Rt

Hence,

When 50% of the molecules are unfolded [D] = [N] and KND = 1, therefore AGN D = 0. The temperature at which this occurs is referred to as the melting temperature, Tm. An increase of Tm is indicative of an increase of stability; for example, T4 lysozyme (lysozyme from bacteriophage T4) is more stable at pH 6.5 where Tm is 65°C than at pH 2 where Tm is 42°C.

Aggregation

Some proteins self-associate in aqueous solution to form oligomers. Insulin, for example, exists in several associated states; the zinc hexamer of insulin is a complex of insulin and zinc which dissolves slowly into dimers and eventually monomers following its subcutaneous administration, so giving it long-acting properties. In most cases, however, it is desirable to prevent association such that only monomeric or dimeric forms are present in the formulations and a more rapid absorption is achieved. Recent studies have been directed towards engineering insulin molecules which are not prone to association,4-6 or the prevention of association through the addition of surfactants.7 Protein self-association is a reversible process, i.e. alteration of the solvent properties can lead to the re-formation of the monomeric native protein. There is an important distinction between this association process and the aggregation of proteins, which relates to the usually nonreversible interaction of protein molecules in their denatured state. Aggregation therefore implies that the proteins have undergone some form of denaturation prior to their interaction.

If an intermediate forms that has a solubility less than that of N or D this can lead to aggregation and eventually to precipitation. For example, the addition of moderate amounts of denaturant to bovine growth hormone (bGH) can generate a partially unfolded intermediate of low solubility which aggregates. Similarly, y-interferon (IFN-y) is inactivated by acid treatment or by the addition of salt because the dimeric native state is converted into monomers, which are partially denatured. For both proteins the formation of intermediates leads to inactivation.

Surface adsorption and precipitation

The adsorption of proteins such as insulin on surfaces such as glass or plastic in giving sets is important as it can reduce the amount of agent reaching the patient. It can also lead to further denaturation, which can then cause precipitation and the physical blocking of delivery ports in insulin pumps, for example. Insulin adsorption on the surface of the containers and the subsequent 'frosting' effect, due to the presence of a finely divided precipitate on the walls, is accelerated by the presence of a large headspace allowing a greater interaction of the insulin with the air/water interface, which facilitates denaturation.

11.2.2 Formulation and protein stabilisation

There are several possible ways in which the physical stability of the protein can be improved through formulation. We will examine methods for minimising this and chemical degradation in the following sections.

Prevention of adsorption

Some measures can be taken to eliminate, or at least minimise, protein denaturation resulting from surface adsorption. The surface of glass is conducive to adsorption and it is preferable in principle to use more hydrophilic surfaces, although this may not be feasible in practice. Alternatively, when the use of glass cannot be avoided, components may be added to the protein solution to prevent adsorption to the glass surface. These additives can act by coating the surface of glass or by binding to the proteins. For example, serum albumin can be included in the formulation since this will compete with the therapeutic protein for the binding sites on the glass surface and so reduce its adsorption. A similar effect can be achieved by the addition of surfactants such as poloxamers and polysorbates to the protein solution. Consideration must, however, be given to the effects of the surfactants on the pharmacology of the protein and to the toxi-cological effects of the surfactant itself.

Minimisation of exposure to air

Significant denaturation of proteins can occur when the protein solutions are exposed at the air/solution interface. In this respect the air interface is behaving as a hydrophobic surface and the extent of denaturation is found to be dependent on the time of exposure of the protein at the interface. Agitation of protein solutions in the presence of air or application of other shear forces, such as those which occur when the solutions are filtered or pumped, may also cause denaturation. Again, the inclusion of surfactants can reduce denat-uration arising from these processes. Stability testing of protein-containing formulations often involves subjecting the solutions to shaking for several hours and the subsequent assessment of the protein configuration. If the protein has retained its native state and has not aggregated, the formulation is considered to be stable against surface or shear-induced denaturation.

Addition of cosolvents

Some excipients and buffer components added to the protein solution are able to minimise denaturation through their effects on solvation. These compounds, including polyethylene glycols and glycerol, are referred to as cosolvents. They may act either by causing the preferential hydration of the protein or alternatively by binding to the protein surface. Preferential hydration results from an exclusion of the cosolvent from the protein surface due to steric effects (as in the case of polyethylene glycols); surface tension effects (as with sugars, salts and amino acids) or some form of chemical incompatibility such as charge effects. As a result more water molecules pack around the protein in order to exclude the additive and the protein becomes fully hydrated and stabilised in a compact form (Fig. 11.8). Alternatively, the cosolvent may stabilise the protein molecule either by binding to it nonspecifically or by binding to specific sites on its surface.

