Factors Influencing the Protein Drugs Stability

Protein stability is a multifactorial issue that must be addressed through careful optimization of formulation components, environmental conditions, and manufacturing processes. Stability-enhancing additives should be judiciously selected, and their potential adverse effects thoroughly assessed to ensure the efficacy and safety of protein-based therapeutics.

The stability of protein formulations is influenced by several key factors, including the formulation composition, environmental conditions, contaminants, and the manufacturing process. Each of these factors is discussed in detail below.

Fig. 2 summarizes the main bottlenecks for protein application while also presenting potential solutions.

How Does Formulation Composition Affect Protein Stability?

One of the critical elements within a formulation is the pH value, which exerts a profound impact on protein stability. By modulating the type, density, and distribution of surface charges on the protein, pH influences both conformational stability and colloidal stability. This effect is not confined to the native state of the protein but also extends to its aggregation rate and the types and rates of chemical degradation. Moreover, pH regulates solubility, viscosity, and the interactions between protein molecules. It is noteworthy that physical changes induced by pH, such as protein oligomerization, are often considered reversible processes.

Another key factor is ionic strength, which directly affects protein stability by neutralizing surface charges on the protein. Similar to pH, ionic strength can influence both the conformational and colloidal stability of proteins. In general, the addition of salts tends to promote protein aggregation due to charge effects. However, the specific outcome depends on both the salt concentration and the pH of the solution. Additionally, changes in ionic strength can alter the osmotic pressure of the solution.

Other excipients or additives present in the solution may also significantly impact protein stability. Buffers are a notable example; their type and concentration can affect various protein properties, including thermal stability, conformational stability, aggregation propensity, osmotic pressure, and viscosity.

To enhance protein stability, stabilizers are commonly included in formulations. However, the introduction of these additives must be carefully evaluated, as certain excipients at specific concentrations may actually reduce protein stability. Examples include sugars, specific amino acids (such as arginine and histidine), surfactants (such as Pluronic F-68 and Tweens), and polymers. Some additives, such as EDTA, may exacerbate protein oxidation through particular mechanisms. Furthermore, certain excipients may promote chemical modifications of the protein via chemical bonding or catalytic degradation. In most cases, the inclusion of preservatives further reduces protein stability.

Environmental Factors and Contaminants

The environmental conditions under which protein formulations are stored or administered play a pivotal role in maintaining their stability. Temperature fluctuations, light exposure, and mechanical agitation can accelerate degradation processes such as denaturation, aggregation, or oxidation. Additionally, contaminants, including trace metals or microbial agents, can catalyze reactions that destabilize the protein, necessitating stringent control of environmental conditions and contamination risks during production and storage.

Manufacturing Process Considerations

The methods used in the production of protein drugs, including purification, filtration, and packaging, also influence stability. During processing, proteins may be exposed to shear forces, temperature variations, or interfaces that can cause structural changes or aggregation. Furthermore, the materials used in packaging and storage, such as plastic or glass containers, can interact with the protein, affecting its long-term stability.

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Environmental Factors Affecting Protein Stability

Two primary environmental factors that significantly influence protein stability are temperature and light exposure. The relationship between temperature and protein stability follows a parabolic pattern, encompassing both cold denaturation and thermal denaturation. Notably, the temperature at which cold denaturation occurs can be higher than 0°C. For instance, a native protein, such as XXX, which exists under physiological pH conditions, exhibits cold denaturation at approximately 0°C and thermal denaturation at around 40°C. When the temperature drops below 12°C, protein precipitation may occur, likely due to cold denaturation. Although proteins in cold and thermal denaturation states share residual secondary structures, there are marked differences in their hydration levels.

Light exposure, on the other hand, induces a variety of oxidative, photolytic, and cross-linking degradation products. These products include oxidized amino acids from susceptible residues such as methionine (Met), tryptophan (Trp), and histidine (His), His-His dimers, adducts formed with buffer components, acidic and basic variants, hydrolytic fragments, and reducible or non-reducible dimers and oligomers, which may eventually form insoluble particles. These aggregates often represent a mixture of oxidation products, potentially leading to visible color changes. The literature has also documented that light exposure can reduce cysteine residues, forming thioacetals, thioethers, and disulfides, thereby influencing product formation.

