Stability Of Peptides And Proteins

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Stability Of Peptides And Proteins

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  • Tissue plasminogen activator
  • Stability Of Peptides And Proteins

    1. 1. STABILITY PROBLEMS ANDPREVENTION IN PROTEINSAND PEPTIDES DRUGDELIVERY SYSTEM B.THILAK CHANDRA M.PHARMACY (PHARMACEUTICS)VAAGDEVI INSTITUTE OF PHARMACEUTICAL SCIENCES
    2. 2. Contents• INTRODUCTION• PEPTIDE AND PROTEIN STRUCTURE• PROPERTIES AFFECTED BY UNSTABILITY• TYPES OF STABILITY PROBLEMS 1. PHYSICAL STABILITY 2. CHEMICAL STABILTY• CONCLUSION• REFERNCES
    3. 3. INTRODUCTION Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. Peptides - 20 amino acids. proteins - 50 or more amino acids. Polypeptides- 20 to 50 amino acids. Peptide chains in peptides and proteins are seldom linear and adapt a variety of specific folded three dimensional patterns and conformations. Conformation in a peptide chain is determined by the covalently bonded amino acids sequence, by disulfide bridges between cysteine residues and by total conformational energy
    4. 4. PEPTIDE AND PROTEINSTRUCTURE1. Primary structure2. Secondary structure3. Tertiary structure4. Quaternary structure
    5. 5. EXAMPLES Insulin Interferon γ Interferon β TPA 5
    6. 6. PROPERTIES AFFECTEDBY UNSTABILITY PHYSICAL PROPERTIES solubility, spectral properties such as circular dichorism. CHEMICAL PROPERTIES alteration of stabilized reactive group or group sterically shield from the reagents. BIOLOGICAL PROPERTIES 3-D structures place catalytic groups into proper orientation for enzymatic activity or place backbone and side-chain groups into proper orientation for hormone receptor interaction. Stability to enzymatic cleavage since some of the amide groups susceptible to proteolysis are deterred due to sterical peptide chain orientation.
    7. 7. TYPES OF STABILITYPROBLEMS
    8. 8. PHYSICAL STABILITY Physical instability involves transformations in the secondary, tertiary, or quaternary structure of the molecule.1. DENATURATION• Any nonproteolytic modification of the unique structure of a native protein that effects definite changes in physical, chemical, and biological properties.• Peptides and proteins are comprised of both polar amino resides and nonpolar amino acid residues.
    9. 9. FACTORS THAT FAVOURTHE DENATURATION When solvent changes from an aqueous to organic solvents or to a mixed solvent. On unfolding hydrophobic and hydrogen bonds are broken.
    10. 10. FACTORS THAT FAVOUR THE DENATURATION pH changes –alters the ionization of the carboxylic acid and amino acids and there by the charges carried by the molecules. Alteration in the ionic strength. Temperature rise.
    11. 11.  Denaturation may be reversible or irreversible Denaturation may lead to decrease in in solubility, alteration in surface tension, loss of crystallizing ability, changes in constituent group reactivity and molecular profile, vulnerability to enzymic degradation, loss or alteration of antigencity and loss of specific biological activity. DENATURING AGENTScategory mechanism examplesPolar and protic Disrupt H-bonds Urea, guanidine HCL, alcohol, acetic acidchemicalssurfactants Hydrophobic disruption Sodium dodecyl sulphate, and charge group polyethylene gltcol, dodecyl separation ammonium chloride
    12. 12. METHODS TO PREVENTDENATURATION Denaturated protein is restored on removal of denaturants. Maintaining pH. Maintaining ionic strength. Maintaining Temperature .
    13. 13. ADSORPTION Peptides and proteins are amphiphilic in nature, hence they tend to adsorb at interfaces such as air-water and air- solid. Example-Insulin Polar – hydrophilic , nonpolar – hydrophobic Conformational rearrangement leading to denaturation can be induced by their interfacial adsorption. After adsorption, they form some short-range bonds (van der Waals, hydrophobic, electrostatic, hydrogen, ion-pair bonds) with the surface resulting into further denaturation of polypeptide moieties.
    14. 14. ADSORPTION
    15. 15.  Adsorption of peptides and proteins at the interfaces are rapid, but the rates of conformational changes are relatively slower. On adsorption there may be a loss or change in biological activity as the molecular structure is rearranged. If peptide and protein drug entities are adsorbed at interfaces there may be a reduction in the concentration of drug available to elicit its function. Such loss of proteinaceous drug(s) may occur during purification, formulation, storage and/or delivery.
