Feasibility and stability evaluation of parabens as an alternative preservative for liquid monoclonal antibody formulations

Abstract

Preservatives are critical for preventing microbial contamination in multi-dose biologics, yet their compatibility with proteins poses a major challenge. Traditional preservatives such as benzyl alcohol are limited by instability, adsorption, and negative effects on protein stability. This study demonstrates that parabens, including methylparaben and propylparaben, are feasible alternatives in trastuzumab liquid formulations. Differential scanning fluorimetry revealed minimal impact on monoclonal antibody conformational and colloidal stability compared with conventional preservatives. Trastuzumab with parabens under stress and photostability testing was systematically characterized using micro-flow imaging, dynamic light scattering, size exclusion chromatography, ion exchange chromatography, capillary electrophoresis, microfluidic modulation spectroscopy, and ultra-high resolution mass spectrometry. Parabens did not significantly affect main peak purity, charge heterogeneity, fragmentation, or secondary structure, and only slightly increased sub-visible particles, while also being suitable for a variety of monoclonal antibodies and fusion proteins. These findings establish parabens as reliable, practical preservatives, offering a new approach to developing safer and more stable multi-dose biologics.

Introduction

Biologics, particularly monoclonal antibodies (mAbs), are a rapidly growing and clinically important class of biologic therapeutics. Multi-dose formulations improve patient convenience, reduce per-dose costs, and simplify medication management, and they now represent a substantial fraction of commercially available protein injectables (Meyer et al., 2007). These formulations require the inclusion of preservatives to prevent microbial contamination during repeated use and to ensure consistent product quality and safety.

Liquid insulin products provide a typical example, with Humalog® (insulin lispro) containing 0.315% m-cresol and Novolog® (insulin aspart) containing 0.172% m-cresol and 0.15% phenol. For mAbs, long-term stability is often achieved through freeze-drying and reconstitution in preservative-containing water for injection (WFI) (Roy et al., 2005). For instance, the multi-dose formulation of Herceptin® (trastuzumab, Tra) is reconstituted with 1.1% benzyl alcohol, while Enbrel® (etanercept) uses 0.9% benzyl alcohol. However, preservatives can induce mAb aggregation in liquid formualtion (Maharjan et al., 2025). Furthermore, many preservatives such as benzyl alcohol possess characteristics such as volatility, hydrophobicity, and low molecular weight, which can lead to a potential decrease in their concentration within multi-dose formulations during long-term storage. Royce et al. reported that storing 1% benzyl alcohol in a 2 mL cartridge resulted in a concentration of only 0.9% after one month (Royce and Sykes, 1957). A decrease in preservative concentration poses a risk of microbial contamination during the long-term use of multi-dose formulations. These limitations highlight the need to identify preservatives that are both effective and protein-compatible, providing a foundation for developing safer and more stable multi-dose biologic formulations.

This study focuses on paraben preservatives. Parabens are non-volatile, allowing them to maintain stable concentrations in multi-dose formulations. Furthermore, they exhibit excellent antimicrobial activity over a broad pH range (4–8). Their aqueous solutions can remain stable for up to four years at room temperature under pH 3–6 (Aalto et al., 1953) and withstand conventional heat sterilization processes (Sunderland and Watts, 1984). In contrast, benzyl alcohol achieves optimal bacteriostatic effect primarily under pH < 5 (Karabit et al., 1986). Commercial protein formulations generally fall within a pH range of 4.6–8.2 (Gervasi et al., 2018). Achieving effective antimicrobial activity within this range often necessitates higher concentrations of benzyl alcohol, which may exacerbate its impact on protein stability. Additionally, benzyl alcohol can slowly oxidize in air to benzaldehyde, which subsequently converts to benzoic acid, posing a potential risk of altering the system pH and triggering stability issues.

Although research on the impact of parabens on protein and mAb stability is not comprehensive, existing research generally suggests that parabens have minimal effects on the vaccine stability. Sawant et al., evaluating the effects of different preservatives on the storage stability of a recombinant P[4] protein antigen in a non-replicating rotavirus vaccine with Alhydrogel® adjuvant, found that parabens and chlorobutanol induced the least instability (Sawant et al., 2022). Miao et al., while screening preservatives for a human papillomavirus (HPV) vaccine, indicated that 0.12% MP was most suitable as a preservative for the multi-dose HPV-2 vaccine, ensuring at least a three-year shelf life (Miao et al., 2022). To date, no dedicated systematic studies have been conducted on the application of parabens in mAb formulations.

