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

传统上，瞬时基因表达用于快速生产重组蛋白，以支持药物开发期间的临床前研究以及病毒生产。这项技术的进步导致了滴度的增加，再加上定义良好的稳健过程，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。研究了该体系的物理化学特性，包括转染剂的类型、DNA与转染剂的比例以及DNA与细胞的比例、转染液(和类型)与培养体积的比例、流动条件等。大多数工作都是在小规模上进行的，最近的结果表明，业绩在不同规模之间存在差异。只有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，并且缺乏对影响可伸缩性的工程因素/参数的了解。工程研究的目的是在宏观和微观尺度上评估支撑混合过程的因素，这对于从机理上理解混合过程是至关重要的，并将使确定最佳比例战略成为可能。同样，需要能够在高分辨率下测量能量耗散的技术，以准确地确定可能影响瞬时转染过程性能的沿细胞路径的应力。该项目建立在成功的UCL-Cytiva COE项目的基础上，该项目侧重于Allegro STR生物反应器的宏观特征。作为后者的一部分，伦敦大学学院提供了两个1L规模的快板原型，分别用于工程和生物学研究。PAL团队将以悬浮和贴壁两种形式带来内部和外部瞬时转基因的直接经验。项目目标将达到1。在1L Scale 2上进行量身定制的生物学和混合研究。开发一种可广泛应用于应力剖面测定的新技术。为瞬时转导过程建立优化方案建议的时间表1年1在1L Allegro STR原型中进行生物和混合研究，以定义设计空间并验证可伸缩性第2年建立HEK293(悬浮适应)培养、转基因和分析第3年用于量化细胞路径上的累积应力的新技术开发第4年的过程优化和规模验证建议的时间表1L Allegro STR原型中的第1年生物和混合研究以确定设计空间和验证可伸缩性第2年建立HEK293(悬浮适应)培养、转基因和分析第3年用于量化细胞路径累积应力的新技术开发Year 4过程优化和验证规模

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