title: Fc domain mediated self-association of an IgG1 monoclonal antibody under a low ionic strength condition.
abstract: Recently, we reported that IgG1 monoclonal antibody A (MAb A) underwent liquid-liquid phase separation and separated into light and heavy phases under a low ionic strength condition. The liquid-liquid phase separation was induced due to self-association of MAb A in the heavy phase when the initial concentration of MAb A was between the two critical concentrations [Nishi et al., Pharm. Res., 27, 1348-1360 (2010)]. Here, we determined the interaction site of MAb A by using proteolytic Fab and Fc fragments of MAb A. The mean hydrodynamic diameter of the Fc fragment increased in a low ionic strength buffer, and furthermore the SPR measurement detected interactions of the Fc fragment with both whole MAb A and the Fc fragment, whereas the Fab fragment interacted with neither whole MAb A nor the Fc fragment. No binding was detected under an isotonic ionic strength condition. Zeta potential of MAb A was significant positive below pH 5.5 and negative above pH 6.5. Between pH 5.5 and 6.5 where the phase separation is significantly induced, MAb A had only a small positive or negative net charge. The isothermal titration calorimetry dilution method revealed that dissociation of MAb A accompanied endothermic heat changes, suggesting that intermolecular interactions among MAb A molecules were attributed to the enthalpically driven process. These results suggest that liquid-liquid phase separation of MAb A is mediated by a weak electrostatic intermolecular interaction among MAb A molecules mainly at Fc portions.
authors.affiliation: Formulation Technology Research Laboratories, Daiichi Sankyo Co, Ltd, 1-12-1 Shinomiya, Hiratsuka, Kanagawa 254-0014, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Nishi
authors.forename: Makoto
authors.initials: M
authors.lastname: Miyajima
authors.forename: Naoki
authors.initials: N
authors.lastname: Wakiyama
authors.forename: Kei
authors.initials: K
authors.lastname: Kubota
authors.forename: Jun
authors.initials: J
authors.lastname: Hasegawa
authors.forename: Susumu
authors.initials: S
authors.lastname: Uchiyama
authors.forename: Kiichi
authors.initials: K
authors.lastname: Fukui
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D009994:Osmolar Concentration
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G