浓度≥100mg/mL的单克隆抗体制剂的高粘度可阻碍其发展为适于皮下递送的产品。疏水和静电分子间相互作用对MAB1溶液行为的影响,MAB1在高浓度下变得不可接受的粘性,通过测试5个单点突变体进行了研究。突变被设计为通过破坏易聚集区(APR)来降低粘度。它还参与了2个疏水表面斑块,或可变区中带负电荷的表面贴片。位于轻链和重链可变结构域界面的APR的破坏,VH和VL,通过L45K突变使MAB1不稳定,并消除了抗原结合。然而,在前面的残基突变(V44K),这也是在同一个APR中,在不牺牲抗原结合或热稳定性的情况下增加MAB1的表观溶解度和降低的粘度。中和带负电荷的表面贴片(E59Y)也增加了MAB1的表观溶解度和降低的粘度,但是在相同位置(E59K/R)的电荷逆转导致不稳定,降低溶解度并导致样品操作困难,从而无法在高浓度下进行粘度测量。V44K和E59Y突变均显示相似的表观溶解度增加。然而,E59Y的粘度曲线明显优于V44K,提供证据证明MAB1中的分子间相互作用是静电驱动的。总之,中和带负电荷的表面贴片可能比电荷逆转或易于聚集的基序破坏更有利于降低高度浓缩的抗体溶液的粘度。
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.