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

被引：6
|
作者：
Puri, Aastha [1 ]
Quan, Yong [1 ]
Narang, Ajit S. [1 ]
Adams, Monica [1 ]
Gandhi, Rajesh [1 ]
Nashine, Vishal C. [1 ]
机构：
[1] Bristol Myers Squibb Co, Drug Prod Sci & Technol, 1 Squibb Dr, New Brunswick, NJ 08901 USA
来源：
AAPS PHARMSCITECH | 2017年 / 18卷 / 03期
关键词：
deamidation; fluorescence; high-throughput; isoaspartate; protein; stability; ADENOSYLMETHIONINE-DEPENDENT METHYLTRANSFERASES; MASS-SPECTROMETRY ANALYSIS; COLORIMETRIC ASSAY; DEAMIDATION; ACID;
D O I：
10.1208/s12249-016-0570-7
中图分类号：
R9 [药学];
学科分类号：
1007 ;
摘要：
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.
引用
收藏
页码：803 / 808
页数：6
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