A
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`Report
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`On
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`Stability of Polypeptides and Proteins
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`SUBMITTED BY:
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`Sr. NO.
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`NAME
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`ID NO.
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`1.
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`Gunja Chaturvedi
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`2008H146101
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`Submitted for the partial fulfillment of the requirements of the course
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`Advanced physical pharmaceutics (PHA G542)
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`BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE
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`PILANI (RAJASTHAN)
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`AUGUST, 2009
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`Page 1
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`NPS EX. 2039
`CFAD v. NPS
`IPR2015-01093
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`Stability of Polypeptides and Proteins
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`Background:
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`Proteins comprise an extremely heterogeneous class of biological macromolecules. They are
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`often unstable when not in their native environments, which can vary considerably among cell
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`compartments and extracellular fluids. Their properties make them particularly difficult to
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`formulate but, with right approach, they can be developed into effective therapies. Proteins
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`and polypeptides are fast becoming an important segment of the pharmaceutical industry.
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`Although there have been tremendous advances in production of the active pharmaceutical
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`ingredient (API), production of the peptide-based drug products is still a significant challenge.
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` Peptides are defined as polypeptides of less than 50 residues or so and lacking any organized
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`tertiary or globular structure. Some do adopt secondary structure, although this tends to be
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`limited, for example a single turn of an α-helix. While their smaller size makes them easier to
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`deliver across biological barriers than larger proteins, their formulation can be problematic.
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`Mainly because of their chemical instability or degradation like by hydrolysis and racemization
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`and physical degradation depending upon their molecular weight, they undergo denaturation,
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`aggregation and precipitation; they are very challenging to be formulated in desired dosage
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`form.
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`Proteins and peptides exhibit the following challenges to the formulation scientists:
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` They exhibit maximal chemical instability.
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` They tend to self associate.
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` They adopt multiple conformers.
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` They can exhibit complex physical instabilities, such as gel formation.
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`Chemical and physical properties of peptides and proteins have been studied extensively and
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`the thermodynamics of protein structure have also been studied in detail and reported. But
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`because of the complicated degradation mechanisms, it is generally more difficult to predict
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`the stability of peptide and protein pharmaceuticals.
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`Proteins and peptides undergo degradation by two mechanisms:
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`a) Physical mechanisms
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`b) Chemical mechanisms
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`PHYSICAL INSTABILITY:
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`Physical instability or noncovalent changes are generally observed in case of larger peptides
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`and proteins. Physical degradation includes denaturation, self association, aggregation,
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`adsorption, and gelation.
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`Denaturation: protein Denaturation is mainly associated with any modification in conformation
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`not accompanied by rupture of peptide bonds and ultimate step might correspond to a totally
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`unfolded polypeptide structure which can be reversible or irreversible. It can also results in loss
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`of bioactivity mainly because of the alteration the tertiary structure of the proteins.
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`Furthermore, exposure of hydrophobic groups upon Denaturation often leads to adsorption on
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`the surfaces, aggregation, and precipitation. Denaturation sometimes also triggers the chemical
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`degradation pathways often not seen with the native or natural tertiary (and/or quaternary)
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`structure. Other effects of Denaturation are:
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` Decreased solubility
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` Altered water binding capacity
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` Destruction of toxins
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`
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`Improved digestibility
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`Increased intrinsic viscosity
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`Inability to crystallize
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` Denatured proteins
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`Causes of protein Denaturation:
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`1. Temperature fluctuation
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`- Effect of increased temperature:
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` Affect interactions of tertiary structure
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`
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`Increased flexibility → reversible
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` H-bonds begin to break → water interaction
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`
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`
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`Increased water binding
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`Increased viscosity of solution
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` Structures different from native protein
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`- Effect of decreased temperature:
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` Can result in Denaturation(for e.g.Gliadins, egg and milk proteins)
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` Remain active( for e.g.Some lipases and oxidases and Release from sub-cellular
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`compartments)
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` Proteins with high hydrophobic/polar amino
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`residues and structures dependent on hydrophobic interactions
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`
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`2. Water content affects heat Denaturation
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`3. Mechanical treatments
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`4. Hydrostatic Pressure
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`5. Irradiation
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`6. Heavy metal salts act to denature proteins in much the same manner as acids and bases.
