Influence of Pressure on the Retention and Separation of Insulin Variants under Linear Conditions

Abstract

The effect of pressure on the retention behavior of insulin variants in RPLC on a YMC-ODS C18 column was investigated under linear conditions. The retention factors of these variants increase nearly 2-fold when the average column pressure is increased from 55 to 250 bar while their separation factors remain nearly unchanged. This effect is explained by a change of the partial molar volume of the insulin variants associated with their adsorption that decreases from -99 to -80 mL/mol for mobile-phase concentrations of acetonitrile increasing from 29 to 33% (v/v). This volume change is much larger than the one observed with low molecular weight compounds. For the same pressure variation, the average number Z of acetonitrile molecules displaced from the protein and the stationary phase upon adsorption increases from 22 to 23.3. The pressure-induced relative increase of the term b[S]/[D0]z (which corresponds to the initial slope of the adsorption isotherm) is approximately twice as large for Lispro than for porcine insulin. Because the binding constant of insulin decreases with increasing pressure, this suggests that the number of binding sites on the stationary phase increases even faster. Finally, it was observed that the column efficiency at flow rates higher than 0.6 mL/min increases slightly with increasing pressure. It is suggested that these observations are also valid for other proteins analyzed in RPLC.

Superficially porous silica particles with wide pores for biomacromolecular separations

“…In contrast, the increase in retention factor for ribonuclease A was a factor of 1.3 with double the flow rate/pressure. This effect has previously been reported [19]. …”

New trends in reversed-phase liquid chromatographic separations of therapeutic peptides and proteins: Theory and applications

“…For large molecules such as proteins, it has been observed that pressure can have a rather strong influence on retention [207][208][209][210][211]. The change of retention can be attributed to the fact that the partial molar volume of the analyte is smaller when it is adsorbed in the stationary phase than when it is in the liquid mobile phase [207][208][209][210][211].…”

“…The change of retention can be attributed to the fact that the partial molar volume of the analyte is smaller when it is adsorbed in the stationary phase than when it is in the liquid mobile phase [207][208][209][210][211]. The conformational change (unfolding or spreading) of a protein molecule upon adsorption is a well-known phenomenon that leads to the exposure of its hydrophobic core [209,210]. At higher pressures, the adsorption of proteins onto the stationary phase is thus more relevant.…”

Mass Transfer Kinetics in the Chromatography of Insulin Variants under Nonlinear Conditions

“…However, if this approach allows an easy separation of proteins, such separations can be modeled only if the isotherm data can be measured accurately, which supposes the possibility to perform their isocratic elution (7). A variety of interferences can be observed, e.g., protein aggregation (8), protein unfolding (9), and isomerization, which prevent the achievement of satisfactory results. Finally, the retention of proteins depends much more on the pressure than that of low molecular weight compounds ( 9 − 11 ).…”

“…In this work, we report on the modeling of the chromatographic behavior of an insulin variant, Lispro, on a conventional packed C 18 ‐bonded silica column, using an acidic solution of acetonitrile in water as the mobile phase. Under the experimental conditions selected for this work, there are no adverse effects due to insulin aggregation (9) while the presence of internal −S−S− bridges blocks the molecule under a globular structure that offers restricted possibility of isomerization. Although it is small (MW = 5808), insulin has a behavior that can be considered as representative of that of many proteins.…”