Systematic evaluation of mobile phase additives for the LC–MS characterization of therapeutic proteins

• Various acidic and basic mobile phase additives were evaluated.
• 10 mM Formate buffer pH 3 was found to be the most promising alternative of TFA.
• Moderate adsorption of proteins at the surface of stationary phase was observed.

Trifluoroacetic acid (TFA) is commonly used as mobile phase additive for the analysis of proteins in reversed phase liquid chromatography (RPLC). Due to its interesting features, it provides symmetrical and narrow peak shapes for proteins, but decreases mass spectrometric sensitivity through ion-pairing and spray destabilizing. Since RPLC–MS is an important technique for the characterization of proteins, some alternative MS-compatible mobile phases may be required. The aim of this study was to evaluate various acidic and basic mobile phase additives for the LC–MS analysis of therapeutic proteins possessing molecular weight between 5 and 150 kDa. At the end, 10 mM formate buffer pH 3 was found to be the most promising alternative, since it provided acceptable peak shapes in most cases, together with an average improvement of MS sensitivity by 5-times, compared to TFA.

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