Protein Biomolecule Preservation: Molecular Instability to Advanced Stabilization Method

Abstract

Proteins are important biomolecules that are finding extensive applications in biotechnology, pharmacology, treatments and diagnostics. The sensitivity of proteins to degradation caused by physical, chemical and biological factors during formulation, storage and transportation is due to their complex physical structure and sensitivity to conformational changes. Thus, to preserve the stability, bioactivity, and therapeutic effects of protein biomolecules, preservation is essential.The physicochemical and structural basis of protein stability including protein folding-unfolding equilibria, thermodynamic and kinetic stability and the importance of non-covalent interactions are discussed in this review. It contains the discussion of the key degradation processes and key variables that influence preservation of proteins, which are temperature, pH, moisture, ionic strength, and mechanical stress. The role of stabilization of excipients and conventional preservation techniques is evaluated in a critical manner. In addition, novel strategies, evaluation procedures of stability, modern challenges and future opportunities of advanced and viable preservation of proteins are addressed..

Keywords

Protein preservation; Protein stability; Biopharmaceuticals; Excipients; Lyophilization

Introduction

1.1 Overview of Protein Biomolecules

Proteins are complex biological macromolecules that consist of exceedingly specific three-dimensional structures of amino acid residues joined through peptide bonds. They are also some of the most functionally diverse classes of macromolecule, performing functions in transport (hemoglobin), signaling (hormones and cytokines), catalysis (enzymes), molecular recognition (antibodies) and structural support (collagen and keratin). The higher-order structures of proteins, such as hydrogen bonds, hydrophobic interactions, electrostatic forces, and covalent disulfide bridges are critical to the biological activity of proteins.On the contrary to small-molecule medications, proteins are relatively low-thermodynamic stability with even minor changes in their environmental conditions such as temperature, pH, ionic strength, etc. severely altering their shape and activity. The sensitivity of protein biomolecules makes them particularly difficult to preserve in the course of production and storage as well as administration. (1)

1.2 Significance of Protein Preservation in Pharmaceuticals and Biotechnology

The preservation of protein biomolecules is one of the most crucial issues in the biotechnology and pharmaceutical sciences. The structural breakdown of therapeutic proteins and peptides is common during their formulation processes, such as purification, freezing, drying, transportation, and long-term storage. Any loss of natural structure can result in reduced bioactivity, high immunogenicity or complete therapeutic failure. (2)

Instability of proteins in the biopharmaceutical sector can result into:

• Biological potency is lost
• Immunogenic hazardous Aggregates are formed
• Reduced shelf life and increased dependence on cold-chain transportation

Effective methods of preserving proteins are essential in order to ensure the quality, safety, efficacy and regulation compliance of these products, particularly in the case of biologics (monoclonal antibodies, enzymes, vaccines and the like) where it is important to guarantee quality, safety, efficacy and regulatory compliance. (3,4)

1.3 uses of Proteins in Nature

Preserved protein biomolecules have many biotechnological and medical applications:Treatment: Monoclonal antibodies, growth hormones, interferons, insulin, and enzyme replacement treatments. Diagnostics: ELISA and antibodies in ELISA, biosensors and immunoassays; Vaccines: Protein antigens and recombinant subunit vaccines which require long-term stability of structure. (5–7)Research and Industrial Enzymes: Proteins Oxidoreductases, polymerases and proteases used in molecular biology, food, and medicine. (8)In each of these applications, functional performance is directly associated with the preservation of the natural protein structure. (9)

1.4 Scope and Objectives of the Review

This review aims to provide a comprehensive description of the fundamental concepts, disaggregation mechanisms, stabilization methods, and the latest technology regarding the preservation of protein biomolecules. Among the goals are:  Physical, chemical, and biological degradation pathways; understanding the mechanisms of structural and physicochemical factors that control protein stability. Comparing conventional and innovative preservation techniques; outlining current concerns, gaps in the research, and future opportunities related to preservation of proteins. (2,10,11)

2. PHYSICOCHEMICAL AND STRUCTURAL BASIS OF PROTEIN STABILITY

2.1.Protein Structure Levels and its role in Stability

Protein stability is determined through four hierarchical-based stages of structure:

• Primary structure Linear amino acid sequence: stability can be permanently affected by chemical modifications like deamidation.
• Secondary structure: a- helices and b- sheets are provided with the help of hydrogen bonds; breaking them leads to the partial disintegration.
• Tertiary structure: Three dimensional folding is as a result of disulfide bonds, salt bridges, and hydrophobic interaction. (12)
• Quaternary structure: The binding of numerous subunits; particularly important in multimeric enzymes and antibodies.

Loss of secondary or tertiary structure is often followed by aggregation and functional inactivation. (13–17)

2.2. Protein Thermodynamic vs. Kinetic Stability

Protein stability can be described in the following way:

• Thermodynamic stability: The difference between unfolded and the free energy of the unfolded state.
• Kinetic stability: An energy barrier which prevents aggregation or unfolding.

A number of therapeutic proteins are trapped kinetically in their natural folds, but only thermodynamically stable to a limited degree. Instead of modifying the intrinsic thermodynamics, the preservation solutions often aim at increasing kinetic impediments. (13,18,19)

2.3.Protein Folding and Unfolding Equilibria

Protein folding is a reversible process that is influenced by different environmental factors. Stressors such as heat, ice, or dehydration may cause the shift of the balance towards the unfolded or partially folded intermediates. Such intermediates promote aggregation and permanent disintegration because they expose the hydrophobic residues. (20)

2.4 The Role of Disulfide Bridges, Hydrogen Bonding and Hydrophobic Interactions

• Hydrogen bonds: stabilize secondary structure.
• Hydrophobic interactions: these promote folding and at the same time promote aggregation upon exposure. (21)
• Disulfide bridges: They provide covalent stability especially in therapeutic and extracellular proteins.

Destruction of these bonds is one of the key contributors to protein instability. (22,23)

3. MECHANISMS OF PROTEIN DEGRADATION

Protein degradation is any physical, chemical or biological process that causes the loss of the original structure, biological activity or therapeutic effect of a protein. In pharmaceutical formulations, degradation directly affects the quality parameters of safety, potency, and shelf life and so is a critical quality issue. These processes of protein breakdown are often interlinked and in the stressful conditions, multiple processes can occur simultaneously. (10,24)

3.1 Physical Instability

Physical instability is defined as protein conformational changes which do not involve covalent bond breaking. This is one of the most common causes of protein failure during preservation. (25)

• Denaturation

Denaturation is the process by which a protein undergoes a loss in its original secondary, tertiary, or quaternary structure without its primary structure. It can be induced by thermal stress, pH changes, freezing, dehydration, exposure to organic solvents and high ionic strength. Denatured proteins often expose hydrophobic regions that are normally located in the protein core leading to aggregation and permanent loss of biological activity. Pharmaceutical products can be subjected to denaturation due to improper storage, lyophilization or spray drying. (26)

• Precipitation and Aggregation

Protein aggregation is the process of joining partially unfolded or misfolded molecules of proteins by hydrophobic interactions, hydrogen bonding or disulfide exchange. The aggregates are either insoluble (precipitates) or soluble (oligomers). Aggregation is particularly an issue in therapeutic proteins due to their ability to increase immunogenicity and reduce efficacy. Such variables as variations in temperature, agitation, freeze-thaw cycles, and high protein concentrations considerably contribute to the aggregation risk. (24,27)

• Surface Adsorption

Some examples of solid surfaces where proteins may adsorb include glass containers, rubber stoppers, stainless steel equipment and air-liquid interfaces. The adsorption often leads to incomplete unfolding because of contact with the hydrophobic or charged surfaces. Since aggregation is encouraged by repeated exposure to interfaces, aggregation is especially significant in pumped or agitated liquid formulations. (23)

3.2 Chemical Instability

Chemical breakdown of Amino acid residues is a covalent modification, which is often accelerated by temperature, pH, light, and oxygen.

