Stability And Characterization Of Protein And Peptide Drugs Case Histories Pdf


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Characterization testing is utilized to gain an understanding of the physical and chemical properties of biopharmaceutical materials. Therefore, a well characterized biopharmaceutical is integral to moving a candidate through development and to the market. Due to the complex nature of these materials, extensive testing utilizing a wide array of techniques is required.

Formulation, Characterization, and Stability of Protein Drugs

To ensure product safety and efficacy, protein therapeutics must meet defined quality characteristics immediately after manufacture as well at the end of their designated shelf lives.

Many physical and chemical factors can affect the quality and stability of biopharmaceutical products, particularly after long-term storage in a container—closure system likely to be subject to variations in temperature, light, and agitation with shipping and handling. Compared with traditional chemical pharmaceuticals, proteins are considerably larger molecular entities with inherent physiochemical complexities, from their primary amino acid sequences through higher-order secondary and tertiary structures — and in some cases, quaternary elements such as subunit associations 1.

Many proteins are glycosylated, and some have other posttranslational modifications such as phosphorylation, which also affects their potential degradation pathways as well as the kinetics of their degradation. Proteins are typically sensitive to slight changes in solution chemistry. They remain compositionally and conformationally stable only within a relatively narrow range of pH and osmolarity, and many require additionally supportive formulation components to remain in solution, particularly over time 2.

Even lyophilized protein products experience degradation 3 , 4. Advances in analytical chemistry have identified many degradation pathways that can occur in recombinant protein therapeutics over time.

These pathways generate either chemical or physical instability. Chemical instability refers to the formation or destruction of covalent bonds within a polypeptide or protein structures.

Chemical modifications of protein include oxidation, deamidation, reduction, and hydrolysis 5. Unfolding, dissociation, denaturation, aggregation, and precipitation are known as conformational or physical instabilities 5.

In some cases, protein degradation pathways are synergistic: A chemical event may trigger a physical event, such as when oxidation is followed by aggregation. Here, we present several protein degradation events: oxidation, photodegradation, disulfide scrambling, deamidation, aggregation, precipitation, dissociation, and fragmentation. We illustrate the biochemistry of each, showing potential means of induction and suggesting formulation considerations for prevention.

In an upcoming issue, Part 2 will conclude with methods of detection and strategies for validation of stability-indicating methods. Our objective is to provide an introduction or refresher to the major degradation pathways of protein products, with references for each. Readers are encouraged to consult those references for expanded details on the basic biochemistry of each pathway, case studies describing experiments with specific proteins, and further information on formulation development strategies.

Proteins and peptides are susceptible to oxidative damage through reaction of certain amino acids with oxygen radicals present in their environment. Methionine, cysteine, histidine, tryptophan, and tyrosine are most susceptible to oxidation: Met and Cys because of their sulfur atoms and His, Trp, and Tyr because of their aromatic rings 6.

It can also induce potential negative effects on potency and immunogenicity depending on the position of oxidized amino acids in a protein relative to its functional or epitope-like domain s.

For example, parathyroid hormone biological activity was differentially affected by a single oxidation of either Met-8 or Met and double oxidation Met-8 with Met when each specie was isolated and testing using in vitro bioassays 7 , 8.

In other cases, oxidation had no measurable impact on protein potency even when substantial structural changes were seen. Mechanism and Factors Involved: Figure 1 shows biochemical pathways for oxidation of methionine and cysteine residues. Methionine is oxidized by atmospheric oxygen and oxygen radicals in solution to form methionine sulfoxide and methionine sulfone. Both species are larger and more polar than nonoxidized methionine, which can alter protein folding and structural stability The rate of methionine oxidation in recombinant human parathyroid hormone rHu-PTH by hydrogen peroxide is enhanced at alkaline pH 8.

Cysteine oxidation is also more prevalent at alkaline pH, which deprotonates thiol groups. In such an environment, cysteine oxidation involves nucleophilic attack of thiolate ions on disulfide bonds, generating new disulfide bonds and different thiolate ions. The new thiolate can then react with another disulfide bond to form cysteine.

Such intermolecular disulfide links formed by protein degradation accumulate mispaired disulfide bonds and scrambled disulfide bridges, which can alter protein conformation and subunit associations 6.

Cysteine residues may also undergo s pontaneous oxidation to form molecular byproducts — sulfinic acid and cysteic acid — in the presence of metal ions or nearby thiol groups For example, human fibroblast growth factor FGF-1 exhibits copper-catalyzed oxidation that can create homodimers Spatial orientation of thiol groups in proteins plays an important role in cysteine oxidation.

