title: Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity.
abstract: There is strong interest in the design of bispecific monoclonal antibodies (bsAbs) that can simultaneously bind 2 distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Multiple bispecific formats have been proposed and are currently under development. Regeneron's bispecific technology is based upon a standard fully human IgG antibody in order to minimize immunogenicity and improve the pharmacokinetic profile. A single common light chain and 2 distinct heavy chains combine to form the bispecific molecule. One of the heavy chains contains a chimeric Fc sequence form (called Fc*) that ablates binding to Protein A via the constant region. As a result of co-expression of the 2 heavy chains and the common light chain, 3 products are created, 2 of which are homodimeric for the heavy chains and one that is the desired heterodimeric bispecific product. The Fc* sequence allows selective purification of the FcFc* bispecific product on commercially available affinity columns, due to intermediate binding affinity for Protein A compared to the high avidity FcFc heavy chain homodimer, or the weakly binding Fc*Fc* homodimer. This platform requires the use of Protein A chromatography in both a capture and polishing modality. Several challenges, including variable region Protein A binding, resin selection, selective elution optimization, and impacts upon subsequent non-affinity downstream unit operations, were addressed to create a robust and selective manufacturing process.
authors.affiliation: a Regeneron Pharmaceuticals Inc. , Tarrytown , NY , USA.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Tustian
authors.affiliation: a Regeneron Pharmaceuticals Inc. , Tarrytown , NY , USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Endicott
authors.affiliation: a Regeneron Pharmaceuticals Inc. , Tarrytown , NY , USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Adams
authors.affiliation: a Regeneron Pharmaceuticals Inc. , Tarrytown , NY , USA.
authors.forename: John
authors.initials: J
authors.lastname: Mattila
authors.affiliation: a Regeneron Pharmaceuticals Inc. , Tarrytown , NY , USA.
authors.forename: Hanne
authors.initials: H
authors.lastname: Bak
mesh_terms: D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D000915:Antibody Affinity; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D013205:Staphylococcal Protein A / Q000276:immunology*
chemical_list: D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D013205:Staphylococcal Protein A
keywords: Affinity chromatography; bispecific antibody; downstream process; elution pH; heterodimeric antibody; protein A chromatography; protein purification