• Deamidation

Deamidation is a similar breakdown process that occurs to asparine or glutamine residues to produce aspartic acid or glutamic acid, respectively. This reaction alters the structure and charge distribution of the protein that can reduce the stability and biological activity. Deamidation is the fastest in neutral to alkaline conditions and is very pH dependent. (28,29)

• Oxidation

The primary targets of oxidation are methionine, cysteine, tryptophan, and tyrosine and other sulfur-containing and aromatic amino acids. Oxidative stress can be caused by dissolved oxygen, sun exposure, pollutants of metal ions or peroxide contaminants in excipients. Oxidation can also reduce enzymatic activity, lose receptors, as well as cause structural changes. (18,30,31)

• Hydrolysis and Isomerization.

Peptide bonds are hydrolyzed by extreme pH conditions or long storage periods. Isomerization, particularly the formation of isoaspartate residues, disrupts secondary structure and places conformational strain on it. These responses are normally delayed and significant in the long term storage. (25,32)

3.3 Biological Degradation

Biological deterioration is caused by microbial infection or enzymatic activity.

• Proteolysis

Proteolytic breakdown is brought about by environmental enzymes or residual host-cell proteases. Even small concentrations of proteins during storage can result in protein fragmentation and loss of activity by cleavage of proteins. (31,33)

• Microbial Contamination

Besides posing safety risk, further growth of microbes accelerates the enzymatic breakdown of proteins. Hence, preservatives and strict aseptic processing are essential in protein composition. (24,34)

4. FACTORS AFFECTING PROTEIN PRESERVATION

Protein biomolecules are naturally unstable due to their weak non-covalent bonds and complex three dimensional structures. During production, storage and transport, proteins are exposed to numerous mechanical and environmental stresses and this can lead to physical, chemicals or even biological degradation. These factors need to be known when designing effective methods of preserving proteins. (20,35)

4.1. The thermal stress and temperature

Temperature is one of the most significant variables that influence the stability of proteins. Proteins are sensitive to temperature and even small changes in temperature eventually lead to unfolding of proteins due to the formation of hydrophobic and hydrogen bonds that stabilize secondary and tertiary structures as well as break down the same structures. Increased temperature accelerates unfolding, aggregation and chemical degradation processes such as oxidation, and deamidation through an increase in molecular mobility. (5,36)Low temperatures do not necessarily stabilize proteins. Alterations in the hydration of hydrophobic residues may cause cold denaturation, and freezing may cause the formation of ice crystals. Frozen matrices Solute cryoconcentration pH changes in frozen matrices. Such events can lead to partial unfolding and aggregation during freeze-thaw cycles. So to be stored long-term, therapeutic proteins are often either cryopreserved (≤ −80 °C) or refrigerated (2–8 °C). Lyophilisation to minimize thermal mobility. (26,37,38)

4.2 Buffer Composition and pH

Due to pH changes in ionization of amino acid side chains, protein stability is highly pH- sensitive. Alteration in the ideal pH of the protein may lead to instability in the protein structure and aggregation because it disrupts the hydrogen bonds, salt bridges, and even the electrostatic interactions. (39)

Extreme pH conditions increase chemical degradation pathways, in particular:

• Hydrolysis with low pH catalyzed by acid
• Decomposition of glutamine and asparanil residues at neutral to alkaline pH

This is the reason why the choice of buffers is very important. Common buffers such as phosphate, histidine and citrate must be selected carefully due to their need.

• Buffers have the ability to bind to protein surfaces
• Freezing Selective crystallization of buffer salts can lead to pH changes

Research has asserted that poor buffer systems have the potential to reduce protein stability in lyophilization and storage drastically.(40)

4.3 Water Activity and Moisture Content

Water performs two roles in protein stability. Solid formulations contain excess moisture, which supports molecular motion, which in turn helps to initiate aggregation and chemical degradation, although hydration is required to maintain original shape.Under the formulation of lyophilated proteins:

Residual moisture reduces the glass transition temperature (Tg) to an amount below the threshold; the more water is active, the faster it hydrolyzes and oxidizes.

Conversely, water loss to extreme dehydration has the potential to result in collapse of structures due to disruption of hydrogen bonding networks. As such bound water would be the best ratio to have a long-term preservation. The fact that protein medicines are moisture-sensitive is due to: Refrigeration, dehumidifiers and humidity-controlled packages.(41)

4.4 Effects of salt and ionic strength

Ionic strength determines the Type of electrostatic interactions and solubility of proteins. Low salt concentrations cause electrostatic repulsion between charged residues that causes the folded structure to become unstable. Excessive ionic strength, on the other hand, can encourage salting-out and the subsequent precipitation and aggregation of proteins.Also, some of the ions can react with functional sites of the proteins altering their stability. Though certain anions might lead to the unstability of tertiary structure, multivalent metal ions can enhance oxidative reactions. Consequently, during formulation the type and amount of salt have to be optimized. (42,43)

4.5 Mechanical Stress (Shear, Agitation, Freeze-thaw Cycles)

The major cause of protein instability is mechanical forces experienced during processing and transportation. Proteins are subjected to agitation, shaking, pumping and filtration with air-liquid interfaces. These pressures can lead to partial aggregation and unfolding especially in monoclonal antibodies and enzymes. The frequent freeze-thaw cycles are extremely dangerous. The likelihood of unfolding and aggregate formation under the influence of ice also rises with every cycle and reduction of the biological activity. Thus, mechanical stress is an important factor to be reduced through careful handling and efficient filling processes. (35,44)

4.6 Exposure to Light and Oxygen

The breakdown of proteins under light exposure, especially UV and visible light can also be performed under photochemical conditions. Examples of aromatic amino acids that absorb light resulting in the production of reactive oxygen species (ROS), which induce oxidation and fragmentation are tryptophan, tyrosine, and phenylalanine.Exposure to oxygen aggravates oxidative degradation particularly in formulations that include: Metal ion, cysteine and methionine contaminants. To reduce such effects, protein formulations are often: Put into amber or light resistant containers; topped with nitrogen, an inert atmosphere; and augmented with chelating agents, and antioxidants.

5.CONVENTIONAL METHODS OF PROTEIN PRESERVATION

The fundamental aims of the conventional methods of protein preservation include the decreased mobility of the molecules, reduced exposure to the destabilizing environment, and maintenance of the natural shape of the proteins in preservation and transportation. Such traditional methods are still the basis of the protein production in biotechnology and pharmaceutical sectors even after developing more advanced methods. (11,45)

5.1 Refrigeration and Cryopreservation

Refrigeration (2-8 ?) is the most commonly used short-term preservation method of protein formulations. Low temperatures reduce the rate of degradation process such as unfolding, aggregation, deamidation and oxidation, since they decrease the kinetic energy. Total elimination of instability is, however, not achieved by refrigeration, and an activity slow down is likely to occur in even long-term storage at these temperatures. (36)Enzymes, antibodies and research grade proteins are commonly stored over a long duration period through a method known as cryopreservation that involves the maintenance of a low temperate of between -20 degC and -80 degC or below. Although chemical degradation is highly inhibited in the cryogenic temperatures, freezing leads to the occurrence of some stressors such as:

• The crystalisation of ice
• Solute cryoconcentration
• local pH variations

These pressures may lead to partial unfolding and aggregation especially in freeze-thaw cycles. Thus, to enhance stability, sugars and glycerol, which are examples of cryoprotectants, are often used. (10)

5.2 Freeze-Drying (Lyophilization)

Freeze-drying (freezing-drying or lyophilization) is the most common technique of preserving medicinal proteins and vaccines. This procedure entails:

Other Frozen solution of the protein.

