Parenteral Delivery of Peptides and Proteins - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 544

Figure 11.5 Methionine oxidation
and formation of methionine sulfone
(chemical instability).
on the conformation of the protein and the resultant exposure of these amino acids to
the solvent and environmental conditions such as the presence of oxygen, light, high
temperature, metal ions, and various free radical initiators [90] .
Chemical degradation such as methionine oxidation ( Fig. 11.5 ) occurs in a number
of polypeptides and proteins, like antistatin, granulocyte colony-stimulating factor,
(G-CSF), antithrombin, EGF, and adrenocorticotropin hormone (ACTH). The prod-
ucts of all these reactions/chemical degradations might be inactive or can cause unpre-
dictable side effects like toxicity or antigenicity. Cystine residues that contain thiol
groups can be oxidized to form disulfide bonds, which can be prevented by maintain-
ing low pH values. Tryptophan oxidation occurs in LHRH, somatostatin, and ACTH.
Effective precautions against oxidation are using chelating agents to eliminate
metal catalysis, increasing ionic strength, eliminating peroxide and metallic contami-
nants in formulation additives, protecting from light, being aware of possible interac-
tion of light exposure and phosphate buffer in forming free radicals, replacing oxygen
with nitrogen or argon during manufacturing, removing oxygen from the headspace
of the final container, and maintaining the formulation at the lowest pH possible while
keeping the desired protein solubility and hydrolytic stability [91] .
Antioxidants tend to terminate free radical reactions by reacting with reactive
radicals. Oxidation may be minimized by the use of antioxidants such as butylated
hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E,
ascorbic acid, platinum, catalase, sodium thiosulfate, and sodium sulfite. Chelating
agents are used to remove metals, such as iron, copper, calcium, manganese, and
zinc, from the formulations and usually tend to protect proteins from metal-catalyzed
reactions. The main chelating agent used is disodium ethylenediaminetetraacetic acid
(DSEDTA). The concentration of DSEDTA is usually very small, such as 0.05%.
Citric acid and thioglycolic acid are also reported as effective chelating agents in
protein formulation. However, while selecting a suitable antioxidant/chelating agent
for P/P formulation, its aqueous solubility and safety parameters must be consid-
ered, which may restrict the choice to very few antioxidant and chelating agents. Of
course, reagents such as EDTA are avoided when protein activity demands the pres-
ence of divalent metal ions [130,131] .
In general, protein incorporation processes that involve interfaces, as in emulsions,
should be avoided. There should be a thorough understanding of the degradation
processes and the macro- and microsurroundings, including stresses generated during
the manufacturing process, which will enable one to find the best suitable precaution.
If the protein can be incorporated in a solid form, such as a lyophilized powder, the
chances of degradation are lessened.