Insight

Bioactive ingredients (also referred to as active substances or drug molecules), particularly proteins, are subjected to a wide range of physicochemical and biological stresses during manufacturing, storage, transportation and administration. These stresses include elevated temperatures, freeze-thaw cycles, mechanical shear forces, non-specific adsorption, interfacial tension, photolysis, various oxidative reactions, formation of covalent and non-covalent interactions between molecules in solution, as well as enzymatic degradation. Such stresses may trigger one or more adverse outcomes: loss of biological activity, degradation, denaturation, aggregation, precipitation and chemical modification of active pharmaceutical ingredients. Among these stress factors, elevated temperature, freeze-thawing, shear force, interfacial adsorption and intermolecular non-covalent interactions can induce conformational changes or aggregation of active components at every stage of production, distribution and application. This article mainly discusses the application of stabilizers and protectants during the manufacturing of protein therapeutics to preserve the native structure of bioactive ingredients.

As the primary therapeutic molecules of biopharmaceuticals, peptides and proteins consist of diverse amino acids and possess multiple hierarchical structures, endowing these biomacromolecules with sophisticated conformational features. The primary structure of biomacromolecules is maintained by covalent bonds, whereas higher-order structures are stabilized via synergistic effects of diverse non-covalent interactions. The global and local domains, internal and surface regions, linkers and structural domains of biomacromolecules demand a specific microenvironment in solution to retain structural integrity throughout manufacturing processes and ultimately exert expected biological functions.

Non-covalent interactions are weak interatomic forces with lower bond energies than covalent bonds (the bond energy of typical covalent bonds in biomolecules generally ranges from 300 to 800 kJ/mol). These interactions do not involve shared electron pairs and are mediated by weak forces between atoms or chemical moieties. The major intramolecular and intermolecular non-covalent interactions for biomacromolecules are described below:

Electrostatic interaction: Attractive or repulsive forces between ionized groups bearing opposite electrical charges. Its effective distance is typically 1–10 nm, with bond energy of 0.2–4.0 kJ/mol at approximately 1 nm. Key influencing factors include the type and ionic strength of inorganic salts, pH, temperature and the dielectric constant of aqueous solvent.

Hydrophobic interaction: Weak forces arising from the tendency of nonpolar groups or molecules to avoid aqueous solvent and aggregate spontaneously. The effective distance is generally 0.4–0.5 nm. Hydrophobic stacking within polypeptide chains normally contributes 4–20 kJ/mol per residue, and temperature constitutes the primary influencing factor.

Hydrogen bond: Non-covalent interaction between hydrogen-containing moieties and highly electronegative atoms (e.g., O, N), characterized by saturation and directionality. It acts over a distance of 0.25–0.35 nm with bond energy ranging from 10 to 40 kJ/mol at around 1 nm. Water and solute molecules extensively participate in hydrogen bond formation.

Van der Waals forces: Intermolecular electrostatic forces encompassing three subtypes. Dispersion force, the most universal form, originates from mutual attraction induced by instantaneous dipoles generated by continuous electron motion. Orientation force occurs between polar molecules via aligned positive and negative poles. Induction force arises when a polar molecule distorts the electron cloud of an adjacent nonpolar molecule to create an attractive dipole. The effective distance is roughly 0.3–0.5 nm. Dispersion force delivers energy of 0.2–2 kJ/mol at ~1 nm, while orientation force and induction force range from 0.5–4 kJ/mol at ~1 nm, and the dielectric constant of solvent serves as a major influencing parameter.

Cation-π interaction: Orbital overlap combined with dispersion forces between cations (e.g., Na⁺, K⁺, guanidinium groups) and aromatic π systems. It works within 0.3–0.6 nm, with bond energy usually between 5 and 20 kJ·mol⁻¹, affected by solute type and concentration.

Biomacromolecules including proteins, DNA and RNA contain abundant potential hydrogen bonding sites, alongside binding sites for ionic bonds, van der Waals forces and hydrophobic interactions. The cumulative effect of these weak intermolecular forces can be remarkably substantial.

Polyols represent a class of alcohols containing two or more hydroxyl groups. Most polyols are water-soluble with strong solubilizing capacity for polar substances; they are low-toxicity, low-volatility viscous liquids or crystalline solids.

Mechanisms of stabilization in aqueous solution at ambient temperature: Stabilization is primarily driven by electrostatic interactions that induce preferential orientation of co-solvents such as glycerol at the protein surface, leading to exclusion of glycerol and formation of a preferential hydration layer. These interactions promote the native protein to adopt a more compact conformation and enhance its thermodynamic stability. Glycerol selectively binds to continuous large hydrophobic regions and acts as an amphipathic interface between hydrophobic surfaces and aqueous solvent. It inhibits protein unfolding and stabilizes aggregation-prone folding intermediates to prevent further aggregation and precipitation.

