title: Understanding the relationship between biotherapeutic protein stability and solid-liquid interfacial shear in constant region mutants of IgG1 and IgG4.
abstract: Relative stability of therapeutic antibody candidates is currently evaluated primarily through their response to thermal degradation, yet this technique is not always predictive of stability in manufacture, shipping, and storage. A rotating disk shear device is proposed that produces defined shear conditions at a known solid-liquid interface to measure stability in this environment. Five variants of IgG1 and IgG4 antibodies were created using combinations of two discrete triple amino acid sequence mutations denoted TM and YTE. Antibodies were ranked for stability based on shear device output (protein decay coefficient, PDC), and compared with accelerated thermal stability data and the melting temperature of the CH2 domain (Tm 1) from differential scanning calorimetry to investigate technique complimentarity. Results suggest that the techniques are orthogonal, with thermal methods based on intramolecular interaction and shear device stability based on localized unfolding revealing less stable regions that drive aggregation. Molecular modeling shows the modifications' effects on the antibody structures and indicates a possible role for Fc conformation and Fab-Fc docking in determining suspended protein stability. The data introduce the PDC value as an orthogonal stability indicator, complementary to traditional thermal methods, allowing lead antibody selection based on a more full understanding of process stability.
authors.affiliation: The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK; MedImmune, Granta Park, Cambridge, CB21 6GH, UK.
authors.forename: Roumteen
authors.initials: R
authors.lastname: Tavakoli-Keshe
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Phillips
authors.forename: Richard
authors.initials: R
authors.lastname: Turner
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Bracewell
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D004355:Drug Stability; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry*; D008958:Models, Molecular; D008970:Molecular Weight; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013499:Surface Properties; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011506:Proteins
keywords: IgG antibody; biopharmaceuticals characterization; calorimetry (DSC); in silico modeling; molecular modeling; protein aggregation; protein structure; thermal analysis