title: Rational design of viscosity reducing mutants of a monoclonal antibody: hydrophobic versus electrostatic inter-molecular interactions.
abstract: High viscosity of monoclonal antibody formulations at concentrations ≥100 mg/mL can impede their development as products suitable for subcutaneous delivery. The effects of hydrophobic and electrostatic intermolecular interactions on the solution behavior of MAB 1, which becomes unacceptably viscous at high concentrations, was studied by testing 5 single point mutants. The mutations were designed to reduce viscosity by disrupting either an aggregation prone region (APR), which also participates in 2 hydrophobic surface patches, or a negatively charged surface patch in the variable region. The disruption of an APR that lies at the interface of light and heavy chain variable domains, VH and VL, via L45K mutation destabilized MAB 1 and abolished antigen binding. However, mutation at the preceding residue (V44K), which also lies in the same APR, increased apparent solubility and reduced viscosity of MAB 1 without sacrificing antigen binding or thermal stability. Neutralizing the negatively charged surface patch (E59Y) also increased apparent solubility and reduced viscosity of MAB 1, but charge reversal at the same position (E59K/R) caused destabilization, decreased solubility and led to difficulties in sample manipulation that precluded their viscosity measurements at high concentrations. Both V44K and E59Y mutations showed similar increase in apparent solubility. However, the viscosity profile of E59Y was considerably better than that of the V44K, providing evidence that inter-molecular interactions in MAB 1 are electrostatically driven. In conclusion, neutralizing negatively charged surface patches may be more beneficial toward reducing viscosity of highly concentrated antibody solutions than charge reversal or aggregation prone motif disruption.
authors.affiliation: a Biotherapeutics Pharmaceutical Sciences Research and Development; Pfizer Inc. ; Andover , MA USA.
authors.forename: Pilarin
authors.initials: P
authors.lastname: Nichols
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Sandeep
authors.initials: S
authors.lastname: Kumar
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Buck
authors.forename: Satish K
authors.initials: SK
authors.lastname: Singh
authors.forename: Sumit
authors.initials: S
authors.lastname: Goswami
authors.forename: Bryan
authors.initials: B
authors.lastname: Balthazor
authors.forename: Tami R
authors.initials: TR
authors.lastname: Conley
authors.forename: David
authors.initials: D
authors.lastname: Sek
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Allen
mesh_terms: D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D017354:Point Mutation; D012995:Solubility; D055672:Static Electricity; D014783:Viscosity
chemical_list: D000911:Antibodies, Monoclonal
keywords: APR, Aggregation Prone Region; ASA, Accessible Surface Area; ASAFv-HPH, hydrophilic accessible surface area of the Fv portion; ASAFv-HYD, hydrophobic accessible surface area of the Fv portion; CE, Capillary Electrophoresis; CH2; CH3, third constant domain in heavy chain; CHO, Chinese Hamster Ovary; D0, diffusion coefficient at infinite dilution; DFv, dipole moment of Fv; DLS, Dynamic Light Scattering; ELISA, Enzyme-Linked Immunosorbent Assay; Fab, fragment antigen binding; Fc, fragment crystallizable; Fv, fragment variable; HC, heavy chain; IgG, immunoglobulin G; LC, light chain; MAB 1 Control, MAB 1 expressed in CHO cells; MD, molecular dynamics; NTU, Nephelometric Turbidity Unit; PEG, polyethylene glycol; Pagg-VH, aggregation propensity of VH domain; Pagg-VL, aggregation propensity of VL domain; RPM, revolutions per minute; SE-HPLC, Size Exclusion High Performance Liquid Chromatography; Tm, thermal transition temperature; VH, variable domain in the heavy chain; VL, variable domain in the light chain; ZDHH, Debye-Huckel Henry Charge; ZFv, net charge of the Fv; ZFv-app, apparent charge of the Fv; aggregation prone regions; cIEF, capillary Isoelectric Focusing; cP, centipoise; high concentration; kD, protein-protein interaction parameter; mAb, monoclonal antibody; molecular modeling; monoclonal antibodies; negatively charged patches; rational design; second constant domain in the heavy chain; solubility; viscosity; ΔGFv, change in Free energy of Fv; η, solution viscosity; η0, solvent viscosity; ηrel, relative viscosity; ξFv, zeta-potential of the Fv