Characterizing various monoclonal antibodies with milder reversed phase chromatography conditions

Reversed phase liquid chromatography (RPLC) of therapeutic monoclonal antibodies (mAbs) is often performed at elevated temperatures (80-90 °C) and in the presence of relatively high concentrations of TFA (0.1%). Under such conditions, it is possible to achieve suitable performance in terms of peak shapes and recoveries. Yet, it is also possible, to cause on-column hydrolysis and the generation of artefact peaks. Interestingly, a wide-pore silica-based superficially porous (SPP) material with a high coverage phenyl bonding was recently introduced, and may offer a chance to perform protein RPLC with milder conditions (lower temperatures and lower TFA concentrations). To evaluate this possibility, 23 mAbs approved by Food and Drug Administration (FDA) and European Medicines Agency (EMA) were analysed on this new column, as well as a reference C4 SPP widepore silica-based column. Separations were performed at various temperatures ranging from 60 to 90 °C using various proportions of TFA and FA ranging from 0.1% TFA to 0.01% TFA/0.09% FA. It appears that temperature can be reduced down to 75 °C for intact mAbs and 65 °C for mAb subunits, using the high coverage phenyl bonded stationary phase. At such temperatures, desirable peak shape and >90% recovery can be observed for each of the studied mAbs. To achieve the same performance with the reference C4 bonded stationary phase, it was necessary to work at 90 °C and 85 °C, respectively. In addition, for mAb subunit analysis, it has been found that combining the phenyl bonded stationary phase with a 0.03% TFA/0.07% FA mobile phase can yield separations similar those obtained with 0.1% TFA, but with an increase of mass spectrometric sensitivity by about 40%. This work provides examples of milder conditions (65 °C and 0.03% TFA + 0.07% FA) being successfully employed in the RPLC(-MS) analysis of mAb subunits. A suitable stationary phase was selected, and milder conditions allow an improved MS sensitivity, while maintaining comparable elution profiles. Moreover, the use of milder conditions may reduce the risk of artefact peaks.