Nvw

Scheme 11.6 Mechanism of disulfide exchange in (a) alkaline or neutral media and (b) acidic media mercuribenzoate, N-ethylmaleimide or copper ions are present. In acidic conditions the disul-fide exchange takes place through a sulfenium cation, which is formed by attack of a proton on the disulfide bond (Scheme 11.6b). The sulfenium cation carries out an electrophilic displacement on a sulfur atom of the disulfide. Addition of thiols can inhibit this exchange by scavenging the sulfenium ions.

11.2.4 Accelerated stability testing of protein formulations

As we have seen from the above discussions, the processes involved in the degradation of proteins may be complex and highly dependent on the proximity of other functional groups in the molecule and also on the protein conformation. As a consequence, the mechanisms of degradation at higher temperatures may not be the same as at lower temperatures and the application of the Arrhenius equation in the prediction of protein stability will be more uncertain than with formulations of small-molecule drugs. Nevertheless, many workers have attempted to use the Arrhenius approach with some degree of success. In general, this approach appears to be applicable when only the activity is monitored. In most of these studies degradation was due to thermal denaturation, and loss of activity was a consequence of conformational changes rather than covalent chemical reaction. Although the product of this reaction may involve many different unfolded forms of the protein, these forms will be inactive and indistinguishable from each other by activity assay. Deviation from the Arrhenius equation occurs, however, if the protein exists in multiple conformational forms that retain activity during unfolding. Where degradation occurs by deamidation or oxidation, it may still be possible to apply the Arrhenius equation if protein activity only is monitored, since the final activity loss will be determined by the fastest reaction.

11.3 Protein formulation and delivery

It has been said25 that 'drug delivery represents the potential Achilles' heel of biotechnology's peptide drug industry'. The reasons for this include the range of instabilities discussed above, the inherent low membrane transport by diffusion, because of molecular size and hydrophilicity, and often the need for temporal and site control of delivery.

11.3.1 Protein and peptide transport

For a series of 11 model peptides in an in vitro intestinal cell monolayer system, a good correlation was found between the permeability coefficient, P, and the log of the partition coefficient of the peptides between heptane and ethylene glycol (rather than octanol and water) (Fig. 11.9), results which also suggest that the principal deterrent to peptide transport is the breaking of hydrogen bonds.26 Molecular volume (or size) will increasingly be a factor as the molecular weight of the peptide increases.

The diffusion of proteins and peptides in solution is dictated by the same considerations as those discussed in section 3.6. The rate of translational movement depends on the size of the molecule, its shape and interactions with solvent molecules. The rate of translational movement is often expressed by a frictional coefficient, f defined in relation to the diffusion coefficient D, by equation (11.4):

where kB is the Boltzmann constant and T is the absolute temperature. Many proteins are nearly spherical in solution, but if their shape deviates from sphericity this is reflected in a frictional ratio, f/f0, above unity, where f0 is the rate of diffusion of a molecule of the same size but of spherical shape. The frictional ratio

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