Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
Institute of Functional Interfaces (IFG), Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, Germany.
Biotechnol J. 2018 Feb;13(2). doi: 10.1002/biot.201700250. Epub 2017 Nov 22.
The availability of clinical-scale downstream processing strategies for cell-based products presents a critical juncture between basic research and clinical development. Aqueous two-phase systems (ATPS) facilitate the label-free, scalable, and cost-effective separation of cells, and are a versatile tool for downstream processing of cell-based therapeutics. Here, we report the application of a previously developed robotic screening platform, here extended to enable a multiplexed high-throughput cell partitioning analysis in ATPS. We investigated the influence of polymer molecular weight and tie-line length on the resolution of five model cell lines in "charge-sensitive" polyethylene-glycol (PEG)-dextran ATPS. We show, how these factors influence cell partitioning, and that the combination of low molecular weight PEGs and high molecular weight dextrans enable the highest resolution of the five cell lines. Furthermore, we demonstrate that the separability of each cell line from the mixture is highly dependent on the polymer molecular weight composition and tie-line length. Using a countercurrent distribution model we demonstrate that our screenings yielded conditions that theoretically enable the isolation of four of the five cell lines with high purity (>99.9%) and yield.
用于基于细胞的产品的临床规模下游处理策略的可用性在基础研究和临床开发之间呈现出一个关键的转折点。双水相系统（ATPS）有助于无标记、可扩展和具有成本效益的细胞分离，并且是基于细胞治疗的下游处理的多功能工具。在这里，我们报告了先前开发的机器人筛选平台的应用，该平台在此基础上扩展到能够在 ATPS 中进行多路高通量细胞分区分析。我们研究了聚合物分子量和连接线长度对“电荷敏感”聚乙二醇（PEG）-葡聚糖 ATPS 中五种模型细胞系分辨率的影响。我们展示了这些因素如何影响细胞分区，并且低分子量 PEG 和高分子量葡聚糖的组合能够实现五种细胞系的最高分辨率。此外，我们证明了每种细胞系从混合物中的分离能力高度依赖于聚合物分子量组成和连接线长度。使用逆流分布模型，我们证明我们的筛选产生了理论上能够以高纯度（>99.9%）和产率分离五种细胞系中的四种的条件。
