**1. Introduction**

The higher-order structure (HOS) of proteins includes the secondary, tertiary, and quaternary structure, and represents a critical quality attribute directly related to the structural integrity and the function of therapeutic proteins. The characterization of HOS represents a significant challenge for biopharmaceuticals and is currently being performed using low- to medium-resolution biophysical methods, such as Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD) spectroscopy, intrinsic fluorescence spectroscopy (FLD), and differential scanning calorimetry (DSC) [1,2]. With the increasing interest in different protein modalities in biopharmaceutical development and the rapidly expanding area of biosimilar development, there is a growing need for new analytical methods with higher specificity than the methods commonly applied. During the development and lifecycle of protein therapeutics, the innovator product will most often go through multiple process changes, in which it is required to show that any process-related drug product variations are within the acceptable criteria, and therefore considered comparable. In a similar fashion, it is required to show similarity between the biopharmaceutical reference product and developed biosimilars. The application of nuclear magnetic resonance (NMR) for the assessment of HOS has been suggested as a technology with the potential to more accurately assess differences in HOS as compared to established methods [3]. This technology, referred to as Profile NMR, is based on a one-dimensional diffusion NMR method, in which the strong signals from excipients are efficiently suppressed by dephasing the signals through gradients due to faster Brownian motions of smaller excipient molecules as compared to larger protein in the sample, leaving a spectrum of the protein product only [4,5]. In

**Citation:** Hwang, T.-L.; Batabyal, D.; Knutson, N.; Wikström, M. Use of the 2D 1H-13C HSQC NMR Methyl Region to Evaluate the Higher Order Structural Integrity of Biopharmaceuticals. *Molecules* **2021**, *26*, 2714. https://doi.org/10.3390/ molecules26092714

Academic Editor: Thomas Mavromoustakos

Received: 29 March 2021 Accepted: 29 April 2021 Published: 5 May 2021

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addition to the 1D NMR method, a 2D 1H-13C HSQC method was introduced [6], which shows great promise for the HOS assessment of monoclonal antibodies (mAbs) [7]. Finally, mass spectrometric methods, such as hydrogen-deuterium exchange experiments, have also gained considerable interest for the assessment of biopharmaceuticals [8,9].

In this study, we have compared two established methods, near-ultraviolet circular dichroism (NUV CD) and intrinsic fluorescence (FLD) spectroscopy, for the assessment of HOS for biopharmaceuticals against a 2D 1H-13C HSQC NMR method modified to suppress signals from excipients. To demonstrate the effect HOS has on each spectroscopic method, we compared the folded and unfolded states of two monoclonal antibody subclasses, IgG1 and IgG2, with about 95% sequence identity.
