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

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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.

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Abbreviations

Asp:

Aspartate

IsoAsp:

Isoaspartate

CEFIA:

Coupled enzymatic fluorescence-based IsoAsp assay

TPM:

Tryptic peptide mapping

PEG:

Polyethylene glycol

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Authors and Affiliations

  1. Drug Product Science & Technology, Bristol-Myers Squibb, Co., 1 Squibb Drive, New Brunswick, New Jersey, 08901, USA

    Aastha Puri, Yong Quan, Ajit S. Narang, Monica Adams, Rajesh Gandhi & Vishal C. Nashine

Authors
  1. Aastha Puri
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  2. Yong Quan
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  3. Ajit S. Narang
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  4. Monica Adams
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  5. Rajesh Gandhi
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  6. Vishal C. Nashine
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Correspondence to Vishal C. Nashine.

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Puri, A., Quan, Y., Narang, A.S. et al. A Fluorescence-Based High-Throughput Coupled Enzymatic Assay for Quantitation of Isoaspartate in Proteins and Peptides. AAPS PharmSciTech 18, 803–808 (2017). https://doi.org/10.1208/s12249-016-0570-7

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  • DOI: https://doi.org/10.1208/s12249-016-0570-7

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