Dataset Information

Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry.

ABSTRACT: Charge variant analysis is a widely used tool to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs). Although it is a powerful technique for revealing mAb heterogeneity, an unexpected outcome, for example the appearance of previously undetected isoforms, requires further, time-consuming analysis. The process of identifying these unknowns can also result in unwanted changes to the molecule that are not attributable to the manufacturing process. To overcome this, we recently reported a method combining highly selective cation exchange chromatography-based charge variant analysis with on-line mass spectrometric (MS) detection. We further explored and adapted the chromatographic buffer system to expand the application range. Moreover, we observed no salt adducts on the native protein, also supported by the optimal choice of MS parameters, resulting in increased data quality and mass accuracy. Here, we demonstrate the utility of this improved method by performing an in-depth analysis of adalimumab before and after forced degradation. By combining molecular mass and retention time information, we were able to identify multiple modifications on adalimumab, including lysine truncation, glycation, deamidation, succinimide formation, isomerisation, N-terminal aspartic acid loss or C-terminal proline amidation and fragmentation along with the N-glycan distribution of each of these identified proteoforms. Host cell protein (HCP) analysis was performed using liquid chromatography-mass spectrometry that verified the presence of the protease Cathepsin L. Based on the presence of trace HCPs with catalytic activity, it can be questioned if fragmentation is solely driven by spontaneous hydrolysis or possibly also by enzymatic degradation.

SUBMITTER: Fussl F

PROVIDER: S-EPMC6343805 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry.

Füssl Florian F Trappe Anne A Cook Ken K Scheffler Kai K Fitzgerald Oliver O Bones Jonathan J

mAbs 20181111 1

Charge variant analysis is a widely used tool to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs). Although it is a powerful technique for revealing mAb heterogeneity, an unexpected outcome, for example the appearance of previously undetected isoforms, requires further, time-consuming analysis. The process of identifying these unknowns can also result in unwanted changes to the molecule that are not attributable to the manufacturing process. To ...[more]

PMID: 30296204

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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

Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry.

Publications

Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry.

Similar Datasets

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