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Comparative Effects of Metal-Catalyzed Oxidizing Systems on Carbonylation and Integrity of Therapeutic Proteins

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ABSTRACT

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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Abbreviations

ATP:

Adenosine triphosphate

DCVJ:

9-(dicyanovinyl)julolidine

DNP:

2,4-dinitrophenyl hydrazone

DNPH:

2,4-dinitrophenyl hydrazine

ELISA:

Enzyme-linked immunosorbent assay

G-CSF:

Granulocyte-colony stimulating factor

HSA:

Recombinant human serum albumin

IgG:

Immunoglobulin G

MFI:

Microfluidic imaging

ROS:

Reactive oxygen species

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ACKNOWLEDGMENTS AND DISCLOSURES

This research was supported by the CDER Critical Path Initiative. We thank Dr. Shen Luo for help with microfluidic imaging. We would like to thank Dr. Hiroshi Uehara and Elliot Rosen (FDA) for critical reading of the manuscript. 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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  1. Laboratory 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

    Dmitry Kryndushkin & V. Ashutosh Rao

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  1. Dmitry Kryndushkin
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  2. V. Ashutosh Rao
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Correspondence to V. Ashutosh Rao.

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Supplementary Fig. S1

Evaluation of oxidative effects of ascorbic acid in the presence of metal chelator EDTA. Each of the three proteins (HSA, IgG and G-CSF) was incubated for 16 h at 25°C alone or with addition of the indicated compounds (5 mM ascorbic acid or 5 mM ascorbic acid with 3 mM EDTA). After oxidation proteins were derivatized with DNPH and analyzed by (A) carbonyl immunoblotting with antibodies against DNP group, (B) carbonyl ELISA to quantify protein carbonylation. Carbonyl values were normalized to the control reactions (protein only) and shown as carbonylation fold increases. Data are shown as mean ± SD. * p < 0.05 (GIF 21 kb)

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Kryndushkin, D., Rao, V.A. Comparative Effects of Metal-Catalyzed Oxidizing Systems on Carbonylation and Integrity of Therapeutic Proteins. Pharm Res 33, 526–539 (2016). https://doi.org/10.1007/s11095-015-1807-y

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