The Effect of Pharmaceutical Excipients on Protein Chemical Degradation Through Deamidation and Isomerization

Abstract

Introduction

Therapeutic proteins are crucial in the treatment of a wide range of diseases. However, the proteins are sensitive to chemical degradation reactions, particularly deamidation and isomerization, which can compromise efficacy and safety. Formulation excipients, such as sugars and non-ionic surfactants, are commonly used to enhance stability, yet their effects on chemical degradation remain insufficiently understood.

Methods

This study investigates how fructose, sucrose, melezitose, and the non-ionic surfactants polysorbate 80 and DDM (n-Dodecyl-β-D-maltoside) affect the structure and chemical stability of the affibody GA-Z, a protein prone to deamidation and isomerization. Chemical degradation and conformational changes were characterized using peptide fingerprinting, Liquid Chromatography-Mass Spectrometry, Titration fluorescence spectroscopy, two-dimensional Nuclear Magnetic Resonance spectroscopy, and Differential Scanning Calorimetry.

Results

All three sugars lowered chemical degradation by stabilizing the folded state of the z-domain and inducing minor structural changes in the albumin-binding domain, thereby lowering the propensity for deamidation and isomerization. Polysorbate 80 showed minimal impact on both degradation and protein structure. In contrast, DDM increased deamidation and isomerization due to surfactant–protein interactions, resulting in structural changes.

Conclusion

These results demonstrate how excipient-induced structural changes affect chemical degradation of proteins in liquid formulations. This study contributes to the understanding and design of more effective formulations for therapeutic proteins, enhancing their stability and safety.

Introduction

Therapeutic proteins are widely used in modern medicine and are increasingly represented among the highest-earning pharmaceuticals. In 2024, five of the ten top-selling drugs worldwide were therapeutic proteins [1]. However, proteins are prone to both physical and chemical degradation in solution, which can reduce drug efficacy and, in some cases, trigger immunogenic responses [2,3,4,5,6,7,8,9,10,11]. To stabilize proteins and reduce denaturation, aggregation, deamidation, isomerization, oxidation, etc., excipients are added to liquid formulations.

Among chemical degradation reactions, deamidation and isomerization are the two most common [2, 3]. Their rates in solution are influenced by physical and chemical properties of the solvent and the protein itself, including, e.g., ionic strength, pH, dielectric constant, and the charge of nearby amino acid residues [12,13,14,15,16,17,18,19,20,21]. Deamidation and isomerization are initiated when a deprotonated peptide backbone amide nitrogen attacks the side chain carbonyl group of asparagine and aspartic acid residues [12]. The primary and higher-order structures can promote these reactions by bringing the reacting groups into close proximity [22]. Additionally, increased peptide backbone mobility increases the likelihood of conformations that shorten the distance between these groups, further enhancing the risk of the reactions [12, 13, 16, 17, 22, 23]. Conversely, secondary and tertiary structures can stabilize proteins by imposing conformational restrictions [18, 24,25,26,27,28]. Furthermore, the peptide backbone amide nitrogen is often involved in hydrogen bonding, in both α-helices and β-turns, which decreases its reactivity [29].

Sugars are often used to enhance the physical stability of therapeutic proteins. Sucrose is the most common sugar excipient and is typically included at concentrations of 25–200 mg/mL in liquid formulations [30, 31]. Sugars stabilize proteins against denaturation and aggregation [32,33,34,35], primarily through preferential exclusion from the protein surface, which drives the protein toward a folded state [36,37,38,39]. They have also been reported to increase enzyme stability and activity [35, 40]. Some studies have been conducted on the effect of sugars on deamidation and isomerization. Sucrose and trehalose lowered the deamidation and isomerization of denatured lysozyme [41] and sucrose and mannitol reduced the deamidation of peptides [33, 42, 43]. Additionally, a study on a protein in an optimized formulation showed that sucrose increased chemical degradation [44]. However, there is a lack of extensive systematic studies analyzing how sugars stabilize proteins from chemical degradation.

Non-ionic surfactants are primarily included in liquid formulations of therapeutic proteins to inhibit aggregation, increase solubility, and reduce surface adsorption. Polysorbate 80 is the most commonly used surfactant, with a critical micelle concentration of approximately 0.013 mM, and typical formulation concentrations of 0.01–2 mg/mL [30, 31]. Another non-ionic surfactant, n-Dodecyl-β-D-maltoside (DDM), is frequently used to solubilize membrane proteins [45] but is not used in therapeutic protein formulations [30, 31]. DDM has a critical micelle concentration of 0.17 mM in water [45]. Despite their widespread use, the effect of surfactants on chemical stability, particularly deamidation and isomerization, remains poorly understood.

This study aims to investigate how different excipients affect the structure and chemical stability of the affibody GA-Z, which is known to degrade through deamidation and isomerization [46]. Three sugars of varying size, fructose, sucrose, and melezitose, and the two non-ionic surfactants, polysorbate 80 and DDM, were added to GA-Z in solution. The chemical stability and the effects of the excipients on degradation were evaluated using peptide fingerprinting and liquid chromatography-mass spectrometry (LC–MS). Structural changes were assessed by titration fluorescence spectroscopy and two-dimensional NMR spectroscopy, while thermal stability was characterized using differential scanning calorimetry (DSC).

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Materials

The affibody GA-Z was provided by Swedish Orphan Biovitrum AB (Stockholm, Sweden) at a stock concentration of 90 mg/mL in a 25 mM sodium phosphate buffer with 125 mM NaCl at pH 7.0 and was stored at − 80°C before use. GA-Z is a small two-domain protein with a molecular weight of 11 900 Da and an isoelectric point (pI) of approximately 4.5. Fructose (≥ 99%), sucrose (≥ 99.5%), melezitose (≥ 99.0%), tris-hydrochloride (Molecular Biology grade, ≥ 99%), urea (BioUltra, ≥ 99%), and trypsin (Porcine, MS grade) were purchased from Sigma-Aldrich (MO, USA). Sodium phosphate dibasic dihydrate (Na2HPO4•2H2O, 99–102%) and sodium phosphate dibasic monohydrate (NaH2PO4•H2O, 98.5–100.5%) were obtained from Merck (NJ, USA). Formic acid (LC–MS graded, 99%) and sodium chloride (100%) were purchased from VWR (PA, USA). Polysorbate 80 (Super Refined) was provided by Croda (UK), and n-Dodecyl-β-D-maltoside (analytical grade) was purchased from Anatrace Products (OH, USA). Acetonitrile (Optima LC–MS) and formic acid (Optima LC–MS) were purchased from Fisher Chemical (NH, USA). Milli-Q grade water (Merck Millipore) was used for all buffers, mobile phases, and sample preparations.

Ramm, I., Diehl, C., Västberg, A. et al. The Effect of Pharmaceutical Excipients on Protein Chemical Degradation Through Deamidation and Isomerization. Pharm Res (2026). https://doi.org/10.1007/s11095-026-04040-4