    16. 16. METHODS TO PREVENTADSORPTION Insulin adsorption may be minimized by the addition of 0.1% to 1% albumin. Excess agitation should prevented during production. The headspace within the confines of the container should be small. Use of surfactants to reduce adsorption. Smooth glass walls best to reduce adsorption or precipitation
    17. 17. Aggregation andPrecipitation The denatured, unfolded protein may rearrange in such a manner that hydrophobic amino acid residue of various molecules associate together to form the aggregates.
    18. 18.  If the aggregation is on a macroscopic scale, precipitation occurs. Interfacial adsorption may be followed by aggregation and precipitation. The extent to which aggregation and precipitation occurs is defined by the relative hydrophilicity of the surfaces in contact with the polypeptide/protein solution.
    19. 19. CAUSES OF AGGREGATIONAND PRECIPITATION The presence of large air-water interface generally accelerates this process. Presence of large headspace within the confines of the container also accelerates the course of precipitation. Insulin forms finely divided precipitates on the walls of the containers, referred to as frosting. The presence of large air-water interface generally accelerates this process.
    20. 20. CAUSES OF AGGREGATIONAND PRECIPITATION Increase in thermal motion of the molecules due to agitation. Solvent composition, solvent dielectric profile, ionic strength pH
    21. 21. METHODS TO PREVENTAGGREGATION ANDPRECIPITATION Organic solvent such as10-15% propylene glycol can suppress the formation of peptide liquid crystals. Excess agitation should prevented during production. The headspace within the confines of the container should be small. The ionic strength, solvent composition, solvent dielectric profile and ph should be carefully controlled at every step in production. Use of surfactants to reduce aggregation.
    22. 22. Chemical instability Involves alteration in the molecular structure producing a new chemical entity, by bond formation or cleavage. The stability of peptide and proteins against a chemical reagent is decided by temperature, length of exposure, and the amino acid composition, sequence and conformation of the peptide/protein.
    23. 23. DEAMIDATION This reaction involves the hydrolysis of the side chain amide linkage of an amino acid residue leading to the formation of a free carboxylic acid. Asparagine glutamine leading to conversion of a neutral residue to a negatively charged residue and primary sequence isomerization. In vivo deamidation is observed with human growth hormone, bovine growth hormone, prolactin, adreno- corticotropic hormone , insulin, lysozyme and secretin.
    24. 24. Factors that favour the rateof deamidation pH temperature ionic strength The deamidation of Asn residues is accelerated at neutral and alkaline pH The tertiary structure of the protein also affects its stability, as observed with trypsin in which the tertiary structure prevents deamidation.
    25. 25. METHODS TO PREVENTDEAMIDATION The use of genetic engineering and by recombinant DNA technology. The Asparagine residues can be selectively eliminated and replaced by other residues, provided conformations and bioactivity of protein can be maintained.
    26. 26. Oxidation and Reduction Major degradation pathways Oxidation commonly occurs during isolation, synthesis and storage of proteins
    27. 27. Factors that favour theOxidation and Reduction The oxidative degradation reactions can even occur in atmospheric oxygen under mild conditions (autoxidation). Temperature, pH, trace amounts of metal ions and buffers influence these reactions. Oxidation may take place involving side chains of histidine (His), lysine (Lys), tryptophan (Trp), and thyronine (Tye) residues in proteins. The thioether group of methionine (Met) is particularly susceptible to oxidation. Under acidic conditions Met residues can be oxidized by atmospheric oxygen.
    28. 28. Factors that favour theOxidation and Reduction(cont…) Oxidizing agents like hydrogen peroxide, dimethylsulphoxide and iodine can oxidize Met-to-Met sulphoxides. Thethiol group of cysteine can be oxidized to sulphonic acid; oxidation by iodine and hydrogen peroxide is catalyzed by metal ions and may occur spontaneously by atmospheric oxygen. Usually the oxidation of amino acid residues is followed by a significant loss of biological activity as observed after oxidation of Met residues in calcitonin, corticotrophin and gastrin. Glucagon is an exception as it retains biological activity even after oxidation.
    29. 29. METHODS TO PREVENTOXIDATION AND REDUCTION Oxidation scavengers may block these acid or base catalyzed oxidations. Example phenolic compounds, propyl gallate. Reducing agents –methionine, ascorbic acid, sodium sulphate, thioglycerol and thioglycolic acid. Chelating agents –EDTA, Citric Acid Nitrogen flush, refrigeration, protection from light and adjustment of ph.
