P. 1
Stability of Peptides and Proteins

Stability of Peptides and Proteins

|Views: 1,975|Likes:
Published by Gunja Chaturvedi
this article gives idea about the various stability issues while formulating proteins & peptides.
this article gives idea about the various stability issues while formulating proteins & peptides.

More info:

Categories:Types, Research, Science
Published by: Gunja Chaturvedi on May 10, 2010
Copyright:Attribution Non-commercial


Read on Scribd mobile: iPhone, iPad and Android.
download as PDF, TXT or read online from Scribd
See more
See less





A Report On

Stability of Polypeptides and Proteins
SUBMITTED BY: Sr. NO. 1. NAME Gunja Chaturvedi ID NO. 2008H146101

Submitted for the partial fulfillment of the requirements of the course

Advanced physical pharmaceutics (PHA G542)



Stability of Polypeptides and Proteins
Background: Proteins comprise an extremely heterogeneous class of biological macromolecules. They are often unstable when not in their native environments, which can vary considerably among cell compartments and extracellular fluids. Their properties make them particularly difficult to formulate but, with right approach, they can be developed into effective therapies. Proteins and polypeptides are fast becoming an important segment of the pharmaceutical industry. Although there have been tremendous advances in production of the active pharmaceutical ingredient (API), production of the peptide-based drug products is still a significant challenge. Peptides are defined as polypeptides of less than 50 residues or so and lacking any organized tertiary or globular structure. Some do adopt secondary structure, although this tends to be limited, for example a single turn of an α-helix. While their smaller size makes them easier to deliver across biological barriers than larger proteins, their formulation can be problematic.

Mainly because of their chemical instability or degradation like by hydrolysis and racemization and physical degradation depending upon their molecular weight, they undergo denaturation, aggregation and precipitation; they are very challenging to be formulated in desired dosage form. Proteins and peptides exhibit the following challenges to the formulation scientists:  They exhibit maximal chemical instability.


  

They tend to self associate. They adopt multiple conformers. They can exhibit complex physical instabilities, such as gel formation.

Chemical and physical properties of peptides and proteins have been studied extensively and the thermodynamics of protein structure have also been studied in detail and reported. But because of the complicated degradation mechanisms, it is generally more difficult to predict the stability of peptide and protein pharmaceuticals.

Proteins and peptides undergo degradation by two mechanisms: a) Physical mechanisms b) Chemical mechanisms

PHYSICAL INSTABILITY: Physical instability or noncovalent changes are generally observed in case of larger peptides and proteins. Physical degradation includes denaturation, self association, aggregation, adsorption, and gelation.

Denaturation: protein Denaturation is mainly associated with any modification in conformation not accompanied by rupture of peptide bonds and ultimate step might correspond to a totally unfolded polypeptide structure which can be reversible or irreversible. It can also results in loss of bioactivity mainly because of the alteration the tertiary structure of the proteins. Furthermore, exposure of hydrophobic groups upon Denaturation often leads to adsorption on the surfaces, aggregation, and precipitation. Denaturation sometimes also triggers the chemical degradation pathways often not seen with the native or natural tertiary (and/or quaternary) structure. Other effects of Denaturation are:    Decreased solubility Altered water binding capacity Destruction of toxins

  

Improved digestibility Increased intrinsic viscosity Inability to crystallize

Denatured proteins Causes of protein Denaturation: 1. Temperature fluctuation Effect of increased temperature:       Affect interactions of tertiary structure Increased flexibility → reversible H-bonds begin to break → water interaction Increased water binding Increased viscosity of solution Structures different from native protein


Effect of decreased temperature:   Can result in Denaturation(for e.g.Gliadins, egg and milk proteins) Remain active( for e.g.Some lipases and oxidases and Release from sub-cellular compartments)   Proteins with high hydrophobic/polar amino residues and structures dependent on hydrophobic interactions


2. Water content affects heat Denaturation 3. Mechanical treatments 4. Hydrostatic Pressure 5. Irradiation 6. Heavy metal salts act to denature proteins in much the same manner as acids and bases. Heavy metal salts usually contain Hg+2, Pb+2, Ag+1 Tl+1, Cd+2 and other metals with high atomic weights. Since salts are ionic they disrupt salt bridges in proteins. The reaction of a heavy metal salt with a protein usually leads to an insoluble metal protein salt. 7. Heavy metals may also disrupt disulfide bonds because of their high affinity and attraction for sulfur and will also lead to the denaturation of proteins

Self association: The propensity of peptides to self-associate is connected with their physical instability. While self-association of peptides for e.g. melittin and corticotrophin – releasing factor (CRF), the relationship between these metastable oligomeric species and larger aggregates has been investigated, but still unclear. Noncovalent aggregation has been suggested for many other proteins, but not always confirmed. For e.g. a conjugate formed between a vinca alkaloid and a monoclonal antibody exhibited aggregation in solution, the mechanism of which (covalent or noncovalent) was not clear. Aggregates formed upon agitation of insulin solutions in the presence of hydrophobic surfaces (Teflon) were dissociated with urea, suggesting noncovalent aggregation.

