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.

中文翻译：

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用超临界二氧化碳和过氧乙酸对表皮生长因子进行灭菌；分析氨基酸和蛋白质水平的变化。

随着医疗设备变得越来越复杂并包括活性生物制剂，无菌处理和最终灭菌变得越来越具有挑战性。出于患者安全和生产成本的考虑，首选终端灭菌。我们旨在确定使用过氧乙酸（PAA）含量低的超临界CO2（scCO2）灭菌如何影响氨基酸和作为模型蛋白的人表皮生长因子（EGF）。在台式反应性测试中，氨基酸蛋氨酸，色氨酸，精氨酸和赖氨酸与溶液中低水平的PAA反应。但是，在用于scCO2灭菌的PAA含量下，质谱法只能鉴定出蛋氨酸和色氨酸上的氧化加合物。暴露于scCO2 / PAA的EGF的质谱分析确定了Met21，Trp49和Trp50残基上的氧化加合物，以及残基Trp49和Trp50之后的低水平截短。重要的是，在含有scCO2的溶液中处理EGF不会影响其天然构象，而经过灭菌的EGF在细胞增殖试验中仍保持其活性。当用scCO2 / PAA冻干形式处理样品时，氨基酸不与PAA反应，蛋氨酸和色氨酸中的加合物的存在被强烈地减少了，无论是作为单个氨基酸还是在EGF中。色氨酸残基后未发生截短。当加工过程中添加水分时，以冻干形式灭菌的EGF保持其活性。这些结果对于维持灭菌的脱细胞支架中的生物学功能以及制造包含治疗性肽或蛋白质的最终灭菌的组合装置的能力具有重要意义。