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Differentiating the Effects of Oxidative Stress Tests on Biopharmaceuticals

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Abstract

Purpose

A rapid and broadly applicable method to assess relevant oxidative damage in biopharmaceuticals is important for lifecycle management of product quality. Multiple methods are currently employed as stress tests to induce oxidative damage for assessment of stability, safety, and efficacy. We compared two common methods for inducing oxidative damage to assess differences in impact on bioactivity and structure of the biopharmaceuticals.

Methods

Biopharmaceuticals were treated with either metal-catalyzed oxidation (MCO) conditions or the reactive-oxygen species (ROS) inducer 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH), then analyzed for changes in structure and bioactivity.

Results

We demonstrate that commonly used chemical methods for assessing oxidation yield distinct oxidation profiles for each of the biotechnology products analyzed, including monoclonal antibodies. We further report oxidant- and product-specific changes in bioactivity under oxidizing conditions, along with differential oxidation on the molecular subunits of monoclonal antibodies.

Conclusion

Our results highlight the need for product-specific optimization and selection of orthogonal, relevant oxidizers when characterizing stress responses in biopharmaceuticals.

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Abbreviations

AAPH:

2,2′-Azobis(2-amidinopropane) dihydrochloride

ABS:

(4-amino)benzenesulfonic acid

ADCC:

antibody-dependent cell-mediated cytotoxicity

Bis-ANS:

4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid

DNPH:

2,4-dinitrophenylhydrazine

DOPA:

dihydroxyphenylalanine

ELISA:

enzyme-linked immunosorbence assay

G-CSF:

granulyte-colony stimulating factor

HSA:

human serum albumin

LC-MS:

liquid chromatography-mass spectrometry

MCO:

metal-catalyzed oxidation

ROS:

reactive oxygen species

RP-HPLC:

reverse phase high-performance liquid chromatography

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TBTA:

tris(benzyltriazolylmethyl)amine

TMB:

3,3′,5,5′-tetramethylbenzidine

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors thank Drs. Christopher J. Chang and Shixian Lin (University of California, Berkeley) for the generous donation of Ox4 probe for these studies, as well as Perry Beamer (FDA) for his assistance in adapting the DOPA formation assay for quantitation. The authors would also like to thank Dr. Dmitry Kryndushkin (FDA) for helpful discussions regarding amyloid-like structures and Dr. Darón Freedburg (FDA) for assistance with the circular dichroism studies. Drs. Rong-Fong Shen and Wells Wu (FDA) are thanked for assistance with mass spectrometry analyses. The authors are further indebted to Dr. Jeffrey Baker for discussions, as well as Drs. Delaram Moshkelani and Baikuntha Aryal (FDA) for critical reading of the manuscript. This work was funded in part by an appointment to the Research Participation Program at the Office of Biotechnology Products, Center for Drug Evaluation and Research at the U.S. Food and Drug Administration administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the FDA. 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. The authors declare that they have no conflicts of interest.

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Authors and Affiliations

  1. Laboratory of Applied Biochemistry, Division of Biotechnology 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

    Geoffrey A. Heinzl, Lo Lai & V. Ashutosh Rao

Authors
  1. Geoffrey A. Heinzl
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  2. Lo Lai
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  3. V. Ashutosh Rao
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Contributions

G.A.H., L.L. and V.A.R. conceived the experiments. G.A.H.and L.L. performed the experiments and analysis. G.A.H. and V.A.R. wrote the manuscript.

Corresponding author

Correspondence to V. Ashutosh Rao.

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Heinzl, G.A., Lai, L. & Rao, V.A. Differentiating the Effects of Oxidative Stress Tests on Biopharmaceuticals. Pharm Res 36, 103 (2019). https://doi.org/10.1007/s11095-019-2627-2

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