Mechanisms under low-temperature aqueous conditions: Glycerol forms hydrogen bonds with water molecules to suppress ice nucleation and crystal growth, lower freezing point, and shield hydrophobic interactions. This prevents cold-induced protein denaturation, aggregation and mechanical damage. High concentrations of glycerol (25%–50%) maintain solutions in liquid state at −20 °C, completely eliminating damage from freeze-thaw cycles. Glycerol is commonly used for enzyme preservation, and its typical working concentration ranges from 5% to 20% (0.678 M–2.71 M) in aqueous formulations, with viscosity of 1.1–2.2 mPa·s at 25 °C. It stabilizes enzymes, refolding proteins, recombinant proteins, cytokines and peptides, and can be applied in lyophilized preparations upon formulation optimization.

Mannitol, with a typical concentration below 5% (<275 mM) and viscosity of 1.0–1.2 mPa·s at 25 °C, shows limited advantages for protein stabilization in liquid phase and cannot function as a cryoprotectant. It acts as a bulking agent and crystalline excipient. When combined with other components, it helps stabilize the overall lyophilization environment and indirectly protects proteins during lyophilization.

Sorbitol is generally formulated at 1%–10% (55 mM–550 mM) with viscosity of 1.0–1.2 mPa·s at 25 °C. It is universally applicable in aqueous systems and can serve as a cryoprotectant or auxiliary anti-aggregation agent. It functions as both bulking agent and stabilizer in lyophilized products.

In solid-state lyophilized formulations: During dehydration, small-molecule polyols penetrate the protein surface and form hydrogen bonds with polar residues, replacing water molecules removed during drying. This avoids hydrogen bond rupture and collapse of spatial conformation caused by water loss. At high concentrations in the late drying stage, glycerol forms a highly viscous amorphous glassy matrix, drastically reducing molecular diffusion rates and inhibiting polypeptide chain mobility, aggregation and conformational loosening.

This category of stabilizers and protectants mainly consists of non-reducing disaccharides. The stabilization mechanism of sugars resembles that of polyols.

In aqueous solution at ambient temperature: Sugar molecules tend to be excluded from the vicinity of protein surfaces, increasing surface tension at the protein-water interface. This drives proteins to fold into tighter native conformations to minimize surface energy, thereby improving thermodynamic stability.

During lyophilization: Hydroxyl groups of sugars can replace water molecules to form hydrogen bonds with polar residues on protein surfaces, maintaining secondary and tertiary structures and preventing conformational collapse upon dehydration. At high concentrations or under dry conditions, sugar molecules form highly viscous amorphous glassy matrices, severely restricting the mobility of protein molecules and suppressing chemical degradation and physical aggregation.

Sucrose is commonly used at 5%–10% (146 mM–292 mM) with viscosity of 1.3 mPa·s. It suits most globular proteins in liquid formulations, cannot deliver cryoprotective effects, and exerts lyoprotection when used alone.

Trehalose is typically applied at 2%–10% (58 mM–292 mM) with viscosity of 1.85 mPa·s. It demonstrates universal and superior stabilizing performance in aqueous solution. It has no cryoprotective property but works independently in lyophilized systems, and is widely recognized as the optimal lyoprotectant to date.

It should be noted that working concentrations of these stabilizers and protectants are frequently expressed as mass percentages, while some studies adopt stabilizer-to-protein ratios (w/v) or molar ratios. Protective efficacy depends on the target protein and storage temperature (e.g., preventing crystallization and precipitation of excipients). Combinations of multiple stabilizers and protectants are commonly deployed.

Based on non-covalent interactions between solutes and biomacromolecules in solution, these reagents can also be utilized during purification to reduce or shield interactions between target proteins and impurities, supporting downstream purification – particularly for matrices with highly complex background components.

Among various protectants and stabilizers, functional pharmaceutical excipients are intentionally added formulation components rather than process impurities. Therefore, the concept of “residual limit” does not apply; instead, assay testing and formulation compliance evaluation are required. In contrast, substances introduced during biomanufacturing that are not intentionally incorporated into the final product are treated as raw materials, for which residual limits shall be established based on safety assessment. Quality specifications shall be established for both raw materials and excipients.

In biopharmaceutical manufacturing practices, the selection of protein stabilizers and protectants requires comprehensive evaluation of protective performance, process manufacturability, safety, qualified supplier availability and material costs. Potential environmental impacts should also be taken into consideration.