Nanoparticulate protectants for the dry storage of protein therapeutics

ABSTRACT

Protein therapeutics are a cornerstone of modern medicine, yet their inherent instability often requires expensive and energy-intensive cold-chain logistics to maintain efficacy. Dry-state formulations offer a viable strategy to enhance protein stability, but the associated drying processes, such as lyophilization and spray drying, can introduce stresses that damage protein molecules. While excipients are widely used to protect proteins during dehydration, conventional formulations often require protein-specific, iterative optimization. Further, protein stabilizing excipients can crystallize during storage, thereby reducing product quality, increase product viscosity beyond thresholds for injection, and raise safety concerns. Nanoparticulate excipients are receiving increasing attention to address these challenges. This article provides an overview of nanoparticle excipients showing promise for stabilizing proteins during drying processes. It is envisioned that nanoparticulates may advance toward universal excipients capable of stabilizing a variety of proteins without the need for cumbersome optimization while also providing robust biocompatibility.

1. Introduction

Protein therapeutics have emerged as a cornerstone of modern medicine, owing to their high specificity, functional versatility, and biocompatibility [1,2]. Their potential for considerable continued future growth is underscored by market projections, which estimate the value of the protein therapeutics market will exceed $653 billion by 2030 [3].

However, protein therapeutics are susceptible to environmental stresses. Exposure to elevated temperature, pH changes, humidity, surface adsorption, or agitation can lead to chemical alteration of the protein or induce a conformational change of the protein that is detrimental to the therapeutic effect. Further, perturbations to the chemical identity or structure of the protein can trigger an immune response, which often poses a serious risk to patient safety [4].

Maintaining protein stability during transport and storage is critical for preserving therapeutic effectiveness. Liquid-state formulations of protein therapeutics are generally more unstable than those in a solid-state (i.e., frozen, dried) because of the potential for hydrolysis, as well as increased aggregation via hydrophobic interactions between highly mobile proteins at the liquid-air and liquid-container interfaces [4–6]. Most often, protein therapeutics are maintained at reduced temperatures throughout the distribution and storage process [6,7]. Considering one −80°C freezer uses a comparable amount of energy as one household per day, maintaining this cold-chain storage is expensive and energy-consuming [8].

To improve protein stability without cold-chain logistics, dry processing holds enormous promise, with lyophilization, spray drying, and desiccation among the most common industrial drying processes. Lyophilization, also known as freeze-drying, is a dehydration method that involves freezing a solution and then reducing the pressure to sublimate the frozen water directly from the solid to the gas phase [9]. As an example, Keytruda® (Pembrolizumab) is a lyophilized monoclonal antibody cancer drug first approved by the United States Food and Drug Administration (FDA) in 2014 [1,6]. Spray drying is a dehydration process that transforms a solution into atomized micron-sized droplets, which are then rapidly dried by a heated, dry gas stream [10]. Afrezza is an inhalable spray-dried insulin for type 1 or type 2 diabetes [11]. Lastly, desiccation is a method for drying bulk solutions in ambient conditions, under vacuum, or with heated air, which finds limited use for drying pharmaceuticals and is instead mostly used for research-grade products [2].

Although many dried protein therapeutics have been approved for clinical use, dehydration can impose stresses that compromise drug activity [1,12,13]. For example, in the lyophilization process, proteins undergo freezing and sublimation, whereas in spray-drying they experience shear stress and rapid solvent evaporation. In both processes, proteins are exposed to fluctuations in temperature, pH, and ionic strength, while solvent removal can promote protein adsorption onto various surfaces [6,7,10]. To prevent damage to protein chemical identity and structure, many pharmaceutical formulations include excipients, additives that can protect the proteins from the various stresses associated with drying processes.

