title: Improved Protein-A separation of V(H)3 Fab from Fc after papain digestion of antibodies.
abstract: Antibody-binding fragments (Fab) are generated from whole antibodies by treatment with papain and can be separated from the Fc component using Protein-A affinity chromatography. Commercial kits are available, which facilitate the production and purification of Fab fragments; however, the manufacturer fails to report that this method is inefficient for antibodies with V(H)3 domains as a result of the intrinsic variable region affinity for Protein-A. A commercially available, modified Protein-A resin (MabSelect SuRe) has been engineered for greater stability. Here, we report that an additional consequence of the modified resin is the ability to purify V(H)3 family Fab fragments, which cannot be separated effectively from other components of the papain digest by traditional Protein-A resin. This improvement of a commonly used procedure is of significance, as increasingly, therapeutic antibodies are being derived from human origin, where V(H)3 is the most abundantly used variable region family.
authors: Mater Medical Research Institute, South Brisbane, Australia.
authors: Therese A
authors: TA
authors: Seldon
authors: Karen E
authors: KE
authors: Hughes
authors: David J
authors: DJ
authors: Munster
authors: David Y
authors: DY
authors: Chin
authors: Martina L
authors: ML
authors: Jones
mesh_terms: D000074323:Alemtuzumab; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification; D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry / Q000302:isolation & purification; D000912:Antibodies, Neoplasm / Q000737:chemistry / Q000302:isolation & purification; D000068258:Bevacizumab; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000302:isolation & purification*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000302:isolation & purification*; D010206:Papain / Q000737:chemistry*; D011485:Protein Binding; D000069283:Rituximab; D013205:Staphylococcal Protein A / Q000737:chemistry*; D000068878:Trastuzumab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived; D000912:Antibodies, Neoplasm; D055767:Immobilized Proteins; D007141:Immunoglobulin Fc Fragments; D007135:Immunoglobulin Variable Region; D013205:Staphylococcal Protein A; D000068258:Bevacizumab; D000074323:Alemtuzumab; D000069283:Rituximab; D010206:Papain; D000068878:Trastuzumab