的项目建立在伦敦大学学院开发的超缩小模型的基础上。该工具和其他工具将用于实现以下目标：-将超规模缩小方法应用于细胞和基因治疗的载体。-在超滤（浓缩）/滤除或无菌过滤过程中表征膜和载体的相互作用。-使用多元数据分析方法，探索制造高产量集中功能向量所需的设计空间。-利用物理和生物表征方法阐明缓冲液选择对过滤和载体稳定性的影响。EPSRC研究领域分类：过程系统：组件和集成，制造技术，医疗保健技术，生物材料和组织工程EPSRC研究主题：制造未来[1]细胞和基因治疗弹射器临床试验数据库2019。[在线]可在：https://ct.catapult.org.uk/sites/default/files/publication/Clinical%20Trials%20Commentary_for%20publication_150120.pdf[2] Valkama， A. J. et al.（2020）“慢病毒载体大规模下游加工的发展”，分子治疗-方法和临床发展。细胞出版社，17，第717-730页。doi: 10.1016 / j.omtm.2020.03.025。[3]Fernandez-Cerezo， L.等人（2019）“在使用超滤膜的切向流过滤过程中研究单克隆抗体处理的超缩小方法”，生物技术与生物工程，116(3)，第581-590页。doi: 10.1002 / bit.26859。

The number of clinical trials for Advanced Therapeutic Medicinal Products in the UK is increasing and with many of these advancing past Phase I/II, there is a need for scalable and robust manufacturing process [1]. A key component in the delivery of many of these therapies is the vector capable of carrying the therapeutic genetic material to the host's cells. More recently, the use of vectors in fighting infectious diseases has put a spotlight on the ability to manufacture such critical components for advanced therapies.The manufacturing of these vectors is broadly subdivided into upstream processing (generation of the vector,) and downstream processing (harvest, clarification and purification steps). A large proportion of loses occurs during the purification and formulation steps [2]. Membrane processing are commonly employed in the preparation and manufacture of vectors for cell and gene therapies: from dead-end filtration during clarification to tangential flow filtration during vector concentration/formulation in ultrafiltration/diafiltration and finally, sterile filtration to produce the final vector. The systematic study of these membrane operations on the functional loss of vectors is yet to be investigated. Currently, no scale-down model exists to investigate these loses and develop an optimised membrane processing system. A recent study has developed an ultra-scale down model to investigate the optimal parameters required for membrane processing for antibody therapeutics [3]. A similar ultra scale-down approach will be applied to gain understanding of the processing of vectors using membranes filters. The overall aim of this PhD project to investigate the effect of the types of membrane materials, cell culture media or buffers on these membrane operations and explore the experimental design space for the robust and effective processing of vectors. Examples of vectors that will be studied are lentiviral vectors and extracellular vesicles, specifically exosomes.The proposed project builds on the ultra-scale down model developed at UCL. This and other tools will be used to achieve the following objectives:- Applying ultra-scale down methodology to vectors for cell and gene therapy.- Characterising membrane and vector interaction during ultrafiltration (concentration)/diafiltration or sterile filtration.- Using a multivariate data analysis approach to explore the design space required to manufacture high yield concentrated functional vector.- Elucidate the effect of buffer choice on filtration and vector stability using physical and biological characterisation methods.EPSRC Research Areas Classification: Process Systems: Components and Integration, Manufacturing Technologies, Healthcare Technologies, Biomaterials and Tissue EngineeringEPSRC Research Theme: Manufacturing the Future [1] Cell and Gene Therapy Catapult Clinical Trials Database 2019. [online] Available at: https://ct.catapult.org.uk/sites/default/files/publication/Clinical%20Trials%20Commentary_for%20publication_150120.pdf[2] Valkama, A. J. et al. (2020) 'Development of Large-Scale Downstream Processing for Lentiviral Vectors', Molecular Therapy - Methods and Clinical Development. Cell Press, 17, pp. 717-730. doi: 10.1016/j.omtm.2020.03.025.[3] Fernandez-Cerezo, L. et al. (2019) 'An ultra scale-down method to investigate monoclonal antibody processing during tangential flow filtration using ultrafiltration membranes', Biotechnology and Bioengineering, 116(3), pp. 581-590. doi: 10.1002/bit.26859.