**3. Discussion**

In this study, we have compared the ability of NUV-CD, FLD, and 2D NMR to measure HOS in two monoclonal antibody subclasses, IgG1 and IgG2. Unlike NUV-CD and FLD, which are only able to infer structural integrity from a limited number of chromophores in a protein, 2D NMR provides structural information about the entire molecule and is hence sensitive to even subtle changes in all levels of HOS. If low-resolution spectroscopic methods such as NUV-CD and FLD currently set the bar for assessing the structural integrity of biopharmaceuticals, we propose that vastly more informative 2D 1H-13C HSQC NMR methods become a replacement in many cases for this type of HOS assessments, and for the product characterization of biopharmaceuticals.

#### **4. Materials and Methods**

#### *4.1. Sample Preparation*

The test solutions were prepared from 100 mg/mL monoclonal antibodies IgG1 and IgG2 in the formulation buffer: 10 mM sodium acetate buffer, 9% (*w/v*) sucrose, at pH 5.2. The sequence identity comparing the IgG1 and Ig2 antibodies is 95% [13]. The IgG1 molecule harbors glycosylation on N302, while the IgG2 molecule contains glycosylation modifications on N298. Stock solutions of intact (folded) IgG1 and IgG2 were prepared at 50 mg/mL in the same formulation buffer. Stock solutions of the denatured (unfolded) IgG1 and IgG2 were prepared at 50 mg/mL in the formulation buffer, with 6M urea and 50 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP).
