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Structural analysis of a therapeutic monoclonal antibody dimer by hydroxyl radical footprinting
| Content Provider | Scilit |
|---|---|
| Author | Deperalta, Galahad Alvarez, Melissa Bechtel, Charity Dong, Ken McDonald, Ross Ling, Victor |
| Copyright Year | 2013 |
| Description | Hydroxyl radical footprinting is a covalent labeling strategy used to probe the conformational properties of proteins in solution. We describe the first application of this high resolution technique for characterizing the structure of a therapeutic monoclonal antibody (mAb) dimer. As monitored by size-exclusion chromatography (SEC), therapeutic mAbs typically contain small amounts of a dimer species relative to the primary monomeric form in its drug substance or drug product. To determine its structural orientation, a sample enriched in an IgG1 mAb dimer was oxidized by hydroxyl radicals generated by exposure of the aqueous solution to synchrotron X-rays in millisecond timescales. The antibody monomer that served as a control was oxidized in a similar fashion. The oxidized samples were digested with trypsin and analyzed by RP-UHPLC-MS. The footprinting data show that peptides displaying decreased rates of oxidation (i.e., regions of increased protection) in the dimer are localized in the light and heavy chains of the Fab domain. The interface region for the monomers comprising the dimer was thus inferred to be between their Fab arms, allowing us to model two possible theoretical dimer orientations: a head-to-head, single arm-bound Fab-to-Fab dimer, and a head-to-head, double arm-bound Fabʹ2-to-Fabʹ2 dimer. Lower resolution fragment-SEC analysis of the dimer and monomer samples treated with papain or FabRICATOR® enzyme provided complimentary evidence to support the Fab/Fab orientation of the IgG1 dimer. |
| Related Links | http://www.tandfonline.com/doi/pdf/10.4161/mabs.22964?needAccess=true https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564890/pdf |
| ISSN | 19420862 |
| e-ISSN | 19420870 |
| DOI | 10.4161/mabs.22964 |
| Journal | mAbs |
| Issue Number | 1 |
| Volume Number | 5 |
| Language | English |
| Publisher | Informa UK Limited |
| Publisher Date | 2013-01-01 |
| Access Restriction | Open |
| Subject Keyword | Analytical Chemistry Fab-to-fab Fabricator® Fabʹ2-to-fabʹ2 Uhplc-ms Aggregates Higher Order Structure Hydroxyl Radical Footprinting Mass Spectrometry Oxidative Footprinting Protein Conformation Radiolysis Size Exclusion Chromatography (sec) Synchrotron Irradiation Therapeutic Mab |
| Content Type | Text |
| Resource Type | Article |
| Subject | Immunology and Allergy Immunology |
