Monoclonal antibody (mAb) pharmaceuticals have attracted significant interest due to their strong therapeutic potency and specificity to treat inflammatory, oncologic, and autoimmune diseases. Because of their intrinsic complexity due to a large number of micro-heterogeneities, there is a crucial need of analytical methods to provide a comprehensive in-depth characterization of candidate molecules.
A plethora of analytical techniques is provided by Creative Biolabs for antibody characterization and homogeneity assessment at all stage of mAb discovery, preclinical and clinical development. Here, we introduce the application of capillary isoelectric focusing (cIEF) for antibody charge heterogeneity analysis. It is widely used for the characterization of therapeutic mAbs. By determining the pI of mAb charge isoforms, cIEF provides information crucial for establishing identity, antibody impurity, post-translational modifications assays, and antibody stability.
cIEF is based on the principle of capillary gel electrophoresis. Electrophoresis is a separation technique based on the differential speed of movement of charged species in an electric field, toward the electrode of opposite charge. To separate molecules based on isoelectric point (pI), a pH gradient inside the gel is established by using ampholyte mixtures. Under the electric field, the molecules migrate along the electrical field until they reach the pH corresponding to the pI, where the positive and negative charges in the molecules are balanced. The UV absorption over the whole capillary is measured throughout the separation, allowing real-time observation and a final quantification.
Fig.1 Principle of isoelectric focusing. (Pergande, 2017)
mAbs are susceptible to various post-translational modifications (e.g., glycosylation, phosphorylation, oxidation, and lysine clipping), leading to charge variants. Some of these charge variants may have a possible effect on biological activity and safety of the molecule to varying degrees. For instance, the glycosylation profile of mAbs highly influences the biological activity of the protein. Consequently, routine monitoring of mAb charge heterogeneity is an integral part of any product quality and lifecycle management plan. It is also one of the most important analytical aspects of biosimilar development and comparability studies. As a charge-based separation technique, cIEF is a powerful tool to characterize the charge profile of the mAbs, because different charge variants can be distinguished according to their pI using internal calibration. As cIEF provides very high reproducibility and specificity, this is one of the most popular tools for mAb analysis.
Creative Biolabs also offers a wide range of antibody function assays to help understand the effects of charge variants on antibody activity and properties, such as effector functions, FcRn, and antigen binding affinity, and PK, thereby elucidating the structure-function relationships. If you are interested in our service, please contact us to discuss your requirements.
Reference
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