Hydroxypropyl-β-cyclodextrin as a multifunctional excipient for enhancing stability and aerosol performance of spray-dried protein formulations

Abstract

Protein-based drugs are precise therapeutic options for various refractory lung diseases, significantly improving patient prognosis and quality of life. In this study, a comparative spray-drying approach was employed to prepare inhalable dry-powder formulations of bovine serum albumin (BSA), a surface-active model protein, using established pharmaceutical excipients, including 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), trehalose (Tre), leucine (Leu), and a Tre-Leu combination. The relative performance of HP-β-CD was systematically benchmarked against Tre, Leu, and dual-excipient systems under identical formulation and processing conditions, with respect to protein protection and aerodynamic properties. After 3 months of storage under accelerated stability condition (40 ± 2 °C and 20 ± 5% relative humidity), the protein monomer content in the HP-β-CD-based formulations was marginally lower than that observed in the Tre- and dual-excipient-containing dry powders but still demonstrated effective protein protection. Moreover, the aerosol performance of HP-β-CD formulations surpassed that of other excipients, indicating its superior potential for pulmonary delivery of protein molecules. Overall, this comparative study highlights the potential of HP-β-CD as an alternative excipient for protein-based aerosol dry-powder formulations.

Introduction

In recent years, the breakthroughs in biopharmaceutical technology have demonstrated the significant therapeutic impact of biologics in treating various pulmonary diseases including asthma (Chung et al., 2014), lung cancer (Plichta et al., 2023), pulmonary fibrosis (Ma et al., 2024), and chronic obstructive pulmonary disease (Passaro et al., 2023). Biologics could provide efficient therapeutic outcomes with minimal side effects compared to traditional small-molecule drugs, primarily through targeting specific inflammatory pathways and biomarkers (Craik et al., 2013, Mahmood and Bernkop-Schnürch, 2019, Wechsler, 2018, Ye et al., 2021). However, the distinctive characteristics of biologics, such as their large molecular size and susceptibility to degradation, pose significant challenges to their effective delivery. Owing to their large molecular weight, high hydrophilicity, and deactivation in the complex gastrointestinal (GI) environment, protein-based drugs typically exhibit an oral bioavailability of less than 1% (Haddadzadegan et al., 2022). Consequently, over 90% of protein therapeutics still rely on invasive parenteral administration, primarily via subcutaneous and intravenous routes. Long-term administration of these biologics via parenteral routes may often lead to adverse effects, including injection pain, an increased risk of infection, and ultimately poor patient compliance (Agrawal et al., 2017).

Pulmonary delivery represents a promising non-invasive route of administration of biologics, facilitating patient self-administration and improving compliance. It also enables the direct delivery of high drug concentrations to the lungs, promoting a rapid onset of therapeutic effects. Compared to other non-invasive routes, pulmonary administration avoids first-pass metabolism and achieves high systemic bioavailability (Naz et al., 2022). Currently, dry powder inhalers (DPIs) are widely used to conveniently deliver biologics to the lungs, providing improved therapeutic benefits (Cun et al., 2021). In this context, the spray drying process is widely utilized to formulate inhalable dry powders of biologics in a single step, producing particles with appropriate aerodynamic size and morphology (Ji et al., 2016). Nevertheless, the dehydration stress during the spray drying process might compromise protein stability by inducing structural denaturation.

