30 February / March 2016
Figure 3: QC chromatograms of trastuzumab (A) and T-DM1 (B) for the IYPTNGYTR unique signature peptide.
Figure 4. Chromatograms demonstrating the presence of the miscleavage peptides in T-DM1 (350 µg/mL), as compared to Trastuzumab (350 µg/mL), and blank rat plasma when digested and extracted using the kit; Panel A: FTISADTSKNTAYLQMNSLR and Panel B: GPSVFPLAPSSKSTSGGTAALGCLVK.
Conclusion
Commercially available digest and clean- up kits were successfully used to purify infliximab from a typical set of standard curve and QC samples in rat plasma. A limit of quantification of 10 ng/mL was readily achieved, while maintaining excellent linearity and single digit precision. The total sample prep time including an affinity purification step was less than 6 hours. The total digest prep time was just over 2 hours.
Figure 5. Chromatogram demonstrating confirmation of the identity of the payload-conjugated miscleavage peptide FTISADTSKNTAYLQMNSLR monitoring different MRM transitions.
of these conjugated peptides in TDM-1 plasma samples (350 µg/mL), as compared to Trastuzumab (350 µg/mL), and blank rat plasma. Presence of the FTISADTSKNTAYLQMNSLR conjugated peptide was confirmed by multiple MRM transitions, and is shown in Figure 5.
Additionally, the area counts from both of these conjugated TDM-1 peptides increased with increasing concentration of T-DM1. This is highlighted in Figure 6, panels A and B.
Additionally, these optimised kits were successfully used to quantify trastuzumab and the ADC T-DM1 from a typical set of standard curve and QC samples in plasma. Through direct digestion of 35 µL of plasma, quantification limits of 0.5 to 1 µg/mL were achieved, while maintaining excellent linearity, precision and accuracy.
By using these kits, whether the analyst is an experienced or novice bioanalytical and/or biomarker researcher, ultra-low detection limits can be achieved with a simple step-wise protocol and a set of standardised, pre-measured reagents, ensuring both the sensitivity required to make time-sensitive and critical project decisions during drug development.
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