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Inhalation OctOber 2024 11 erefore, it is of paramount importance to ensure that the administered formulation does not con- tain protein aggregates. Bioprocessing of proteins should be thoroughly characterized and refined to reduce aggregation risks and any aggregates formed should be eliminated from the finished product [10]. While protein aggregations depend on various fac- tors including the type of proteins, composition of the buffer and nature of stresses experienced, it has been reported that different IgG molecules form sim- ilar aggregates when subjected to the same external stress [3], affording some predictability in aggregate formation. In situations where the removal of aggre- gates is impractical, for instance, when aggregation occurs during nebulization or spray drying, appro- priate formulation design is an effective strategy to mitigate aggregation risks [11]. Surfactant excipients such as polysorbates can be used to limit aggregation through protein displacement at interfaces [5, 8]. Sugars and oligosaccharides like trehalose and cyclo- dextrins also serve as protein stabilizers when protein is dried, through purported mechanisms of vitrifica- tion and water (hydrogen bond) replacement. Analytical techniques for characterization of protein aggregates Aggregation can result in a wide range of particles with various sizes and numbers, exerting different implications on the quality and safety of inhaled pro- tein formulations. A single analytical technique is insufficient in characterizing the heterogenous aggre- gates, especially during the initial development phase where prior knowledge is limited. erefore, orthog- onal methods, which work by diverse fundamen- tal principles with different resolution and range of detection, ought to be employed in combination to give a comprehensive understanding of the aggregate profile. Among these, analytical techniques that have been frequently employed in inhaled protein formu- lations are elaborated in the following sections as well as in Table 1. Visible particles Visual inspection Naturally, the detection of visible particles is achieved through visual inspection of liquid samples. ere is compendial guidance (e.g., Ph. Eur. method 2.9.20 Particulate Contamination) that standardizes this vir- tually simple method. Visual inspection requires the sample to be viewed for about 5 seconds against both a vertical, matte, black panel and a vertical, non- glare, white panel over a horizontal, non-glare, white surface, upon gentle swirling or inverting the con- tainer without introducing air bubbles. e sample should be illuminated by a suitable, shaded, white light source (e.g., LED) with an appropriate diffuser that gives an illumination intensity between 2,000 and 3,750 lux at the viewing point. A longer obser- (such as heating, cooling and freeze-thaw cycles), mechanical stress (such as agitation, vortex mixing and shear stress) and oxidative stress, while examples of the latter include changes in pH, ionic strength or osmolarity of the buffer, and changes of the protein concentration. Aggregation of proteins can therefore occur at all stages of product life cycle, including (bio-)synthesis and subsequent purification, formu- lation, transportation, storage, drug reconstitution and administration to end users, during which the proteins are inevitably exposed to various stresses. While many of these steps are generally relevant to all protein formulations, inhalation of protein formu- lations adds yet another significant source of desta- bilization, in the form of atomization. Pulmonary administration requires the medication formulation to be presented as an aerosol (either liquid or solid aerosol) of appropriate particle size distribution. For liquid formulation, aerosolization is achieved by nebulization at the time of administration, in which nebulizers atomize drug in the reservoir into inhal- able mists. Depending on the drying method used, the manufacture of dry powder formulations may also involve atomization of the protein solution. In spray drying, which is a commonly used particle engineering technique to prepare inhalable powders at both laboratory and industrial scale, the feedstock solution is atomized into fine liquid droplets, which are then rapidly dried into small particles. In both situations, the atomization of a protein solution fea- tures the creation of large, nascent air-liquid inter- faces, thereby exerting stress on the dissolved protein that promotes unfolding and aggregation [5]. e stress may possibly be aggravated by the accompany- ing temperature rise, as well as solvent evaporation during drying that changes buffer composition and concentrates the protein. e presence of protein aggregates in a formulation is undesirable. Aggregated proteins often lose their intended bioactivity due to protein unfolding and denaturation. e secondary and tertiary structures of proteins, on which their bioactivity depends, can be disrupted. Protein aggregates also exhibit reduced solubility and lead to a reduction in the effective pro- tein concentration. e relatively stochastic nature of protein aggregation implies that protein aggre- gates can exist in mixed sizes. Large aggregates (in the micrometer size range) may precipitate in solution and become solid particulates that may interfere with inhaler devices. For instance, these particles have diameters comparable to, or even larger than, that of the apertures found on the mesh of vibrating mesh nebulizers [6, 7]. In contrast, subvisible, submicronic particles or soluble aggregates may be more concern- ing because they are challenging to detect. ey are small enough to enter the body, making them more immunogenic and increasing the risks of unexpected adverse drug reactions [8, 9].