Aggregation can lead to either amorphous or ordered forms. Ordered aggregates usually take the form of fibrils; these fibrillar structures are the basis for the most common type of the aggregation seen for peptides, namely gelation.Gelation is the last step in a pathway that starts with the formation of peptides protofibrils that exhibit β-sheet structure. The protofibrils then associate to form mature fibrils, which propagate and intertwine, resulting in gelation. Detection of aggregates: Insoluble aggregates can be detected by FTIR, Raman, and electron spin resonance spectroscopy, or light scattering techniques (UV absorption). Soluble aggregates
can be detected by HP-SEC (High Performance Size Exclusion Chromatography), found in many proteins like hGH, insulin, interferon-2 (lL-2), anti trypsin-a1,IFN-g, basic fibroblast growth factor and IFN-b.

Adsorption: The interaction of proteins with the surface of their storage containers is potentially a significant problem. The amphiphilic nature of the protein molecule results in their adsorption to a wide variety of surfaces and also both their loss and destabilization. Adsorption of protein on surfaces is an important phenomenon, which should be considered while formulating and selecting container and closure for pharmaceutical products. This is extremely important in low dose drugs. Adsorption to a surface is problematic in parenteral administration. Detection of adsorption of proteins: X-ray and neutron reflection are used to study the adsorption of protein at liquid-gas and solid-liquid interfaces, and parameters like adsorbed amount, total thickness of the adsorbed layer, pH, and excipients.


is the process that converts a fluid solution into a semi-solid mass. Microscopic

examination reveals that the gel is composed of multiple peptide fibrils, intertwined in a complex mesh. It is known that pH, temperature and ionic strength all affect the rate of gelation, as well as the physical properties of the gel –for e.g. Transparency, gel stiffness, reversibility and so on. These factors all suggest that colloidal stability plays an important role in gel formation. Colloidal stability determines whether peptide molecules are attached to each other or repelled. Low colloidal stability means that the net forces between peptide molecules are attractive overall, which leads to decreased solubility and increased likelihood to assemble into larger structures, such as fibrils. Conversely, increased colloidal stability indicates net repulsive forces between peptides, which improves solubility and diminishes growth of organised fibrils. Stabilization: The problem of aggregation can be overcome, by modulating the solution conditions such as pH, buffer composition and ionic strength and by addition of other excipients. Like Cyclodextrins have been shown to improve the physical stability of peptides by shielding hydrophobic amino acids. For Glucagon the addition of cyclodextrins was found to delay the formation of insoluble aggregates. Similarly, the addition of sucrose has been shown to improve the physical stability of bioactive peptides. To overcome the problem of adsorption


of proteins to surfaces, the adsorption of an inert protein like serum albumin to saturate the container surface, or compounds that reduce surface interactions such as surfactants, carbohydrates or aminoacids, can be employed. In formulation, surfactant addition can reduce adsorption losses e.g., Tween 80 and Pluronic F68 have been shown to reduce the adsorption of calcitonin to a glass surface. The preservatives and surfactants are sometimes essential in protein formulation for prevention of microbial growth, and to prevent aggregation and adsorption.For avoiding the problem of Denaturation, Proteins and peptides are often formulated with excipients such as polyalcohols and polymers, to protect them during freezedrying and storage. Polymers are also used to form a matrix, for controlled release. Excipients such as heparin, and anionic polymers, decreased the rate of covalent aggregation in recombinant human keratinocyte growth factor (rhKGF), at elevated temperatures.Polyhydric alcohols like mannitol, sorbitol, and non reducing sugars like dextrose, sucrose, and trehalose, are the most commonly used excipients in lyophilized protein and peptide formulations. Also the optimum conditions of temperature, pH , ionic strength and moisture are need for the stabilization of the proteins and peptides from the problem of gelation.

