Essential Physics of Low Temperature and Freezing - Freeze-drying of Pharmaceuticals and Biopharmaceuticals - page 22

10

1

undercooled

0.1

frozen

-10 -5 0 5 10 15 20 25

Temperature (deg C)

Figure 2 Oxygen uptake by a diapausing silk moth pupa (Hyalophora cecropia) in the
chilled (undercooled) and frozen states, showing the injurious effects of freezing

at or near 01C. The indications are therefore that the haemolymph does
not freeze but that chill reduces the insect's metabolic activity, as would
be expected. The process is fully reversible, so that upon rewarming, the
oxygen uptake increases and the insect recovers from a quasi-dormant
state. If, however, the larva is held at or below 01C and touched with an
ice crystal, the rate of oxygen uptake exhibits a sharp discontinuity,
dropping rapidly upon cooling. The haemolymph has frozen and nor-
mal oxygen uptake is not recovered upon thawing, i.e. freezing has
caused the death of the insect. Thus, chill causes a slowdown of metabolic
processes, enabling the insect to survive winter, whereas freezing is lethal.
The same conclusion can be reached from in vitro studies, illustrated in
Figure 3 for the temperature dependence of the specific activity of
isolated lactate dehydrogenase (LDH) enzyme in a dilute aqueous
solution at pH 7. 14 When stored at room temperature, the decrease in
specific enzyme activity shows a typically biphasic behaviour, dropping
rapidly by 80% during the first week and more slowly thereafter.
Storage in a refrigerator at 41C (chill) reduces the rate of activity loss,
but when exposed to deep chill but unfrozen conditions, say 201C, the
activity loss is practically inhibited. The kinetics of inactivation exactly
follow the Arrhenius equation. Within the normal experimental error
for enzyme assays, LDH has been recovered at close to 100% of its
original activity, even after 6 years of storage. By comparison, freezing
the enzyme solution at 201C leads to an almost immediate and
irreversible loss of 80% of its activity, with a 100% loss observed after
1 week of frozen storage. Here again, therefore, chill is seen to stabilise,
while freezing causes an irrecoverable loss of biochemical activity.