Use of imaged capillary isoelectric focusing technique in development of diphtheria toxin mutant CRM197

Polysaccharide based-vaccines have been successful in providing protection in adults from bacterial infections, however they are not as effective in infants or young children. To enhance the immune response in these high risk groups, the polysaccharide is conjugated with a carrier protein such as cross-reacting material 197 (CRM197). The CRM197 protein has been well-characterized biochemically and biophysically using various analytical techniques however, none of these have been CE-based methods. Of the various CE techniques, imaged capillary isoelectric focusing (icIEF) is a method that has been used extensively in the field of protein-based drug development as a tool for product identification, stability monitoring, and characterization. Applications of icIEF technique using Convergent Bioscience icIEF instrumentation with whole-field imaging technology are presented and discussed in this paper. These applications include rapid method development to establish a CRM197 identity test for product release, a concentration assay for upstream and downstream in-process product development, and CRM197 stability with respect to its charge heterogeneity under accelerated temperature stress. The data presented demonstrates the utility of the icIEF method as a multifunctional assay because it can screen for better product candidates during early stage clonal selection as well as support in-process and final product characterization throughout CRM197 development.