A high-throughput UPLC method for the characterization of chemical modifications in monoclonal antibody molecules.

Development of high-throughput release and characterization assays is critical for the effective support of the rapidly growing biologics pipeline for biotherapeutics. Clipping of polypeptide chains is commonly monitored during process optimization, formulation development, and stability studies. A reduced capillary electrophoresis-sodium dodecyl sulfate (rCE -SDS) method is often used as a purity release assay for monitoring clips in monoclonal antibodies (mAbs); however, it has a cycle time of approximately 40 min, which is not suited for high-throughput screening. Additionally, the characterization of clips and variants from electropherograms is not straightforward and takes significant time. Reduced reversed-phase (RP) chromatography has been a popular assay for the characterization and identification of clips and variants because it can be directly coupled with online mass spectrometric analysis. However, the high-column temperature and low pH required for RP assays can induce on-column cleavage and therefore skew the results. To minimize on-column degradation, we have developed a high-throughput method with a significantly shorter cycle time of 5 min. The short cycle time was achieved using an ultra-high-pressure liquid chromatography (UPLC) system with a 1.7 μm phenyl column. This UPLC method allowed quantitation of hinge clipping in an IgG1 molecule and acid induced aspartic acid/proline (D/P) clip in an IgG2 molecule. The results from the UPLC method were comparable to those obtained with rCE-SDS. Additionally, the phenyl column offered partial resolution of oxidation and other chemical modifications, making this technique an attractive assay for high-throughput process characterization and formulation screens.