Assessing the Oxidation of Biopharmaceutical Compounds by Capillary Isoelectric Focusing (cIEF) (CAT#: STEM-B-0406-CJ)

cIEF is based on the principle of capillary gel electrophoresis (cGE). In general, electrophoresis is a separation technique based on the migration of charged molecules in response to an electric field, toward the electrode of opposite charge. It is performed mainly in polyacrylamide gels. To separate molecules based on pI, a pH gradient inside the gel is established by ampholyte mixtures. The molecule of interest migrates along the electrical field until it reaches the pH corresponding to its pI, where it has a net charge of zero and stops migrating. The UV absorption over the whole capillary is measured throughout the separation, allowing real-time observation as well as a final quantification.

Biopharmaceutica

Two-step and single-step IEF methods are available in capillary format. In two-step cIEF, protein samples (c. 500 μg ml−1) are dissolved in carrier ampholytes containing TEMED and methylcellulose at 2–3% concentrations. A sample solution is introduced into a chemically modified capillary that suppresses EOF. The anodic and cathodic ends are immersed in 10 mM phosphoric acid and 20 mM NaOH solution, respectively. By applying voltage at both ends of the capillary, pH gradient is generated and the analytes move and stop at the regions of their pI values. After focusing, the anolyte solution is changed to 20 mM NaCl, and voltage is again applied. Focused protein bands move and pass through the detector in the order of decreasing (or increasing) pI values.

To eliminate the problem, cIEF is often performed in a bare capillary in the single-step mode. Formation of pH gradient and focusing of proteins as well as migration of entire liquid in a capillary to the cathodic end through a detection window occur simultaneously. However, this method often meets the passing of proteins through a detection window before completion of focusing step.

• Sample: Peptides, Proteins, Vaccines, Virus-like particles
• Equipment: Capillary Isoelectric Focusing (cIEF)

When compared to conventional isoelectric focusing (IEF) in slab gels, cIEF allows for higher resolution, faster sample analysis and has an approximately 10-fold lower limit of detection.