**5. Conclusions**

The results show that 2D 1H-13C HSQC NMR is incredibly sensitive to primary, secondary, tertiary, and quaternary structures, and provides unique fingerprints for both the IgG1 and IgG2 subclasses used. Near-ultraviolet circular dichroism (NUV-CD) is also able to differentiate between the two IgG subclasses, while intrinsic fluorescence (FLD) is only able to distinguish between the folded and unfolded states of each protein, but not able to distinguish IgG1 from IgG2. When the 2D NMR methyl fingerprints are visually compared to the results from NUV-CD and FLD, the degree of HOS information captured by 2D NMR is vastly superior to that of either currently established method. Our findings therefore exemplify the superiority of NMR in the assessment of higher-order structural attributes of biopharmaceuticals.

**Author Contributions:** Conceptualization, M.W.; Formal analysis, D.B. and N.K.; Investigation, T.-L.H., D.B. and N.K.; Methodology, T.-L.H.; Resources, T.-L.H.; Supervision, M.W.; Writing original draft, D.B. and N.K.; Writing—review and editing, M.W.; Manuscript reviewing, all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors would like to acknowledge the technical contributions of Jette Wypych.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

