Design of Co-lyophilised Ternary Insulin-Sucrose-Polymer Systems with Enhanced Amorphous Glass stability

Abstract

The glass stability of lyophilized amorphous peptide formulations, intended for incorporation into solid oral dosage forms, require stabilisation against the challenges of manufacturing, storage and handling temperature and humidity. High glass transition temperature (Tg) polymers, polyvinylpyrrolidone (PVP) and polyvinylpyrrolidone-vinyl acetate (PVPVA), were added to insulin-sucrose formulations to enhance glass stability when exposed to temperature and humidity. Tg and onset glass transition humidity (RHg) parameters were experimentally determined as indicators of formulation glass stability with respect to temperature and humidity, respectively. A mixture design of experiment approach was employed to determine the influence of insulin, sucrose and polymer composition on formulation Tg and RHg. Statistical regression models were established to evaluate the relationship between formulation composition and the corresponding glass transition parameters, Tg and RHg. Phase separation noted for PVPVA-containing formulations, undermined regression model goodness of fit. Insulin content was shown to have a negative effect on both formulation Tg and RHg. Formulation Tg appeared to be influenced by insulin’s dynamical temperature rather than a previously reported insulin Tg value. Insulin-sucrose and insulin-polymer interactive effects resulted in increased Tg and RHg values, indicating enhanced formulation glass stability. Formulation optimization for maximized Tg and RHg identified a formulation composed of 26% w/w insulin, 40% w/w sucrose, and 34% w/w PVP, with a predicted Tg of 82°C and RHg of 60% RH. The enhanced glass stability of the ternary insulin-sucrose-polymer formulations offers potential advantages for the manufacture, storage and handling of peptide containing oral dosage forms.

Introduction

Therapeutic peptides are an important class of drugs for the treatment of various diseases (Afonso et al., 2025, Wang et al., 2022). Peptides are predominantly administered parenterally, as subcutaneous or intravenous injections. This approach is favoured as it facilitates peptide systemic delivery. Despite its efficacy, the parenteral route of administration presents drawbacks, including the costs associated with aseptic sterile production. These costs, coupled with patient discomfort and the risks of infection posed by parenteral administration (Patel et al., 2014, Antosova et al., 2009), have motivated the development of non-parenteral dosage forms for systemic peptide delivery. Among these non-parenteral dosage forms, oral dosage forms have received considerable interest due to patients’ preferences for oral administration (Limenh et al., 2024).

To date, research into oral peptide delivery has predominantly focused on overcoming barriers to bioavailability, such as peptide metabolism within the gastrointestinal tract (Haddadzadegan et al., 2022, Pawar et al., 2014) and poor permeation across the intestinal epithelial barrier (Lipinski et al., 2001). Advances in formulation strategies, such as the use of protective excipients, enzyme inhibitors, permeation enhancers, nanoparticles, microcapsules, and enteric-coated dosage forms, have addressed these challenges (Bashir et al., 2023, Zhu et al., 2021). However, peptide stability can also be undermined during drug product manufacture, storage, and during patient handling prior to administration. These potential challenges during drug product development warrant investigation as they also pose a barrier to the development, commercial manufacture, and clinical translation of solid oral peptide dosage forms.

Peptides are incorporated into solid oral dosage forms in a solid state. In the solid state, reduced molecular mobility can enhance peptide stability (Chang and Pikal, 2009). Lyophilization, also referred to as freeze drying, is a commonly employed process to convert protein and peptide solutions into solid formats. Non-reducing disaccharides, such as sucrose and trehalose, are commonly added to lyophilised peptide formulations to mitigate the destabilisation stresses experienced during lyophilisation (Gervasi et al., 2018).

These disaccharides preserve peptide conformational stability during freezing (cryoprotection) and drying (lyoprotection) (Karunnanithy et al., 2024, Thakral et al., 2021). Two key mechanisms are proposed which contribute to disaccharide stabilisation of peptides during lyophilisation and in solid-state (Mensink et al., 2017).

One mechanism is the classic vitrification theory, based on the formation of an amorphous glass disaccharide matrix which immobilizes peptide molecules, retarding physical and chemical degradation (Chang et al., 2005). Peptide stabilisation via the vitrification theory is highly dependent on the relationship between an amorphous solid’s glass transition temperature (Tg) and the temperature exposure during processing or storage. Tg is the temperature at which the amorphous solid changes from a high viscosity, rigid, glassy state to a lower viscosity, softer, rubbery state with increased molecular mobility. Increased matrix molecular mobility at temperatures above the system’s Tg can undermine peptide stability in the solid matrix due to increased peptide molecular mobility, and potential for crystallisation of the thermodynamically unstable amorphous disaccharide state. Therefore, maintaining a disaccharide glass matrix formed via lyophilisation is considered critical for stabilisation of peptide and proteins (Mensink et al., 2017). In this study the maintenance of an amorphous glassy state is referred to as ‘glass stability’.

The resulting solid-state formed by lyophilisation (amorphous or crystalline) is determined by the interplay of formulation chemistry and process parameters. Amorphous solid phase arises when molecular mobility is restricted during the solution freezing and drying stages, producing a disordered amorphous matrix. Crystalline solid phases arise when molecules have sufficient mobility during processing to organize into ordered lattices, often influenced by excipients with strong crystallization tendencies such as mannitol or certain salts. Therefore, the formulation constituents, the thermal and pressure profile applied during lyophilization, and the level of residual moisture all contribute to the final solid-state phases obtained (Thakral et al., 2021).

