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AAPS PharmSciTech

, Volume 18, Issue 3, pp 803–808 | Cite as

A Fluorescence-Based High-Throughput Coupled Enzymatic Assay for Quantitation of Isoaspartate in Proteins and Peptides

  • Aastha Puri
  • Yong Quan
  • Ajit S. Narang
  • Monica Adams
  • Rajesh Gandhi
  • Vishal C. NashineEmail author
Research Article

Abstract

Formation of isoaspartate (IsoAsp) from spontaneous asparagine (Asn) deamidation or aspartate (Asp) isomerization is one of the most common non-enzymatic pathways of chemical degradation of protein and peptide pharmaceuticals. Rapid quantitation of IsoAsp formation can enable rank-ordering of potential drug candidates, mutants, and formulations as well as support shelf life prediction and stability requirements. A coupled enzymatic fluorescence-based IsoAsp assay (CEFIA) was developed as a high-throughput method for quantitation of IsoAsp in peptides and proteins. In this note, application of this method to two therapeutic candidate proteins with distinct structural scaffolds is described. In addition, the results obtained with this method are compared to those from conventional assays.

Keywords

deamidation fluorescence high-throughput isoaspartate protein stability 

Abbreviations

Asp

Aspartate

IsoAsp

Isoaspartate

CEFIA

Coupled enzymatic fluorescence-based IsoAsp assay

TPM

Tryptic peptide mapping

PEG

Polyethylene glycol

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Copyright information

© American Association of Pharmaceutical Scientists 2016

Authors and Affiliations

  • Aastha Puri
    • 1
  • Yong Quan
    • 1
  • Ajit S. Narang
    • 1
  • Monica Adams
    • 1
  • Rajesh Gandhi
    • 1
  • Vishal C. Nashine
    • 1
    Email author
  1. 1.Drug Product Science & TechnologyBristol-Myers Squibb, Co.New BrunswickUSA

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