LC-MS Method for the Quantitation of Two Monoclonal Antibodies by Multiple Signature Peptides in Monkey Serum (original) (raw)
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The AAPS Journal, 2013
We have recently developed a general liquid chromatography-tandem mass spectrometric (LC-MS/MS) method using a stable isotope-labeled (SIL) monoclonal antibody (mAb) as an internal standard (IS) for single-analyte quantification of mAb (Li et al. Anal Chem 84(3):1267-1273, 2012). The method offers an advantage over ligand binding assay in reducing the time and resources needed for bioanalytical support in preclinical stages of drug development. In this paper, we report another marked increase in assay efficiency for multi-analyte bioanalysis using unique surrogate peptides for each analyte and the strategic choice of the SIL-IS peptide. The method was qualified for the simultaneous determinations of four mAbs in rat plasma and applied to samples from discrete-and cassette-dosed rats. The pharmacokinetic parameters of the four mAbs of cassette dosing were comparable to those of discrete dosing and of enzyme-linked immunosorbent assay results. Although there may be limitations and special considerations for cassette-dosing of biologics, these results demonstrate the robust performance of the multi-analyte LC-MS/MS method allowing cassette-dosing that would ultimately reduce animal use and improve efficiency.
Analytical Chemistry, 2013
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.
Analytical Chemistry, 2008
The development of a quantification method for monoclonal antibodies in serum has been accomplished by high-performance liquid chromatography multiple reactions monitoring mass spectrometry. A human monoclonal antibody (HmAb) was used as the model protein for method development and validation. A peptide from the CDR3-region of its heavy chain was selected and used for quantifying the entire mAb. This signature peptide served as a template for the internal standard. Prior to mass spectrometric analysis approximately 50% of the total serum protein content was removed by albumin depletion. The accuracy of the method ranged between 99 and 112% in cynomolgus monkey serum. The intraassay coefficient of variation (CV) was lower than 4% at 4 µg/mL and 200 µg/mL HmAb (n) 3). The CV at 400 µg/mL corresponded to 9% (n) 3). In addition, the interassay variation was investigated in a male cynomolgus serum pool and in a female cynomolgus serum pool. The CV for the male cynomolgus pool at 4 µg/mL HmAb was 7% (n) 3). The CV obtained from the female pool was 8% (n) 3), at 4 µg/mL. The dynamic range of the method was 3 orders of magnitude. After albumin depletion of 25 µL of serum, a lowest limit of quantification of 2 µg/mL HmAb was reached in both human and cynomolgus monkey samples.
A general process for the development of peptide-based immunoassays for monoclonal antibodies
Cancer Chemotherapy and Pharmacology, 2010
Purpose Monoclonal antibodies (mAb) are an important and growing class of cancer therapeutics, but pharmacokinetic analyses have in many cases been constrained by the lack of standard and robust pharmacologic assays. The goal of this project was to develop a general method for the production of immunoassays that can measure the levels of therapeutic monoclonal antibodies in biologic samples at relevant concentrations. Methods Alemtuzumab and rituximab are monoclonal approved for the treatment of B-cell malignancies and were used as a model system. Phage-displayed peptide libraries were screened for peptide sequences recognized by alemtuzumab (anti-CD52) or rituximab (anti-CD20). Synthetic biotinylated peptides were used in enzyme-linked immunosorbent assays (ELISA). Peptides directly synthesized on polymer resin beads were used in an immunoXuorescent-based assay. Results Peptide mimetope sequences were recovered for both mAb and conWrmed by competitive staining and kinetic measurements. A peptide-based ELISA method was developed for each. The assay for rituximab had a limit of detection of 4 g/ml, and the assay for alemtuzumab had a limit of detection of 1 g/ml. Antibody-speciWc staining of peptide conjugated beads could be seen in a dose-dependent manner. Conclusion Phage-displayed peptide libraries can be a source of highly speciWc mimetopes for therapeutic mAb. The biotinylated forms of those peptides are compatible with conventional ELISA methods with sensitivities comparable to other assay methods and suYcient for pharmacological studies of those mAb given at high dose. The process outlined here can be applied to any mAb to enable improved pharmacokinetic analysis during the development and clinical use of this class of therapies.
The AAPS Journal, 2011
The predominant driver of bioanalysis in supporting drug development is the intended use of the data. Ligand-binding assays (LBA) are widely used for the analysis of protein biotherapeutics and target ligands (L) to support pharmacokinetics/pharmacodynamics (PK/PD) and safety assessments. For monoclonal antibody drugs (mAb), in particular, which non-covalently bind to L, multiple forms of mAb and L can exist in vivo, including free mAb, free L, and mono-and/or bivalent complexes of mAb and L. Given the complexity of the dynamic binding equilibrium occurring in the body after dosing and multiple sources of perturbation of the equilibrium during bioanalysis, it is clear that ex vivo quantification of the forms of interest (free, bound, or total mAb and L) may differ from the actual ones in vivo. LBA reagents and assay formats can be designed in principle to measure the total or free forms of mAb and L. However, confirmation of the forms being measured under the specified conditions can be technically challenging. The assay forms and issues must be clearly communicated and understood appropriately by all stakeholders as the program proceeds through the development process. This paper focuses on monoclonal antibody biotherapeutics and their circulatory L that are either secreted as soluble forms or shed from membrane receptors. It presents an investigation into the theoretical and practical considerations for total/free analyte assessment to increase awareness in the scientific community and offer bioanalytical approaches to provide appropriate PK/PD information required at specific phases of drug development.
Bioanalysis, 2018
Aim: The requirements for developing antibody biotherapeutics benefit from understanding the nature and relevant aspects of the entire molecule. The method presented herein employs on-line multidimensional LC–quadrupole time-of-flight (QTOF)-MS for the quantitative determination of an antibody isolated from biological samples while maintaining the intact native biologically active conformation of the antibody. Results: Following method optimization for a model antibody, an incurred biotherapeutic in cynomologus monkey was quantified in its intact top-down native conformation. A partial method validation demonstrated acceptable precision and accuracy although improved sensitivity requires further studies. Conclusion: An on-line multidimensional LC–MS approach presents a proof-of-principle example for quantifying an intact, native antibody isolated from an incurred biological sample via immunoaffinity techniques coupled with top-down QTOF LC–MS bioanalysis.