• Primary drying (lower pressure ice sublimation)
• Secondary drying (removal of bound water)

Lyophilization is used to enhance the stability of proteins at ambient temperature or even in the refrigerator at a long period of time by converting them into an amorphous form with highly reduced molecular mobility. But when proteins are subjected to the process of the lyophilization, they are exposed to various challenges, including the dehydration stress and the denaturation associated with the freezing. Loss of hydrogen bonding networks in the drying process may cause instability of the secondary and tertiary structures and in the reconstitution process, aggregation may occur. Lyoprotectants such as sucrose and trehalose are applied to preserve the structure of the protein by replacing the water molecules and providing a glassy matrix around the protein to overcome these challenges. (7,35,36)

5.3 Spray Drying

Another preservation technique in solid state known as spray drying sprays the protein solution into a hot drying gas, resulting in rapid evaporation of the solvent and formation of particles. Compared to lyophilization,

• Spray drying is quicker
• More expandable
• Inexpensive

High temperatures and shear stress during atomization however have a serious negative impact on protein stability. Provided that process parameters are not adjusted correctly, proteins can undergo thermal denaturation and aggregation. Spray drying is, in its turn, more suitable to moderately stable proteins or when combined with the protective excipients that minimize the effect of heat. (46,48)

5.4 Preservative and Stabilizing Agents

Stabilizing agents and preservatives are often added to protein formulations to enhance stability upon storage as well as with multi-dose usage. Some of the commonly used stabilizers are:

• Polyols (glycerol, sorbitol)
• Amino acids (glycine, arginine)
• Sugars (sucrose, trehalose)
• Surfactants (polysorbate 20 and 80)

These are active as they retain hydration shells around proteins molecules, prevent aggregation and reduce surface adsorption. Antimicrobial preservatives may also be used to prevent microbial contamination although their compatibility with protein structure must be taken into careful consideration. (49–52)

5.5 Limitations of Traditional Methods of Preservation

Most common methods of preserving proteins are conventional methods, yet they have several limitations. Refrigeration and cryopreservation require strict cold-chain maintenance but this increases the costs and limits its accessibility especially in developing countries. In the case of frozen formulations, freeze-thaw sensitivity remains a critical issue.Lyophilization is notwithstanding effective because:energy intensive, time consuming and formulation dependent.Spray drying cannot be used to dry highly labile biomolecules, and exposes proteins to heat stress. Also, all the conventional methods do not completely prevent chemical degradation or accumulation and this highlights the importance of improved and more comprehensive preservation methods. (8)

6. EXCIPIENTS IN THE STABILIZATION OF PROTEINS

Excipients are necessary during processing and storage to minimise the physical and chemical degradation of proteins. They stabilize proteins through the change of the microenvironment, slowing down molecular mobility, and preventing aggregation. (53,54)

6.1 Sugars and Polyols

The stabilizers contain sugars such as lactose, sucrose and trehalose and polyols such as sorbitol and glycerol. These excipients protect proteins by:

• The replacement process of water (hydrogen bonding to the protein residues)
• Vitrification, which is a procedure that fixes proteins in a glassy material.

The elevated glass transition temperature and chemical inertness of trehalose result in its special effectiveness. (38,55,56)

6.2 Stabilizers ( Amino Acids and Proteins )

Amino acids such as arginine, glycine and histidine prevent aggregation by inhibiting protein-protein interactions. Arginine is especially helpful when eliminating aggregation in the event of refold and freeze-thaw stress.

6.3 Polymers and Surfactants

Polymers (PEG, dextran) and non-ionic surfactants (polysorbate 20/80) prevent: Adsorption and interfacial denaturation on the surface. The monoclonal antibody compositions require the presence of surfactants to prevent agitation-induced aggregation.

6.4 Anti-oxidants and Chelating Agents

Antioxidants (methionine, ascorbic acid) and chelators (EDTA) reduce oxidative degradation by scavenging the free radicals and binding metal ions respectively. (42,51)

6.5. Mechanisms of Excipient-Protein Interaction

The stabilization of proteins by excipients takes place through steric hindrance, hydrogen bonding, preferential exclusion, and reduction of interfacial stress (30,38,57)

7. ADVANCED AND EMERGING TECHNIQUES ON PROTEIN PRESERVATION

Studies have focused more on novel strategies to enhance protein stability, shelf-life, and in some cases permit room-temperature storage because of the limitations of conventional preservation technologies (such as cold-chain reliance and partial stability). Formulation science, molecular engineering, and nanotechnology are used to combine these advanced technologies to protect the structural integrity and biological activity of proteins in a wide range of applications, such as medicines and diagnostics. (13,45,58,59)

7.1 Protein Engineering and Mutational Stabilization

Protein engineering aims at enhancing intrinsic stability by changing the amino acid sequence of the protein. Engineering changes the structure of the protein to reduce its susceptibility to unfolding and degradation, instead of acting externally as formulation techniques. (30,55,60)

Important Methods:

• Directed evolution:Variants that are more stable are generated through random or semi-random mutagenesis, and improved clones are selected. (61)
• Rational design: Mutational stabilization additions are performed by structural knowledge (e.g. substituting long loops, forming disulfide bonds).
• Computational design: Algorithms predict mutations that enhance packing or low unfolding propensity.
• Such methods have been commonly applied with enzymes in industrial and pharmaceutical applications to enhance their thermostability and functional stability. The use of computational design and molecular evolution together tremendously enhances protein strength in stress conditions of relevance to formulation and storage.  (62)

Importance in Preservation:

Engineering also reduces the requirements of stabilizing chemicals and cold storage by enhancing thermodynamic and kinetic stability. (18,60,63)

7.2 Encapsulation Techniques

The protective microenvironment of encapsulation helps the protein molecules to avoid denaturation, aggregation and enzyme degradation. Also, the controlled release is enabled through encapsulation that is particularly useful in delivery systems. (64–67)

• Nanoparticles

Nanoparticles which are made of polymers or themselves are capable of encapsulating proteins and this enhances their resistance to environmental stressors. Protein nanoparticles can be prepared to:

• Boost bioavailability
• Reduce proteolytic degradation.
• Offer targeted delivery

As an example, therapeutic proteins and therapeuticss have been incorporated into albumin-based nanoparticles to enhance stability and controlled release as well as maintaining or enhancing functional properties. (25)

• Liposomes

The phospholipids are assembled into spherical vesicles called liposomes. They also protect proteins against hydrolytic and proteolytic degradation in addition to targeting distribution to specific tissues or cells. (68,69)

• Polymeric Carriers

Biodegradable polymer systems like chitosan and PLGA are used to encapsulate the proteins and stabilize them and authenticate their long-term release. The polymer matrix hinders and shields the movement of proteins against stressors.

Uses: Nanocarriers are of special importance in the case of therapeutic protein delivery, where controlled release and preservation of circulation are essential requirements. (68,70)

7.3 Preservation of Solid-State Proteins

Solid-state preservation includes the conversion of proteins into amorphous forms solid with minimal mobility of molecules. Among the methods are:

• Lyophilization (freeze-drying), the process where water is removed to form stable solids.
• Spray drying: Rapid drying to prepare the particle of protein-encapsulation.
• Ensilication: A state-of-the-art sol-gel process that functions by coating individual proteins with silica to protect them against the harsh environment such as heat and humidity enabling ambient-temperature stability and eliminating the use of cold chains to administer vaccinations.

Oxidative and hydrolytic degradation processes are reduced by solid forms and, therefore, a solid can be more stable than liquid. (7,71,72)

7.4 Ionic Liquids and Deep Eutectic Solvents

Proteins can be stabilized through ionic liquids (ILs) and deep eutectic solvents (DESs), which are a novel type of unconventional solvents that can stabilize proteins by forming hydrogen-bond networks and regulated polarity. (64)DESs, mixtures of two or more components that form a eutectic with the lower melting point than the components, can be used as protein-compatible environments, preventing protein damage during freezing and preventing the formation of ice. Owing to their adaptable physicochemical properties, biodegradability, and lower toxicity compared to traditional organic solvents, DESs are gaining growing popularity as non-toxic and cleaner and greener media of processing proteins. (64)Mechanistically, DESs hold protein structure by minimizing denaturation reaction and stabilizing natural folding by use of hydrogen bonding and electrostatic interactions.Emerging Role: DESs could be used as a cold-chain-free preservation medium since they can stabilize proteins during processing and storage as well as be applied to extraction. (35,40,43)

7.5 Nature-Inspired and Biomimetic Preservation Methods

Nature gives indications of stability through species that endure high levels of dehydration or temperatures.

For example:

• The Anhydrobiotic organisms stabilize the cellular proteins without use of water by vitrified through use of sugars such as trehalose.(73)
• Similar techniques, including the addition of natural stabilizers or mimicking of protective matrixes that lower the mobility of molecules are called biomimetic techniques, and used to recapitulate these strategies in protein preparations.

These approaches aim at reducing the use of refrigeration and long cold chains by stabilizing the solutions at room temperature. (74–76)

8. STABILIZATION OF BIOPHARMACEUTICALS AND THERAPEUTIC PROTEINS

Therapeutic proteins and biopharmaceuticals are one of the fastest-growing branches in the pharmaceutical industry. Compared to conventional small-molecule drugs, these products, monoclonal antibodies, enzymes, hormones, cytokines, and vaccines have outstanding specificity and efficacy. Conservation is however a major challenge in scientific and technological matters because their nature is intrinsically unsteady and structurally a complex affair. (77–80)Unlike chemical drugs, therapeutic proteins are large, flexible molecules, and their ability to act biologically is only possible when their original shape is maintained. The importance of preservation methods is that the slightest alterations in the structure could lead to immunogenic reactions, altered pharmacokinetics, or inactivity. (54,55,73)

8.1 Monoclonal Antibodies (mAbs)

Monoclonal antibodies are one of the most common biopharmaceuticals that are utilized to treat inflammatory diseases, autoimmune diseases, and cancers. Structurally, mAbs are large (~150 kDa), multidomain glycoproteins comprising of two heavy and two light chains linked together by disulfide bonds. (45,81)

Stability Issues:

Particularly, monoclonal antibodies are susceptible to:

• Semi- unfolding or interfacial stress-induced aggregation
• Oxidation of methionine residues at the Fc region
• Process of deamidation of asparagine residues
• Hydrolysis or proteolysis which results in fragmentation.

Aggregation is the critical issue since it is closely associated with a high level of immunogenicity that can pose dangerous risks to patients. (82–85)

Preservation Techniques:

To minimize chemical degradation, mAbs are maintained in optimized buffer system, e.g. histidine buffer at slightly acidic pH.Lyophilization is applied in long-term stability particularly when the product is to be distributed throughout the world; controlled storing temperature (2-8 degC) is employed to slow down the rate of degradation; surfactants such as polysorbate 20 or 80 are included to prevent aggregation caused by agitation and adsorption at interfaces. More and more complicated methods such as protein engineering, solid-state stabilization, as well as high-concentration formulations are explored to increase shelf life and reduce dependence on the cold chain. (71,81,86)

8.2 Enzymes and Hormones

• Enzymes

Therapeutic enzymes are used in thrombolytic therapy, in metabolic diseases and in enzyme replacement therapy (ERT). Some examples include alteplase, asparaginase and glucocerebrosidase. (10)

Stability Issues:

Enzymes are highly sensitive to:

• Proteolytic degradation
• Change of temperature and pH.
• Conformational active site changes.
• Oxidative stress

Even a small percentage of structural integrity may be lost and catalytic activity is lost completely.

Preservation Techniques:

• Enzymes are commonly preserved through the use of Lyophilization with sugar stabilizers

• Cryopreservation is used to store enzymes in large quantities
• In polymeric matrices or nanoparticles, proteins prevent the denaturation and degradation of enzymes by encapsulation
• It is possible to stabilize the active conformation by use of cofactors or substitutes of the substrate. (74,87)

• Hormones

Protein and peptide hormones such as insulin, growth hormone and glucagon are often used in the treatment of chronic diseases. (32)

Stability Issues:

Hormones can lead to:

• Aggregation, e.g. insulin fibrillation
• Chemical degradation (oxidation and degradation)

• Adsorption on surfaces of containers.

In agitation and high temperatures, especially insulin, is known to form fibrils thereby decreasing its effectiveness.

Examples of preservation methods are:

• Preservation of insulin hexamers by using zinc ions and phenolic preservatives
• Minimizing ph to reduce aggregation.
• Cold storage and light protection.
• Lyophilated formulations are used in the long-term stability.

8.3 Vaccines and Antigenic proteins

Antigenic structure has to be maintained to achieve effective immune response by the protein-based and subunit vaccines. Vaccine preservation is necessary because structural degradation may cause a loss of immunogenicity without any observable alterations. (58)

Stability Issues:

Thermal sensitivity that causes antigen denaturation; aggregation; reduction in antigen presentation; hydrolysis and oxidation in storage; and cold-chain dependence that is especially problematic in developing areas.

Preservation Techniques:

• Lyophilization is one of the popular ways to enhance the stability of vaccines.
• The sugar-based stabilizers such as trehalose and sucrose protect the antigenic epitopes.
• Adjuvant systems can also be useful in stabilization.
• Advanced solid-state methods and biomimetic preservation methods are also being explored to allow preservation of vaccines at room temperature.

Improving vaccine preservation is among the international health agenda, particularly during mass immunization exercises and during pandemic preparedness. (88,89)

8.4 Regulatory Concerns on Protein Stability

Regulatory authorities such as FDA, EMA and ICH put a high value on stability of proteins all through the product life.

Significant Regulatory Expectations:

• Massive stability tests in accelerated and real-time
• Observing aggregation, degradation products and potency
• Evaluation of freeze thaw stability
• Protecting storage conditions and shelf life

ICH Q5C in particular covers stability testing of biotechnological products with a special emphasis on the need to ensure that preservation methods ensure safety, effectiveness, and consistency. (2)

There should be comparability tests that demonstrate that the quality of products is not subject to alteration by formulation or preservation methods. (82,90,91)

9. EVALUATION OF PROTEIN STABILITY BY ANALYTICAL TECHNIQUES

Protein stability is a major factor in the development of protein formulations and their quality control. Protein stability cannot be fully assessed by a single method of analysis because protein degradation may occur through a variety of mechanisms such as chemical, biological and physical. Instead, the structural integrity, aggregation state, chemical alterations, and biological activity are determined through the combination of a wide range of free analytical methods. (24)Protein stability testing is considered to be one of the quality attributes (CQA) of the biopharmaceuticals and therapeutic proteins by regulatory bodies. Consequently, analytical techniques are used throughout the product life cycle, such as post-approval changes, production, stability testing, and formulation development. (65,92–94)

9.1 Spectroscopic Methods

Since spectroscopic techniques are rapid, sensitive, and in most cases non-destructive, they are commonly used to determine the structure of both liquid and solid proteins.

• UV-Visible Spectroscopy

UV- vis spectroscopy is commonly used to determine:

• Protein concentration (induced absorption at 280 nm)
• Turbidity and aggregation (greater light scattering)
• Transformations that are chemical and affect aromatic residues

UV absorbance profiles may also indicate protein unfolding, aggregation or oxidation. Even though UV-Vis is not very specific, it is useful in routine monitoring and preliminary screening. (95)

• Circular Dichroism Spectroscopy (CD)

CD spectroscopy is one of the useful ways of analyzing secondary and tertiary protein structure.

• Near UV CD (250-320 nm): Provides information regarding tertiary structure and aromatic side chain environment
• Far-UV CD (190-250 nm): Measures a-helix and b-sheet content.

CD is widely used to:

• Following transitions in track folding-unfolding.

• Answer: investigate the effects of high temperatures, pH and excipients on structure
• Compare sample protein and stressed protein.

The loss or shift in CD spectra is a sign of conformational instability. (95,96)

• Fourier Transform Infrared (FTIR) Spectrophotometer

The FTIR spectroscopy provides information on secondary structure by studying amide I and amide II bands.

• responsible to hydrogen bond changes
• suitable in solid-state analysis (e.g. lyophilized proteins)
• capable of sensing aggregation and conformational shifts in the course of drying or storing.

