A single liquid chromatography–quadrupole time‐of‐flight mass
spectrometric method for the quantification of total antibody,
antibody‐conjugated drug and free payload of antibody–drug
Byeong ill Lee
Young G. Shin
College of Pharmacy, Chungman National
University, Daejeon, Republic of Korea
Young G. Shin, College of Pharmacy,
Chungman National University, Daejeon,
Republic of Korea.
Ministry of Food and Drug Safety, Grant/
Award Number: 17172MFDS211
A single hybrid affinity‐captured‐LC‐TOF‐MS/MS method was developed and
applied for the quantification of total antibody, antibody conjugated drug and free
payload of antibody drug conjugate (ADC). Adcetris®, a valine–citrulline
monomethyl auristatin E conjugated ADC, was used as a model ADC compound.
A quadratic regression (weighted 1/concentration) was used to fit calibration curves
over the concentration range 30.65–613.00 ng/mL with an equation y = ax
+ bx + c
for the antibody‐conjugated drug of Adcetris®. The qualification run met the accep-
tance criteria of ±25% accuracy and precision values for quality control samples.
For the analysis of total antibody, a signature peptide (TTPPVLDSDGSFFLYSK,
molecular weight 1874) was used after affinity capture using magnetic beads and
on‐bead trypsin digestion. A quadratic regression (weighted 1/concentration) was
used to fit calibration curves over the concentration range 5.00–100.00 μg/mL with
an equation y = ax
+ bx + c for total antibody. For free payload analysis of
monomethyl auristatin E, a protein precipitation method followed by LC‐TOF‐MS/
MS analysis was used. A quadratic regression (weighted 1/concentration) was used
to fit calibration curves over the concentration range 1.01–2200 ng/mL with an
equation y = ax
+ bx + c for free payload. Pharmacokinetic study samples and
in vitro stability samples in rat were successfully analyzed by this a hybrid affinity‐
captured‐LC‐TOF‐MS/MS method. This single platform method is a useful comple-
mentary method for the pharmacokinetics study of ADC with valine–citrulline linker
at the early drug discovery stage.
asingleLC‐ESI‐TOF/MS platform, ADC, Adcetris®, quantification, valine‐citrulline linker
Antibody–drug conjugate (ADC) consists of a monoclonal antibody,
linkers and cytotoxic drugs for direct delivery of the cytotoxic payload
to the target (Zolot, Basu, & Million, 2013). When compared with
Abbreviations: acDrug, antibody‐conjugated drug; ADC, antibody–drug
conjugate; DAR, drug–antibody ratio; EDTA, ethylenediaminetetraacetic acid;
MMAE, monomethyl auristatin E; PBS, phosphate buffer solution.
Received: 20 December 2017 Revised: 22 February 2018 Accepted: 26 February 2018
Biomedical Chromatography. 2018;32:e4229.
Copyright © 2018 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc 1of9