Development and validation of an LC-MS/MS assay for the quantitation of a PEGylated anti-CD28 domain antibody in human serum: overcoming interference from antidrug antibodies and soluble target.
OBJECTIVE
To support drug development of a PEGylated anti-CD28 domain antibody, a sensitive and robust LC-MS/MS assay was developed for the first in-human multiple ascending dose study.


METHODS
The procedure consists of a protein precipitation with acidified acetonitrile, followed by trypsin digestion of the supernatant. A surrogate peptide from the complementarity determining region was quantified with an LC-MS/MS assay using a stable isotope-labeled internal standard with flanking amino acids. An acid dissociation step was found to be essential to achieve full analyte recovery in the presence of antidrug antibodies and soluble target CD28.


CONCLUSIONS
The fully validated LC-MS/MS assay demonstrates good accuracy (% deviation ≤6.3) and precision (%CV ≤5.2) with an lower limit of quantitation of 10 ng/ml.
Analytical & Bioanalytical Development, Bristol-Myers Squibb, Route 206 & Province Line Road, Princeton, NJ 08543, USA.
Chao
C
Gong
Jianing
J
Zeng
Billy
B
Akinsanya
Hao
H
Jiang
Johanna
J
Mora
Shannon
S
Chilewski
Janice
J
Gambardella
Alban
A
Allentoff
Carol
C
Gleason
Anne-Francoise
AF
Aubry
Binodh S
BS
DeSilva
Mark E
ME
Arnold
D000097:Acetonitriles / Q000737:chemistry; D001774:Blood Chemical Analysis / Q000379:methods* / Q000592:standards; D018106:CD28 Antigens / Q000737:chemistry / Q000276:immunology*; D002138:Calibration; D011232:Chemical Precipitation; D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D004364:Pharmaceutical Preparations; D011092:Polyethylene Glycols / Q000737:chemistry*; D059748:Proteolysis; D012015:Reference Standards; D061905:Single-Domain Antibodies / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D000097:Acetonitriles; D018106:CD28 Antigens; D004364:Pharmaceutical Preparations; D061905:Single-Domain Antibodies; D011092:Polyethylene Glycols; D014357:Trypsin; C032159:acetonitrile