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 using nuclear magnetic resonance (NMR), the method comprising:

• providing a 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;
• and
• applying a cycle of signal processing steps, the cycle comprising:
• applying a radio frequency (RF) pulse;
• applying a gradient pulse having a pulse length less than or equal to 1000 μs;
• wherein said gradient pulse accompanies an echo/anti-echo scheme;
• and
• applying a water suppression technique (WET) to suppress the third NMR signal,
• wherein the first NMR signal, the second NMR signal, and the third NMR signal are located in a region of NMR spectra in a defined ppm range of 13C methyl signal;
• wherein the first NMR signal is a NMR signal related to 13C methyl of a therapeutic molecule, the second NMR signal is a signal related to 13C sucrose, and the third NMR signal is a signal related to 1H acetate or another excipient;
• repeating the cycle 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.