Abstract
Capillary isoelectric focusing (CIEF) is a common choice for separation and analysis of the charge variants and impurities of therapeutic proteins. In this study, we developed a sensitive CIEF analysis method for determining the charge heterogeneity of therapeutic monoclonal antibody (mAb) using Beckman PA800 plus platform. The mixture of 5% Pharmalyte 8β10.5 and 1% Pharmalyte 3β10 was used to overcome the limitation of using single Pharmalyte 3β10 in detecting charge heterogeneity of basic mAb. This approach largely improved the resolution of the heterogeneous peaks. In addition, 3βM urea and 50βmM arginine (Arg) were used to improve the separation as solubilizer and cathodic stabilizer, respectively. Under optimized condition, both acidic and basic peaks of the mAb were separated well. Method qualification results showed good specificity, precision, and linearity within the concentration range of 0.03β0.20βmg/mL for mAb R1. The method was then used for Cβterminal lysine (Lys) variants characterization and glycosylation profiles analysis. Furthermore, it also had a wide application in the clone screening process. The highly sensitive and repeatable results highlighted the wide application prospects of this method in biopharmaceutical industry.