Characterization of recombinant cytokine fragments using isotachophoresis-capillary zone electrophoresis, reversed-phase high performance liquid chromatography, and mass spectrometry

Purpose. Capillary zone electrophoresis with isotachophoretic sample precon centration (ITP-CZE) and reversed-phase high performance liquid chromatogra phy (RP-HPLC) with UV detection and on-line coupling to electrospray-ioniza tion mass spectrometry were investigated for their potential to separate an d identify fragments of recombinant human interleukin-6 formed during acidi c stress of the parent protein. Results. Based on the orthogonal separation principles governing ITP-CZE an d RP-HPLC, different peak patterns were observed using both methods. The se lectivity of ESI-MS allowed identification of several co-migrating compound s. Data obtained by on-line ESI-MS were compared to results from off-line i nvestigations by MALDI-TOF-MS performed with single fractions collected fro m the RP-HPLC system. Cleavage of the protein backbone occurred preferably at acid-labile Asp-sites. The total amount of rhIL-6 needed for ITP-CZE-ESI -MS identification of all fragments was only in the upper femtomole range, while RP-HPLC required amounts of protein three orders of magnitude higher. On the other hand, the low CE sample volume opposes the collection of frac tions to perform off-line analysis. Conclusions. Growing acceptance of CE with on-line MS detection for pharmac eutical quality control of proteins is expected.