title: High-throughput micro-scale cultivations and chromatography modeling: Powerful tools for integrated process development.
abstract: Upstream processes are rather complex to design and the productivity of cells under suitable cultivation conditions is hard to predict. The method of choice for examining the design space is to execute high-throughput cultivation screenings in micro-scale format. Various predictive in silico models have been developed for many downstream processes, leading to a reduction of time and material costs. This paper presents a combined optimization approach based on high-throughput micro-scale cultivation experiments and chromatography modeling. The overall optimized system must not necessarily be the one with highest product titers, but the one resulting in an overall superior process performance in up- and downstream. The methodology is presented in a case study for the Cherry-tagged enzyme Glutathione-S-Transferase from Escherichia coli SE1. The Cherry-Tag™ (Delphi Genetics, Belgium) which can be fused to any target protein allows for direct product analytics by simple VIS absorption measurements. High-throughput cultivations were carried out in a 48-well format in a BioLector micro-scale cultivation system (m2p-Labs, Germany). The downstream process optimization for a set of randomly picked upstream conditions producing high yields was performed in silico using a chromatography modeling software developed in-house (ChromX). The suggested in silico-optimized operational modes for product capturing were validated subsequently. The overall best system was chosen based on a combination of excellent up- and downstream performance.
authors.affiliation: Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Science, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
authors.forename: Pascal
authors.initials: P
authors.lastname: Baumann
authors.affiliation: Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Science, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Hahn
authors.affiliation: Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Science, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany. juergen.hubbuch@kit.edu.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hubbuch
mesh_terms: D001688:Biological Products / Q000302:isolation & purification* / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods*; D002845:Chromatography / Q000379:methods*; D003124:Colorimetry; D004926:Escherichia coli / Q000254:growth & development* / Q000378:metabolism*; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000302:isolation & purification / Q000378:metabolism; D057166:High-Throughput Screening Assays; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001688:Biological Products; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase
keywords: BioLector; UV absorption modeling; high-throughput screening; ion exchange chromatography; micro-scale cultivation