In situ probing of insulin aggregation in chromatography effluents with spectroturbidimetry

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 lambda(-4). Insulin at 25 degrees C in 3.5 N acetic acid is mainly monomeric (non-aggregated). At 25 degrees 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 degrees 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 degrees C, insulin exists mainly in monomer form. In situ probing of size exclusion chromatography, SEC, effluents in 3.5 N acetic acid at 4 degrees 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 degrees 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 degrees 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 degrees C, n(m) ranges from 14 to 27, in order of elution. Analytical HPLC results at 25 degrees C imply that the aggregates are reversible.