Development of a Reverse Phase HPLC Assay to Characterize a Monoclonal Antibody

Monoclonal antibodies (mAbs) represent the largest class of therapeutic proteins. One major challenge for biopharmaceutical industry is to understand the mAb microheterogeneity which could affect its therapeutic efficacy. mAb microheterogeneity could result from post translational modifications such as deamidation, oxidation and clipping. The reverse-phase HPLC (RP-HPLC) analysis is one of the widely used separation techniques to monitor the purity and stability of mAbs during manufacturing and storage. In this study, a RP-HPLC assay was developed to characterize the purity of a mAb. The mAb was reduced and separated on a RP HPLC column to reveal the Heavy Chain (HC), Light Chain (LC) and impurities. Oxidation and clipping induced impurities were identified by LC-MS. Reduction condition, columns, separation temperature and gradient were optimized to achieve the best separation and minimize assay induced artifacts. The RP-HPLC assay was validated for its accuracy, precision, linearity, specificity and range following FDA and ICH guidelines.