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, a n immunoglobulin fusion protein. Two different LC approaches, size-exc lusion (SEC) and ''mixed-mode'' ion-exchange (ABx), were developed alo ng with a CE method that uses a micellar electrokinetic chromatographi c buffer consisting of berate ions, sodium dodecyl sulfate and acetoni trile. These assays measure the presence of several CTLA4Ig-related sp ecies, and the observed changes resulting from multiple modes of degra dation. In an attempt to identify possible degradation products, colle ctions were taken from the ABx and SEC liquid chromatographic systems and further analyzed by matrix-assisted laser desorption time-of-fligh t (MALDI TOF) mass spectrometry. Multiple species were detected coveri ng a wide molecular mass range. In addition, the CE method was used to study conformational kinetics between two forms of CTLA4Ig and to est imate the activation energy of the conformer-conformer transition. Pse udo-first-order reaction kinetics were demonstrated for CTLA4Ig sample s stressed with papain, H2O2, and sodium dodecyl sulfate/heat.