Overview of protein and peptide degradation pathways

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Overview of protein and peptide degradation pathways

Overview of protein and peptide degradation pathways. In order to ensure product safety and efficacy, protein therapeutic preparations must be able to achieve the specified quality characteristics after manufacture and at the end of the specified shelf life.

Many physical and chemical factors will affect the quality and stability of biopharmaceutical products, especially after the biological agent is stored in a closed container system for a long time, temperature, light, and agitation during transportation and handling may change the biological agent.

Compared with traditional chemical drugs, proteins have more complex physical and chemical characteristics and larger molecular entities, ranging from its primary amino acid sequence to higher-level secondary and tertiary structures, and in some cases also include subunits such as Concord four-level elements.

Many proteins are glycosylated, and some proteins have other forms of post-translational modifications, such as phosphorylation, which are also related factors that affect their potential degradation pathways and their degradation kinetics. Proteins are usually sensitive to small changes in the chemical properties of the solution.

They only maintain composition and conformation stability within a relatively narrow range of pH and osmotic pressure, and many solutions also require additional supportive formulation ingredients to keep the protein in the solution stably, especially over time, even Even freeze-dried protein products can also be degraded.

Advances in analytical chemistry have determined that over time, many degradation pathways may occur in recombinant protein therapeutic products. These pathways produce chemical or physical instability. Chemical instability refers to the formation or destruction of covalent bonds within the structure of a polypeptide or protein. Chemical modification of proteins includes oxidation, deamidation, reduction, and hydrolysis.

The unfolding, dissociation, denaturation, aggregation and precipitation of proteins are called conformational or physical instability. In some cases, protein degradation pathways have a synergistic effect: chemical events may trigger physical events, such as aggregation after oxidation.

Here, we introduced several protein degradation pathways: oxidation, photodegradation, disulfide bond changes, deamidation, aggregation, precipitation, dissociation and cleavage. We exemplified the biochemical characteristics of each, showed the potential means of inducing changes, and proposed precautions for preparations for prevention. And end with the detection method and the strategy of verifying the stability indicator method.

Our goal is to provide an introduction (or review) for the main degradation pathways of protein products, and to provide references for each pathway. We encourage readers of “Yao Shikong” to consult these references for expanded details on the basic biochemistry of each pathway, case studies describing specific protein experiments, and more information on formulation development strategies.

Oxidation, photodegradation and disulfide changes

Certain amino acids react with oxygen free radicals in the environment, and proteins and peptides are susceptible to oxidative damage. Methionine, cysteine, histidine, tryptophan and tyrosine are most easily oxidized: Met and Cys are oxidized due to their sulfur atoms, while His, Trp and Tyr are oxidized due to their aromatic rings. Oxidation can change the physicochemical properties of proteins (such as folding and subunit association) and cause aggregation or fragmentation. Depending on the position of the oxidized amino acid in the protein relative to its functional or epitope-like domain, it may also have a potentially negative impact on potency and immunogenicity.

For example, the difference in the biological activity of parathyroid hormone is affected by either Met-8 or Met-18 and double-oxidized mono-oxidation (MET-8 is Met-18) when separated from each species and used in in vitro bioassays for detection law. Similarly, the oxidation of Met-36 and Met-48 in human stem cell factor (huSCF) derived from Escherichia coli reduced their potency by 40% and 60%, respectively, and at the same time increased the dissociation rate constant of the SCF dimer by 2 to 3 times, indicating an influence on subunit binding and tertiary structure. In other cases, even if substantial structural changes are seen, oxidation has no measurable effect on protein performance. For example, the oxidized Met-111 in interferon α-2b affects the primary, secondary and tertiary structure of the molecule, and prevents the recognition of site-specific epitopes by monoclonal antibodies (MAb) without changing the in vitro Biological activity.

