Microrheology of Monoclonal Antibodies during Gelation under Low pH Conditions

Non-native aggregation of monoclonal antibodies (mAbs) during downstream processing can reduce their efficacy (e.g., in immunotherapies) and the total yield. The pH of mAb solutions was lowered, similar to steps during viral inactivation and protein A chromatography, and a continuous phase transition to a physically cross-linked gel was observed via microrheology. The dynamic power-law scaling of the shear modulus on the frequency, G′(ω) ∝ G″(ω) ∝ ωn, was calculated using time-cure superposition, yielding a dynamical critical exponent, n = 0.52 ± 0.05. Dynamic light scattering showed a similar power law scaling exponent of μ = 0.49 ± 0.04 about the gel point which is related to the fractal dimension. Circular dichroism showed large increases in the β-sheet content of the mAbs at low pHs combined with a large increase in fluorescence of a ThT stained sample. Thus, mAb gelation seems to occur via the formation of amyloid fibrils that cause a continuous phase transition that is well described by a dynamic scaling model for percolation.

单克隆抗体（monoclonal antibodies, mAbs）在下游加工过程中发生的非天然聚集，会降低其效力（如在免疫治疗中的应用效果）及总收率。将单克隆抗体溶液的pH值调低（该操作与病毒灭活、蛋白A层析过程中的步骤类似），通过微流变学观测到体系发生了向物理交联凝胶的连续相转变。利用时间-固化叠加法计算得到剪切模量随频率的动态幂律标度关系$G'(omega) propto G''(omega) propto omega^n$，得到动力学临界指数$n=0.52pm0.05$。动态光散射结果显示，在凝胶点附近同样存在幂律标度关系，对应的标度指数$mu=0.49pm0.04$，该指数与分形维数相关。圆二色谱检测结果显示，在低pH条件下单克隆抗体的β折叠含量显著升高，同时经硫黄素T（Thioflavin T, ThT）染色的样品荧光强度大幅增强。综上，单克隆抗体的凝胶化过程似乎是通过淀粉样原纤维的形成引发的，该过程所对应的连续相转变可通过渗滤动态标度模型得到良好的描述。