title: Stress selections on domain antibodies: 'what doesn't kill you makes you stronger'.
abstract: In addition to the desired specificity and affinity for their respective therapeutic targets, antibody-based drugs must also demonstrate an ability to be manufactured and formulated at the concentrations needed for therapeutic application and to remain resistant to aggregation during storage to reduce the risk of induced immunogenicity. Improvements to the thermodynamic stability of the folded state of the protein are considered to be critical for decreasing the aggregation propensity of the protein. In this work, we have improved the biophysical properties of a number of human domain antibodies (dAbs) by identifying mutations which decrease the propensity for dAb self-aggregation without compromising the affinity for their respective target antigen. The mutations were identified by subjecting phage-displayed error-prone PCR-generated libraries to a variety of generic environmental conditions (temperature, pH and protease) followed by antigen capture, facilitating selection for improved thermodynamic stability of the protein. The results indicate that sufficient sequence diversity usually exists within the complementarity determining regions of dAbs to allow for mutations that lead to improvements to biophysical properties with full retention of parent lead biochemical and biological properties. Improved biophysical properties were often accompanied by higher apparent melting temperature values, while alternative selection pressures often identified similar features, suggesting generic nature of these mutations.
authors: Biopharm Innovation, RD Biopharm R&D, GlaxoSmithKline Plc., 315 Cambridge Science Park, Cambridge CB4 0WG, UK carrie.l.enever@gsk.com.
authors: C
authors: C
authors: Enever
authors: Biopharm Innovation, RD Biopharm R&D, GlaxoSmithKline Plc., 315 Cambridge Science Park, Cambridge CB4 0WG, UK.
authors: M
authors: M
authors: Pupecka-Swider
authors: Biopharm Innovation, RD Biopharm R&D, GlaxoSmithKline Plc., 315 Cambridge Science Park, Cambridge CB4 0WG, UK.
authors: A
authors: A
authors: Sepp
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000627:therapeutic use*; D001709:Biotechnology / Q000379:methods; D002152:Calorimetry, Differential Scanning; D022801:Complementarity Determining Regions / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D007834:Lasers; D008027:Light; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D017472:Receptors, Interleukin-1 / Q000737:chemistry; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D017472:Receptors, Interleukin-1; D047888:Receptors, Tumor Necrosis Factor, Type I; D042461:Vascular Endothelial Growth Factor A
keywords: biophysical properties; domain antibody; library; phage display