LIQUID-CHROMATOGRAPHIC AND CAPILLARY ELECTROPHORETIC EXAMINATION OF INTACT AND DEGRADED FUSION PROTEIN CTLA4IG AND KINETICS OF CONFORMATIONAL TRANSITION
Kf. Greve et al., LIQUID-CHROMATOGRAPHIC AND CAPILLARY ELECTROPHORETIC EXAMINATION OF INTACT AND DEGRADED FUSION PROTEIN CTLA4IG AND KINETICS OF CONFORMATIONAL TRANSITION, Journal of chromatography, 723(2), 1996, pp. 273-284
Citations number
10
Categorie Soggetti
Chemistry Analytical","Biochemical Research Methods
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.