Professional Documents
Culture Documents
Stability of Peptides and Proteins
Stability of Peptides and Proteins
Report
On
SUBMITTED BY:
1
Stability of Polypeptides and Proteins
Background:
2
They tend to self associate.
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.
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.
3
Improved digestibility
Increased intrinsic viscosity
Inability to crystallize
Denatured proteins
4
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
5
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.
6
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
Deamidation
Oxidation
Diketopiperazine formation
Photodegradation of proteins
7
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.
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.
8
Scheme showing the deamidation, isomerization, and racemization of peptides
having
asparagine oraspartic acid residues
9
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.
Detection: Peptide maps are convenient for detecting methionine oxidation, and
MS.RP- HPLC
is used to separate the oxidized forms.
10
contain disulfide bonds, probably reflecting the need for the increased stability
of such
proteins.
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.
11
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.
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.
12
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.
13
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.
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.
14
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.
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.
15
Additives:
Many compounds may be added to protein solutions to lengthen shelf life:
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)
16