Methods of fingerprinting therapeutic proteins via a two-dimensional (2d) nuclear magnetic resonance technique at natural abundance for formulated biopharmaceutical products

A method of fingerprinting a specific molecule in a com position using nuclear magnetic resonance (NMR), the method comprising:

• providing the composition comprising at least a first molecule having a first NMR signal, a second molecule having a second NM Rsignal, and a third molecu le having a third NMR signal, wherein each of the signals arises from each of the respective molecules having a nuclear spin differing from zero;
• and
• applying a cycle of signa I processing steps, the cycle comprising:
• applying a radio frequency (RF) pulse;
• and
• applying a gradient pulse;
• wherein the first NMR signal, the second NMR signal, and the third NMR signal are located in a region of NM Rspectra I window from about 5 ppm to about 150 ppm;
• repeating the cycle for at least 3 times to acquire an enhanced signal of the composition;
• and
• fingerprinting the specific molecule based on the enhanced signal of the composition.

A method of fingerprinting a specific molecule in a composition using nuclear magnetic resonance (NMR), the method comprising:

• providing the composition comprising at least a first molecule having a first NMR signal, a second molecule having a second NMR signal, and a third molecule having a third NMR signal, wherein each of the signals arises from each of the respective molecules having a nuclear spin differing from zero;
• applying a radio frequency (RF) pulse to the com position to excite the first NMR signal while suppressing the second NM Rsignal, the RF pulse comprising at least one of a Reburp pulse, a combination of a broad band inversion pulse and a Gaussian inversion pulse, and an asymmetric adiabatic pulse, applying a gradient pulse having a pulse length less than or equal to 1000 ps;
• applying a water suppression tech nique (WET) sequence to suppress the third NMR signa l;
• acquiring an enhanced signal of the composition;
• and
• fingerprinti ng the specific molecule based on the enhanced signal of the composition.