Faustinelli Barrios, Valentina; (2025) Exploring the role of mass spectrometry analytical methods in nanobody therapeutic development. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Mass spectrometry (MS) is a highly versatile analytical technique commonly utilised in bioprocessing field. This study focuses on the comprehensive applications of MS for characterising challenges in large-scale manufacturing and upstream development of nanobodies, using 2XV6 nanobody against HIV p24 capsid as the model protein. The first challenge involves large-scale nanobody production, where high-yield demands can lead to nanobody expression in the cytoplasm, rather than the conventional periplasm. MS-techniques were employed to assess the effects of these two expression environments on critical quality attributes (CQAs) such as structure, stability and function. The second challenge centres on optimising process design by identifying stabilising and destabilising features of the 2XV6 nanobody, while evaluating the use of MS as an orthogonal method for assessing stability alongside standard low-resolution biophysical techniques. A wide range of MS techniques have been utilised to asses these two challenges including, native-MS, collision induced unfolding (CIU), hydrogen deuterium exchange mass spectrometry (HDX-MS), coupled with computational tools such as Rosetta and molecular dynamics (MD) simulations. In order to characterise the effects of cytoplasmic expression during large-scale production, nanobodies were expressed at small-scale in the cytoplasm or periplasm. Intact mass analysis revealed that cytoplasmic expression resulted in two forms of the nanobody: one containing disulphide bonds and one lacking them, with the latter being more prevalent. In contrast, periplasmic expression produced a single nanobody form containing disulphide bonds. Stability assessments by CIU demonstrated that the disulphide-containing nanobodies exhibited better stability profiles compared to those lacking the disulphide bond. Functional activity was assessed through ligand titration using native-MS, which showed that the periplasmically expressed nanobody had stronger binding affinity. HDX-MS was then employed to examine potential differences in epitope and paratope location, results revealed similar binding interfaces in both nanobody forms. However, the cytoplasmically expressed nanobody exhibited lower protection in the binding interfaces, consistent with its reduced binding strength observed in ligand titration experiments. To explore the reasons behind this, differential HDX-MS and MD simulations were used to analyse the protein dynamics, confirming that the absence of disulphide bonds significantly increased nanobody flexibility, which ultimately affects its function. Furthermore, stabilising and destabilising mutations were introduced into the nanobody, guided by computational predictions from Rosetta and MD simulations. Stability features were confirmed through standardised biophysical techniques in conjunction with CIU. Comparative analysis of these methods offered valuable insights into the correlation between experimental stability measurements and computational predictions, providing a framework for optimising streamlined bioprocess designs. Overall, this study highlights the pivotal role of mass spectrometry in uncovering and characterising critical challenges in bioprocessing.

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