Characterising Antibody Conformation and Glycosylation using Mass Spectrometry Techniques

  • Alice Upton

Student thesis: Phd


Monoclonal antibody (mAb)-based therapeutics are dominating the pharmaceutical market for the targeted treatment of a wide range of diseases. With this market dominance comes an increase in competition from the market manufacturers who want a share of the ~$100 billion world-wide sales. As originator products come off patent the opportunity opens up for other manufacturers to step-in and develop a biosimilar (biologically equivalent) product. However, rigorous biosimilarity testing is required before such products can be approved for clinical use. In order to define acceptance criteria for any biosimilar candidate, a full characterisation of multiple originator products must be completed. The use of mammalian cell lines during the manufacturing process; however, can lead to an inherently heterogeneous mAb product, making biosimilarity approval a non-trivial process. The presence of Post-Translational Modifications (PTMs) such as glycosylation, are essential for the therapeutic efficacy of many mAb products and therefore the glycan profiles must be identified and quantified in full. Mass Spectrometry (MS) has evolved into an important tool for studying the critical quality attributes of mAbs thanks to low sample consumption, high sensitivity and high throughput. Combined with Ion Mobility separation (IM-MS), the range of conformational ensembles adopted by different proteins can also be studied. The rotationally averaged Collision Cross Section (CCS) of a protein is indicative of its structural diversity and hierarchy. In addition, the solution-phase labelling technique, Hydrogen-Deuterium Exchange (HDX), can be coupled with MS for the study of conformational dynamics. These three techniques (MS, IM-MS and HDX-MS) are used extensively throughout this thesis to assess lot-to-lot variation between Herceptin mAb products. A ‘LC’-MS method combined with enzymatic digestions was developed for the rapid identification and quantification of glycan profiles and for the determination of glycan occupancy and high mannose content. These results were coupled with orthogonal techniques to link afucosylation to therapeutic efficacy. IM-MS and HDX-MS were used to simultaneously assess lot-to-lot variations and the effects of glycosylation in terms of the conformational spread and dynamic properties of Herceptin. The use of activated IM-MS offered insights into the stabilisation induced by the glycans. Finally, IM-MS and Variable Temperature (VT) IM-MS was used to probe the effects of cold storage upon the conformational flexibility of mAbs. Initial findings suggested a significant restriction upon the global framework of Herceptin following storage at -80 °C cf. storage at 4 °C.
Date of Award1 Aug 2019
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorDavid Knight (Supervisor) & Perdita Barran (Supervisor)


  • Glycosylation
  • Ion Mobility
  • Mass Spectrometry
  • Antibody
  • Biosimilar

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