Excipient blends consisting of small molecules, surfactants, and polymers are routinely included in protein therapeutic formulations to improve drug stability during processing and storage. Despite measurable effectiveness with many protein therapeutics, existing excipients can degrade over time, they can undergo crystallization in the dry state that can drive protein unfolding, they can increase the product viscosity, making administration by injection difficult or impossible, and they may pose a risk of toxicity. For example, the common disaccharide excipient trehalose can crystallize during freezing [14], while sucrose can crystallize via residual water – think cotton candy on a humid day [15], which creates surfaces that can promote protein unfolding and can lead to poor reconstitution of dry protein products. The surfactant Tween-80 is susceptible to hydrolysis via residual product water [16]. Linear polymers often used to improve protein bioavailability [17], such as poly(ethylene glycol) (PEG), are often poor dry-state excipients because they entangle at high densities [18], leading to high product viscosities that compromise product end-use [19,20]. Finally, sugars and surfactants can alter the molecular, micro-, and macro-scale behavior of each other during the freezing step and the dehydration step, further complicating the product formulation process [21,22]. As a result, formulating excipients for dry-state protein therapeutic production remains a complex, iterative process that can greatly increase product development costs and significantly extend the product development timeline. As a result, the vast majority of protein therapeutics are formulated for long-term cold storage despite the known inefficiencies and burdens of cold-chain logistics.

Nanoparticulate excipients are emerging as a viable alternative to established small molecule, surfactant, and polymer composite formulations. For instance, the polyacrylamide-derived copolymer poly(acryloylmorpholine-co-N-isopropylacrylamide) (MoNi) has been employed to achieve ultra-high protein concentrations that exceed the capabilities of conventional excipients [23]. Phytoglycogen extracted from sweet corn has been shown to stabilize various proteins during lyophilization when formulated in a physiologically-relevant buffer that is often incompatible with sucrose and trehalose, suggesting potential to reduce to the complexity of excipient screening processes [24].

In this Special Report, we will provide a brief overview of the mechanisms by which excipients are presently thought to stabilize proteins in the dry state, which is an area of active research [4], as well as present a survey of existing and emerging drying methods used in the manufacturing of protein therapeutics. We also highlight examples of nanoparticulate protectants that are showing promise for addressing the complex formulation processes, high viscosity, instability, and safety risks of existing excipient formulations that are currently used to develop dry-state protein therapeutics in industrial-scale manufacturing.

2. Drying techniques and conventional excipients used in dry-state formulations

2.1. Drying methods – examples and unmet challenges

Lyophilization is widely applied in pharmaceutic manufacturing because of key advantages over other drying methods (Figure 1(a)). Under ideal processing conditions, the sublimation process results in a physically stable uniform cake that has a similar shape as the storage vial, and which lacks obvious cracks or bubbles indicative of boiling. In turn, this cake can be quickly and readily reconstituted into a dosage form upon addition of water, without appreciable product aggregation or crystallization. The removal of water from the product prevents protein collisions that can lead to unfolding or aggregation, as well as the chemical reactions that can compromise protein activity. Finally, the dry solid-state product can often be maintained at ambient and elevated temperatures to reduce or eliminate the need for cold-chain logistics. Nonetheless, there are also some notable challenges with lyophilization, including the costs associated with the sequential, batch-wise freezing and drying processes, complicated scale-up from pilot to mass production due to batch variability and risk of contamination in open processing steps that require strict air quality settings, and, most importantly, the potential for damage to sensitive protein molecules due to freezing and drying stresses [7,25–28].

Figure 1.

Schematic description of various dehydration techniques. (a) Lyophilization (freeze drying), (b) Spray drying, (c) Air drying or convection drying, (d) Vacuum drying, (e) Spray freeze drying, and (f) Supercritical fluid drying.

Spray drying, in which a dry product can be produced through the rapid evaporation of an atomized solution using a hot gas, has many advantages for industrial-scale manufacturing (Figure 1(b)). It is a continuous process that is simple, fast, inexpensive, and offers higher throughput than lyophilization [29,30]. Despite these advantages, though, the hot dry air can damage drug products via thermal stress, in addition to the dehydration stresses and interfacial adsorption that is likewise seen with lyophilization [31,32]. Loss of powder on the drying chamber wall can also lead to variable product yields. As with lyophilization, many excipients have been used to improve the quality of spray-dried pharmaceutic products through similar theoretical mechanisms, described in section 2.2 below.

