Liquid chromatographic and capillary electrophoretic examination of intact and degraded fusion protein CTLA4Ig and kinetics of conformational transition

Methods have been developed for the CE and HPLC analysis of CTLA4Ig, an immunoglobulin fusion protein. Two different LC approaches, size-exclusion (SEC) and ''mixed-mode'' ion-exchange (ABx), were developed along with a CE method that uses a micellar electrokinetic chromatographic buffer consisting of berate ions, sodium dodecyl sulfate and acetonitrile. These assays measure the presence of several CTLA4Ig-related species, and the observed changes resulting from multiple modes of degradation. In an attempt to identify possible degradation products, collections were taken from the ABx and SEC liquid chromatographic systems and further analyzed by matrix-assisted laser desorption time-of-flight (MALDI TOF) mass spectrometry. Multiple species were detected covering a wide molecular mass range. In addition, the CE method was used to study conformational kinetics between two forms of CTLA4Ig and to estimate the activation energy of the conformer-conformer transition. Pseudo-first-order reaction kinetics were demonstrated for CTLA4Ig samples stressed with papain, H2O2, and sodium dodecyl sulfate/heat.