A Universal Dual Peptide Assay for Antibody-Based Drug Candidates in Animal PK Studies Using a Full-Length Stable Isotope Labeled Internal Standard

By: Kevin Ray and Pegah R. Jalili, Sigma-Aldrich, St. Louis, MO

Abstract

There is a growing demand for reliable LC–MS/MS assays to support the bioanalysis of a human monoclonal antibody-based drug candidates in preclinical studies. Traditional LC-MS/MS assays rely on analyte specific stable isotope labeled (SIL) peptides that provide poor absolute quantitation due to late introduction of the internal standard peptide. We have developed a stable isotope labeled human IgG1 kappa monoclonal antibody standard which may be introduced early in the analytical workflow. This full-length internal standard enables a universal dual peptide approach because of its overlap with conserved sequences on both the Fc and kappa light chain regions of candidate antibodies. We demonstrate here that the utilization of immunoaffinity enrichment, rapid tryptic digestion, microflow LC/MS/MS, and the SILuMab-Kappa internal standard in a dual peptide assay allows significant improvements in throughput, sensitivity, accuracy, and reproducibility when assessing the pharmacokinetic properties of antibody-based drug candidates in preclinical studies.

Methods

SILuMab-Kappa was expressed in CHO cells which were grown in serum-free 13C615N4 Arg / 13C615N2 Lys enriched media.

The SILuMab-Kappa was analyzed at the intact protein level and after trypsin digestion. Intact mass analysis (SEC-MS) was used to confirm the amino acid composition of the protein and level of glycosylation. The sequence and isotope incorporation were determined at the peptide level after trypsin digestion.

For immunoaffinity enrichment and quantification, samples were prepared by spiking 5 μg/mL of SILuMab-Kappa as an IS into cynomonkey plasma containing 0.1 - 10 μg/mL of Humira (adalimumab). Samples were enriched using cross adsorbed goat anti-human IgG biotin conjugate antibody bound to magnetic beads co-immobilized with trypsin. The enriched samples were digested at 70°C for 2 hours and peptides were separated on a Supelco BIOshell A160 Peptide C18, 2.7 um fused core particle column; 10 cm x 500 μm. Detection was performed in MRM mode on an AB Sciex QTRAP 5500 system.

Table 1. Tryptic peptides liberated from conserved regions of heavy chain and light chain of SILuMab-Kappa
 

Universal Peptide Sequence Location
SGTASVVCLLNNFYPR Light Chain (kappa)
VDNALQSGNSQESVTEQDSK Light Chain (kappa)
DSTYSLSSTLTLSK Light Chain (kappa)
FNWYVDGVEVHNAK Heavy Chain (IgG1)
VVSVLTVLHQDWLNGK Heavy Chain (IgG1, IgG3, IgG4)
GFYPSDIAVEWESNGQPENNYK Heavy Chain (IgG1, IgG4)

Workflow

Workflow for quantitation of antibody comprised of surrogate peptide selection, immunoaffinity enrichment, rapid tryptic digestion, and LC-MRM analysis.

Figure 1. Workflow for quantitation of antibody comprised of surrogate peptide selection, immunoaffinity enrichment, rapid tryptic digestion, and LC-MRM analysis.

Results

UV trace and deconvoluted mass spectra resulting from intact LCUV-MS analysis of the SILuMab-Kappa standard

Figure 2. UV trace and deconvoluted mass spectra resulting from intact LCUV-MS analysis of the SILuMab-Kappa standard.

 

Incorporation of 13C615N2 labeled Lysine for a selected peptide of SILuMab-Kappa. Incorporation was demonstrated to be greater than 99%

Figure 3. Incorporation of 13C615N2 labeled Lysine for a selected peptide of SILuMab-Kappa. Incorporation was demonstrated to be greater than 99%.

 

Extracted ion chromatogram (XIC) of SILUMab-Kappa peptides

Figure 4. Extracted ion chromatogram (XIC) of SILUMab-Kappa peptides.

 

Calibration curves of three representative SILUMab-Kappa peptides obtained from immunoaffinity enriched samples

Figure 5. Calibration curves of three representative SILUMab-Kappa peptides obtained from immunoaffinity enriched samples.

 

A lower limit of quantitation (LLOQ) of 0.1 μg/mL was obtained, with accuracy of 85% -115% and CV of < 15%

Figure 6. A lower limit of quantitation (LLOQ) of 0.1 μg/mL was obtained, with accuracy of 85% -115% and CV of < 15%.

Summary

  • Stable isotope labeled full-length IgG1-Kappa antibody has been produced with high purity and isotopic incorporation > 99%
  • SILuMab-Kappa can be used as an universal internal standard in a dual peptide assay of therapeutic antibodies in a wide variety of pre-clinical species, providing more confidence in quantitative data obtained for PK study samples
  • A lower limit of quantitation of 0.1 μg/mL was obtained using immunoaffinity enrichment and rapid tryptic digestion

Materials

     
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