In situ probing of insulin aggregation in chromatography effluents with spectroturbidimetry

Abstract

Probing protein aggregation in situ is quite important for analyzing and developing chromatographic protein purification processes. A spectroturbidimetry method with a photodiode array detector is developed and tested for probing insulin aggregation in solution and determining the aggregation number, n m . All aggregates examined are in the Rayleigh light scattering regime, where the turbidity between 400 and 350 nm is proportional to λ −4 . Insulin at 25 °C in 3.5 N acetic acid is mainly monomeric (non-aggregated). At 25 °C and lower acetic acid concentrations, from 0.1 to 1 N, the average insulin aggregation number n m ranges from 2.9 to 1.6. Aggregates, with n m = 2 – 3 , are found in 2.6 N acetic acid with 20 vol% acetonitrile. In 0.8 N acetic acid with 20 vol% denatured ethanol, n m = 1.2 . At 4 °C, as acetic acid concentration decreases from 3.5 to 0.1 N, n m decreases from 2.4 to 1.8. In 2.8 N acetic acid with 20 vol% denatured ethanol at 4 °C, insulin exists mainly in monomer form. In situ probing of size exclusion chromatography, SEC, effluents in 3.5 N acetic acid at 4 °C shows n m = 1.6 at the fronting portion (a mixture of monomers and dimers or other oligomers) and n m = 1.1 (mostly monomers) at the tailing portion of the main peak. In another example, for LysPro-insulin in reversed phase chromatography at 4 °C, complex elution patterns and broad peaks are due to substantial aggregation. For a linear gradient of acetonitrile from 10 to 60 vol% at 4 °C, n m ranges from 2.2 to 12, in order of elution. For a linear gradient of ethanol from 30 to 50 vol% at 4 °C, n m ranges from 14 to 27, in order of elution. Analytical HPLC results at 25 °C imply that the aggregates are reversible.