Rapid analysis of charge variants of monoclonal antibodies using non-linear salt gradient in cation-exchange high performance liquid chromatography

• Rapid (4 min), analytical CE–HPLC method for analysis of charge heterogeneity in mAbs.
• Use of non-linear sigmoidal shape gradient for reduction of analysis time.
• Use of phosphate buffer gradient as a suitable analytical CE–HPLC method for mAbs.
• Application of this short analytical method as a potential PAT tool.

A method is proposed for rapid development of a short, analytical cation exchange high performance liquid chromatography method for analysis of charge heterogeneity in monoclonal antibody products. The parameters investigated and optimized include pH, shape of elution gradient and length of the column. It is found that the most important parameter for development of a shorter method is the choice of the shape of elution gradient. In this paper, we propose a step by step approach to develop a non-linear sigmoidal shape gradient for analysis of charge heterogeneity for two different monoclonal antibody products. The use of this gradient not only decreases the run time of the method to 4 min against the conventional method that takes more than 40 min but also the resolution is retained. Superiority of the phosphate gradient over sodium chloride gradient for elution of mAbs is also observed. The method has been successfully evaluated for specificity, sensitivity, linearity, limit of detection, and limit of quantification. Application of this method as a potential at-line process analytical technology tool has been suggested.