CHEMICAL STABILITY Occurs through the following mechanisms:           Deamidation Oxidation Cystine destruction and thiol- disulfide exchange Hydrolysis at aspartic acid residue C- terminal succinimide formation at asparagines residue Diketopiperazine formation Deglycosylation and desialylation Photodegradation of proteins Enzymatic proteolysis and autolysis Proteases activity during fermentation and cell culture

Deamidation: Deamidation is a common post-translational modification resulting in the conversion of an asparagine residue to a mixture of isoaspartate and aspartate. Deamidation of glutamine residues can occur but does so at a much lower rate. Deamidation can occur under acidic, neutral or alkaline conditions, although the chemical mechanism of hydrolysis is strongly dependent of pH. Deamidation has been observed and characterized in a wide variety of proteins. It has been shown to regulate some time-dependent biological processes and to correlate with others, such as development and aging. Deamidation can make protein prone to proteases and denaturation. This can affect the in vivo half-life, activity, and conformation of protein, and also increase the immunogenicity of certain protein. For e.g In insulin formulation lyophilized from acidic solutions (pH3-5), the rate determining first step involves intermolecular nucleophilic attack of the C-terminal AsnA 21 carboxylic acid onto the side chain amide carbonyl, to release ammonium, and to form reactive cyclic anhydride intermediate which can further react with various nucleophiles. The protein deamidation process involves the conversion of the amide side-chain moieties of asparagine and glutamine residues to carboxyl groups. This conversion is an unusual form of protein modification in that it requires catalysis by an intramolecular reaction where both the substrate (asparagine and glutamine side chains) and "catalytic site" (the peptide nitrogen of the succeeding residue) are constituents of several consecutive residues along the polypeptide chain. Deamidation of asparaginyl (Asn) and glutaminyl (Gln) residues to produce aspartyl (Asp) and glutamyl (Glu) residues causes structurally and biologically important alterations in peptide and protein structures. At neutral pH, deamidation introduces a negative charge at the reaction site and can also lead to structural isomerization. The rates of deamidation depend on primary sequence, three-dimensional (3D) structure, pH, temperature, ionic strength, buffer ions, and other solution properties.


Scheme showing the deamidation, isomerization, and racemization of peptides having asparagine oraspartic acid residues

Detection: It is detected by charge, molecular weight, and formation of succinimide residues or isoaspartic acid residues and peptides maps, capillary electrophoresis, isoelectric focusing, and enzyme catalyzed radio labeling of the isoaspartyl sites. Also recently an advanced technique has been used which is probing Deamidation events by using anion exchange and RP- HPLC to isolate two deamidated forms of recombinant hirudin at pH 3 and 37o C . Stabilization: Formulation approaches include lowering of pH (desialylation can occur, therefore optimization essential), compatibility studies in presence of various buffers, because deamidation is also affected by buffer composition.


Oxidation: Oxidation generally occurs in Methionine, cystine, (more common) tryptophan, tyrosine residues. The oxidation of methionine residues has been associated with the loss of biological activity in a number of peptides and proteins. Its oxidation results in conversion of the thioether to its sulphoxide counterpart. But this is a reversible reaction in which the methionine residue can be generated either by reducing agents or enzymatically. Oxygen radicals can be generated in vitro by compounds commonly used in protein folding/unfolding studies. For e.g. small amount of copper in presence of glucose oxidizes a particular methionine residue in α1 – proteinase inhibitor, whereas the autooxidation of the reducing sugars can inactivate the enzyme rhodanese with a concomitant loss in sulfhydryl titer. In addition, air oxidation of DTT can lead to H2O2 generation and subsequent protein oxidation. Detection: Peptide maps are convenient for detecting methionine oxidation, and MS.RP- HPLC is used to separate the oxidized forms. Stabilization: Formulation approaches include addition of anti oxidants, (sodium thiosulphate, catalase, or platinum), and adjustment of environmental conditions (pH, or temperature). Cystine oxidation can be prevented by keeping low pH. Other Formulation approaches include, maintaining acidic pH, and avoiding potential reducing agents (like anti-oxidant excipients), lyophilization, substituting non critical cystine residues with other residues to reduce the potential instability of free thiols in presence of disulphide e.g. human interferon (IF-N) beta analogue. Cystine destruction and thiol- disulfide exchange: Cystine residues (disulfides) are naturally occurring crosslinks that covalently connect polypeptide chains either intra – or intermolecularly. Disulfides are formed by oxidation of thiol groups of cysteine residues by either thiol disulfide interchange or direct oxidation. Intracellular proteins usually lack such crosslinks and their atypical presence commonly reflects a role in enzyme’s catalytic mechanism or involvement in the regulation of its activity. In contrast, extracellular proteins frequently

contain disulfide bonds, probably reflecting the need for the increased stability of such proteins.

