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

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.