Protein stability during nebulization: Mind the collection step

[Display omitted] Inhaled protein therapeutics meet a growing interest for the treatment of respiratory diseases. In liquid aerosols, proteins face stresses that may generate instabilities, such as physicochemical denaturations, aggregation and loss of activity. Monitoring protein stability is thus crucial but implies collection of aerosol droplets before analysis. Many aerosol collection methods may be used, still their interference on protein stability is unknown. In this study, we compared the impact of six aerosol samplers on the stability of a model monoclonal antibody (Ig1), aerosolized with a mesh nebulizer. Ig1 stability was assessed for aggregation and biological activity. The six aerosol samplers generated distinct aggregation profiles for Ig1 at all size scales; counts of micron-sized particles varied by a factor of 100. The heterogeneity did not impact Ig1 activity, which was not significantly changed after nebulization. To extrapolate these results, we evaluated the impact of two samplers on three other proteins. Depending on the protein, samplers gave discordant aggregation and/or activity profiles, sometimes in the reverse trend as compared to Ig1. In conclusion, aerosol samplers interfere with protein stability; this impact depends both on the samplers and the protein, highlighting the importance of using the same collection device throughout the aerosol development process.