FabRICATOR in an Evaluation of Mobile-phase Additives for LC-MS Characterization of mAbs

Biopharmaceuticals, including monoclonal antibodies (mAbs), have become an important class of therapeutics. The manufacturing procedure of mAbs is complex, and many possible variants of a particular mAb can be generated as a result of enzymatical and chemical modifications. Some of these modifications are critical for the efficacy and safety of the therapeutic mAb and are known as critical quality attributes (CQAs). CQAs need to be thoroughly monitored to ensure the quality and safety of the therapeutic agent.


LC-MS has become the gold standard to characterize mAbs. Two denaturing chromatographic methods are generally applied and coupled to MS; reversed phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC). The solute retention in RPLC is principally controlled by hydrophobic interactions and in HILIC, it is more complex. The differences in retention mechanism between RPLC and HILIC result in differences in elution order and selectivity, making the two approaches highly complementary in the characterization of a mAb. To improve the chromatographic separation, mobile-phase additives are used. However, one of the most effective additives used in RPLC and HILIC, trifluoroacetic acid (TFA) is a strong suppressor of the MS signal. Therefore, its use is often limited in hyphenated techniques.
In this study, a wide range of acidic additives were evaluated in order to find alternatives to TFA with comparable chromatographic performance and improved MS sensitivity. It was found that stronger acidic additives were required for intact level analysis compared to subunit level analysis, and that the nature of the additive had a larger impact on the chromatographic separation in HILIC compared to RPLC. For the subunit level analysis, FabRICATOR (IdeS) was used to digest the mAb into F(ab’)2 and Fc/2 fragments. After reduction, Fd’, LC and sFc fragments were obtained and analyzed using RPLC- and HILIC-MS.
The authors conclude that multiple alternatives for TFA are available for both RPLC and HILIC conditions. However, the coupling to MS requires a careful additive selection by taking into account the required selectivity and MS sensitivity for analyzing the mAb of interest.


Lardeux et al., 2021. Alternative mobile phase additives for the characterization of protein biopharmaceuticals in liquid chromatography – Mass spectrometry. Analytica Chimica Acta doi: 10.1021/acs.analchem.0c04685






FabRICATOR – Cystine protease for digestion of mAbs, ADCs and Fc-fusion proteins at a specific site below the hinge.