Prototype sphere-on-sphere silica particles for the separation of large biomolecules

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.