FTIR is also useful when evaluating freeze-dried formulations in which there are few solution-based techniques. (21,97)

9.2 Thermal Analysis

Thermal methods determine the stability of proteins with temperature, and this is used to inform the understanding of the unfolding transitions and stability in formulating a product. (98)

DSC is the abbreviation of Differential Scanning Calorimetry:DSC is considered as the gold standard technique when considering the thermal stability of proteins. (99)

DSC metrics:

• The melting temperature (Tm) of a protein is the temperature at which it bars
• The enthalpy of unfolding (DH) is a sign of structural stability.

Generally, increased stability is associated with increased Tm. DSC is commonly applied in:

Test the pH and ionic strength, compare formulations and evaluate the ionic strength stabilizing properties of excipients.Glass transition temperature (Tg) is determined using DSC and is critical in predicting the stability of the formulation in the long-term when used in the lyophilized form. (49,82,89)

• Thermogravimetric Analysis TGA

TGA is mainly used to:

• Determine moisture residual values
• Determine thermal decadence in solid formulations
• Determine loss of weight with temperature

TGA is an important supplemental technique because moisture is one of the major factors affecting the stability of proteins. (99)

9.3 Chromatographic Techniques

Chromatography is applied in the quality control and it is essential in the determination of chemical degradation and aggregation. (2)

• Size Exclusion Chromatography (SEC-HPLC)

SEC-HPLC is the most common technique used to analyze the aggregate. It becomes possible to identify proteins as monomers, dimers, high-molecular-weight aggregates, and fragmentation products by separating them based on their molecular size.Since the presence of protein aggregates is closely associated with immunogenic risk, SEC is crucial as it relates to regulatory compliance.(90)

• Reverse Phase High-performance Liquid Chromatography (RP-HPLC)

RP-HPLC is employed in the identification of oxidation, deamidation, truncated or modified species and proteins depending on their hydrophobicity. RP-HPLC provides high-resolution and can be used in conjunction with mass spectrometry to characterize it in depth. (29,100)

• Ion Exchange Chromatography (IEX)

IEX can be used to identify charge variations, deamidation products and chemical heterogeneity by distinguishing between the proteins using charge differences.Charge heterogeneity is particularly essential in the monoclonal antibody compositions. (101)

9.4 Particle Size and Aggregation Analysis

The analysis of particle size is important because an aggregation is an important instability process.(102)

• Dynamic Light Scattering (DLS)

DSL measures the hydrodynamic radius of solution and the spread of particle size in the solution.

Applications include:

• Predicting the onsets of aggregate formation at a very small scale
• Kinetic aggregation monitoring
• Comparison of formulation stability

Even though DLS does not give much structural information, it is very sensitive to minute aggregates. (27,96)

• Microscopic Methods

1. Optical microscopy
2. Electron microscopy (TEM/SEM)

The methods are capable of seeing large aggregates and particles, in particular in injectable formulations, where the particulate matter poses safety concerns. (1)

9.5 Bioactivity and Functional Assays

There is no direct relationship between biological activity and structural integrity. The functional experiment is thus essential in confirming the stability of proteins. (59,103)

• Enzyme Activity Assays

Catalytic activity assays directly test both the performance of enzymes. Active-site or structural degradation is manifested by loss of activity.

• Binding and Potency Assays

Concerning hormones and monoclonal antibodies:Receptor-binding assays, cell-based bioassays and immunological assays (ELISA).A conservation of proteins observed by such experiments is expected and indicates the expected biological activity.(103,104)

9.6 Integrated Analytical Strategy

In practice, the analysis of protein stability is conducted on an orthogonal basis. The method is a combination of structural methods (CD, FTIR), thermal methods (DSC), separation methods (SEC, HPLC), bioactivity tests or functional tests. This comprehensive characterization and regulatory compliance is ensured. (41,46)

10. LIMITATIONS AND PROBLEMS IN PROTEIN PRESERVATION

The preservation of effective protein biomolecules remains a critical issue despite the enormous progress of biotechnology and formulation science. Protein complexity and manufacturing, storage and regulatory constraints are barriers in the production of globally stable protein formulations. (48,105)

10.1 Trade-Off between Stability and Bioactivity

One of the most significant issues in the preservation of protein is finding a balance between bioactivity and structural integrity.

• Techniques That can be used to improve physical stability: Chemical modification, crosslinking, use of heavy excipients, however may also change:

1. Sites that are active
2. Regions that bind receptors
3. Flexibility in conformity

• Excessive stabilization may cause reduced biological activity on enzymes and receptor-mediated therapy.

Preservation methods should thus ensure that proteins are not only preserved in their structure but they are also preserved in their functional activity which is often difficult to predict and control. (25)

10.2 Scale-Up and Manufacturing Issues

At scale-up to industrial levels, the preservation methods that have been effective in the laboratory are often faced with difficulties.Some of the significant issues include shear stress during pumping and filtering, freeze-thaw stress during bulk storage, batch-to-batch variability and the cost and complexity of advanced excipients or encapsulation system. (64,67)As an example, the limiting capabilities of heat and mass transport can make it difficult to apply lyophilization cycles that are optimized on a small scale to large-scale production. (8)

10.3 Cold-Chain Dependence, Transportation and Storage

Most protein-based products require storage at 2-8 degC thus cold-chain logistics is very important.Changes in temperatures during transit, absence of cold-chain system in low and middle-income countries, increased distribution costs and unintended freezing, which may lead to aggregation.Cold-chain-dependencies also represent a harsh limitation to accessibility in the global context, particularly in the case of vaccines and biologics used in public health programs.

11. CURRENT GAPS IN RESEARCH AND INNOVATION OPPORTUNITIES

The field of protein preservation has a number of significant gaps that remain unexplored after decades of research, which provides an opportunity of innovation.(106,107)

11.1 Limitations of the Preservation Technologies

The existing strategies often:

• Only target specific pathways of degradation
• Require complex processing
• High dependence on refrigeration
• Do not have general applicability to different classes of proteins.

The need to have platform technologies can be explained by the fact that no single preservation method can be used on all proteins. (15,35,45,108)

11.2 Requirement of Room-Temperature Stable Protein Formulations

One of the largest unmet needs is the formation of thermostable protein compositions. Some of the advantages of room-temperature stability include the removal of cold-chain logistics, reduced cost, improved patient compliance and distribution across the globe.This has been vigorously tackled by studies on vitrification, solid-state stabilization, and biomimetic methods, but has not yet been put into practice. (92)

11.3 Absence of Universal Preservation Platforms

Proteins differ in size, structure, charge and function. This leads to time extension in development, complexity in manufacturing, and often formulations require to be formulated on a case-by-case basis.The absence of universal preservation platforms is still one of the largest challenges to the development of biopharmaceuticals.

11.4 Multidisciplinary Approaches Opportunities

Further development requires integration:

Protein chemistry, pharmaceutics sciences, materials science, computational biology and nanotechnology.Multidisciplinary approaches can be used to accelerate the creation of innovative preservation systems that have the potential to resolve multiple stability problems simultaneously. (109,110)

12. FUTURE PERSPECTIVES

The future of protein preservation is smart, predictive, and sustainable solutions that reduce the dependence of traditional methods of preservation. (106,111,112)

12.1 Smart Preservation Systems

Smart systems respond in a dynamic way whenever they are stressed by the environment through:

• Encapsulation Polymers or responsive matrix
• Releasing stabilizers in case of need
• Alteration of the microenvironment of proteins

These devices have controlled protection under different storage and transport conditions.

