Mechanistic Modeling of Reversed-Phase Chromatography of Insulins with Potassium Chloride and Ethanol as Mobile-Phase Modulators

Abstract

The purpose of this study was to investigate the adsorption mechanism in reversed-phase chromatography (RPC) of proteins and to develop a model for the effect of dual mobile phase modulators—a salt and an organic solvent—on this process. Two different adsorption mechanisms were considered: (1) pure association of a protein molecule and one or more ligands and (2) displacement of the organic modulator, with which the adsorbent is saturated, by the protein upon association with one or more ligands. One model was then derived from each of the two considered mechanisms, combining thermodynamic theories on salting-in, RPC, and the solubility of proteins. The model was then applied to chromatographic data from an earlier report as well as supplementary data for solubility and vapor–liquid equilibria, and case-specific simplifications were made. We found that an adaptation of Kirkwood’s electrostatic theories to hydrophobic interaction chromatography describes the observed effect of KCl well. Combining chromatographic and solubility data for one of the insulins, we concluded that the variation in the activity coefficient of the insulin with respect to the concentration of ethanol alone cannot describe its effect on retention. Consequently, one or more other phenomena must affect the adsorption process. Our second model fits the retention data well, supporting the hypothesis that ethanol is directly involved in the adsorption mechanism in this case. Using additional experiments at a high-protein load, we extended the linear-range equilibrium model into a dynamic model for preparative conditions. This model shows good agreement with the high-load data for one of the insulin variants, without any additional effects of the modulator concentrations on the adsorption capacity.

Model‐based monitoring of industrial reversed phase chromatography to predict insulin variants

“…Mechanistic models of reversed phase chromatography have been extensively published, including the behavior of insulin in RPC. Most studies have investigated parameters influencing the efficiency of the RPC, such as column materials, elution conditions, different gradients, various buffer, and temperature conditions . As monitoring has not been the focus in those studies typically one protein is simulated, or mixtures with a few selected proteins, but not real process material and validations have been rarely performed.…”

“…As monitoring has not been the focus in those studies typically one protein is simulated, or mixtures with a few selected proteins, but not real process material and validations have been rarely performed. However, the elution behavior in RPC can be easily affected, especially by the abovementioned parameters, for example, the effect of potassium chloride or ethanol on the retention …”

“…Most studies have investigated parameters influencing the efficiency of the RPC, such as column materials, elution conditions, different gradients, various buffer, and temperature conditions. 9,34,35 As monitoring has not been the focus in those studies typically one protein is simulated, or mixtures with F I G U R E 6 Finding the optimal pooling criteria based on constraints. a) Constraint 1 of Variant A Insulin < 0:03% at all pooling criteria options ν 1 and ν 2 .…”

“…However, the elution behavior in RPC can be easily affected, especially by the abovementioned parameters, for example, the effect of potassium chloride or ethanol on the retention. 9 As demonstrated by Degerman et al 29 and Johansson et al 37 when the UV absorbance is used for model calibration to simulate artificial mixtures of product and impurities, the UV absorbance profile needs to display well pronounced shoulders to allow the simulation of the impurities. 29,37 Even then, the prediction of variants might still be challenging.…”

Monitoring of product variants in biopharmaceutical downstream processing : Mechanistic and data-driven modeling approaches

“…Mechanistic models of the various chromatography systems, such as ion exchange, hydrophobic interaction, and a nity, have been extensively published for process development [105][106][107][108][109][110][111][112][113][114]. Also, reversed phase chromatography and the separation of insulin on RPC have been modeled to evaluate column material, bu ers and temperature e ects [115][116][117]. Usually, mechanistic models are calibrated to the UV absorbance which needs to display a well-pronounced shoulder to model the impurity [82,118].…”