Both visible and ultraviolet (UV) light generate reactive oxygen species (ROS) and free radicals, which accelerate oxidative and cleavage processes. Tryptophan residues in proteins exhibit high reactivity in transferring light energy to oxygen, significantly enhancing degradation. This explains why the presence of tryptophan exacerbates histidine oxidation within the same molecule and methionine oxidation in other molecules upon light exposure. Further complicating this issue, immunoglobulins can catalyze reactions between O₂ and water, producing hydrogen peroxide, which accelerates oxidation. Light is also a crucial factor in promoting protein aggregation and solid-state oxidation.

Impact of Contaminants on Protein Stability

Various potential contaminants introduced during manufacturing or derived from raw materials can significantly impact protein stability. One of the most common contaminants is metal ions, which may originate from metal processing equipment or the raw materials themselves. Metal contaminants can bind directly to proteins, thereby destabilizing their conformation and accelerating metal-catalyzed oxidation, carbonylation, cleavage, or aggregation. To mitigate the leaching of metals from processing equipment, the use of disposable components has emerged as an effective strategy.

Residual organic solvents can also compromise protein stability, with the extent of this effect dependent on the solvent quantity and the protein's sensitivity. Studies have shown that solvents such as glycerol, ethanol, and 2,2,2-trifluoroethanol can significantly promote protein aggregation under specific conditions. Even trace amounts of disinfectant residues, such as hypochlorous acid and peracetic acid, may induce protein aggregation through oxidation.

It is also noteworthy that residual proteases can catalyze protein fragmentation, forming particulate matter and promoting the hydrolysis of polysorbate, indirectly contributing to the destabilization of protein formulations.

In addition, potential leachables from product contact surfaces, along with impurities from commonly used excipients, such as nanoparticles in sucrose, peroxides and formaldehyde in polyethylene glycol (PEG), and free fatty acids in polysorbates, can affect protein stability to varying degrees.

The Multifaceted Impact of Formulation Processes on Protein Stability

The stability of protein-based therapeutics is profoundly influenced by several critical processes during manufacturing. These processes include, but are not limited to, agitation, pumping, freeze/thaw cycles, and lyophilization. A detailed analysis of these factors is provided below:

Agitation

Various forms of agitation, such as rotation, shaking, stirring, mixing, and vortexing, may have a direct effect on protein stability. During these processes, mechanical forces can induce denaturation, unfolding, or aggregation of the protein. The addition of surfactants at appropriate stages can mitigate protein adsorption at the air/water interface, which is a key factor in aggregation induced by surface interactions. However, it is important to note that even lyophilized protein products, which have undergone drying to enhance stability, may experience instability and aggregation when subjected to mechanical agitation during transportation or storage.

Freeze/Thaw Cycles

The freeze/thaw process, a critical step in many biopharmaceutical production workflows, poses significant challenges to protein stability. Freezing can perturb the protein structure, promoting adsorption at the ice/liquid interface or entrapment within the ice phase. Furthermore, the process often involves cryoconcentration, leading to excipient crystallization, solute concentration changes, and shifts in pH, all of which can synergistically induce protein aggregation or chemical degradation. Therefore, optimizing the freezing and thawing rates, along with maintaining control over the final frozen state temperature, is essential for preserving protein stability.

Additionally, proteins are more susceptible to chemical degradation, such as oxidation, during frozen storage due to the concentration of reactants. This phenomenon arises from the reduced mobility of the surrounding water, which increases the local concentration of reactive species, thereby accelerating oxidative processes. Consequently, special attention must be given to the formulation and storage conditions to minimize these degradation pathways.

The manufacturing processes of agitation and freeze/thaw cycles represent critical points where protein instability may be introduced. Optimizing each step, from the addition of surfactants to the rate of freezing and thawing, is essential for maintaining the structural integrity and therapeutic efficacy of protein drugs. Effective process control not only mitigates physical instability, such as aggregation, but also reduces the risk of chemical degradation, ensuring the longevity and quality of the final product.

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