PURPOSE: Capillary zone electrophoresis with isotachophoretic sample preconcentration (ITP-CZE) and reversed-phase high performance liquid chromatography (RP-HPLC) with UV detection and on-line coupling to electrospray-ionization mass spectrometry were investigated for their potential to separate and identify fragments of recombinant human interleukin-6 formed during acidic stress of the parent protein. RESULTS: Based on the orthogonal separation principles governing ITP-CZE and RP-HPLC, different peak patterns were observed using both methods. The selectivity of ESI-MS allowed identification of several co-migrating compounds. Data obtained by on-line ESI-MS were compared to results from off-line investigations by MALDI-TOF-MS performed with single fractions collected from 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 fractions to perform off-line analysis. CONCLUSIONS: Growing acceptance of CE with on-line MS detection for pharrmaceutical quality control of proteins is expected.
PURPOSE: Capillary zone electrophoresis with isotachophoretic sample preconcentration (ITP-CZE) and reversed-phase high performance liquid chromatography (RP-HPLC) with UV detection and on-line coupling to electrospray-ionization mass spectrometry were investigated for their potential to separate and identify fragments of recombinant humaninterleukin-6 formed during acidic stress of the parent protein. RESULTS: Based on the orthogonal separation principles governing ITP-CZE and RP-HPLC, different peak patterns were observed using both methods. The selectivity of ESI-MS allowed identification of several co-migrating compounds. Data obtained by on-line ESI-MS were compared to results from off-line investigations by MALDI-TOF-MS performed with single fractions collected from 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 fractions to perform off-line analysis. CONCLUSIONS: Growing acceptance of CE with on-line MS detection for pharrmaceutical quality control of proteins is expected.
Authors: H M Lazarus; E F Winton; S F Williams; D Grinblatt; M Campion; B W Cooper; H Gunn; S Manfreda; R E Isaacs Journal: Bone Marrow Transplant Date: 1995-06 Impact factor: 5.483
Authors: T Andus; T Geiger; T Hirano; H Northoff; U Ganter; J Bauer; T Kishimoto; P C Heinrich Journal: FEBS Lett Date: 1987-08-31 Impact factor: 4.124