Improved drug-like properties of therapeutic proteins by directed evolution

• Published in: Protein engineering, design and selection Vol. 25; no. 10; pp. 631 - 638
• Main Authors: Buchanan, Andrew, Ferraro, Franco, Rust, Steven, Sridharan, Sudharsan, Franks, Ruth, Dean, Greg, McCourt, Matthew, Jermutus, Lutz, Minter, Ralph
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.10.2012
• Subjects: Animals; directed evolution; Directed Molecular Evolution - methods; Drug development; Drugs; Erythropoietin; Erythropoietin - chemistry; Erythropoietin - genetics; Erythropoietin - pharmacokinetics; Escherichia coli; Escherichia coli - genetics; Granulocyte colony-stimulating factor; Granulocyte Colony-Stimulating Factor - chemistry; Granulocyte Colony-Stimulating Factor - genetics; Humans; Mice; Models, Molecular; Original; Protein Stability; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - pharmacokinetics; Ribosomes; Ribosomes - genetics; Shelf life; Solubility; ribosome display; developability; expression; stability; directed evolution
• ISSN: 1741-0126; 1741-0134; 1741-0134
• DOI: 10.1093/protein/gzs054

Many natural human proteins have functional properties that make them useful as therapeutic drugs. However, not all these proteins are compatible with large-scale manufacturing processes or sufficiently stable to be stored for long periods prior to use. In this study, we focus on small four-helix bundle proteins and employ ribosome display in conjunction with three parallel selection pressures to favour the isolation of variant proteins with improved expression, solubility and stability. This in vitro evolution strategy was applied to two human proteins with known drug development issues, granulocyte colony-stimulating factor (G-CSF) and erythropoietin (EPO). In the case of G-CSF, the soluble expression levels in Escherichia coli were improved 1000-fold, while for EPO the level of aggregation in an accelerated shelf-life study was reduced from over 80% to undetectable levels. These results exemplify the general utility of our in vitro evolution strategy for improving the drug-like properties of therapeutic proteins.