Physical Degradation

Larger peptides and proteins are susceptible to noncovalent or physical changes (so-called 'physical degradation') in addition to chemical degradation. Physical degradation includes denaturation, aggregation, adsorption, and precipitation. Denaturation, that is, an alteration of tertiary (and/or quaternary) structure, generally results in loss of bioactivity. Furthermore, exposure of hydrophobic groups upon denaturation often leads to adsorption onto surfaces, aggregation, and precipitation. Denaturation may also prompt chemical degradation pathways often not seen with the native or natural tertiary (and/or quaternary) structure. Therefore, there is considerable interest in preventing denaturation while formulating protein drugs.

Cross-linkages via disulfide bond formation cause aggregation of peptides and proteins, as described in Section 5.1.1.4. Hydrophobic bond formation, on the other hand, causes aggregation without covalent changes. Freeze-dried human growth hormone exhibited noncovalent aggregation in addition to chemical degradation via methionine oxidation and deamidation of asparagine residues.825 Noncovalent aggregation was also observed with P-galactosidase in aqueous solution, although freeze-dried P-galactosidase, having limited moisture, exhibited aggregation via disulfide bond formation. Size-exclusion chromato-grams indicated that the aggregates formed in solution were dissociated by guanidine hydrochloride, a hydrophobic-bond-breaking reagent (Fig. 201).811

Figure 201. Size-exclusion chromatograms of aggregates formed during storage of P-galactosidase solution at 50°C. Duration of storage: (1) 0, (2) 30, (3) 240, (4) 360 min. (a) No additive; (b) in the presence of dithiothreitol; (c) in the presence of guanidine hydrochloride; (d) in the presence of dithiothreitol and guanidine hydrochloride. (Reproduced from Ref. 811 with permission.)

Figure 201. Size-exclusion chromatograms of aggregates formed during storage of P-galactosidase solution at 50°C. Duration of storage: (1) 0, (2) 30, (3) 240, (4) 360 min. (a) No additive; (b) in the presence of dithiothreitol; (c) in the presence of guanidine hydrochloride; (d) in the presence of dithiothreitol and guanidine hydrochloride. (Reproduced from Ref. 811 with permission.)

Noncovalent aggregation has been suggested for many other proteins, but not always confirmed. A conjugate formed between a vinca alkaloid and a monoclonal antibody exhibited aggregation in solution, the mechanism of which (covalent or noncovalent) was not clear.826 Aggregates formed upon agitation of insulin solutions in the presence of hydrophobic surfaces (Teflon) were dissociated with urea, suggesting noncovalent aggregation.827

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