Sterilization of epidermal growth factor with supercritical carbon dioxide and peracetic acid; analysis of changes at the amino acid and protein level

Aseptic processing and terminal sterilization become increasingly challenging as medical devices become more complex and include active biologics. Terminal sterilization is preferred for patient safety and production costs. We aimed to determine how sterilization using supercritical CO₂ (scCO₂) with low levels of peracetic acid (PAA) affects amino acids and human epidermal growth factor (EGF) as a model protein. In a benchtop reactivity test, the amino acids methionine, tryptophan, arginine and lysine reacted with low levels of PAA in solution. At PAA levels used for scCO₂ sterilization, however, mass spectrometry only identified oxidative adducts on methionine and tryptophan. Mass spectrometry analysis of EGF exposed to scCO₂/PAA identified oxidative adducts on residues Met₂₁, Trp₄₉ and Trp₅₀, as well as a low level of truncations after residues Trp₄₉ and Trp₅₀. Importantly, processing of EGF in solution with scCO₂ did not affect its native conformation, and sterilized EGF maintained its activity in cell proliferation assays. When processing samples in lyophilized form with scCO₂/PAA, amino acids did not react with PAA and the presence of adducts was strongly reduced on methionine and tryptophan, both as single amino acids and in EGF. Truncation after tryptophan residues did not occur. EGF sterilized in the lyophilized form retained its activity when processing occurred with added moisture. These results have significant implications for the maintenance of biological function in sterilized decellularized scaffolds and the ability to manufacture terminally sterilized combination devices containing therapeutic peptides or proteins.