Conformational dynamics of pneumococcal polysaccharide conjugates drive UV-vis quantification errors and a strategy to enable UV-vis spectrophotometer quantification.

Accurate quantification of polysaccharide content is indispensable for the development and quality control of glycoconjugate vaccines. Conventional methods are often laborious, require large sample volumes, or are susceptible to interference. Ultraviolet-visible (UV-Vis) spectrophotometry presents an attractive alternative but suffers from an unexplained nonlinear concentration-absorbance relationship, precluding its reliable use. Here, we reveal that molecular behavior of pneumococcal polysaccharide conjugates including the solution conformation dynamics is the principal cause of this quantification anomaly. We demonstrate that polysaccharide chains undergo concentration-dependent conformational transitions: from an extended state with enhanced absorbance at low concentrations, to a protein-coated form, and finally to aggregates at high concentrations. This molecular behavior, characterized by dynamic light scattering and diffusion-ordered NMR spectroscopy, directly causes nonlinear absorbance and spectral shifts. By modeling this relationship with a sine wave-based function, we identified a stable quantitative window and established a robust UV-Vis method for direct polysaccharide quantification. The developed method is proven to have acceptable recovery in conjugate samples with VLP carrier proteins that are in a stable conformation. Our findings provide a mechanistic understanding of a long-standing analytical challenge in vaccinology. The developed method, validated for its precision, accuracy, enables rapid and simple quantification of polysaccharide content with minimal sample consumption. This work not only facilitates accelerated conjugate vaccine development but also offers a generalizable framework for analyzing complex biomolecular therapeutics.