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Chapter: Pharmaceutical Drugs and Dosage: Protein and peptide drug delivery

Protein pharmaceuticals commonly exhibit both physical and chemical 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.

Physical instability

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 and precipitation

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.

Surface adsorption

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

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 exchange

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.

Maillard reaction

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 open chain allows nucleophilic attack of the amine group of a basic amino acid side chain on the carboxyl carbon. Sucrose, even though a nonreduc-ing sugar, is a disaccharide and can get hydrolyzed at acidic pH into reduc-ing sugars.

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.

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