Introduction to Imaged Capillary Isoelectric Focusing (iCIEF)

Imaged capillary isoelectric focusing (iCIEF) is a capillary electrophoresis (CE) technique used to study physical properties of proteins.

Proteins are made up of amino acid with charged functional groups. As such, the sum of the charge for all of these groups gives the protein an overall charge. This summed charge is referred to as the isoelectric point (pI).

In isoelectric focusing (IEF), a pH gradient is created by applying an electric field to a solution containing carrier ampholytes (CAs). CAs are composed of small molecules with many different pIs. The focusing is when the protein of interest also present in the solution distribute themselves along the pH gradient within a gel medium to where the pH is equal to their pI. Reference standards of known pI are also added to verify the identity of each protein peak. In IEF, the voltage is stopped after focusing and the proteins are dyed for visualization.

More recently, IEF has been performed within a small capillary (CIEF) to decrease sample volume to increase sensitivity and decrease focusing time. CIEF further saves time by not needing to stain the gel the run. However, without the gel matrix the focused proteins remix once the electric field is removed. CIEF separations are visualized by pushing each peak past a single detection point equipped with an ultraviolet (UV) light to measure absorbance.

CIEF is much faster than gel IEF but the resolution of the separation may be damaged while the peak is being pushed. Imaged CIEF (iCIEF) instruments and cartridges employ whole-column imaging detection (WCID) technology that allows CIEF process to be “imaged” in real time. iCIEF separations do not require mobilization so, when compared to traditional CIEF with single-point detection, sample analysis is faster and more precise.

iCIEF is a valuable method because protein biopharmaceuticals, such as monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs), make up an increasingly large portion of top treatments for many conditions and diseases.

When making protein biopharmaceuticals, some will have a small difference in charge compared to the main “target” protein. The presence of these charge isoforms has the potential to alter the safety and efficacy of the drug and must be regulated; the charge heterogeneity of protein biologics is a critical quality attribute (CQA) for drug submission.

iCIEF generates a profile of the different isomers separated by charge, thus visualizing the presence of these charge variants relative to the target protein. With a full understanding of the importance and potential of iCIEF, the CEInfinite Analytical and Preparative instrument systems were created to meet the current needs of the biopharmaceutical scientists for a robust, high-throughput instrument for charge variant analysis and characterization.

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