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Metal-mediated protein oxidation: applications of a modified ELISA-based carbonyl detection assay for complex
Hiroshi Uehara1, V Ashutosh Rao
1Laboratory of Chemistry, Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 29 Lincoln Drive Bldg 29A, Room 2A-11, Bethesda, Maryland, 20892, USA.
Purpose:
Therapeutic proteins are prone to oxidative modification during manufacturing, processing, and storage that may lead to degradation, aggregation, and immunogenicity. Protein carbonylation is an irreversible oxidative modification and has been identified as a hallmark of severe oxidative stress but not extensively studied for its impact on the stability and activity of therapeutic proteins.
Methods:
We describe the application of a modified ELISA-based method to quantify global levels of carbonyl modification of complex proteins. We investigated protein oxidation of large protein molecules (transferrin, rabbit IgG, or β-glucosidase) and complex protein samples (human plasma) that were either stored in different buffer formulations, with varying amounts of divalent iron, or under different storage temperatures to determine the impact of different physicochemical stresses on carbonyl modifications.
Results:
The modified ELISA allows for sensitive and specific carbonyl quantification with measurements that closely match those determined with the conventional spectrophotometric method. The method was useful for complex protein mixtures such as cell lysates without the need for additional procedures to remove DNA and RNA. Our findings demonstrate significant oxidative modification of each of the proteins stored in commonly used buffers and excipients at 37°C, 23°C, and 4°C. The carbonyl levels were further exacerbated with addition of trace amounts of Fe(2+). We also measured the extent of protein aggregation under oxidizing conditions.
Conclusions:
Collectively, our results indicate the importance of better characterizing carbonyl modification of proteins during their storage and use.
Insights
Protein carbonylation, an irreversible oxidative modification, impacts therapeutic protein stability. This study developed a sensitive ELISA to quantify carbonyls, revealing significant oxidation in proteins under common storage conditions, exacerbated by iron.
Area of Science:
- Biochemistry and Molecular Biology
- Protein Chemistry
- Analytical Chemistry
Background:
- Therapeutic proteins are susceptible to oxidative modifications during production and storage.
- Protein carbonylation is a significant oxidative stress marker but its impact on therapeutic proteins is understudied.
- Oxidation can lead to protein degradation, aggregation, and immunogenicity, affecting therapeutic efficacy.
Purpose of the Study:
- To develop and apply a modified ELISA for quantifying global carbonyl modifications in complex proteins.
- To investigate the impact of various physicochemical stresses (buffer formulation, iron concentration, temperature) on protein carbonylation.
- To assess the relationship between oxidative modification and protein aggregation.
Main Methods:
- A modified ELISA-based assay was employed for sensitive and specific quantification of protein carbonyls.
- Large proteins (transferrin, IgG, β-glucosidase) and human plasma were subjected to different storage conditions.
- Physicochemical stresses included varying buffer formulations, divalent iron (Fe2+) levels, and storage temperatures (4°C, 23°C, 37°C).
Main Results:
- The modified ELISA demonstrated high sensitivity and specificity, correlating well with spectrophotometric methods.
- Significant carbonyl modifications were observed in proteins stored under common conditions (buffers, excipients) at various temperatures.
- Addition of trace Fe2+ significantly increased carbonyl levels, and protein aggregation was observed under oxidizing conditions.
Conclusions:
- Characterizing protein carbonyl modification is crucial for understanding therapeutic protein stability during storage and use.
- Common storage conditions can induce significant oxidative damage, highlighting the need for protective strategies.
- The developed ELISA method provides a valuable tool for assessing oxidative stress in complex protein samples.
