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Rapid Characterization of Antibodies via Automated Flow Injection Coupled with Online Microdroplet Reactions and Native-pH Mass Spectrometry.

ABSTRACT: Microdroplet reactions have aroused much interest due to significant reaction acceleration (e.g., ultrafast protein digestion in microdroplets could occur in less than 1 ms). This study integrated a microdroplet protein digestion technique with automated sample flow injection and online mass spectrometry (MS) analysis, to develop a rapid and robust method for structural characterization of monoclonal antibodies (mAbs) that is essential to assess the antibody drug's safety and quality. Automated sequential aspiration and mixing of an antibody and an enzyme (IdeS or IgdE) enabled rapid analysis with high reproducibility (total analysis time: 2 min per sample; reproducibility: ∼2% coefficient of variation). Spraying the sample in ammonium acetate buffer (pH 7) using a jet stream source allowed efficient digestion of antibodies and efficient ionization of resulting antibody subunits under native-pH conditions. Importantly, it also provided a platform to directly study specific binding of an antibody and an antigen (e.g., detecting the complexes mAb/RSFV antigen and F(ab')₂/RSVF in this study). Furthermore, subsequent tandem MS analysis of a resulting subunit from microdroplet digestion enabled localizing post-translational modifications on particular domains of a mAb in a rapid fashion. In combination with IdeS digestion of an antibody, additional tris(2-carboxyethyl)phosphine (TCEP) reduction and N-glycosidase F (PNGase F) deglycosylation reactions that facilitate antibody analysis could be realized in "one-pot" spraying. Interestingly, increased deglycosylation yield in microdroplets was found, simply by raising the sample temperature. We expect that our method would have a high impact for rapid characterization of monoclonal antibodies.

SUBMITTER: Gunawardena HP

PROVIDER: S-EPMC10492509 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Rapid Characterization of Antibodies via Automated Flow Injection Coupled with Online Microdroplet Reactions and Native-pH Mass Spectrometry.

Gunawardena Harsha P HP Ai Yongling Y Gao Jinshan J Zare Richard N RN Chen Hao H

Analytical chemistry 20230119 6

Microdroplet reactions have aroused much interest due to significant reaction acceleration (e.g., ultrafast protein digestion in microdroplets could occur in less than 1 ms). This study integrated a microdroplet protein digestion technique with automated sample flow injection and online mass spectrometry (MS) analysis, to develop a rapid and robust method for structural characterization of monoclonal antibodies (mAbs) that is essential to assess the antibody drug's safety and quality. Automated ...[more]

PMID: 36656670

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Project description:Characterization of charge heterogeneity in monoclonal antibodies (mAbs) is needed during developability assessment and downstream development of drug candidates. Charge heterogeneity can come from post-translational modifications like deamidation, isomerization, and sialylation. Elucidation of charge variants with mass spectrometry (MS) has historically been challenging. Due to the nonvolatility and high ionic strength of conventional buffer systems, labor-intensive offline fractionation followed by MS analysis is routinely used. Here, we describe an alternative strategy that directly couples strong cation exchange (SCX) chromatography to high-resolution Orbitrap MS for online native MS analysis (SCX-MS). A combined pH and salt gradient was used for universal separation of mAbs from a wide range of pI values (6.38 ~ 9.2), including infliximab (Remicade®, chimeric IgG1/kappa), NISTmab (humanized IgG1/kappa) and trastuzumab (Herceptin®, humanized IgG1/kappa), without tailoring of chromatographic profiles. Liquid chromatography and MS parameters were optimized to achieve high-quality spectra and enhanced detection of low abundant species under high flow rate conditions. Genedata Expressionist, a vendor agnostic software, was used for data processing. This integrated strategy allows unbiased characterization of numerous charge variant species and low molecular weight fragments (<0.05%) without post-column flow splitting. The application was further expanded with middle-up approaches for subdomain analysis, which demonstrated the versatility of the strategy for analysis of various construct types. With our analysis of mAbs during developability assessment and forced degradation studies, which aimed at assessing potential critical quality attributes in antibody drug molecules, we provide, for the first time, direct visualization of molecular alterations of mAbs at intact level. Furthermore, strong correlation was observed between this novel MS approach and analysis by capillary isoelectric focusing.

Dataset Information

Rapid Characterization of Antibodies via Automated Flow Injection Coupled with Online Microdroplet Reactions and Native-pH Mass Spectrometry.

Publications

Rapid Characterization of Antibodies via Automated Flow Injection Coupled with Online Microdroplet Reactions and Native-pH Mass Spectrometry.

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