Protein and Peptide Delivery through Respiratory Pathway - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 431

physical structure. Freeze-drying (lyophilization) is frequently used to remove water
from protein solutions, but the spray-drying method is faster, with a lesser extent of
degradation. Water plays a key role in maintaining the higher, noncovalent structure
of proteins [9] . Dried protein preparations must be rehydrated to resume their cor-
rect secondary, tertiary, and quaternary structures and biological properties. Drying
processes must be optimized to get optimal residual water content for preserving the
protein structure while maintaining the required lung deposition characteristics.
9.3.4.2 Chemical Stability of Peptide and Protein Drugs
Proteins and peptides are degraded or the biological activity is changed by many
chemical reactions, such as deamidation, hydrolysis, oxidation, and racemization, and
by physical factors like pH.
9.3.4.2.1 Deamidation and Racemization
Both asparagine and aspartic acid residues undergo deamidation via the formation of
succinimide intermediates at neutral and slightly alkaline pH, resulting in the forma-
tion of isoaspartate linkages and racemized aspartate residues [10] . Glutamine and
glutamic acid residues also undergo deamidation via a glutaramide intermediate in a
slower manner. Formation of succinimide and glutaramide intermediates is the pre-
dominant mode for protein racemization at physiologic pH and degradation of the
primary structure of peptides and proteins at neutral to moderately alkaline pH.
9.3.4.2.2 Reactions of Disulfides and Thiols
Disulfide linkages between cysteine residues are primary stabilizers of protein
secondary structures. Cysteine disulfides are unstable in alkaline environments,
decomposing to dehydroalanine and cysteine oxidation products, such as sulfenic,
sulfinic, and sulfonic acids [11] . This reaction is another major degradative pathway
contributing to the chemical instability of peptides and proteins at neutral to slightly
alkaline pH.
9.3.4.2.3 Proteolysis and Hydrolysis
Peptides and proteins are readily decomposed by group of enzymes called prote-
ases that are generated by microorganisms. Chemical hydrolysis of peptide bonds
is accomplished with either an acid or base catalysis; exposing these compounds to
extreme pH ultimately leads to complete destruction of the primary structure. Peptide
bonds adjacent to aspartate residues are especially sensitive to hydrolytic cleavage at
mildly acidic pH.
9.3.4.2.4 Oxidation
Cysteine, methionine, tryptophan, histidine, and tyrosine residues are subject to oxi-
dative destruction [12] . Degradation of methionine residues to methionine sulfone is
commonly observed for peptides and proteins stored in a solution exposed to air and
long-wave ultraviolet or fluorescent light. Photocatalyzed air oxidation of methionine
residues generally proceeds faster at neutral to moderately alkaline pH. Such peptide