title: Charge-mediated Fab-Fc interactions in an IgG1 antibody induce reversible self-association, cluster formation, and elevated viscosity.
abstract: Concentration-dependent reversible self-association (RSA) of monoclonal antibodies (mAbs) poses a challenge to their pharmaceutical development as viable candidates for subcutaneous delivery. While the role of the antigen-binding fragment (Fab) in initiating RSA is well-established, little evidence supports the involvement of the crystallizable fragment (Fc). In this report, a variety of biophysical tools, including hydrogen exchange mass spectrometry, are used to elucidate the protein interface of such non-covalent protein-protein interactions. Using dynamic and static light scattering combined with viscosity measurements, we find that an IgG1 mAb (mAb-J) undergoes RSA primarily through electrostatic interactions and forms a monomer-dimer-tetramer equilibrium. We provide the first direct experimental mapping of the interface formed between the Fab and Fc domains of an antibody at high protein concentrations. Charge distribution heterogeneity between the positively charged interface spanning complementarity-determining regions CDR3H and CDR2L in the Fab and a negatively charged region in CH3/Fc domain mediates the RSA of mAb-J. When arginine and NaCl are added, they disrupt RSA of mAb-J and decrease the solution viscosity. Fab-Fc domain interactions between mAb monomers may promote the formation of large transient antibody complexes that ultimately cause increases in solution viscosity. Our findings illustrate how limited specific arrangements of amino-acid residues can cause mAbs to undergo RSA at high protein concentrations and how conserved regions in the Fc portion of the antibody can also play an important role in initiating weak and transient protein-protein interactions.
authors: a Department of Pharmaceutical Chemistry , University of Kansas , Lawrence , KS , USA.|b Macromolecule and Vaccine Stabilization Center, University of Kansas , Lawrence , KS , USA.
authors: Jayant
authors: J
authors: Arora
authors: a Department of Pharmaceutical Chemistry , University of Kansas , Lawrence , KS , USA.|b Macromolecule and Vaccine Stabilization Center, University of Kansas , Lawrence , KS , USA.
authors: Yue
authors: Y
authors: Hu
authors: c Department of Formulation Sciences , MedImmune LLC , Gaithersburg , MD , USA.
authors: Reza
authors: R
authors: Esfandiary
authors: c Department of Formulation Sciences , MedImmune LLC , Gaithersburg , MD , USA.
authors: Hasige A
authors: HA
authors: Sathish
authors: c Department of Formulation Sciences , MedImmune LLC , Gaithersburg , MD , USA.
authors: Steven M
authors: SM
authors: Bishop
authors: a Department of Pharmaceutical Chemistry , University of Kansas , Lawrence , KS , USA.|b Macromolecule and Vaccine Stabilization Center, University of Kansas , Lawrence , KS , USA.
authors: Sangeeta B
authors: SB
authors: Joshi
authors: a Department of Pharmaceutical Chemistry , University of Kansas , Lawrence , KS , USA.|b Macromolecule and Vaccine Stabilization Center, University of Kansas , Lawrence , KS , USA.
authors: C Russell
authors: CR
authors: Middaugh
authors: a Department of Pharmaceutical Chemistry , University of Kansas , Lawrence , KS , USA.|b Macromolecule and Vaccine Stabilization Center, University of Kansas , Lawrence , KS , USA.
authors: David B
authors: DB
authors: Volkin
authors: a Department of Pharmaceutical Chemistry , University of Kansas , Lawrence , KS , USA.|d Department of Chemistry and R.N. Adams Institute of Bioanalytical Chemistry , University of Kansas , Lawrence , KS , USA.
authors: David D
authors: DD
authors: Weis
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D022801:Complementarity Determining Regions / Q000737:chemistry; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D014783:Viscosity
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G
keywords: Hydrogen exchange; monoclonal antibody; protein aggregation; protein-protein interactions; reversible self-association