Milena Opacic

Background: ExPEC9V was a 9-valent vaccine candidate intended for the prevention of invasive extraintestinal pathogenic Escherichia coli (ExPEC) disease (IED). Here, we describe more than a decade-long formulation development trajectory of this vaccine candidate aimed at establishing a stable, robust and scalable drug product that maintains long-term stability while addressing potential manufacturing challenges and increasing the probability of successful global deployment. Methods: Selected formulation development studies of the ExPEC multivalent vaccine candidate are summarized, spanning formulation screening, confirmation, and design of experiments (DoE)-based robustness, stability and compatibility studies. A formulation initially developed for an early low-valency vaccine candidate was subsequently tested and confirmed for candidates with additional serotypes incorporated based on antigen heterogeneity evidence. Contact materials employed included primary packaging—polycarbonate (PC) and polyethylene terephthalate glycol (PETG) bottles, borosilicate glass vials, stoppers, and prefilled syringes; vessel types—bags and stainless steel vessels used in drug substance (DS) and drug product (DP) manufacturing; and varying concentrations of tungsten and hydrogen peroxide. An evolving analytical panel was applied to assess attributes such as purity, protein concentration and degree of O-acetylation. Results: A phosphate-based formulation containing sorbitol, methionine, and polysorbate 80 showed superior stability in screening and was confirmed as fit for purpose across increasing vaccine valency. The ExPEC 9V drug product displayed remarkable thermal and formulation robustness, long-term (3 years) stability at 2–8 °C in glass vials and prefilled syringes, and compatibility with assessed primary containers and manufacturing materials. DoE-based robustness studies defined acceptable excipient and pH ranges, supporting a wide formulation design space. Conclusions: The development trajectory of the ExPEC9V vaccine candidate demonstrates that early prioritization of a robust, scalable formulation that remains fit for purpose across valency evolution supports a stable late-stage manufacturable drug product.

Background: Predictive modeling of vaccine stability is an essential tool for accelerating development and ensuring product quality, particularly when long-term data are limited. To ensure high-quality input for accurate stability predictions, it is often necessary to allocate substantial analytical resources. Methods: This study demonstrates that integrating high-throughput screening (HTS) techniques such as ultraviolet–visible spectrophotometry (UV-VIS), intrinsic fluorescence and dynamic light scattering (DLS) with Advanced Kinetics Modeling (AKM) results in a synergistic approach, facilitating the development of robust predictive stability models. Results: Here, we applied AKM to a glycoconjugate vaccine targeting extra-intestinal pathogenic Escherichia coli (ExPEC). The aggregation processes observed via size-exclusion high-performance liquid chromatography (SE-HPLC), DLS, turbidity by UV-Vis absorbance and local changes in tryptophan microenvironment captured by intrinsic fluorescence were effectively described by the developed kinetic models. Conclusions: Using accelerated stability data across multiple temperatures, AKM successfully described key degradation pathways. Furthermore, the HTS assay results showed strong correlation with SE-HPLC data, indicating that these assays provide an efficient alternative requiring minimal analytical resources, material, and time.

The trimeric light-harvesting complexes II (LHCII) of plants and green algae are pigment-protein complexes involved in light harvesting and photoprotection. Different conformational states have been proposed to be responsible for their different functions. At present, detergent-solubilized LHCII is used as a model for the “light-harvesting conformation”, whereas the “quenched conformation” is mimicked by LHCII aggregates. However, none of these conditions seem to perfectly reproduce the properties of LHCII in vivo. In addition, several monomeric LHC complexes are not fully stable in detergent. There is thus a need to find conditions that allow analyzing LHCs in vitro in stable and, hopefully, more native-like conformations. Here, we report a study of LHCII, the major antenna complex of plants, in complex with amphipols. We have trapped trimeric LHCII and monomeric Lhcb1 with either polyanionic or non-ionic amphipols and studied the effect of these polymers on the properties of the complexes. We show that, as compared to detergent solutions, amphipols have a stabilizing effect on LHCII. We also show that the average fluorescence lifetime of LHCII trapped in an anionic amphipol is ~30 % shorter than in α-dodecylmaltoside, due to the presence of a conformation with 230-ps lifetime that is not present in detergent solutions.