NMR studies of formulation stability and liquid-liquid phase separation in biopharmaceuticals

  • Jack Bramham

Student thesis: Phd


Biopharmaceuticals, including antibodies (Abs), are the fastest growing segment of the pharmaceutical industry, and offer unique treatment possibilities for a range of diseases, including neurodegenerative and immune diseases, and cancers. Abs are currently typically administered by expensive and time consuming intravenous injection in a clinical setting, and there is growing interest in faster administration by subcutaneous injection, potentially by the patient themselves at home. However, subcutaneous injection volumes are typically limited to < 2 mL, requiring the development of high-concentration protein formulations (> 100 mg/mL). Such high concentrations promote physical instabilities, such as reversible self-association, irreversible aggregation, and liquid-liquid phase separation (LLPS). Characterising these instabilities by conventional biophysical techniques is challenging, particularly in situ at high concentration. In this Thesis, we demonstrate the use of NMR spectroscopy to study the stability and phase separation of Abs and other model proteins. In the first half of the Thesis, we show that 1H NMR spectroscopy can detect the degradation of both the protein and small molecule components of Ab formulations in situ without sample manipulation. Additionally, we employ a typical formulation approach, namely the addition of an excipient (Arg·Glu) to the phase separated dense fraction, to show that LLPS is suitable as an alternative method to concentrate Ab solutions. In the second half of the Thesis, we detail the development of two new 19F NMR approaches to study protein self-association and LLPS. We show that 19F Dark-state Exchange Saturation Transfer (DEST) NMR can be used to detect the self-association and clustering of 19F tagged Abs in otherwise NMR invisible dark states. Finally, we demonstrate a novel experimental approach, with a fluorinated probe molecule combined with spatially-selective and conventional bulk-detection NMR, to study the kinetics of phase separation. We first develop this approach in a model system (bovine serum albumin), before applying it to a biopharmaceutical Ab. In conclusion, we have developed a number of NMR approaches to study Abs and phase separation. These approaches may encourage the use of NMR spectroscopy in the biopharmaceutical industry.
Date of Award31 Dec 2021
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorJames Warwicker (Supervisor) & Alexander Golovanov (Supervisor)


  • biopharmaceuticals
  • NMR spectroscopy
  • monoclonal antibodies
  • liquid-liquid phase separation
  • formulation

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