Effects of formulation conditions on protein biologics behaviour

  • Maria Laura Greco

Student thesis: Phd


Antibodies developed against tumour cell-specific surface antigens often display limited therapeutic activity, hence alternate strategies have been explored to enhance their activity including antibody drug conjugates (ADCs). ADCs have emerged from the combination of an antibody with a highly potent cytotoxic agent that lacks target specificity, thereby mAb and small molecule limitations can be overcome. As a result, ADCs present a synergic therapeutic effect, where mAbs low potency is enhanced by the presence of the cytotoxic agent and off- site toxicity of the drug is minimised by mAbs target specificity. Despite their immense potential, only six ADCs have been approved by regulatory authorities to date, due mainly to the substantial challenges encountered during their complex development. The interplay between the antibody, the linker and the payload suggests that a fine balance is essential to guarantee efficacy. It becomes, therefore, evident why rational design approaches are required to simultaneously address the issues pertaining to their development. In this work, several approaches were pursued to generate a pool of rationally-designed ADCs by varying several factors, such as the site and amount of drug molecules conjugated to cysteine-engineered Trastuzumab, the linker functionalisation by inclusion of hydrophilic polymeric moieties and the formulation composition. Ad hoc mutations were introduced at the CH2 (i239C), CH1 (A118C) and CH3 (S442C) domains of Trastuzumab, which allowed the site- specific conjugation of four different Pyrrolobenzodiazepines (PBDs)-payloads from the Spirogen library (Spirogen Ltd, a member of the AstraZeneca Group). This in turn led to the generation of a novel panel of ADCs, which were subsequently characterised in different formulations with respect to their (bio)physical properties. Conformational stability was investigated through differential scanning fluorimetry (DSF) and dynamic light scattering (DLS), whilst colloidal stability was evaluated by static light scattering (SLS) . In addition, pre-formulation and formulation stability studies provided useful insights on the effect of different excipients (NaCl, ArgGlu and PEG400) on the physical and chemical stability of the ADCs in the liquid state. The influence of the mutations on the conformational and colloidal properties of Trastuzumab both pre- and post-conjugation were investigated. This revealed significant alterations in the transition of unfolding for the mutants and ADCs that underwent mutation at the CH2 domain and increased self-association propensity for the other mutants and their respective ADCs in the NaCl-containing formulation. In addition, pre-formulation studies enabled the rational design of the formulation, which was composed of histidine at pH 6.8 and also contained either 154 mM NaCl, 120 mM ArgGlu or 7% v/v PEG400. The physical and chemical stability of the in- house ADCs was then investigated in real-time (6 months at 5 °C), accelerated (6 months at 25 °C), and stressed (3 weeks at 37 °C) conditions. Finally, excipient screening studies (NaCl, ArgGlu and PEG400) allowed the identification of the factors involved in the purity reduction and payload deconjugation. Hydrophobic interactions were found to be the driving force for aggregation and the hydrolysis of the succinimide ring at the linker moiety seemed to be the best approach to ensure linker stability in the liquid state. Eventually this work covered all the different stages involved in drug design, generation and development of such a complex system as an ADC, thus enabling a deeper understating of the different behaviours observed in the biophysical characterisation and formulation development. Among the parameters under investigation (e.g., conjugation site, chemical composition of the payload and formulation composition), the modulation of the conjugation site was deemed the most effective at improving the ADC's stability.
Date of Award1 Aug 2021
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorRobin Curtis (Supervisor) & Alain Pluen (Supervisor)

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