2D J-correlated proton NMR experiments for structural fingerprinting of biotherapeutics

J Magn Reson. 2019 Oct;307:106581. doi: 10.1016/j.jmr.2019.106581. Epub 2019 Aug 20.


The higher order structure (HOS) of protein therapeutics is essential for drug safety and efficacy and can be evaluated by two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy at atomic resolution. 1Hn-15N amide correlated and 1H-13C methyl correlated NMR spectroscopies at natural isotopic abundance have been demonstrated as feasible on protein therapeutics as large as monoclonal antibodies and show great promise for use in establishing drug substance structural consistency across manufacturing changes and in comparing a biosimilar to an originator reference product. Spectral fingerprints from 1Hn-1Hα correlations acquired using 2D homonuclear proton-proton J-correlated NMR experiments provide a complementary approach for high-resolution assessment of the HOS of lower molecular weight (<25 kDa) protein therapeutics. Here, we evaluate different pulse sequences (COSY, TOCSY and TACSY) used to generate proton-proton J-correlated NMR spectral fingerprints and appraise the performance of each method for application to protein therapeutic HOS assessment and comparability.

Keywords: 2D NMR; Biologic; COIN-TACSY; COSY; Fingerprinting; Higher order structure; TOCSY.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Biological Factors / therapeutic use*
  • Egg Proteins / chemistry
  • Indicators and Reagents
  • Magnetic Resonance Spectroscopy
  • Muramidase / chemistry
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Peptide Mapping / methods*
  • Protons


  • Biological Factors
  • Egg Proteins
  • Indicators and Reagents
  • Protons
  • Muramidase