American Chemical Society
ac401455f_si_001.pdf (1.41 MB)

Nondenaturing Size-Exclusion Chromatography-Mass Spectrometry to Measure Stress-Induced Aggregation in a Complex Mixture of Monoclonal Antibodies

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journal contribution
posted on 2013-07-02, 00:00 authored by Jonathan Woodard, Hollis Lau, Ramil F. Latypov
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.