Chemical Stresses

In general, proteins and peptides are not robust molecules; they can be readily oxidized, deamidated, cleaved, and aggregated into forms with reduced biological activity when handled in vitro. All of these same events could occur following their SC injection, providing multiple potential pathways for destruction and clearance in vivo. It is important to remember that most proteins and peptides produced by cells in the body are broken down and cleared in minutes to hours; only a select few seem to last for days to weeks. Thus, the desire to identify protein and peptide formulations with multiyear stability and 100% bioavailability following SC injection is certainly a tremendous challenge. A clear picture of the SC environment and how it affects injected protein and peptide formulations does not exist at present. Having this picture would expedite efforts to find optimal formulation strategies to protect injected proteins or peptides from chemical decomposition. While it is possible to anticipate some chemical stressors confronting a protein or peptide therapeutic on the basis of in silico and in vitro studies, it is unclear how much of this information will ultimately define events in the SC space.

Proteins and peptides can also undergo a variety of amino acid-specific chemical modifications that can affect their structural and biological properties, including aggregation and/or diminished bioactivity (76). Oxidation of amino acids, in particular tryptophan, methionine, reduced cysteine, tyrosine, and phenylalanine residues, can alter structural and biological properties of protein and peptide therapeutics (77). Similarly, protein oxidation can be detrimental; excessive oxidation of serum proteins has been associated with pathological states (78). Deamidation of asparagine at neutral pH can lead to the production of an isoaspartic acid structure with backbone scission as one potential outcome (79).

More frequently, asparagine and glutamine can undergo deamidation to produce aspartic acid and glutamic acid, respectively, both in vitro and in vivo (80). As deamidation events appear to be a normal process of proteins in vivo (81), the suggestion has been made that proteins use this modification as a sort of molecular clock (82). Several disease states have been correlated with protein deamidation, including prion disease (83), Alzheimer's disease (84), and cataracts (85).

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