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Pharmaceutical Dosage Forms and Drug Delivery
of size, shape, and solution properties of proteins through direct and indirect techniques that include the
following:
• tography, gel electrophoresis, and/ or viscometry.
• Thermodynamic methods such as microcalorimetry and surface plasma resonance can help delineate the state of protein association and interactions with other molecules in solution.
• Particulate formation is caused by protein self-association or interaction with other components in
a solution by dynamic light scattering (DLS).
• copy can help determine the stability of protein conformation in solution.
25.3.2 Physicochemical Characterization
25.3.2.1 Solubility
uble. The water solubility of a protein requires interactions, such as hydrogen bonding and electrostatic
interactions, of protein surface with the aqueous medium. The hydrophilic interactions, which are stronger
solubility of proteins and peptides is dependent on the pH of the solution.
of all its functional groups. A protein is usually positively charged at a low pH and negatively charged at
a high pH. Protein solubility increases as the pH of the solution moves away from the isoelectric point
(IEP) (Figure 25.5
protein at its IEP lead to a greater tendency for self- association. As the net charge on the protein changes
the aqueous environment increases, and the protein molecules also exert greater electrostatic repulsion
among each other, thus preventing them from self- associating. This increases their aqueous solubility.
However, extremes of pH can cause the protein to unfold with the exposure of hydrophobic groups and
protein self- association at their exposed hydrophobic regions, leading to precipitation.
The phase behavior of protein solutions, whether a protein solution is a single- phase solution or has
protein separation (solid and liquid), is affected by pH, ionic strength, and temperature ().
FIGURE 25.5
isoelectric point.
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Protein and Peptide Drug Delivery
FIGURE 25.6 Phase behavior of proteins in solution formulation. Typical phases of physical instability of protein in
solution with the addition of a precipitating agent (such as salt) or change of a precipitation- inducing phenomenon (such as
temperature).
437
(Figure 25.5). This phenomenon is called the salting out effect. This phenomenon is used to concentrate
dilute solutions of proteins and to separate a mixture of proteins (if one of the proteins salts out at a lower
salt concentration than the other). The added salt can then be removed by dialysis.
Organic solvents tend to decrease the solubility of proteins by lowering the dielectric constant of the
solution (). The presence of other highly water- soluble polymers in the solution (cosolutes) also
tends to reduce protein solubility by their interactions with solvent molecules, thus tying up the solvent and
reducing protein– solvent interactions. This phenomenon is known as the volume exclusion effect.
25.3.2.2 Hydrophobicity
Different amino acids have different degrees of hydrophobicity (Figure 25.4). The overall hydrophobicity
or hydrophilicity of a protein is determined by the nature of functional groups exposed on the surface of
the protein. These groups contribute to protein– solvent and, protein– protein interactions.
In an aqueous solution, hydrophobic regions of a polypeptide tend to point away from the hydrophilic
aqueous environment to achieve the thermodynamically least energy state of greatest stability. In doing
so, the hydrophobic surfaces of a protein tend to cluster together on the inside of the protein and form
multiple weak van der Waals interactions. These multiple simultaneous weak hydrophobic interactions
and ionic interactions, hydrophobic interactions within and among a protein’s polypeptide chains sta-
sequences in synthetic peptides are at the optimum distances in space, the molecules coil with the hydrophobic amino acids on the inside of each coil and the hydrophilic ones on the outside. Thus, secondary,
tertiary, and quaternary structures of polypeptide chains are important in determining the net hydrophobic
or hydrophilic nature of the protein.
25.4 Instability
Protein pharmaceuticals commonly exhibit both physical and chemical instability. Physical instability
refers to changes in the higher- order structure that do not include covalent bond cleavage or formation.
or bond cleavage (e.g., deamidation), yielding a new chemical entity. Physical instability often results in
protein denaturation (loss of natural conformation), which can lead to adsorption to surfaces, aggregation,
and precipitation.
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25.4.1 Physical Instability
Pharmaceutical Dosage Forms and Drug Delivery
Protein denaturation is a result of the change in higher- order folding or conformation that commonly
exhibits as a change in the surface exposure of functional groups. An increase in surface hydrophobicity
due to protein denaturation can lead to aggregation, precipitation, and/ or adsorption to the surface of the
container or closure.
25.4.1.1 Denaturation
Protein native structure represents the least overall thermodynamic free energy of interaction of different
residues of the polypeptide(s) with the solvent (water) and with themselves. This determines the
native state of protein structure. The three- dimensional structure of a protein is held together by weak
such as solution composition and temperature. For example, a change in the solvent medium can result
in a different structure, which is the lower, thermodynamically least free energy state of protein conformation. For example, the addition of salt or organic solvent would reduce the propensity for hydrophilic
interactions on the protein surface.
