Characterization of recombinant cytokine fragments using isotachophoresis-capillary zone electrophoresis, reversed-phase high performance liquid chromatography, and mass spectrometry.
OBJECTIVE
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.
Gysler
J
J
Division of Analytical Chemistry, Leiden/Amsterdam Center for Drug Research, The Netherlands. gysler@chem.leidenuniv.nl
Helk
B
B
Dambacher
S
S
Tjaden
U R
UR
van der Greef
J
J
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003574:Cytochrome c Group / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D015850:Interleukin-6 / Q000032:analysis* / Q000737:chemistry*; D009113:Muramidase / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D017421:Sequence Analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D003574:Cytochrome c Group; D015850:Interleukin-6; D010446:Peptide Fragments; D011994:Recombinant Proteins; D009113:Muramidase