Microrheology of Monoclonal Antibodies during Gelation under Low pH Conditions

Abstract

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.

Keywords: acetic acid; aggregation; circular dichroism; downstream purification; dynamic light scattering; gel; microrheology; monoclonal antibody; protein A elution; sol−gel transition; thioflavin T; viral inactivation.