The Detrimental Effects of Crystalline Excipients: How They Jeopardize the Long-Term Stability of Freeze-Dried Polypeptide Formulations

Abstract

Introduction

In recent decades, advances in synthesis and delivery technologies for polypeptide-based drugs have significantly promoted their development as promising therapeutic agents. Notable examples include insulin, semaglutide, and tirzepatide [1,2,3,4]. Semaglutide, in particular—approved for glucose lowering and weight management—ranked among the top-ten best-selling drugs worldwide by revenue in 2024 [5,6,7]. However, due to their intermediate molecular size, which falls between that of conventional small-molecule drugs (SMDs) and monoclonal antibodies (mAbs), therapeutic polypeptides often exhibit inferior stability compared to SMDs. Conventional perspectives frequently assumed that polypeptides possess stability comparable to that of SMD, thereby overlooking instability mechanisms shared with proteins and mAbs, such as susceptibility to aggregation and degradation. Like mAbs, polypeptides drugs are vulnerable to various external stressors during manufacturing, transportation, and storage, which can induce both chemical and physical degradation [8,9,10,11].

Chemical instability in polypeptides typically involves covalent modification, such as oxidation, deamidation or polypeptide backbone hydrolysis [12]. In contrast, physical instability, a hallmark of biopharmaceuticals, primarily arises from aggregation, unfolding, adsorption, precipitation, or fragmentation [12,13]. Owing to low oral bioavailability, most polypeptide and protein therapeutics require repeated administration via injection, typically formulated as solutions or suspensions. However, polypeptides in solution exhibit limited shelf life. Moreover, physical aggregation can lead to elevated levels of subvisible particles, raising potential safety concerns for injection [14]. The stability profile of each therapeutic polypeptide is uniquely determined by its amino acid sequence. For instance, residue such as cysteine, methionine, or tryptophan are susceptible to oxidation, a process accelerated by freeze–thaw cycles and elevated pH conditions, ultimately compromising therapeutic efficacy [15]. Additionally, aspartic acid residues are prone to hydrolysis, while aromatic amino acids such as phenylalanine and tryptophan undergo photochemical degradation [16]. Even minor alterations in the unique amino acid sequence and chemical structure of a polypeptide can potentially lead to a complete loss of its therapeutic function.

Freeze-drying (FD) has traditionally been employed to improve the long-term storage stability of unstable therapeutic high-molecular-weight biopharmaceuticals, particularly protein drugs, by mitigating their inherent instability [17,18,19]. FD process commonly consists of freezing, primary drying, and secondary drying, with an optional annealing step if necessary. Throughout the FD process, the removal of water molecules disrupts the original hydrogen bonds between proteins and water, which may lead to protein inactivation unless appropriate formulation strategies are adopted. Therefore, incorporating excipients (such as disaccharides, amino acids, buffers, and surfactants) into protein formulations is considered a critical strategy in FD design to mitigate interfacial stresses and maintain protein stability during freezing, drying, and long-term storage [20,21,22].

Traditionally, nonreducing disaccharides such as trehalose and sucrose have been widely used as an essential stabilizer in freeze-dried protein formulations. Studies have concluded that these sugars can effectively inhibit the protein unfolding and maintain the native structure of proteins during the freezing and drying. More importantly, they form a stable glassy matrix in the solid state, which is critical for extending the shelf life of lyophilized products. On the other hand, crystalline excipients such as mannitol are commonly incorporated as bulking agents in freeze-dried formulations of both SMD and mAbs. Owing to its favorable physicochemical characteristics, mannitol is utilized in various solid dosage forms. Key applications include: (i) serving as a diluent in tablet formulations, owing to its non-hygroscopic property and brittle fracture behavior under compression, and (ii) incorporation into chewable tablets due to its cooling effect, sweet taste, and pleasant mouthfeel [23,24,25,26,27]. Yet during the freezing step of the FD process, solute concentration can induce the crystallization of mannitol, potentially disrupting the native structure of proteins at the ice–water interface. Moreover, the crystalline forms generated—particularly mannitol hemihydrate (MHH)—have been reported to adversely affect the long-term stability of mAbs. Multiple studies indicate that the presence of MHH in the lyophilized cake can compromise the stability of thermolabile therapeutic components [28,29,30,31].