If the enthalpy barrier from the native state to the altered lower thermodynamic free energy state
can be met (e.g., by heating the protein solution), the protein conformation might change to the new
form of thermodynamically least free energy. This loss of a natural or native state of a protein is termed
denaturation. Protein denaturation refers to a disruption of the tertiary and secondary structure of a pro-
chemicals. Protein denaturation is often associated with the increased hydrophobic surface of a protein.
Several protein molecules in the solution might self- associate and exclude the solvent in such cases. This
phenomenon is termed aggregation. If the aggregates separate from the solution and become visible, the
phenomenon is called protein precipitation.
Protein denaturation can also lead to protein unfolding. It can be reversible or irreversible.
Reversible denaturation can be caused by temperature or exposure to chaotropic agents, such as
noncovalent 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.
25.4.1.2 Aggregation and Precipitation
Aggregation of proteins refers to the 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, leading to thermodynamically unfavorable surroundings of the hydrophobic protein. This drives intermolecular interactions
between exposed hydrophobic regions of different protein molecules, leading to association and, thus,
aggregation.
When insoluble protein aggregates are visually evident, the protein is said to have precipitated. Protein
precipitation is a macroscopic process producing a visible change in the protein solution, such as turbidity/ clouding of the solution or formation of visible particulates. On the other hand, the accumulation of
soluble protein aggregates 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.
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25.4.1.3 Surface Adsorption
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protein surface interaction with nonpolar surfaces. 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
adsorption to a 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
25.4.2 Factors Affecting Physical Stability
25.4.2.1 Peptide Concentration
plateau. During the lag phase, a number of oligomeric species, including nucleating species, and short
then slows as a plateau is reached, which represents either the point where monomers are completely
depleted or an equilibrium point.
25.4.2.2 Amino Acid Sequence
an aggregation- prone region (APR). Prediction of aggregation propensity relies on factors like gate-
25.4.2.3 pH and Net Charge
Electrostatic interactions are crucial for peptide self- association and forming aggregates. The higher
the net charge, the slower aggregation and the lower the propensity to aggregate. Solution conditions
affect the rate and extent of aggregation. For example, the aggregation kinetics of GLP- 1 into amyloid-
and acylphosphatase. Aggregation kinetics can be different around the IEP of peptides and proteins,
concentrations.
25.4.2.4 Chemical Degradation
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Pharmaceutical Dosage Forms and Drug Delivery
biologic aggregation. These pathways alter physical properties like hydrophobicity, secondary and tertiary structure, and thermodynamic and kinetic barriers to unfold, affecting the aggregation of proteins.
25.4.2.5 Effect of ß- Elimination and Racemization
been observed in Asp residues in crystallin, as described in several studies.
25.4.2.6 Chemical Modifications
on stability remains unclear. Recent biophysical studies on liraglutide and novicidin showed that lipidated
peptides can form oligomers in solution, with a pH- sensitive equilibrium observed near pH 7. Acylation
-
tion and increased oligomer formation.
25.4.2.7 Surfaces and Interfaces
Surface adsorption is a physical degradation process where peptide molecules accumulate and adhere
to a surface without penetration, causing them to become insoluble. Factors driving adsorption include
intra- molecular forces, hydrophobicity, and ionic or electrostatic interactions. Research on human insulin
and protein adsorption rate is typically governed by diffusion and concentration, with surface- adsorbed
species increasing over time due to further peptide aggregation at the site.
25.4.2.8 Excipients
sugars, and carbohydrates have been used to reduce aggregation in various systems, with their effects on
used buffers in pharmaceutical development being acetate, citrate, histidine, phosphate, tris, and glycine.
stability. Their effects vary based on the surface charge of the peptide or protein. The overall effect of
salts on physical stability is a balance of various interactions with water and biomolecules, including
Hofmeister effects and Debye- Hückel effects.
Surfactants, such as Tween 20 and Tween 80, are used in pharmaceutical development to prevent aggregation or adsorption in peptide solutions. However, their chemical stability is crucial, as they undergo
oxidation and cleavage at their ethylene oxide subunits and hydrolysis of the fatty acid ester bond. This
can promote precipitation in some peptide formulations. For example, Tween 80 in the protein IL- 2
formulation inhibited shaking- induced aggregation but also affected protein oxidation and aggregation
during liquid formulation storage. To prevent this issue, methionine and tryptophan are used in formulation development to prevent Tween from oxidative degradation.
to prevent bacterial growth during storage. However, they can sometimes cause protein aggregation, such
as recombinant human interleukin- 1 receptor aggregation and cytochrome c aggregation, as reported in
some studies.
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25.4.2.9 Impurities
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tion rates. These impurities can originate from raw materials, manufacturing processes, and degradation
during manufacturing or storage and can include deletions, truncations, diastereoisomers, substitutions,
Most synthetic peptides are manufactured using solid- phase procedures, and the resulting product usually
contains TFA salts, which affect the physico- chemical properties of the peptidic material. The presence
grade glucagon.
