Evaluating the Main Peak Isoelectric Point of a Panel of Therapeutic mAbs: A Methodological Comparison Across Three icIEF Platforms

Monoclonal antibodies (mAbs) charge heterogeneity in Quality Control (QC) is commonly assessed using imaged capillary IEF (icIEF). icIEF separates proteins based on their isoelectric point (pI) within a capillary, facilitated by a stabilised pH gradient under the application of an electric field. Compared to conventional cIEF, icIEF offers faster separation, higher resolution, improved repeatability, and a simpler method development process. Although charge heterogeneity is considered a Critical Quality Attribute, the pI value is typically not included in biotherapeutic release specifications as an identity parameter due to marked variability observed across different analytical platforms. Instead, mAb QC usually involves comparing the charge variant profile of the sample drug product with that of its reference standard. Recently, the Ph. Eur. published a general text describing horizontal (i)cIEF methods for mAb charge variant analysis to be used as a standard starting point for the development of validated methods, underlining the need for transversal analytical platforms. To meet the demand, two distinct strategies — based on the One Factor at a Time and Analytical Quality by Design approaches, respectively — have been developed. To evaluate the performance of each method, the main peak pI values of a panel of therapeutic mAbs were compared against values obtained through product-specific industrial methods. Despite differences in sample matrix composition, both proposed methods produced pI values in close agreement with reference standards, demonstrating high analytical performance. These findings suggest that the developed methods are suitable for accurate pI determination and may be applicable as horizontal analytical platforms for identity testing within routine QC workflows for biotherapeutics, reinforcing pI value measurements as a reliable and objective identity parameter.