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A

Report

On

Stability of Polypeptides and Proteins

SUBMITTED BY:

Sr. NO. NAME ID NO.

1. Gunja Chaturvedi 2008H146101

Submitted for the partial fulfillment of the requirements of the course

Advanced physical pharmaceutics (PHA G542)

BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE

PILANI (RAJASTHAN)

AUGUST, 2009

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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.

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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

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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

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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.

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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.

Gelation 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

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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 freeze-

drying 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

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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.

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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.

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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

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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 reversed-

phase 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

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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 L-

cyclic imide. The L-cyclic imide intermediate is capable of undergoing racemization to the D-

cyclic imide and thus forms the D-aspartic acid peptide and the D-iso-aspartic acid peptide on

hydrolysis.

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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

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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.

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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 single-

use 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 low-

level 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.

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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 0.02-

0.05% (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.

REFERENCES:

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)