Variance of Commercial Powdered Milks Analyzed by Proton Nuclear Magnetic Resonance and Impact on Detection of Adulterants.

Proton nuclear magnetic resonance spectra for 66 commercial powdered milk samples were analyzed by principal component analysis, soft independent modeling of class analogy, and pooled, crossed analysis of variance. It was found that the sample type (skim milk powder or non-fat dry milk), the supplier, the production site, the processing temperature (high, medium, or low temperature), and the day of analysis provided statistically significant sources of variation. Interestingly, inexact alignment (deviations of ±0.002 ppm) of the spectral reference peak was a significant source of variation, and fine alignment was necessary before the variation arising from the other experimental factors could be accurately evaluated. Using non-targeted analysis, the lowest detectable adulteration for dicyandiamide, melamine, and sucrose was 0.05%, the lowest detectable adulteration for maltodextrin and urea was 0.5%, the lowest detectable adulteration for ammonium sulfate and whey was 5%, and the lowest adulteration for soy protein isolate was undetectable using methods described herein. The measurement of variance and detection of adulteration were relatively unaffected by the resolution. Similar results were obtained with unbinned data (0.0003 ppm resolution) and binning of 333 data points (0.1 ppm resolution).

Application of 2D-NMR with room temperature NMR probes for the assessment of the higher order structure of filgrastim.

The higher order structure (HOS) of biotherapeutics is a critical quality attribute that can be evaluated by nuclear magnetic resonance (NMR) spectroscopy at atomic resolution. NMR spectral mapping of HOS can be used to establish HOS consistency of a biologic across manufacturing changes or to compare a biosimilar to an innovator reference product. A previous inter-laboratory study performed using filgrastim drug products demonstrated that two-dimensional (2D)-NMR 1HN-15NH heteronuclear correlation spectroscopy is a highly robust and precise method for mapping the HOS of biologic drugs at natural abundance using high sensitivity NMR 'cold probes.' Here, the applicability of the 2D-NMR method to fingerprint the HOS of filgrastim products is demonstrated using lower sensitivity, room temperature NMR probes. Combined chemical shift deviation and principal component analysis are used to illustrate the performance and inter-laboratory precision of the 2D-NMR method when implemented on room temperature probes.