Dissecting the molecular basis of high viscosity of monospecific and bispecific IgG antibodies

Abstract

Some antibodies exhibit elevated viscosity at high concentrations, making them poorly suited for therapeutic applications requiring administration by injection such as subcutaneous or ocular delivery. Here we studied an anti-IL-13/IL-17 bispecific IgG₄ antibody, which has anomalously high viscosity compared to its parent monospecific antibodies. The viscosity of the bispecific IgG₄ in solution was decreased by only ~30% in the presence of NaCl, suggesting electrostatic interactions are insufficient to fully explain the drivers of viscosity. Intriguingly, addition of arginine-HCl reduced the viscosity of the bispecific IgG₄ by ~50% to its parent IgG level. These data suggest that beyond electrostatics, additional types of interactions such as cation-π and/or π-π may contribute to high viscosity more significantly than previously understood. Molecular dynamics simulations of antibody fragments in the mixed solution of free arginine and explicit water were conducted to identify hotspots involved in self-interactions. Exposed surface aromatic amino acids displayed an increased number of contacts with arginine. Mutagenesis of the majority of aromatic residues pinpointed by molecular dynamics simulations effectively decreased the solution's viscosity when tested experimentally. This mutational method to reduce the viscosity of a bispecific antibody was extended to a monospecific anti-GCGR IgG₁ antibody with elevated viscosity. In all cases, point mutants were readily identified that both reduced viscosity and retained antigen-binding affinity. These studies demonstrate a new approach to mitigate high viscosity of some antibodies by mutagenesis of surface-exposed aromatic residues on complementarity-determining regions that may facilitate some clinical applications.

Keywords: Bispecific IgG; IgG; viscosity.

Figure 1.

(a) Cartoon representation of monospecific and bispecific antibody formats investigated. The isotype for the anti-IL-17 antibody is IgG₁, whereas the anti-IL-13 and BsIgG antibodies are both IgG₄. All antibodies have κ light chains. Half-antibodies containing knobs-into-holes mutations³⁴ (‘knob’ mutation (T366W) in the anti-IL-13 arm or ‘hole’ mutations (T366S:L368A:Y407V) in the anti-IL-17arm) were expressed and purified separately before assembly in vitro as described previously.^35,36 A hinge stabilizing mutation (S228P) was introduced into the IgG₄ to attenuate Fab arm exchange.^37,38 (b) Viscosity of fragments (F(ab’)₂ and Fab) or full-length anti-IL-13/IL-17 bispecific or its parent antibodies are shown. (c) The effect of 150 mM NaCl or 150 mM Arg-HCl on solution viscosity is shown. The dotted line represents the viscosity of parent IgG antibody level as a reference. Data shown are the mean values (n = 3, ± SD) measured by rheometer at 23ºC and 150 mg/mL total protein concentration in 20 mM His-HCl pH 6.0 (buffer) in the absence or presence of excipients.

Figure 2.

X-ray crystallographic structures of anti-IL-13 (lebrikizumab)⁴⁰ and anti-IL-17 (MCAF5352A⁴¹) antibody Fabs complexed with their respective ligands displaying aromatic residues chosen for mutational studies. (a) View of anti-IL-13/IL-13 interface depicting important viscosity reducing residues (PDB 4I77).⁴⁰ The anti-IL13 V_H is cyan and V_L is light pink, with dark cyan and magenta side chains within 4 Å of IL-13. IL-13 is gray with yellow epitope within 4 Å of anti-IL-13. (b) View of anti-IL-17/IL-17F interface depicting important residues (PDB 6PPG). Anti-IL-17F V_H is cyan and V_L is light pink, with dark cyan and magenta side chains within 4 Å of IL-17. IL-17F is gray with yellow epitope within 4 Å of anti-IL-17F. See the Supplementary Materials for the sequences for the variable domains for the anti-IL-13 (Figure S6) and anti-IL17 (Figure S7) antibodies.

Figure 3.

MD simulations of (a) anti-IL-13 (lebrikizumab⁴⁰) and (b) anti-IL-17 (MCAF5352A⁴¹) variable heavy (V_H) and variable light (V_L) domains in the presence of arginine. Data shown are the mean number of arginine contacts from 20 independent simulations. Residues that show >2σ or >3σ deviations from the mean (μ) number of arginine contact number of all residues in chains are highlighted. Kabat numbering is used.^42.

Figure 4.

Viscosity of antibody variants. Monospecific anti-IL-13 IgG₄ and anti-IL-17 IgG₁ antibody variants were mixed with the parent anti-IL-17 IgG₁ and anti-IL-13 IgG₄ antibody counterparts, respectively, at a 1:1 ratio and a final total concentration of 150 mg/mL. The viscosity of the solutions was then measured by rheometer at 23ºC. (a) Viscosity of mixtures of anti-IL-13 charged variants and the anti-IL-17 parent antibody. (b) Viscosity of mixtures of anti-IL-17 charged variants and the anti-IL-13 parent antibody. (c) Viscosity of mixtures of anti-IL-13 and anti-IL-17 aromatic variants with their parent IgG antibody counterpart. (d) Viscosity of anti-IL-13/IL-17 BsIgG₄ variants at 150 mg/mL final concentration. Data shown are the mean values (n = 2, ± SD) measured in 20 mM His-HCl buffer at pH 6.0. The dotted line shows the parent antibody viscosity level as a reference.