Optimization of Sugar-Derivatives Mixtures for Stabilizing Polyclonal Immunoglobulin G in Spray-Dried Inhalable Powders During Processing and Long-Term

Abstract

Background/Objectives: The development of dry powder formulations for pulmonary delivery of therapeutic antibodies requires careful stabilization strategies to preserve protein integrity during spray-drying and long-term storage. This study investigates the impact of various sugar-derivatives, a polyol (D-mannitol), a disaccharide (D-sucrose) and a polysaccharide (dextran 10kDa) used individually or in combination, on the physical stability of bovine polyclonal immunoglobulin G (pAb) in dry powders for inhalation (DPIs).

Methods: A design of experiments (DoE) approach was employed to evaluate the effects of these excipients on residual moisture (RM), low-order aggre-gates (LOA) and high-order aggregates (HOA), immediately after spray-drying (T0) and after 10 months of storage at room temperature (T10) in a desiccator.

Results: All DPIs exhibited a high amorphous content and a favorable glass transition temperature, with RM decreasing over time. A combination of D-mannitol and dextran 10kDA (DPI-MD) demonstrated the best stabilization, minimizing LOA and HOA formation, both at T0 and T10. A ternary mixture, including also D-sucrose (DPI-MSD), showed enhanced short-term stability, but was less stable over time. The aerodynamic perfor-mance of these carrier-free DPIs, assessed via laser diffraction and a Next Generation Impactor, confirmed that DPI-MD and DPI-MSD formulations produced aerosol with suitable size distribution and fine particle fractions (FPFn of 70 ± 5% for DPI-MSD), for deep pulmonary deposition.

Conclusions: These findings highlight the importance of combining excipients with complementary physical properties to achieve robust pro-tein stabilization. The DPI-MD emerged as the most promising candidate for pAb lung delivery, balancing protein integrity, powder stability, and aerodynamic efficiency.

Introduction

Since the pioneering development of the hybridoma technology by Kohler and Milstein in 1975 [1], monoclonal antibodies (mAbs) have gained importance as therapeutic agents because of their high affinity and specificity for target molecules/receptors [2]. Despite these advantages, therapeutic mAbs face several challenges, primarily due to their high-molecular weight (mw) and complex tertiary structure. Their production remains time-consuming and expensive, as it relies exclusively on biological systems [3], and the subsequent purification process often involves multiple steps [4]. Another major challenge lies in the structural instability of mAbs. Their tertiary structure is crucial for biological activity, but can be easily disrupted by external factors, such as heat, adsorption at air/water interfaces, or pH fluctuations for examples [5]. Once unfolded, hydrophobic amino acid residues become exposed, promoting intermolecular interactions that can lead to aggregate formation [6]. These aggregates not only compromise therapeutic efficacy but also raise concerns regarding immunogenicity [7]. Aggregates can range from reversible low-order aggregates (LOA), such as dimers or trimers, to irreversible high-order aggregates (HOA), which are typically insoluble and often originate from LOA [8].

To limit aggregation and enhance protein stability, formulation strategies commonly employ stabilizing excipients. Moreover, transitioning proteins to a dry state offers several advantages over liquid formulations. Dry forms extend shelf life and eliminate water-mediated degradation pathways, such as deamidation or hydrolysis in the hinge region [9,10]. Removing 95–99% of the water also decreases transportation costs and permits ambient temperature shipping, in contrast to liquid protein formulations that often require cold-chain logistics due to limited long-term stability [9].

Among the scalable drying techniques, spray-drying offers notable benefits over freeze-drying (lyophilization), such as cost-effectiveness, speed, and the ability to engineer particles in a single step. Spray-drying transforms a liquid feed into dry particles, and various process parameters can be adjusted to influence particle size, shape, density, crystallinity, and residual solvent content [9,11]. This technique is particularly suitable for developing pharmaceutical formulations intended for noninjectable routes, such as pulmonary delivery [9,12].

Pulmonary administration via inhalation is a non-invasive route that enables direct drug delivery to the lungs, especially in the treatment of respiratory diseases. This localized delivery reduces systemic exposure and side effects, while maintaining therapeutic efficacy at lower doses. It also facilitates the delivery of hydrophilic and large molecules, such as proteins, to their site of action in the bronchial or alveolar lumen, bypassing biological barriers, such as endothelial and epithelial membranes encountered with systemic routes [12].

