The higher order structure of complex biological therapeutics such as polysaccharide-containing vaccines, recombinant proteins, and monoclonal antibodies is an important quality attribute of biopharmaceutical products. The relationship between higher order structure and product efficacy is a crucial issue in comparability studies, be they assessments of manufacturing changes or follow-on biologics. Several biophysical methods, such as circular dichroism, fluorescence, and bioassays, are typical for assessing structure but none provide information at high resolution. Nuclear magnetic resonance (NMR) spectroscopy is a well-established technique for biomolecular structure determination, albeit underused in characterizing biotherapeutics. NMR is misperceived as too expensive or complicated and therefore is excluded from the methods toolbox to assess higher order structure. In this paper, we show simple applications of NMR that provide detailed information on higher order structure. We also address the complexity and cost of including NMR in the process and quality control environments.
Carbohydrates Polysaccharide Vaccines
NMR spectroscopy is a versatile method that can be used to characterize molecules at the atomic level. After its use in characterizing small molecules (MW ≤1,000), NMR spectroscopy was used to characterize polysaccharide vaccines. Despite their high molecular weights, many of these heterogeneous molecules display very sharp lines in their 13 C and 1 H NMR spectra. Because NMR spectroscopy can yield information at atomic resolution, the components of complex molecules can be identified and quantified. Presently, 1 H NMR is used to provide identity, ascertain O-acetyl and N-acetyl proportions, determine water content, and monitor stability (through decomposition) of pneumococcal,1 meningococcal, and Haemophilus influenzae polysaccharide vaccines. Reliable quantification is easily accomplished as long as the fundamental principles of NMR, such as the guidelines of accurate data acquisition and nuclear relaxation, are followed.2 The dynamic range of NMR has increased, so impurities present even at the level of parts per billion can be detected.