title: Improved assembly of bispecific antibodies from knob and hole half-antibodies.
abstract: A process was developed for large-scale assembly of IgG1 and IgG4 bispecific antibodies from knob and hole half-antibodies. We optimized assembly conditions such as pH, temperature, stabilizers, and reducing agent. We also identified and exploited structural changes unique to knob and hole half-antibodies with the result of improving assembly outcome, specifically storing half-antibodies at higher pH will condition them to assemble more rapidly and produce fewer high molecular-weight species (HMWS). Application of heat to the assemblies resulted in an acceleration of assembly rate, with optimal formation of bispecific achieved at 37°C. IgG4 half-antibodies were unusually sensitive to temperature-dependent formation of HMWS in pre-assembly conditioning as well as during assembly. We selected l-histidine and Polyvinylpyrrolidone (PVP) as stabilizers to prevent HMWS formation in IgG4 , and achieved rapid and high-efficiency assemblies. Using optimized assembly conditions, we developed and scaled up a method for assembling bispecific antibody with 90% assembly efficiency over 6 h with minimal impact to product quality, generating a pool with bispecific antibody for downstream processing.
authors.affiliation: Dept. of Purification Development, Pharma Technical Development, Genentech, South San Francisco, CA.
authors.forename: Ambrose Jon
authors.initials: AJ
authors.lastname: Williams
authors.affiliation: Dept. of Purification Development, Pharma Technical Development, Genentech, South San Francisco, CA.
authors.forename: Glen
authors.initials: G
authors.lastname: Giese
authors.affiliation: Dept. of Purification Development, Pharma Technical Development, Genentech, South San Francisco, CA.
authors.forename: Josefine
authors.initials: J
authors.lastname: Persson
mesh_terms: D018033:Antibodies, Bispecific / Q000737:chemistry*; D005978:Glutathione; D006639:Histidine / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry; D008970:Molecular Weight; D011205:Povidone / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*
chemical_list: D018033:Antibodies, Bispecific; D007074:Immunoglobulin G; D006639:Histidine; D011205:Povidone; D005978:Glutathione
keywords: assembly; bispecific antibody; half-antibody; knob and hole