Irès van der Zwaan1, Kouamé Kan Firmin Akoumia6, Bernard Ucakar6, Johanna Salomon1, Steven Oag2, Aida Edvardsson1, Erica Bäckström3, Sandra Gracin1, Tanvir Tabish4, Markus Fridén1,5 & Rita Vanbever6
1 AstraZeneca; Pharmaceutical Technology & Development; Global Product Development; Gothenburg, Sweden
2 AstraZeneca; R&D Clinical Pharmacology & Safety Sciences; Animal Science & Technology; Gothenburg, Sweden
3 AstraZeneca; R&D Biopharmaceuticals; Respiratory & Immunology; Drug Metabolism & Pharmacokinetics Gothenburg, Sweden
4 AstraZeneca; R&D Biopharmaceuticals; Early Stage Formulation Sciences; Cambridge, UK
5 Uppsala University, Department of Pharmacy, Uppsala, Sweden
6 Université catholique de Louvain (UCLouvain), Louvain Drug Research Institute (LDRI), Advanced Drug Delivery & Biomaterials (ADBB), Brussels 1200, Belgium.
Summary
One of the main challenges in developing inhaled biologics is the risk of protein aggregation, which can occur during the aerosolizing of liquid formulations or during spray-drying processes used to manufacture dry powder inhalation (DPI). Aggregates of protein therapeutics are generally considered as a risk for increased immunogenicity and potentially also increase lung clearance mechanisms, reducing availability at target sites. While various in vitro techniques can detect the absence/presence of potential aggregates, the in vivo fate of protein aggregates inside the lungs remain unclear. This study aimed to develop an in vivo assay to visualize inhaled proteins and their aggregates in rat lungs using confocal microscopy. An antibody fragment (Fab) was fluorescently labelled and prepared both as a solution and as a spray-dried powder, with the powder formulation not specifically optimized to minimize protein aggregation. Formulations were dosed to rats via intratracheal instillation or through inhalation. Aggregates were characterized in vitro using size-exclusion chromatography, dynamic light scattering, and micro-flow imaging, revealing minimal aggregation in the solution and significantly higher levels of aggregates in the spray-dried powder. Confocal microscopy demonstrated successful detection of the protein and its aggregates both in vitro and in rat lung tissue after dosing. At time point 0, the solution showed a homogeneous distribution of the protein within the tissue. Initial visual image inspection for the DPI formulation indicated additional and more concentrated spots (supposedly aggregates), as may have been expected from in vitro characterisation. Confocal imaging captured both diffuse and aggregated protein signals, confirming the technique’s sensitivity in the lung tissue. This novel imaging assay paves the way for better understanding and optimization of inhaled biologics, with ongoing work aimed at quantitative image analysis for more accurate formulation comparison.