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Default Application of Natural Isotopic Abundance (1)H-(13)C- and (1)H-(15)N-Correlated Two-Dimensional NMR for Evaluation of the Structure of Protein Therapeutics.

Application of Natural Isotopic Abundance (1)H-(13)C- and (1)H-(15)N-Correlated Two-Dimensional NMR for Evaluation of the Structure of Protein Therapeutics.

Related Articles Application of Natural Isotopic Abundance (1)H-(13)C- and (1)H-(15)N-Correlated Two-Dimensional NMR for Evaluation of the Structure of Protein Therapeutics.

Methods Enzymol. 2016;566:3-34

Authors: Arbogast LW, Brinson RG, Marino JP

Abstract
Methods for characterizing the higher-order structure of protein therapeutics are in great demand for establishing consistency in drug manufacturing, for detecting drug product variations resulting from modifications in the manufacturing process, and for comparing a biosimilar to an innovator reference product. In principle, solution NMR can provide a robust approach for characterization of the conformation(s) of protein therapeutics in formulation at atomic resolution. However, molecular weight limitations and the perceived need for stable isotope labeling have to date limited its practical applications in the biopharmaceutical industry. Advances in NMR magnet and console technologies, cryogenically cooled probes, and new rapid acquisition methodologies, particularly selective optimized flip-angle short transient pulse schemes and nonuniform sampling, have greatly ameliorated these limitations. Here, we describe experimental methods for the collection and analysis of 2D (1)H(N)-(15)N-amide- and (1)H-(13)C-methyl-correlated spectra applied to protein drug products at natural isotopic abundance, including representatives from the rapidly growing class of monoclonal antibody (mAb) therapeutics. Practical aspects of experimental setup and data acquisition for both standard and rapid acquisition NMR techniques are described. Furthermore, strategies for the statistical comparison of 2D (1)H(N)-(15)N-amide- and (1)H-(13)C-methyl-correlated spectra are detailed.


PMID: 26791974 [PubMed - in process]



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