Characterization of high-molecular-weight nonnative aggregates and aggregation kinetics by size exclusion chromatography with inline multi-angle laser light scattering.

摘要

Size exclusion chromatography with an inline multi-angle light scattering detector (SEC-MALS) was assessed as a means to characterize and monitor the formation of soluble, high-molecular-weight (HMW) protein aggregates so as to better quantify and model nonnative aggregation kinetics. Assay configuration and robustness were tested with respect to sample preparation, column type, and assay variability. Independent comparison of SEC-MALS with batch light scattering analysis indicates good agreement between the two methods. Weight-average molecular weight (M(w)), radius of gyration (R(g)), apparent polydispersity, and mass fraction monomer (m) together are shown to provide qualitative and quantitative experimental signatures to distinguish high-MW aggregate growth via chain polymerization versus that via aggregate-aggregate condensation. Mechanistic treatment of aggregation kinetics monitored by SEC-MALS is illustrated by data regression using a recently developed Lumry-Eyring Nucleated Polymerization model that explicitly accounts for aggregate nucleation and competing growth via chain- and condensation-polymerization. The combination of time-dependent M(w) and m data are shown to provide a convenient and robust means to separate and quantify characteristic time scales or rate coefficients for concurrent stages of irreversible aggregation. In addition, the scaling of R(g) with M(w) for HMW aggregates provides useful insights into aggregate morphology and mechanisms of aggregate growth.