2.1.3. Protein HOS

The protein DS may be formulated at concentration of about 1 mM or less (Table 1). The 1D 1H NMR spectrum is the most sensitive NMR method to characterize protein HOS in DP formulations. The spectra need to be vertically enlarged by 2–4 orders of magnitude in order to visualize the lower intensity protein peaks (Figure 1, right panels). Among the tested DPs, the 3.43 kDa calcitonin-salmon is formulated at the lowest concentration of 9.7 μM. Calcitonin's sharp and dispersed amide peaks suggested that calcitonin-salmon adopts a folded monomeric HOS in formulation (Figure 1A). The 3.75 kDa liraglutide has a similar M.W. to calcitonin-salmon, however, broadened amide peaks were observed in liraglutide's spectra (Figure 1B), suggesting oligomerization of the protein in formulation.

**Figure 1.** The 1D 1H NMR spectra of protein drug products of Miacalcin® (**A**), Saxenda® (**B**), Forteo® (**C**), Byetta® (**D**), Lantus® (**E**) and Rituxan® (**F**) collected using an 850 MHz spectrometer. The spectra on the left are in full scale and those on the right are vertically enlarged and horizontally cut to display protein peaks. Signals from major excipients, drug substances and the leachable compounds dimethylsilanediol (d), trimethylsilanol (t) and polydimethylsiloxane (s) are annotated.

The 4.12 kDa teriparatide had sharp and dispersed amide peaks, suggesting a folded HOS in the formulation (Figure 1C). For the 4.19 kDa exenatide, much broadened peaks were observed while the detected number of peaks was much less (Figure 1D), suggesting the peptide was undergoing intermediate exchange broadening [37]. The observed exchange broadening is associated with exenatide in equilibrium between several HOS states and the exchange kinetics occur over a similar time scale of the chemical shift difference between different states, usually in the range of μs-ms exchange.

For the 6.06 kDa insulin glargine, the detected dispersed peaks suggest well folded HOS in the formulation at pH 4 (Figure 1E). Finally, the observed broadened peaks of the 145 kDa rituximab were due to its large M.W., but the dispersed amide peaks suggest the monoclonal antibody has a folded HOS (Figure 1F).
