8 February / March 2019


Imaged Capillary Iso-Electric Focussing: Background, Status, and Perspectives


by Gerard Rozing, ROZING.COM Consulting, Karlsruhe, Germany


Iso-electric focusing (IEF) has proven to be a very versatile separation method for the characterisation, identification, purity determination, and quantitation of amphoteric substances such as peptides and proteins. It is an indispensable and invaluable analytical tool in proteomics research, in the development of new biotherapeutics and in quality- and process-control in their manufacturing. The method is based on arranging and separating proteins according to their isoelectric points or pI, allowing to establish changes in the charge state of the molecules after structural modifications such as deamidation, oxidation, isomerisation, N-terminus modification, disulfide formation, and glycosylation. Modifications that change biochemical characteristics of biotherapeutics like their isoelectric point (pI), reduce their safety and long-term stability, influence drug efficacy, and increase undesired immunogenicity.


Nowadays, iso-electric focusing is performed in two different formats:


Slab Gel IEF


Early iso-electric focusing was executed on a flat substrate (glass or plastic plate) coated with a mixture of polyacrylamide or agarose to suppress hydrodynamic or electro-osmotic flow and so-called carrier ampholytes to establish a pH gradient. Carrier ampholytes (CA, amphoteric electrolytes) are oligomeric molecules with MW between 300-1000 obtained by the reaction of polyamines with acrylic acid (or similar structured unsaturated sulphonic acids). Each carrier ampholyte molecule, therefore, will contain several ionisable acidic and basic groups which determine the pI of each ampholyte molecule. A mixture of structurally different CA’s will cover a range of pI’s. The plate is flanked with trenches containing a low pH solution (anolyte) and a high pH solution (catholyte).


Laborious gel preparation with ampholyte, precise sample application, the visualisation of the separated bands, and the unstable pH gradient made this IEF method moderately reproducible and presented the user with ambiguous separation interpretations.


A significant improvement of the IEF method was obtained with the first description of Immobilised pH Gradient (IPG) gels by Bjellqvist et al [1] which makes the polymer gel itself amphoteric. Since the ampholyte


molecules on an IPG are static, better reproducibility is obtained. These IPGs were introduced as a commercial product by LKB of Sweden (now GE Healthcare). The IPGs are prepared on small plates or strips the latter of which is used as the first dimension in 2D Gel Electrophoresis.


Capillary Iso-Electric Focusing (CIEF)


Despite the improvement of IEF by using prefab IPG gel plates, the method remained laborious, lacked automation, on-line detection with electronic data processing and documentation. The introduction of instrumentation for Capillary Electrophoresis (CE) followed quickly, employing IEF in a capillary. However, IEF, as described before, is a spatial separation method which takes some time for focusing to complete and sample molecules have congregated as separate bands or spots at a specific location. This in contrast to CE, which is a temporal separation method, in which the time is measured from the moment t0 when the sample molecules enter the capillary until a time ti


when a compound passes the point of detection (PoD).


The biggest challenge in implementing IEF in a capillary, is to focus all the sample molecules in front of the point of detection during the focusing stage and once focusing has been completed, to move the focussed zones past the PoD. In addition, one must realise, that with IPG gels, the sample


application is a discrete independent step after the pH gradient has been established, which in principle can take place at any physical location along the gradient on the gel plate or over the length of an IPG strip. Conventional CE sample introduction occurring at the inlet end of the separation capillary after the pH gradient has been formed is not meaningful in CIEF.


Hjertén was the first to describe IEF in a CE capillary [2] in 1985 followed by many practical and theoretical reports in peer reviewed journals and textbooks [3,4,5,6] . Despite the significant improvements in the methodology on CE-instrumentation, CIEF still requires significant user experience and expertise. The work by Mack et al [7], describing a systematic study on the usage of so-called blocking agents that confined the focussing to occur in front of the PoD, has been a major step forward in improving the method.


Principle of CIEF [8]


It is appropriate to first look into details of the CIEF process to appreciate the improvement with imaged CIEF.


For CIEF a coated fused silica (FS) capillary is used, typically 50 µm i.d. and 30-40 cm depending on the brand of the CE instrument used. The internal capillary coating (fluorocarbon, polyvinyl alcohol, DB-WAX) suppresses the electro endo- osmotic flow (EOF). At the beginning of a


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