Engineering High-Performance Inhalable Monoclonal Antibody Powders via Spray Freeze Drying

Abstract

Spray freeze-drying (SFD) is a cutting-edge technique for producing dry powders, ideal for pulmonary drug delivery, owing to its ability to generate porous microparticles with low density and excellent aerodynamic properties. The SFD process, involving atomisation, freezing, and drying, is particularly suited for formulating biologics, e.g., monoclonal antibodies (mAbs), due to its low-temperature conditions. However, the method poses challenges, including the potential denaturation of mAbs due to shear, freezing, and dehydration stresses during processing. Here, we investigated how formulation modulates mAb stability and delivery performance during and after SFD processing. A model mAb was spray freeze-dried in the presence of selected stabilizing excipients, including sugars/polyols, amino acids, cyclodextrins, and surfactants. MAb chemical stability was assessed by size exclusion chromatography after the SFD process to study the strength of the formulations in each stage. The resulting inhalable mAb powders demonstrated favourable aerodynamic properties, rapid dissolution rates, and stability under various storage conditions. Notably, trehalose combined with either leucine or HPβCD effectively preserved mAb stability throughout the SFD process, ensuring high fine particle fractions (50-60%), and good physical stability of the powders. The optimised dry powder was successfully administered to rats via inhalation, as evidenced by lung deposition and systemic exposure, supporting the suitability of inhaled mAb-based therapeutics. This work elucidates key formulation-process interactions governing the pulmonary delivery of inhalable mAbs and demonstrates how rational excipient design enables the production of stable, inhalable biologic dry powders via SFD.

1. Introduction

Approved for human use in 1986, monoclonal antibodies (mAbs) play a pivotal role in the pharmaceutical industry [1]. The recent pandemic significantly increased the commercial interest on mAbs, since many drugs for COVID-19 treatment or prevention were mAb-based [1]. To date, over 50 mAbs are currently approved and more than 500 mAb-based therapies are under clinical trial [2]. MAbs are employed in many therapeutic areas, including oncology, cardiovascular, respiratory, inflammatory, and infectious diseases [3]. Although systemic delivery (intravenous and intramuscular ones) is still standard practice, the last years have witnessed a huge growth of mAb-based drugs for inhalation therapies. Systemic delivery has drawbacks, such as the poor mAb bioavailability in the lung, where the therapeutic effect should be exerted. Indeed, 500-10000 times higher mAb concentrations are usually detected in serum than in bronchoalveolar lavage fluid [4]. The pulmonary delivery route represents a more effective, convenient, and cheaper alternative to systemic administration for respiratory infections [5]. Inhaled mAbs can escape first-pass metabolism and enzymatic degradation, enhancing their therapeutic effectiveness. Moreover, the inhalation route offers high targeting efficiency, which allows for reduced drug doses and side-effects [6].

However, pulmonary delivery of mAbs presents many challenges, such as clearance mechanisms aimed at preventing the entrance of external material into the respiratory system [7], which can reduce the local drug concentration in the lungs. Consequently, to maintain the effective drug concentration at the action sites, patients should take the drugs frequently, which may result in poor patient compliance and adherence [8]. Also, mAbs are highly sensitive to chemical and physical degradation after exposure to environmental stresses due to their large size and complex structure.

Despite their therapeutic potential, full-length mAbs present intrinsic challenges for pulmonary administration due to their large molecular size, complex structure, and limited formulation flexibility. To overcome some of these limitations, increasing attention has been directed towards smaller engineered formats, including Fab fragments (∼50 kDa) and single-domain antibodies (sdAbs or nanobodies, 12-15 kDa), which have shown promising properties for inhaled delivery [9], [10]. Their reduced molecular size can improve formulation properties, including lower viscosity at high concentrations compared with mAbs, potentially facilitating aerosolisation and high-dose delivery [9]. However, the reduced size of mAb fragments is associated with important pharmacokinetic differences compared with full-length mAbs. Fab fragments and sdAbs lack the Fc domain responsible for neonatal Fc receptor (FcRn)-mediated recycling, resulting in limited systemic persistence and more rapid clearance following absorption into the circulation [10]. While this property may reduce systemic exposure and potential toxicity, it also eliminates a mechanism that contributes to the prolonged pulmonary residence of mAb molecules. Therefore, the development of effective pulmonary mAb therapies requires a careful balance between achieving sufficient local retention, maintaining mAb activity, and ensuring adequate formulation stability [11]. Generally, drying processes are employed to improve the stability of proteins and mAbs by entrapping them into a solid matrix. Spray drying (SD) and spray freeze-drying (SFD) have been used to design inhalable powders. Although SD is a well-established technique to produce inhalable microparticles (MPs), it is often associated with thermal exposure and increase in protein concentration and interfacial stress that occurs as solvent evaporates, which can promote mAb aggregation. Given its operation at low temperatures and the elimination of shrinkage, SFD presents a less invasive alternative for handling mAbs. SFD consists of three steps, i.e., atomisation, freezing, and drying [12]. The resulting MPs are called large porous particles as they are larger than spray dried ones, but porous due to the sublimation of ice crystals during drying. Such features make spray freeze-dried powders potentially suitable for pulmonary delivery since the internal porosity provides the MPs with low density and optimal aerodynamics [13].

