Protein pharmaceuticals commonly exhibit both physical and chemical instability.
Instability
Protein
pharmaceuticals commonly exhibit both physical and chemical instability.
Physical instability refers to changes in the higher order struc-ture that does
not include covalent bond cleavage or formation, whereas chemical instability
refers to modification of proteins via bond formation (e.g., oxidation) or bond
cleavage (e.g., deamidation), yielding a new chemi-cal entity. Physical
instability often results in protein denaturation (loss of natural
conformation), which can lead to adsorption to surfaces, aggrega-tion, and
precipitation.
Protein
denaturation is a result of change in higher order folding or con-formation
that commonly exhibits as a change in the surface exposure of functional
groups. Increase in surface hydrophobicity due to protein dena-turation can
lead to aggregation, precipitation, and/or adsorption to the surface of the
container or closure.
Protein
native structure represents the least overall thermodynamic free energy of
interaction of different residues of the polypeptide(s) with the sol-vent
(water) and with themselves. This determines the native state of pro-tein structure. The three-dimensional structure
of a protein is held together by weak noncovalent interactions, is flexible,
and relatively unstable. It can be modified by environmental factors, such as
solution composition and temperature. For example, a change in the solvent
medium can result in a different structure being the lower, thermodynamically
least free energy state of protein conformation. For example, addition of salt
or organic sol-vent would reduce the propensity for hydrophilic interactions on
the pro-tein surface.
If
the enthalpy barrier from the native state to the altered lower thermo-dynamic
free energy state can be met (e.g., by heating the protein solution), the protein
conformation might change to the new form of thermodynami-cally least free
energy. This loss of natural, or native, state of a protein is termed denaturation. Protein denaturation
refers to disruption of the ter-tiary and secondary structure of a protein or
peptide. It can be caused by heating, cooling, freezing, extremes of pH, and
contact with organic chem-icals. Protein denaturation is often associated with
increased hydrophobic surface of a protein. In such cases, several protein
molecules in solution might self-associate and exclude the solvent. This
phenomenon is termed aggregation. If
the aggregates separate from the solution and become vis-ible, the phenomenon
is called protein precipitation.
Protein
denaturation can also lead to protein unfolding. It can be revers-ible or
irreversible. Reversible denaturation can be caused by temperature or exposure
to chaotropic agents, such as urea
and guanidine hydrochlo-ride. The chaotropic agents interfere with stabilizing
intramolecular nonco-valent interactions in proteins, including hydrogen
bonding, van der Waals forces, and hydrophobic effects. In the case of reversible denaturation, if the
denaturing condition is removed, the protein will regain its native state and
maintain its activity. Irreversible denaturation
implies that the unfolding process disrupted the native protein structure to
the extent that the native structure cannot be regained simply by changing the
denaturing condition (such as temperature). The ease of protein denaturation
depends on the strength and number of intermolecular interactions that keep the
protein in its native conformation.
Aggregation
of proteins refers to nonreversible interaction and clustering of two or more
protein molecules. Protein aggregates may
be soluble or insoluble. Protein
aggregation is driven by the unfolding process, which exposes the interior hydrophobic region to the solvents, usually
water, lead-ing to thermodynamically unfavorable surroundings of the
hydrophobic protein. This drives intermolecular interactions between exposed
hydro-phobic regions of different protein molecules, leading to association
and, thus, aggregation.
Several
factors may lead to protein aggregation. For example:
·
Shear forces: Shearing and
shaking of protein solutions during formu-lation and shipment may lead to
aggregation.
·
Temperature: An increase in
temperature results in greater flexibility
of proteins and an increased tendency to form aggregates.
·
Ionic strength: An increase in the
ionic strength may lead to neutral-ization of the surface charge of the protein
molecules, which may lead to aggregation.
·
pH: Charge
neutralization and subsequent aggregation can also occur when the pH of the solution approaches the IEP of the protein.
·
Moisture: An optimal residual
moisture level is required to maintain the
stability of lyophilized protein formulations, the absence of which may lead to
protein aggregation. Thus, hydration in formulated pro-teins must be ensured by
either increasing residual moisture content or by adding water-substituting
excipients.
