Reduced subunit analysis of a PDB based antibody drug conjugate

19th Jul 2022

Subunit analysis of Zynlonta using a SCIEX X500B Mass Spectrometer

Antibody-drug conjugates (ADCs) are a class of biopharmaceuticals combining the highly targeted nature of monclonal antibodies (mAbs) with cytotoxic payloads. There are many new upcoming ADCs in clinical trials exploring innovative and novel payloads. ADCs present unique analytical challenges due to the inherent heterogeneity of the mAb scaffolds combined with varying numbers of conjugated drug. Drug-antibody-ratio (DAR) for ADCs isa critical quality attribute (CQA) affecting the efficacy and safety of the final product. Some highly potent drugs require a low DAR to balance toxicity, whereas others have a higher DAR with less potent warheads.

Here we outline an analysis strategy to confirm the DAR of an ADC with a pyrrolobenzodiazepine (PBD) dimer payload bound to cysteine residues of a CD19 targeting mAb (loncastuximab tesirine). All data was collected on a SCIEX X500B Mass Spectrometer with an EXION HPLC using a bioZen Intact XBC8 column (3.6 ┬Ám, 50 x 2.1 mm) with a 10 minute gradient of water and acetonitrile (0.1% formic acid).

Figure 1: Total ion chromatograph of all chains. Separation of conjugated species is possible with near baseline separation. A small variant peak is observed at 3.0 minutes but has not been assigned. See more here.

Figure 2: Reconstructed spectra of loncastuximab tesirine light chain. The DAR of the light chain was calculated as 0.4 with some in- source fragmentation of payload (23835). See more here.

Figure 3: Reconstructed spectra of loncastuximab tesirine heavy chain. The DAR of the heavy chain was calculated as 0.75. See more here.

PBDs are hydrophobic molecules and can be difficult to analyse by analytical methods. In Figure 1 we can observe the heavy and light chains with near baseline separation between the peaks.

Reconstructions are shown in Figure 2 for L0and L1 and Figure 3 for H0, 1, 2, and 3. DAR can be calculated for each chain individually through reconstructed peak area, with heavy chain peaks including assigned glycans G0F, G1F, and G2F with the presence of C-terminal lysine clipping and N-terminal pyroglutamate formation.

The DAR for the heavy chain was calculated at 0.75 and the light chain at 0.4 giving a total of 2.3, hydrophobic interaction chromatography was conducted on the same sample which was in agreement.

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