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.

中文翻译：

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剖析单特异性和双特异性IgG抗体高粘度的分子基础。

一些抗体在高浓度下表现出升高的粘度，从而使其不适用于需要通过注射施用的治疗应用，例如皮下或眼部递送。在这里，我们研究了一种抗IL-13 / IL-17双特异性IgG4抗体，与它的亲本单特异性抗体相比，它具有异常高的粘度。在存在NaCl的情况下，溶液中双特异性IgG4的粘度仅降低了约30％，这表明静电相互作用不足以完全解释粘度的驱动因素。有趣的是，加入精氨酸HCl可使双特异性IgG4的粘度降低至其母体IgG水平约50％。这些数据表明，除了静电以外，其他类型的相互作用（如阳离子-π和/或π-π）可能比以前理解的更显着地有助于高粘度。在游离精氨酸和清水的混合溶液中进行抗体片段的分子动力学模拟，以识别参与自我相互作用的热点。暴露的表面芳香族氨基酸显示出与精氨酸接触的数量增加。通过分子动力学模拟指出的大多数芳香族残基的诱变有效降低了溶液的粘度（通过实验测试）。降低双特异性抗体粘度的这种突变方法已扩展到具有较高粘度的单特异性抗GCGR IgG1抗体。在所有情况下，都容易鉴定出既降低了粘度又保留了抗原结合亲和力的点突变体。