Application of imaging capillary IEF for characterization and quantitative analysis of recombinant protein charge heterogeneity.

In this work several aspects of imaging capillary IEF (icIEF) application for charge heterogeneity analysis of recombinant proteins and monoclonal antibodies have been discussed. Advantages of the method as compared with traditional approaches for determination of biomolecule charge heterogeneity, such as gel and IEC, have been demonstrated. Correlation of icIEF-detected protein isoforms with the charge heterogeneity determined by IEC has been shown for a representative recombinant monoclonal antibody. Identification of charged variants collected from IEC has been performed by ESI-MS. Qualification of an icIEF method for use in quality control environment for quantitative analysis of recombinant protein charge heterogeneity and monitoring protein stability has also been discussed. The intermediate precision for determination of pI of main or main acidic species was </=0.2% RSD. Relative % peak areas for acidic, main and basic species were reproducible within 1.9, 0.9 and 16.6% RSD, respectively. Based on the assay performance evaluation, icIEF assay has been shown to allow for fast method development, short analysis time and high sample throughput. Some aspects of the method specificity for use as an identity test in biopharmaceutical development have been discussed.