Understanding TFF operations in cell and gene therapy bioprocessing

背景：切向流过滤（TFF）通常用于生产细胞和基因治疗（CGT）的生物过程序列中的几个步骤。这些通常用于超滤（浓缩），滤除或缓冲交换，有时用于澄清和/或颗粒去除。该单元操作正在成为回收CGT产品，包括其材料（例如，用于成分DNA或RNA纯化，或脂质纳米颗粒生产）的宝贵工作机器。目前生产中常用的格式是中空纤维，有不同的长度，内径，纤维厚度和膜类型。另一种用于TFF但在CGT应用程序中不常见的格式是平板格式。目前缺乏关于某些CGT应用的更好格式的信息，也没有系统的研究来比较两种格式和不同的设计参数，这些参数在应用于CGT时影响它们的性能。这两种格式的放大也略有不同。例如，通过保持流道长度，同时增加膜宽度以增加表面积，可以使平板膜的放大变得坚固。湍流启动子的使用是常见的，但这些对细胞或病毒载体的影响是未知的。中空纤维膜的放大是不同的，如果在工艺开发的不同阶段使用不同的长度，可能会证明稍微具有挑战性。鉴于对CGT产品的需求不断增加，也需要增加对所涉及的单元操作的理解，以实现稳健的工艺设计。本研究旨在探讨TFF手术在细胞和基因治疗中的应用。项目描述：该项目将建立在我们对单克隆抗体在平板膜上的TFF按比例缩小特性的研究基础上，并将其扩展到使用中空纤维膜的TFF。目前，Rayat小组正在进行慢病毒载体生产方面的一些工作，重点是在离子交换色谱之前进行TFF。然而，TFF应用于过程的不同点，用于不同的模式（例如，病毒载体，RNA）或过程组件（例如，DNA， LNP），因此有必要在流程的不同位置调查TFF。有机会获得各种模式的各种数据，同时，使用计算建模来研究TFF设计的方面，这可能会影响细胞和基因治疗应用的性能。该项目所产生的工艺知识和新方法将有利于生物加工界。目的：本研究的主要目的是表征和优化细胞和基因治疗生物处理中的TFF操作。具体而言，该项目旨在：评估不同格式的TFF在生产率、产品稳定性、回收率和杂质去除率方面的性能。确定可能影响每种格式TFF性能的关键参数（例如，中空纤维长度，膜类型，孔径，平板湍流促进剂等）确定关键的TFF条件，使不同CGT产品和成分（例如，RNA， DNA, LNPs，病毒载体）的最佳回收率。

Background:Tangential flow filtration (TFF) is often used in several steps within a bioprocess sequence to produce cell and gene therapies (CGT). These are typically for ultrafiltration (concentration), diafiltration or buffer-exchange, and sometimes for clarification and/or particulates removal. This unit operation is becoming an invaluable workhorse for the recovery of CGT products, including their materials (e.g., for component DNA or RNA purification, or lipid nanoparticle production). The current format commonly used in production are hollow fibres which come in different lengths, internal diameters, fibre thickness and membrane type. Another format used for TFF, but not often seen in CGT application, is the flat-sheet format. There is currently a lack of information as to the better format for certain CGT applications, nor are there systematic studies that compare the two formats and the different design parameters that affect their performance when applied to CGT. Scale-up of these two formats are also slightly different. For example, scale-up of flat sheet membranes have been made robust by maintaining the flow-path length while increasing the membrane width for increased surface area. The use of turbulence promoters is common but the effect of these on cells or viral vectors are unknown. Scale-up of hollow fibre membranes is different and may prove slightly challenging if different lengths are used in the different stages of process development. Given the increasing demand for CGT products, the need also grows for increased understanding of the unit operations involved to achieve a robust process design. This study aims to investigate the TFF operation for cell and gene therapy applications.Project Description:The project will build on our work on TFF scale-down characterisation for Mabs on flat-sheet membranes and extend this to TFF using hollow-fibre membranes. There is some work currently underway in the Rayat Group on lentiviral vector production focusing on TFF prior to ion exchange chromatography. However, TFF is applied at different points of the process and for different modalities (e.g., viral vectors, RNA) or process components (e.g., DNA, LNP) and so there is a need to investigate TFF at different places of the flowsheet. There is an opportunity to obtain a wide variety of data for various modalities, and at the same time, to use computation modelling to look at the aspect of TFF design which can affect performance in cell and gene therapy applications. The process knowledge and novel methods this project can generate will benefit the bioprocessing community.Objectives: The main aim of this study is to characterise and optimise TFF operations in cell and gene therapy bioprocessing. Specifically, the project aims to:Evaluate the performance of the different formats of TFF in terms of productivity, product stability and recovery and impurity removal.Apply novel modelling methodologies for the bioprocess development of TFFDetermine the key parameters which may affect the TFF performance for each format (e.g., hollow fibre length, membrane type, pore size, flat-sheet turbulence promoters etc)Identify the key TFF conditions that enable the optimal recovery of different CGT products and components (e.g., RNA, DNA, LNPs, viral vectors)