Downstream processing - Pharmaceutical Biotechnology: Concepts and Aapplications - page 163

Table 6.6 Some of the most commonly employed protease
inhibitions and the specifi c classes of proteases they inhibit

Inhibitor Protease class inhibited

Phenylmethylsulfonyl fl uoride Serine proteases

Some cysteine proteases

Benzamidine Serine proteases

Pepstatin A Aspartic proteases

EDTA Metallo-proteases

Minimizing processing times obviously limits the duration during which proteases may come
into direct contact with the protein product. Processing at low temperatures (often 4 C) reduces
the rate of proteolytic activity. Inclusion of specifi c proteolytic inhibitors in processing buffers, in
particular homogenization buffers, can be very effective in preventing uncontrolled proteolysis.
Although no one inhibitor will inhibit proteases of all mechanistic classes, a number of effective
inhibitors for specifi c classes are known ( Table 6.6). The use of a cocktail of such inhibitors is thus
most effective. However, the application of many such inhibitors in biopharmaceutical processing
is inappropriate due to their toxicity.
In most instances, instigation of precautionary measures protecting proteins against proteolytic
degradation is of prime importance during the early stages of purifi cation. During the later stages,
most of the proteases present will have been removed from the product stream. A major aim of
any purifi cation system is the complete removal of such proteases, as the presence of even trace
amounts of these catalysts can result in signifi cant proteolytic degradation of the fi nished product
over time.
As discussed in Chapter 2, many therapeutic proteins are glycosylated, and the sugar side
chains can infl uence protein function, structure and stability. Chemical or enzymatic modi-
fi cation of a protein's glycocomponent, therefore, could affect its therapeutic properties. The
presence of glycosidase enzymes in crude preparations, for example, could lead to partial
degradation of sugar side chains. Generally, however, such eventualities may be effectively
minimized by carrying out downstream processing at lower temperatures and as quickly as
possible.

6.9.1.2 Protein deamidation

Deamidation and imide formation can also negatively infl uence a protein's biological activ-
ity. Deamidation refers to the hydrolysis of the side chain amide group of asparagine and/or
glutamine, yielding aspartic acid and glutamic acid respectively ( Figure 6.19). This reaction
is promoted especially at elevated temperatures and extremes of pH. It represents the major
route by which insulin preparations usually degrade. Imide formation occurs when the -

amino nitrogen of either asparagine, aspartic acid, glutamine or glutamic acid attacks the
side chain carbonyl group of these amino acids. The resultant structures formed are termed
aspartimides or glutarimides respectively. These cyclic imide structures are, in turn, prone
to hydrolysis.

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