Effects of formulation conditions on protein biologics behaviour

UoM administered 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, while minimising undesired side-effects and off-site toxicity. 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 sitespecific 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.


Original languageEnglish
Awarding Institution
Award date14 Jun 2021