posted on 2013-07-02, 00:00authored byJonathan 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.