Hydrophobic interaction chromatography coupled with dynamic surface tension detection for the determination of surface active species in protein formulations

Abstract

The measurement and analysis of surface active species (surfactants) in protein formulations by hydrophobic interaction chromatography (HIC) coupled with gradient elution and dynamic surface tension detection (DSTD) is presented. The DSTD is based upon the measurement of the pressure inside a repeating drop formed when the eluate of a liquid chromatographic separation flows through the end of a pointed stainless steel capillary tube. The DSTD provides a measure of the dynamic surface tension at the air–liquid interface, and in some applications, information on adhesive interactions at the solid–liquid interface. A method for extraction of the desired surface tension signal from the raw pressure data is reported. High-salt gradient HIC separations are demonstrated and shown to be compatible with DSTD. With the gradient pumping system, optimum signal sensitivity and stability (low noise) were achieved when the pressure measurement is taken when the drop size is a minimum. Implementation of HIC–DSTD with salt gradient elution revealed that the noise of DSTD was slightly higher in the gradient mode, due to higher flow-rate imprecision with a dual pump system, as compared to employing isocratic elution. The combination of HIC with DSTD provides a selective methodology for the analysis of surface active species in commercially available protein formulations. Calibration of the HIC–DSTD instrument was achieved using surface active and/or surface tension lowering standards (2-propanol, 1-hexanesulfonic acid, and N-decyl- N, N-dimethyl-3-ammonio-1-propane-sulfonate), that were separated by gradient HIC with sequential ultraviolet (UV) absorbance detection (at 250 nm) and DSTD. The standards were detected only by DSTD. A commercial ribonuclease A formulation was analyzed. The data revealed one primary peak by UV absorbance detection, while two significant peaks with retention times earlier than the primary UV absorbance peak were observed with the DSTD. The identity of the primary UV absorbing peak was confirmed as ribonuclease A by enzyme assay. The first two surface active species observed with the DSTD show no enzyme activity. The utility of the DSTD coupled with HIC separations is demonstrated, opening several new avenues of investigation for the researcher interested in gaining a new perspective on the surface active species in complex biological samples.