(W1430-02-06) Application of a Novel Affinity Purified Chicken IgY in an Immunoaffinity LC-MS/MS Assay for Rapid Quantitation of Monoclonal Antibody Therapeutics and ADCs in Nonclinical Species

Purpose: Today monoclonal antibody-based therapeutics (mAbs) serve as important targeted treatment modalities across numerous therapeutic areas.  Thus, to facilitate development of new agents there is a need to increase the quality and throughput of bioanalysis to expedite assessments of nonclinical PK prior to initiating Phase I evaluation in humans.  Accordingly, we have adapted an affinity purified chicken IgY with pan-specificity for human IgG subclasses which displays negligible cross-reactivity to IgG from common nonclinical species, e.g. cynomolgus monkey, for use in an immunoaffinity LC-MS/MS assay workflow to establish a bioanalytical method for rapid quantitation of monoclonal antibody-based therapeutics, including antibody drug conjugates (ADCs).

Methods: The immunoaffinity capture is performed by combining the following components in a protein LoBind Eppendorf tube and incubating for at least 2 hours at RT with gentle mixing: PBS pH 7.4 containing 0.2% BSA + 3% PEG (8kDa mol. wt.) binding buffer, a suspension of high-capacity streptavidin coated magnetic beads (~ 1 mg) with immobilized biotinylated affinity purified chicken IgY (~ 0.025 mg), and cynomolgus monkey serum or diluted standards (e.g. 10-100 µL) containing the candidate mAb therapeutic. After incubation the beads are washed 3 times with a high salt buffer (20 mM Tris + 750 mM NaCl + 0.1% Tween-20 pH 7.4) to remove albumin and unwanted serum proteins. The final bead wash is performed using 20 mM Tris + 150 mM NaCl + 0.02% NaN₃ pH 7.4. The washed beads are stored refrigerated in this same buffer prior to LC-MS/MS evaluation. Following capture of Adalimumab (model mAb), on-bead digestion is performed using Trypsin/LysC to digest the captured human IgG1 into signature peptides to be analyzed by LC-MS/MS. Two signature peptides (VVSVLTVLHQSWLNGK and DSTYSLSSTLTLSK) are followed with MRM transitions 603.7/805.4 and 752.0 to 836.0, respectively. A stable isotope labeled version of Adalimumab was used as the internal standard (IS), which produced heavy labeled signature peptides that aligned with the peptides used for quantitation. MRM transitions of 606.3/809.4 and 756.0/844 were used to monitor the IS. Quantitation was performed using peak area ratios. A 6500+ Sciex mass spectrometer was used coupled with a 30 series UPLC and autosampler from Shimadzu. Chromatographic separation of the peptides was performed using a Phenomenex Kinetex Biphenyl (2.1x30 mm, 2.6 µm). The column was eluted using a gradient mobile phase mixture of acetonitrile/water and 0.1% formic acid at a flow rate of 0.5 mL/min. Signature peptides were quantitated down to a lower limit of quantitation of 0.2 µg/mL.

Results: Following affinity purification, the purity and reactivity of the chicken IgY reagent were assessed (Figure 1). The titer assessment against both human IgG and cynomolgus IgG indicated that the reagent was highly specific for human IgG (10.4 ng/mL EC₅₀) vs. cynomolgus IgG (3490 ng/mL EC₅₀, ~ 0.3% residual cross-reactivity). Purity analysis by SDS-PAGE showed a single band at approximately 180 kDa which is consistent with chicken IgY molecular weight and demonstrating high purity. Signature peptides monitored were linear and reproducible and appropriate for accurate quantitation of the intact mAb down to 0.2 µg/mL.

Conclusion: The new hybrid LC-MS/MS assay will permit rapid and sensitive quantitation of human mAbs in serum from sera of nonclinical species, including cynomolgus monkey. Moreover, the broad specificity of the chicken antiserum for human IgG subclasses will allow this assay to function in a plug-and-play manner with minimal optimization for efficient support of nonclinical PK of different monoclonal-based therapeutics and ADCs across nonclinical species. This novel workflow will drastically increase the lead time for bioanalysis since the LC-MS/MS workflow can be applied to all human IgG based therapeutics.

(A) ELISA cross-reactivity assessment to human and cynomolgus IgG and (B) purity of the reagent chicken IgY by SDS-PAGE.

LC-MS/MS chromatogram of VVSVLTVLHQSWLNGK (left panel) at 0.2 µg/mL and SIL-VVSVLTVLHQSWLNGK (right panel) at 20 µg/mL