Most therapeutic proteins are vulnerable to physicochemical degradation as exposed to pH change and digestive enzymes, and hence exhibit limited absorption in the gastrointestinal tract. Therefore, they are mainly administered through parenteral routes, especially injections. There still exist limitations associated with the injection such as infection risks, treatment cost and time, and needle-phobia [1]. To improve the patients’ compliance, many products are developed in prefilled syringes or pen types so that the patients inject the therapeutics for themselves through subcutaneous route. Nevertheless, liquid-filled vials and syringes need be stored under refrigerated conditions to prevent any damages on the therapeutics during storage and handling [[2], [3], [4]]. The development of therapeutic proteins such as antibodies in dry powder form may have advantages with respect to stability and give an opportunity to pursue different administration routes such as lungs and other respiratory tract with a dry powder inhaler (DPI) [5,6].

The location where inhaled powders delivered within the lungs is dependent on their in vitro aerodynamic characteristics. In case of DPI formulations, particles with an aerodynamic diameter ranging 1–5 μm are typically targeted for delivery into lungs and other respiratory tract [[7], [8], [9], [10]]. Size larger than the range will be deposited in the upper airways, whereas submicron particles (<0.5 μm) may reach the alveoli [11,12] or be exhaled [13,14]. Moreover, the effects of particle shape and surface characteristics on aerodynamic performance cannot be overlooked, given their influence on particle flowability. Understanding of particle engineering might be necessary for precisely delivering therapeutics to the lungs. Drug retention in the mouth–throat can decrease delivery efficiency and increase the cost of treatment [15]. Furthermore, high drug deposition into non-targeted tissues may cause undesirable effects [16,17]. Recently, several studies have investigated the pulmonary drug delivery and aerodynamic performance of the protein powder [[18], [19], [20], [21], [22], [23], [24], [25]]. However, only a few studies focused on particle engineering for protein dry powders [18,22,23]. To ensure in vivo performance of DPIs, it is crucial to consider particle engineering and powder processing [14,24,[26], [27], [28], [29], [30]]. By improving the aerodynamic performance of protein powders, DPI may overcome the burdens of intravenous administration of biopharmaceuticals.

Protein dry powders are mainly manufactured through spray drying techniques, including cryogenic techniques like spray freeze drying and spray freeze into liquid [15,18]. The FDA-approved insulin inhalation powder (Exubera®), which was withdrawn in 2007, was prepared using the spray drying process [31]. However, since spray drying relies on heated gas to generate dried particles, it is not suitable for thermally unstable proteins because it may denature and/or aggregate proteins [6,32,33]. Moreover, the freeze drying process may cause irreversible damage to proteins, resulting in a loss of structural or biological activity [34,35]. For example, ice-induced denaturation of proteins can occur during freezing [[36], [37], [38]]. Alternatively, protein precipitation with an organic solvent could obtain interests as an alternative drying method at room temperature [[39], [40], [41], [42], [43]].

Using suitable solvents for protein precipitation can effectively preserve protein stability. This approach enables swift dehydration of proteins within an immiscible solvent, bypassing the need for freezing or exposing them to high temperatures. While this method addresses stability concerns, challenges persist in particle engineering. Notably, conventional precipitation techniques yield particles with a wide size distribution. In a prior investigation, a novel method of protein precipitation was introduced with Shirasu porous glass (SPG) membrane emulsification, and it was used to prepare protein microparticles (microbeads) that exhibited uniformity and robust biophysical stability upon rehydration in Table 1 [4,44].

The main objective of this study was to explore the development of microbead formulations and their potential as protein dry powders for inhalation, using intravenous immunoglobulin (IVIG) as a model protein. This study also aimed to improve previous method of protein microbead preparation with respect to the physicochemical properties of model drug for efficient pharmaceutical applications (Table 1). In addition, due to the compatibility issue of conventional pharmaceutical excipients, an alternative would be necessary to develop a drug product. This investigation encompassed comparative analysis of IVIG-loaded microbeads with varying concentrations of trehalose as a stabilizer to determine its effects on protein stability. Moreover, various preparation processes of microbeads were evaluated to achieve optimum conditions.