title: High-throughput downstream process development for cell-based products using aqueous two-phase systems.
abstract: As the clinical development of cell-based therapeutics has evolved immensely within the past years, downstream processing strategies become more relevant than ever. Aqueous two-phase systems (ATPS) enable the label-free, scalable, and cost-effective separation of cells, making them a promising tool for downstream processing of cell-based therapeutics. Here, we report the development of an automated robotic screening that enables high-throughput cell partitioning analysis in ATPS. We demonstrate that this setup enables fast and systematic investigation of factors influencing cell partitioning. Moreover, we examined and optimized separation conditions for the differentiable promyelocytic cell line HL-60 and used a counter-current distribution-model to investigate optimal separation conditions for a multi-stage purification process. Finally, we show that the separation of CD11b-positive and CD11b-negative HL-60 cells is possible after partial DMSO-mediated differentiation towards the granulocytic lineage. The modeling data indicate that complete peak separation is possible with 30 transfers, and >93% of CD11b-positive HL-60 cells can be recovered with >99% purity. The here described screening platform facilitates faster, cheaper, and more directed downstream process development for cell-based therapeutics and presents a powerful tool for translational research.
authors: Karlsruhe Institute of Technology (KIT), Institute of Process Engineering in Life Science, Section IV: Biomolecular Separation Engineering (MAB), Engler-Bunte-Ring 3, 76131 Karlsruhe, Germany.
authors: Sarah
authors: S
authors: Zimmermann
authors: Karlsruhe Institute of Technology (KIT), Institute of Process Engineering in Life Science, Section IV: Biomolecular Separation Engineering (MAB), Engler-Bunte-Ring 3, 76131 Karlsruhe, Germany; Karlsruhe Institute of Technology (KIT), Institute of Functional Interfaces (IFG), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.
authors: Sarah
authors: S
authors: Gretzinger
authors: Karlsruhe Institute of Technology (KIT), Institute of Process Engineering in Life Science, Section IV: Biomolecular Separation Engineering (MAB), Engler-Bunte-Ring 3, 76131 Karlsruhe, Germany; DIARECT AG, Department of Quality Assurance and Quality Control, Bötzinger Str. 29 B, 79111 Freiburg, Germany.
authors: Marie-Luise
authors: ML
authors: Schwab
authors: Karlsruhe Institute of Technology (KIT), Institute of Process Engineering in Life Science, Section IV: Biomolecular Separation Engineering (MAB), Engler-Bunte-Ring 3, 76131 Karlsruhe, Germany.
authors: Christian
authors: C
authors: Scheeder
authors: Karlsruhe Institute of Technology (KIT), Institute of Process Engineering in Life Science, Section IV: Biomolecular Separation Engineering (MAB), Engler-Bunte-Ring 3, 76131 Karlsruhe, Germany.
authors: Philipp K
authors: PK
authors: Zimmermann
authors: Karlsruhe Institute of Technology (KIT), Institute of Process Engineering in Life Science, Section IV: Biomolecular Separation Engineering (MAB), Engler-Bunte-Ring 3, 76131 Karlsruhe, Germany; Boehringer Ingelheim Pharma GmbH & Co. KG, Global Bioprocess & Pharmaceutical Development, Birkendorfer Straße 65, 88397 Biberach an der Riss, Germany.
authors: Stefan A
authors: SA
authors: Oelmeier
authors: Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces (IBG 5), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.
authors: Eric
authors: E
authors: Gottwald
authors: Novo Nordisk A/S, Biopharm Purification Development & Virology, Hagedornsvej 1, 2820 Gentofte, Denmark.
authors: Are
authors: A
authors: Bogsnes
authors: Novo Nordisk A/S, Diabetes Research, Novo Nordisk Park, 2760 Måløv, Denmark.
authors: Mattias
authors: M
authors: Hansson
authors: Novo Nordisk A/S, CMC Project Planning & Management, Smørmosevej 17, 2880 Bagsværd, Denmark.
authors: Arne
authors: A
authors: Staby
authors: Karlsruhe Institute of Technology (KIT), Institute of Process Engineering in Life Science, Section IV: Biomolecular Separation Engineering (MAB), Engler-Bunte-Ring 3, 76131 Karlsruhe, Germany. Electronic address: juergen.hubbuch@kit.edu.
authors: Jürgen
authors: J
authors: Hubbuch
mesh_terms: D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D064987:Cell- and Tissue-Based Therapy; D002477:Cells / Q000737:chemistry* / Q000166:cytology; D006801:Humans; D011092:Polyethylene Glycols / Q000737:chemistry
chemical_list: D011092:Polyethylene Glycols
keywords: Aqueous two-phase systems (ATPS); Downstream processing of cell-based products; High throughput screening (HTS); High-throughput flow cytometry; Label-free cell separation