Cosolvent effects such as this can be analysed from a thermodynamic point of view. Addition of cosolvents which cause preferential hydration of the protein stabilises the compact conformations of the protein because the co-solvent results in an increase in the free energy of the system. To reduce the free energy, the surface area of the protein is minimised.

Optimisation of pH

In order to avoid stability problems arising from charge neutralisation and to ensure adequate solubility, a pH must be selected which is at least 0.5 pH units above or below the iso-electric point. This is often difficult to achieve, however, since a pH range of 5-7 is usually required to minimise chemical breakdown and this frequently coincides with the iso-electric point.

Characterisation of degradation

Finally, if the formulation does not prevent denaturation and aggregation of the protein, then the pharmacology, immunogenicity, and toxicology of the denatured or aggregated protein must be studied to determine its safety and efficacy. Several studies must be performed to determine the extent of degradation that is acceptable for administration. If the aggregates are soluble there may be a significant effect on the pharmacokinetics and immunogenicity of the protein. On the other hand, insoluble aggregates are generally unacceptable and instructions are usually given

Preferential binding O Water A Solvent additive

Preferential hydration

Preferential binding O Water A Solvent additive

Preferential hydration

Figure 11.8 Schematic illustration of preferential binding and preferential hydration by solvent additives. In preferential binding the additive occurs in the solvation shell of the protein at a greater local concentration than in the bulk solvent, while preferential hydration results from the exclusion of the additive from the surface of the protein.

Reproduced from S. N. Timasheff and T. Arakawa, in Protein Structure: A Practical Approach (ed. T. E. Creighton), pp. 331 -345, IRL Press, Oxford, 1989.

not to administer protein solutions containing precipitates.

11.2.3 Chemical instability

Chemical instability can involve one or more of a variety of chemical reactions including:

• Deamidation, in which the sole chain linkage in a glutamine (Gln) or asparagine (Asn) residue is hydrolysed to form a carboxylic acid.

• Oxidation: the side-chains of histidine (His), methionine (Met), cysteine (Cys), tryptophan (Trp) and tyrosine (Tyr) residues in proteins are potential oxidation sites.

• Racemisation: all amino acid residues except glycine (Gly) are chiral at the carbon atom bearing the side-chain and are subject to base-catalysed racemisation.

• Proteolysis, involving the cleavage of peptide (ZCO-NHY) bonds.

• Beta elimination: high-temperature treatment of proteins leads to destruction of disulfide bonds as a result of ^-elimination from the cystine residue.

• Disulfide formation: the interchange of disul-fide bonds can result in an altered three-dimensional structure.

Table 11.5 lists the amino acids or sequences which are subject to chemical degradation and lists the formulation approvals used to overcome the problems.

Protein deamidation8

In the deamidation reaction, the side-chain amide linkage in a glutamine (Gln) or asparagine (Asn) residue is hydrolysed to form a free carboxylic acid; the Asn peptides being more susceptible to deamidation than the Gln peptides.

The deamidation of Asn and Gln residues of proteins is an acid- and base-catalysed hydrolysis reaction which can occur rapidly under physiological conditions. As with all acid-base catalysed reactions, there will be an optimum pH for stability; for example, the deamidation of glutaminyl residues of pentapeptides occurs at a minimum rate at pH 6. The rate of deamidation is strongly influenced by the sequence of residues in the peptide molecule. Different mechanisms are responsible for deamidation in neutral/alkaline and in acidic aqueous solution. The acid-catalysed deamidation reaction involves protonation of the amide leaving-group to form aspartate directly as the major degradation product, as outlined in Scheme 11.1. At neutral to alkaline pH, deamidation is believed to proceed through a cyclic imide intermediate by attack of the peptide nitrogen on the C-terminal side of the asparagine on the side-chain carbonyl group, producing isoaspartate and aspartate as outlined in Scheme 11.2.

The hydrolysis reactions are completely reversible, i.e. the final products of deamida-tion can interconvert through the cyclic imide

Table 11.5 lists the amino acids or sequences which are subject to chemical degradation and lists the formulation approvals used to overcome the problems.

Table 11.5 Amino acids or sequences susceptible to chemical degradation, together with formulation strategies to reduce degradation"

Amino acid or sequence

Mechanism of degradation

Formulation strategy

Cysteine-cysteine

Glutamine, asparagine Tryptophan, methionine, cysteine, tyrosine, histidine Methionine Tryptophan Lysine-threonine

Asparagine-proline, asparagine-tyrosine

Aggregation

Deamidation Oxidation

Oxidation Photodecomposition Copper induced cleavage Hydrolysis

Addition of surfactants, polyalcohols and other excipients pH 3-5 pH < 7

Protect from oxygen Protect from light Chelating agents pH > 7

a Reproduced from D. A. Parkins, U. T. Lashmar, PSST, 3,

129-137 (2000).

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