Identity Profiling of Complex Mixtures of Peptide Products by Structural and Mass Mobility Orthogonal Analysis

Identity is a critical quality attribute that must be determined before releasing batches of medicinal and dietary products. However, the identities of peptide-derived products composed of a large number of diverse molecules is challenging since most analytical techniques cannot analyze multiple molecules simultaneously. Here, we proposed the determination of the weight-average molecular weight (M_w) and polydispersity index (PDI) by mass spectrometry for control quality for the batch release of complex products, namely, glatiramer acetate (Copaxone), collagen hydrolysate (Colagenart), and a human dialyzable leucocyte extract (Transferon). The M_w and PDI values were orthogonally determined by PFG-STE-H₂O(presaturation)-DOSY-NMR analysis. To the best of our knowledge, this is the first time that MS and NMR spectra have been combined to determine the PDI of complex products derived from protein hydrolysis that are not monodisperse. The performance of each method was evaluated by comparing the obtained results to those reported for glatiramer acetate using MALLS, the technique commonly employed to determine PDI. This combined approach demonstrates the ability of these techniques to separate peptide populations from complex mixtures to establish their identity through their mass distribution profiles.