Measurement of DAR of trastuzumab-vcE ADC after incubation in IgG depleted serum for seven days
Stability of biologics is a critical quality attribute (CQA) of ADC stability, affecting the levels of dosing and efficacy.
- Measurement = can be complicated by the inherent heterogeneity present within the protein as well as the payload-linker
- Mass Spectrometry can measure loss of payloads on the protein level or as a released drug.
- Intact measurements have the benefit of minimising sample handling after incubation compared to enzymatic release methods.
Plasma stability protocol
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 IgG depleted rat (Sprague Dawley (BioIVT)) serum for time periods up to seven days. The ADC was then captured on Protein A magnetic beads (BioRad),before release and reduction. Data was collected over two pooled biological replicates and three technical replicates. All data was collected on a SCIEX X500B Mass Spectrometer with a bioZen Intact XB-C8 Column (3.6 µm, 50 x 2.1 mm).
Figure 1: TIC of reduced trastuzumab vcE conjugate before (blue) and after (red) incubation. Separation between each conjugated form is observed with changes in extremes after incubation. See more here.
Figure 2: Mass Spectrum of trastuzumab vcE H0 andH3 main glycoforms. Overlaid spectra show changes in relative intensity and oxidation corresponding with incubation in serum.. See more here.
Figure 3: Average DAR versus incubation time. Overall heavy and light chain DAR decreases after incubation in serum. Error bars from technical replicates. See more here.
Loss of payload over time
The ADC at t=0 was observed in H1 through to H3with very little H0, DAR was calculated at just over 4.5 matching Hydrophobic Interaction Chromatography. Figure 1 shows the TIC of all the subunit peaks after reduction at t=0 (blue) and after seven days incubation in IgG depleted serum (red). Overall, a decrease in all drug bound species and an increase inL0 and H0 was observed.
The overall DAR of both light and heavy chains, including glycoforms and modifications, was recorded in BPV Flex and the output is displayed in Figure 3 where we see a decrease from DAR ~4.5 to DAR ~3 over seven days.
Figure 2 shows the mass spectrum of the first glycoform of H0 and H3 normalised against L1. We can clearly track the decrease and oxidation of H3 and the corresponding increase of H0.
Frequently asked questions
Plasma stability involves incubating ADCs in plasma or serum to observe the behaviour of the payload-linker. Some linkers can be prematurely cleaved by enzymes in certain plasmas or others can be lost at the conjugation site.
If you have a high potency payload, any loss prior to reaching its target could result in reduced efficacy or increased toxicity.
Plasma and serum are complex matrices, therefore samples require some level of clean up before they can be analysed. Clean up strategies have to be balanced between how specific they are, versus flexibility for a variety of samples
Not every lab has access to high-resolution mass spectrometry or the expertise to perform complex analytics. CDMOs allow organisations to benefit from a wide variety of equipment without the associated capital investments.
At Sterling, we are more than a traditional CDMO. We are a PDMO®, or partnership development and manufacturing organisation. This means we are uniquely responsive to your needs, committed to your product, and capable of adding a deep level of scientific value and we are differentiated by the three core characteristics of service, passion, and science.
