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.
Highlights
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]
If low-resolution spectroscopic methods such as near-ultraviolet circular dichroism (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 nuclear magnetic resonance (NMR) methods become a replacement in many cases for this type of HOS assessments, and for the product characterization of biopharmaceuticals
Summary
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 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]. Mass spectrometric methods, such as hydrogen-deuterium exchange experiments, have gained considerable interest for the assessment of biopharmaceuticals [8,9]
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