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The availability of preparative-scale downstream processing strategies for cell-based products presents a critical juncture between fundamental research and clinical development. Aqueous two-phase systems (ATPS) present a gentle, scalable, label-free, and cost-effective method for cell purification, and are thus a promising tool for downstream processing of cell-based therapeutics. Here, the application of a previously developed robotic screening platform that enables high-throughput cell partitioning analysis in ATPS is reported. In the present case study a purification strategy for two model cell lines based on high-throughput screening (HTS)-data and countercurrent distribution (CCD)-modeling, and validated the CCD-model experimentally is designed. The obtained data are shown an excellent congruence between CCD-model and experimental data, indicating that CCD-models in combination with HTS-data are a powerful tool in downstream process development. Finally, the authors are shown that while cell cycle phase significantly influences cell partitioning, cell type specific differences in surface properties are the main driving force in charge-dependent separation of HL-60 and L929 cells. In order to design a highly robust purification process it is, however, advisable to maintain constant growth conditions.

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.