Ensuring structural integrity of Hib capsular polysaccharide in conjugate vaccine manufacturing by ¹H NMR and...

The structural integrity of Haemophilus influenzae type b (Hib) capsular polysaccharide (polyribosylribitol phosphate, PRP) is a critical determinant of glycoconjugate vaccine quality, yet conventional pharmacopoeial methods lack the resolution to monitor atomic-level alterations during manufacturing. This study employed an orthogonal analytical strategy integrating solution-phase ¹H NMR spectroscopy and HPSEC-MALLS/RI to rigorously evaluate the preservation of PRP's structure across three consecutive production batches, encompassing purified polysaccharide, activated intermediate, ADH-derivatized polymer, and the final tetanus toxoid (TT) conjugate. ¹H NMR analysis demonstrated high-degree of batch-to-batch consistency, with invariant spectral fingerprints in the diagnostic saccharide region (δ 3.4-4.4 ppm) confirming the core ribose and ribitol moieties remained entirely undisturbed through chemical activation and conjugation. HPSEC-MALLS/RI revealed consistent weight-average molecular weight (Mw) through activation and derivatization, ruling out depolymerization or aggregation, while an expected Mw increase post-conjugation quantified the successful removal of unconjugated species and enrichment of the high-molecular-weight glycoconjugate. The final lyophilized product maintained molecular size stability. These results demonstrate the preservation of PRP's structural and macromolecular integrity throughout the manufacturing process. We propose ¹H NMR and HPSEC-MALLS/RI as essential quality control tools for in-process monitoring and lot-release, ensuring the structural consistency, safety, and efficacy of Hib conjugate vaccines.