12.2 Artificial Intelligence and Computational Protein Stability Prediction

Some of the increasing applications of artificial intelligence and machine learning include predicting degradation pathways, optimizing formulation compositions, identifying stabilizing mutations and reducing experimental trial-and-error.Computational methods have the potential of increasing the success rates in formulations and significantly shortening development times. (12)

12.3 Green and Sustainable Preservation TechniquesIn response to environmental concern, green excipients, biodegradable polymers, ionic liquids, deep eutectic solvents and energy efficient drying methods are all being developed.International aims to reduce environmental impact and maintain the quality of pharmaceuticals are consistent with sustainable preservation methods.(47)

CONCLUSION

Protein biomolecules are a critical component of modern biotechnology, medicines and diagnostics. Nevertheless, preservation has serious concerns because of their complex structure and vulnerability to environmental pressure. Protein preservative and biological activity: To preserve the protein integrity and preserve the biological activity, one needs to know the mechanisms involved in protein degradation, the stable factors essential in the preservation of proteins as well as the processes involving the use of appropriate preservation methods. Although older methods, such as lyophilization and refrigeration, are still frequently used, alternative approaches such as protein engineering, encapsulation, biomimetic systems and advanced solvent methods all offer a potential alternative to overcome the existing limitations. Analytical tools provide important tools of evaluating stability and ensuring compliance to the regulations.To develop high-quality, low-cost, and broadly available protein formulations, which ultimately will enhance patient outcomes and biopharmaceutical science, additional research and interdisciplinary invention is needed. (8,11,61,75)

