Impact of protein aggregation on the immunogenicity of a human monoclonal antibody following pulmonary administration in mice.

Spray drying is a widely employed method for generating dry powder formulations for inhalation. Yet, it presents substantial challenges when applied to therapeutic proteins due to stability issues. The formation of protein aggregates during the atomization and the heating steps can diminish protein activity and raise immunogenicity concerns. Here, we assessed the impact of varying levels of protein aggregates generated during spray-drying on the fate and the immunogenicity of the human monoclonal antibody NIP228 following intratracheal administration in mice. Aggregate-free rhodamine labelled NIP228 was spray-dried with or without 1% polysorbate 80 surfactant, resulting in the generation of powder formulations with associated low and high protein aggregate levels, respectively. Confocal imaging highlighted the presence of aggregates in the lungs for both powders but not for the solution, while flow cytometry analysis designated alveolar macrophages as the main immune cells taking up rhod-NIP228 in the lungs with very little involvement of dendritic cells and interstitial macrophages following a single dose administration. Notably, repeated intratracheal administration of the three formulations in mice did not impact the magnitude of the anti-drug antibody response in sera or broncho-alveolar lavages. The pulmonary route appeared to evoke a more robust immune response when compared to subcutaneous administration. Overall, the level of NIP228 aggregation in this study did not appear to be the primary driver of NIP228 immunogenicity following delivery to the lungs in mice, shedding new light on the interplay between protein aggregation and immunogenicity in the context of the pulmonary delivery of therapeutic proteins.

Background:

Spray drying is a widely employed method for generating dry powder formulations for inhalation. Yet, it presents substantial challenges when applied to therapeutic proteins due to stability issues. The formation of protein aggregates during the atomization and the heating steps can diminish protein activity and raise immunogenicity concerns.

Methods:

Here, we assessed the impact of varying levels of protein aggregates generated during spray-drying on the fate and the immunogenicity of the human monoclonal antibody NIP228 following intratracheal administration in mice. Aggregate-free rhodamine labelled NIP228 was spray-dried with or without 1% polysorbate 80 surfactant, resulting in the generation of powder formulations with associated low and high protein aggregate levels, respectively.

Results:

Confocal imaging highlighted the presence of aggregates in the lungs for both powders but not for the solution, while flow cytometry analysis designated alveolar macrophages as the main immune cells taking up rhod-NIP228 in the lungs with very little involvement of dendritic cells and interstitial macrophages following a single dose administration. Notably, repeated intratracheal administration of the three formulations in mice did not impact the magnitude of the anti-drug antibody response in sera or broncho-alveolar lavages.

Data Summary:

The pulmonary route appeared to evoke a more robust immune response when compared to subcutaneous administration.

Conclusions:

Overall, the level of NIP228 aggregation in this study did not appear to be the primary driver of NIP228 immunogenicity following delivery to the lungs in mice, shedding new light on the interplay between protein aggregation and immunogenicity in the context of the pulmonary delivery of therapeutic proteins.

Practical Significance:

shedding new light on the interplay between protein aggregation and immunogenicity in the context of the pulmonary delivery of therapeutic proteins.