Fully Validated
LC-MS/MS Assay for the Simultaneous
Quantitation of Coadministered Therapeutic Antibodies in Cynomolgus
Monkey Serum
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Abstract
An LC-MS/MS assay was developed and
fully validated for the simultaneous
quantitation of two coadministered human monoclonal antibodies (mAbs),
mAb-A and mAb-B of IgG4 subclass, in monkey serum. The total serum
proteins were digested with trypsin at 50 °C for 30 min after
methanol denaturation and precipitation, dithiothreitol reduction,
and iodoacetamide alkylation. The tryptic peptides were chromatographically
separated with a C18 column (2.1 × 100 mm, 1.7 μm) with
mobile phases of 0.1% formic acid in water and acetonitrile. Four
peptides, a unique peptide for each mAb and two confirmatory peptides
from different antibody domains, were simultaneously quantified by
LC-MS/MS in the multiple reaction-monitoring mode. Stable isotopically
labeled peptides with flanking amino acids on C- and N-terminals were
used as internal standards to minimize the variability during sample
processing and detection. The LC-MS/MS assay showed lower limit of
quantitation (LLOQ) at 5 μg/mL for mAb-A and 25 μg/mL
for mAb-B. The intra- and interassay precision (%CV) was within 10.0%
and 8.1%, respectively, and the accuracy (%Dev) was within ±5.4%
for all the peptides. Other validation parameters, including sensitivity,
selectivity, dilution linearity, processing recovery and matrix effect,
autosampler carryover, run size, stability, and data reproducibility,
were all evaluated. The confirmatory peptides played a critical role
in confirming quantitation accuracy and the integrity of the drugs
in the study samples. The robustness of the LC-MS/MS assay and the
data agreement with the ligand binding data demonstrated that LC-MS/MS
is a reliable and complementary approach for the quantitation of coadministered
antibody drugs