The stabilizing and destabilizing capacities of various excipients in different high-molecule-weight protein formulations and the mechanism for such stabilization were reported by extensive literature [32,33,34,35,36]. In the field of freeze-dried mAb formulations, crystalline excipients such as mannitol are conventionally utilized as bulking agents. However, their crystallization tendency may disrupt the native structure of mAbs at the ice-water interface and fail to provide a protective amorphous matrix during long-term storage. In contrast, amorphous stabilizers like sucrose are preferentially employed in freeze-dried mAb formulations, where they form a stable amorphous matrix that effectively mitigates protein–protein interactions, thereby ensuring enhanced long-term stability. Nevertheless, there is limited literature describing the excipients effects on the stability of polypeptide drugs in the freeze-dried formulations [8,37,38,39,40]. In one study, Santana et al. reported that the degradation rate of in recombinant human epidermal growth factor assessed by reversed-phase-high performance liquid chromatography could decrease 100 times at 37 °C and 70 times at 50 °C in freeze-dried with respect to aqueous formulation [41]. And an increase in freeze-dried recombinant human epidermal growth factor stability was observed with the increase in protein concentration from 25 to 250 μg/vial, which demonstrating the polypeptide stability may correlated with the concentration [42]. In addition, through a screening of combinations comprising amorphous excipients and surfactants, Fang et al. concluded that polypeptides may exhibit distinct behaviors compared to proteins due to their smaller molecular size and less ordered secondary structure [8]. Although a limited number of studies have investigated freeze-dried polypeptides, a systematic comparison of the stabilization and destabilization mechanisms induced by crystalline excipients in polypeptide versus monoclonal antibody formulations remains lacking. To address this research gap, this study aims to systematically investigate the long-term stability of freeze-dried polypeptides.

Table 1. The overview of FD formulations.

In this study, insulin and glucagon were selected as model polypeptide for FD and long-term accelerated stability experiments. These hormones represent foundational therapeutics in their respective clinical areas and rank among the most commercially successful polypeptide-based drugs to date. Insulin, a 51-amino acid (AA) polypeptide, plays a critical role in maintaining blood glucose homeostasis and serves as an essential therapy for both type I and II diabetes [43,44]. Glucagon, a 29-AA polypeptide secreted by pancreatic α-cells in response to hypoglycemia, elevates blood glucose levels by promoting hepatic gluconeogenesis and glycogenolysis [45,46].

The present work systematically investigates the effects of key excipients (such as crystalline/amorphous stabilizers, surfactants, and arginine) on the stability of freeze-dried formulations using insulin and glucagon as model systems. Our central hypothesis posits that the stabilization mechanisms conferred by excipients differ substantially between low-molecular-weight polypeptides and high-molecular-weight mAbs, owing to inherent differences in molecular size and structural complexity. The polypeptide aggregation was monitored using a complementary set of techniques, including size exclusion-high performance liquid chromatography (SE-HPLC), dynamic light scattering (DLS), and micro-flow imaging (MFI). Concurrently, polypeptide degradation was assessed by reversed-phase-high performance liquid chromatography (RP-HPLC). Through a systematic, cross-scale comparison, this study aims to elucidate these distinct interaction mechanisms. The findings are anticipated to establish a critical theoretical foundation for rational formulation design principles specifically tailored to polypeptide-based therapeutics.

Download the full article as PDF here The Detrimental Effects of Crystalline Excipients

or continue reading here

Materials

Glucagon (purity: 98%) was purchased from Science Polypeptide Biological Technology Co., Ltd. (Shanghai, China). Insulin (purity: 98%) was purchased from Hisun Pharmaceutical Co., Ltd. (Taizhou, China). Mannitol was obtained from Aladdin Industrial Corporation (Shanghai, China). Sodium chloride, and hydrochloric acid were purchased from Sinopharm Chemical Reagent (Shanghai, China). Trehalose was obtained from Sinozyme Biological Technology Co., Ltd. (Nanjing, China). Arginine hydrochloride was obtained from GPC Biological Technology Co., Ltd. (Beijing, China). HES was obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). Sorbitol was obtained from Alpha Hi-tech Pharmaceutical Co., Ltd. (Jiangxi, China). Polysorbate 20 was obtained from JTBaker (Lardner, IL, USA). We purchased 0.22 μm filter membranes from Millipore Co., Ltd. (Nantong, China). Gray butyl stoppers (13 mm) and borosilicate type I scintillation vials (2 mL) were purchased from West Pharmaceuticals (Singapore) and Schott (Suzhou, China), respectively.

Gao, H.; Ouyang, J.; Hu, Z.-B.; Fang, W.-J. The Detrimental Effects of Crystalline Excipients: How They Jeopardize the Long-Term Stability of Freeze-Dried Polypeptide Formulations. Pharmaceutics 2025, 17, 1543. https://doi.org/10.3390/pharmaceutics17121543

Read also our introduction article on Mannitol here: