ABSTRACT
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.
(Photo Courtesy of DASGIP)
|
From the start, the quantitative characterization and analysis of biologics has been a daunting undertaking. This task is
complicated by the varied nature of biologics and compounded because many analytical methods have not been well suited to
characterize this complex set of products. Consequently, most pharmaceutical companies rely on bioassays to demonstrate consistency
with material used in the clinical trials where efficacy was proven.
In this paper, we first show how nuclear magnetic resonance (NMR) has been used to successfully characterize many aspects
of polysaccharide vaccines, then expand our scope to polysaccharide conjugate vaccines, and finally, indicate how NMR can
be used in the characterization of complex biologics.
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.
Figure 1. The 1H NMR spectrum of two different polysaccharides (PS) whose cognate antibodies cross-react. The spectra show differences between
these two polysaccharides in the anomeric region (6.0 to 4.5 ppm), the acetyl region (2.5 to 1.8 ppm), and the methyl region
(1.7 to 1.0 ppm).
|
An example of 1 H NMR use can be seen in Figure 1. The 1 H NMR spectra displays two different pneumococcal serotypes, 17A and 17F. The presence of either polysaccharide induces protective
cross-reactive immune responses, thus rendering a typical ELISA assay inadequate to identify either polysaccharide in a vaccine.3 In sharp contrast to the ELISA identity assay, 1 H NMR can easily detect the difference between the two polysaccharides. The spectra in Figure 1 shows differences in the
anomeric region and in the number of rhamnose and acetyl groups.