The rate of oxidation is inversely proportional to the distance between those thiol groups This can eventually lead to formation of large oligomers or nonfunctional monomers, as with basic fibroblast growth factor bFGF , which contains three cysteines that are easily oxidized and form intermolecular or intramolecular disulfide bonds Histidine residues are highly sensitive to oxidation through reaction with their imidazole rings, which can subsequently generate additional hydroxyl species 6.

It may be a transient moiety because it can trigger protein aggregation and precipitation, which can obscure isolation of 2-O-His as an individual degradant Oxidation of tyrosine may result in covalent aggregation through formation of bityrosine Spatial factors may also affect tyrosine and histidine oxidation. Adjacent negatively charged amino acids accelerate tyrosine oxidation because they have high affinity to metal ions, whereas positively charged amino-acid residues disfavor the reaction 17 , If an adjacent amino acid is bulky, it may mask oxidation of neighboring amino acids and prevent them from getting oxidized.

It has been observed that histidine present in a sequence markedly increases both the peptide oxidation rate and methionine sulfoxide production. The strong metal binding affinity of the imidazole ring on the histidine side chain brings oxidizing species close to the substrate methionine 6.

Light Degradation: Photooxidation can change the primary, secondary, and tertiary structures of proteins and lead to differences in long-term stability, bioactivity, or immunogenicity Exposure to light can trigger a chain of biochemical events that continue to affect a protein even after the light source is turned off. These effects depend on the amount of energy imparted to a protein and the presence of environmental oxygen.

Photooxidation is initiated when a compound absorbs a certain wavelength of light, which provides energy to raise the molecule to an excited state. The excited molecule can then transfer that energy to molecular oxygen, converting it to reactive singlet oxygen atoms.

This is how tryptophan, histidine, and tyrosine can be modified under light in the presence of O 2 6. Tyrosine photooxidation can produce mono-, di-, tri-, and tetrahydroxyl tyrosine as byproducts Aggregation is observed in some proteins due to cross-linking between oxidized tyrosine residues Photooxidation reaction is predominately site specific For example, in human growth hormone treated with intense light, oxidation is carried out predominantly at histidine In addition, the peptide backbone is also a photodegradation target Alternatively, the energized protein itself can react directly with another protein molecule in a photosensitized manner, typically via methionine and tryptophan residues at low pH 6.

Excipients and leachables can synergistically affect the oxidation and hence, degradation of a protein. Formulation components influence the rate of photooxidation in some instances: e. Metal-ion—catalyzed oxidation depends on concentration of metal ions in the environment. The presence of 0. Oxidation can be exacerbated in the presence of a reducing agent such as ascorbate. Ascorbic acid increased oxidation of human ciliary neurotrophic factor Excipients such as polyols and sugars involved in stabilizing protein structure can decrease the rate of oxidation 6.

Oxidative modification depends on intrinsic structural features such as buried and exposed amino acids. In the case of human growth hormone, Met and Met are readily oxidized by H 2 O 2 because they are exposed to the surface of the protein, whereas Met in its buried position can be oxidized only when the molecule is unfolded Also, atmospheric oxygen can cause protein oxidation over time. Oxidation can be induced during protein processing and storage by peroxide contamination resulting from polysorbates and polyethylene glycols PEGs , both commonly used as pharmaceutical excipients.

A correlation has been observed between the peroxide content in Tween and the degree of oxidation in rhG-CSF, and peroxide-induced oxidation appeared more severe than that from atmospheric oxygen Peroxide can also leach from plastic or elastomeric materials used in primary packaging container—closure systems, including prefilled syringes 27 , Substitution of methionine of epidermal growth factor EGF with a nonnaturally occurring norleucine also prevented oxidative degradation Removal of headspace oxygen by degassing may be effective for preventing oxidation in some cases.

Filling steps are carried out under nitrogen pressure, and vial headspace oxygen is replaced with an inert gas such as nitrogen to prevent oxidation 21 , With some oxidation-sensitive proteins, processing is carried out in the presence of an inert gas such as nitrogen or argon.

For multidose drug preparations, use of cartridges with negligible headspace overcomes oxidation and related consequences Care must be exercised when container—closure changes are considered. Many such changes for protein therapeutics from vials to prefilled syringes or prefilled syringes to pen devices, for example are considered to enhance patient convenience and ease of use.

But historical experience with container—closure systems based only on chemical ph armaceuticals should be reevaluated when the same materials are used with protein-based products because of potential for unexpected, unique impacts on protein degradation.

Cysteine oxidation often can be controlled by maintaining the correct redox potential of a protein formulation, such as with addition of thioredoxin and glutathione.

