Temperature Dependence of the Degradation Rate of Peptide and Protein Drugs

Stability prediction for peptide and protein drugs under accelerated testing conditions is possible if the temperature dependence of the degradation rate is determined and found to be well behaved. The temperature dependence can often be represented by the Arrhenius equation, as was seen with small-molecule drugs. Linear Arrhenius plots and the values of apparent activation energy calculated from the slopes have been reported for chemical degradation of various peptides in aqueous solutions. Values of approximately 20 kcal/mol

Figure 209. Weibull plots for the inactivation of two different lipases in the solid state. a: Fraction inactivated. A, Pancreatic lipase; O, Rhizopus lipase. (Reproduced from Ref. 881 with permission.)
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Figure 210. Arrhenius plots for chemical degradation of two Asn-hexapeptides and salmon calcitonin in aqueous solution. A, Val-Tyr-Pro-Asn-Gly-Ala, pH 7.5; □, Val-Tyr-Pro-Asn-Val-Ala, pH 7.5; salmon calcitonin, pH 6.0. (Reproduced from Refs. 790,791, and 876 with permission.)

for deamidation of a hexapeptide having an asparagine residue in a neutral region790 791 and 96 kJ/mol(22.9 kcal/mol) for hydrolysis of gonadorelin and triptorelin,883 each having 10 amino acid residues, were reported. Other values include 24 kcal/mol for the hydrolysis of LH-RH801; 20-33 kcal/mol for alkaline racemization of casein799; 15 kcal/mol for deamidation of calcitonin at pH 6876; and 21 kcal/mol for hydrolysis of cholecystokinin-B receptor antagonist in the acidic pH region.884 Figure 210 shows some typical Arrhenius plots for

Figure 211. Arrhenius plots for the inactivation of a-chymotrypsin (O) and bromelain^); at pH 7.4. (Reproduced from Ref. 879 with permission.)

Figure 211. Arrhenius plots for the inactivation of a-chymotrypsin (O) and bromelain^); at pH 7.4. (Reproduced from Ref. 879 with permission.)

Figure 212. Anhenius plots of k,, k-2, and ¿3, for kallikrein inactivation in aqueous solution at pH 7.4. Rate constants k,, k2, and k3 were calculated according to the reaction scheme in Eq. (5.1). (Reproduced from Ref. 879 with permission.)

Figure 212. Anhenius plots of k,, k-2, and ¿3, for kallikrein inactivation in aqueous solution at pH 7.4. Rate constants k,, k2, and k3 were calculated according to the reaction scheme in Eq. (5.1). (Reproduced from Ref. 879 with permission.)

three peptides. When Arrhenius behavior is adhered to, accelerated-temperature testing of solutions or formulations is possible, whereas accelerated testing cannot be applied even to chemical degradations when the mechanism varies with temperature. This is the case for degradation of interleukin-ip, which exhibits deamidation as its primary degradation pathway below 30°C and oxidation at higher temperatures.855

Even when mechanisms and pathways are unknown, it is sometimes possible to use Arrhenius or empirical equations to determine apparent rate constants at other temperatures. Apparent first-order rate constants for a-chymotrypsin and bromelain inactivation exhibited linear Arrhenius plots (Fig. 21 1).879 Apparent rate constants for kallikrein inactivation calculated according to the reaction scheme in Eq. (5.1) resulted in the temperature dependence seen in Fig. 212.850 For inactivation of various peptide and protein pharmaceuticals

Figure 213. Arrhenius plots of the apparent zero-order rate constant for urokinase inactivation in the presence (□) land absence (x) of human serum albumin. (Reproduced from Ref. 838 with permission.)

Figure 214. Arrhenius plots for the inactivation of marketed solid digestive enzyme formulations. a-Chy-motrypsin troche; A, a-chymotrypsin tablet; P-galactosidase powder; V, ▼ bromelain tablets; O, "kallikrein capsule. (Reproduced from Ref. 888 with permission.)

Figure 214. Arrhenius plots for the inactivation of marketed solid digestive enzyme formulations. a-Chy-motrypsin troche; A, a-chymotrypsin tablet; P-galactosidase powder; V, ▼ bromelain tablets; O, "kallikrein capsule. (Reproduced from Ref. 888 with permission.)

including interferon,886 accelerated testing appears to be possible. Activation energies have been reported for inactivation of digestive enzymes (23-58 kcal/mol)880 881 and horse serum cholinesterase (25 kcal/mol).882

Apparent activation energy values have also been reported for solid-state degradation of peptide and protein pharmaceuticals. The reported values include 1-2 kcal/mol for human interferon-P formulated with human serum albumin887; 15 kcal/mol for freeze-dried urokinase (Fig. 213)838; and 11-18 kcal/mol for a-chymotrypsin and bromelain tablets, kallikrein capsules, and P -galactosidase powder (Fig. 214).888

The kinetics of denaturation of peptide and protein drugs have not been extensively treated. Some studies on the temperature dependence of denaturation rates have been reported. A kinetic study of the denaturation of G actin in solution using DSC yielded linear Arrhenius plots, from which values for the activation energy and the frequency factor of 23 1 kJ/mol (55.2 kcal/mol) and 76.8 s-1, respectively, were obtained (Fig. 215).889 Linear

Figure 215. Arrhenius plot for the denaturation of G actin in aqueous solution at pH 8.0. (Reproduced from Ref. 889 with permission.)

Figure 215. Arrhenius plot for the denaturation of G actin in aqueous solution at pH 8.0. (Reproduced from Ref. 889 with permission.)

Arrhenius plots were also observed for the denaturation of P-galactosidase in solution.890 These linear Arrhenius 'plots suggest that prediction of denaturation rate by accelerated testing is possible if the denaturation mechanism does not change in the temperature range in question. Because peptide and protein drugs generally denature via complicated mechanisms (thermal denaturation, denaturation at interfaces, etc.), denaturation at lower temperature may occur via mechanisms different from those at higher temperature. This makes it difficult to predict the stability of peptide and protein drugs by accelerated testing. In cases where only thermal denaturation occurs, however, prediction of denaturation rate by accelerated testing may be possible.

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