Immunoaffinity purification using anti-PEG antibody followed by two-dimensional liquid chromatography/tandem mass spectrometry for the quantification of a PEGylated therapeutic peptide in human plasma.
Anal Chem
; 82(16): 6877-86, 2010 Aug 15.
Article
em En
| MEDLINE
| ID: mdl-20704377
ABSTRACT
Quantification of a PEGylated peptide in human plasma using LC-MS/MS to support clinical studies presented challenges in terms of assay sensitivity, selectivity, and ruggedness. To ensure specific recognition of PEGylated species, an immunoaffinity purification method (IAP) using anti-PEG antibody followed by two-dimensional (2D) LC-MS/MS was developed for MK-2662, an investigational peptide containing 38 amino acids with a 40 kDa branched PEG [poly(ethylene glycol)] at C-terminus. Biotinylated anti-PEG antibody, bound to streptavidin-coated magnetic beads, was used to capture MK-2662 and its stable-isotope-labeled internal standard from human plasma. After on-bead digestion with trypsin, the supernatant was injected on a 2D high-performance liquid chromatography (HPLC) system constructed with strong cation-exchange and reversed-phase columns, followed by MS/MS detection of the surrogate N(1-12)-mer of MK-2662 on an API5000. The assay ruggedness was improved by optimizing the trypsin digestion and sample storage conditions. The intraday validation, conducted in parallel with protein precipitation (PPT) assay, demonstrated 94.8-105.8% accuracy with <9.76% coefficient of variation (CV) for IAP, and 99.0-101.0% accuracy with <3.43% CV for PPT, over a dynamic range of 2-200 nM and 1-1000 nM, respectively. A cross comparison, performed using clinical samples, showed that the values obtained from IAP assay were about 15-30% lower than those from PPT method, which supports more specific PEG recognition provided by IAP.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
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Polietilenoglicóis
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Cromatografia Líquida de Alta Pressão
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Espectrometria de Massas em Tandem
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Anticorpos
Limite:
Humans
Idioma:
En
Ano de publicação:
2010
Tipo de documento:
Article