Identifying protein aggregation mechanisms and quantifying aggregation rates from combined monomer depletion and continuous scattering

Abstract

Parallel temperature initial rates (PTIR) from chromatographic separation of aggregating protein solutions are combined with continuous simultaneous multiple sample light scattering (SMSLS) to make quantitative deductions about protein aggregation kinetics and mechanisms. PTIR determines the rates at which initially monomeric proteins are converted to aggregates over a range of temperatures, under initial-rate conditions. Using SMSLS for the same set of conditions provides time courses of the absolute Rayleigh scattering ratio, IR(t), from which a potentially different measure of aggregation rates can be quantified. The present report compares these measures of aggregation rates across a range of solution conditions that result in different aggregation mechanisms for anti-streptavidin (AS) immunoglobulin gamma-1 (IgG1). The results illustrate how the two methods provide complementary information when deducing aggregation mechanisms, as well as cases where they provide new mechanistic details that were not possible to deduce in previous work. Criteria are presented for when the two techniques are expected to give equivalent results for quantitative rates, the potential limitations when solution non-idealities are large, as well as a comparison of the temperature dependence of AS-IgG1 aggregation rates with published data for other antibodies.

Keywords: Aggregation mechanisms; Aggregation rates; Laser scattering; Protein stability.