Mechanisms and factors involved: Figure 1 below shows the biochemical pathways of oxidation of methionine and cysteine residues. Methionine is oxidized by atmospheric oxygen and oxygen free radicals in the solution to form methionine sulfoxide and methionine sulfone. Both of these are larger and more polar than unoxidized methionine, which can change the folding and structural stability of the protein. At alkaline pH, hydrogen peroxide can increase the oxidation rate of methionine in recombinant human parathyroid hormone (rHu-PTH).

Overview of protein and peptide degradation pathways Figure 1 Biochemical pathways of oxidation of methionine and cysteine residues

The oxidation of cysteine is also more common at alkaline pH, deprotonating sulfhydryl groups. The oxidation of cysteine induces disulfide bond breakage in a reducing environment (Figure 1, bottom panel). In such an environment, cysteine oxidation involves the nucleophilic attack of thiolate ions on disulfide bonds, resulting in new disulfide bonds and different thiolate ions. The new thiolate can then react with another disulfide bond to form cysteine.

This intermolecular disulfide bond formed by protein degradation will accumulate mismatched disulfide bonds and chaotic disulfide bonds, thereby changing protein conformation and subunit association.

In the presence of metal ions or nearby sulfhydryl groups, cysteine residues may also spontaneously oxidize to form molecular by-products sulfinic acid and cysteine. For example, human fibroblast growth factor (FGF-1) exhibits copper-catalyzed oxidation and can produce homodimers.

The spatial orientation of sulfhydryl groups in proteins plays an important role in the oxidation of cysteine. The rate of oxidation is inversely proportional to the distance between those thiol groups. Like basic fibroblast growth factor (bFGF), this may eventually lead to the formation of large oligomers or non-functional monomers, while basic fibroblast growth factor (bFGF) contains three molecules that are easily oxidized and formed Intermolecular or intramolecular disulfide bond cysteine.

This oxidation usually induces conformational modification of the protein, because the cysteine disulfide bond increases the side chain volume inside the protein and leads to unfavorable van der Waals interactions, thereby maintaining the original structure.

Histidine residues are highly sensitive to oxidation through the reaction with the imidazole ring and can subsequently generate other hydroxyl groups. Oxidized histidine can be degraded by light source and/or metal oxidation (to produce asparagine/aspartic acid and 2-oxo-histidine (2-O-His) as degradation products. It may be a transient part , Because it can trigger protein aggregation and precipitation, which makes the separation of 2-O-His (as a separate degradation product) difficult to understand.

The oxidation of tyrosine can lead to covalent aggregation through the formation of tyrosine. Space factor It may also affect the oxidation of tyrosine and histidine. Neighboring negatively charged amino acids accelerate the oxidation of tyrosine because they have a high affinity for metal ions, while positively charged amino acid residues are not conducive to the reaction. Large adjacent amino acids may mask the oxidation of adjacent amino acids and prevent them from being oxidized.

It has been observed that the presence of histidine in sequence significantly increases the oxidation rate of peptides and the production of methionine sulfoxide. The strong metal binding affinity of the imidazole ring on the side chain of histidine makes the oxidizing substance close to the substrate methionine.

Photodegradation: Photooxidation can change the primary, secondary, and tertiary structure of proteins and cause differences in long-term stability, biological activity, or immunogenicity. Exposure to light will trigger a series of biochemical events, even after turning off the light source, these biochemical changes will continue to affect the protein. These effects depend on the energy imparted to the protein and the presence of ambient oxygen.

When a compound absorbs a certain wavelength of light, it triggers photooxidation, which provides energy to elevate the molecule to an excited state. The excited molecules can then transfer this energy to molecular oxygen, converting it into reactive singlet oxygen atoms. This is how tryptophan, histidine and tyrosine can be modified under light in the presence of O. The photooxidation of tyrosine can produce mono-, di-, tri- and tetra-hydroxy tyrosine as a by-product. Due to cross-links between oxidized tyrosine residues, aggregation is observed in certain proteins.

