Cell Separation in Aqueous Two‐Phase Systems − Influence of Polymer Molecular Weight and Tie‐Line Length on the Resolution of Five Model Cell Lines

Abstract

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.

Microbioreactor Systems for Accelerated Bioprocess Development

“…In the past, the integration of robotic systems for downstream process development led to significant increases in the throughput of several standard operation procedures. For example, cell lysis has been optimized for improved release of intracellular products, the screening of chromatographic material and binding/elution conditions was facilitated, and the identification of optimal compositions of aqueous two‐phase systems or precipitation conditions has become experimentally feasible. So far only a few but highly interesting studies have been reported that combine high‐throughput upstream and downstream bioprocess development .…”

Characterization and optimization of immunoaffinity aqueous two‐phase systems with PEGylated CD133/2‐biotin antibody in route to stem cell separation

“…They are formed when two hydrophilic compounds such as polymers, salts or ionic liquids are combined above critical concentrations, resulting in the formation of two immiscible phases . The resulting phases have different concentrations of both compounds and thus different electrochemical and hydrophobic characteristics, allowing the partition of biomolecules according to their intrinsic properties . However, such systems lack specificity when the differences between the component of interest and the contaminants (red blood cells, non‐viable cells, cell debris and other mononuclear cells) are small.…”