Rapid quantification of a cleavable antibody-conjugated drug by liquid chromatography/tandem mass spectrometry with microwave-assisted enzymatic cleavage.
Antibody-drug conjugates (ADCs) play an increasingly important role for targeted cancer treatment. One class of ADCs has attracted particular interest in drug development. These ADCs employ a cleavable chemistry linkage for drugs and utilize the reduced interchain disulfide cysteine residues for conjugation. In this work, a novel bioanalytical method for the quantification of a cleavable antibody-conjugated drug in plasma was developed, qualified, and implemented. This novel method significantly improves throughput by combining a microwave-assisted, enzymatic cleavage of conjugated drugs from ADCs with a 96-well based sample preparation procedure to immunocapture ADCs in plasma. The released drug is subsequently quantified using a LC/MS/MS method. Our results represent a high-throughput, generic, and sensitive quantification method for antibody-conjugated microtubule inhibitors (such as MMAE) for preclinical PK/PD studies. The linear range of the standard curve for antibody conjugated drug (MMAE) was from 2.01 to 2010ng/mL with an excellent linearity (r(2)>0.997). The intra-run precision was below 8.14% and accuracy was from -7.71% to -1.08%. No matrix effect or carryover was observed for this method. This method was successfully used to measure the level of conjugated drug in a preclinical PK/PD study in mice.
Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited, 35 Landsdowne Street, Cambridge, MA 02139, United States. Electronic address: Ling.xu@takeda.com.
Ling
L
Xu
Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited, 35 Landsdowne Street, Cambridge, MA 02139, United States. Electronic address: laura.packer@takeda.com.
Laura E
LE
Packer
Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited, 35 Landsdowne Street, Cambridge, MA 02139, United States. Electronic address: mark.qian@takeda.com.
Mark G
MG
Qian
Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited, 35 Landsdowne Street, Cambridge, MA 02139, United States. Electronic address: Jing-Tao.wu@takeda.com.
Jing-Tao
JT
Wu
D000818:Animals; D001386:Azides / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002401:Cathepsin B / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004798:Enzymes / Q000737:chemistry; D005260:Female; D006868:Hydrolysis; D018796:Immunoconjugates / Q000032:analysis* / Q000493:pharmacokinetics*; D051379:Mice; D016513:Mice, SCID; D008868:Microtubule Proteins / Q000037:antagonists & inhibitors; D008872:Microwaves; D009842:Oligopeptides / Q000737:chemistry; D011412:Propanolamines / Q000737:chemistry; D011786:Quality Control; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
D001386:Azides; D004798:Enzymes; D018796:Immunoconjugates; D008868:Microtubule Proteins; D009842:Oligopeptides; D011412:Propanolamines; D001710:Biotin; C032509:4-azidobenzylcarazolol; D002401:Cathepsin B; C495575:monomethyl auristatin E
Antibody-conjugated drug; Drug antibody ratio; Immunocapture mass spectrometry; MMAE; Microwave-assisted hydrolysis