REFERENCES

1. Baud F, Karlin S. Measures of residue density in protein structures. Proc Natl Acad Sci. 1999 Oct 26;96(22):12494–9.
2. Salvatore L. A Comprehensive Review of Protein Purification Techniques: Advancements, Challenges, and Future Prospects.
3. Woods EJ, Thirumala S, Badhe-Buchanan SS, Clarke D, Mathew AJ. Off the shelf cellular therapeutics: Factors to consider during cryopreservation and storage of human cells for clinical use. Cytotherapy. 2016 June;18(6):697–711.
4. Büttel IC, Chamberlain P, Chowers Y, Ehmann F, Greinacher A, Jefferis R, et al. Taking immunogenicity assessment of therapeutic proteins to the next level. Biologicals. 2011 Mar;39(2):100–9.
5. Mamipour M, Yousefi M, Hasanzadeh M. An overview on molecular chaperones enhancing solubility of expressed recombinant proteins with correct folding. Int J Biol Macromol. 2017 Sept;102:367–75.
6. Uchio T, Baudyš M, Liu F, Song SC, Kim SW. Site-specific insulin conjugates with enhanced stability and extended action profile. Adv Drug Deliv Rev. 1999 Feb;35(2–3):289–306.
7. Burnett MJB, Burnett AC. Therapeutic recombinant protein production in plants: Challenges and opportunities. PLANTS PEOPLE PLANET. 2020 Mar;2(2):121–32.
8. Daqun L. Food Preservation by Using Traditional Techniques and Modern Industrial Techniques.
9. Engelking LR. Protein Structure. In: Textbook of Veterinary Physiological Chemistry [Internet]. Elsevier; 2015 [cited 2026 Jan 8]. p. 18–25. Available from: https://linkinghub.elsevier.com/retrieve/pii/B9780123919090500049
10. Naing AH, Kim CK. A brief review of applications of antifreeze proteins in cryopreservation and metabolic genetic engineering. 3 Biotech. 2019 Sept;9(9):329.
11. Rk I. Conventional and Modern Methods of Preservation of Foods. Food Sci Nutr Technol. 2019 Sept 19;4(5):1–6.
12. Robinson NE, Robinson AB. Prediction of protein deamidation rates from primary and three-dimensional structure. Proc Natl Acad Sci. 2001 Apr 10;98(8):4367–72.
13. Deutscher MP. Chapter 10 Maintaining Protein Stability. In: Methods in Enzymology [Internet]. Elsevier; 2009 [cited 2026 Jan 8]. p. 121–7. Available from: https://linkinghub.elsevier.com/retrieve/pii/S007668790963010X
14. Chen Y, Ling J, Li M, Su Y, Arte KS, Mutukuri TT, et al. Understanding the Impact of Protein–Excipient Interactions on Physical Stability of Spray-Dried Protein Solids. Mol Pharm. 2021 July 5;18(7):2657–68.
15. Teufl M, Zajc CU, Traxlmayr MW. Engineering Strategies to Overcome the Stability–Function Trade-Off in Proteins. ACS Synth Biol. 2022 Mar 18;11(3):1030–9.
16. Lan T, Dong Y, Jiang L, Zhang Y, Sui X. Analytical approaches for assessing protein structure in protein-rich food: A comprehensive review. Food Chem X. 2024 June;22:101365.
17. Fija?kowski P, Pryshchepa O, Van Eldik R, Pomastowski P. Ovotransferrin – Multifunctional protein: Structure, bioactivity, and industrial potential. Int J Biol Macromol. 2025 June;317:144810.
18. Goldenzweig A, Fleishman SJ. Principles of Protein Stability and Their Application in Computational Design. Annu Rev Biochem. 2018 June 20;87(1):105–29.
19. Brandts JF. The Thermodynamics of Protein Denaturation. 11. A Model of Reversible Denaturation and Interpretations Regarding the Stability of Chymotrypsinogen.
20. Kauzmann W. Some Factors in the Interpretation of Protein Denaturation. In: Advances in Protein Chemistry [Internet]. Elsevier; 1959 [cited 2026 Jan 8]. p. 1–63. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0065323308606087
21. Ohtake S, Kita Y, Arakawa T. Interactions of formulation excipients with proteins in solution and in the dried state. Adv Drug Deliv Rev. 2011 Oct;63(13):1053–73.
22. Anfinsen CB. Principles that Govern the Folding of Protein Chains. Science. 1973 July 20;181(4096):223–30.
23. Akers MJ. Excipient–Drug Interactions in Parenteral Formulations. J Pharm Sci. 2002 Nov;91(11):2283–300.
24. Krause ME, Sahin E. Chemical and physical instabilities in manufacturing and storage of therapeutic proteins. Curr Opin Biotechnol. 2019 Dec;60:159–67.
25. Zapadka KL, Becher FJ, Gomes Dos Santos AL, Jackson SE. Factors affecting the physical stability (aggregation) of peptide therapeutics. Interface Focus. 2017 Dec 6;7(6):20170030.
26. Tanford C. PROTEIN DENATURATION.
27. Moorthy BS, Schultz SG, Kim SG, Topp EM. Predicting Protein Aggregation during Storage in Lyophilized Solids Using Solid State Amide Hydrogen/Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS). Mol Pharm. 2014 June 2;11(6):1869–79.
28. Robinson NE. Protein deamidation. Proc Natl Acad Sci. 2002 Apr 16;99(8):5283–8.
29. Mycek MJ, Waelsch H. The Enzymatic Deamidation of Proteins. J Biol Chem. 1960 Dec;235(12):3513–7.
30. Ji JA, Zhang B, Cheng W, Wang YJ. Methionine, tryptophan, and histidine oxidation in a model protein, PTH: Mechanisms and stabilization. J Pharm Sci. 2009 Dec;98(12):4485–500.
31. Davies MJ. The oxidative environment and protein damage. Biochim Biophys Acta BBA - Proteins Proteomics. 2005 Jan;1703(2):93–109.
32. Lambeth TR, Riggs DL, Talbert LE, Tang J, Coburn E, Kang AS, et al. Spontaneous Isomerization of Long-Lived Proteins Provides a Molecular Mechanism for the Lysosomal Failure Observed in Alzheimer’s Disease. ACS Cent Sci. 2019 Aug 28;5(8):1387–95.
33. Speck O, Speck T. An Overview of Bioinspired and Biomimetic Self-Repairing Materials. Biomimetics. 2019 Mar 20;4(1):26.
34. Ramos R, Bernard J, Ganachaud F, Miserez A. Protein?Based Encapsulation Strategies: Toward Micro? and Nanoscale Carriers with Increased Functionality. Small Sci. 2022 Mar;2(3):2100095.
35. Wang W, Nema S, Teagarden D. Protein aggregation—Pathways and influencing factors. Int J Pharm. 2010 May;390(2):89–99.
36. Robinson NE, Robinson AB. Deamidation of human proteins. Proc Natl Acad Sci. 2001 Oct 23;98(22):12409–13.
37. Dozier J, Distefano M. Site-Specific PEGylation of Therapeutic Proteins. Int J Mol Sci. 2015 Oct 28;16(10):25831–64.
38. Mensink MA, Frijlink HW, Van Der Voort Maarschalk K, Hinrichs WLJ. How sugars protect proteins in the solid state and during drying (review): Mechanisms of stabilization in relation to stress conditions. Eur J Pharm Biopharm. 2017 May;114:288–95.
39. Taghavi F, Habibi-Rezaei M, Amani M, Saboury AA, Moosavi-Movahedi AA. The status of glycation in protein aggregation. Int J Biol Macromol. 2017 July;100:67–74.
40. Yang K, Bian C, Ma X, Mei J, Xie J. Recent advances in emerging techniques for freezing and thawing on aquatic products’ quality. J Food Process Preserv [Internet]. 2022 Sept [cited 2026 Jan 8];46(9). Available from: https://onlinelibrary.wiley.com/doi/10.1111/jfpp.16609
41. Kamerzell TJ, Esfandiary R, Joshi SB, Middaugh CR, Volkin DB. Protein–excipient interactions: Mechanisms and biophysical characterization applied to protein formulation development. Adv Drug Deliv Rev. 2011 Oct;63(13):1118–59.
42. Hawkins CL, Davies MJ. Generation and propagation of radical reactions on proteins. Biochim Biophys Acta BBA - Bioenerg. 2001 Apr;1504(2–3):196–219.
43. P?otka-Wasylka J, De La Guardia M, Andruch V, Vilková M. Deep eutectic solvents vs ionic liquids: Similarities and differences. Microchem J. 2020 Dec;159:105539.
44. Goldsmith M, Aggarwal N, Ashani Y, Jubran H, Greisen PJr, Ovchinnikov S, et al. Overcoming an optimization plateau in the directed evolution of highly efficient nerve agent bioscavengers. Protein Eng Des Sel. 2017 Apr;30(4):333–45.
45. Kintzing JR, Filsinger Interrante MV, Cochran JR. Emerging Strategies for Developing Next-Generation Protein Therapeutics for Cancer Treatment. Trends Pharmacol Sci. 2016 Dec;37(12):993–1008.
46. Chen Y, Mutukuri TT, Wilson NE, Zhou Q (Tony). Pharmaceutical protein solids: Drying technology, solid-state characterization and stability. Adv Drug Deliv Rev. 2021 May;172:211–33.
47. Chen Y, Mutukuri TT, Wilson NE, Zhou Q (Tony). Pharmaceutical protein solids: Drying technology, solid-state characterization and stability. Adv Drug Deliv Rev. 2021 May;172:211–33.
48. Pinto JT, Faulhammer E, Dieplinger J, Dekner M, Makert C, Nieder M, et al. Progress in spray-drying of protein pharmaceuticals: Literature analysis of trends in formulation and process attributes. Dry Technol. 2021 Aug 2;39(11):1415–46.
49. Nick Pace C, Scholtz JM, Grimsley GR. Forces stabilizing proteins. FEBS Lett. 2014 June 27;588(14):2177–84.
50. Bowie J. Stabilizing membrane proteins. Curr Opin Struct Biol. 2001 Aug 1;11(4):397–402.
51. Jamróz E, Kopel P. Polysaccharide and Protein Films with Antimicrobial/Antioxidant Activity in the Food Industry: A Review. Polymers. 2020 June 4;12(6):1289.
52. Nakauchi Y, Nishinami S, Shiraki K. Glass-like protein condensate for the long-term storage of proteins. Int J Biol Macromol. 2021 July;182:162–7.
53. Zajac JWP, Muralikrishnan P, Heldt CL, Perry SL, Sarupria S. Towards stable biologics: understanding co-excipient effects on hydrophobic interactions and solvent network integrity. Mol Syst Des Eng. 2025;10(6):432–46.
54. Mensink MA, Frijlink HW. How sugars protect proteins in the solid state and during drying (review): Mechanisms of stabilization in relation to stress conditions.
55. Karunnanithy V, Abdul Rahman NHB, Abdullah NAH, Fauzi MB, Lokanathan Y, Min Hwei AN, et al. Effectiveness of Lyoprotectants in Protein Stabilization During Lyophilization. Pharmaceutics. 2024 Oct 21;16(10):1346.
56. Lee J, Lin EW, Lau UY, Hedrick JL, Bat E, Maynard HD. Trehalose Glycopolymers as Excipients for Protein Stabilization. Biomacromolecules. 2013 Aug 12;14(8):2561–9.