The impact of preservatives on mAb stability is often related to their hydrophobic properties. More hydrophobic preservatives tend to bind to surface regions of the protein, potentially causing local conformational perturbations and thereby inducing aggregation (Hutchings et al., 2013). For mAbs, the CH2 domain is often a target site for preservatives. A reduction in the conformational stability of this region translates to a decrease in the overall stability (Karunaratne et al., 2023). Among different preservatives, their tendency to induce mAb aggregation generally follows: m-cresol > phenol > chlorobutanol > benzyl alcohol > propylparaben (PP) > methylparaben (MP); among them, m-cresol, phenol, and benzyl alcohol can even cause mAb precipitation (Gupta and Kaisheva, 2003). Preservatives containing benzene ring structures might promote aggregate formation by perturbing local protein conformation through non-covalent interactions like π-π stacking or cation-π interactions (Zhang et al., 2004). However, in specific protein systems, parabens might exert a stabilizing effect. Li et al., using molecular dynamics simulations, found that after binding of parabens to bovine serum albumin, the root mean square fluctuation and solvent accessible surface area decreased, suggesting reduced structural flexibility accompanied by local folding, thereby enhancing overall structural stability (Li et al., 2025).

Notably, some studies have reported potential endocrine-disrupting toxicity associated with paraben preservatives, which has limited their widespread application (Nowak et al., 2018). However, research by Barr et al. showed no statistical correlation between breast cancer patients and the MP content in their breast tissue (Stroppel et al., 2023). The European Medicines Agency’s Committee for Medicinal Products for Human Use also stated that 0.2% MP poses no risk to humans, including children (Stroppel et al., 2023). Furthermore, multiple studies confirm that paraben lacks teratogenicity, mutagenicity, and carcinogenicity, and definitive evidence for its toxicity in humans has not been established (Petric et al., 2021). The incidence of allergic reactions induced by paraben preservatives is extremely low, and they remain among the preservatives with the lowest sensitizing potential. Public misconceptions about their safety have often led to their replacement by preservatives with higher allergenic potential (Fransway et al., 2019, Sasseville, 2004).

Table 1 presents the toxicological and pharmacokinetic data of parabens and conventional preservatives, derived from toxicity studies in rats and mice via oral administration. Subchronic and reproductive/developmental toxicity results are expressed as the no observed adverse effect level (NOAEL), while acute toxicity results are presented as the median lethal dose (LD50). A comprehensive comparison indicates that the intrinsic toxicity of parabens is lower than that of commonly used preservatives such as benzyl alcohol, phenol, and m-cresol. Furthermore, in terms of the concentrations required to achieve effective antimicrobial activity in formulations, MP (0.18%) and PP (0.02%) are significantly lower than benzyl alcohol (0.9%–1.1%), phenol (0.2%–0.5%), and m-cresol (0.25%–0.5%). Therefore, under the premise of ensuring equivalent preservative efficacy, parabens result in lower human exposure and offer a higher safety margin. However, it is important to note that PP poses a higher risk than MP, as PP may accumulate in the body and cause endocrine disruption.

This study references the Handbook of Pharmaceutical Excipients and selects commonly used concentrations of parabens, specifically 0.18% MP and 0.02% PP, with Tra as the model monoclonal antibody to evaluate the stability of parabens in biologics. First, differential scanning fluorimetry (DSF) was employed to assess the effects of five preservatives (benzyl alcohol, phenol, m-cresol, chlorobutanol, and parabens) on the thermal stability of Tra, highlighting the stability advantages of parabens among preservatives. The impact of parabens on the aggregation of Tra under stress conditions systematically evaluated using micro-flow imaging (MFI), dynamic light scattering (DLS), and size exclusion chromatography (SEC). Charge heterogeneity was analyzed by ion exchange chromatography (IEC). Fragmentation variants were detected by capillary electrophoresis (CE). Secondary structure changes were monitored by microfluidic modulation spectroscopy (MMS). Substitution reaction was characterized using ultra-high-resolution mass spectrometry (HRMS). Different types of proteins were then selected to verify the general applicability of parabens. This study aims to systematically assess the application potential of parabens in biologics, providing new perspectives and a scientific basis for the rational selection of preservatives in biologics.

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Materials

Tra, Tra-DM1, FP-1, mAb-2, and mAb-3 by Zhejiang Bioray Biopharmaceutical Co. Ltd. (Zhejiang, China). Trehalose was purchased from Pfanstiehl (Waukegan, Illinois, USA). Polysorbate (PS) 20 (Tween 80) and PS80 were purchased from JT Baker (Radnor Township, IL, USA). L-Histidine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrochloric acid, Karl Fischer reagent, anhydrous methanol, sodium chloride, and sodium dihydrogen phosphate dihydrate were purchased from Sinopharm Chemical Reagent Co., Ltd.

Zi-Ting Xu, Yan-Rong Gao, Jia-Yi Lv, Huan-Fang Xie, Yusheng Pan, Wei-Jie Fang, Feasibility and stability evaluation of parabens as an alternative preservative for liquid monoclonal antibody formulations, International Journal of Pharmaceutics, 2026, 126907, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2026.126907.

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