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Metal-Mediated Protein Oxidation: Applications of a Modified ELISA-Based Carbonyl Detection Assay for Complex Proteins

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ABSTRACT

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 Fe2+. 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.

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ACKNOWLEDGMENTS AND DISCLOSURES

This research was supported by the CDER Critical Path Initiative. We thank Dr. Joseph Kotarek (FDA) for help in analyzing protein aggregates. We would like to thank Elliot Rosen (FDA), Dr. Shen Luo (FDA), Dr. Nancy B. Wehr (NIH/NHLBI) and Dr. Rodney L. Levine (NIH/NHLBI) for critical reading of the manuscript or suggestions. The authors have no competing financial interests to disclose. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the U.S. Food and Drug Administration and the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

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Authors and Affiliations

  1. Laboratory 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

    Hiroshi Uehara & V. Ashutosh Rao

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  1. Hiroshi Uehara
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  2. V. Ashutosh Rao
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Correspondence to V. Ashutosh Rao.

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Supplementary Figure S1

Validation of DNPH removal step for carbonyl content determination. Following DNP derivatization, free unbound DNPH was removed either by ethanol/ethyl acetate extraction as described in Materials and Methods section (sample 1) or by size exclusion chromatography (sample 2). The Y axis indicates relative carbonyl content (%) in samples 2 compared to samples 1 for each protein preparation. These data were average of three separate experiments. Size exclusion chromatography utilizing Bio-Gel P-6 (Micro BioSpin chromatography columns, Bio-Rad Laboratories, Harcules, CA) : 100 μl of oxidized protein samples were derivatized with DNPH, precipitated with final 20% TCA, briefly rinsed with ethanol/ethyl acetate (1:1 v/v) and air dried. The precipitate was resolved in 70 μl of 6M Gu-HCl solution and the proteins were separated from free un-reacted DNPH by size exclusion column chromatography utilizing Bio-Gel P-6 spin column. (GIF 66 kb)

High resolution image (EPS 402 kb)

Supplementary Figure S2

Analysis of aggregate formation in rabbit IgG (RIgG) or transferrin stored at various temperature with or without Fe 2+ /H 2 O 2 /ascorbic acid. A. Analysis by Zetasizer dynamic light scattering. Y-axis denotes derived count rate (Kcps: kilocounts per second). B. Binding of molecular rotor dye. 25μg of RIgG or transferrin was mixed with Proteostat dye and RFU (excitation 550nm/emission 600nm) was measured following manufacturer’s instruction. (GIF 51 kb)

High resolution image (EPS 431 kb)

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Uehara, H., Rao, V.A. Metal-Mediated Protein Oxidation: Applications of a Modified ELISA-Based Carbonyl Detection Assay for Complex Proteins. Pharm Res 32, 691–701 (2015). https://doi.org/10.1007/s11095-014-1496-y

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  • DOI: https://doi.org/10.1007/s11095-014-1496-y

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