Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational, structure-guided Fv engineering

Abstract

Undesired solution behaviors such as reversible self-association (RSA), high viscosity, and liquid-liquid phase separation can introduce substantial challenges during development of monoclonal antibody formulations. Although a global mechanistic understanding of RSA (i.e., native and reversible protein-protein interactions) is sufficient to develop robust formulation controls, its mitigation via protein engineering requires knowledge of the sites of protein-protein interactions. In the study reported here, we coupled our previous hydrogen-deuterium exchange mass spectrometry findings with structural modeling and in vitro screening to identify the residues responsible for RSA of a model IgG1 monoclonal antibody (mAb-C), and rationally engineered variants with improved solution properties (i.e., reduced RSA and viscosity). Our data show that mutation of either solvent-exposed aromatic residues within the heavy and light chain variable regions or buried residues within the heavy chain/light chain interface can significantly mitigate RSA and viscosity by reducing the IgG's surface hydrophobicity. The engineering strategy described here highlights the utility of integrating complementary experimental and in silico methods to identify mutations that can improve developability, in particular, high concentration solution properties, of candidate therapeutic antibodies.

Keywords: AC-SINS; Antibody engineering; DLS; antibody developability; homology modeling; monoclonal antibody; reversible self-association; viscosity.

Figure 1.

Single CDR mutations prevent mAb-C self-association. (A) AC-SINS analysis of self-association of mAb-C variants in PBS, pH 7.4. mAb-C exhibits high self-association in PBS (25 nm plasmon shift). Mutation of histidine 35 of the VH to alanine, but not asparagine, substantially decreases self-association. Mutation of tryptophan 50 of the VH to arginine, and tyrosine 49 and leucine 54 of the VL to aspartate, reduce self-association to varying extents. Plasmon shifts are normalized to the plasmon wavelength of each antibody incubated with control nanoparticles that lack anti-Fc capture antibody. (B) DLS determination of mAb-C variant hydrodynamic size as a function of concentration. The H35A, W50R, Y49D, and L54D mutations prevent mAb-C interaction at up to 10 mg/mL. H35N also decreases hydrodynamic size, but to a much lesser extent. Error bars indicate standard deviation of triplicate measurements.

Figure 2.

Charge and hydrophobicity analysis of mAb-C variants. (A) Capillary isoelectric focusing of mAb-C variants shows only minor differences in pI (indicated above peaks) and charge heterogeneity. (B) Hydrophobic interaction chromatography analysis indicates that the H35A, W50R, Y49D, and L54D mutations substantially reduce surface hydrophobicity of mAb-C.

Figure 3.

Thermostability analysis of mAb-C variants by differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) show that the introduced mutations have a negligible effect on physical stability.

Figure 4.

MAb-C VH and VL mutations reduce viscosity. Antibody viscosity was measured at 70 mg/mL in PBS, pH 7.4 at 4°C by an Anton-Paar MCR-301 cone-and-plate rheometer.

Figure 5.

Cartoon of mAb-C homology model depicting location of mutated VH and VL residues. The HDX-identified binding interface (green) includes VH FR2, a segment of CDR H2, CDR L2, and a segment of VL FR2 and FR3. (A) H35 (magenta) is positioned at the VH/VL interface beneath adjacent aromatic side chains, whereas W50 (orange), Y49 (pink), and L54 (purple) are exposed. Mutation of H35 to a small, non-polar, amino acid (e.g., alanine) may create a cavity that allows W50 or other nearby hydrophobic residues to become more buried into the cleft between the VH/VL interface. VH and VL framework are in blue and gray, respectively. CDRs excluded from the HDX interface are labeled cyan (H1), light blue (H3), yellow (L1), and brown (L3). (B) Aggregation surface analysis of mAb-C Fv. Aggregation prone regions of the surface are depicted in red. A predicted aggregation patch composed of W50 and Y33 is located on the VH (top image, green oval). On the VL, Y49 and L54 compose an aggregation surface (bottom image, green oval).