ArticlemAbs
Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis.
Article in mAbs. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers.
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Who cites it
18 citing papers in PubMed, 51 citations in OpenAlex.
- Article
- Native Top-Down Mass Spectrometry Combined with High-Resolution Charge Variant Analysis of Trastuzumab Originator and Biosimilars.Journal of the American Society for Mass Spectrometry · 2026Article
- Article
- Developments in Ion Exchange Chromatography-Mass Spectrometry for the Characterization of Intact Proteins and Proteoforms.Journal of separation science · 2025Review
- Enzymatic Desialylation Enables Reliable Charge Variant Characterization of Highly Glycosylated and Sialylated Fc Fusion Proteins.ACS pharmacology & translational science · 2025Article
- Identification of Surfactant Impact on a Monoclonal Antibody Characterization via HPLC-Separation Based and Biophysical Methods.Pharmaceutical research · 2024Article
- Article
- Quantitative Recoveries of Exosomes and Monoclonal Antibodies from Chinese Hamster Ovary Cell Cultures by Use of a Single, Integrated Two-Dimensional Liquid Chromatography Method.Analytical chemistry · 2023Article
- Top-Down Proteomics and the Challenges of True Proteoform Characterization.Journal of proteome research · 2023Review
- Enhancing Selectivity of Protein Biopharmaceuticals in Ion Exchange Chromatography through Addition of Organic Modifiers.International journal of molecular sciences · 2023Article
- Challenges and Strategies for a Thorough Characterization of Antibody Acidic Charge Variants.Bioengineering (Basel, Switzerland) · 2022Article
- Method Development and Qualification of pH-Based CEX UPLC Method for Monoclonal Antibodies.Biotech (Basel (Switzerland)) · 2022Article
- Multi attribute method implementation using a High Resolution Mass Spectrometry platform: From sample preparation to batch analysis.PloS one · 2022Article
- Rapid Analysis of Biotherapeutics Using Protein A Chromatography Coupled to Orbitrap Mass Spectrometry.Analytical chemistry · 2021Article
- Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics.International journal of molecular sciences · 2021Article
- Combining ion chromatography with mass spectrometry and inductively coupled plasma-mass spectrometry: Annual review 2020.Analytical science advances · 2021Review
- Toward Drug-Like Multispecific Antibodies by Design.International journal of molecular sciences · 2020Review
- Article
Corrections and comments
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Recently, cation exchange chromatography (CEX) using aqueous volatile buffers was directly coupled with mass spectrometry (MS) and applied for intact analysis of therapeutic proteins and antibodies. In our study, chemical modifications responsible for charge variants were identified by CEX-UV-MS for a monoclonal antibody (mAb), a bispecific antibody, and an Fc-fusion protein. We also report post-CEX column addition of organic solvent and acid followed by mixing at elevated temperatures, which unfolded proteins, increased ion intensity (sensitivity) and facilitated top-down analysis. mAb stressed by hydrogen peroxide oxidation was used as a model system, which produced additional CEX peaks. The on-line CEX-UV-MS top-down analysis produced gas-phase fragments containing one or two methionine residues. Oxidation of some methionine residues contributed to earlier (acidic), some to later (basic) eluting peaks, while oxidation of other residues did not change CEX elution. The abundance of the oxidized and non-oxidized fragment ions also allowed estimation of the oxidation percentage of different methionine residues in stressed mAb. CEX-UV-MS measurement revealed a new intact antibody proteoform at 5% that eluted as a basic peak and included paired modifications: high-mannose glycosylation and remaining C-terminal lysine residue (M5/M5 + K). This finding was confirmed by peptide mapping and on-column disulfide reduction coupled with reversed-phase liquid chromatography - top-down MS analysis of the collected basic peak. Overall, our results demonstrate the utility of the on-line method in providing site-specific structural information of charge modifications without fraction collection and laborious peptide mapping.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.