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12 used three of these approaches (in-gel digestion, RPLC fractionation, and gel-free fractionation) to investigate an unknown 10-kDa fragment and a concomitant shoulder of the monomer peak in nonreduced CE-SDS (nrCE-SDS) of a heat-stressed mAb.įragmentation is a well-characterized degradation pathway of therapeutic antibodies and is usually monitored by capillary electrophoresis–sodium dodecyl sulfate (CE-SDS).

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Owing to difficulties in direct fraction collection or online coupling to a mass spectrometer, studies on CE-SDS peak identification largely rely on: 1) prior knowledge of possible species under certain conditions 2) sodium dodecyl sulfate-polyacrylamide gel electrophoreses (SDS-PAGE) separation, gel band excision, and in-gel digestion peptide mapping 3) gel-free fractionation, intact mass, and peptide mapping 12 4) SEC-based fractionation with offline intact mass, peptide mapping, and CE-SDS 10,13,14 and 5) reversedphase liquid chromatography (RPLC) based fractionation, intact mass, top-down tandem mass spectrometry (MS/MS), or offline peptide mapping and CE-SDS.

In contrast, capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) methods are better suited for fragment quantitation at all stages of pharmaceutical development for lot release, stability, in-process testing, characterization, and investigations.