Potential of hydrophilic interaction chromatography for the analytical characterization of protein biopharmaceuticals

Abstract

A new stationary phase based on wide-pore hybrid silica bonded with amide ligand has been used to explore the utility of HILIC for the analytical characterization of protein biopharmaceuticals. Various, highly-relevant samples were tested, including different insulins, interferon α-2b and trastuzumab. This work shows that HILIC can be successfully employed for the analysis of therapeutic proteins and mAbs, using mobile phase compositions comprised of between 65 and 80% ACN and 0.1% TFA. In terms of elution order and selectivity, these HILIC separations have proven to be highly orthogonal to RPLC, while the kinetic performance remains comparable. In the case of characterizing trastuzumab, HILIC was uniquely able to resolve several important glycoforms at the middle-up level of analysis (fragments of 25-100kDa). Such a separation of glycoforms has been elusive by other separation mechanisms, such as RPLC and IEX. Besides showing orthogonality to RPLC and improved separations of glycoforms, HILIC offers several additional benefits for biopharmaceutical characterization: i) an inherent compatibility with MS, ii) a reduced requirement for very high mobile phase temperatures that are otherwise needed in RPLC to limit undesirably strong adsorption to the surface of the stationary phase, and iii) the possibility to couple several columns in series to improve resolving power, thanks to comparatively low mobile phase viscosity.

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

“…It is worth noting that addition of acetonitrile may potentially effect the conformation and so the retention behaviour of the solute being analyzed. In this case this effect seemed to be negligible, since the chromatograms obtained with 60% organic solvent composition was highly comparable with the reference chromatograms using the initial gradient ramp and aqueous sample solvent [6]. Flourescence emission spectra of peaks eluting close to column dead time were compared to protein peaks and confirmed as not being of protein origin.…”

“…This is due to the high solvent strength of water in HILIC conditions which may decrease the peak focusing effect at the column inlet. In our previous studies, it was shown that a fast and steep gradient ramp at the beginning of the chromatographic run can compensate for solvent incompatibility [6,7]. The initial highly organic mobile phase composition supported the appropriate adsorption of the sample components at the column inlet, which were then eluted in regular, symmetrical peaks.…”

“…Thanks to recent developments in column technology, widepore HILIC phases are now commercially available and enables the separation of peptides, protein fragments and intact proteins with high efficiency [6,7]. Several highly-relevant samples such as insulins, interferon alpha-2b and monoclonal antibody (mAb) have been recently analysed in HILIC [6].…”

“…Several highly-relevant samples such as insulins, interferon alpha-2b and monoclonal antibody (mAb) have been recently analysed in HILIC [6]. It was shown that a mobile phase composition between 80 and 65% acetonitrile in presence of 0.1% trifluoroacetic acid (TFA) provided optimal conditions to retain proteins and obtain appropriate peak shapes.…”

“…HILIC offers several additional benefits for biopharmaceutical characterization, as inherent compatibility with MS (better sensitivity in many cases compared to RPLC [3,11]), the use of moderate mobile phase temperature for several proteins that are poorly recovered in RPLC, and the possibility to couple several columns in series to improve resolving power (peak capacity), thanks to comparatively low mobile phase viscosity [6].…”