Design and characterisation of a scale-down perfusion platform for mammalian cell process development

该项目旨在通过解决两个相关挑战来优化灌注过程，即：-新型细胞保留方法（AWS）的缩小和工艺验证-了解稳态并最大化工艺性能和生产率目标-设计和构建一个小规模的AWS系统版本，能够与250mL灌注生物反应器一起工作并完全连接。-了解AWS灌注过程的“操作窗口”的限制和特征。对其他细胞保留技术进行基准测试。了解细胞的稳定状态：细胞大小分布，细胞周期分布，初级代谢，特定生产力。基于模型的过程优化（灌注介质，灌注率，稳态目标VCD）。Pall开发的声波分离器（AWS）技术具有许多有前景的优点，例如：(a)不易结垢，(b)可调，允许细胞分离和条件出血，(c)对细胞造成的压力较小。然而，在小批量自动化生物反应器中，不存在适合高通量工艺开发的缩小版本。我们的目标是与Pall合作开发一种缩小版的AWS，作为小体积生物反应器中的细胞保留装置，并了解使用AWS技术的连续过程的“操作窗口”的限制和特征。实验研究将基于伦敦大学学院最近开发的完全可控的按比例缩小的灌注生物反应器。该新型250ml生物反应器专为哺乳动物细胞灌注培养而设计，配有Levitronix泵和TFF过滤器，用于细胞保留。1VVD的灌注运行已成功运行，并且在10天内具有良好的再现性。颇尔在AWS及其运营方面的专业知识将是选择合适的扩展标准的关键。稳定状态的数学分析和表征将基于UCL最近开发的一系列代谢建模和系统生物学技术。将在稳态下进行数学和实验研究的过程变量包括：细胞大小分布、细胞周期分析、初级代谢摄取和分泌率、氨基酸摄取和分泌率、细胞活力和活细胞密度。

This project seeks to optimise perfusion processes via addressing two related challenges, namely:- Scale-down and process verification of novel cell retention methodologies (AWS)- Understanding steady state and maximise process performance and productivityObjectives- Design and build a small scale version of the AWS system, able to work with and fully connected to a 250mL perfusion bioreactor. - Understand the limits of and characterize the "operating window" for AWS perfusion processes. Benchmark against other cell retention technologies.- Understand steady state with respect to: cell size distribution, cell cycle distribution, primary metabolism, specific productivity. Model based process optimisation (perfused media, perfusion rate, target VCD at steady state).Project DescriptionThe Acoustic Wave Separator (AWS) technology developed by Pall offers a number of promising benefits such as: (a) not being susceptible to fouling, (b) being tuneable to allow for cell fractionation and conditional bleeding and (c) causing less stress to the cells. However, a scale down version amenable to high-throughput process development in small volume automated bioreactors does not exist. The aim is to develop, in collaboration with Pall, a scale down version of the AWS as a cell retention device in small volume bioreactors and to understand the limits of and characterize the "operating window" for continuous processes using the AWS technology. Experimental investigations will be based on a fully controlled scale-down perfusion bioreactor recently developed at UCL. This novel 250ml bioreactor, specifically designed for mammalian cell cultivation under perfusion mode, is equipped with a Levitronix pump and TFF filter, used for cell retention. Perfusion runs at 1VVD have been successfully run and showed good reproducibility over 10 days. Expertise from Pall on the AWS and its operation will be key to select suitable scaling criteria.Mathematical analysis and characterisation of the steady state will be based on a series of metabolic modelling and Systems Biology techniques recently developed at UCL. Process variables that will be studied both mathematically and experimentally at steady state include: cell size distribution, cell cycle analysis, primary metabolic uptake & secretion rates, amino acid uptake & secretion rates, cell viability and viable cell density.