Capillary Isoelectric Focusing

Abstract

Capillary isoelectric focusing (CIEF) has proven to be a very versatile separation method for characterization, identification, purity determination, and quantitation of amphoteric substances such as peptides and proteins. CIEF has become an indispensable and invaluable analytical tool in proteomics research, in the development of new biotherapeutics and in quality and process control of their manufacturing. The method is based on arranging and separating proteins according to their isoelectric point (pI), allowing to determine changes in the charge state of proteins after structural modifications such as deamidation, oxidation, isomerization, N‐terminus modification, disulfide formation, and glycosylation. Modifications that change biochemical characteristics of biotherapeutics such as their isoelectric point (pI) reduce their safety and long‐term stability, influence drug efficacy, and increase undesired immunogenicity.

Integrating ultra‐high‐performance liquid chromatography tandem mass spectrometry and imaged capillary isoelectric focusing for in‐depth characterization of complex fusion proteins

“…17 Imaged capillary isoelectric focusing (icIEF) has become an indispensable tool in therapeutic protein development and manufacturing because of its high analytical throughput, ease of use, fast method development, and excellent reproducibility. [17][18][19] Additionally, high-performance liquid chromatography (HPLC) highresolution tandem mass spectrometry (HRMS/MS) has been widely utilized for in-depth characterization. [20][21][22] The complex structure of the etanercept used in this study brings great challenges to quality evaluation.…”