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Site-Specific Tryptophan Oxidation Induced by Autocatalytic Reaction of Polysorbate 20 in Protein Formulation

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ABSTRACT

Purpose

Tryptophan (Trp) oxidation leading to atypical degradation of a protein (Fab) formulated with polysorbate 20 (PS20) was investigated. Such atypical Trp oxidation was discussed in relation to a kinetic model that involves initiation of oxidizing free radical through an autocatalytic reaction.

Methods

Ion-exchange chromatography and peptide mapping were used to determine Trp oxidation. Peroxides in PS20 and free radicals in Fab samples were detected by fluorometric assay and electron paramagnetic resonance (EPR), respectively.

Results

PS20 with increased peroxides level led to degradation of Fab stored at 30°C. Degradation was characterized as Trp50 oxidation, which was not observed in a Fab variant where His31 was replaced. EPR peaks related to known spin adducts of 5,5 dimethylpyrroline N-oxide were detected in Fab exhibiting Trp oxidation, indicating free radicals were present. Trp oxidation of Fab observed in several drug product lots with different degradation rates fits an autocatalytic reaction model that involves free radicals. EDTA, catalase, and free tryptophan prevented oxidation.

Conclusions

A metal-binding amino acid, His31, was responsible for Trp50 oxidation of Fab induced by peroxides in PS20 present in the protein formulation. Oxidation was induced by autocatalytic degradation of PS20 and could be inhibited by antioxidants.

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ACKNOWLEDGMENTS

The authors would like to thank Marian Eng and Lin Luis for their technical assistance. We would also like to thank Yvonne Chen, Manda Wong and Dr. Henry Lowman for synthesizing the Fab variant, and Dr. Lipika Basumallick for reviewing the EPR study.

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Authors and Affiliations

  1. Department of Pharmaceutical and Processing Development, Genentech, Inc., 1 DNA Way, South San Francisco, California, 94080, USA

    Xanthe M. Lam, William G. Lai, Edwin K. Chan & Chung C. Hsu

  2. Department of Protein Analytical Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, California, 94080, USA

    Victor Ling

Authors
  1. Xanthe M. Lam
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  2. William G. Lai
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  3. Edwin K. Chan
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  4. Victor Ling
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Correspondence to Xanthe M. Lam.

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Lam, X.M., Lai, W.G., Chan, E.K. et al. Site-Specific Tryptophan Oxidation Induced by Autocatalytic Reaction of Polysorbate 20 in Protein Formulation. Pharm Res 28, 2543–2555 (2011). https://doi.org/10.1007/s11095-011-0482-x

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  • DOI: https://doi.org/10.1007/s11095-011-0482-x

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