Although lyophilization and spray drying are the major methods to manufacture dry protein therapeutic products, there are other drying methods, such as air drying, convection drying, vacuum drying, spray freeze drying, and supercritical fluid drying, with potential for industrial-scale use. Air drying is a method that uses natural evaporation of solvent in ambient conditions (Figure 1(c)). A recent study reported minimalistic tripeptides (composed of tryptophan – lysine – tyrosine (WKY)) to encapsulate biomolecules in microparticles. These tripeptides form highly soluble, dynamic ensembles stabilized by multivalent side-chain interactions. Upon drying, these tripeptide assemblies undergo a sequential phase transition, from liquid-liquid phase separation to solidification, spontaneously forming a film of stiff, porous microparticles. Enhanced green fluorescent protein (EGFP) or lysozyme formulated with WKY tripeptides remained active after storage at ambient temperatures for 120 hours or 15 days, respectively [33].

Convection drying or oven drying uses heated air to evaporate solvent from the sample (Figure 1(c)), but because of the thermal stress associated with increased temperature, it is not common in the therapeutic protein industry, and instead finds most use in food science or laboratory-scale experiments [34]. Vacuum drying works by lowering the solvent boiling point through a reduction in pressure, which is attractive for heat-sensitive molecules like proteins (Figure 1(d)). However, because this method requires a constant vacuum, it typically has a long drying time and introduces the risk of product oxidation [35,36]. This suggests that nanomaterials that prevent oxidation or reduce solvent boiling points could be effective excipients to advance the use of vacuum drying.

Spray freeze drying and supercritical fluid drying are relatively recent technologies compared to air, convection, or vacuum drying. Spray freeze drying is a combination of spray drying and freeze drying (lyophilization) (Figure 1(e)). The sample is sprayed into a cryogenic liquid, and then the atomized droplets rapidly freeze. Subsequently, the solvent is removed from the droplets by sublimation in the same way as in the lyophilization process. The sequential process of atomization, fast freezing, and drying allows the sample to sublimate rapidly, which minimizes interaction with the air-water interface [2,7,10]. In a recent study, hIgG was spray-freeze-dried with a combination of trehalose and various amino acids (leucine, phenylalanine, arginine, cysteine, or glycine). hIgG formulated with trehalose, and either leucine, phenylalanine, or glycine demonstrated stable monomer characteristics for 2 months at 40°C [37]. Supercritical fluid drying dehydrates samples using a supercritical fluid (i.e., ethylene, methanol, or carbon dioxide) (Figure 1(f)). For example, carbon dioxide (CO₂) transitions to a supercritical fluid above its critical temperature (31°C) and pressure (73 bar). Either by using the supercritical fluid as an extraction medium to remove water, or as a propellant for atomization, supercritical fluid drying allows for the creation of particles with uniform and adjustable sizes under mild conditions [2,7,10,38,39].

2.2. Mechanisms of dry-state protein stabilization by excipients

Proteins can experience various stresses during drying processes. For example, in the freezing step, ice crystal formation creates new solid interfaces (e.g., ice-liquid and ice-container) onto which proteins may adsorb. Further, ice crystal formation leads to the concentration of all solutes in the formulation, which can increase the relative protein concentration, induce solute crystallization, and alter the pH and ionic strength of the environment around the protein. During the dehydration step, the protein loses hydrogen bonds with water, which may cause a conformational change in the protein. Excipients are commonly included in pharmaceutic formulations as inactive ingredients that can mitigate these stresses.

Ideal excipients share key features: be inert to the other ingredients, nontoxic, cost-effective, and enhance product performance [40,41]. The most commonly used industrial lyophilization excipients include disaccharides (e.g., sucrose, trehalose, and maltose), polysorbates, such as Tween-80 ®, which restrict protein adsorption onto solid surfaces, microcrystalline cellulose or lactose, which can act as a bulking agent, and lastly, buffering agents (e.g., L-histidine) [42–46].

