Essential Chemistry and Biochemistry Associated with Low Temperature and Freezing - Freeze-drying of Pharmaceuticals and Biopharmaceuticals - page 47

demixing, i.e. their miscibility with water decreases with increasing tem-
perature, giving rise to the well-known closed-loop liquid-liquid phase
diagrams, e.g. water-polyethylene glycol. 46 Evidence for such phase
separations, as they develop during freeze-concentration, has been cited, 47
but the phenomenon remains to be definitely established. If such phase
separation can indeed play a role during freezing, some current views on
formulation development would need to be significantly modified.
5.2 Cold Inactivation of Proteins
The diverse phenomena surrounding protein stability in solution have
been studied in great detail but are still subject to several unresolved
problems. In the context of this monograph, emphasis is placed on the
cold inactivation of proteins, a process that is quite unrelated to freez-
ing. The possibility of its existence was first suggested by Brandts, 48
based on detailed studies of the combined effects of pH, urea concen-
tration and high temperature on the reversible denaturation of chymo-
trypsinogen, as shown in Figure 1.
The parabolic nature of the thermal stability profile, which for obvi-
ous reasons terminates at the freezing point of water, led Brandts to
Figure 1 The effect of pH on the thermal stability of chymotrypsinogen according to
Brandts. 48 Circular symbols refer to a range of pH values; triangles refer to 2.3
M urea. T H is the well-characterised thermal denaturation temperature. T L is
the experimentally determined denaturation temperature in undercooled water
Franks 48