*2.2. 2D 1H-13C Spectroscopy*

## 2.2.1. Protein Specificity

As an alternative to 1D 1H spectra, 2D 1H-13C heteronuclear single quantum coherence (HSQC) spectra are highly specific to protein sequence and HOS. The HSQC spectrum of the methyl chemical shift region, which was 12–27 ppm along the 13C axis and 0–1.5 ppm along the 1H axis, has higher sensitivity due to the 3 C-H correlations and fast internal rotational dynamics of the methyl group. In addition, major excipient or solvent peaks, if observed in a methyl HSQC spectrum, can be readily identified because of their strong intensity and unique 13C chemical shifts, e.g., ethanol in insulin DPs of HumulinR®, Humalog® and Basaglar® (Figure 3). Therefore, methyl-HSQC spectra can be an ideal high-resolution spectrum for HOS assessment.

**Figure 2.** The superimposed 1D 1H NMR spectra of representative rituximab drug products (DP) of Rituxan® and Reditux® collected using 850 MHz (**A**, left) and 600 MHz (**B**, left) spectrometers. The spectral regions in gray of 0–0.2 ppm, 0.85–0.95 ppm, 1.15–1.45 ppm, 1.55–1.65 ppm, 1.9–2.1 ppm, 2.2–2.9 ppm, 3.3–6 ppm and 8.45–8.47 ppm were excluded before principal component analysis (PCA). The resulting PCA scores for each DP lot from both 850 MHz data (**A**, right) and 600 MHz data (**B**, right) were plotted along the PC1 and PC2 axes. The 90% confidence ellipses are drawn for Rituxan® lots only (A/B, right).

The amino acids possessing one or two methyl groups are Alanine (Ala), Methionine (Met), Threonine (Thr), Valine (Val), Leucine (Leu) and Isoleucine (Ile). For insulin, methyl peaks of Ala, Thr, Val, Leu and Ile observed in the DP spectra of HumulinR® and Humalog® (Table 1) can be approximately assigned by referencing the literature assignment for insulin human [39] and insulin lispro [40] (Figure 3A). Due to formulation or buffer differences, the assignment can be confidently made for residues of Ile at positions A2 and A10, Ala at position B14 and Thr at positions A8, B27 and B30; ambiguities remain for the Val and Leu clusters in the 13C chemical shift ranges of 21–22 ppm and 23–27 ppm, respectively (Figure 3A). Nevertheless, large chemical shift differences were observed between insulin human and insulin lispro, both of which have an identical M.W. of 5808 Da. The insulin lispro sequence differs from insulin human by two amino acid residues at positions B28–B29, Pro-Lys in insulin human and Lys-Pro in insulin lispro. Thus, the sequence difference introduced large changes in chemical shifts for almost every methyl peak (Figure 3A), which is consistent with a large change of HOS in the formulated API arising from only a two amino acids swap.

Shown in Figure 3B is the superimposed spectra between Lantus® and Basaglar®, both of which contain the same DS insulin glargine. The chemical shift assignment was not transferrable from other insulins because the spectra are significantly different (Figure 3A,B). The methyl peaks of insulin glargine were labeled with possible amino acid type and alphabetic letters. The total number of identified peaks (*s*/*n* > 10) was 48, which is more than the expected 28 methyl peaks calculated from the insulin glargine sequence. The increased peak number is attributed to some of the methyl groups adopting at least two slow exchange conformations in the formulation, e.g., Ala(B14) had two peaks of Ala-a and Ala-b at 13C chemical shift of 19 ppm (Figure 3B). Overall, the methyl HSQC spectra between the two DPs are highly similar, suggesting that insulin glargine is folded in similar HOS for the two formulations.

**Figure 3.** The superimposed 2D 1H-13C HSQC NMR spectra between insulin drug products of HumulinR® and Humalog® (**A**) and between Lantus® and Basaglar® (**B**) collected using a 600 MHz spectrometer. The plotting threshold of intensity was at a signal to noise ratio of 5 and 10 for (**A**) and (**B**), respectively. The methyl spectra of insulin human and insulin lispro were approximately assigned according to the published assignments; ambiguities were observed in the Valine and Leucine clusters shown in dashed boxes (**A**). The spectra of insulin glargine cannot be definitively assigned due to a large change in the observed chemical shifts and each peak was labeled for possible amino acid type and with an alphabetic letter (**B**).
