title: Improved drug-like properties of therapeutic proteins by directed evolution.
abstract: 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.
authors.affiliation: MedImmune Research, Granta Park, Cambridge CB21 6GH, UK. buchanana@medimmune.com
authors.forename: Andrew
authors.initials: A
authors.lastname: Buchanan
authors.forename: Franco
authors.initials: F
authors.lastname: Ferraro
authors.forename: Steven
authors.initials: S
authors.lastname: Rust
authors.forename: Sudharsan
authors.initials: S
authors.lastname: Sridharan
authors.forename: Ruth
authors.initials: R
authors.lastname: Franks
authors.forename: Greg
authors.initials: G
authors.lastname: Dean
authors.forename: Matthew
authors.initials: M
authors.lastname: McCourt
authors.forename: Lutz
authors.initials: L
authors.lastname: Jermutus
authors.forename: Ralph
authors.initials: R
authors.lastname: Minter
mesh_terms: D000818:Animals; D019020:Directed Molecular Evolution / Q000379:methods*; D004921:Erythropoietin / Q000737:chemistry / Q000235:genetics* / Q000493:pharmacokinetics; D004926:Escherichia coli / Q000235:genetics; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics; D012270:Ribosomes / Q000235:genetics; D012995:Solubility
chemical_list: D011994:Recombinant Proteins; D004921:Erythropoietin; D016179:Granulocyte Colony-Stimulating Factor