High Resolution CIEF Fractionation: CEInfinite Preparative iCIEF

Capillary isoelectric focusing (CIEF) is an indispensable tool for the development of new protein pharmaceutical products, as well as their formulation and quality control (QC). CIEF, including imaged (iCIEF), indicates the presence of charge isomers and their distribution along a pH gradient.

However, the identity of the individual isomers cannot be confirmed without the use of other techniques, such as mass spectrometry, which require a relatively pure fraction.

In response to this need for isomers separated with sufficient purity, Advanced Electrophoresis Solutions Ltd. has developed the CEInfinite Preparative iCIEF instrument system.

Once the proteins are focused, the mobilization process begins and the peaks are carefully pushed toward the cathodic end of the separation capillary. This is visualized in real time on-screen via the whole column image detection (WCID) of iCIEF, which is not present in single-point detection CIEF.

During mobilization, the electric field is still applied to the separation capillary and the proprietary capillary diameter transfer technology (CDTT) maintains the separation resolution of the sample into the narrower transfer capillary for collection.

Figure 1. Illustration of capillary diameter transfer technology (CDTT) maintaining the separation of focused peaks
Figure 1. Illustration of capillary diameter transfer technology (CDTT) maintaining the separation of focused peaks

Using the CEInsight software, the start and end of each peak can be easily marked and program will calculate when that peak is available for collection.

After collection, a portion of the fraction can be reinjected for Analytical (non-Preparative) iCIEF to confirm peak purity.

A potential limitation of this fractionation, using mAbs as an example, is that due to the direction of peak mobilization and collection, it is faster to obtain a fraction of the basic peaks than of the acidic peaks. To remedy this issue, the CEInfinite Preparative instrument offers the capability to reverse the polarity for focusing, allowing more acidic peaks to be on the side of the instrument where fractions are collected.

Automatic Fraction Collection (AFC)

Identify up to 9 peaks of interested to be automatically collected by the instrument and if more sample is needed than one separation run provides, multiple separations of the sample can be fractionated using the same initial marking details.

The coordination of the CEInfinite instrument and CEInsight software delivers pure, isolated charge isomers in quantities suitable that allow for many more characterization analyses than before.

Figure 2. CEInfinite Preparative iCIEF Instrument System
Figure 2. CEInfinite Preparative iCIEF Instrument System

The CEInfinite Preparative iCIEF Instrument System aims to eliminate solves the challenges of micro-preparation, purification, and characterization of proteins and peptides for biopharmaceutical applications.

If you have questions or think that iCIEF fractionation could solve a problem in your lab, please contact us.