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

Publication date: Available online 29 November 2019Source: Biochimica et Biophysica Acta (BBA) - Proteins and ProteomicsAuthor(s): David M. Bednarski, Ellen E. Lantz, Cedric E. Bobst, Anthony R. Eisenhut, Stephen J. Eyles, Julien P. FeyAbstractAseptic 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. ...
Source: Biochimica et Biophysica Acta (BBA) Proteins and Proteomics - Category: Biochemistry Source Type: research