Comprehensive assessment of protein and excipient stability in bio-pharmaceutical formulations using 1H NMR spectroscopy

Biopharmaceutical proteins are important drug therapies in the treatment of a range of diseases. Proteins, such as antibodies (Abs) and peptides, are prone to chemical and physical degradation, particularly at the high concentrations currently sought for subcutaneous injections, and so formulation conditions, including buffers and excipients, must be optimised to minimise such instabilities. Therefore, both the protein and small molecule content of biopharmaceutical formulations, and their stability are critical to a treatment’s success. However, assessing all aspects of protein and small molecule stability currently requires a large number of analytical techniques, most of which involve sample dilution or other manipulations which may themselves distort sample behaviour. Here, we demonstrate the application of 1H nuclear magnetic resonance (NMR) spectroscopy to study both protein and small molecule content and stability in situ in high concentration (100 mg/mL) Ab formulations. We show that protein degradation (aggregation or fragmentation) can be detected as changes in 1D 1H NMR signal intensity, whilst apparent relaxation rates are specifically sensitive to Ab fragmentation. Simultaneously, relaxation filtered spectra reveal the presence and degradation of small molecule components such as excipients, as well as changes in general solution properties, such as pH. 1H NMR spectroscopy can thus provide a holistic overview of biopharmaceutical formulation content and stability, providing a preliminary characterisation of degradation and acting as a triaging step to guide further analytical techniques.