title: A Fluorescence-Based High-Throughput Coupled Enzymatic Assay for Quantitation of Isoaspartate in Proteins and Peptides.
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.
authors.affiliation: Drug Product Science & Technology, Bristol-Myers Squibb, Co., 1 Squibb Drive, New Brunswick, New Jersey, 08901, USA.
authors.forename: Aastha
authors.initials: A
authors.lastname: Puri
authors.affiliation: Drug Product Science & Technology, Bristol-Myers Squibb, Co., 1 Squibb Drive, New Brunswick, New Jersey, 08901, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Quan
authors.affiliation: Drug Product Science & Technology, Bristol-Myers Squibb, Co., 1 Squibb Drive, New Brunswick, New Jersey, 08901, USA.
authors.forename: Ajit S
authors.initials: AS
authors.lastname: Narang
authors.affiliation: Drug Product Science & Technology, Bristol-Myers Squibb, Co., 1 Squibb Drive, New Brunswick, New Jersey, 08901, USA.
authors.forename: Monica
authors.initials: M
authors.lastname: Adams
authors.affiliation: Drug Product Science & Technology, Bristol-Myers Squibb, Co., 1 Squibb Drive, New Brunswick, New Jersey, 08901, USA.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Gandhi
authors.affiliation: Drug Product Science & Technology, Bristol-Myers Squibb, Co., 1 Squibb Drive, New Brunswick, New Jersey, 08901, USA. vishal.nashine@bms.com.
authors.forename: Vishal C
authors.initials: VC
authors.lastname: Nashine
mesh_terms: D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D057075:Enzyme Assays / Q000379:methods; D005453:Fluorescence; D026581:Isoaspartic Acid / Q000737:chemistry*; D007536:Isomerism; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*
chemical_list: D026581:Isoaspartic Acid; D010455:Peptides; D011506:Proteins; D001224:Aspartic Acid; D001216:Asparagine
keywords: deamidation; fluorescence; high-throughput; isoaspartate; protein; stability