Principal Component Analysis of 1D 1H Diffusion Edited NMR Spectra of Protein Therapeutics.

The one-dimensional (1D) diffusion edited proton NMR method, Protein Fingerprint by Lineshape Enhancement (PROFILE) has been demonstrated to be suitable for higher order structure (HOS) characterization of protein therapeutics including monoclonal antibodies. Recent reports in the literature have demonstrated its advantages for HOS characterization over traditional methods such as circular dichroism and Fourier-transform infrared spectroscopy. Previously, we have demonstrated that the PROFILE method is complementary with high resolution 2D methyl correlated NMR methods and how both may be deployed as a multi-modal platform to further the utility of NMR for HOS characterization. A major limitation of the PROFILE method remains its need for high signal to noise data due to its reliance on convolution difference processing and linear correlation metrics to assess spectral similarity. Here we present an alternative method for analyzing 1D diffusion edited spectra, which overcomes this limitation by using nonlinear iterative partial least squares (NIPALS) principal component analysis, and which we dub PROtein Fingerprint Observed Using NIPALS Decomposition (PROFOUND). We demonstrate that results from the PROFOUND method are robust with respect to instrument, operator and in the presence of high experimental noise and how it may be employed to provide quantitative assessment of spectral similarity.

Comparative Analysis of One-Dimensional Protein Fingerprint by Line Shape Enhancement and Two-Dimensional 1H,13C Methyl NMR Methods for Characterization of the Higher Order Structure of IgG1 Monoclonal Antibodies.

The use of NMR spectroscopy has emerged as a premier tool to characterize the higher order structure of protein therapeutics and in particular IgG1 monoclonal antibodies (mAbs). Due to their large size, traditional 1H-15N correlation experiments have proven exceedingly difficult to implement on mAbs, and a number of alternative techniques have been proposed, including the one-dimensional (1D) 1H protein fingerprint by line shape enhancement (PROFILE) method and the two-dimensional (2D) 1H-13C methyl correlation-based approach. Both 1D and 2D approaches have relative strengths and weaknesses, related to the inherent sensitivity and resolution of the respective methods. To further increase the utility of NMR to the biopharmaceutical community, harmonized criteria for decision making in employing 1D and 2D approaches for mAb characterization are warranted. To this end, we have conducted an interlaboratory comparative study of the 1D PROFILE and 2D methyl methods on several mAbs samples to determine the degree to which each method is suited to detect spectral difference between the samples and the degree to which results from each correlate with one another. Results from the study demonstrate both methods provide statistical data highly comparable to one another and that each method is capable of complementing the limitations commonly associated with the other, thus providing a better overall picture of higher order structure.