The photooxidation reaction is mainly site-specific. For example, in human growth hormone treated with strong light, oxidation mainly occurs at histidine. In addition, the peptide backbone is also a target for photodegradation. Alternatively, the energized protein itself can directly react with another protein molecule in a photosensitive manner, usually through methionine and tryptophan residues at low pH.

Excipients and leachables can synergistically affect the oxidation (and therefore degradation) of proteins.

In some cases, formula ingredients can affect the rate of photooxidation: for example, phosphate buffers can promote the degradation rate of methionine more than other buffer systems. The oxidation catalyzed by metal ions depends on the concentration of metal ions in the environment. The presence of 0.15-ppm chloride salt of Fe3+, Ca2+, Cu2+, Mg2+ or Zn2+ does not affect the oxidation rate of human insulin-like growth factor, but when the metal concentration increases to 1 ppm, a significant increase in oxidation is observed. In the presence of reducing agents (such as ascorbate), oxidation may be intensified. Ascorbic acid increases the oxidation of human ciliary neurotrophic factor. Likewise, the presence of denaturing/unfolding reagents in the solution will increase the degree of protein oxidation. Excipients involved in stabilizing protein structure (such as polyols and sugars) can reduce the rate of oxidation.

Oxidative modification depends on inherent structural features, such as buried and exposed amino acids. In the case of human growth hormone, Met-14 and Met-125 are easily oxidized by H2O2 because they are exposed on the surface of the protein, while Met-170 in a hidden position can only be oxidized when the molecule is oxidized. Unfold. Similarly, oxygen in the atmosphere will cause protein oxidation over time. Headspace oxygen in a multi-dose vial of tuberculin purified protein (TPP) caused a 50% loss of potency within four months.

During protein processing and storage, peroxide contamination usually caused by polysorbate and polyethylene glycol (PEG) used as pharmaceutical excipients can lead to oxidation. It has been observed that there is a correlation between the content of peroxide in Tween-80 and the degree of oxidation in rhG-CSF, and peroxide-induced oxidation appears to be more severe than oxidation from atmospheric oxygen. Peroxide can also be obtained from plastic or elastomeric materials used in primary packaging container closure systems, including pre-filled syringes.

Preventive measures: A molecular engineering strategy to minimize oxidative degradation is to replace oxygen-labile amino acids with oxygen-resistant amino acids if the nature of the protein permits. In therapeutic interferon β (IFN-β), the 17th cysteine is replaced by serine, because the former loses its antiviral activity during storage, leading to oxidation and disulfide bond competition. Replacing epidermal growth factor (EGF) methionine with non-naturally occurring norleucine can also prevent oxidative degradation.

In some cases, removing headspace oxygen by degassing may be effective in preventing oxidation. The filling step is performed under nitrogen pressure, and the oxygen in the headspace of the vial is replaced with an inert gas, such as nitrogen, to prevent oxidation. For some proteins that are sensitive to oxidation, they are treated in the presence of an inert gas (such as nitrogen or argon). For multi-dose pharmaceutical formulations, the use of cartridges with negligible headspace can overcome oxidation and related consequences.

When considering changing the tightness of the container, extreme care must be taken. Many of these changes in protein therapeutics (for example, from vials to pre-filled syringes or from pre-filled syringes to pen-type devices) are thought to improve patient convenience and ease of use. However, when the same material is used with protein-based products, the historical experience of container closure systems based solely on chemicals should be reassessed, as this may have unexpected and unique effects on protein degradation.

Controlling or enhancing factors such as pH, temperature, exposure, and buffer composition can also reduce oxidation by affecting the protein’s environment. The oxidation of cysteine can usually be controlled by maintaining the correct redox potential of the protein preparation, such as the addition of thioredoxin and glutathione. Antioxidants and metal chelating agents can also be used to prevent oxidation in protein formulations. Antioxidants are chemical “sacrifice targets” that have a strong tendency to oxidize and consume chemicals that promote oxidation.

