Nichols Pilarin, Li Li, Kumar Sandeep, Buck Patrick M, Singh Satish K, Goswami Sumit, Balthazor Bryan, Conley Tami R, Sek David, Allen Martin J
a Biotherapeutics Pharmaceutical Sciences Research and Development; Pfizer Inc. ; Andover , MA USA.
MAbs. 2015;7(1):212-30. doi: 10.4161/19420862.2014.985504.
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.
浓度≥100 mg/mL的单克隆抗体制剂的高粘度会阻碍其作为适合皮下给药产品的开发。通过测试5个单点突变体，研究了疏水和静电分子间相互作用对高浓度下变得不可接受地粘稠的单克隆抗体1（MAB 1）溶液行为的影响。这些突变旨在通过破坏易于聚集的区域（APR）来降低粘度，该区域也参与2个疏水表面斑块，或者破坏可变区中的带负电荷的表面斑块。通过L45K突变破坏位于轻链和重链可变域VH和VL界面处的APR会使MAB 1不稳定并消除抗原结合。然而，位于同一APR中的前一个残基（V44K）处的突变增加了MAB 1的表观溶解度并降低了粘度，而不会牺牲抗原结合或热稳定性。中和带负电荷的表面斑块（E59Y）也增加了MAB 1的表观溶解度并降低了粘度，但在同一位置的电荷反转（E59K/R）导致不稳定、溶解度降低并导致样品处理困难，从而无法在高浓度下测量其粘度。V44K和E59Y突变均显示表观溶解度有类似增加。然而，E59Y的粘度曲线比V44K的粘度曲线要好得多，这证明MAB 1中的分子间相互作用是由静电驱动的。总之，中和带负电荷的表面斑块可能比电荷反转或破坏易于聚集的基序对降低高浓度抗体溶液的粘度更有益。
