Evaluation of a new wide pore core–shell material (Aeris™ WIDEPORE) and comparison with other existing stationary phases for the analysis of intact proteins

Abstract

The separation of large biomolecules such as proteins or monoclonal antibodies (mAbs) by RPLC can be drastically enhanced thanks to the use of columns packed with wide-pore porous sub-2μm particles or shell particles. In this context, a new wide-pore core–shell material has been recently released under the trademark Aeris WIDEPORE. It is made of a 3.2μm solid inner core surrounded by a 0.2μm porous layer (total particle size of 3.6μm). The aim of this study was to evaluate the performance of this new material, compare it to other recently developed and older conventional wide-pore columns and demonstrate its applicability to real-life separations of proteins and mAbs. At first, the traditional hmin values of the Aeris WIDEPORE column were determined for small model compounds. The hmin values were equal to 1.7–1.8 and 1.4 for the 2.1 and 4.6mm I.D. columns, respectively, which are in agreement with the values reported for other core–shell materials. In the case of a small protein Insulin (5.7kDa), the achievable lowest h value was below 2 and this impressive result confirms that the Aeris WIDEPORE material should be dedicated to protein analysis. This column was then compared with five other commercially available wide-pore and medium-pore stationary phases, in the gradient elution mode, using various flow rates, gradient steepness and model proteins of MW=5.7–66.8kDa. The Aeris WIDEPORE material often provided the best performance, in terms of peak capacity, peak capacity per time and pressure unit (PPT) and also based on the gradient kinetic plot representation. Finally, real separations of filgrastim (18.8kDa) and its oxidized and reduced forms were performed on the different columns and the Aeris WIDEPORE material provided the most impressive performance (peak capacity>100 for tgrad<6min). Last but not least, this new material was also evaluated on digested and reduced mAb and powerful, high-throughput separations were also attained.