TY - JOUR
T1 - Development of an efficient mAb quantification assay by LC-MS/MS using rapid on-bead digestion
AU - Chiu, Huai Hsuan
AU - Tsai, Yun Jung
AU - Lo, Chiao
AU - Lin, Ching Hung
AU - Tsai, I. Lin
AU - Kuo, Ching Hua
N1 - Funding Information:
This study was supported by the Ministry of Science and Technology of Taiwan (107-2113-M-002-016-MY3). The authors thank the NTU Integrated Core Facility for Functional Genomics of the National Research Program for Genomic Medicine of Taiwan for technical assistance.
Funding Information:
This study was supported by the Ministry of Science and Technology of Taiwan ( 107-2113-M-002-016-MY3 ). The authors thank the NTU Integrated Core Facility for Functional Genomics of the National Research Program for Genomic Medicine of Taiwan for technical assistance.
Publisher Copyright:
© 2021 Elsevier B.V.
PY - 2022/2/8
Y1 - 2022/2/8
N2 - 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 μL of digestion buffer at 60 °C for a 60 min digestion. The calibration curve was generated from 10 to 200 μg 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, respectively. 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 μg 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.
AB - 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 μL of digestion buffer at 60 °C for a 60 min digestion. The calibration curve was generated from 10 to 200 μg 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, respectively. 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 μg 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.
KW - High temperature trypsin digestion
KW - Liquid chromatography–tandem mass spectrometry (LC-MS/MS)
KW - Monoclonal antibody (mAb)
KW - On-bead trypsin digestion
KW - Protein G purification
KW - Therapeutic drug monitoring (TDM)
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U2 - 10.1016/j.aca.2021.339319
DO - 10.1016/j.aca.2021.339319
M3 - Article
C2 - 35058007
AN - SCOPUS:85120166351
SN - 0003-2670
VL - 1193
JO - Analytica Chimica Acta
JF - Analytica Chimica Acta
M1 - 339319
ER -