Understanding the impact of the engineering environment on transient transfection and process scale-up

瞬时基因表达（TGE）传统上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。该技术涉及将质粒DNA引入哺乳动物细胞中，然后在有限的时间内（通常为两周）表达目的蛋白。近年来，该技术取得了一些进展，使滴度从mg/L增加到g/L。这些进展，加上一个强大的，可扩展的和明确的过程，提供了一个机会，探索TGE作为一种替代工具，用于快速生产临床材料（例如，在流行病的情况下或在个性化药物的背景下）。本研究的目的是阐明转染剂添加（体积、浓度等）、搅拌模式、生物反应器几何形状和操作条件对瞬时转染过程中细胞生长、DNA摄取和生产率的影响，并将研究结果应用于定义最佳缩放方法，该方法有效性高达200 L规模。该提案是使用最准确地反映几何形状和流动条件的缩小规模的生物反应器系统来研究该过程。这建立在UCL已经提供的缩小规模的模型和流体动力学表征方法的基础上（见下文），以实现快速进展和产生结果。博士生将为该项目做出贡献，并将专注于以下目标：通过定制系统中的高级工程表征提高对过程的理解。混合动力学将使用基于当前工艺的模型系统在摇动和搅拌生物反应器配置中评估不同操作条件下的混合动力学，并且将使用工业系统的缩小模拟物评估流体流变学和进料位置的影响。这也将使转染条件在不同生物反应器几何形状之间的可靠规模转换成为可能，即从波动到搅拌配置。将建立一个瞬时转染过程模型与相关的分析和实验将使用搅拌策略，通风和进料位置运行的表征研究所告知。该项目将是卓越的UCL-AstraZeneca中心（CoE），是伦敦（UCL）和阿斯利康之间的联合合作的一部分。该中心的目标是为上游和下游生物制药加工活动生成一套预测决策工具。这涉及微尺度和高通量研究的应用，包括相关性开发，多变量数据分析（MVDA），过程经济学和离散事件优化。候选人将加入一个由博士和博士后研究人员组成的团队，在整个上游和下游处理以及建模工具方面在CoE工作。该项目将由伦敦大学学院生物化学工程系主办，并与阿斯利康在剑桥（英国）和盖瑟斯堡（美国）的工厂合作。

Transient gene expression (TGE) is traditionally used for rapid production of recombinant proteins to support preclinical studies during drug development. This technology involves introducing plasmid DNA into mammalian cells which then express the protein of interest over a limited period of time, typically two weeks. In recent years, there have been some advances in this technology which have led to an increase in titres from mg/L to g/L. These advances, combined with a robust, scalable and well defined process, provide an opportunity to explore TGE as an alternative tool for rapid production of clinical material (e.g. in a pandemic situation or in the context of personalised medicines). The purpose of this research is to elucidate the effect of transfection agent addition (volume, concentration etc), agitation mode, bioreactor geometry and operating conditions on cell growth, DNA uptake, and productivity in a transient transfection process and apply the findings to define an optimal scaling methodology valid up to 200L scale. The proposal is to study the process using scale-down bioreactor systems that most accurately reflect the geometry and flow conditions This builds upon scale-down models and fluid dynamics characterisation methods already available at UCL (see below) to enable rapid progress and generation of results. The doctoral candidate will contribute to the project and will focus on the following objectives:Improve process understanding through advanced engineering characterization in customised systems. Mixing kinetics will be evaluated for different operating conditions using a model system based on current processes, in both rocked and stirred bioreactor configurations, and the impact of fluid rheology and feed location will be evaluated using scale-down mimics of the industrial systems.Establish optimum transfection conditions in specified bioreactor geometries. This will also enable reliable scale translation of transfection conditions between different bioreactor geometries i.e. wave to stirred configurations.Conduct biological verification experiments at scale. A transient transfection process model will be established with associated analytics and experiments will be run using agitation strategy, aeration and feed location as informed by the characterization studies.This project will be part of the UCL-AstraZeneca Centre of Excellence (CoE) that is a joint collaboration between University College London (UCL) and AstraZeneca. The centre's aim is to generate a set of predictive decisional tools for both upstream and downstream biopharmaceutical processing activities. This involves the application of micro-scale and high-throughput studies involving correlation development, multivariate data analysis (MVDA), process economics and discrete-event optimisation. The candidate will join a team of PhD and postdoctoral researchers working in the CoE across upstream and downstream processing as well as modelling tools. The project will be hosted by the department of Biochemical Engineering at University College London in collaboration with AstraZeneca sites across Cambridge (UK) and Gaithersburg (US).