For this reason, scavengers such as L-methionine and ascorbic acid are used in biotherapeutics. In the absence of metal ions, cysteine as a free amino acid can act as an effective antioxidant. As chelating agents, EDTA and citrate may form complexes with transition metal ions and inhibit metal-catalyzed site-specific oxidation. Due to the complexation of sugars and polyols with metal ions, the addition of sugars and polyols can also prevent metal-catalyzed oxidation. Human relaxin was used to observe the protective effects of glucose, mannitol, glycerol and ethylene glycol on the catalytic oxidation of metals. The use of primary or secondary packaging systems for physical protection from UV/white light may require protection of light-labile proteins from photo-oxidation.

Deamidation

For many recombinant proteins, changes in the structure of peptides and proteins can be observed through non-enzymatic deamidation of glutamine and asparagine residues. This may affect their physiochemical and functional stability. It has been observed that the deamidation of hGH alters the proteolytic cleavage of human growth hormone. It is reported that the deamidation of IFN-β increases its biological activity. It has been determined that compared with natural peptides, the deamidation of the peptide growth hormone releasing factor in aspartyl and isoaspartyl forms reduces the biological activity by 25-fold and 500-fold, respectively. The deamidation of Asn-Gly site in hemoglobin changes its affinity for oxygen. Deamidation of asparagine interferes with antigen presentation on the class II major histocompatibility complex molecules. It is reported that the isomerization of Asp 11 in human epidermal growth factor leads to a five-fold decrease in its mitogenic activity. The deamidation of the two Asn-Gly sequences in phosphotriose phosphate isomerase leads to the dissociation of subunits.

Mechanisms and factors involved: Deamidation is a chemical reaction in which the amide functional group is removed from the amino acid. Consequences include protein isomerization, racemization and truncation. Figure 2 below shows the mechanism of deamidation and degradation of asparagine.

Overview of protein and peptide degradation pathways Figure 2 shows the mechanism of asparagine degradation by deamidation.

Isomerization: The protein solution in the isomerization of aspartic acid to isoaspartic acid residues is a non-enzymatic deamidation (the most commonly observed result.
Racemization: The succinimide intermediate formed in the process of asparagine deamidation is very easy to racemize and convert to d-asparagine residues. The racemization of amino acids other than glycine was observed at alkaline pH.
Truncation: At low pH, the amide group on the asparagine side chain of peptides and proteins undergoes a deprotonation reaction, and then the nitrogen atom of the amidase nucleophilic attack on the peptide carbonyl carbon of the asparagine residue. This produces peptide chain cleavage by forming succinimide peptide fragments. Subsequent hydrolysis of the succinimide ring can produce asparaginyl and β-asparaginyl peptides.

The mechanism of aspartic acid-isoaspartic acid deamidation and isomerization reactions are similar because they both proceed through intramolecular cyclic imide intermediates. The deamidation rate of each amide residue depends on its primary sequence and three-dimensional (3D) structure and the properties of the solution, such as pH, temperature, ionic strength and buffer ions (45). The deamidation rate of glutamine residues is generally lower than that of asparagine residues.

If the pH is> 5.0, deamidation occurs through the formation of a very unstable cyclic imide intermediate, which will undergo spontaneous hydrolysis. Under strongly acidic conditions (pH 1-2), the direct hydrolysis of amide side chains is more advantageous than the formation of cyclic imides. Peptide bond cleavage occurs to a greater extent in the direct hydrolysis of amides. At neutral pH, deamidation can lead to structural isomerization.

