Utility of isotachophoresis-capillary zone electrophoresis, mass spectrometry and high-performance size-exclusion chromatography for monitoring of interleukin-6 dimer formation

utility of isotachophoresis-capillary zone electrophoresis (ITP-CZE) and hi gh-performance size-exclusion chromatography (HPSEC) was investigated for d etermination of dimeric and monomeric recombinant human interleukin-6 (rhIL -6). Using ITP-CZE heterogeneity of dimeric rhIL-6 could be revealed resolv ing two peaks in the electropherograms, while with HPSEC dimeric rhIL-6 elu ted as one homogeneous fraction. Both protein forms were monitored during i ncubation of monomeric rhIL-6 at different pH and temperature. The selectiv ity of counterflow ITP-CZE in conjunction with the low concentration determ ination limits enabled reanalysis of HPSEC fractions for identification of the dimer in the electropherograms. Both ITP-CZE and HPSEC were shown to be suitable to monitor the dimerization of rhIL-6, similar monomer-to-dimer p eak area ratios were obtained throughout the incubation. Dimer formation ki netics increased with decreasing pH and with increasing temperature, it was entirely suppressed at neutral pH and room temperature. in contrast to HPS EC, ITP-CZE enabled separation of further still unidentified artifacts appa rently formed during incubation of rhIL-6. CZE analysis in conjunction with electrospray ionization mass spectrometry revealed the non-covalent bindin g character of the dimeric rhIL-6 complex and facilitated interpretation of the electropherograms. (C) 1999 Elsevier Science B.V. All rights reserved.