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

在英国，高级治疗药品的临床试验数量正在增加，其中许多试验已经过了I/II期，因此需要可扩展且稳健的生产工艺[1]。许多这些疗法的递送中的关键组分是能够将治疗性遗传物质携带到宿主细胞的载体。近年来，利用载体防治传染病使人们关注生产先进治疗所需关键成分的能力，这些载体的生产大致分为上游加工（载体的产生）和下游加工（收获、澄清和纯化步骤）。大部分损失发生在纯化和配制步骤中[2]。膜处理通常用于制备和生产用于细胞和基因治疗的载体：从澄清期间的死端过滤到超滤/渗滤中的载体浓缩/配制期间的切向流过滤，最后，无菌过滤以产生最终载体。这些膜操作对载体功能丧失的系统研究尚待研究。目前，还没有缩小规模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种超缩小模型，以研究抗体治疗剂膜加工所需的最佳参数[3]。一个类似的超规模缩小的方法将被应用于获得使用膜过滤器的矢量处理的理解。该博士项目的总体目标是研究膜材料，细胞培养基或缓冲液类型对这些膜操作的影响，并探索载体稳健有效处理的实验设计空间。将被研究的载体的例子是慢病毒载体和细胞外囊泡，特别是exosomes.The拟议的项目建立在UCL开发的超缩小模型。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载体。表征超滤（浓缩）/渗滤或无菌过滤过程中的膜和载体相互作用。使用多元数据分析方法探索制造高产量浓缩功能向量所需的设计空间。使用物理和生物学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。[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）“慢病毒载体大规模下游加工的开发”，分子治疗-方法和临床开发。Cell Press，17，pp. 717-730. doi：10.1016/j.omtm.2020.03.025。[3]费尔南德斯-塞雷索湖等人（2019）“使用超滤膜在切向流过滤过程中研究单克隆抗体处理的超缩小方法”，生物技术和生物工程，116（3），pp. 581-590. 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.