Development of an efficient mAb quantification assay by LC-MS/MS using rapid on-bead digestion

The use of monoclonal antibody (mAb) therapeutics is increasing rapidly, but mAb concentrations vary widely between individuals and might subsequently affect mAb exposure and treatment response. Precision medicine has gained much attention in recent years, but little is known about the personalized dosage of mAb therapeutics. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been demonstrated as a selective and sensitive approach to quantify mAb therapeutics in biological samples, but current methods to quantify mAbs are usually time-consuming and require tedious sample preparation. This study developed an efficient LC-MS/MS method using an on-bead trypsin digestion procedure at a higher digestion temperature. Five mAbs, bevacizumab, evolocumab, nivolumab, pembrolizumab, and trastuzumab, used for treating different diseases, were selected for method development. Tocilizumab was selected as the internal standard. The result of the on-bead digestion protocol was compared to the conventional low-pH elution method, and it showed better sensitivity and reproducibility for most mAbs. The optimized on-bead digestion protocol used 75 mu L of digestion buffer at 60 degrees C for a 60 min digestion. The calibration curve was generated from 10 to 200 mu g mL(-1). The accuracies at the three QC levels of the 5 mAbs were all within 94.5 +/- 5.2% to 111.6 +/- 3.7%. The repeatability and intermediate precision of the 5 mAbs were all lower than 6.1 and 9.5% RSD, respec-tively. The newly developed method was successfully applied to quantify trastuzumab in six breast cancer patients under different treatment cycles, and the concentrations ranged from 66.4 to 173.2 mu g mL(-1). In conclusion, the developed method is more efficient and more practical for real-world analysis of a large number of clinical samples, it could be used for routine therapeutic drug monitoring, and it could contribute to personalized mAb treatment. (C) 2021 Elsevier B.V. All rights reserved.

Automated summary

This study developed an efficient LC-MS/MS method using an on-bead trypsin digestion procedure to quantify mAb therapeutics. The method showed better sensitivity and reproducibility, making it suitable for real-world analysis of a large number of clinical samples and contributing to personalized mAb treatment.