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Pulmonary delivery of therapeutic proteins for the management of respiratory diseases has been receiving research attention, particularly after the COVID-19 pandemic. In contrast to parenteral injection (the most common administration route for proteins), inhalation is a non-invasive administration route that permits a high drug concentration to be localized at the site of action, thereby minimizing systemic exposure that could lead to adverse drug reactions. 10 OctOber 2024 Inhalation Analytical techniques for detecting and characterizing protein aggregates in inhaled formulations Including emerging techniques not widely used with inhaled formulations Michael Y. T. Chow, PhD and Jenny K. W. Lam, PhD UCL School of Pharmacy, University College London Pulmonary delivery of therapeutic proteins for the management of respiratory diseases has been receiv- ing research attention, particularly after the COVID- 19 pandemic. In contrast to parenteral injection (the most common administration route for proteins), inhalation is a non-invasive administration route that permits a high drug concentration to be localized at the site of action, thereby minimizing systemic expo- sure that could lead to adverse drug reactions. e relatively mild environment in the respiratory tract, with reduced metabolizing activity compared to the gastrointestinal tract or the liver, also favors the phar- macokinetics of inhaled proteins. ere are multiple types of therapeutic proteins with diverse functions, such as enzymes, cytokines, anti- bodies and subunit vaccines. e first inhaled protein medicine, nebulized dornase alfa (Pulmozyme, Genen- tech) (molecular weight approximately 29 kDa), obtained regulatory approval from the United States Food and Drug Administration (FDA) in 1993. Dor- nase alfa is a recombinant human deoxyribonuclease I, an enzyme that is indicated for the symptomatic management of cystic fibrosis through cleaving extra- cellular DNA and thinning mucus. Monoclonal anti- bodies (or immunoglobulins, IgG), constituting a major class of therapeutic proteins, have also demon- strated potential in treating a wide array of lung dis- orders, including chronic inflammatory lung diseases, respiratory infections and lung cancers. Despite the clinical possibilities offered by these pro- teins, nebulized dornase alpha and two inhalable insulin powder formulations have remained the only approved inhaled protein or peptide formulations for decades, and a few candidates have entered Phase III clinical trials [1], testifying to the tremendous under- lying development barriers [2]. Among these obsta- cles is the propensity of proteins to form aggregates, which poses a major challenge to product stability. In this article, a summary on protein aggregation is pre- sented, illustrating its significance and the challenges associated with protein drug development. It is fol- lowed by an overview of various analytical techniques that have been used for the detection and characteri- zation of protein aggregates in inhaled formulations. An overview of protein aggregation Protein aggregation refers to the self-association of protein molecules resulting from physical and/or chemical reactions in a non-specific fashion. is non-specificity distinguishes protein aggregates from native dimers or oligomers of proteins that are nat- urally occurring for their biological activity and/or stability (e.g., a hemoglobin tetramer). Protein aggre- gates can be broadly categorized by their size, solu- bility, morphology, reversibility, extent of protein unfolding, state of conformation (native or dena- tured), or whether a covalent bond (e.g., disulfide bond between two cysteine residues) or hydrophobic interaction is involved [3, 4]. ere is no strong con- sensus on how these classifications should be defined and working definitions are often used. e tendency of proteins (typically in a solution where the protein molecules are mobile) to form aggregates increases when they are subjected to external stresses or an alteration of the buffer system occurs. Examples of the former include thermal stress