Enhancing Peak Pattern Stability and Reproducibility in Capillary Isoelectric Focusing (cIEF) by Plugging Capillary Column During Focusing (CE Pharm 2011)

In isoelectric focusing (IEF), at the end of the focusing process, all components in a sample are focused and stop at their pI points. In order to perform IEF in a capillary column (cIEF), two forces within the column that interfere with the focusing process have to be eliminated: electroosmotic flow (EOF) and hydrodynamic flow. In commercial cIEF instruments, the EOF is substantially reduced by column coatings and the hydrodynamic flow is eliminated by placing both ends of the column at the same level during the IEF process. In ProteinSimple's iCE280 IEF Analyzer, the hydrodynamic flow within the separation column is eliminated by using a specially designed, constant fluid level waste vial at the outlet of the column and a balancing vial at the column inlet in the autosampler that has the same fluid level as the waste vial. This design constantly provides equal fluid levels at both ends of the column regardless of the waste volume dumped into the waste vial. However, some high concentration additives could generate an unbalancing force within the column during IEF for some unknown reasons. One example is high concentration urea. When these high concentration additives are used, the peak pattern sometimes is pushed back or forth within the separation column during the IEF process, making the peak pattern unstable. This can reduce separation resolution and reproducibility. We found that this problem could be solved by plugging one end of the column during the IEF process with a microswitch valve. The new design will be adapted in ProteinSimple's new model iCE instrument. In this presentation, we will compare the peak pattern stability of a monoclonal antibody on the iCE of the new design and the existing design when 8 M urea is used as the additive.