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`Heavy metal salts usually contain Hg+2, Pb+2, Ag+1 Tl+1, Cd+2 and other metals with high
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`atomic weights. Since salts are ionic they disrupt salt bridges in proteins. The reaction of
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`a heavy metal salt with a protein usually leads to an insoluble metal protein salt.
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`7. Heavy metals may also disrupt disulfide bonds because of their high affinity and
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`attraction for sulfur and will also lead to the denaturation of proteins
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` Self association: The propensity of peptides to self-associate is connected with their physical
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`instability. While self-association of peptides for e.g. melittin and corticotrophin – releasing
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`factor (CRF), the relationship between these metastable oligomeric species and larger
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`aggregates has been investigated, but still unclear. Noncovalent aggregation has been
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`suggested for many other proteins, but not always confirmed. For e.g. a conjugate formed
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`between a vinca alkaloid and a monoclonal antibody exhibited aggregation in solution, the
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`mechanism of which (covalent or noncovalent) was not clear. Aggregates formed upon
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`agitation of insulin solutions in the presence of hydrophobic surfaces (Teflon) were dissociated
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`with urea, suggesting noncovalent aggregation.
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`Aggregation can lead to either amorphous or ordered forms. Ordered aggregates usually take
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`the form of fibrils; these fibrillar structures are the basis for the most common type of the
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`aggregation seen for peptides, namely gelation.Gelation is the last step in a pathway that starts
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`with the formation of peptides protofibrils that exhibit β-sheet structure. The protofibrils then
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`associate to form mature fibrils, which propagate and intertwine, resulting in gelation.
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`Detection of aggregates: Insoluble aggregates can be detected by FTIR, Raman, and electron
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`spin resonance spectroscopy, or light scattering techniques (UV absorption). Soluble aggregates
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`can be detected by HP-SEC (High Performance Size Exclusion Chromatography), found in many proteins
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`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
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`potentially a significant problem. The amphiphilic nature of the protein molecule results in their
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`adsorption to a wide variety of surfaces and also both their loss and destabilization. Adsorption
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`of protein on surfaces is an important phenomenon, which should be considered while
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`formulating and selecting container and closure for pharmaceutical products. This is extremely
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`important
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`in
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`low dose drugs. Adsorption to a surface
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`is problematic
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`in parenteral
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`administration.
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`Detection of adsorption of proteins: X-ray and neutron reflection are used to study the
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`adsorption of protein at liquid-gas and solid-liquid interfaces, and parameters like adsorbed
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`amount, total thickness of the adsorbed layer, pH, and excipients.
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`Gelation is the process that converts a fluid solution into a semi-solid mass. Microscopic
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`examination reveals that the gel is composed of multiple peptide fibrils, intertwined in a
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`complex mesh. It is known that pH, temperature and ionic strength all affect the rate of
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`gelation, as well as the physical properties of the gel –for e.g. Transparency, gel stiffness,
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`reversibility and so on. These factors all suggest that colloidal stability plays an important role in
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`gel formation.
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`Colloidal stability determines whether peptide molecules are attached to each other or
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`repelled. Low colloidal stability means that the net forces between peptide molecules are
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`attractive overall, which leads to decreased solubility and increased likelihood to assemble into
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`larger structures, such as fibrils. Conversely, increased colloidal stability indicates net repulsive
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`forces between peptides, which improves solubility and diminishes growth of organised fibrils.
