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Updated: Mar 31, 2026
Author Spotlight: In Silico Creation and Impact of Carbonylated Amino Acids on Protein Structure and Function
Published on: April 26, 2024
Comparative Effects of Metal-Catalyzed Oxidizing Systems on Carbonylation and Integrity of Therapeutic Proteins
Dmitry Kryndushkin1, V Ashutosh Rao2
1Laboratory of Applied Biochemistry, Division of Biotechnology Products Research and Review III, Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, 20993, USA.
Purpose:
Ascorbic acid has been considered as a potential radical scavenging excipient for pharmaceutical formulations. However, under certain circumstances, ascorbic acid can generate reactive oxygen species via redox cycling. The objective of this study was to investigate ascorbic acid-induced oxidative carbonylation of therapeutic proteins and correlate the increase in carbonylation with protein aggregation.
Methods:
An optimized ELISA for quantifying carbonyl levels was used to compare the oxidizing potentials of ascorbic acid and hydrogen peroxide by testing four pharmaceutically-relevant proteins (human serum albumin, immunoglobulin G, granulocyte-colony stimulating factor and calcitonin). Several transition metals at micromolar concentrations were evaluated for their ability to enhance ascorbic acid-induced protein carbonylation. Protein aggregation under oxidative conditions, with or without free radical scavengers, was measured by aggregate binding fluorescent dye and confirmed by microfluidic imaging.
Results:
Addition of ascorbic acid alone resulted in higher increases in carbonylation than addition of hydrogen peroxide. The presence of trace amounts (>75 ppb) of copper enhanced oxidative effects of ascorbic acid, whereas other tested metals did not comparably promote oxidation. During oxidation, protein destabilization indicated by loss of the full-length protein, positively correlated with the increase in protein aggregation. However, levels of aggregation did not always correlate with the levels of protein carbonylation. At comparable carbonylation levels, addition of copper produced greater protein destabilization and aggregation than addition of iron.
Conclusions:
The results strongly suggest that ascorbic acid with traces of metals, especially copper, can promote therapeutic protein carbonylation and potentially aggregation. At similar carbonylation levels, some oxidative conditions may lead to greater protein destabilization than others.
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