Coupling a glycan antigen to an immunogenic carrier protein produces a glycoconjugate vaccine that can generate an immune response and induce T-cell memory booster responses upon revaccination. Cross Reactive Material 197 (CRM197) is a nontoxic mutant of diphtheria toxin that is very effective and widely used as a carrier protein for this purpose.
Isolation of the pathogen glycans from natural sources is often a difficult technical challenge due to their large size and heterogeneity. To overcome this issue, vaccine developers are now focusing on incorporating well-defined, synthetic glycans into glycoconjugate vaccines. The coupling process involves synthesis of the glycan with a spacer, followed by addition of a linker molecule, and finally conjugation of the glycan-linker construct to a primary amine on the carrier protein. To date, only one semi-synthetic glycoconjugate vaccine has actually been approved for market.
The problem is that carrier proteins such as CRM197 have many primary amines with which the construct might react, resulting in heterogeneous products with heterogeneous vaccine effectiveness. Therefore, methods that can accurately and reliably define the conjugation sites on the carrier protein and characterize the end products are critical for assuring the quality of semi-synthetic vaccines and the eventual creation of completely defined conjugates.
A novel, mass spectrometry-based strategy was recently described for the in-depth characterization of a series of glycoconjugates produced using synthetic glycans and CRM197. The researchers used three orthogonal approaches for assessing the conjugates: MALDI-TOF MS of intact protein, middle-down LC-MALDI-TOF MS, and bottom-up LC-ESI-MS.
The bottom-up strategy produced insufficient sequence coverage and uncontrollable heterogeneity of the proteolytic glycopeptide products, so the middle-down approach was developed to overcome these limitations. The conjugates were cleaved with cyanogen bromide, and the resulting glycopeptides were analyzed by LC-MALDI-TOF MS on a Bruker ultrafleXtreme system. This new method resulted in virtually complete sequence coverage and provided a good overview of the conjugate modifications. Furthermore, comparisons of batch-to-batch conjugations were simplified.
Using this approach to analyze the conjugation of various synthetic glycans with CRM197, the researchers found that of the 40 primary amines that could potentially react with the glycan-linker complex, only a few were preferentially conjugated. Results also indicated that steric accessibility, the local amino acid environment and the protein secondary structure are the factors most likely to influence which residues react during the conjugation.
This novel technique now makes it possible to comprehensively characterize CRM197 glycoconjugate vaccine candidates. The ability to fully characterize these candidates along with any potential batch-to-batch variation in their creation brings us one step closer to the reality of rational vaccine design and will likely play an essential role in assuring a reliable supply of safe and effective vaccines.
Meet the ultrafleXtreme spectrometer
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