ArticleThe AAPS journal2012
Conformational analysis of therapeutic proteins by hydroxyl radical protein footprinting.
Article in The AAPS journal, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 30 papers.
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Who cites it
30 citing papers in PubMed.
- Multiplex Trifluoromethyl and Hydroxyl Radical Chemistry Enables High-Resolution Protein Footprinting.Analytical chemistry · 2025Article
- Mapping conformational changes on bispecific antigen-binding biotherapeutic by covalent labeling and mass spectrometry.Journal of pharmaceutical analysis · 2024Article
- Basic regulatory science behind drug substance and drug product specifications of monoclonal antibodies and other protein therapeutics.Journal of pharmaceutical analysis · 2024Review
- Multiplex Chemical Labeling of Amino Acids for Protein Footprinting Structure Assessment.Analytical chemistry · 2022Article
- Complementary Structural Information for Antibody-Antigen Complexes from Hydrogen-Deuterium Exchange and Covalent Labeling Mass Spectrometry.Journal of the American Society for Mass Spectrometry · 2022Article
- Carbocation Footprinting of Soluble and Transmembrane Proteins.Analytical chemistry · 2021Article
- Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins.Journal of visualized experiments : JoVE · 2021Article
- Covalent Labeling/Mass Spectrometry of Monoclonal Antibodies with Diethylpyrocarbonate: Reaction Kinetics for Ensuring Protein Structural Integrity.Journal of the American Society for Mass Spectrometry · 2020Article
- Higher-Order Structure Influences the Kinetics of Diethylpyrocarbonate Covalent Labeling of Proteins.Journal of the American Society for Mass Spectrometry · 2020Article
- Rapid Quantification of Peptide Oxidation Isomers From Complex Mixtures.Analytical chemistry · 2020Article
- Compensated Hydroxyl Radical Protein Footprinting Measures Buffer and Excipient Effects on Conformation and Aggregation in an Adalimumab Biosimilar.The AAPS journal · 2019Article
- Covalent Labeling with Diethylpyrocarbonate: Sensitive to the Residue Microenvironment, Providing Improved Analysis of Protein Higher Order Structure by Mass Spectrometry.Analytical chemistry · 2019Article
- Article
- Fast Photochemical Oxidation of Proteins Coupled with Mass Spectrometry.Protein and peptide letters · 2019Review
- Effect of freeze drying on stability, thermo-responsive characteristics, andResearch in pharmaceutical sciences · 2018Article
- Towards high-throughput fast photochemical oxidation of proteins: Quantifying exposure in high fluence microtiter plate photolysis.Analytical biochemistry · 2018Article
- Real Time Normalization of Fast Photochemical Oxidation of Proteins Experiments by Inline Adenine Radical Dosimetry.Analytical chemistry · 2018Article
- Quality Comparison of Biosimilar and Copy Filgrastim Products with the Innovator Product.Pharmaceutical research · 2018Article
- Covalent labeling-mass spectrometry with non-specific reagents for studying protein structure and interactions.Methods (San Diego, Calif.) · 2018Review
- Protein Footprinting Comes of Age: Mass Spectrometry for Biophysical Structure Assessment.Molecular & cellular proteomics : MCP · 2017Review
Corrections and comments
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Authors and funding
2 authors.
Funding
Abstract
Unlike small molecule drugs, therapeutic protein pharmaceuticals must not only have the correct amino acid sequence and modifications, but also the correct conformation to ensure safety and efficacy. Here, we describe a method for comparison of therapeutic protein conformations by hydroxyl radical protein footprinting using liquid chromatography-mass spectrometry (LC-MS) as an analytical platform. Hydroxyl radical protein footprinting allows for rapid analysis of the conformation of therapeutic proteins based on the apparent rate of oxidation of various amino acids by hydroxyl radicals generated in situ. Conformations of Neupogen®, a patented granulocyte colony-stimulating factor (GCSF), were compared to several expired samples of recombinant GCSF, as well as heat-treated Neupogen®. Conformations of different samples of the therapeutic proteins interferon α-2A and erythropoietin were also compared. Differences in the hydroxyl radical footprint were measured between Neupogen® and the expired or mishandled GCSF samples, and confirmed by circular dichroism spectroscopy. Samples that had identical circular dichroism spectra were also found to be indistinguishable by hydroxyl radical footprinting. The method is applicable to a wide variety of therapeutic proteins and formulations through the use of separations techniques to clean up the protein samples after radical oxidation. The reaction products are stable, allowing for flexibility in sample handling, as well as archiving and reanalysis of samples. Initial screening can be performed on small amounts of therapeutic protein with minimal training in LC-MS, but samples with structural differences from the reference can be more carefully analyzed by LC-MS/MS to attain higher spatial resolution, which can aid in engineering and troubleshooting.
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