Evaluation of strategies for overcoming trifluoroacetic acid ionization suppression resulted in single-column intact level, middle-up, and bottom-up reversed-phase LC-MS analyses of antibody biopharmaceuticals

• Published in: Talanta (Oxford) 2021-10, Vol.233, p.122512-122512, Article 122512
• Main Authors: Khalikova, Maria A., Skarbalius, Laimonas, Naplekov, Denis K., Jadeja, Siddharth, Švec, František, Lenčo, Juraj
• Format: Article
• Language: English
• Subjects: Ionization suppression; LC-MS; Multilevel analyses; Protein biopharmaceuticals; Separation efficiency; Therapeutic monoclonal antibodies; Trifluoroacetic acid
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2021.122512

A wide range of strategies for efficient chromatography and high MS sensitivity in reversed-phase LC-MS analysis of antibody biopharmaceuticals and their large derivates has been evaluated. They included replacing trifluoroacetic acid with alternative acidifiers, relevancy of elevated column temperature, use of dedicated stationary phases, and counteraction of the suppression effect of trifluoroacetic acid in electrospray ionization. At the column temperature of 60 °C, which significantly reduces in-column protein degradation, the BioResolve RP mAb Polyphenyl, BioShell IgG C4 columns performed best using mobile phases with full or partial replacement of trifluoroacetic acid with difluoroacetic acid in the analysis of intact antibodies. Similarly, 0.03% trifluoroacetic acid in combination with 0.07% formic acid is a good alternative in analyzing antibody chains at 60 °C. Collectively, the addition of 3% 1-butanol to the mobile phase acidified with 0.1% formic acid was the most efficient approach to simultaneously achieving good chromatographic separation and MS sensitivity for intact and reduced antibody biopharmaceuticals. Moreover, this mobile phase combined with the BioResolve RP mAb Polyphenyl column was subsequently demonstrated to provide excellent results for peptide mapping of antibody biopharmaceuticals fully comparable with those obtained using a state-of-the-art column for peptide separation, thus opening an avenue for a single-column multilevel analysis of these biotherapeutics. [Display omitted] •Strategies to overcome TFA-related ionization suppression were thoroughly evaluated.•A method for safe efficient chromatography and high MS sensitivity of mAbs was determined.•Analysis of intact and reduced mAbs without changes in LC-MS settings was proved at 60 °C.•Polyphenyl stationary phase allows single-column analyses of mAbs at various mass levels.