During storage environmental stressors such as elevated temperature and humidity can accelerate peptide chemical degradation pathways, compromising peptide structural integrity in the solid state (Fagan et al., 2025). To mitigate the destabilising potential of environmental humidity, commercial peptide oral tablets, such as Rybelsus® (oral semaglutide), are supplied in aluminium–aluminium blister packaging with the patients advised to retain tablets in their original packaging until use (European Medicines Agency 2026). Environment humidity can also undermine the amorphous glass stability of lyophilised disaccharide systems. These amorphous disaccharide systems are hygroscopic due to their polar chemistry combined with their high surface area architecture, a feature of lyophilised products (Duralliu et al., 2018 Jun 1). Absorbed water acts as a plasticizer increasing the free volume within the amorphous matrix, thereby lowering the systems glass transition temperature (Tg) (Santitewagun and Zeitler, 2025). This plasticizing effect “loosens” the system, enhancing molecular mobility and accelerating degradation reactions (Zografi et al., 2025). Kilburn et al. (Kilburn et al., 2005) demonstrated that even small amounts of sorbed water disrupt hydrogen‑bonded carbohydrate networks, producing a marked expansion of nanoscopic free volume elements and increasing matrix mobility well below the macroscopic Tg. More recent work on polysaccharide-disaccharide mixtures has reinforced these mechanistic insights. Li et al. (Li et al., 2022) reported that water sorption behaviour, monolayer hydration capacity, and Tg depression in dextran-sugar blends depend strongly on molecular compatibility and the extent to which water perturbs packing density.

The plasticising effects of absorbed moisture are minimised for lyophilised parenteral peptide products due to their packaging operation in sealed primary packaging conducted in the extremely low humidity environment of the lyophiliser chamber (Lyophilization of Parenteral 2014). Lyophilised formulations incorporated into solid dosage formats, are exposed to environmental humidity during manufacture and patient handling which can undermine their amorphous glass stability (Ng et al., 2022). Primary packaging materials, such as aluminium–aluminium blister packaging, can protect against humidity stress during long term storage. However, environmental humidity exposure during manufacturing and packaging operations, in-process storage, and patient handling prior to administration, is more challenging to eliminate or control. Therefore, the motivation for this study was to design optimised lyophilised peptide-disaccharide formulations that would stabilise their amorphous glass solid-state during tablet manufacture (typically performed at ambient temperature and 30–65% RH) (Haycocks et al., 2021), and patient handling between removal from packaging and administration.

To mitigate these environmental challenges to amorphous glass stability, ternary lyophilised systems were evaluated consisting of insulin, as a model peptide, sucrose, as a cryo- and lyo-protectant, and a high Tg polymer, polyvinylpyrrolidone (PVP) or polyvinylpyrrolidone-vinyl acetate (PVPVA). The inclusion of these polymers has been shown to increase the Tg of lyophilised sugar matrices, making them more resistant to collapse or phase separation when exposed to heat or humidity (Shamblin et al., 1998). A previous study by our group demonstrated that incorporation of these high Tg polymers in lyophilized formulations effectively stabilized the glassy state of binary lyophilised polymer-disaccharide matrices by reducing the systems’ hygroscopicity, enhancing Tg, and through disaccharide-polymer hydrogen-bonding interactions (Giannachi et al., 2024). The hypothesis evaluated in this study was that this stabilisation effect would also be observed in ternary systems containing a peptide component. To design ternary formulations with optimised glass stability, a mixture design of experiments (DOE) was employed. The effects of varying proportions of insulin, sucrose, and polymer (PVP or PVPVA) on key glass stability parameters was determined. Specifically, the DOE was designed to maximize the Tg and the onset glass transition humidity (RHg). The RHg represents the transition point humidity where absorbed water acts as a plasticizer, causing a shift in sorption characteristics due to increased bulk absorption as material mobility increases above its glass transition (Duralliu et al., 2018 Jun 1).

Download the full article as PDF here Design of Co-lyophilised Ternary Insulin-Sucrose-Polymer Systems with Enhanced Amorphous Glass stability

or continue reading here

Materials

Sucrose and polymers (PVP K30 and PVPVA 64) were donated by Pfanstiehl Inc. (USA), and BASF Inc. (Germany), respectively. Recombinant human insulin with a molecular weight (Mw) of 5.8 KDa was supplied by SAFC (Switzerland). Lyophilization 10 mL ISO Clear Type I Tubular glass vials, and 20 mm grey silicone stoppers were supplied by Adelphi Healthcare Packaging (UK). All other chemicals were reagent grade and solvents HPLC grade.

Claudia Giannachi, Evin Allen, Sonja Vucen, Abina Crean, Design of Co-lyophilised Ternary Insulin-Sucrose-Polymer Systems with Enhanced Amorphous Glass stability, European Journal of Pharmaceutical Sciences, 2026, 107450, ISSN 0928-0987, https://doi.org/10.1016/j.ejps.2026.107450.

Read more on World Diabetes Day here: