Historical Background - Freeze-drying of Pharmaceuticals and Biopharmaceuticals - page 9

C-terminal deamidation

β-elimination

ASP transpeptidation

MET oxidation

deamidation

ASP peptide cleavage various oxidations

Disaccharide browning Disulfide shuffling
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Acid Neutral Base

pH

Figure 1 Susceptibility of protein inactivation reactions to pH

the native state is considered to be in dynamic equilibrium with several
unfolded states, which means that, over time, every molecule samples
several states; the degree of occupancy of these states, averaged over
time, is given by an equilibrium constant K, where
K ¼½ N = Y ½ D i ð 1 Þ

where [N] refers in the concentration of the unique native (bioactive)
state and [D i ] the concentration of the ith denatured state. Little is as yet
known about the numbers and structures of such D states, let alone their
respective concentrations. A caveat needs to be entered here: Protein
stability studies are generally performed in dilute solutions. It is ques-
tionable whether, or how, the results and conclusions from such studies
can be immediately related to reactions in supersaturated, highly viscous
solutions, such as are produced during the freezing process, or in the
formulated, dried state.
It might be thought that freezing, followed by thawing, constitutes a
reversible processing cycle. Although frozen foods are an example of this
technology, the frozen product, when reconstituted, rarely matches the
original state in quality. The main reason is that freezing (i.e. drying) is a
process that takes place at very high rates in most substrates and one that
can hardly be controlled. Thus, thawing cannot be regarded as a reversal,
at least in chemical terms, of the freezing process. The consequences are