During the development of protein-based DPIs, several sugar-based stabilizers are routinely employed to attenuate adverse processing effects, minimize physical and chemical degradation of proteins during storage, and achieve optimal aerodynamic performance (Zhang et al., 2024a). Trehalose (Tre) is a commonly utilized carbohydrate-based stabilizer for protein therapeutics; it forms a rigid, amorphous glassy matrix owing to its high glass transition temperature (∼120 °C), effectively immobilizing biomolecules to prevent aggregation (Chang and Chan, 2022). However, amorphous Tre is prone to recrystallization upon moisture uptake. It has been reported that phase separation induced by sugar recrystallization could adversely affect the stability of biologics (Shirakashi and Takano, 2018). To address this shortfall, Tre can be combined with leucine (Leu), a naturally occurring hydrophobic amino acid. Leu exhibits surfactant-like properties due to its amphiphilic structure. During the spray drying process, Leu migrates to droplet surfaces, forming a protective hydrophobic shell that shields biologics from moisture-induced degradation (Chang et al., 2019). Previous studies have demonstrated Leu’s efficacy as a moisture-protective excipient in spray-dried formulations containing lysozyme and Tre, where it inhibits recrystallization of the amorphous sugar matrices (Mah et al., 2019, Zhang et al., 2024b). Concurrently, Leu can reduce interparticle cohesion, thereby enhancing powder dispersibility during inhalation. Despite these advantages, formulating spray-dried DPIs with dual excipients (i.e., Tre and Leu) may introduce process complexities. This necessitates extensive screening of drug-to-excipient ratios (e.g., elevated Leu concentrations may induce crystallization, adversely affecting protein powder stability), posing significant challenges for robust formulation development (Ji et al., 2017, Zhang et al., 2024b).

Against this backdrop, 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) emerges as a next-generation pharmaceutical excipient with distinct technical advantages (Wu et al., 2021). It is a highly water-soluble β-cyclodextrin derivative produced via a hydroxypropylation protocol (Santos et al., 2025). In addition to the water- displacement and vitreous mechanisms common to spray-drying protectants (i.e., Tre), HP-β-CD exhibits a unique surfactant-like property (Serno et al., 2011). At the spray-dried droplet interfaces, its hydroxypropyl substituents can reduce surface tension through preferential adsorption, competing with protein drugs. This could effectively minimize contact between protein drugs and the gas–liquid interface, thereby lowering the risk of protein inactivation due to unfolding and exposure of hydrophobic groups (Dieplinger et al., 2023, Pan et al., 2022). Furthermore, pioneering studies indicated that the aerodynamic properties of protein drugs conferred an enhanced fine particle fraction (FPF) with increasing HP-β-CD content, whereas a higher Tre content can reduce FPF (Ramezani et al., 2017). Moreover, it could effectively enhance the drug permeability and bioavailability (Hao et al., 2021). It also displays favorable in vitro and in vivo safety profiles (Matilainen et al., 2008, Santos Gomes et al., 2025, Vartiainen et al., 2017).

While HP-β-CD, Leu, and Tre are well-established materials and their application in inhalable dry powder formulations has been studied previously, such studies typically examine them in isolation or under different formulation and processing conditions. In contrast, our work provides side-by-side benchmarking of HP-β-CD against Leu, Tre, and a Leu-Tre combination under identical spray-drying conditions, enabling a meaningful evaluation of relative performance in terms of protein integrity and aerosolization behavior. To the best of our knowledge, this type of comparative assessment has not been previously reported and is particularly relevant to formulation development and industrial decision-making.

The aim of this study was to provide a direct, side-by-side comparison of HP-β-CD with Leu, Tre, and a Leu-Tre system for spray-dried inhalable protein powders under identical formulation and processing conditions. By focusing on comparative physicochemical and aerosolization performance using bovine serum albumin (BSA), a surface-active model protein (Arte et al., 2024), this work aimed to generate formulation-relevant insights to support rational excipient selection rather than to introduce new excipients or analytical methodologies.

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Materials

Bovine serum albumin (BSA, purity ≥ 97%) and (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD, purity ≥ 98%) were received from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). D(+)-Trehalose dihydrate (Tre, purity ≥ 98%) and L-leucine (Leu, purity ≥ 98%) were procured from MeilunBio® (Dalian, China). All other chemicals and reagents used in the study were of analytical grade.

Yuchen Yang, Qingyi Zhu, Hriday Bera, Yasir Faraz Abbasi, Liping Wei, Xiaoxi Deng, Dongmei Cun, Mingshi Yang,
Hydroxypropyl-β-cyclodextrin as a multifunctional excipient for enhancing stability and aerosol performance of spray-dried protein formulations, International Journal of Pharmaceutics, Volume 694, 2026, 126729, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2026.126729.

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