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An Efficient and Rapid Method to Monitor the Oxidative Degradation of Protein Pharmaceuticals: Probing Tyrosine Oxidation with Fluorogenic Derivatization

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Abstract

Purpose

The loss of potency of protein therapeutics can be linked to the oxidation of specific amino acid residues leading to a great variety of oxidative modifications. The comprehensive identification of these oxidative modifications requires high-resolution mass spectrometry analysis, which requires time and expensive resources. Here, we propose a fluorogenic derivatization method of oxidized Tyr and Phe yielding benzoxazole derivatives, as an orthogonal technique for the rapid screening of protein oxidation.

Methods

Four model proteins, IgG1, human growth hormone (hGH), insulin and bovine serum albumin (BSA) were exposed to oxidation via peroxyl radicals and metal-catalyzed reactions and efficiently screened by fluorogenic derivatization of Tyr and Phe oxidation products. Complementary LC-MS analysis was done to identify the extent of methionine oxidation in oxidized proteins.

Results

The Fluorogenic derivatization technique can easily be adapted to a 96-well plate, in which several protein formulations can be screened in short time. Representatively for hGH, we show that the formation of benzoxazole parallels the oxidation of Met to methionine sulfoxide which enables estimation of Met oxidation by just recording the fluorescence.

Conclusions

Our rapid fluorescence based screening allows for the fast comparison of the stability of multiple formulations.

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Abbreviations

AAPH:

2, 2′-azobis (2-methylpropionamidine) dihydrochloride

ABS:

4-(aminomethyl) benzenesulfonic acid

BMS:

bis (2-mercaptoethyl) sulfone

BSA:

Bovine serum albumin

DOCH:

2-amino-3-(3, 4-dioxocyclohexa-1, 5-dien-1-yl) propanoic acid

DOPA:

3, 4-dihydroxyphenylalanine

hGH:

Human growth hormone

IAA:

Iodoacetamide

IgG:

Immunoglobulin G

LTQ-FT:

Linear ion trap quadrupole-Fourier transform

MCO:

Metal catalyzed oxidation

Q-TOF:

Quadrupole time-of-flight

SEC:

Size exclusion chromatography

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Author information

Author notes

  1. Olivier Mozziconacci

    Present address: Analytical Sciences, Merck & Co., Inc., Kenilworth, New Jersey, USA

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, Kansas, 66047, USA

    Rupesh Bommana, Olivier Mozziconacci & Christian Schöneich

  2. Late Stage Pharmaceutical Development, Genentech, S. San Francisco, California, 94080, USA

    Y. John Wang

Authors
  1. Rupesh Bommana
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  2. Olivier Mozziconacci
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  3. Y. John Wang
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  4. Christian Schöneich
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Corresponding author

Correspondence to Christian Schöneich.

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Bommana, R., Mozziconacci, O., John Wang, Y. et al. An Efficient and Rapid Method to Monitor the Oxidative Degradation of Protein Pharmaceuticals: Probing Tyrosine Oxidation with Fluorogenic Derivatization. Pharm Res 34, 1428–1443 (2017). https://doi.org/10.1007/s11095-017-2159-6

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  • DOI: https://doi.org/10.1007/s11095-017-2159-6

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