Article Figures & Data
Figures
- Fig. 1.
Analysis of a liver lysosomal incubation sample of T-DM1 by LC-UV/HRMS. (A) LC/UV profile. (B) TIC of the full scan MS dataset. (C) Background subtraction–processed TIC of the full scan MS dataset. (D) PIF (m/z 547.2207)-processed TIC of full scan MS/MS dataset. (E) Extracted ion chromatogram of all detected metabolite. T-DM1 was incubated at a concentration of 0.5 mg/ml for 48 hours.
- Fig. 2.
Product ion spectra and proposed structures of payload-containing components released from T-DM1 in incubation with lysosomes. (A) L7. (B) L9. (C) L5. (D) L1.
- Fig. 3.
LC-HRMS detection of released payload-containing catabolites of ADC-1 in monkey liver S9 incubation for 48 hours. (A) A base peak chromatogram of the ADC-1 incubation. (B) A base peak chromatogram of the control antibody incubation in monkey liver S9 for 48 hours. (C) A base peak chromatogram of the ADC-1 incubation after background subtraction using the LC-HRMS data of the control antibody incubation. (D) A base peak chromatogram of the ADC-1 incubation after sequential background subtractions by LC-HRMS data sets of the control antibody and the ADC-1 incubation sample at 0 hours.
- Fig. 4.
Mass spectral data of the ADC-1 incubation in monkey liver S9 for 48 hours. Full scan MS spectrum at RT 26.39 minutes (M1) from the LC-MS dataset of the ADC-1 incubation without background subtraction (A) and with background subtraction (B). (C) MS/MS spectrum of M1. (D) MS/MS spectrum of M4.
- Fig. 5.
Detection and characterization of a released payload-containing component in incubation of ADC-1 in tumor cells for 48 hours. (A) Total ion chromatogram of the raw LC-MS dataset. (B) Background subtraction–processed total ion chromatogram.
Tables
- TABLE 1
A summary of payload-containing components released in incubation of T-DM1 in human liver lysosomes for 48 hours
Metabolite RT (min) Observed Mass (m/z) Identification Theoretical Mass (m/z) Mass Error (ppm) L1 7.39 684.8181 (2+) Lys-His-Lys-MCC-DM1 684.8191 (2+) −1.5 L2 7.54 684.8184 (2+) Lys-His-Lys-MCC-DM1 684.8191 (2+) −1.0 L3 7.99 673.8077 (2+) Arg-Ser-Lys-MCC-DM1 673.8088 (2+) −1.6 L4 8.15 673.8091 (2+) Arg-Ser-Lys-MCC-DM1 673.8088 (2+) 0.4 L5 8.14 671.2960 (2+) His-Thr-Lys-MCC-DM1 671.2955 (2+) 0.7 L6 8.30 671.2954 (2+) His-Thr-Lys-MCC-DM1 671.2955 (2+) −0.1 L7 9.39 1103.4731 Lys-MCC-DM1 1103.4772 −3.7 L8 9.57 1103.4734 Lys-MCC-DM1 1103.4772 −3.4 L9 10.50 1138.4425 Tyr-MCC-DM1 1138.4456 −2.7 L10 10.62 1138.4429 Tyr-MCC-DM1 1138.4456 −2.4 L11 11.68 975.3793 MCC-DM1 975.3823 −3.1 L12 11.80 975.3788 MCC-DM1 975.3823 −3.6 - TABLE 2
Detection and characterization of payload-containing components released from incubations of ADC-1 with rat, monkey, and human liver S9 for 48 hours
Metabolite RT (min) Molecular Formula m/z Identification Rat Monkey Human M1 26.39 C58H95N9O14S 587.8433 (+2)
1174.6792 (+1)Lys-Cys-mc-MMAF 0.19% 0.81% 1.11% M2 27.62 C45H76N6O9 423.2910 (+2)
845.5747 (+1)Hydrolysis of M4 1.44% 2.16% 0.83% M3 28.91 C56H90N8O15S 574.3196 (+2)
1147.6320 (+1)Thr-Cys-mc-MMAF 0.10% 2.65% 0.98% M4 29.09 C52H83N7O13S 523.7958 (+2)
1046.5842 (+1)Cys-mc-MMAF 98.27% 94.38% 97.08%
Data Supplement
- Supplemental Figures -
Supplemental Figure S1. Product ion spectra and proposed structures of payload-containing catabolites of T-DM1
in incubation with lysosomes. (A) L3. (B) L11.Supplemental Figure S2. Mass spectra and proposed structures of catabolites formed in ADC-1 incubation in
monkey liver S9 for 48 h. (A) MS2 spectrum of M2. (B) MS2 spectrum of M3.
- Supplemental Figures -
