Accelerating microfluidic capillary electrophoresis-mass spectrometry for charge-variant and glycoform analysis of intact monoclonal antibodies

The analytical determination of critical quality attributes (CQAs) of monoclonal antibodies (mAbs) is often time-consuming. Hence, increasing analysis throughput can shorten biopharmaceutical development timelines and enables rapid decision-making. Microfluidic capillary electrophoresis coupled to mass spectrometry (MCE-MS) allows fast analysis of charge variants and glycoforms of mAb samples. In a recently published MCE-MS method, we used a microfluidic chip with a 22-cm separation channel length, permitting analysis of 96 samples per day. To further increase the throughput of the method and better meet the needs of the biopharmaceutical industry, we propose the use of a chip with a distinctly shorter channel length (10 cm). However, a substantial drop in electrophoretic resolution was observed due to the increased laminar flow caused by the pressure put on the shortened channel during analysis. To mitigate this phenomenon, the MCE setup was modified to allow independent adjustment of the gas pressure on the separation channel inlet. The mAb charge-variant resolution significantly improved by reducing the inlet pressure from 2.0 to 0.75 psi. The performance of the modified setup was assessed by analysis of mAb samples from cell lines, mimicking a typical biosimilar clone screening. The original (22-cm channel, 2.0 psi on inlet) and modified (10-cm channel, 0.75 psi on inlet) setups yielded comparable separation profiles and number of MS-identified proteoforms across all clone mAbs. Overall, the new MCE-MS method reduces total analysis time per sample by 3.3-fold, which conceptually facilitates the analysis of more than 300 samples per day with minimal loss in separation performance.