Formation of a disulfide bond through oxidation of cysteine residues Covalent bond formation, other than disulfide bond formation, is also involved in other intermolecular cross-linkages. The covalent linkages in the aggregates of freeze-dried ribonuclease A appeared to result from the participation of lysine, asparagine, and glutamine residues as suggested by amino acid analysis of the aggregates. Detection: By a systematic approach using UV spectroscopy, size-exclusion HPLC, and reversedphase chromatography. By running reduced and non reduced gel electrophoresis; SDS-PAGE ,ellman’s reagents for thiols detection, peptide mapping and matrix assisted Laser Desorption Ionization MS. Stabilization: By avoiding the use of potential reducing agents and avoiding moisture i.e the proteins should be kept in anhydrous conditions.

C- Terminal succinimide formation at asparagines residue: Succinimide formation at the asparagines residues can potentially lead to the spontaneous cleavage of polypeptide chains. In this case, the side chain amide nitrogen attacks the peptide bond to form a C- terminal succinimide residue and newly formed amino terminal. Diketopiperazine formation: Peptides and proteins that possess an N- terminal sequence in which ‘Pro’ is the penultimate residue undergo non – enzymatic hydrolysis yielding a Diketopiperazine (DKP),which arises from the first two amino acids , and truncated polypeptide. The mechanism of DKP formation involves nucleophilic attack of N- terminal nitrogen on carbonyl carbon of the peptide bond between the second and third amino acid residues in the primary sequence. This intramolecular aminolysis reaction occurs readily in aqueous solutions

and was shown to be catalyzed in both acidic and basic conditions. DKP formation was reported to occur in human growth hormone, bradykinin and histrelin.

Diketopiperazine formation in proteins Detection: The DKP products can be detected by N- terminal sequence analysis ,MS and tryptic mapping. But before that the DKP products are separated by using hydrophobic interaction chromatography. Hydrolysis at aspartic acid residue: Hydrolysis is a pathway often observed during peptide and protein degradation. As shown in scheme. Aspartic acid residues in particular are susceptible to hydrolysis in theacidic pH range.for e.g. Secretin, apart from undergoing isomerization, also undergoes degradation by hydrolysis of its aspartic acid residues at position-3 and position-15. Hydrolysis of aspartic acid residues under acidic conditions has also been observed with recombinant human macrophage colony-stimulating factor,recombinant human interleukin-11 ,and a hexapeptide. Hydrolysis may also occur at serine and histidine residues. Peptides and proteins having an aspartic acid residue also undergo isomerization, and racemization via cyclic imide formation L-aspartic acid peptide can isomerize to L-iso-aspartic acid peptide via its Lcyclic imide. The L-cyclic imide intermediate is capable of undergoing racemization to the Dcyclic imide and thus forms the D-aspartic acid peptide and the D-iso-aspartic acid peptide on hydrolysis.


Pathways proposed for the hydrolysis of peptides at aspartic acid residues Deglycosylation and desialylation: In glycoproteins, sugars are attached either to the amide nitrogen atom in the side chain of asparagines (termed N-linkage), or to the oxygen atom in the side chain of serine or threonine (termed O-linkage). An asparagine residue can accept an oligosaccharide only, if the residue is part of an Asn-X-Thr sequence, where X can be any residue. Thus, a potential glycolisation site can be detected within aminoacid sequences. There are a number of glycosylated proteins that have sugar and sialic acid molecules covalently linked to peptide structure. e.g., IFN-beta has greater stability to aggregation than corresponding protein produced by bacterial fermentation; in the non-glycosylated form.Desialylation can occur at acidic pH on storage. Differing sialic acid content has shown to be responsible for variability in the biological activity of highly purified pituitary lutinizing hormone isoforms. The modification of human insulin by the covalent attachment of monosaccharide moieties to insulin amino groups altered the aggregation and self association behavior, and improved both the pharmaceutical stability and biological response.

Detection: Change in glycocylation can be detected by various gel methods including flurophore - assisted carbohydrate electrophoresis (FACE) and MS. Change in sialic acid content can be detected by measurement of free sialic acid. Oligosaccaride structure can be analyzed by


normal phase HPLC combined with MS, and high resolution of normal phase, by high pH anion exchange chromatography combined with MS. Photodegardation of proteins: Both ionizing and non ionizing radiations can cause protein inactivation. The effects of different types of ionizing radiations (γ- rays , X –rays ,electrons and α- particles)on protein molecule ( both in solid and solution states). Non –ionizing radiations like UV rays also may cause irreversible damage to the protein molecules. These effects are of particular concern biologically in understanding the mechanism of cataract formation and sunburn damage. The amino acids tryptophan, tyrosine and cysteine are particularly

susceptible to UV-A (320- 400 nm) and UV- B ( 250 – 320nm) photolysis. The absorption of photon leads to the photoionization and the formation of photodegaradation products through either direct interaction with an amino acid or indirectly via various sensitizing agents (such as dyes,riboflavin or oxygen). Commonly observed photodegardation product in an aerated, neutral pH, aqueous protein solution include S-S bond fission , conversion of tyrosine to DOPA, 3-(4- hydroxyphenyl)lactic acid and dityrosine as well as fragmentation byproducts and the conversion of tryptophan residues to kynurenine and N- formyl- kynurenine . It is also important
to take into account, potential damage to the protein during analysis using circular dichorism (CD), UV or fluorescent measurements, where incident radiation is being used.