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`Stabilization: The problem of aggregation can be overcome, by modulating the solution
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`conditions such as pH, buffer composition and ionic strength and by addition of other
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`excipients. Like Cyclodextrins have been shown to improve the physical stability of peptides by
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`shielding hydrophobic amino acids. For Glucagon the addition of cyclodextrins was found to
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`delay the formation of insoluble aggregates. Similarly, the addition of sucrose has been shown
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`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
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`container surface, or compounds that reduce surface interactions such as surfactants,
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`carbohydrates or aminoacids, can be employed. In formulation, surfactant addition can reduce
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`adsorption losses e.g., Tween 80 and Pluronic F68 have been shown to reduce the adsorption of
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`calcitonin to a glass surface. The preservatives and surfactants are sometimes essential in
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`protein formulation for prevention of microbial growth, and to prevent aggregation and
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`adsorption.For avoiding the problem of Denaturation, Proteins and peptides are often
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`formulated with excipients such as polyalcohols and polymers, to protect them during freeze-
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`drying and storage. Polymers are also used to form a matrix, for controlled release. Excipients
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`such as heparin, and anionic polymers, decreased the rate of covalent aggregation in
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`recombinant human keratinocyte growth factor (rhKGF), at elevated temperatures.Polyhydric
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`alcohols like mannitol, sorbitol, and non reducing sugars like dextrose, sucrose, and trehalose,
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`are the most commonly used excipients in lyophilized protein and peptide formulations. Also
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`the optimum conditions of temperature, pH , ionic strength and moisture are need for the
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`stabilization of the proteins and peptides from the problem of gelation.
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`CHEMICAL STABILITY
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`Occurs through the following mechanisms:
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` Deamidation
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` Oxidation
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` Cystine destruction and thiol- disulfide exchange
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` Hydrolysis at aspartic acid residue
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` C- terminal succinimide formation at asparagines residue
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` Diketopiperazine formation
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` Deglycosylation and desialylation
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` Photodegradation of proteins
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` Enzymatic proteolysis and autolysis
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` Proteases activity during fermentation and cell culture
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`Deamidation: Deamidation is a common post-translational modification resulting in the
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`conversion of an asparagine residue to a mixture of isoaspartate and aspartate. Deamidation of
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`glutamine residues can occur but does so at a much lower rate. Deamidation can occur under
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`acidic, neutral or alkaline conditions, although the chemical mechanism of hydrolysis is strongly
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`dependent of pH.
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`Deamidation has been observed and characterized in a wide variety of proteins. It has been
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`shown to regulate some time-dependent biological processes and to correlate with others, such
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`as development and aging. Deamidation can make protein prone to proteases and
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`denaturation. This can affect the in vivo half-life, activity, and conformation of protein, and also
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`increase the immunogenicity of certain protein. For e.g In insulin formulation lyophilized from
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`acidic solutions (pH3-5), the rate determining first step involves intermolecular nucleophilic
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`attack of the C-terminal AsnA 21 carboxylic acid onto the side chain amide carbonyl, to release
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`ammonium, and to form reactive cyclic anhydride intermediate which can further react with
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`various nucleophiles.
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`The protein deamidation process involves the conversion of the amide side-chain moieties of
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`asparagine and glutamine residues to carboxyl groups. This conversion is an unusual form of
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`protein modification in that it requires catalysis by an intramolecular reaction where both the
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`substrate (asparagine and glutamine side chains) and "catalytic site" (the peptide nitrogen of
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`the succeeding residue) are constituents of several consecutive residues along the polypeptide
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`chain. Deamidation of asparaginyl (Asn) and glutaminyl (Gln) residues to produce aspartyl (Asp)
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`and glutamyl (Glu) residues causes structurally and biologically important alterations in peptide
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`and protein structures. At neutral pH, deamidation introduces a negative charge at the reaction
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`site and can also lead to structural isomerization. The rates of deamidation depend on primary
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`sequence, three-dimensional (3D) structure, pH, temperature, ionic strength, buffer ions, and
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`other solution properties.
