Measurement of ADC stability in plasma via released payload quantitation
28th Mar 2022
Measurement of payload concentration of trastuzumab-vcE ADC after incubation in plasma
Antibody drug conjugates represent highly heterogenous molecules with the inherent variations of an antibody combined with the addition of cytotoxic payloads and linkers. When measuring these samples for stability, either in plasma or during storage, there needs to be robust analytical methods to monitor specific critical quality attributes (CQAs). If the linker is designed to be cleaved this can be used to measure the amount of payload present via a quantitative MRMHR type method.
Here we outline an analysis strategy to determine the stability of a trastuzumab val-cit PABC MMAE conjugate (DAR 4.5).The samples were incubated in rat plasma (Sprague Dawley (BioIVT)) for time periods up to seven days. The ADC was then captured on Protein A magnetic beads (BioRad), before enzymatic linker cleavage. Data was collected over two pooled biological replicates and three technical replicates. All data was collected on a SCIEX X500B Mass Spectrometer with an EXION HPLC monitoring the 718 m/z ion with an internal standard of MMAF (925 m/z). The chromatography was performed with a Kinetix XB-C18 Column (1.7 µm, 50 x2.1 mm).
Figure 1: MSMS fragmentation of MMAE payload. Red arrow indicates 718 m/z precursor. Ion 686 m/z was used as the quantifier ion with 152 m/z. See more here.
Figure 2: Calibration curve of MMAE over four orders of magnitude. r2 was >0.99 with an internal standard of MMAF at 2.5 ng/ml. See more here.
Figure 3: XIC of MMAE payload ion 686 m/z. Intensity of peak is reduced over time as payload is lost during incubation in plasma. See more here.
The payload was observed in a single peak eluting at 1.5 minutes on a five-minute gradient of water and acetonitrile (both with 0.1% formic acid). Figure 1 shows the MSMS spectrum of the payload with a number of fragments which could be positively assigned.
A calibration curve was generated using SCIEXOS software and plasma incubated samples were compared to this. At time zero the concentration of released MMAE from the ADC was 280± 35 ng which is equivalent to a DAR 4.5 conjugate with an~30% capture.
Figure 3 shows the XIC peak area of MMAE over the five time points. We are able to observe a decrease in response over time due to loss of the linker by enzyme activity in the plasma. The peak areas were compared to the calibration curve to give the values shown in Table 1. There was overall a 75% reduction in bound drug over time.
