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Improving Mass Spectral Quality of Monoclonal Antibody Middle-Up LC-MS Analysis by Shifting the Protein Charge State Distribution
- Ding, Wei, Qiu, Difei, Bolgar, Mark S., Miller, Scott A.
- Analytical chemistry 2018 v.90 no.3 pp. 1560-1565
- drugs, gases, liquid chromatography, mass spectrometry, monoclonal antibodies, proteins
- Monoclonal antibodies (mAbs) are experiencing accelerated development in the pharmaceutical industry. Utilization of middle-up LC-MS methodology can provide detailed characterization of mAbs via reduction and/or enzymatic cleavage of the mAb into smaller protein fragments. However, under typical LC-MS conditions, these fragments, especially the more heterogeneous heavy chain, can present charge state distributions (CSD) featuring a severe interference in the low mass-to-charge (m/z) region in the mass spectrum, adversely impacting spectral quality of these proteins and ultimately the deconvoluted mass spectrum. Here, we introduce a novel method to characterize protein fragments by partially reducing mAbs and using acidic mobile phases (MPs) with a trace amount of base additive. Gas-phase charge stripping occurs with the basic MP additive, causing the CSD to shift to a higher m/z region resulting in high-quality mass spectra with enhanced resolution of protein charge states. Subsequently, high-quality deconvoluted spectra and accurate mass measurement of the fragments are achieved. This method has been applied to the intact mass measurement of mAbs and antibody drug conjugates.