*Article* **Use of the 2D 1H-13C HSQC NMR Methyl Region to Evaluate the Higher Order Structural Integrity of Biopharmaceuticals**

**Tsang-Lin Hwang †, Dipanwita Batabyal †, Nicholas Knutson and Mats Wikström \***

Attribute Sciences, Amgen Inc., Thousand Oaks, CA 91320, USA; thwang@amgen.com (T.-L.H.); dbatabya@amgen.com (D.B.); nsk2@g.uclas.edu (N.K.)

**\*** Correspondence: matsw@amgen.com

† Equal contributions.

**Abstract:** The higher-order structure (HOS) of protein therapeutics is directly related to the function and represents a critical quality attribute. Currently, the HOS of protein therapeutics is characterized by methods with low to medium structural resolution, such as Fourier transform infrared (FTIR), circular dichroism (CD), intrinsic fluorescence spectroscopy (FLD), and differential scanning calorimetry (DSC). High-resolution nuclear magnetic resonance (NMR) methods have now been introduced, representing powerful approaches for HOS characterization (HOS by NMR). NMR is a multi-attribute method with unique abilities to give information on all structural levels of proteins in solution. In this study, we have compared 2D 1H-13C HSQC NMR with two established biophysical methods, i.e., near-ultraviolet circular dichroism (NUV-CD) and intrinsic fluorescence spectroscopy, for the HOS assessments for the folded and unfolded states of two monoclonal antibodies belonging to the subclasses IgG1 and IgG2. The study shows that the methyl region of the 1H-13C HSQC NMR spectrum is sensitive to both the secondary and tertiary structure of proteins and therefore represents a powerful tool in assessing the overall higher-order structural integrity of biopharmaceutical molecules.

**Keywords:** higher-order structure; tertiary structure; fluorescence; circular dichroism; NMR; HOS by NMR; product characterization; biopharmaceuticals