During SFD, the mAb is exposed to shear stress, freezing stress, and dehydration stress [14]. Also, the formation of an interface between the N₂ gas and the liquid can expose the hydrophobic surfaces of the protein, thus inducing mAb denaturation. Excipients should be finely selected to protect the mAb from the stresses encountered during the process and prevent mAb aggregation during storage to guarantee long-term stability. Moreover, excipients should promote powder aerosolisation and possess safety, biodegradability, biocompatibility, and mucoadhesive properties. Chitosan, hyaluronic acid, poly(lactic-co-glycolide acid) [15], lactose [16], mannitol (MAN) [17], L-leucine (LL), and magnesium stearate [18] have been previously employed while producing inhalable powders. Among them, some polymers, amino acids, sugars, polyols, surfactants, and cyclodextrins are examples of widespread classes of excipients also employed for protein stabilisation [11]. However, their suitability and regulatory acceptance must be carefully considered. For instance, while α-lactose monohydrate is the most widely accepted carrier in commercial inhalation products, its reducing nature makes it chemically incompatible with peptides and proteins. Consequently, non-reducing alternatives like sucrose, trehalose (TRE), and MAN are preferred as bulking agents and cryo-lyoprotectants. TRE mainly operates through water replacement and vitrification, while MAN acts as a bulking agent, cryoprotectant, and lyoprotectant [19]. Amino acids, such as phenylalanine, LL, and glycine, also showed a stabilising effect on a spray freeze-dried model immunoglobulin G (IgG), while cysteine and arginine (Arg) had an opposite effect [20]. On the contrary, Arg acted as a good stabiliser for spray dried IgG4 [21], stressing out how the optimal formulation depends on the technique and molecule. Cyclodextrins have been receiving much attention due to their surfactant-like behaviour and stabilising action through water replacement and vitrification [14]. Particularly, 2-hydroxypropyl-beta-cyclodextrin (HPβCD) holds potential for its non-reducing nature, amphiphilic nature, and availability of hydrogen bonds [14]. Surfactants, e.g., polysorbate 20 and polysorbate 80 (PS80), are also employed to protect biologicals molecules from air-water, solid-water, and oil-water interfaces [22].

While mAbs have revolutionised the treatment of respiratory diseases, their conventional liquid formulations remain severely limited by physical instability and strict cold-chain storage requirements. To overcome these barriers, this study engineered the first solid-state formulation of a clinically relevant IgG for pulmonary delivery using a highly optimised SFD process. Converting the formulation from liquid to solid offers several advantages, including enhanced long-term stability, extended shelf life, and improved patient compliance [11]. Moreover, solid formulations provide more efficient pulmonary delivery by reducing product loss during administration and allowing for lower effective doses, contributing to greater therapeutic efficiency and cost-effectiveness [23]. We employed a design of experiments (DoE) methodology to synergistically integrate five strategically selected excipients. Such an approach allowed for a complete study of the effect of single and combined excipients, providing further insights into the formulation of stable mAbs powders. This represents a critical shift from previous studies, which frequently evaluate single excipients in isolation, towards a comprehensive, process-specific stabilisation strategy. Moreover, the process-induced mAb aggregation was investigated for each stage of SFD, i.e., atomisation, freezing, and drying, to better identify the drawbacks of this technique. This enabled a comprehensive, stepwise deconstruction of the SFD process, where process-induced mAb aggregation was investigated at each individual stage to definitively identify and overcome the technique’s physical bottlenecks. The mechanistic understanding of isolated stresses in complex biopharmaceuticals such as mAbs is still in its early stages and requires further experimental data. While a recent study identified the air-ice interface generated during freezing as the main cause of IgG aggregation [24], our results recognised the atomisation stage as the primary driver of aggregation. This discrepancy underscores that stabilisation strategies must be specifically tailored to the molecule’s sensitivity, shifting the focus towards mitigating air-liquid interface stresses using HPβCD and TRE. A key innovation of this work is the application of advanced characterisation tools to fill the void between laboratory results and clinical efficacy. We utilise the RespiCell™ dissolution system, which replicates the lung’s air-liquid interface and limited fluid volume to provide biorelevant data on the dissolution behaviour of inhaled particles. Moreover, the spatial distribution of the mAb in lung tissue was investigated using MALDI-MS Imaging (MSI), which allows for the precise mapping of mAb deposition, distinguishing between bronchial localisation and deep alveolar penetration. By integrating biorelevant in vitro testing with spatially resolved in vivo analysis, this work aims to bridge the gap between physicochemical properties of inhalable powders and their aerodynamic performance, an aspect which was not sufficiently covered by previous studies. We believe this comprehensive approach establishes a robust platform for overcoming the current limits of SFD in engineering inhalable mAb therapies.

2. Materials and Methods

2.1. Material

A mAb, a humanised IgG4 created from a genetically engineered DG44 CHO cell line (150 kDa) and commonly used in the treatment of a pulmonary disease, was buffered in 20 mM L-histidine (HIS, C₆H₉N₃O₂, 98.5+%, Sigma-Aldrich, MO, USA), pH 5.5. D-MAN (C₆H₁₄O₆, 98+%. Chem-Lab NV, Zedelgem, Belgium), D-(+)-TRE dihydrate (98+%, C₁₂H₂₂O₁₁, Sigma-Aldrich, MO, USA), LL (Sigma-Aldrich, MO, USA Sigma-Aldrich, MO, USA), HPβCD (Sigma-Aldrich, MO, USA), and PS80 (Sigma-Aldrich, MO, USA) were employed as excipients.

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Lorena Pasero, Adamo Sulpizi, Elettra Pinetti, Salvatore Sauli, Viola Geminati, Giulia Marenghi, Giulia Carretta, Lara Benoldi, Tomaso Guidi, Roberto Pisano, Engineering High-Performance Inhalable Monoclonal Antibody Powders via Spray Freeze Drying, European Journal of Pharmaceutics and Biopharmaceutics, Volume 227, 2026, 115202, ISSN 0939-6411,
https://doi.org/10.1016/j.ejpb.2026.115202.