Abstract
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 optimized to minimize 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 behavior. 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, while 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 characterization of degradation and acting as a triaging step to guide further analytical techniques.
Highlights
Biopharmaceutical antibodies (Abs), such as monoclonal antibodies and, more recently, bispecific antibodies (BsAbs), are increasingly important therapies in the treatment of a wide range of diseases, including cancer, arthritis, and diabetes
Having spectroscopically separated protein and small molecule components, we show that the signal intensities and apparent relaxation rates of Abs can be used to monitor protein stability and the occurrence of degradation, such as aggregation and fragmentation, over a 12-week period
We show here that 1H nuclear magnetic resonance (NMR) spectroscopy can be used as an orthogonal technique to simultaneously characterize the content and stability of both protein and small molecule formulation components, perhaps as a triaging approach to inform decisions on which specialized techniques should be employed to quantify or study particular forms of degradation in more detail
Summary
Biopharmaceutical antibodies (Abs), such as monoclonal antibodies (mAbs) and, more recently, bispecific antibodies (BsAbs), are increasingly important therapies in the treatment of a wide range of diseases, including cancer, arthritis, and diabetes. There is considerable interest in the development of high-concentration (>100 mg/mL) protein formulations to enable subcutaneous administration of the lowest possible volume injection,[1,2] potentially by the patient themselves.[3] Such administration strategies result in lower treatment costs and better patient experience, in the treatment of chronic conditions such as autoimmune disorders.[4] high protein concentrations are associated with increased levels of physical instabilities, such as self-association,[5,6] aggregation,[7] and liquid−liquid phase separation (LLPS),[8−10] in addition to chemical degradation, such as fragmentation[11,12] and oxidation. In practice, a wide range of techniques is deployed,[19−21] and these techniques typically require manipulation of highconcentration formulations, such as dilution, addition of a probe molecule, or salt removal, potentially leading to changes in protein and small molecule structure and behavior
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
