31
Figure 6: Chromatograms demonstrating increase of the miscleavage peptides of T-DM1 with increasing T-DM1 concentration in plasma, when digested and extracted using the kit; Panel A: FTISADTSKNTAYLQMNSLR and Panel B: GPSVFPLAPSSKSTSGGTAALGCLVK
Appendix
MRM conditions Infl iximab
Peptide
DILLTQSPAILSVSPGER* SINSATHYAESVK* DSTYSLSSTLTLSK
SVSELPIMHQDWLNGK (ISTD)
*Unique signature peptide Protein
T-DM1/ Trastuzumab Trastuzumab Trastuzumab
Trastuzumab T-DM1
miscleavage with small
molecule drug attached
Murine mAb (IS)
MRM Transition 633.10>731.80 469.6>603.80 751.88>836.47 618.64>834.41
Cone Voltage (V) 31 40 31 16
Collision Energy (eV) 21 10 24 12
Cone Peptide IYPTNGYTR FTISADTSK DTYIHWVR GPSVFPLAPSSK* FTISADTSKNTAYLQMNSLR MRM Transition
542.77>249.16 542.77>808.40 485.20>721.40 485.20>608.30 543.30>597.30 545.30>710.40 593.83>699.40 1073.17>547.20 1073.17>485.22
GPSVFPLAPSSKSTSGGTAALGCLVK 1073.17>547.20
SVSELPIMHQDWLNGK VNSAAFPAPIEK
*Generic IgG signature peptide
Analytical method conditions LC system: Waters ACQUITY UPLC®
MS detection: Waters Xevo® Spectrometer, ESI+
Data management: Waters MassLynx® Software (v4.1)
Column: Waters ACQUITY UHPLC Peptide BEH C18, 300Å, 1.7 µm, 2.1 x 150 mm
Column temp: 55°C Sample temp: 10°C Injection volumes: Infl iximab, 10 µL; Trastuzumab and T-DM1, 5 µL
Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in
acetonitrile System TQ-S Mass
618.64>834.41 622.30>654.44
Gradient:
Flow rate (mL/min)
0.3 0.3 0.3
0.3
MS conditions Capillary: Cone:
Time (min)
0.0 1.0 7.0
8.0 Profile
%A %B 100 0
100 0 50 50
10 90
3 kV 30 V
Source offset: 50 V Source temp:
150°C
Desolvation temp: 600°C Cone gas fl ow: 150 L/hr Collision gas fl ow: 0.15 mL/min
Nebuliser gas fl ow: 7 Bar
Produced in association with the Chromatography Society
Curve
6 6 6
6
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or call us on: +44 (0)1727 855574
Voltage (V) 36 12 28 28 28 28 31 35 35
35
16 28
Collision Energy (eV) 16 16 22 22 24 28 21 38 38
38
12 16
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www.chromatographytoday.com
References
1. McKinsey and Company; Data Source: Evaluate Pharma, US Patent Expiration Dates.
2. ProteinWorks Care and Use Guide, Waters Corporation, 2016.
3. Peddi PF, Hurvitz SA. Trastuzumab emtansine: the fi rst targeted chemotherapy for treatment of breast cancer. Future oncology (London, England). 2013;9(3):10.2217/fon.13.7. doi:10.2217/ fon.13.7.
4. BarokM, JoensuuH, IsolaJ. Trastuzumab emtansine: mechanisms of action and drug resistance. Breast Cancer Res. 2014 Mar5;16(2):
209.doi:10.1186/bcr3621
5.
http://www.drugbank.ca/drugs/DB05773
6. FDA Guidance for Industry for Bioanalytical Method Validation, CDER
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