Thermally induced denaturation of lyophilized bovine somatotropin and lysozyme as impacted by moisture and excipients (original) (raw)

1995, Journal of Pharmaceutical Sciences

The endothermic thermal transitions (i.e., denaturation) of lyophilized recombinant bovine somatotropin (rbSt) and lysozyme as seen via differential scanning calorimetry were evaluated with respect to moisture and excipients. The denaturation temperature, Tm, of rbSt and lysozyme decreased with increasing moisture irrespective of the excipient. However, the magnitude of the decrease elicited by moisture was dependent on the type of excipient. Furthermore, the effect of the excipient was dependent on the moisture content; excipients decreased Tm in low moisture solids (i.e., <5% moisture) and increased it in hydrated solids (i.e., >15% moisture). In the dry state (<1% moisture), the addition of 50% sucrose, sorbitol, or glycerol lowered the Tm of rbSt from 161 °C to 136, 120, and 83 °C, respectively, indicating a destabilizing mechanism. Likewise, the Tm of lysozyme decreased from 156 °C to 142, 128, and 97 °C due to the addition of sucrose, sorbitol, and glycerol, respectively. At higher moisture contents, the excipients promoted a higher transition temperature at a given moisture content than the pure protein systems, indicating a stabilizing mechanism. An increase in the enthalpy of unfolding for dehydrated lysozyme was noted with increasing levels of moisture and/or excipient, despite the observed decrease in Tm. The thermal stability, or Tm, of the dehydrated proteins appeared to be correlated to the glass transition temperature (Tg) of the excipient, which in turn should be related to the Tg of the system. The lower the Tg of the excipient, the greater was the degree of destabilization. This result suggests different modes of conformational stabilization by excipients may exist for proteins in the solid state depending on the moisture content.