April 9, 2024
The stability of proteins determines their ability to maintain their functional conformation under different environmental conditions, such as changes in temperature, pH, and solvent composition. It is an important aspect in pharmaceutical manufacturing, as the proteins (and resulting drug substances) derived from elaborate production processes must preserve their structural integrity until they have accomplished the task they were designed for.
In the course of this article, we will not only provide a brief overview of what protein stability is, but also discuss factors that have an impact on protein structure. Furthermore, we will highlight methods to assess protein stability, along with optimization opportunities for overall stability throughout protein manufacturing.1
Proteins, also referred to as the building blocks of life, exhibit remarkable structural diversity crucial for their function. The sequence of amino acids, forming polypeptide chains (multiple peptides linked together) as the primary structure, sets the basis for protein folding. Via this folding process, the secondary structures are created, including alpha helices and beta sheets. The secondary structure also determines the three-dimensional tertiary structure, and in some cases, proteins even assemble into quaternary structures, composed of multiple subunits.
The stability of a protein's folded conformation after synthesis is essential for its biological activity. Various interactions, including those between amino acid side chains, hydrogen bonding, hydrophobic and protein-protein interactions, contribute to maintaining this stability.
Naturally, not all proteins are the same. Globular proteins, for instance, often display comparatively low conformational stability – biochemistry, however, holds measures that aid in increasing the stability of proteins.2 3
In biopharmaceuticals, protein stability is paramount for ensuring the efficacy and safety of therapeutic proteins. Stable proteins maintain their native conformation, preserving their biological activity during manufacturing, storage, and administration. Enzymes and antibodies, for example, rely on stability for maintaining their catalytic activity and binding specificity.
In vitro, stable proteins enable reliable assays and high-throughput screening, while in vivo, they ensure proper pharmacokinetics and therapeutic outcomes. Mutations or destabilizing factors can compromise protein function, leading to reduced efficacy of these biologics or adverse effects.
Various factors influence the stability of proteins, impacting their structural integrity and functional activity. Chemical factors such as pH, ionic strength, and the presence of denaturants or ligands can disrupt protein folding and stability. Physical factors including temperature, pressure, and agitation can also affect protein stability by altering the balance of interactions within the protein structure.
Additionally, the composition of the solvent environment can impact protein stability, particularly for hydrophobic interactions. Mutations or modifications in the protein sequence can introduce destabilizing changes, leading to misfolding or aggregation.
Chemical factors play a significant role in influencing protein stability. Denaturation, caused by changes in pH, temperature, or the presence of denaturing agents, disrupts the native structure of proteins, leading to loss of function.
The solvent environment, including polarity and ionic strength, can affect protein stability by altering interactions between amino acid side chains. Binding of ligands or small molecules to proteins can either stabilize or destabilize their structure, impacting their functional activity. Additionally, chemical factors can promote protein aggregation, a common cause of instability in biopharmaceuticals.
Strategies to enhance protein stability include the formation of disulfide bonds, the use of stabilizing agents or chaperones, mutagenesis to introduce stabilizing mutations, and the manipulation of cysteine residues in wild-type proteins.
Hydrophobic residues, which tend to aggregate in the protein core, play a significant role in stabilizing the protein structure through hydrophobic interactions. Additionally, hydrogen bonds, e.g. formed between polar amino acid side chains, contribute to maintaining the protein's secondary and tertiary structure. Van der Waals interactions, arising from transient fluctuations in electron density, also contribute to overall stability.4 5
Physical factors are critical determinants of protein stability. Electrostatic interactions between charged amino acid residues further influence stability, especially in environments with varying ionic strength. Furthermore, entropy, a measure of disorder, can impact protein stability by affecting conformational flexibility.
Considering the multiple mechanisms that ensure protein stability, it does not surprise that this balance can be easily disturbed not only by chemical, but also by physical forces. Mechanical stress, such as agitation or shear forces during handling and processing, can disrupt protein structure and lead to denaturation. Gentle handling techniques are essential to maintain protein stability.
Furthermore, thermodynamic stability of proteins is seen as a major challenge: Their thermal stability is limited – too low or too high temperatures during protein storage can lead to degradation and product loss.
To make sure that a biopharmaceutical product will be safe and effective, protein stability must be granted. After all, a drug must not only look well on paper or under lab conditions, but maintain its qualities throughout the supply chain. For that aim, several methods have been established to assess the stability of proteins.
Thermodynamic approaches: Thermodynamic methods involve measuring changes in free energy associated with protein folding and unfolding. These include calorimetric techniques, such as differential scanning calorimetry (DSC), and equilibrium unfolding studies.
Spectroscopic techniques: Fluorescence spectroscopy is commonly used to monitor changes in protein conformation and stability by measuring fluorescence emission upon excitation. Additionally, techniques such as circular dichroism (CD) spectroscopy provide information about protein secondary structure and stability.
Structural methods: X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy offer insights into the three-dimensional structure of proteins, allowing for the characterization of folded and unfolded states.
Computational simulations: Molecular dynamics simulations and other computational methods provide valuable insights into protein stability by modeling protein folding pathways and interactions. These simulations are complemented by bioinformatics tools for analyzing protein sequences and predicting stability-related properties.
Temperature plays a crucial role in determining protein stability, with fluctuations often leading to denaturation and loss of protein function. One effective method to mitigate temperature-related instability is through precise control of freezing processes. Plate freezing, especially when implemented with the support of innovative solutions like those offered by Single Use Support, can significantly optimize protein production and improve protein stability.
Plate freezing technology ensures controlled rapid and uniform freezing of drug substances, thereby minimizing the formation of ice crystals and maintaining the integrity of protein structures. By employing plate freezers tailored for biopharmaceutical applications, such as the RoSS.pFTU platform by Single Use Support, consistent and controlled freezing conditions can be achieved across various batch sizes throughout biopharmaceutical processes.
The RoSS.pFTU platform offers scalability, accommodating both small-scale laboratory experiments and large-scale commercial production. Its advanced features include fully automated processes, and compatibility with single-use bags of all sizes.
Covered in protective packaging, the single-use bags can thereafter be stored in high-performance ultra-low temperature freezers like Single Use Support’s RoSS.FRDG. It offers high storage density, and the implemented manufacturing execution systems (MES) enable manufacturers to precisely monitor and control storage temperature.
Incorporating plate freezing and ULT storage technology from Single Use Support into biopharmaceutical manufacturing processes enhances product quality, minimizes protein aggregation, and preserves the functionality of therapeutic proteins in cold chains. This innovative approach not only improves the stability of proteins but also contributes to the overall efficiency and reliability of bioproduction workflows.
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