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`Scheme showing the deamidation, isomerization, and racemization of peptides having
`asparagine oraspartic acid residues
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`Detection: It is detected by charge, molecular weight, and formation of succinimide residues or
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`isoaspartic acid residues and peptides maps, capillary electrophoresis, isoelectric focusing, and
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`enzyme catalyzed radio labeling of the isoaspartyl sites. Also recently an advanced technique
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`has been used which is probing Deamidation events by using anion exchange and RP- HPLC to
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`isolate two deamidated forms of recombinant hirudin at pH 3 and 37o C .
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`Stabilization: Formulation approaches include lowering of pH (desialylation can occur,
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`therefore optimization essential), compatibility studies in presence of various buffers, because
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`deamidation is also affected by buffer composition.
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`Oxidation: Oxidation generally occurs in Methionine, cystine, (more common) tryptophan,
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`tyrosine residues. The oxidation of methionine residues has been associated with the loss of
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`biological activity in a number of peptides and proteins. Its oxidation results in conversion of
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`the thioether to its sulphoxide counterpart. But this is a reversible reaction in which the
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`methionine residue can be generated either by reducing agents or enzymatically.
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`Oxygen radicals can be generated in vitro by compounds commonly used in protein
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`folding/unfolding studies. For e.g. small amount of copper in presence of glucose oxidizes a
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`particular methionine residue in α1 – proteinase inhibitor, whereas the autooxidation of the
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`reducing sugars can inactivate the enzyme rhodanese with a concomitant loss in sulfhydryl
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`titer. In addition, air oxidation of DTT can lead to H2O2 generation and subsequent protein
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`oxidation.
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`Detection: Peptide maps are convenient for detecting methionine oxidation, and MS.RP- HPLC
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`is used to separate the oxidized forms.
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`Stabilization: Formulation approaches include addition of anti oxidants, (sodium thiosulphate,
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`catalase, or platinum), and adjustment of environmental conditions (pH, or temperature).
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`Cystine oxidation can be prevented by keeping low pH. Other Formulation approaches include,
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`maintaining acidic pH, and avoiding potential reducing agents (like anti-oxidant excipients),
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`lyophilization, substituting non critical cystine residues with other residues to reduce the
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`potential instability of free thiols in presence of disulphide e.g. human interferon (IF-N) beta
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`analogue.
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`Cystine destruction and thiol- disulfide exchange: Cystine residues (disulfides) are naturally
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`occurring crosslinks
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`that covalently connect polypeptide chains either
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`intra – or
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`intermolecularly. Disulfides are formed by oxidation of thiol groups of cysteine residues by
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`either thiol disulfide interchange or direct oxidation. Intracellular proteins usually lack such
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`crosslinks and their atypical presence commonly reflects a role in enzyme’s catalytic mechanism
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`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
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`proteins.
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`Formation of a disulfide bond through oxidation of cysteine residues
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`Covalent bond formation, other than disulfide bond formation, is also involved in other
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`intermolecular cross-linkages. The covalent
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`linkages
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`in the aggregates of freeze-dried
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`ribonuclease A appeared to result from the participation of lysine, asparagine, and glutamine
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`residues as suggested by amino acid analysis of the aggregates.
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`Detection: By a systematic approach using UV spectroscopy, size-exclusion HPLC, and reversed-
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`phase chromatography. By running reduced and non reduced gel electrophoresis; SDS-PAGE
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`,ellman’s reagents for thiols detection, peptide mapping and matrix assisted Laser Desorption
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`Ionization MS.
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`Stabilization: By avoiding the use of potential reducing agents and avoiding moisture i.e the
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`proteins should be kept in anhydrous conditions.
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`C- Terminal succinimide formation at asparagines residue: Succinimide formation at the
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`asparagines residues can potentially lead to the spontaneous cleavage of polypeptide chains. In
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`this case, the side chain amide nitrogen attacks the peptide bond to form a C- terminal
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`succinimide residue and newly formed amino terminal.
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`Diketopiperazine formation: Peptides and proteins that possess an N- terminal sequence in
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`which ‘Pro’ is the penultimate residue undergo non – enzymatic hydrolysis yielding a
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`Diketopiperazine (DKP),which arises from the first two amino acids , and truncated polypeptide.