The rate of deamidation is also affected by the secondary structure of the protein. The increase in the helical structure will reduce the deamidation rate of certain proteins. The relationship between the rate of deamidation in several growth hormone releasing factor analogues and the α-helical structure induced by methanol was investigated. The addition of methanol increases the level of α-helix and reduces the rate of deamidation. In its natural structure, RNAase can resist deamidation. This may be because the relatively rigid backbone in the ring is stabilized by the disulfide bond between Cys-8 and Cys-12 and the β-turn at residues 66-68. This may prevent the formation of ring structures. However, if it is reduced and denatured, it will refold to produce aspartic acid and isoaspartic acid forms, indicating different enzymatic activities. Substituting Iso-Asp-67 for Asp-67 shows that the refolding rate of the isoaspartic acid form is half that of the fully amidated form.

In the presence of certain biological buffers, storage temperature will affect the rate of protein deamidation. Because amine buffers (such as Tris and histidine) have a high temperature coefficient, storage at a temperature different from the preparation temperature may change the pH of the formulation. Deamidation and isomerization reactions are pH-sensitive processes, so those changes in formulation pH may change the rate of deamidation. Another indirect effect of temperature is the dissociation constant of water: the hydroxide ion concentration of water can change with temperature, thereby affecting the rate of deamidation.

Preventive measures: The pH value of the solution will seriously affect the deamidation effect. Preparations with a pH of 3-5 can minimize peptide deamidation. AsnA-21 and AsnB-3 of insulin form isoaspartic acid or aspartic acid according to the pH of the solution. Insulin is rapidly deaminated in a low pH solution of Asn A-21. The steric hindrance also affects the speed of the deamidation reaction: a large number of residues after asparagine may inhibit the formation of succinimide intermediates in the deamidation reaction. Replacing glycine residues with larger leucine or proline residues will cause this ratio to decrease by a factor of 30 to 50. In lyophilized formulations, the deamidation rate generally decreases, which may be due to the limited availability of free water in which the reaction can occur.

Formulations containing organic co-solvents can reduce the rate of deamidation, because the addition of organic solvents will reduce the dielectric constant of the solution. Reducing the dielectric strength of the solvent-by adding cosolutes such as glycerol, sucrose and ethanol in a protein solution-results in a significantly lower rate of isomerization and deamidation. Reducing the dielectric strength of the medium from 80 (water) to 35 (PVP/glycerol/water formula) resulted in a reduction in the peptide deamidation rate by about six times. The lower deamidation rate is attributed to the poorly stable ionic intermediates formed during the cyclization process in the asparagine deamidation pathway. Insulin prepared in a neutral solution containing phenol showed reduced deamidation, which may be due to its stabilizing effect on the tertiary structure (α-helix formation) surrounding the deamidated residues, thereby reducing the formation of intermediates Possibility of imide.

Aggregation and precipitation

Aggregates are an important concern for biopharmaceutical products because they may be related to decreased biological activity and increased immunogenicity. The macromolecular protein complex can trigger the patient’s immune system to recognize the protein as “non-self” and trigger an antigen response. Large polymer aggregates can also affect fluid dynamics in organ systems such as the eyes.

Aggregation is a common problem encountered during protein production and storage. Exposure of proteins to liquid-gas, liquid-solid, and even liquid-liquid interfaces generally enhances the potential for aggregation forms. The mechanical stress of agitation (shaking, stirring, pipetting or pumping through a test tube) can cause protein aggregation. Freezing and thawing can also promote it. Solution conditions (such as temperature, protein concentration, pH, and ionic strength) affect the rate and number of aggregates observed. When sucrose is hydrolyzed, the agent in sucrose increases aggregation over time due to protein glycosylation. The presence of certain ligands (including certain ligands) may enhance aggregation. The interaction with the metal surface can lead to epitaxial denaturation, which triggers the formation of aggregates. Impurity particles in the environment, manufacturing process, or container closure system (for example, silicone oil) can also induce polymerization. Even processing protein products in compound pharmacies can induce aggregation, the amount of which is 10 times higher than initially observed.

