Utility of isotachophoresis-capillary zone electrophoresis, mass spectrometry and high-performance size-exclusion chromatography for monitoring of interleukin-6 dimer formation.
The utility of isotachophoresis-capillary zone electrophoresis (ITP-CZE) and high-performance size-exclusion chromatography (HPSEC) was investigated for determination of dimeric and monomeric recombinant human interleukin-6 (rhIL-6). Using ITP-CZE heterogeneity of dimeric rhIL-6 could be revealed resolving two peaks in the electropherograms, while with HPSEC dimeric rhIL-6 eluted as one homogeneous fraction. Both protein forms were monitored during incubation of monomeric rhIL-6 at different pH and temperature. The selectivity of counterflow ITP-CZE in conjunction with the low concentration determination 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 peak area ratios were obtained throughout the incubation. Dimer formation kinetics increased with decreasing pH and with increasing temperature, it was entirely suppressed at neutral pH and room temperature. In contrast to HPSEC, ITP-CZE enabled separation of further still unidentified artifacts apparently formed during incubation of rhIL-6. CZE analysis in conjunction with electrospray ionization mass spectrometry revealed the non-covalent binding character of the dimeric rhIL-6 complex and facilitated interpretation of the electropherograms.
Gysler
J
J
Leiden/Amsterdam Center for Drug Research, Division of Analytical Chemistry, The Netherlands. gysler@chem.leidenuniv.nl
Mazereeuw
M
M
Helk
B
B
Heitzmann
M
M
Jaehde
U
U
Schunack
W
W
Tjaden
U R
UR
van der Greef
J
J
D002850:Chromatography, Gel / Q000379:methods*; D019281:Dimerization; D004586:Electrophoresis / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015850:Interleukin-6 / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D013696:Temperature
D015850:Interleukin-6