Chemical Stability

The major pathways of chemical degradation are, among others, hydrolysis and oxidation as shown in Table 2. An unfolded polypeptide chain in a protein is more prone to chemical degradation than the native and folded protein (46). These chemical degradation reactions may be modifications involving changes in the covalent bonds, for example, deamidation, oxidation, or disulfide bond shuffling (47), and the reactions are usually irreversible (28). The breaking of peptide bonds is referred to as proteolysis and can often

Table 4 Sequences of Amino Acids, Which are Susceptible to Chemical Degradation and Formulation Strategies to Prevent Degradation (29,30,40,62)

Amino acid sequence

Mechanism of degradation Formulation strategy cys-cys lys, -thr glu, asp asp-pro, asp-tyr asn, gin trp, met, cys, tyr, his met trp cys, ser, thr, phe, and lys s-s reduction

Copper-induced cleavage

Deamidation

Hydrolysis

Hydrolysis

Oxidation

Oxidation

Photodegradation

Elimination

Addition of surfactant, polyalcohols and other excipients Chelating agents pH 3-5 pH > 7

Protect from oxygen Protect from light Acidic pH

lead to extensive configurational changes (23,28). In Table 4, the amino acids and amino acid sequences that are especially prone to chemical alterations or to cause physical alterations are listed. In addition, the formulation strategy to prevent degradation is included.

Hydrolysis most frequently occurs in the side chains of Asn, Gin, and the peptide bond at the C-terminal side of Asp and Pro. Hydrolysis involves the reaction of water and can lead to deamidation. The pH has a huge influence on the deamidation rates, since the rate is much slower at acidic pH than at neutral or alkaline pH (30). Deamidation does not always have an effect on the biological activity of the protein but it may affect, for instance, the clearance rate and thereby also the efficacy of the protein (30,62). Oxidation is one of the most common degradation pathways of proteins. The amino acids most likely to be influenced are those with sulphur (Cys and Met) and those with aromatic rings (His, Tyr, and Trp). The underlying mechanism involves oxygen and reactive oxygen species, which react with the protein or peptide. The reaction occurs with or without catalysts such as metal ions and it is pH dependent (30). The reaction is also catalyzed by light and is then referred to as photodegradation or photooxidation. The amino acids that primarily undergo photooxidation are Trp, Tyr, Phe, and Cys (63).

Other chemical degradation pathways include ^-elimination, which can lead to racemization and disulphide exchange reactions. The amino acids that may undergo P-elimination include Cys, Ser, Thr, Phe, and Lys (40) and occur especially at alkaline pH (64). The reduction and oxidation of the disulphide bonds are often accompanied by a considerable change in the protein conformation (23,33). An example is the secondary structure of insulin that is disrupted or completely lost when the disulphide bonds are broken (23). The disulphide bond disruption or interchange can also result in an altered three-dimensional structure and therefore a possible loss of activity (40) or aggregation (29). For further details, the reader is directed to the following reviews and book chapters (30,40,62,65).

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