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.

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The goal of this study was to evaluate the possibilities offered by a prototype HPLC column packed with ∼2.5μm narrow size distribution sphere-on-sphere (SOS) silica particles bonded with C4 alkyl chains, for the analytical characterization of large biomolecules. The kinetic performance of this material was evaluated in both isocratic and gradient modes using various model analytes. The data were compared to those obtained on other widepore state-of-the-art fully core-shell and fully porous materials commonly employed to separate proteins moreover to a reference 5μm wide pore material that is still often used in QC labs. In isocratic mode, minimum reduced plate height values of hmin=2.6, 3.3 and 3.3 were observed on butylparaben, decapeptide and glucagon, respectively. In gradient elution mode, the SOS column performs very high efficiency when working with fast gradients. This prototype column was also comparable (and sometimes superior) to other widepore stationary phases, whatever the gradient time and flow rate, when analyzing the largest model protein, namely BSA. These benefits may be attributed to the SOS particle morphology, minimizing the intra-particle mass transfer resistance. Finally, the SOS column was also applied for the analytical characterization of commercial monoclonal antibody (mAb) and antibody-drug conjugate (ADC) samples. With these classes of proteins, the performance of SOS column was similar to the best widepore stationary phases available on the market.

The goal of this work is to understand the phenomenon behind the poor injection repeatability often observed with protein biopharmaceuticals. All the measurements were carried out in size exclusion chromatography (SEC) mode, using fifteen commercially available therapeutic monoclonal antibodies (mAbs). First of all, we proved that the variation of peak areas between consecutive injections was much more critical with highly concentrated mAb samples (up to 80mg/mL), while the SEC measurements of commercial mAbs having concentrations below 5mg/mL were reliable. Second, we emphasized that the shapes, volumes and materials of the sample vial insert also contribute to the change of peak areas observed during consecutive injections. In this study, six different insert models were studied and the most critical were the ones possessing the narrowest conical shape at the bottom. Furthermore, the homogenization of samples (with pipette mixing rather than vortex) prior to analysis was of great interest and allows a significant improvement in injection repeatability. Finally, because the on-wall (on-insert) adsorption of mAbs reaches its equilibrium in around 50min, it is advised to add a specified residence time prior to injection to achieve repeatable injection.