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biopharmaceuticals biochemistry and biotecnology - Walsh G.

Walsh G. biopharmaceuticals biochemistry and biotecnology - John Wiley & Sons, 2003. - 572 p.
ISBN 0-470-84327-6
Download (direct link): biochemistryandbiotechnology2003.pdf
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Proteolytic degradation
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 usually promoted by the presence of trace quantities of proteolytic enzymes, but can also be caused by some chemical influences.
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).
Table 3.21. Some of the most commonly employed protease inhibitions and the specific classes of proteases they inhibit
Inhibitor Protease class inhibited
PMSF Serine proteases, some cysteine
Benzamidine Serine proteases
Pepstatin A Aspartic proteases
EDTA Metallo-proteases
PMSF = Phenylmethylsulphonyl fluoride. EDTA = Ethylenediaminetetra-acetic acid
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 (catalysing the sequential cleavage of peptide bonds beginning 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 proteolysis 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 degradation of the protein product, these include:
• minimizing processing times;
• processing at low temperatures;
• use of specific protease inhibitors.
Minimizing processing times obviously limits the time 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 specific proteolytic inhibitors in processing buffers, in particular homogenization buffers, can be very effective in preventing uncontrolled proteolysis. While no one inhibitor will inhibit proteases of all mechanistic classes, a number of effective inhibitors for specific classes are known (Table 3.21). 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, the instigation of precautionary measures protecting proteins against proteolytic degradation is of prime importance during the early stages of purification. During the later stages, most of the proteases present will have been removed from the product stream. A major aim of any purification system is the complete removal of such proteases, as the presence of even trace amounts of these catalysts can result in significant proteolytic degradation of the finished product over time.
Protein deamidation
Deamidation and imide formation can also negatively influence a protein’s biological activity. Deamidation refers to the hydrolysis of the side-chain amide group of asparagine and/or
Figure 3.21. Deamidation of asparagine and glutamine, yielding aspartic acid and glutamic acids, respectively. This process can often be minimized by reducing the final product pH to 4-5
glutamine, yielding aspartic and glutamic acid, respectively (Figure 3.21). 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 a-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.
Oxidation and disulphide exchange
The side chains of a number of amino acids are susceptible to oxidation by air. Although the side chains of tyrosine, tryptophan and histidine can be oxidized, the sulphur atoms present in methionine or cysteine are by far the most susceptible. Methionine can be oxidized by air or more potent oxidants, initially forming a sulphoxide and, subsequently, a sulphone (Figure 3.22). The sulphur atom of cysteine is readily oxidized, forming either a disulphide bond or (in the presence of potent oxidizing agents) sulphonic acid (Figure 3.22). Oxidation by air normally results only in disulphide bond formation. The oxidation of any constituent amino acid residue can (potentially) drastically reduce the biological activity of a polypeptide.
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