Lysine as a multifunctional excipient for protein-based biopharmaceutical formulations

Abstract

Lysine (Lys) is a nutritionally essential amino acid with a well-established parenteral safety record, yet its potential as a multifunctional excipient for protein-based biopharmaceutical formulation has not been the subject of a dedicated comprehensive review. The physicochemical properties of Lys include bifunctional amine structure, stable cationic charge under formulation relevant pH conditions, and low molecular weight suitable for free-volume anti-plasticization. These characteristics are discussed in accordance with protein formulation requirements. Thermal characterization reveals that the glass transition temperatures of frozen and dried Lys-based solids are markedly responsive to counter-ion selection, enabling rational process optimization. Stability data from liquid and freeze-dried protein systems demonstrate concentration-dependent aggregation suppression, viscosity reduction in concentrated monoclonal antibody formulations, and lyoprotective efficacy across multiple therapeutic protein classes. Four FDA-approved protein-based biologics currently incorporate Lys in their formulations. Protective mechanisms span ice nucleation inhibition, water replacement, vitrification, beta-relaxation suppression, and direct ion–dipole interactions with protein surfaces. Notably, Lys functions as an essential nutrient in upstream cell culture and as a process stabilizer throughout downstream purification, suggesting its potential applicability across multiple stages of the biopharmaceutical manufacturing lifecycle. Practical limitations including Maillard reactivity and thermal constraints are addressed. Future priorities include systematic counter-ion characterization, mechanistic studies using advanced spectroscopic methods, and integration of Lys derivatives into computationally guided formulation design.

Introduction

Therapeutic protein formulations have become a cornerstone of modern medicine, encompassing monoclonal antibodies (mAbs), enzymes, cytokines, and recombinant hormones that collectively constitute one of the fastest-growing segments of the global pharmaceutical market (Manning et al., 2010, Rahban et al., 2023). Despite their clinical promise, proteins are inherently unstable molecules. They are prone to a wide range of chemical and physical degradation pathways. Common routes include hydrolysis, deamidation, oxidation, disulfide bond rearrangement, structural unfolding, and molecular aggregation (Cleland et al., 1993, Wang, 1999). All of these processes may impair therapeutic efficacy and compromise patient safety (Cleland et al., 1993, Wang, 1999). Of these, aggregation is particularly consequential. Even trace quantities of aggregated protein can trigger unwanted immunogenic responses in vivo, leading to anti-drug antibody formation, loss of clinical response, and potential adverse events (Ratanji et al., 2014, Wang et al., 2012).

To mitigate protein degradation and aggregation and avoid such clinical risks, freeze-drying is widely adopted as a strategy to convert labile protein solutions into solid forms with improved long-term stability (Wang, 2000). However, the process is far from benign. Proteins must survive multiple stress vectors. Freezing concentrates solutes in the unfrozen matrix and may induce cold denaturation, pH shifts, and phase separation. During primary drying, bulk ice is removed by sublimation, resulting in progressive dehydration and increased intermolecular interactions within the freeze-concentrated matrix. Secondary drying subsequently removes nonfrozen and more tightly bound water associated with the protein and excipient matrix, which can disrupt the native hydration shell and reduce molecular mobility buffering. Excessively low residual moisture may further accelerate solid-state chemical degradation pathways, including deamidation, oxidation, and aggregation (Carpenter et al., 1999, Hsu et al., 1992). Moreover, the resulting freeze-dried product must remain stable across diverse storage and distribution conditions, including environments where rigorous cold-chain maintenance cannot be guaranteed (Mensink et al., 2017). To address these challenges, stabilizing excipients are routinely incorporated into protein formulations. Disaccharides such as sucrose and trehalose, alone or combined with amino acid, represent the benchmark stabilizers. These compounds protect proteins via two established mechanisms: water replacement, in which stabilizers form hydrogen bonds with protein surfaces to replace lost hydration interactions, and vitrification, in which proteins are immobilized within a rigid amorphous glass matrix to restrict molecular mobility and slow degradation (Carpenter and Crowe, 1989, Lv et al., 2024, Pikal, 2004). However, these classical stabilizers have limitations. They offer a fixed set of physicochemical properties, limited ability to modulate solution viscosity in high-concentration liquid formulations, and, in the case of reducing sugars, a risk of Maillard-type chemical degradation (Ohtake et al., 2011).

