Protein A does not induce allosteric structural changes in an IgG1 antibody during binding
| Autor: | David D. Weis, Douglas D. Richardson, Juan Pablo Rincon Pabon, Brent A. Kochert, Yan-Hui Liu |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
medicine.drug_class
Allosteric regulation Pharmaceutical Science 02 engineering and technology Plasma protein binding Matrix (biology) Monoclonal antibody 030226 pharmacology & pharmacy Mass Spectrometry 03 medical and health sciences 0302 clinical medicine Affinity chromatography medicine Staphylococcal Protein A Proa biology Chemistry Antibodies Monoclonal 021001 nanoscience & nanotechnology biology.organism_classification Biochemistry Immunoglobulin G biology.protein Hydrogen–deuterium exchange 0210 nano-technology Protein A Protein Binding |
| Zdroj: | Journal of pharmaceutical sciences. 110(6) |
| ISSN: | 1520-6017 |
| Popis: | Affinity chromatography is widely used for antibody purification in biopharmaceutical production. Although there is evidence suggesting that affinity chromatography might induce structural changes in antibodies, allosteric changes in structure have not been well-explored. Here, we used hydrogen exchange-mass spectrometry (HX-MS) to reveal conformational changes in the NIST mAb upon binding with a protein A (ProA) matrix. HX-MS measurements of NIST mAb bound to in-solution and resin forms of ProA revealed regions of the CH2 and CH3 domains with increased protection from HX upon ProA binding, consistent with the known ProA binding region. In-solution ProA experiments revealed regions in the Fab with increased HX uptake when the ProA:mAb molar ratio was increased to 2:1, suggesting an allosterically induced increase in backbone flexibility. Such effects were not observed with lower ProA concentration (1:1 molar ratio) or when ProA resin was used, suggesting some kind of change in binding mode. Since all pharmaceutical processes use ProA bound to resin, our results rule out reversible allosteric effects on the NIST mAb during interaction with resin ProA. However, irreversible effects cannot be ruled out since the NIST mAb was previously exposed to ProA during its original purification. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|