13
Conclusions and Outlook of iCIEF
With the introduction of iCIEF by the pioneers of Convergent Biosciences in the early 2000’s, iso-electric focusing has become a very versatile, easy-to-use, robust and cost-effective analysis technique for protein research and biopharmaceutical development and manufacturing.
Figure 10: Time course of the dissociation of the protein-ligand complex (red asterix). The protein is marked with a blue Asterix. The ligand is not detected.
Voltage is provided from the MS with the sprayer needle set at ground voltage.
The focussing process results in a higher concentration in the focussed zone. In order to become detected in the MS, the protein must remain dissolved also in the path between the CE instrument and the ESI interface.
Many reports have been published describing solutions to overcome these challenges for CIEF-MS, unfortunately this has not led to the introduction of commercial product that supports CIEF-MS however it is beyond the scope of this paper to review these.
Advanced Electrophoresis Solutions has taken its iCIEF one step further by allowing fraction collection for further processing e.g. by MALDI-MS resp. on-line coupling with ESI-MS. A pictorial representation of the system is given in Figure 11.
The sample injection valve connects with an additional syringe pump which is filled with water or a dilute solution of methylcellulose. After sample injection and focusing has completed, this syringe delivers the contents of the iCIEF separation capillary towards a collection vial, MALDI target or via a transfer capillary to the ESI interface. Since the focusing voltage remains while pumping, focussing continues and resolution is not lost. In the transfer capillary there is no field and some zone broadening may occur. This is counteracted by using a narrow i.d. wall- coated FS capillary.
Collected protein fractions can be used for in-vial tryptic mapping followed by nanoLC- ESI MS. Recent work by Neusüß et al. demonstrated the on-line coupling by using a valve to collect fractions and direct ESI-MS or CE-MS [15].
Especially in the field of proteomics and biomarker discovery or in development of manufacturing processes for HMW biopharmaceuticals, many techniques are available for determination of purity, charge heterogeneity, post- translational modfications, presence of isoforms etc. Besides CIEF, Ion- Exchange Chromatography and recently chromatofocusing are regarded alternatives for these analyses. The analyst choice for a measurement technique not only depends on the abilities and performance of an instrumental method, but also on availability of the required equipment and the levels of experienced staff in the lab.
iCIEF has been established as a routine method for protein characterisation and purity determination. The market entrance of Advanced Electrophoresis Solutions is fortunate since it will stimulate competition to the advantage of analysts and users. Moreover, AES is committed to expand the technology as a platform for iCIEF-MS and application in neighbouring fields of application
Acknowledgements
The author would like to thank Agilent Technologies, Sciex Separations, Advanced Electrophoresis Solutions and Isogen Lifescience for permission to publish figures. Dr Hanno Stutz for providing Figure 5. Professor Guyla Vigh is acknowledged for invaluable suggestions and explanations that precise the description of the CIEF process.
1. B. Bjellqvist, K. Ek, P. G. Righetti, E. Gianazza, A. Görg, R. Westermeier, and W. Postel, J. of Biochemical and Biophysical Methods, 6 (1982) 317-339.
2. S. Hjertén and M. Zhu, Journal of Chromatography, 346 (1985) 265.
3. R. Rodriguez-Diaz, T. Wehr and M. Zhu, Electrophoresis, 1997, 18, 2134-2144
Figure 9: Pictorial representation of iCIEF fractionation system.
4. K. Shimura, Electrophoresis. 2002, 23,3847 5. L.H.H. Silvertand, J. SastreToraño,
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