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Investigation of a Degradant in a Biologics Formulation Buffer Containing L-Histidine

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ABSTRACT

Purpose

An unknown UV 280 nm absorbing peak was observed by SEC for protein stability samples formulated in L-histidine during a stress stability study. Understanding the source would enhance the confidence in the SEC results. We identified the unknown peak, studied the cause, and evaluated ways to eliminate it.

Methods

The unknown peak was fractionated by preparative size exclusion chromatography separations, and subsequently analyzed by Hydrophilic Interaction Chromatography (HILIC) coupled with Time-of-Flight (TOF) high resolution mass spectrometry. The possible degradation was also studied with the presence of different excipients, including metal cations, chelating agents, and amino acids.

Results

The unknown peak was identified to be trans-urocanic acid, a degradant of histidine, based on evidences from HILIC retention time, UV profile, accurate mass measurement, trans-cis isomerization, and pI measurement. The degradation from histidine to urocanic acids was not affected by the presence of Fe2+, but slightly activated by Mn2+. The chelating agents, EDTA and DTPA, counteracted the Mn2+ effects. This degradation was evidenced to be caused by contamination. Adding alanine or cysteine as an excipient was found to reduce this degradation by 97 and 98%, respectively.

Conclusions

L-histidine formulation buffer can be contaminated to induce histidine degradation to trans-urocanic acid, which shows a large UV 280 nm absorbing peak at the total permeation volume under SEC conditions. Amino acids alanine and cysteine effectively inhibit this histidine degradation.

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Abbreviations

2-IA:

2-imidazolecarboxaldehyde

4-IA:

4(5)-imidazolecarboxaldehyde

DTPA:

Diethylene triamine pentaactic acid

EDTA:

Ethylenediamine teracetic acid

TBHP:

Tert-butylhydroperoxide

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors would like to thank Dr. Adrienne Tymiak, Bethanne Warrack and Dr. Daniel O’Malley for reviewing the manuscript and providing helpful suggestions.

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Authors and Affiliations

  1. Bioanalytical and Discovery Analytical Sciences, Research & Development, Bristol-Myers Squibb Company, Route 206 and Province Line Road, Princeton, New Jersey, 08543, USA

    Chunlei Wang, Jun Dai, Sike Chen & Yingru Zhang

  2. Department of Discovery Protein Science, Research & Development, Bristol-Myers Squibb Company, Route 206 and Province Line Road, Princeton, New Jersey, 08543, USA

    Aaron Yamniuk & Noah Ditto

  3. Discovery Pharmaceutics, Research & Development, Bristol-Myers Squibb Company, Route 206 and Province Line Road, Princeton, New Jersey, 08543, USA

    Paul Stetsko

Authors
  1. Chunlei Wang
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  2. Aaron Yamniuk
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  3. Jun Dai
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  4. Sike Chen
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  5. Paul Stetsko
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  6. Noah Ditto
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  7. Yingru Zhang
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Correspondence to Chunlei Wang.

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Wang, C., Yamniuk, A., Dai, J. et al. Investigation of a Degradant in a Biologics Formulation Buffer Containing L-Histidine. Pharm Res 32, 2625–2635 (2015). https://doi.org/10.1007/s11095-015-1648-8

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  • DOI: https://doi.org/10.1007/s11095-015-1648-8

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