    30. 30. METHODS TO PREVENTOXIDATION AND REDUCTION Avoiding vigorous stirring and exclusion of air by degassing solvents can prevent air initiated oxidation.
    31. 31. PROTEOLYSIS The hydrolysis of peptide bonds within the polypeptide or protein destroys or at least reduces its activity. The vulnerability of peptide bonds to cleavage is dependent on the other residues involved. In comparison to other residues, Asn residues are unstable and in particular the Asn-Proline bond
    32. 32. FACTORS THAT FAVOUR THEPROTEOLYSIS AND PREVENTION  Proteolysis may occur on exposing the proteins to harsh conditions, such as prolonged exposure to extremes of pH or high temperature or proteolytic enzymes.  Bacterial contamination is the most common source of proteases. This can be avoided by storing the protein in the cold under sterile conditions.  Proteases may also gain access during the isolation, purification and recovery of recombinant proteins from cell extracts or culture fluid.
    33. 33. FACTORS THAT FAVOUR THEPROTEOLYSIS AND PREVENTION  This problem can be minimized by the manipulation of the solution conditions during the stage of purification and/or by addition of protease inhibitors.  Some proteins even have autoproteolytic activity. This property aids in controlling the level or function of protein in vivo .
    34. 34. DISULPHIDE EXCHANGE Thiol-disulfide exchange showing the linear intermediate in which the charge is shared among the three sulfur atoms. The thiolate group (shown in red) attacks a sulfur atom (shown in blue) of the disulfide bond, displacing the other sulfur atom (shown in green) and forming a new disulfide bond. Cystine Disulphide bonds may break and reform with incorrect pairings. This results in an alteration in the three- dimensional structure followed by a resultant change in biological activity.
    35. 35.  A peptide chain with more than one disulphide can enter into disulphide exchange reactions, leading to scrambling of disulphide bridges and thereby a change in conformation. By analogous reactions, trimers and dimers can be formed. The reaction is concentration dependent, particularly for oligomer formation. These oligomers appear at low Rf value on TLC and are readily removed by gel filtration.
    36. 36. METHODS TO PREVENTDISULPHIDE EXCHANGE BY THIOL SCAVENGERS SUCH AS P-MERCURIBENZOATE N-EHYLMALEIMIDE COPPER IONS
    37. 37. RACEMIZATION Racemization is the alteration of L-amino acids to D,L- mixtures. With the exception of Gly, all the mammalian amino acids are chiral at the carbon bearing chain and are susceptible to base-catalyzed racemization. Racemization may form peptide bonds that are sensitive to proteolytic enzymes. This reaction can be catalyzed in neutral and alkaline media by thiols, which may arise as a result of hydrolytic cleavage of disulphides.
    38. 38. METHODS TO PREVENTRACEMIZATION The thiolated ions carry out nucleophilic attack on a sulphur atom of the disulphide. Addition of thiol scavengers such as p-mercuribenzoate, N- thylmaleimide and copper ions, may prevent susceptible sulphur and disulphide.
    39. 39. BETA-ELIMINATION The mechanism involved in the beta-elimination is similar to the racemization, i.e. it proceeds through a carbanion intermediate. Higher elimination rate prevails under alkaline conditions which ultimately lead to loss of biological activity. Protein residues susceptible to beta-elimination under alkaline conditions include Cys, Lys, Phe, Ser.
    40. 40.  Stabilize proteins against thiol-disulfide exchange by chemically block the thiol group(s) involved in the process. For example, S-alkylating the Cys-34 of albumin stabilizes the protein not only during high temperature and high humidity storage, but also when loaded within a polymeric matrix.
    41. 41. CONCLUSION Therapeutic peptides and proteins can degrade by several physical and chemical pathways. In most cases, more than one pathway of physical or chemical instability is responsible for the degradation of peptides and proteins. The primary structure will often reveal potential sites of chemical degradation. Physical instability is more difficult to predict from primary structure.
    42. 42. CONCLUSION However, if most residues are hydrophobic amino acids, it does suggest a strong tendency toward adsorption and aggregation. For proteins, the secondary and tertiary structures may be more useful predictors of physical stability. Comparability protocols for well-characterized biologics will allow the introduction of biogenerics into the market.
    43. 43. REFERENCES CONTROLLED DRUG DELIVERY S.P.VYAS AND ROOP K.KHAR Therapeutic Peptides and Proteins Ajay K. Banga, Ph.D. Biochemistry U.Satyanarayana http://en.wikipedia.org

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