Qualification and application of a liquid chromatography–quadrupole time‐of‐flight mass spectrometric method for the determination of trastuzumab in rat plasma

Abstract

An liquid chromatography-quadrupole time-of-flight (QqTOF) mass spectrometric method was developed for the determination of humanized or human monoclonal antibodies in rat plasma at the early drug discovery stage. Trastuzumab was used as a model monoclonal antibody. The method consisted of immunoprecipitation followed by tryptic digestion for sample preparation and LC-TOF-MS/MS analysis of specific signature peptides in the positive ion mode using electrospray ionization for analysis. A stable isotope-labeled signature peptide was also used as internal standard. A quadratic regression (weighted 1/concentration(2) ), with an equation y = ax(2) + bx + c, was used to fit calibration curves over the concentration range of 0.500-100 µg/mL for trastuzumab. Samples from a pharmacokinetic study in rat were analyzed by this qualified LC-TOF-MS/MS method and concentrations were compared with those generated by enzyme linked immunosorbent assays method. The LC-TOF-MS/MS method was accurate and precise, with quantitative results comparable with those of ELISA. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control samples. Within-run accuracy ranged from 1.53 to 9.20% with precision values ≤10.29%. This LC-TOF-MS/MS method approach could be used as a complementary method for humanized or human monoclonal antibodies at the early drug discovery stage.

Rapid and simultaneous quantification of a mixture of biopharmaceuticals by a liquid chromatography/quadrupole time‐of‐flight mass spectrometric method in rat plasma following cassette‐dosing

“…The calibration curves had quadratic regressions for all antibodies over the studied concentration range with correlation coefficients r > 0.990 and an LLOQ of 1 μg/mL was easily achieved by this method. The sensitivity was generally enough for most preclinical PK studies to cover the expected concentration range throughout a PK time course to calculate PK parameters for antibody drugs . Using a larger volume (≥40 μL) in the sample preparation method, more optimized conditions of LC/TOF‐MS/MS or a more sensitive instrument (e.g.…”

“…The previous method was modified to incorporate on‐bead digestion to quantify many antibodies. Each 24‐μL aliquot of plasma study samples, QCs and STDs was separately mixed with 370 μL of PBS containing 0.1% Tween 20 and 30 μL of magnetic bead suspension.…”

“…Traditionally, pharmacokinetic (PK) evaluation of mAbs has been mainly performed by immunoassays . Immunoassays have several advantages such as high sensitivity, robust performance, and high throughput . However, immunoassays also offer several disadvantages such as non‐specific binding as well as time‐consuming, labor‐intensive reagent development and cross‐reactions which make assay development challenging at the early stage of bioanalysis …”

An Overall Comparison of Small Molecules and Large Biologics in ADME Testing

“…In recent comparative studies, Peng et al developed and validated a simple, sensitive, specific and precise LC-MS/MS assay for quantitation of infliximab PK in human serum by using isotopelabeled signature peptide as the internal standard (IS), which achieved an excellent correlation with ELISA detection [73]. ParK et al applied LC-MS/MS quadruple time-of-flight mass spectrometric method (QTOF) for the determination of trastuzumab in rat plasma with good sensitivity (LLOD 0.5 µg/ml) and results in consistence with ELISA assay (R 2 =0.9104) [74]. Zhang et al reported a generic automated LC-MS/MS method for the pharmacokinetic study of a mAb in cynomolgus monkey with comparable data by the immunoassay data as well [75].…”

Ultra‐performance liquid chromatography/multiple reaction monitoring mass spectrometry quantification of trastuzumab in human serum by selective monitoring of a specific peptide marker from the antibody complementarity‐determining regions

“…Although for the trastuzumab deamidation monitoring a peptide marker included in the CDR is used, the signature peptide proposed for trastuzumab quantification in the same work is located in the Fab heavy chain region outside the CDR. In addition, the development of a generic method for quantifying mAbs in animal plasma by LC/TOF‐MS/MS has been reported . However, since the specific signature peptides chosen for trastuzumab determination in rat plasma are located within the Fc and are also present in human immunoglobulins, this methodology is not amenable to the quantification of trastuzumab in human plasma and/or serum.…”