DSC reveals the excipient impact on aggregation propensity of pharmaceutical peptides during freezing

Introduction

Aside from classical biopharmaceuticals such as monoclonal antibodies, pharmaceutical peptides have become increasingly important. Although being smaller representatives of biopharmaceuticals, peptides face the same or even increased challenges regarding their short and long-term stability in aqueous solution. With degradation often observed after hours, peptides are classically either stored in the frozen state, or freeze-dried (Joshi et al., 2000; Matilainen et al., 2008). Storage in the frozen state is employed during early stages of formulation and drug product development, if freeze-drying is not applicable, or if the stability of the biopharmaceutics in the refrigerated liquid is not sufficient (Avis & Wagner, 2010). Although degradation of peptides is slowed down in the frozen state, aggregation of the peptides can be amplified due to the crystallization of water during freezing. The peptide is subjected to interfacial stress during crystallization through the formation of ice/solution interfaces (Authelin et al., 2020). To inhibit aggregation during storage in the frozen state, excipients such as disaccharides, e.g., sucrose are added to the peptide solution (Connolly et al., 2015). During freezing, these excipients retain a liquid phase, also called freeze concentrate, to stabilize the peptide, subsequently termed amorphous phase. As long excipients do not crystallize and are kinetically stabilized, this amorphous phase is in meta-stable equilibrium with the solid water, i.e., ice phase (Steven L. Nail et al., 2002). The peptides, the excipient, and the residual unfrozen water are confined to this amorphous phase, with the residual water concentration in the amorphous phase given by the solubility of water (Steven L. Nail et al., 2002). As soon as the temperature is lowered below the glass transition temperature of the amorphous phase (classically termed Tg’), further crystallization of water is kinetically inhibited, and no change of the composition in the amorphous phase occurs. The corresponding concentration of solutes (excipient and peptide combined) at and below Tg’ is called wg’. Tg’ is dependent on the composition of excipients and peptides used. A phase with peptide in the monomeric form has a different Tg’ than a phase with peptides in aggregated form, due to its different mean molar mass (Levine & Slade, 1988).

Three possible stabilization theories have been proposed to describe the behavior of excipients used as stabilizers during freezing and thawing: (1) the vitrification theory, (2) the water replacement theory, or (3) a combination of the two aforementioned mechanisms. For the vitrification theory, kinetic stabilization of the peptide in the amorphous phase is assumed. Within this theory, aggregation tendency of the biopharmaceutical is connected to the molecular mobility governed by global and local relaxation within the lyophilizate. (Chang et al., 2005; Cicerone & Douglas, 2012; Groёl et al., 2021; Wang, Tchessalov, Cicerone, et al., 2009; Yoshioka & Aso, 2007). For the water replacement theory, interactions such as hydrogen bonding between biopharmaceutical and excipients are assumed to be responsible for stabilization in solution. The interactions with the water molecules need to be replaced by interactions with the excipient molecules when the amorphous phase is formed upon water removal/crystallization (Andya et al., 1999; Arsiccio & Pisano, 2018; Cleland et al., 2001; Pisano et al., 2024; Wang, Tchessalov, Cicerone, et al., 2009; Wang, Tchessalov, Warne, & Pikal, 2009).

Few works describing the stabilizing capabilities of excipients on peptides are reported in literature. Fang et al. investigated the influence of excipients on the stability of the pharmaceutical peptide glucagon during freeze-drying and subsequent storage using mass spectrometry and chromatography. They showed a superior stability for formulations containing trehalose in comparison to those including β-cyclodextrins and hydroxyethyl starch. However, the impact of freezing-induced stresses on peptide stability was not investigated alone.(Fang et al., 2012) Wewer Albrechtsen et al. studied the aggregation behavior of glucagon and the glucagon-like peptide 1 in human plasma after short and long-time storage and after freeze-thawing using activity assays. During their studies no significant effect of freeze-thawing on the aggregation of glucagon was observed.(Wewer Albrechtsen et al., 2015) Matilainen et al. studied the influence of various cyclodextrins on the chemical stability of glucagon in liquid and freeze-dried state using mass spectrometry and chromatography. They found stabilizing capabilities of cyclodextrins to be superior to lactose.(Matilainen et al., 2008; Matilainen et al., 2009)

The aim of the work is to present a simple and easy approach to assess the stability of peptides during freezing, by monitoring the change in the glass-transition temperature of the amorphous phase (Tg’) over repeated freeze-thawing cycles using differential scanning calorimetry (DSC). This method allows for simultaneous application of freeze-thaw stressing, detection of aggregation, evaluation of cryo-protective properties of excipients, and determination of the solubility of water, all in one measurement. Furthermore, this method only requires a minimal amount of material (few µ-liters). Experiments were performed using a model peptide in combination with two excipients. Glucagon was used as model peptide, as it is known to aggregate under stresses and mishandling (Onoue et al., 2004; Pedersen, 2010). Sucrose was not considered as excipient, as at low pH, this excipient induces hydrolysis (US Pharmacopeia 24, 1999). Instead, trehalose was chosen as excipient, as it does not undergo acid hydrolysis. Lactose was considered as excipient as recent studies found its capabilities in stabilizing lyophilized glucagon through molecular dynamics and microfluidic modulation spectroscopy (Pisano et al., 2024). The solubility of water of the peptide/excipient formulations was also modeled using the equation of state Perturbed-Chain Statistical Association Fluid Theory (PC-SAFT). This method allows for a low effort and fast differentiation of stabilizing capabilities of excipients and may aid in future preliminary formulation screenings.

3.2.1. Trehalose as excipient

Experimental results on Tg’ for a 1:3 glucagon:trehalose mixture are shown in Figure 2 (left). Thawing cycle one is shown in green. Thawing cycle twelve is shown in red. The cycles in between are gradually colored. This color code is used for all following figures. Thermograms for the starting eight cycles are almost identical. Consequently, the measured Tg’ as well as its position in the thermogram are also nearly identical. After nine cycles, a shift of Tg’ to lower temperatures can be observed. In addition, there is a widening of the glass transition temperature region. Tg’ is reduced by 0.22 K after cycle nine compared to cycle eight. The shift after cycle ten, eleven, and twelve is even more pronounced with a difference in Tg’ to cycle eight of 0.25 K, 0.61 K, and 1.69 K respectively. Tg’ of the individual cycles of all experiments are given in Table 1. Even after twelve cycles, the heat flow curve in the thermogram was still shifting, and no superimposition of the last two cycles was observable.

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