The mechanisms by which excipients protect proteins during processing and drying remain an area of active research [4]. One theory is preferential exclusion, which proposes that excipient molecules are excluded from the protein surface in exchange for preferential protein hydration. This exclusion of the excipient molecules is thermodynamically unfavorable, so it maintains proteins in the most compact and native structure, hindering denaturation. A second theory is water replacement, wherein hydrogen bonds formed between a protein and an excipient are thought to stabilize protein structure by mimicking the effect of protein-water bonding. A third theory is water entrapment, which suggests that excipients can indirectly interact with protein molecules to hold water around the protein surface during the sublimation process. A fourth theory is vitrification, which theorizes that excipients with a high glass transition temperature (T_g) can provide an amorphous ‘glassy’ matrix that restricts protein mobility during and after drying [4,6].

2.3. Conventional excipients as stabilizers of biotherapeutic-loaded nanoparticle delivery vehicles

Poly(lactic-co-glycolic acid) (PLGA) nanoparticles have found broad use as carriers for the sustained release of protein therapeutics [47]. However, fabricating PLGA nanoparticles loaded with therapeutic proteins can compromise protein activity due to the need for harsh processing steps and the use of organic solvents [48,49]. Informed by the effectiveness of excipients to stabilize proteins during processing from the wet to dry state, a recent study analyzed various excipients, including arginine (Arg), glycine (Gly), lactose, Poloxamer 188 (P188), and PEG, as stabilizers of proteins during PLGA particle fabrication. Each excipient was co-formulated with bovine serum albumin (BSA) that was loaded into PLGA nanoparticles using an established emulsion method. Guanidine compound excipients (Arg and urea) had a positive impact on BSA stability throughout much of the PLGA nanoparticle fabrication process, while other additives offered more specific benefits. For example, lactose prevented aggregation during the lyophilization step [50]. Similarly, 100 nm-sized chitosan nanoparticles loaded with bromelain via ionic crosslinking could be stabilized during lyophilization by including maltose as an excipient [51]. Notably, including maltose in the formulation resulted in only 8.1% activity loss of bromelain during dry-state storage at 5°C for 90 days. Beyond protein delivery, mRNA-loaded lipid nanoparticle (LNP) vaccines have also been successfully lyophilized with disaccharide excipients, including sucrose, trehalose, and maltose [52–54].

2.4. Nanoparticulates as alternative excipients to stabilize biotherapeutics in the dry state

Increasing studies show that nanoparticulates can serve as excipients to enhance protein stability during processing, transport, and storage. In the following subsections, we will introduce examples of nanoparticulate excipients to stabilize proteins during lyophilization and spray-drying.

2.4.1. Nanoparticulate excipients to stabilize proteins during lyophilization

A protein-based thermostable exoshell (tES) was recently reported to stabilize horseradish peroxidase (HRP). The tES, produced by recombinant protein expression in Escherichia coli (E. coli), has a hydrodynamic diameter of 15 nm and an internal cavity of 8 nm into which HRP can be encapsulated. The lyophilized tES-HRP and HRP were stored at room temperature (RT), 4°C, and −20°C, respectively. At all temperature conditions, tES-HRP samples retained 70% of their activity after a month of storage, while HRP lacking an excipient lost 95% of its activity over the same duration [55]. It was hypothesized that two mechanisms provide a protective effect. First, the protein is protected by a thermostable cage that serves as a physical barrier. Second, the internal surface of the cage complements the surface charge of the target protein, providing energetic stabilization similar to the water replacement theory.

Phytoglycogen, a hyperbranched glucose polymer derived from sweet corn, has also been shown to protect proteins during lyophilization [24]. For example, phytoglycogen excipients maintained between 60–100% activity of green fluorescent protein (GFP), lysozyme, β-galactosidase (β-gal), and HRP over several cycles of lyophilization, versus less than 20% activity for all protein formulations lacking an excipient. The effectiveness of phytoglycogen was shown to depend on its density in the solution-state prior to freezing and drying, with cake being formed in solutions having more than 0.1% w/v phytoglycogen during lyophilization. Notably, phytoglycogen has also been shown to be cytocompatible and is expected to be biocompatible, given its abundance in a widely consumed food [56]. Furthermore, because phytoglycogen is ~30–50% of the mass of dry sweet corn kernels, and because sweet corn is already produced at massive industrial-scale through well-established agricultural practices, phytoglycogen excipients are anticipated to be a cost-effective alternative to conventional excipients.

One plausible mechanism of the excipient activity of phytoglycogen is a through a combination of vitrification and water replacement. Phytoglycogen forms an amorphous glass at high densities, suggesting that it may stabilize proteins via vitrification [57]. Indeed, mixtures of phytoglycogen and proteins were reported to have a similar freeze-concentrate glass transition temperature (Tg′) as other common excipients. On top of that, the hairy surface of flexible glucose-rich chains that are a characteristic of phytoglycogen structure may easily interact with protein molecules, thereby providing for water replacement or water entrapment in the dry state.

2.4.2. Spray-drying composite protein-polymer nanoparticulates

The product of a spray-drying process is a micro- to nano-scale particle consisting of the active pharmaceutical ingredient, carriers, and excipients. Spray-dried protein therapeutics can be delivered via pulmonary routes via inhalation, aerosolization, or nebulization when formulated with biocompatible polymers, including natural materials like gelatin, alginate, chitosan, and albumin, or synthetic polymers such as PLGA, polyvinyl alcohol (PVA), and PEG as a carrier [58–63]. These spray-dried protein formulations also often incorporate excipients, including disaccharides, surfactants to prevent protein adsorption, and amino acids or buffer salts to maintain pH [5,30,32,64], similar to lyophilized products.

A recent study evaluated spray-dried lysozyme mixed with combinations of erythritol, mannitol, trehalose, or sucrose as a primary excipient, with pullulan, NaCl, glycine, or poloxamer 407 (Pluronic F127) as a secondary excipient. Upon comprehensively considering the fine particle fraction, bioactivity values, and aerosolization-promoting features, the optimal formulation was identified as trehalose (20% w/w), NaCl (5% w/w), and L-leucine (15–20% w/w), which maintained 95% of lysozyme after spray drying into 2 μm particles [65].

Oligosaccharides and polysaccharides have also been evaluated as excipients for spray-drying of protein therapeutics. For example, inulin reduced the aggregation of recombinant human deoxyribonuclease I (rhDNase I) formulated for inhalation via spray drying, leading to a product with a good fine particle fraction and robust ex vivo performance [66]. Similarly, 2-hydroxypropyl-β-cyclodextrin (HPβCD) has been used to protect a monoclonal antibody (mAb) by suppressing the crystallization of trehalose that can occur during spray drying. After 4 weeks of storage at 40°C and 25°C, the activity of the mAb formulated with HPCD and trehalose was preserved [67].

Finally, synthetic polymers can be used as excipients for spray-dried protein therapeutic formulations. For example, adding Pluronic F-127 to lysozyme maintained protein structure during spray drying and yielded a product that maintained enzymatic activity after 20 weeks of storage at room temperature [68]. Likewise, the polyacrylamide-derived copolymer poly (acryloylmorpholine-co-N-isopropylacrylamide) (MoNi) was shown to stabilize ultra-high concentrations of BSA, human Immunoglobulin G (hIgG), or an anti-COVID mAb. Ultra-high concentration formulations (>100 mg/mL) of proteins spray-dried with MoNi formed 5–20 µm-sized particles without aggregate formation. In vivo experiments showed that the subcutaneous injection of these spray-dried particles had comparable biological effectiveness as a large volume bolus injection of soluble protein therapeutic [23].

3. Future perspectives

Ideal excipients should be inert to other ingredients, nontoxic, cost-effective, and enhance product performance. The development of better excipients is needed to reduce the need for iterative optimization and enable the mass production of a wide range of protein therapeutics. A novel excipient should not only meet industrial benchmarks [69], but also outperform existing excipients.

The examples presented here and summarized in Table 1 suggest that nanoparticulate excipients may address limitations of existing excipient formulations. However, as a relatively new area of research, there is still much left to understand about how nanoparticulate excipients act to stabilize proteins during processing, drying, and storage. While it is possible that nanoparticulate excipients may share some mechanistic aspects of protein stabilization with conventional small molecule, surfactant, and polymer excipients, it is likely that the distinct size and surface characteristics of nanoparticulate excipients also contribute new ways of stabilizing proteins in the dry state. To move nanoparticulate excipients into industrial use, there remain many practical considerations that should be addressed. One such consideration is the need for more rigorous testing of nanoparticulate excipients across existing industrial drying modalities. Further, clear opportunities exist to assess the performance of nanoparticulate excipients as stabilizers in emerging drying modalities. Another is the need to robustly evaluate the effectiveness of nanoparticulate excipients in long-term storage at ambient and elevated conditions to eliminate cold-chain requirements. Key here will be direct comparisons against existing, industry-standard excipient formulations. Additionally, it will be critical to demonstrate nanoparticulate synthesis, extraction, and purification can be moved from laboratory- to industrial-scale, without compromising performance. Finally, it will be necessary to demonstrate that nanoparticulate excipients are safe and well-tolerated when included in protein therapeutic formulations.

Table 1.

Summary of excipients for stabilizing protein therapeutics during various dehydration techniques.

Dehydration Technique	Excipients	Target	References
Lyophilization	Poly(lactic-co-glycolic acid) (PLGA) with arginine, glycine, lactose, Poloxamer 188, polyethylene glycol (PEG)	Bovine serum albumin (BSA)	[31]
	Chitosan, maltose, glycine	Bromelain	[32]
	Thermostable exoshell (tES)	Horseradish peroxidase (HRP)	[33]
	Phytoglycogen	Green fluorescent protein (GFP), lysozyme, β-galactosidase (β-gal) and HRP	[34]
Spray drying	Trehalose, NaCl, and L-leucine	Lysozyme	[46]
	Inulin	Recombinant human deoxyribonuclease I (rhDNase I)	[47]
	2-hydroxypropyl-β-cyclodextrin (HPβCD), trehalose	Monoclonal antibody	[48]
	Pluronic F-127	Lysozyme	[49]
	Polyacrylamide-derived copolymer poly (acryloylmorpholine-co-N-isopropylacrylamide) (MoNi)	BSA, human Immunoglobulin G (hIgG), or an anti-COVID mAb	[14]
Air drying	Tryptophan – lysine – tyrosine (WYK)	Enhanced green fluorescent protein (EGFP) or lysozyme	[50]
Spray freeze drying	Trehalose with leucine, phenylalanine, or glycine	hIgG	[54]

4. Conclusion

Nanoparticulates are showing promise as excipients that can address long-standing challenges in formulating proteins for dry-state storage. Advances in the use of nanoparticulate excipients will benefit from more robust comparison against conventional excipient formulations that have shown effectiveness, but also limitations, over the last 15  years of dry-state protein therapeutic development. It is envisioned that nanoparticulates may eventually advance toward universal excipients capable of stabilizing a variety of protein therapeutics without the need for cumbersome, iterative optimization to accelerate product development and reduce product cost.

Funding Statement

This manuscript was funded by National Institutes of Health award R35 GM133697. The funders had no role in the decision to publish nor the preparation of the manuscript.

Article highlights

• Nanoparticulates are gaining interest as additives (i.e., “excipients”) that can stabilize fragile protein therapeutics in a dried state, thereby reducing dependence on cold-chain logistics.

• Existing excipient formulations, which typically comprise mixtures of small molecules and surfactants, are often challenged by the need for iterative optimization processes for each new protein drug, as well as concerns regarding their safety and toxicity.

• Recent reports suggest that nanoparticulates may lead to more universal excipients that stabilize different proteins without the need for extensive formulation optimization, while also providing robust biocompatibility, all while providing comparable or better dry-state storage.

• Key next steps will include testing nanoparticulate excipients across existing and emerging industrial drying modalities, robustly evaluating their effectiveness relative to industry-standard formulations at ambient and elevated conditions to eliminate cold-chain requirements, and ensuring that their synthesis, extraction, and purification are scalable from the laboratory to industrial scale without compromising performance.

Author contributions statement

Junha Park – writing – original draft.

Gregory A. Hudalla - supervision, writing - review and editing

Marcio F. R. Resende Jr - writing - review and editing

Disclosure statement

The University of Florida has filed patents related to the use of phytoglycogen as an excipient that name G.A.H., M.F.R.R., and J.P. as inventors. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.