Historical Background - Freeze-drying of Pharmaceuticals and Biopharmaceuticals - page 8

unique molecular shape in which one or several polypeptide chains with
specific amino acid sequences adopt a specific configuration in which the
amino acid residues are held in position by non-covalent, short-range
interactions with one another, with ions and with a solvent (water).
Apparently the only covalent links, apart from the peptide bonds
between amino acids, which contribute to protein stability are disulfide
bonds between cysteine residues.
Instability arises when the so-called native (bioactive) protein state
loses its unique folded configuration. This process is referred to as
''denaturation''. It can be brought about by changes in pH, some salts,
temperature, pressure and certain organic additives. Here the important
point is that, in principle, denaturation should be reversible, with the
native state being reconstituted as the original environmental conditions
are re-established; with careful experimentation this can often be
achieved, even for quite complex structures. 12 Indeed, controlled dena-
turation, followed by renaturation, forms the basis of several down-
stream processing and purification protocols.
In practice, extreme care must be taken in the various processing
stages, including freeze-drying, if high recovery levels of native, bioac-
tive protein are to be achieved. The main reason for irreversibility is that
in their denatured states, proteins become more vulnerable to the
various chemical deterioration mechanisms, already briefly discussed.
Additional inactivation mechanisms include the formation of intra- or
interpeptide bonds (aggregation) and disulfide bond rearrangements,
both of which are essentially irreversible. Some common chemical
reactions that lead to inactivation are summarised in Table 1. Most of
them are pH-sensitive, as shown schematically in Figure 1. In solution,

Table 1 Common chemical reactions leading to the transient or permanent
inactivation of proteins

Reaction Location Notes

Oxidation MET Most susceptible

CYS Catalysed by metals

HIS, TYR, MET, CYS,
TRP

Photooxidation, pH-dependent

S-S interchange Catalysed by -SH

Hydrolysis Deamidation at ASX,

GLX

Buffer concentration effects

Peptide bond cleavage ASP-PRO worst case, acid catalysed

Beta-elimination CYS, SER, THR, PHE,

LYS

Accelerated by high pH and metals

Racemisation MET, ASP Much faster in proteins than in free

amino acids