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`The mechanism of DKP formation involves nucleophilic attack of N- terminal nitrogen on
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`carbonyl carbon of the peptide bond between the second and third amino acid residues in the
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`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
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`to occur in human growth hormone, bradykinin and histrelin.
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`Diketopiperazine formation in proteins
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`Detection: The DKP products can be detected by N- terminal sequence analysis ,MS and tryptic
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`mapping. But before that the DKP products are separated by using hydrophobic interaction
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`chromatography.
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`Hydrolysis at aspartic acid residue: Hydrolysis is a pathway often observed during peptide and
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`protein degradation. As shown in scheme. Aspartic acid residues in particular are susceptible to
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`hydrolysis in theacidic pH range.for e.g. Secretin, apart from undergoing isomerization, also
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`undergoes degradation by hydrolysis of its aspartic acid residues at position-3 and position-15.
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`Hydrolysis of aspartic acid residues under acidic conditions has also been observed with
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`recombinant human macrophage colony-stimulating factor,recombinant human interleukin-11
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`,and a hexapeptide. Hydrolysis may also occur at serine and histidine residues. Peptides and
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`proteins having an aspartic acid residue also undergo isomerization, and racemization via cyclic
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`imide formation L-aspartic acid peptide can isomerize to L-iso-aspartic acid peptide via its L-
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`cyclic imide. The L-cyclic imide intermediate is capable of undergoing racemization to the D-
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`cyclic imide and thus forms the D-aspartic acid peptide and the D-iso-aspartic acid peptide on
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`hydrolysis.
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`Pathways proposed for the hydrolysis of peptides at aspartic acid residues
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`Deglycosylation and desialylation: In glycoproteins, sugars are attached either to the amide
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`nitrogen atom in the side chain of asparagines (termed N-linkage), or to the oxygen atom in the
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`side chain of serine or threonine (termed O-linkage). An asparagine residue can accept an
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`oligosaccharide only, if the residue is part of an Asn-X-Thr sequence, where X can be any
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`residue. Thus, a potential glycolisation site can be detected within aminoacid sequences. There
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`are a number of glycosylated proteins that have sugar and sialic acid molecules covalently
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`linked to peptide structure. e.g.,
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`IFN-beta has greater stability to aggregation than
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`corresponding protein produced by bacterial
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`fermentation;
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`in the non-glycosylated
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`form.Desialylation can occur at acidic pH on storage. Differing sialic acid content has shown to
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`be responsible for variability in the biological activity of highly purified pituitary lutinizing
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`hormone isoforms. The modification of human insulin by the covalent attachment of
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`monosaccharide moieties to insulin amino groups altered the aggregation and self association
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`behavior, and improved both the pharmaceutical stability and biological response.
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`Detection: Change in glycocylation can be detected by various gel methods including
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`flurophore - assisted carbohydrate electrophoresis (FACE) and MS. Change in sialic acid content
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`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
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`exchange chromatography combined with MS.
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`Photodegardation of proteins: Both ionizing and non ionizing radiations can cause protein
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`inactivation. The effects of different types of ionizing radiations (γ- rays , X –rays ,electrons and
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`α- particles)on protein molecule ( both in solid and solution states). Non –ionizing radiations
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`like UV rays also may cause irreversible damage to the protein molecules. These effects are of
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`particular concern biologically in understanding the mechanism of cataract formation and
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`sunburn damage. The amino acids tryptophan, tyrosine and cysteine are particularly
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`susceptible to UV-A (320- 400 nm) and UV- B ( 250 – 320nm) photolysis. The absorption of
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`photon leads to the photoionization and the formation of photodegaradation products through
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`either direct interaction with an amino acid or indirectly via various sensitizing agents (such as
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`dyes,riboflavin or oxygen). Commonly observed photodegardation product in an aerated,
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`neutral pH, aqueous protein solution include S-S bond fission , conversion of tyrosine to DOPA,
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`3-(4- hydroxyphenyl)lactic acid and dityrosine as well as fragmentation byproducts and the
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`conversion of tryptophan residues to kynurenine and N- formyl- kynurenine . It is also important
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`to take into account, potential damage to the protein during analysis using circular dichorism (CD), UV or
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`fluorescent measurements, where incident radiation is being used.
