Protein Stability & Formulation

Succinimide formation is a critical degradation pathway in antibody therapeutics, typically leading to aspartate and isoaspartate products. In my talk, I will describe the experimental identification of an unusually stable succinimide intermediate in an antibody therapeutic. To understand the structural and dynamic basis of this stability, we performed molecular dynamics simulations based on an X-ray structure, revealing features that stabilise the succinimide ring and influence its degradation pathway.

Multivalent NANOBODY molecules face colloidal stability challenges which are not predictable from their monovalent building blocks. With specific data gathering campaigns, we could show that kD-DLS (diffusion interaction coefficient) correlates well with colloidal stability. We then developed a machine learning model predicting kD-DLS from sequence descriptors and formulation conditions. This enables early high-throughput screening of constructs compatible with high-concentration subcutaneous administration, in turn mitigating developability risks.

Photodegradation of antibody-drug conjugates (ADCs) is an often-overlooked stability liability. Using camptothecin-based payloads, we combine quantum mechanical calculations, molecular dynamics simulations, and experimental photostress data to characterise photodegradation pathways and their consequences for ADC quality. In silico approaches rationalise observed discolouration, aggregation, and charge heterogeneity, linking degradation products to their physicochemical properties. This integrated computational-experimental framework offers a generalisable strategy for early-stage photostability risk assessment of ADC drug products.

Protein aggregation poses a challenge in biotherapeutic development, where heterogeneous particle populations and stochastic kinetics complicate mechanistic understanding and formulation design. I will introduce two methodologies: chemical fingerprinting of single protein particles via Raman microscopy, enabling compositional discrimination of micrometre species, and spatially resolved plate-reader fluorescence for quantitatively mapping the role of interfaces in driving protein aggregation. These tools offer a path toward predictive, mechanism-informed strategies for developing biotherapeutic formulations.

This talk explores strategies for suppressing Fab fragment aggregation during nebulisation. Our data shows that high-intrinsic thermostability and high-concentration formulations are critical for stability during aerosolisation. We introduce a qualitative model explaining the counterintuitive protective effect of high concentrations and discuss broader stability implications. These findings provide clear guidance for achieving virtually aggregation-free nebulisation, aimed to minimise immunogenicity risks and facilitate the development of next-generation inhaled protein therapeutics.

Thermodynamic stability assessed by differential scanning calorimetry (DSC) is a key indicator in antibody formulation; however, thermodynamic parameters alone cannot elucidate excipient-induced stabilization mechanisms at the molecular level. We report molecular dynamics simulations revealing the molecular origins of DSC parameter changes in arginine formulations with different counterions. Counterion-dependent reorganization of hydrogen-bond networks and component distributions provides molecular descriptors for interpreting DSC trends.

Our aim is to convert any clinical antibody or protein therapeutic, all of which are injectables, into oral pill formulations. We’ve developed a bispecific protein therapeutic (UTAC) consisting of a human pancreatic enzyme, which retro-transcytoses from gut lumen into blood, fused to anti-IgG moieties. Co-formulating UTAC with a clinical antibody, and leveraging Intract’s core pharmacy technologies, achieves high antibody bioavailability in blood in mice after intraduodenal administration. Finally, we’ll present impressive UTAC pilot data from cynomolgus macaque.

Therapeutic enzymes remain limited by instability, aggregation, and short circulating half life, with PEGylation often used at the expense of tolerability and safety. We present INCYPRO, a structure guided protein stabilization approach, that reinforces native protein folds omitting polymer conjugation. Using asparaginase as a case study, we show how topological stabilization enables a long acting, non PEG enzyme format with preserved catalytic function and improved developability for the treatment of acute lymphoblastic leukemia.

The development and formulation of therapeutic antibodies is a highly complex optimisation task requiring significant time and resources. Specially engineered antibody formats, such as fragments and bispecifics, suffer from developability issues, hampering their translation into successful therapeutics. To address these challenges, we have developed an efficient method to accelerate formulation design based on Bayesian optimisation.