Influence of pressure on the chromatographic behavior of insulin variants under nonlinear conditions

The effect of pressure on the chromatographic behavior of two insulin variants in RPLC was investigated on a YMC-ODS C18 column, under nonlinear conditions. The adsorption isotherm data of porcine insulin and Lispro were measured at average column pressures ranging from 52 to 242 bar. These data fit well to the Toth and the bi-Langmuir isotherm models. The saturation capacity increases rapidly with increasing pressure while the affinity (or equilibrium) constant and the parameter characterizing the surface heterogeneity decrease. It is noteworthy that the distribution coefficient of the insulin variants increases with increasing pressure whereas their equilibrium constant b decreases for porcine insulin and increases for Lispro. The association constant b(ds), which characterizes the adsorption and desorption equilibrium of insulin in the system, increases with increasing pressure. The excellent agreement between the experimental overloaded profiles recorded under different pressures and those calculated using the POR model suggests that the chromatographic behavior of insulin is controlled more by equilibrium thermodynamics than by the mass transfer kinetics. The latter seems to be nearly independent of the average column pressure. Thus, increasing the average column pressure is an efficient, albeit costly, way to increase the loading capacity of the column, hence the production rate in preparative chromatography.