57. Wen L, Zheng X, Wang X, Lan H, Yin Z. Bilateral Effects of Excipients on Protein Stability: Preferential Interaction Type of Excipient and Surface Aromatic Hydrophobicity of Protein. Pharm Res. 2017 July;34(7):1378–90.
58. Chodankar S, Aswal VK, Kohlbrecher J, Vavrin R, Wagh AG. Structural evolution during protein denaturation as induced by different methods. Phys Rev E. 2008 Mar 4;77(3):031901.
59. Kheto A, Adhikary U, Dhua S, Sarkar A, Kumar Y, Vashishth R, et al. A review on advancements in emerging processing of whey protein: Enhancing functional and nutritional properties for functional food applications. Food Saf Health. 2025 Jan;3(1):23–45.
60. Jones BJ, Lim HY, Huang J, Kazlauskas RJ. Comparison of Five Protein Engineering Strategies for Stabilizing an α/β-Hydrolase. Biochemistry. 2017 Dec 19;56(50):6521–32.
61. Lane MD, Seelig B. Advances in the directed evolution of proteins. Curr Opin Chem Biol. 2014 Oct;22:129–36.
62. Roberts CJ. Therapeutic protein aggregation: mechanisms, design, and control. Trends Biotechnol. 2014 July;32(7):372–80.
63. Alber T. Mutational Effects on Protein Stability.
64. Tang J, Liu J, Zheng Q, Li W, Sheng J, Mao L, et al. In?Situ Encapsulation of Protein into Nanoscale Hydrogen?Bonded Organic Frameworks for Intracellular Biocatalysis. Angew Chem Int Ed. 2021 Oct 4;60(41):22315–21.
65. Moreira A, Lawson D, Onyekuru L, Dziemidowicz K, Angkawinitwong U, Costa PF, et al. Protein encapsulation by electrospinning and electrospraying. J Controlled Release. 2021 Jan;329:1172–97.
66. Blocher McTigue WC, Perry SL. Protein Encapsulation Using Complex Coacervates: What Nature Has to Teach Us. Small. 2020 July;16(27):1907671.
67. Mohan A, Rajendran SRCK, He QS, Bazinet L, Udenigwe CC. Encapsulation of food protein hydrolysates and peptides: a review. RSC Adv. 2015;5(97):79270–8.
68. Hawe A, Wiggenhorn M, Van De Weert M, Garbe JHO, Mahler H christian, Jiskoot W. Forced Degradation of Therapeutic Proteins. J Pharm Sci. 2012 Mar;101(3):895–913.
69. Colletier JP, Chaize B, Winterhalter M, Fournier D. Protein encapsulation in liposomes: efficiency depends on interactions between protein and phospholipid bilayer. BMC Biotechnol. 2002 May 10;2(1):9.
70. Ramos R, Bernard J, Ganachaud F, Miserez A. Protein?Based Encapsulation Strategies: Toward Micro? and Nanoscale Carriers with Increased Functionality. Small Sci. 2022 Mar;2(3):2100095.
71. Wang W. Lyophilization and development of solid protein pharmaceuticals. Int J Pharm. 2000 Aug;203(1–2):1–60.
72. Moreira A, Lawson D, Onyekuru L, Dziemidowicz K, Angkawinitwong U, Costa PF, et al. Protein encapsulation by electrospinning and electrospraying. J Controlled Release. 2021 Jan;329:1172–97.
73. Saboury AA, Miroliae M, Nemat-Gorgani M, Moosavi-Movahedi AA. Kinetics denaturation of yeast alcohol dehydrogenase and the effect of temperature and trehalose. An isothermal microcalorimetry study. Thermochim Acta. 1999 Feb;326(1–2):127–31.
74. Acharya VV, Chaudhuri P. Modalities of Protein Denaturation and Nature of Denaturants. Int J Pharm Sci Rev Res [Internet]. 2021 Aug 15 [cited 2026 Jan 8];69(2). Available from: https://globalresearchonline.net/journalcontents/v69-2/02.pdf
75. Teufl M, Zajc CU, Traxlmayr MW. Engineering Strategies to Overcome the Stability–Function Trade-Off in Proteins. ACS Synth Biol. 2022 Mar 18;11(3):1030–9.
76. Blocher McTigue WC, Perry SL. Protein Encapsulation Using Complex Coacervates: What Nature Has to Teach Us. Small. 2020 July;16(27):1907671.
77. Roberts CJ. Therapeutic protein aggregation: mechanisms, design, and control. Trends Biotechnol. 2014 July;32(7):372–80.
78. Song JG, Lee SH, Han HK. The stabilization of biopharmaceuticals: current understanding and future perspectives. J Pharm Investig. 2017 Nov;47(6):475–96.
79. Khodabandehloo A, Chen DDY. Particle Sizing Methods for The Detection of Protein Aggregates in Biopharmaceuticals. Bioanalysis. 2017 Feb;9(3):313–26.
80. Elsayed YY, Kühl T, Imhof D. Regulatory Guidelines for the Analysis of Therapeutic Peptides and Proteins. J Pept Sci. 2025 Mar;31(3):e70001.
81. Guziewicz NA, Massetti AJ, Perez-Ramirez BJ, Kaplan DL. Mechanisms of monoclonal antibody stabilization and release from silk biomaterials. Biomaterials. 2013 Oct;34(31):7766–75.
82. Jahn EM, Schneider CK. How to systematically evaluate immunogenicity of therapeutic proteins – regulatory considerations. New Biotechnol. 2009 June;25(5):280–6.
83. Dingman R, Balu-Iyer SV. Immunogenicity of Protein Pharmaceuticals. J Pharm Sci. 2019 May;108(5):1637–54.
84. Miklos AE, Kluwe C, Der BS, Pai S, Sircar A, Hughes RA, et al. Structure-Based Design of Supercharged, Highly Thermoresistant Antibodies. Chem Biol. 2012 Apr;19(4):449–55.
85. Miklos AE, Kluwe C, Der BS, Pai S, Sircar A, Hughes RA, et al. Structure-Based Design of Supercharged, Highly Thermoresistant Antibodies. Chem Biol. 2012 Apr;19(4):449–55.
86. Pham NB, Meng WS. Protein aggregation and immunogenicity of biotherapeutics. Int J Pharm. 2020 July;585:119523.
87. Fontana A, De Filippis V, De Laureto PP, Scaramella E, Zambonin M. Rigidity of Thermophilic Enzymes. In: Progress in Biotechnology [Internet]. Elsevier; 1998 [cited 2026 Jan 8]. p. 277–94. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0921042398800439
88. Huerta-Viga A, Woutersen S. Protein Denaturation with Guanidinium: A 2D-IR Study. J Phys Chem Lett. 2013 Oct 17;4(20):3397–401.
89. Ma B, Guan X, Li Y, Shang S, Li J, Tan Z. Protein Glycoengineering: An Approach for Improving Protein Properties. Front Chem. 2020 July 23;8:622.
90. Limpikirati PK, Mongkoltipparat S, Denchaipradit T, Siwasophonpong N, Pornnopparat W, Ramanandana P, et al. Basic regulatory science behind drug substance and drug product specifications of monoclonal antibodies and other protein therapeutics. J Pharm Anal. 2024 June;14(6):100916.
91. Wang W. Integration of Regulatory Guidelines into Protein Drug Product Development. PDA J Pharm Sci Technol. 2016 Jan 1;70(1):2–11.
92. Wang W, Ohtake S. Science and art of protein formulation development. Int J Pharm. 2019 Sept;568:118505.
93. Bolje A, Gobec S. Analytical Techniques for Structural Characterization of Proteins in Solid Pharmaceutical Forms: An Overview. Pharmaceutics. 2021 Apr 11;13(4):534.
94. Tatford OC, Gomme PT, Bertolini J. Analytical techniques for the evaluation of liquid protein therapeutics. Biotechnol Appl Biochem. 2004 Aug;40(1):67–81.
95. Eyes TJ, Austerberry JI, Dearman RJ, Johannissen LO, Kimber I, Smith N, et al. Identification of B cell epitopes enhanced by protein unfolding and aggregation. Mol Immunol. 2019 Jan;105:181–9.
96. Creighton TE. Experimental studies of protein folding and unfolding. Prog Biophys Mol Biol. 1979;33:231–97.
97. Barth A. Infrared spectroscopy of proteins. Biochim Biophys Acta BBA - Bioenerg. 2007 Sept;1767(9):1073–101.
98. Bommana R, Chai Q, Schöneich C, Weiss WF, Majumdar R. Understanding the Increased Aggregation Propensity of a Light-Exposed IgG1 Monoclonal Antibody Using Hydrogen Exchange Mass Spectrometry, Biophysical Characterization, and Structural Analysis. J Pharm Sci. 2018 June;107(6):1498–511.
99. Schön A, Clarkson BR, Jaime M, Freire E. Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry. Proteins Struct Funct Bioinforma. 2017 Nov;85(11):2009–16.
100. Davies MJ. Protein oxidation and peroxidation. Biochem J. 2016 Apr 1;473(7):805–25.
101. Madhavai S. A REVIEW ON CHROMATOGRAPHIC TECHNIQUES AND THEIR APPLICATION IN ANALYSIS OF PHARMACEUTICALS. 2024;9(2).
102. Bee JS, Goletz TJ, Ragheb JA. The future of protein particle characterization and understanding its potential to diminish the immunogenicity of biopharmaceuticals: A shared perspective. J Pharm Sci. 2012 Oct;101(10):3580–5.
103. Lafarga T, Hayes M. Bioactive protein hydrolysates in the functional food ingredient industry: Overcoming current challenges. Food Rev Int. 2017 May 4;33(3):217–46.
104. Saboti? J, Bayram E, Ezra D, Gaudêncio SP, Haznedaro?lu BZ, Janež N, et al. A guide to the use of bioassays in exploration of natural resources. Biotechnol Adv. 2024 Mar;71:108307.
105. Kamerzell TJ, Esfandiary R, Joshi SB, Middaugh CR, Volkin DB. Protein–excipient interactions: Mechanisms and biophysical characterization applied to protein formulation development. Adv Drug Deliv Rev. 2011 Oct;63(13):1118–59.
106. Carter PJ. Introduction to current and future protein therapeutics: A protein engineering perspective. Exp Cell Res. 2011 May;317(9):1261–9.
107. Song JG, Lee SH, Han HK. The stabilization of biopharmaceuticals: current understanding and future perspectives. J Pharm Investig. 2017 Nov;47(6):475–96.
108. Devlieghere F, Vermeiren L, Debevere J. New preservation technologies: Possibilities and limitations. Int Dairy J. 2004 Apr;14(4):273–85.
109. Neurath H. THE CHEMISTRY O F PROTEIK DESATURATION.
110. Sridhar A, Ponnuchamy M, Kumar PS, Kapoor A. Food preservation techniques and nanotechnology for increased shelf life of fruits, vegetables, beverages and spices: a review. Environ Chem Lett. 2021 Apr;19(2):1715–35.
111. Deller MC, Kong L, Rupp B. Protein stability: a crystallographer’s perspective. Acta Crystallogr Sect F Struct Biol Commun. 2016 Feb 1;72(2):72–95.
112. Song JG, Lee SH, Han HK. The stabilization of biopharmaceuticals: current understanding and future perspectives. J Pharm Investig. 2017 Nov;47(6):475–96.