Comparative Analysis of 1D PROFILE and 2D (1)H,(13)C 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 (1)H-(15)N correlation experiments have proven exceedingly difficult to implement on mAbs and a number of alternative techniques have been proposed, including the 1D (1)H PROFILE method and the 2D (1)H-(13)C 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 is 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.