NMR Assays for Estimating the O-Acetyl Content of Meningococcal Polysaccharide Serogroup A in Quadrivalent Conjugate Vaccine Formulation

Abstract

The use of multivalent glycoconjugate vaccines has dramatically contributed to reduce the incidence of meningococcal infectious disease. The advanced structural characterization of polysaccharide conjugates leads to enhancements in the quality and control of the products. Here, we report a novel nuclear magnetic resonance (NMR) method to confirm the identity and structural conformity (e.g., O-acetyl content) of saccharide antigens that comprise a licensed tetravalent meningococcal serogroups A, C, W, and Y vaccine. For the first time, the NMR methodology is applied on a formulation (licensed vaccine) containing a large excess of excipient (i.e., sucrose) without analytical sample pretreatment. This work confirms the applicability of a rapid and easy NMR assay on a multivalent conjugate vaccine, which might be extended to other combination vaccines that are already licensed or in clinical development.

Figure 1

Structural representation of MenA-, MenC, MenW, and MenY-CRM₁₉₇ conjugates (R: H, acetyl). The protons used as the label for the identity test of each antigen are highlighted in blue.

Figure 2

¹H NMR spectra of the quadrivalent licensed Menveo vaccine. Labels to facilitate the peak assignment for MenA, MenC, MenW, and MenY antigens are shown.

Figure 3

(a) ¹H–³¹P HMBC spectrum of the MenA-CRM₁₉₇ sample; (b) ¹H NMR spectral window where H₁^MAnAc of MenA-antigen resonates. The vertical bars show the integral regions identified for measuring the OAc and non OAc forms. Labels to facilitate the peak assignment are shown.

Figure 4

¹H spectrum of the monovalent MenA-CRM₁₉₇ sample. Labels to facilitate the peak assignment are shown.