*2.3. 2D 1H-15N Spectroscopy*

The 2D 1H-15N spectrum may be a more specific NMR experiment than 1H-13C HSQC to evaluate protein HOS because the amide 1H and 15N chemical shifts are exclusively sensitive to peptide backbone conformation. However, the 1H-15N HSQC experiment is at least one order of magnitude less sensitive than the methyl 1H-13C HSQC experiment. Thus, 15N spectra via indirect detection in formulated DP samples can be challenging to collect when the DS concentration is less than 1 mM. The previously developed NMR pulse sequence of sofast-heteronuclear multi-quantum correlation (HMQC) has the advantage of shorter recycle delay without perturbing water resonances [43]. The sofast-HMQC experiment allows the 2D 1H-15N correlation spectrum to be collected within 24 h for DPs with protein concentrations as low as 0.06 mM. Representative spectra of Forteo®, Byetta® and Lantus® are shown in Figure 5. Amide peaks of the protein backbone and Asn/Gln side chains are observed in the 15N chemical shift range of 108–129 ppm and the 1H chemical shift range of 7.4–9.1 ppm. The number of detected peaks for teriparatide in Forteo® was 29 (Figure 5A), while a total of 44 peaks are expected. The 66% coverage suggests the teriparatide adopts a well-defined HOS in the formulation, which is consistent with the 1D 1H spectral pattern (Figure 1C). By contrast, only six peaks were detected for exenatide in Byetta® (Figure 5B), whereas a total of 39 peaks are expected. The 15% coverage suggests the exenatide resonances are in intermediate exchange between different HOS forms, which is, again, consistent with the broadening in the 1D 1H spectrum (Figure 1D). For insulin glargine in Lantus®, the 54 peaks detected account for 87% of the expected 62 peaks (Figure 5C). The results for insulin glargine suggest the existence of a single or fast averaged backbone HOS in the formulation.


**Table 2.** The relative peak height comparison of insulin glargine DPs.

n/a: not available.

**Figure 5.** The 2D sofast 1H-15N HMQC NMR spectra of teriparatide in Forteo® (**A**), exenatide in Byetta® (**B**) and insulin glargine in Lantus® (**C**) collected using an 850 MHz spectrometer. The plotting threshold of intensity was at a signal to noise ratio of 5.
