Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins.
Product analytics is the bottleneck of most processes in bioprocess engineering, as it is rather time-consuming. Real-time and in-line product tracing without sample pre-treatment is only possible for few products. The Cherry-Tag™ (Delphi Genetics, Belgium) which can be fused to any target protein allows for straightforward product analytics by VIS absorption measurements. When the fused protein becomes unstable or insoluble, the chromophore function of the group is lost, which makes this technology an ideal screening tool for solubility and stability in up- and downstream process development. The Cherry-Tag™ technology will be presented for the tagged enzyme glutathione-S-transferase (GST) from Escherichia coli in a combined up- and downstream process development study. High-throughput cultivations were carried out in a 48-well format in a BioLector system (m2p-Labs, Germany). The best cultivation setup of highest product titer was scaled up to a 2.5L shake flask culture, followed by a selective affinity chromatography product capturing step. In upstream applications the tag was capable of identifying conditions where insoluble and non-native inclusion bodies were formed. In downstream applications the red-colored product was found to be bound effectively to a GST affinity column. Thus, it was identified to be a native and active protein, as the binding mechanism relies on catalytic activity of the enzyme. The Cherry-Tag™ was found to be a reliable and quantitative tool for real-time tracking of stable and soluble proteins in up- and downstream processing applications. Denaturation and aggregation of the product can be detected in-line at any stage of the process. Critical stages can be identified and subsequently changed or replaced.
Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany. Electronic address: pascal.baumann@kit.edu.
Pascal
P
Baumann
Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
Nicolai
N
Bluthardt
Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
Sarah
S
Renner
Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
Hannah
H
Burghardt
Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany. Electronic address: anna.osberghaus@kit.de.
Anna
A
Osberghaus
Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany. Electronic address: juergen.hubbuch@kit.edu.
Jürgen
J
Hubbuch
D002846:Chromatography, Affinity; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry*; D002479:Inclusion Bodies; D012995:Solubility
D005982:Glutathione Transferase
BioLector; Cherry-Tag™; Micro-cultivation; Native protein; Product analytics