title: Antibody variable region interactions with Protein A: implications for the development of generic purification processes.
abstract: In this paper, a wide range of antibodies from various subclasses and subfamilies are employed to evaluate the creation of generic separation processes using Protein A chromatography. The reasons for elution pH differences amongst several IgG1s, IgG2s, antibody fragments, and Fc-fusion proteins during Protein A chromatography are investigated using several complimentary techniques. The results indicate that variable region interactions play a major role in determining elution pH for VH3 subfamily antibodies while using traditional protein A chromatographic materials. On the other hand, experiments with a resin which employs a ligand consisting solely of B domain of Protein A indicate that variable region interactions can be mitigated, enabling the use of a single elution pH for a range of antibodies. Finally, the moderation of elution conditions associated with this engineered ligand are shown to minimize problems associated with low pH induced aggregation. It is expected that the findings reported in this paper will facilitate faster process development cycle times for this important class of human therapeutics.
authors.affiliation: Purification Process Development, Amgen Incorporation, Seattle, Washington, USA.
authors.forename: Sanchayita
authors.initials: S
authors.lastname: Ghose
authors.forename: Martin
authors.initials: M
authors.lastname: Allen
authors.forename: Brian
authors.initials: B
authors.lastname: Hubbard
authors.forename: Clayton
authors.initials: C
authors.lastname: Brooks
authors.forename: Steven M
authors.initials: SM
authors.lastname: Cramer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D001666:Binding Sites, Antibody; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods*; D006224:Cricetinae; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000302:isolation & purification / Q000378:metabolism; D007074:Immunoglobulin G / Q000302:isolation & purification / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A