Antioxidants and metal chelating agents also can be used to prevent oxidation in protein formulations. Scavengers such as L-methionine and ascorbic acid are used for this purpose in biotherapeutic formulations In the absence of metal ions, cysteine as a free amino acid may act as an effective antioxidant. As chelating agents, EDTA and citrate might form complexes with transition metal ions and inhibit metal-catalyzed, site-specific oxidation 6. Addition of sugars and polyols may also prevent metal-catalyzed oxidation because of their complexion with the metal ions.

Protective effects of glucose, mannitol, glycerol, and ethylene glycol against metal-catalyzed oxidation has been observed with human relaxin With many recombinant proteins, changes in peptide and protein structure are observed through the nonenzymatic deamidation of glutamine and asparagine residues. This can have varying effects on their physiochemical and functional stability 33 , It has been observed that deamidation of hGH alters proteolytic cleavage of the human growth hormone And it was reported that deamidation of IFN-beta increased its biological activity It has been determined that deamidation of peptide growth-hormone—releasing factor leading to aspartyl and iso-aspartyl forms reduces the bioactivity by and fold, respectively, as compared with the native peptide Deamidation at an Asn-Gly site in hemoglobin changes its affinity to oxygen Asparagine deamidation perturbs antigen presentation on Class II major histocompatibility complex molecules It was reported that isomerization of Asp 11 in human epidermal growth factor led to a fivefold reduction in its mitogenic activity And deamidation at two Asn-Gly sequences in triose-phosphate isomerase resulted in subunit dissociation Mechanism and Factors Involved: Deamidation is a chemical reaction in which an amide functional group is removed from an amino acid.

Consequences include isomerization, racemization, and truncation of proteins. Figure 2 shows the mechanism of asparagine degradation by deamidation.

Isomerization: Isomerization of aspartate to isoaspartate residues in a protein solution is the most commonly observed outcome of nonenzymatic deamidation 41 , Racemization: Succinimide intermediates formed during asparagine deamidation are highly prone to racemization and convert to D-asparagine residues 41 , Racemization of other amino acids, except glycine, is observed at alkaline pH

Stability and Characterization of Protein and Peptide Drugs

Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Crystallization is the bottleneck in macromolecular crystallography; even when a protein crystallises, crystal packing often influences ligand-binding and protein—protein interaction interfaces, which are the key points of interest for functional and drug discovery studies. The human hypoxia-inducible factor prolyl hydroxylase 2 PHD2 readily crystallises as a homotrimer, but with a sterically blocked active site. We explored strategies aimed at altering PHD2 crystal packing by protein modification and molecules that bind at its active site and elsewhere.

The information in THPdb has been compiled from research publications, 70 patents and other resources like DrugBank. The current version of the database holds a total of entries, providing comprehensive information on US-FDA approved therapeutic peptides and proteins and their drug variants. The information on each peptide and protein includes their sequences, chemical properties, composition, disease area, mode of activity, physical appearance, category or pharmacological class, pharmacodynamics, route of administration, toxicity, target of activity, etc. In addition, we have annotated the structure of most of the protein and peptides. A number of user-friendly tools have been integrated to facilitate easy browsing and data analysis. To assist scientific community, a web interface and mobile App have also been developed.

To ensure product safety and efficacy, protein therapeutics must meet defined quality characteristics immediately after manufacture as well at the end of their designated shelf lives. Many physical and chemical factors can affect the quality and stability of biopharmaceutical products, particularly after long-term storage in a container—closure system likely to be subject to variations in temperature, light, and agitation with shipping and handling. Compared with traditional chemical pharmaceuticals, proteins are considerably larger molecular entities with inherent physiochemical complexities, from their primary amino acid sequences through higher-order secondary and tertiary structures — and in some cases, quaternary elements such as subunit associations 1. Many proteins are glycosylated, and some have other posttranslational modifications such as phosphorylation, which also affects their potential degradation pathways as well as the kinetics of their degradation. Proteins are typically sensitive to slight changes in solution chemistry.


Stability and Characterization of Protein and Peptide Drugs. Case Histories. Editors: Pearlman, Rodney, Wang, Y. John (Eds.) Free Preview.


Stability and Characterization of Protein and Peptide Drugs

Drug design , often referred to as rational drug design or simply rational design , is the inventive process of finding new medications based on the knowledge of a biological target. In the most basic sense, drug design involves the design of molecules that are complementary in shape and charge to the biomolecular target with which they interact and therefore will bind to it. Drug design frequently but not necessarily relies on computer modeling techniques.

Schematic represents virus-induced host immune system response and viral processing within target cells. Proposed targets of select repurposed and investigational products are noted. Conflicts of interest comprise financial interests, activities, and relationships within the past 3 years including but not limited to employment, affiliation, grants or funding, consultancies, honoraria or payment, speaker's bureaus, stock ownership or options, expert testimony, royalties, donation of medical equipment, or patents planned, pending, or issued.

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