Understanding mixing, scaling and stress-related challenges in transient transfection processes

了解瞬时转染过程中的混合、缩放和与压力相关的挑战

• 批准号: 2881234
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2881234
• 关键词: Understanding; mixing; scaling; stress; related

Transient gene expression is traditionally used for rapid production of recombinant proteins to support preclinical studies during drug development as well as for virus production. Advances in this technology have led to an increase in titer and these, combined with a well defined robust process, provides an opportunity for transient gene expression to be an alternative tool for rapid production of clinical material. Studies have addressed the physicochemical characterisation of the system including type of transfection reagent, DNA to transfection reagent ratio and transfection conditions e.g. DNA to cell ratio, ratio of transfection solution (and type) to culture volume, flow conditions. Most of the work have been carried out at the small scale and recent results have indicated variability across scales in performance. Very limited publications have focused on the scale-up of a transient transfection process at industrially-relevant scale and there is a lack of understanding of the engineering factors/parameters which affects scalability. Engineering studies aimed at the evaluation of factors underpinning mixing process at the macro and microscale are essential to the mechanistic understanding of the process and will enable definition of optimal scaling strategies. Similarly, techniques able to measure energy dissipation at high resolution are needed to accurately determine the stress along the cell path which might affect the transient transfection process performance. This project builds on a successful UCL-Cytiva CoE project focusing on the macroscale characterisation of the Allegro STR Bioreactor. As part of the latter, two 1L scale prototypes of the Allegro for engineering and biological studies, respectively, are available at UCL. The Pall team will bring direct experience of internal and external transient transfections in both suspension and adherent format. The project objectives will be1. To conduct tailored biological and mixing studies at 1L scale2. To develop a novel technique which could be widely used for stress profiles determination3. To establish optimisation protocols for transient transfection processesProposed timelineYear 1 Biological and mixing studies in the 1L Allegro STR prototype to define design space and verify scalabilityYear 2 Establishment of HEK293 (suspension-adapted) culture, transfection and analyticsYear 3 Novel technique development for quantification of cumulative stress along cell pathlinesYear 4 Process optimisation and verification at scaleProposed timelineYear 1 Biological and mixing studies in the 1L Allegro STR prototype to define design space and verify scalabilityYear 2 Establishment of HEK293 (suspension-adapted) culture, transfection and analyticsYear 3 Novel technique development for quantification of cumulative stress along cell pathlinesYear 4 Process optimisation and verification at scale

瞬时基因表达传统上用于重组蛋白的快速生产，以支持药物开发期间的临床前研究以及用于病毒生产。该技术的进步已导致滴度增加，这些与明确定义的稳健工艺相结合，为瞬时基因表达提供了机会，成为快速生产临床材料的替代工具。研究已经解决了系统的物理化学表征，包括转染试剂的类型、DNA与转染试剂的比率和转染条件，例如DNA与细胞的比率、转染溶液（和类型）与培养体积的比率、流动条件。大多数工作都是在小规模上进行的，最近的结果表明，在不同规模的性能变化。非常有限的出版物集中在工业相关规模的瞬时转染过程的放大上，并且缺乏对影响可扩展性的工程因素/参数的理解。工程研究的目的是在宏观和微观尺度上的混合过程的基础因素的评价是必不可少的过程的机械理解，并将使最佳缩放策略的定义。类似地，需要能够以高分辨率测量能量耗散的技术来精确地确定可能影响瞬时转染过程性能的沿细胞路径沿着的应力。该项目建立在一个成功的UCL-Cytiva CoE项目的基础上，该项目专注于Allegro STR生物反应器的宏观表征。作为后者的一部分，两个1 L规模的Allegro原型分别用于工程和生物研究，可在UCL获得。Pall团队将带来悬浮和粘附形式的内部和外部瞬时转染的直接经验。该项目的目标将是1.以1 L规模进行定制的生物学和混合研究2。开发一种可广泛应用于应力分布测定的新技术3。建立瞬时转染工艺的优化方案拟定时间表第1年在1 L Allegro STR原型中进行生物学和混合研究，以确定设计空间并验证可扩展性第2年HEK 293的建立（悬浮适应）培养，转染和分析第3年开发新技术，用于量化沿着细胞路径的累积应力第4年工艺优化和大规模验证拟定时间线第1年生物学在1 L Allegro STR原型中进行混合研究，以确定设计空间并验证可扩展性第2年建立HEK 293（悬浮适应）培养、转染和分析第3年开发用于定量沿着细胞路径累积应力的新技术第4年工艺优化和大规模验证