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`Detection: UV spectroscopy can be used to study changes in secondary and tertiary structures of
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`proteins. As protein is denatured, differences are observed in the absorption characteristics of the
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`peptide bonds due to the disruption of the exciton system.
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`Enzymatic proteolysis and autolysis: Some of enzymes have been identified in vivo that
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`specifically interact with covalently modified proteins, including carboxymethyl transferases
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`(which methylates isoaspartyl residues) and alkaline proteases (which degrades oxidized
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`proteins). It has been proposed that covalent changes caused by in vivo protein oxidation are
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`primarily responsible for the accumulation of catalytically compromised and structurally altered
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`enzymes during aging.
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`Proteases activity during fermentation and cell culture: Presence of protease enzyme can
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`result in the cleavage of recombinant protein. Protease inhibitors can minimize this to a certain
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`extent.
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`GENERAL CONSIDERATIONS FOR PROTEIN STORAGE
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`Temperature:
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` Generally, proteins are best stored at ≤ 4°C in clean, autoclaved glassware or
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`polypropylene tubes. Storage at room temperature often leads to protein degradation
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`and/or inactivity, commonly as a result of microbial growth. For short term storage (1
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`day to a few weeks), many proteins may be stored in simple buffers at 4°C.
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`
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` For long term storage for 1 month to 1 year, some researchers choose to bead single-
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`use aliquots of the protein in liquid nitrogen for storage in clean plastic containers under
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`liquid nitrogen. This method involves adding the protein solution drop wise (about 100
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`μl each) into a pool of liquid nitrogen, then collecting the drop-sized frozen beads and
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`storing them in cryovials under liquid nitrogen.
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` Frozen at -20°C or -80°C is the more common form of cold protein storage. Because
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`freeze-thaw cycles decrease protein stability, samples for frozen storage are best
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`dispensed and prepared in single-use aliquots so that, once thawed, the protein solution
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`will not have to be refrozen. Alternatively, addition of 50% glycerol or ethylene glycol
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`will prevent solutions from freezing at -20°C, enabling repeated use from a single stock
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`without warming (i.e., thawing).
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`Protein Concentration:
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`Dilute protein solutions (< 1 mg/ml) are more prone to inactivation and loss as a result of low-
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`level binding to the storage vessel. Therefore, it is common practice to add “carrier” or “filler”
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`protein, such as purified bovine serum albumin (BSA) to 1-5 mg/ml (0.1-0.5%), to dilute protein
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`solutions to protect against such degradation and loss.
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`Additives:
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`Many compounds may be added to protein solutions to lengthen shelf life:
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` Cryoprotectants such as glycerol or ethylene glycol to a final concentration of 25-50%
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`help to stabilize proteins by preventing the formation of ice crystals at -20°C that
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`destroy protein structure.
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` Protease inhibitors prevent proteolytic cleavage of proteins like Benzamidine for Serine
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`proteases, Pepstatin A for Acid proteases , Leupeptin for Thiol proteases etc.
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` Anti-microbial agents such as sodium azide (NaN3) at a final concentration of 0.02-
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`0.05% (w/v) or thimerosal at a final concentration of 0.01 % (w/v) inhibit microbial
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`growth.
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` Metal chelators such as EDTA at a final concentration of 1-5 mM avoid metal-induced
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`oxidation of –SH groups andhelps to maintain the protein in a reduced state.
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` Reducing agents such a dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) at final
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`concentrations of 1-5 mM also help to maintain the protein in the reduced state by
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`preventing oxidation of cysteines.
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`REFERENCES:
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` 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)
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` 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)
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