When
insoluble protein aggregates are
visually evident, the protein is said to have precipitated. Protein
precipitation is a macroscopic process produc-ing a visible change of the
protein solution, such as turbidity/clouding of the solution or formation of
visible particulates. Accumulation of soluble
protein aggregates, on the other
hand, is evident by the changes in solution
properties of proteins, such as viscosity.
Native,
folded proteins may precipitate under certain conditions, most notably salting
out and isoelectric precipitation. Protein precipitation can be a result of
both covalent and noncovalent aggregation pathways.
The
adsorption of proteins and peptides to the surfaces of the container, closure,
or filter results from protein surface interaction with nonpolar sur-faces.
This can cause proteins to expose their hydrophobic interior, leading to
adherence or adsorption to the surfaces of the containers. Alterations in the
pH and ionic strength of the media can significantly enhance or reduce the
protein’s tendency to adsorb. Protein adsorption to neutral or slightly charged
surface is greatest at its IEP.
The
effect of surface adsorption on the amount of administered drug can be substantial
when the initial concentration of the protein in solution is low, leading to a
high proportion of drug loss due to adsorption. The extent and reversibility of
protein adsorption are dependent on the conformational state of the protein,
the pH and ionic strength of the solution, the nature of the exposed surface,
surface area, and time of exposure. Poly(oxyethylene oxide) (Teflon)-coated
surfaces and siliconized rubber stoppers for vials can minimize the likelihood
of protein adsorption at the surface. Certain for-mulation strategies, such as
increase in the concentration of surfactant, and prerinse of the IV
administration tube-set and filter with the diluent can also minimize protein
adsorption to surfaces.
Chemical
instability of proteins and peptides generally involves one or more of the
following chemical reactions.
Proteolysis
is the hydrolysis of the peptide bond between amino acids in a peptide or
protein. At an extreme pH and temperature, the peptide bond can undergo rapid
proteolysis resulting in protein degradation and/or fragmentation. The most
commonly observed proteolytic reactions in proteins and peptides involve the
side-chain amide groups of asparagine (Asn) and glutamine (Gln), and the
peptide bond on the C-terminal side of an aspartic acid (Asp) or a proline
(Pro) residue. Several therapeutic proteins are known to degrade through
hydrolysis. These include luteinizing hormone-releasing hormone (LHRH),
macrophage colony-stimulating fac-tor (M-CSF), human growth hormone, and
vasoactive intestinal peptide (VIP).
Hydrolysis
leading to protein fragmentation generally compromises pro-tein efficacy and
may produce toxicities or immunogenicity as well.
Protein
degradation by hydrolysis can be observed during stability test-ing by the
formation of charge variants (by isoelectric focusing) or size variants (by
size exclusion chromatography [SEC]). Isolation of these new peaks followed by
their size determination by mass spectroscopy (MS) and/or composition
determination by tryptic peptide mapping (TPM) helps identify the exact size
and sequence of degradants, and the location of hydrolysis.
Deamidation
is one of the main chemical degradation pathways of pro-teins in which the
side-chain linkage in a glutamine (Gln) or asparagine (Asn) residues is
hydrolyzed to form a carboxylic acid. The hydrolysis changes the asparaginyl
residue into an aspartyl or isoaspartyl residue. The deamidation of Asn and Gln
residues of proteins is an acid and base-catalyzed hydrolysis reaction, which
can occur rapidly under physiologi-cal conditions.
Deamidation
may or may not impact protein efficacy, safety, and immu-nogenicity. Thorough
characterization and understanding of the sites and extent of deamidation,
nonetheless, are critical to clinical comparability of the dosed drug
substance.
Deamidation
is generally detected by the change in the size and charge variants, and the
location of deamidation is confirmed by TPM.
Solution
pH optimization and lyophilization are frequently used to minimize deamidation
in proteins. However, residual moisture pres-ent in the lyophilized formulation
can still allow deamidation to take place. In some cases, protein engineering
to replace Asn residue with Ser can be used if it does not affect protein
conformation and biologi-cal activity.