Detection: UV spectroscopy can be used to study changes in secondary and tertiary structures of
proteins. As protein is denatured, differences are observed in the absorption characteristics of the peptide bonds due to the disruption of the exciton system.

Enzymatic proteolysis and autolysis: Some of enzymes have been identified in vivo that specifically interact with covalently modified proteins, including carboxymethyl transferases (which methylates isoaspartyl residues) and alkaline proteases (which degrades oxidized proteins). It has been proposed that covalent changes caused by in vivo protein oxidation are primarily responsible for the accumulation of catalytically compromised and structurally altered enzymes during aging.


Proteases activity during fermentation and cell culture: Presence of protease enzyme can result in the cleavage of recombinant protein. Protease inhibitors can minimize this to a certain extent. GENERAL CONSIDERATIONS FOR PROTEIN STORAGE Temperature:  Generally, proteins are best stored at ≤ 4°C in clean, autoclaved glassware or polypropylene tubes. Storage at room temperature often leads to protein degradation and/or inactivity, commonly as a result of microbial growth. For short term storage (1 day to a few weeks), many proteins may be stored in simple buffers at 4°C.

For long term storage for 1 month to 1 year, some researchers choose to bead singleuse aliquots of the protein in liquid nitrogen for storage in clean plastic containers under liquid nitrogen. This method involves adding the protein solution drop wise (about 100 μl each) into a pool of liquid nitrogen, then collecting the drop-sized frozen beads and storing them in cryovials under liquid nitrogen.

Frozen at -20°C or -80°C is the more common form of cold protein storage. Because freeze-thaw cycles decrease protein stability, samples for frozen storage are best dispensed and prepared in single-use aliquots so that, once thawed, the protein solution will not have to be refrozen. Alternatively, addition of 50% glycerol or ethylene glycol will prevent solutions from freezing at -20°C, enabling repeated use from a single stock without warming (i.e., thawing).

Protein Concentration: Dilute protein solutions (< 1 mg/ml) are more prone to inactivation and loss as a result of lowlevel binding to the storage vessel. Therefore, it is common practice to add “carrier” or “filler” protein, such as purified bovine serum albumin (BSA) to 1-5 mg/ml (0.1-0.5%), to dilute protein solutions to protect against such degradation and loss.


Additives: Many compounds may be added to protein solutions to lengthen shelf life:  Cryoprotectants such as glycerol or ethylene glycol to a final concentration of 25-50% help to stabilize proteins by preventing the formation of ice crystals at -20°C that destroy protein structure.  Protease inhibitors prevent proteolytic cleavage of proteins like Benzamidine for Serine proteases, Pepstatin A for Acid proteases , Leupeptin for Thiol proteases etc.  Anti-microbial agents such as sodium azide (NaN3) at a final concentration of 0.020.05% (w/v) or thimerosal at a final concentration of 0.01 % (w/v) inhibit microbial growth.  Metal chelators such as EDTA at a final concentration of 1-5 mM avoid metal-induced oxidation of –SH groups andhelps to maintain the protein in a reduced state.  Reducing agents such a dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) at final concentrations of 1-5 mM also help to maintain the protein in the reduced state by preventing oxidation of cysteines.

    Stability of drug and dosage form (Sumie Yoshioka and Valentino J. Stella) Protein stability and folding,Theory and practice (Bret A. Shirley) Pharmaceutical formulation development of peptides and proteins (Sven Frokjaer and Lars Hovgaard) Stability of proteins in aqueous solution and solid state( S.Jacob , AA Shirwaikar,KK Srinivasan; Manipal college of pharmaceutical sciences) IJPS(Year : 2006 ; Volume : 68 ; Issue : 2 ; Page : 154-163) Deamidation in Proteins and Peptides(Glen Teshima) Amino Acid Degradation (Bryant Miles)

 


You're Reading a Free Preview

/*********** DO NOT ALTER ANYTHING BELOW THIS LINE ! ************/ var s_code=s.t();if(s_code)document.write(s_code)//-->