Capillary Isoelectric Focusing (cIEF)

Capillary isoelectric focusing (cIEF) is a powerful tool to characterize charge profile of the antibody drugs, because different charge variants can be distinguished according to their isoelectric points using internal calibration. cIEF provides very high reproducibility and specificity, and can be used for routine analysis of various antibody therapeutics, such as monoclonal antibodies, antibody drug conjugates (ADCs), and fusion proteins. Therefore, capillary isoelectric focusing is one of the most popular tools for antibody drug analysis. Creative Proteomics provides advanced capillary isoelectric focusing (cIEF) technology and related analytical services. We have a team of scientists with extensive experience in developing antibody drug characterization tests using cIEF technology. Through highly accurate cIEF analysis tools, we accurately analyze charge heterogeneity and other key quality attributes of antibody drugs, providing a reliable basis for understanding their properties.

Introduction of Capillary Isoelectric Focusing (cIEF)

Capillary Isoelectric Focusing (CIEF) is commonly used to characterize CQA of antibody drugs, such as charge variants, identification, and positional isomers/purity etc. Similar to the development of CGE according to SDS-PAGE, cIEF is a method based on the mechanism of IEF slab gel. The separation of the proteins is based on their isoelectric points (pIs) in a capillary. The classical workflow consists of the use of a basic catholyte, generally sodium hydroxide, placed at the cathode and an acidic anolyte, generally phosphoric acid, placed at the anode. A mixture of ampholytes and sample filled up the whole capillary. Applying an electrical field and due to ampholytes properties, a pH gradient is established in the capillary. As a first step, protein focused along the pH gradient until the region with an equal value between the local pH and it own pI. In this region, the electrophoretic mobility of the sample is zero due to the absence of apparent charge bearing by the protein. As a second step, electrophoretic or hydrodynamic mobilization is implemented to force the sample to move toward the UV detection.

Fig. 1 Isoelectric focusing-mass spectrometry with ultrahigh resolution for characterizing microheterogeneity and isoelectric points of intact protein complexes. (Xu, T. et al., 2022)

Applications of cIEF in Antibody Drug Characterization

• Identification and quantification of charge variants in antibody drugs
• Assessment of product quality attributes, such as aggregation and deamidation
• Investigation of critical quality attributes (CQAs) impacting antibody efficacy and safety
• Comparison of biosimilar and innovator antibody drugs using cIEF

Advantages of Capillary Gel Electrophoresis

• High-resolution separation and highly sensitive detection.
• Quantitative detection of charge variants in antibody drugs.
• Simple development, high throughput and quantitative reproducibility.
• Easy to develop platform methods.

Our Capabilities

• Provides development and validation of antibody drug characterization methods based on cIEF technology. This includes selection and optimization of key parameters, hyphenation of cIEF with MS or other complementary technologies, method improvement, and method reliability validation services.
• Detailed steps for performing cIEF analysis, including sample preparation techniques to ensure optimal results and analysis of assay results, are provided based on the specific assay.
• Provides information on instrument requirements and operating conditions.

Creative Proteomics is pleased to offer advanced solutions for antibody drug characterization using our powerful capillary isoelectric focusing technology. Contact us to learn more about our service and we will be happy to serve you.

References

1. Xu, T.; et al. Automated capillary isoelectric focusing-mass spectrometry with ultrahigh resolution for characterizing microheterogeneity and isoelectric points of intact protein complexes. Analytical Chemistry. 2022, 94(27): 9674-9682.
2. Gahoual, R.; et al. Cutting-edge capillary electrophoresis characterization of monoclonal antibodies and related products. Journal of Chromatography B. 2016, 1032: 61-78.