Dry powder inhalers are among the most common devices for pulmonary drug delivery. They are environmentally friendly and rely on the patient’s inspiratory airflow to aerosolize the powder [13]. However, the powder must possess suitable aerosolization and dispersion properties, which can be compromised by humidity [13]. Therefore, maintaining low and stable residual moisture (RM) is critical for long-term performance [12,14,15]

Currently, four protein-based dry powder formulations, produced via spray-drying, received Food and Drug Administration (FDA)-approval: Exubera® (FDA approval 2006 but retrieved since 2007 [16]), Trelstar® (FDA approval in 2010 [17]), Somatuline® (FDA approval in 2007 [18]), Raplixa® (FDA approval in 2015 [19]) and Inbrija® (FDA approval in 2018[20]).

While spray-drying can affect protein structure, the use of stabilizing excipients may help minimizing degradation [12,21] during the three key steps of spray-drying: atomization, drying and particle separation from the gas phase. Among the stresses encountered during spray-drying, dehydration is particularly critical. Sugars and their derivatives are well-known for their protective role during protein dehydration and are commonly used as cryoprotectants in freeze-drying [22–24]. Although extensively employed in dry-state processes, the mechanisms underlying their stabilizing effects are still being elucidated. Furthermore, the impact of combining different sugars and derivatives with varying physical properties remains underexplored, especially in the context of spray-drying.

Therefore, this study aims to evaluate the effect of various sugar derivatives, each with distinct physical properties, on the stability of a model protein (bovine polyclonal IgG, pAb) following spraydrying and during long-term storage at room temperature (RT) in a desiccated environment. Protein stability in dry powder for inhalation (DPI) was assessed based on the residual moisture (RM) content, which impacts different physical and aerodynamic properties, and the contents of LOA and HOA, which are critical indicators of pAb stability. A rational mixture design of experiments (DoE) was employed to evaluate both the individual and interaction effects of sugar derivatives. The selected excipients, D-mannitol (M), D-sucrose (S) and dextran 10kDA (D), used as controllable input factors, were chosen based on their physical properties (such as mw, glassy transition temperature (Tg) and hydrogen bonding potential) and their known tolerability in pulmonary applications:

1. D-mannitol (C6H14O6) is a small polyol (mw: 182.2 Da) with 12 potential sites of hydrogenbonds (H-bonds) per molecule [25] and a very low Tg of 13°C [26], which is authorized for inhalation by FDA [27].
2. Dextran 10 kDa (mw: 10 000 Da), a polysaccharide of D-glucose is a larger molecule with a very high Tg (213°C) [22,28] and 310 potential H-bonds. Moreover it showed promising results in terms of lung tolerability [29].
3. Because of the lack of lung toxicity data for non-reducing disaccharides, D-sucrose (C12H22O11), an inexpensive intermediate size sugar (mw 342.3 Da), which presents a very low oral toxicity, is widely used in both food and pharmaceutical industries, [30] and, was tested. D-sucrose possesses 14 potential H-bonds per molecule [30] and an intermediate Tg (63°C).

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Materials

pAb was obtained as a lyophilized powder from Equitech (Kerrville, Texas, USA). Citric acid monohydrate and NaH2PO4 were purchased from Merck (Darmstadt, Germany). D-Sucrose (S) and L-Arginine were bought from Sigma-Aldrich (Saint-Louis, Missouri, USA). Trisodium citrate was purchased from Alfa Aesar (Haverhill, Massachusetts, USA), Dextran T10 (D) was obtained from Pharmacosmos (Holbaek, Denmark), and Pearlitol 200SD-Mannitol (M) from Roquette (Lestrem, France). Na2HPO4 anhydrous, sodium hydroxide, sodium azide and silica gel were purchased from VWR Chemicals (Oud-Heverlee, Belgium). The bicinchoninic acid (BCA) protein assay kit, the microBCA kit and related globulin standard ampules were purchased from Thermofisher (Waltham, Massachusetts, USA). Millex polyvinylidene fluoride syringe filters, Durapore®, were purchased for Sigma-Aldrich. Bridged Ethylene Hybrid (BEH) size exclusion chromatography (SEC) standards and SEC standards were purchased from Waters (Antwerp, Belgium) or besides SEC standard from Biorad (Temse, Belgium). The ultrapure water was produced with a Purelab system (Elga LabWater, Wycombe, UK).

Philippe Gevenois, Le Van Bui, Thami Sebti, Yvan Vander Heyden, Karim Amighi, N. Wauthoz, Optimization of Sugar-Derivatives Mixtures for Stabilizing Polyclonal Immunoglobulin G in Spray-Dried Inhalable Powders During Processing and Long-Term, Posted Date: 18 March 2026, doi: 10.20944/preprints202603.1485.v1

Read also our introduction article on Mannitol here: