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.
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