2.1.1. Excipients

Excipients in protein formulations can function as preservatives (e.g., phenol and *m*-cresol), tonicity agents (e.g., mannitol), pH buffering agents (e.g., acetate) or protein stabilizers (e.g., polysorbate 80) [28]. The excipients are mostly small molecules at high concentration relative to the API. Due to the fast tumbling of excipients in liquid formulations, excipient peaks generally were sharper and more intense in the NMR spectra (Figure 1, left panels). Most excipient peaks were located in the high field region between 1 and 4 ppm, while preservatives with aromatic moieties had peaks between 6.5 and 7.5 ppm. The peaks were readily assignable with the help of chemical shift databases [29,30] or 2D 1H-13C spectra. The excipient polysorbate 80 (PS80) had a more complicated spectrum, with major peaks at 3.7, 2.3, 2.0, 1.6, 1.3 and 0.9 ppm [31]. Importantly, all excipient peaks should be excluded when protein HOS comparison is performed.