Oxidation
is one of the major causes of chemical degradation in proteins and peptides.
The functional groups in proteins
that can undergo oxidation include the following (Figure
25.9):
·
Sulfhydryl in cysteine (Cys)
·
Imidazole in histidine (His)
·
Thiolether in methionine (Met)
·
Phenol in tyrosine (Tyr)
·
Indole in tryptophan (Trp)
Figure 25.9 Side-chain oxidation products of oxidizable amino acid residues in a protein.
Factors that increase
oxidative degradation in proteins include the following:
·
Atmospheric oxygen, which alone can lead to oxidation of Met
resi-dues, producing the corresponding sulfoxide.
·
Peroxides, such as hydrogen peroxide, can modify indole,
sulfhydryl, disulfide, imidazole, phenol, and thioether groups of proteins at
neu-tral or slightly alkaline pH. The source of peroxides in formulation is
often the hydrophilic polymeric excipients used.
·
Oxidation can be catalyzed by metal contaminants (e.g., Fe2+/Fe3+
and Cu+/Cu2+), light, acid/base, and free radicals.
·
Solution pH, nature of buffers, presence of metal ions and
metal chelators, and neighboring amino acid residues of susceptible amino acids
influence oxidation in solution.
· Light, which may photoactivate triplet ground state oxygen to the excited, more reactive singlet state.
Stabilization
strategies to
prevent or minimize oxidative degradation of
proteins include the following:
·
Low temperature storage or refrigeration to reduce reaction
rates.
·
Nitrogen overlay in packaging to minimize the impact of
headspace air/oxygen exposure.
·
Protection from light by the use of amber glass containers
for storage.
·
pH optimization.
·
Use of antioxidants and chelating agents. Antioxidants
terminate free-radical reactions. Chelating agents sequester free metals, such
as iron and copper from the formulations.
·
Lyophilization.
·
Certain sugars might prevent or minimize protein oxidation
by com-plexation with metal ions or hydrogen bonding on the protein surface to
preserve its native conformation.
Racemization
can affect protein conformation. All amino acid residues except glycine (Gly)
are chiral at the carbon atom bearing the side chain and are subject to
base-catalyzed racemization. The rate of racemization depends on the particular
amino acids and is influenced by temperature, pH, ionic strength, and metal ion
chelation. Aspartic acid and serine resi-dues are most prone to racemization.
Disulfide
bonds provide covalent structural stabilization in proteins. Cleavage and
subsequent rearrangement of these bonds can alter the tertiary structure,
thereby affecting protein conformation, stability, and biological activity.
Disulfide exchange is catalyzed by thiols, which can arise by initial reduction
of disulfide bond, or β-elimination
in neutral or alkaline media. Disulfide thiol exchange reactions can be
inhibited by the addition of effi-cient thiol scavengers, such as p-mercuribenzoate and N-ethylmaleimide. Figure 25.10 illustrates a
cysteine–disulfide exchange reaction.
Figure 25.10 An illustration of the effect of cysteine disulfide exchange on protein conformation.
The
use, or presence as impurities, of reducing
sugars (e.g., glucose, lac-tose, fructose, maltose, xylose) in a protein
formulation can result in the Maillard browning
reaction, which involves nonenzymatic glycation of the protein at the basic
protein residues such as lysine, arginine, asparagine, and glutamine. Reducing
sugars have an open chain (with an aldehyde or ketone group) and a closed chain
(cyclic oxygen) form coexisting in solu-tion in equilibrium. The presence of
the aldehyde or the ketone group in the
Maillard
reaction results in the formation of a Schiff base (R1R 2C=N–R3),
which can further rearrange to form products with π-electron cloud con-jugation, which
are colored products—hence the name browning
reaction. Maillard reaction could be minimized or prevented by removing
reactive substrate (reducing sugars), pH adjustment, chelation of trace metals,
use of antioxidant, reducing water content (thus minimizing the plasticity and
solute reactivity in the lyophilized solid matrix), and storage at low
temperatures.
Related Topics
TH 2019 - 2024 pharmacy180.com; Developed by Therithal info.