Alternative mobile phase additives for the characterization of protein biopharmaceuticals in liquid chromatography – Mass spectrometry

When analyzing large complex protein biopharmaceuticals, ion-pairing agents imparting low pH are widely used as mobile phase additives to improve the chromatographic performance. However, one of most effective in RPLC and HILIC, trifluoroacetic acid (TFA), is known a strong suppressor MS signal limits its use hyphenated techniques. In this study, we evaluated wide range acidic find alternatives TFA that provided comparable performance improved sensitivity. It was observed stronger were required for intact level analysis compared subunit additive nature had larger impact on HILIC mode RPLC. Therefore, four identified valuable mode, namely, difluoroacetic (DFA), dichloroacetic (DClAA), trichloroacetic (TClAA), methanesulfonic (MSA). Only these acceptable TClAA. After evaluation performance, TClAA discarded due apparent loss intensity both RPLC-MS HILIC-MS mode. Together, results demonstrate remains gold standard additive. DFA found promising alternative could play an important role development methods characterization increasingly biopharmaceuticals.