The effect of aggregation on the potency of the product varies according to the physiochemical properties of each protein relative to its functional domain and the nature of the measured activity. After shaking, enzymes such as urease and catalase may lose up to 50% of their effectiveness. After shear stress, the coagulation activity of fibrinogen will decrease, and the aggregation produced by shaking and shearing will seriously affect the recombinant IL -2 and recombinant interferon activity. Aggregation also affects the mass balance of the protein solution, reducing the concentration of the target protein. During the storage process, over time, the micro-aggregated sub-visible particles produced anywhere in the manufacturing process may develop into larger particles over time. After operation in the pharmacy, the bevacizumab drug product lost 50% of the active IgG, which resulted in significant growth of micron-sized particles in the repackaged solution.

Aggregates can be soluble or insoluble, reversible or irreversible, covalent or non-covalent. Soluble aggregates are usually reversible: for example, by changing solution conditions (for example, changing temperature or osmotic strength) or by slight physical destruction (for example, vortexing or filtering). Insoluble aggregates are usually irreversible. Under severe physical interference (for example, agitation or freezing and thawing) or during storage, over time, they may grow into particles that may eventually settle. When monomeric proteins are chemically cross-linked, for example through disulfide bonds, covalent aggregates are formed. Although covalent bonds are necessary to stabilize the natural tertiary structure of most polypeptide proteins, those formed by degradation can produce undesirable cross-links between protein parts, leading to irreversible aggregation. When proteins are combined and bound based on structural regions of charge or polarity, non-covalent aggregates are formed. Since such associations are weak (as opposed to covalent bonds), they are sensitive to solution conditions and are usually reversible.

Mechanisms and factors involved: Since many physical and chemical operations are required in upstream production and downstream processing, followed by compounding and filling operations, it can be used in almost every step of the process (including fixed points, transportation and transportation) Induce the aggregation of protein biopharmaceuticals. For long-term storage, agitating (for example, shaking, stirring, and shearing) the protein solution can promote aggregation at the gas-liquid interface, where protein molecules may align and unfold, exposing their hydrophobic regions to charge-based associations in. Shock-induced aggregation has been found in many protein products, including recombinant factor XIII, human growth hormone, hemoglobin and insulin. During manufacturing (and during product use), minimizing foam caused by agitation is essential to prevent significant loss of protein activity or the production of visible particulate matter.

Protein concentration can also promote aggregation, regardless of whether an agitation event occurs. The results obtained from two PEGylated proteins and one Fc fusion protein showed that there is a direct correlation between protein concentration and aggregation under non-agitated (still) conditions, but the researchers found that under shaking, vortexing and simulated transport conditions There is an inverse correlation between protein concentration and aggregation.

Antibacterial preservatives used in multi-dose formulations can also induce protein aggregation. For example, benzyl alcohol promotes the aggregation of rhGCSF because it favors the partially unfolded conformation of the protein. The increase in the content of antibacterial preservatives may increase the hydrophobicity of the preparation and may affect the water solubility of the protein.

Phenol and mcresol can significantly destroy proteins: phenol promotes the formation of soluble and insoluble aggregates, while mcresol can precipitate proteins.

Freezing and thawing-which may occur multiple times during the production and use of protein therapeutics-can greatly affect protein aggregation. The water ice crystals produced on the periphery of the container (where heat transfer is greatest) will produce a “salting out” effect, causing the protein and excipients to become increasingly concentrated in the slower freezing center of the container. High salt and/or high protein concentration can cause precipitation and aggregation during freezing, which cannot be completely reversed during thawing. The effect can be seen by hormones that stimulate the thyroid: when stored at −80°C, 4°C or 24°C for up to 90 days, it remains stable, but when frozen to −20°C it loses> 40% The effectiveness of that period was due to the dissociation of Aki. Multiple freezing and thawing cycles will exacerbate this effect and lead to a cumulative effect on the production and growth of sub-visible particles and visible particles. The change in pH may come from the crystallization of buffer components during freezing. In one study, the pH change of potassium phosphate buffer when frozen was much smaller than that of sodium phosphate buffer.

