Downstream processing - Pharmaceutical Biotechnology: Concepts and Aapplications - page 162

Table 6.5 The various molecular alterations that usually
result in loss of a protein's biological activity

Non-covalent alterations
Partial/complete protein denaturation
Covalent alterations
Hydrolysis Oxidation

Deamidation Disulfi de exchange

Imine formation Isomerization

Raceimization Photodecomposition

6.9.1.1 Proteolytic degradation and alteration of sugar side-chains

Proteolytic degradation of a protein is characterized by hydrolysis of one or more peptide (amide)
bonds in the protein backbone, generally resulting in loss of biological activity. Hydrolysis is usu-
ally promoted by the presence of trace quantities of proteolytic enzymes, but can also be caused
by some chemical infl uences.
Proteases belong to one of six mechanistic classes:

• serine proteases I (mammalian) or II (bacterial);

• cysteine proteases;

• aspartic proteases;

• metalloproteases I (mammalian) and II (bacterial).

The classes are differentiated on the basis of groups present at the protease active site known
to be essential for activity, e.g. a serine residue forms an essential component of the active site of
serine proteases. Both exo-proteases (which catalyse the sequential cleavage of peptide bonds be-
ginning at one end of the protein), and endo-proteases (cleaving internal peptide bonds, generating
peptide fragments) exist. Even limited endo-or exo-proteolytic degradation of biopharmaceuticals
usually alters/destroys their biological activity.
Proteins differ greatly in their intrinsic susceptibility to proteolytic attack. Resistance to prote-
olysis seems to be dependent upon higher levels of protein structure (i.e. secondary and tertiary
structure), as tight packing often shields susceptible peptide bonds from attack. Denaturation thus
renders proteins very susceptible to proteolytic degradation.
A number of strategies may be adopted in order to minimize the likelihood of proteolytic deg-
radation of the protein product, these include:

• minimizing processing times;

• processing at low temperature;

• use of specifi c protease inhibitors.