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Research ArticleResearch

Qualification of a Quantitative Method for Monitoring Aspartate Isomerization of a Monoclonal Antibody by Focused Peptide Mapping

Mingyan Cao, Wenjun (David) Mo, Anthony Shannon, Ziping Wei, Michael Washabaugh and Patricia Cash
PDA Journal of Pharmaceutical Science and Technology November 2016, 70 (6) 490-507; DOI: https://doi.org/10.5731/pdajpst.2015.006239
Mingyan Cao
Analytical Biotechnology Department, MedImmune Inc, Gaithersburg, MD
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Wenjun (David) Mo
Analytical Biotechnology Department, MedImmune Inc, Gaithersburg, MD
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Anthony Shannon
Analytical Biotechnology Department, MedImmune Inc, Gaithersburg, MD
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Ziping Wei
Analytical Biotechnology Department, MedImmune Inc, Gaithersburg, MD
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Michael Washabaugh
Analytical Biotechnology Department, MedImmune Inc, Gaithersburg, MD
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  • For correspondence: michael.washabaugh@theraproteins.com Patriciacash8@gmail.com
Patricia Cash
Analytical Biotechnology Department, MedImmune Inc, Gaithersburg, MD
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  • For correspondence: michael.washabaugh@theraproteins.com Patriciacash8@gmail.com
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Abstract

Aspartate (Asp) isomerization is a common post-translational modification of recombinant therapeutic proteins that can occur during manufacturing, storage, or administration. Asp isomerization in the complementarity-determining regions of a monoclonal antibody may affect the target binding and thus a sufficiently robust quality control method for routine monitoring is desirable. In this work, we utilized a liquid chromatography–mass spectrometry (LC/MS)-based approach to identify the Asp isomerization in the complementarity-determining regions of a therapeutic monoclonal antibody. To quantitate the site-specific Asp isomerization of the monoclonal antibody, a UV detection–based quantitation assay utilizing the same LC platform was developed. The assay was qualified and implemented for routine monitoring of this product-specific modification. Compared with existing methods, this analytical paradigm is applicable to identify Asp isomerization (or other modifications) and subsequently develop a rapid, sufficiently robust quality control method for routine site-specific monitoring and quantitation to ensure product quality. This approach first identifies and locates a product-related impurity (a critical quality attribute) caused by isomerization, deamidation, oxidation, or other post-translational modifications, and then utilizes synthetic peptides and MS to assist the development of a LC-UV–based chromatographic method that separates and quantifies the product-related impurities by UV peaks. The established LC-UV method has acceptable peak specificity, precision, linearity, and accuracy; it can be validated and used in a good manufacturing practice environment for lot release and stability testing.

LAY ABSTRACT: Aspartate isomerization is a common post-translational modification of recombinant proteins during manufacture process and storage. Isomerization in the complementarity-determining regions (CDRs) of a monoclonal antibody A (mAb-A) has been detected and has been shown to have impact on the binding affinity to the antigen. In this work, we utilized a mass spectrometry–based peptide mapping approach to detect and quantitate the Asp isomerization in the CDRs of mAb-A. To routinely monitor the CDR isomerization of mAb-A, a focused peptide mapping method utilizing reversed phase chromatographic separation and UV detection has been developed and qualified. This approach is generally applicable to monitor isomerization and other post-translational modifications of proteins in a specific and high-throughput mode to ensure product quality.

  • Monoclonal antibody (mAb)
  • Complementarity-determining regions (CDRs)
  • Aspartic acid isomerization
  • Focused peptide mapping
  • Method qualification

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PDA Journal of Pharmaceutical Science and Technology: 70 (6)
PDA Journal of Pharmaceutical Science and Technology
Vol. 70, Issue 6
November/December 2016
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Qualification of a Quantitative Method for Monitoring Aspartate Isomerization of a Monoclonal Antibody by Focused Peptide Mapping
Mingyan Cao, Wenjun (David) Mo, Anthony Shannon, Ziping Wei, Michael Washabaugh, Patricia Cash
PDA Journal of Pharmaceutical Science and Technology Nov 2016, 70 (6) 490-507; DOI: 10.5731/pdajpst.2015.006239
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Keywords

  • Monoclonal antibody (mAb)
  • Complementarity-determining regions (CDRs)
  • Aspartic acid Isomerization
  • Focused peptide mapping
  • Method qualification

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Qualification of a Quantitative Method for Monitoring Aspartate Isomerization of a Monoclonal Antibody by Focused Peptide Mapping
Mingyan Cao, Wenjun (David) Mo, Anthony Shannon, Ziping Wei, Michael Washabaugh, Patricia Cash
PDA Journal of Pharmaceutical Science and Technology Nov 2016, 70 (6) 490-507; DOI: 10.5731/pdajpst.2015.006239

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