Reduced subunit analysis of a therapeutic monoclonal antibody
28th Mar 2022
Subunit analysis of Trastuzumab using a SCIEX X500B Mass Spectrometer
Biologics represent a growing class of pharmaceuticals being developed to target a variety of disease and illness vectors. As well as established monoclonal antibodies, new modalities are emerging such as bispecific antibodies with differing heavy or light chains. Some of these features such as stoichiometry and glycan profiling can be defined as critical quality attributes (CQAs). Use of subunit analysis through reduction or specific cleavage enzymes like IdeS can be used to simplify the observed spectra to better qualify these attributes.
Here we outline an analysis strategy to confirm the molecular weight of the light and heavy chain of an mAb scaffold targeting the HER2 receptor, trastuzumab, after reduction. In addition, the glycans and C-terminal clips will be relatively quantified. All data was collected on a SCIEX X500B Mass Spectrometer with an EXION HPLC using a bioZen Intact XB-C8column (3.6 µm, 50 x 2.1 mm) with a 10 minute gradient of water and acetonitrile (0.1% formic acid).
Figure 1: Reconstructed spectra of trastuzumab light chain. High resolution MS allows isotopic reconstruction of species with high accuracy. See more here.
Table 1: Observed trastuzumab proteoform masses. Reconstructed masses match previously published data. See more here.
Baseline separation between the light chain (LC) and heavy chain (HC) is achieved eluting at 2.7 and 3.16 minutes respectively. Figure 1 shows the reconstructed data from the LC with isotopic resolution providing excellent mass accuracy.
Figure 2: Reconstructed spectra of trastuzumab heavy chain. Various glycoforms have been assigned based upon accurate mass in line with previously published data. See more here.
Figure 2 shows the reconstructed HC, with four major glycoforms visible, which can be assigned to G0, G0F, G1F, and G2F. The mass accuracy of these is displayed in Table 1 along with their relative intensities. Major species are observed without C-terminal lysine’s however minor peaks are observed attributable to the non-clipped forms. Calculations based on the reconstructed areas estimate 85% of the protein is present without C-terminal clipping.