25.4.2.10 Temperature/ Pressure/ Agitation/ Lyophilization
toward smaller- volume structures, acting on cavities in hydrophobic cores of natively folded proteins
been observed, and low temperatures combined with high pressures can dissociate aggregates and
and protein aggregation to accelerate kinetics and improve experimental results. However, little is
known about the shear forces produced and their effects on different steps in the aggregation reaction. Comparing systems studied in different research groups is also challenging due to variations in
stirring/ shaking.
25.4.3 Chemical Instability
Chemical instability of proteins and peptides generally involves one or more of the following chemical
reactions.
25.4.3.1 Hydrolysis
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
mone (LHRH), macrophage colony- stimulating factor (M- CSF), human growth hormone, and vasoactive intestinal peptide (VIP).
toxicities or immunogenicity as well.
Protein degradation by hydrolysis can be observed during stability testing by the formation of charge
degradants and the location of hydrolysis.
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25.4.3.2 Deamidation
Pharmaceutical Dosage Forms and Drug Delivery
Deamidation is one of the main chemical degradation pathways of proteins. The side- chain linkage in
changes the asparaginyl residue into an aspartyl or isoaspartyl residue. The deamidation of Asn and Gln
physiological conditions.
comparability of the dosed drug substance.
place. In some cases, protein engineering to replace Asn residue with Ser can be used if it does not affect
protein conformation and biological activity.
25.4.3.3 Oxidation
functional
groups in proteins that can undergo oxidation include the following (Figure 25.7):
• Sulfhydryl in cysteine (Cys)
•
• Thiolether in methionine (Met)
• Phenol in tyrosine (Tyr)
• Indole in tryptophan (Trp)
Factors that increase oxidative degradation in proteins include the following:
• Atmospheric oxygen alone can lead to the oxidation of Met residues, producing the corresponding
sulfoxide.
•
and thioether groups of proteins at neutral or slightly alkaline pH. The source of peroxides in the
formulation is often the hydrophilic polymeric excipients used.
•
3+
/ Fe
and Cu+ / Cu
2+
), light, acid/ base,
2+
and free radicals.
• Solution pH, nature of buffers, presence of metal ions and metal chelators, and neighboring amino
• Light, which may photoactivate triplet ground state oxygen to the excited, more reactive singlet state.
Stabilization strategies
• Low- temperature storage or refrigeration to reduce reaction rates.
•
• Protection from light by the use of amber glass containers for storage.
•
•
agents sequester free metals, such as iron and copper from the formulations.
•
•
hydrogen bonding on the protein surface to preserve its native conformation.
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Protein and Peptide Drug Delivery
FIGURE 25.7
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25.4.3.4 Racemization
Pharmaceutical Dosage Forms and Drug Delivery
25.4.3.5 Disulfide Exchange
ment of these bonds can alter the tertiary structure, thereby affecting protein conformation, stability, and
pN- ethylmaleimide.
Figure 25.8
25.4.3.6 Maillard Reaction
The use, or presence as impurities, of reducing sugars (e.g., glucose, lactose, fructose, maltose, xylose)
in a protein formulation can result in the Maillard browning reaction
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 solution in equilibrium. The presence of the aldehyde or the ketone group
in the open chain allows nucleophilic attack of the amine group of a basic amino acid side chain on the
FIGURE 25.8
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at acidic pH into reducing sugars.
Maillard reaction results in the formation of a Schiff base (R1R2C= N– R3), which can further rearrange
browning reaction
at low temperatures.
25.5 Antigenicity and Immunogenicity
The ability of a protein to generate an immune response, triggering the production of antibodies, is
referred to as immunogenicity
response due to low concentration or longer time required for the humoral and cellular immune processes.
Repeated protein administration may often lead to the formation of antibodies, causing an immune
reaction.
Antigenicityepitopes) on the protein to recog-
lead to an immune reaction if the host immune system has antibodies against the foreign protein epitope.
When antibodies are developed upon repeated protein administration (immunogenicity), the protein may
host has formed mature antibodies against the protein.
self- protein
and normally do not elicit an immune response, misfolded or denatured forms of self- proteins may be
immunogenic. Thus, immunogenicity may be prevented by maintaining the molecule in the properly
the recombinant DNA- produced proteins are likely to be relatively more immunogenic compared to
murine antibodies to make chimeras have greatly improved their therapeutic potential by reducing or
eliminating their immune response.
25.6 Drug Product Formulation and Process
commercially available protein pharmaceuticals are administered by parenteral routes. Parenteral pro-
for example, insulin, can also be delivered by inhalation for absorption through the alveolar mucosal
membrane.
Parenterally administered proteins are rapidly cleared from circulation by the reticuloendothelial
with a hydrophilic polymer such as PEG (PEGylation), as discussed earlier. Protein bioavailability from
higher when administered by the SC route. At the same time, SC administration is preferred because it
allows for patient self- administration— as compared to IV administration, which must be carried out by
a healthcare provider.
25.6.1 Route of Administration
Selection of the appropriate route of administration for a protein drug depends on several factors,
including the disease state, the desired onset and duration of drug absorption/ action, drug dose,
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