In general, the currently limited number of particles ≥ 10 microns and sizes ≥ 25 microns can be present in injectable pharmaceutical preparations. But what level of sub-visible particles. Similarly, there are no standard regulations for visible particles in protein drugs. Some biotech products have specifications for the visual appearance of drug solutions, including notes such as “essentially free of visible particles” or “some translucent particles may be present”).

Preventive measures: By optimizing the pH and ionic strength of the solution, the aggregation and precipitation of the protein solution can usually be stabilized. Add sugars, amino acids and/or polyols; and use surfactants. Comprehensive evaluation of optimal pH and osmotic conditions is a key element in the development of formulations to prevent protein aggregation or precipitation). Surfactants, polyols or sugars can prevent irreversible aggregation due to denaturation.

In many cases, adding non-ionic detergents (surfactants) can improve stability and prevent aggregation. The protein-surfactant interaction is hydrophobic, so these compounds stabilize the protein by reducing the surface tension of the solution and binding to hydrophobic sites on its surface, thereby reducing protein-protein interactions that may lead to the formation of aggregates possibility. Non-ionic detergents Tween 20 and Tween 80 can prevent the formation of soluble protein aggregates with a surfactant concentration below the critical micelle concentration (CMC). Polysorbate (Tween) 80 added to the IgG solution can stabilize small aggregates and prevent them from growing into larger particles. Chelating agents can also be used to prevent metal-induced protein aggregation.

Fragmentization

Multimeric proteins with two or more subunits can be dissociated into monomers, and monomers (or single peptide chain proteins) can be degraded into peptide fragments. Non-enzymatic fragmentation is usually carried out by hydrolysis of peptide bonds between amino acids, thereby releasing polypeptides with a molecular weight lower than that of the intact parent protein. The aspartate-glycine peptide bond and Asp-Pro are the most susceptible to cleavage by hydrolyzed proteins. Antibody hydrolysis usually occurs in the hinge region, which is the most flexible domain of antibodies.

However, lowering the pH from 9 to 5 can change the peptide hydrolysis site of the recombinant monoclonal antibody, showing increased cleavage outside this region.

The presence and location of oligosaccharides also affect the rate of peptide hydrolysis at low pH. According to the position, although the fragments in the CH 2 domain are reduced, the cleavage of the hinge region is not affected. Acidic and alkaline hydrolysis do not necessarily have the same effect on the hydrolytic cleavage of peptide bonds. Recombinant human macrophage colony stimulating factor produces different peptide fragments in acidic and alkaline pH solutions. The proteolytic activity of the residual or contaminating protease may cause the cleavage of the enzyme protein or, in some cases, the self-proteolysis of the enzyme protein.

Preventive measures: For each type of protein, proper buffering of the formulation to keep the pH of its solution within an appropriate range is the key to minimizing fragmentation by hydrolysis. For example, calcitonin undergoes hydrolysis at alkaline pH, but this degradation is not observed at pH 7 even at room temperature. The composition of the buffer may also affect the hydrolysis. Under the same pH and ionic strength, fragmentation of recombinant human macrophage colony stimulating factor was observed in phosphate buffer, but not in histidine buffer. It is also important to minimize the potential presence of proteases in protein purification from internal sources in the production process (such as host cell proteins) or exogenous contamination sources (such as adventitious microorganisms).

The natural structure of protein molecules is the result of balancing effects, such as covalent bonds, hydrophobic interactions, electrostatic interactions, hydrogen bonds and van der Waals forces. Protein stability is controlled by numerous internal and external factors, but the main factors are primary sequence, 3D structure, subunit association and post-translational modification. The external influencing factors include pH, osmolality, protein concentration, formulation excipients, and the physical pressure of the product under temperature, light and/or stirring. Leachables and environmental pollution (for example, metals and proteases) in the container closure system also exacerbate product degradation. In summary, all these features make protein degradation a very complex physical and chemical phenomenon, so optimizing formulations is a key aspect of biotechnology product development.

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