Some influences that can alter the biological activity of proteins

A number of different influences can denature or otherwise modify proteins, rendering them less active/inactive. As all protein products are marketed on an activity basis, every precaution must be taken to minimize loss of biological activity during downstream processing and subsequent storage. Disruptive influences can be chemical (e.g. oxidizing agents, detergents, etc.), physical (e.g. extremes of pH, elevated temperature, vigorous agitation) or biological (e.g. proteolytic degradation). Minimization of inactivation can be achieved by minimizing the exposure of the product stream to such influences, and undertaking downstream processing in as short a time as possible. In addition, it is possible to protect the protein from many of these influences by the addition of suitable stabilizing agents. The addition of such agents to the final product is often essential in order to confer upon the product an acceptably long shelf life. During initial development, considerable empirical study is undertaken by formulators to determine what excipients are most effective in enhancing product stability.

A number of different molecular mechanisms can underpin the loss of biological activity of any protein. These include both covalent and non-covalent modification of the protein molecule, as summarized in Table 6.5. Protein denaturation, for example, entails a partial or complete alteration of the protein's three-dimensional shape. This is underlined by the disruption of the intramolecular forces that stabilize a protein's native conformation, namely hydrogen bonding, ionic attractions and hydrophobic interactions (Chapter 2). Covalent modifications of protein structure that can adversely affect its biological activity are summarized below.

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

Deamidation

Imine formation

Raceimization

Oxidation Disulfide exchange Isomerization Photo decomposition

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 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).

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 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 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 degradation of the protein product, these include:

• minimizing processing times;

• processing at low temperature;

• use of specific protease inhibitors.

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

Inhibitor

Phenylmethylsulfonyl fluoride

Benzamidine Pepstatin A EDTA

Protease class inhibited

Serine proteases Some cysteine proteases Serine proteases Aspartic proteases 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 specific 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 specific 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 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.

As discussed in Chapter 2, many therapeutic proteins are glycosylated, and the sugar side chains can influence protein function, structure and stability. Chemical or enzymatic modification 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 influence a protein's biological activity. 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 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.

Deamination

C00H

Asparagine residue

Aspartic acid residue

Deamidation

Glutamine residue

C00H

Glutamic acid residue

Figure 6.19 Deamidation of asparagine and glutamine, yielding aspartic acid and glutamic acid respectively. This process can often be minimized by reducing the final product pH to 4-5

6.9.1.3 Oxidation and disulfide 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 sulfur atoms present in methio-nine or cysteine are by far the most susceptible. Methionine can be oxidized by air or more potent oxidants, initially forming a sulfoxide and, subsequently, a sulfone (Figure 6.20). The sulfur atom of cysteine is readily oxidized, forming either a disulfide bond or (in the presence of potent oxidizing agents) sulfonic acid (Figure 6.20). Oxidation by air normally results only in disulfide bond formation. The oxidation of any constituent amino acid residue can (potentially) drastically reduce the biological activity of a polypeptide.

Oxidation of methionine is particularly favoured under conditions of low pH, and in the presence of various metal ions. Methionine residues on the surface of a protein are obviously particularly susceptible to oxidation. Those buried internally in the protein are less accessible to oxidant. hGH contains three methionine residues (at positions 14, 125 and 170). Studies have found that oxidation of methionine 14 and 125 (the more readily accessible ones) does not greatly effect hGH activity. However, oxidation of all three methionine residues results in almost total inactivation of the molecule.

Oxidation can be best minimized by replacing the air in the headspace of the final product container with an inert gas such as nitrogen, and/or the addition of antioxidants to the final product.

Disulfide exchange can also sometimes occur, and prompt a reduction in biological activity (Figure 6.21). Intermolecular disulfide exchange can result in aggregation of individual polypeptide molecules.

Methionine side chain

Sulphoxide

Sulphone

Cysteine residues

Disulphide bond formation c c c

so3h

Cysteic acid (sulphonic acid)

Figure 6.20 Oxidation of (a) methionine and (b) cysteine side chains, as can occur upon exposure to air or more potent oxidizing agents (e.g. peroxide, superoxide, hydroxyl radicals or hypochlorite). Refer to text for specific details

Figure 6.21 Diagram representing the molecular process of intrachain (a) and interchain (b) disulfide exchange. Refer to text for specific details. (—O— are amino acid residues in the polypeptide)

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