(T1330-02-14) An Advanced LC-QTOF-MS Method for Accurate Characterisation of a Next Generation Vaccine

Purpose: Analytical testing is a critical requirement for the development and manufacture of new biopharmaceuticals. In particular, regulatory agencies require thorough analytical characterisation of novel biotherapeutics to demonstrate drug product safety, purity and efficacy as well as batch to batch consistency. This study demonstrates how an integrated peptide mapping workflow was employed to provide crucial in-depth structural characterisation of a nine-component next generation mosaic-8b vaccine nanoparticle currently under development to broaden protection against both as-yet-unknown SARS-CoV-2 variants and other animal sarbecoviruses with potential to infect humans¹.

Methods: The mosaic 8b nanoparticle comprises 60 mi3 subunits that self-assemble to form a central hollow cage that can incorporate and display eight different SARS-like betacoronavirus spike receptor binding domain (RBD) spike proteins randomly conjugated to 60 sites across the nanoparticle surface. Mosaic-8b was first synthesised and purified. MS analytical characterisation was performed using a high-resolution Agilent 6454XT QTOF LC-MS system, enabling high-throughput peptide mapping workflows.  An optimised trypsin digestion protocol was developed to ensure high digestion efficiency and complete proteolysis of both the nanoparticle scaffold and the displayed RBDs.  Following digestion, samples were subjected to chromatographic separation and MS analysis using optimised LC-MS method parameters.  Sequence analysis of the individual RBD spike proteins was achieved by identifying unique tryptic peptide biomarkers, which were initially confirmed at the MS1 level based on accurate mass of targeted precursor ions and isotopic profiling.  Targeted tandem MS/MS fragmentation was used to increase confidence in peptide assignment through spectral matching.  Data acquisition focused on identifying and quantifying these unique peptides specific to each of the eight RBD spike proteins.  Reference standards were used to normalise differences in ionisation efficiencies and correct for matrix effects and instrument variability.  The extracted ion abundance of these unique peptides was then used to determine the relative distribution of the individual RBD spike proteins displayed across the mosaic 8b surface.  Batch-to-batch consistency was evaluated by analysing multiple production lots prepared by different laboratories at different times.

Results: LC-QTOF-MS analysis successfully confirmed the presence of all RBD spike proteins displayed across the nanoparticle surface indicating efficient and controlled assembly of the mosaic-8b drug substance. Comparative analysis of three separate production batches revealed high reproducibility in terms of extracted ion abundances (and relative % distribution) for each of the RBD peptides as shown in Figure 3. The peptide mapping results confirmed primary amino acid structures and identities for all RBD spike proteins present in the mosaic-8b drug substance.  Our method yielded sequence coverage values from 86% to 100% with minimal post translational modifications (including phosphorylation, oxidation and deamidation adducts) observed.   Interestingly, the MS data consistently revealed higher abundances of RBD 1, 2 and 7 respectively, implying differences in conjugation efficiencies for specific spike proteins during mosaic-8b assembly might be occurring.  The results clearly demonstrate batch-to-batch consistency was achieved highlighting the robustness of the mosaic-8b manufacturing process and the analytical power of LC-QTOF-MS for complex biopharmaceutical characterisation.

Conclusion: The findings from this study unequivocally demonstrate the successful utilisation of LC-QTOF-MS as a powerful analytical tool for the comprehensive characterisation of a highly complex vaccine nanoparticle. In particular, peptide mapping is a crucial analytical technique that plays a vital role in meeting regulatory requirements. As the number of new biotherapeutic drug products prevalent in pre-clinical and clinical trials rapidly increases, the application of LC-QTOF-MS methods such as the one we have developed will be of great assistance to laboratories that require fast and accurate protein characterisation. Compared to other traditional analytical methods (deletion) such as SDS-PAGE, our MS/MS method offers several advantages including high throughput, high mass accuracy and sensitivity, and robust sample preparation protocols that can be easily adapted to accommodate specific analytical requirements for a range of biotherapeutic targets to accelerate drug product development.

References: 1. Cohen et al., 2024, Cell 187, 5554–5571

Figure 1 – Mosaic-8b peptide mapping workflow

Figure 2 – Representative model of mosaic-8b

Figure 3 – Comparison of normalised RBD extracted ion abundances and corresponding % relative distribution for 3 separate batches of Mosaic-8b