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.

Molecular basis of high viscosity in concentrated antibody solutions: Strategies for high concentration drug product development

“…This option requires an advanced understanding of sequencestructure properties of the drug candidates and access to protein engineering and molecular biology facilities. 75 …”

“…Use of full-length antibody models may further improve accuracy of the viscosity prediction methods due to improved descriptions of electrostatic multipole effects that arise from the differences in charges on individual antibody domains. In three closely related studies, Kumar and coworkers 42,71,75 identified the molecular attributes that correlate with viscosity in highly concentrated antibody solutions, rationalized their observations using a putative mechanism involving antibody network formation gleaned from coarse-grained simulations, and then used this knowledge to rationally design viscosity reducing mutants of an antibody. They obtained concentration-dependent viscosity curves of eleven different antibodies under standardized formulation conditions, 71 and interpreted this experimental data in terms of sequence and structural attributes of the mAbs and their components.…”

“…Initial attempts to rationally design such antibody variants have been successful. 73,75,77 Further delineating such rational strategies will be very useful toward suitably improving solution behaviors of highly valuable lead candidates. As discussed earlier, a large and positive osmotic second virial coefficient is a good indicator of lower viscosity of antibody solutions at 150 mg per mL concentration.…”

“…The findings from this data analysis 71 and coarse grained simulations 42 were combined to rationally design viscosity lowering variants of an antibody that demonstrated high viscosities at concentrations above 100 mg per mL. 75 A negatively charged patch and an aggregation-prone region (APR) on the Fv portion of the viscosity-challenged antibody were disrupted via single point mutations. The APR was disrupted via two different single-point mutations, V44K and L45K.…”

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

“…9,15,[18][19][20] In addition to the multitude of distinct domain surfaces that can compose the binding interfaces, the nature of the driving forces behind antibody selfassociation can also be diverse, with both electrostatic and hydrophobic interactions playing key roles. 6,7,18,21,23 Potential strategies to mitigate self-association and other undesired high concentration solution properties may include protein engineering approaches, 20,[24][25][26] as well as formulation development and optimization efforts. [5][6][7]9,18,19,21,23,27 While the former can be limited due to the degree of knowledge of sites of interactions, the latter can be time and resource intensive and may not always be fully successful to a desired degree for all antibodies.…”