Free amino acids have emerged as a versatile and chemically diverse class of excipients capable of complementing or, in some cases, replacing conventional sugar-based stabilizers (Forney-Stevens et al., 2016, Mattern et al., 1999). Among amino acids, the basic subclass consisting of arginine (Arg), histidine (His), and lysine (Lys) has attracted particular attention. Their positively charged side chains at physiological or weakly acidic pH confer distinctive interactions with protein surfaces and co-solutes that differ mechanistically from the preferential exclusion characteristic of sugars and polyols (Carpenter and Crowe, 1988). Arg, in particular, has been the most extensively studied and is now well-established in protein formulation science (Stärtzel, 2018). His has found widespread application primarily as a buffer component and conformational stabilizer (Lv et al., 2024, Zbacnik et al., 2017). Lys, however, despite sharing the fundamental properties of this basic amino acid class, has received comparatively limited review.

The cryoprotective potential of Lys as a pharmaceutical excipient was identified as early as 1990. Seguro and coworkers reported that Lys-hydrochloride (Lys-HCl) protected lactate dehydrogenase from freeze-induced inactivation, an effect attributable to its zwitterionic nature and modulation of supercooling behavior and ice nucleation capacity (Seguro et al., 1990). Subsequent work confirmed that Lys, like Arg and His, remains at least partially amorphous following freeze-drying, a prerequisite for effective protein stabilization in the solid-state (Izutsu et al., 2009). In liquid formulations, the positive charge carried by the ε-amino group of Lys has been shown to stabilize the three structural domains of immunoglobulin G (IgG) through transient surface interactions that reduce the energetic favorability of unfolding and aggregation (Falconer et al., 2011). More recently, Lys derivatives such as N-acetyl Lys and bis-acetyl Lys have been identified as effective viscosity-reducing agents for concentrated mAb formulations, outperforming Arg in some systems by controlling protein–protein interactions and inhibiting deamidation (Srivastava et al., 2022).

From a pharmaceutical development perspective, the safety credentials of Lys as a parenteral excipient are well established. As a chemically defined, synthetically produced material of unambiguous composition (Paik et al., 2012), Lys carries none of the contamination risks associated with biologics-derived stabilizers such as albumin. Lys is a nutritionally essential amino acid that the human body cannot synthesize and must therefore obtain from dietary sources (Holeček, 2025). It is a standard component of commercial amino acid solutions for parenteral nutrition (e.g., Aminosyn, Synthamin), which have been administered intravenously for decades without intrinsic amino acid–associated safety concerns (Holeček, 2025). Lys-HCl is included in total parenteral nutrition formulations across all major regulatory jurisdictions. This extensive clinical precedent places Lys in a substantially stronger regulatory position than many novel excipients, facilitating its incorporation into injectable protein drug products with minimal additional toxicological burden.

Taken together, these findings suggest that Lys may serve as a versatile excipient, active across both the liquid and solid phases of protein drug products, with functional roles spanning cryoprotection, lyoprotection, aggregation suppression, viscosity reduction, and buffering. Yet, unlike Arg, for which a dedicated body of reviews exists (Stärtzel, 2018), no consolidated account of Lys’s physicochemical properties, thermal behavior, mechanistic actions, and formulation applications has been published. We discuss the chemical and physical characteristics of Lys that are relevant to protein formulation, summarize its thermal behavior in frozen and dried states, review available stability data from Lys-containing protein systems, and examine the mechanisms through which it confers protection in both liquid and freeze-dried formats. The goal is to provide a comprehensive, evidence-based framework for the rational use of Lys in the design of stable protein-based biopharmaceutical formulations.

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Section snippets

Jia-Yi Lv, Shang-Yin Wu, Tian-Yi Zhang, Wei-Jie Fang, Lysine as a multifunctional excipient for protein-based biopharmaceutical formulations, International Journal of Pharmaceutics, 2026, 127192, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2026.127192.

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