Light scattering coupled with reversed phase chromatography to study protein self-association under separating conditions

• New in-line method using reversed phase chromatography and static light scattering.
• Measurements can be used to investigate association across the elution peak.
• The refractive index increments were strongly depending on the solvent used.
• Insulin is an excellent reference molecule for molecular mass determinations in water and mixed solvents.

An on-line method, coupling reversed phase chromatography with static light scattering, was developed to determine the association state of freshly eluted proteins. Under downstream process conditions, human insulin desB30 and human insulin AspB28 were tested at concentrations up to 8.5 mg/mL. The refractive index increment (dn/dc) for insulin was found to depend strongly on the solvent used. A refractive index increment of 0.184 ± 0.003 mL/g was found in an aqueous buffer, pH 7.4, whereas the value was 0.155 ± 0.003 mL/g in 30%, w/w ethanol. The methodology combines on-line SLS and UV measurements with the pre-determined refractive index increment values. The developed on-line method was verified by standard off-line measurements establishing the association state at concentrations between 0.2 and 6.0 mg/mL. The equipment was calibrated utilizing insulin under conditions reported to ensure either monomer or hexamer forms. The self-association of human insulin desB30 was found to be strongly suppressed in 30%, w/w ethanol at pH 7.4 in which the monomer predominates. When stabilized by zinc ions in 30%, w/w ethanol at pH 7.4, an average association number of 3.7 was found. These data demonstrate the effect of ethanol to lower strongly the energy advantage by protein self-association. Potassium chloride and/or calcium chloride in the eluents were found to be of no consequence to the association state.