Nondenaturing Size-Exclusion Chromatography-Mass Spectrometry to Measure Stress-Induced Aggregation in a Complex Mixture of Monoclonal Antibodies
| Autor: | Jonathan Woodard, Ramil F. Latypov, Hollis Lau |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
chemistry.chemical_classification
Chromatography Chemistry Sample complexity medicine.drug_class Size-exclusion chromatography Stress induced Antibodies Monoclonal Mass spectrometry Monoclonal antibody Mass Spectrometry Analytical Chemistry chemistry.chemical_compound Monomer Enzyme Stress Physiological Chromatography Gel medicine Humans Molecule |
| Zdroj: | Analytical Chemistry. 85:6429-6436 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/ac401455f |
| Popis: | During therapeutic candidate selection, diverse panels of monoclonal antibodies (mAbs) are routinely subjected to various stress conditions, and assayed for biophysical and biochemical stability. A novel high throughput method has been developed to differentiate candidate molecules in a mixture based on their propensity for forming aggregates when subjected to agitation (vortexing) stress. Protein monomers are separated from soluble and insoluble aggregates using size exclusion chromatography, under nondenaturing conditions, and the individual components in the mixture are identified by mass spectrometry and quantitated relative to an unstressed control. An internal standard was added to the mixture after stress, and used to correct for differences in ionization between samples. Treatment of the samples with the enzyme IdeS (FabRICATOR) significantly reduces sample complexity, and allows for a large number of candidate molecules to be assessed in a single analysis. Simple and robust, the method is well suited for measuring relative aggregation propensity (RAP) in conjunction with molecule selection and coformulation development. |
| Databáze: | OpenAIRE |
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