(M0930-06-39) Enhanced Pharmacokinetic Bioanalysis of Antibody-Drug Conjugates Using Hybrid Immunoaffinity Capture and Microflow LC-MS/MS

Purpose: In theory, antibody-drug conjugates (ADCs) are an elegant solution to the challenges that arise from more traditional chemotherapies (i.e. tumor targeting with less toxicity). However, significantly larger therapeutic indices have yet to be realized, and therefore better understanding of ADCs at the preclinical stage of development is critical to future success in the clinic. Moreover, as the field of ADCs moves toward more intricate and diverse designs, there is also a consequent need for advanced bioanalytical support. Thus, in order to assess in vivo behavior of ADCs, a hybrid immunoaffinity capture and microflow LC-MS/MS (hybrid µLC-MS/MS) approach was developed and applied to a preclinical, pharmacokinetic (PK) murine study. The hybrid >µLC-MS/MS workflow was optimized, and data generated from this workflow were compared to those obtained from traditional ligand binding assays (LBA). Assessments of data quality included analytical figures of merits (i.e. linearity, sensitivity, robustness, etc.) as well as which types of data were available (i.e. measurements of antibody, ADC, payload, etc.). Additionally, other factors were considered from a more holistic approach, such as amount of sample needed per technique and throughput.

Methods: Samples were collected from a PK study conducted in CB17 severe combined immunodeficiency (SCID) mice, which was approved by AstraZeneca’s Institutional Animal Care and Use committee. Mice were bolus IV dosed with 5 mg/kg of ADC and plasma samples were collected at 1, 6, 24, 72, 168, 240, 336, and 504 hours post-injection. Samples were flash-frozen and stored at -80 °C until downstream analysis. Downstream analysis consisted of either in-house ELISA or the hybrid µLC-MS/MS workflow. Briefly, for the ELISA assay, affinity-purified anti-human IgG antibody was coated on a 96-well plate. Following a 1:10 MRD, standards, QCs, and samples were added to the plate and incubated for 1 hr at RT. Wells were then washed and the detection antibody, HRP-labeled goat-anti-human IgG, was added to the plate. After incubation for 1 hr at RT, the plate was washed and tetramethylbenzidine (TMB) was added to each well. The reaction was quenched with 0.2N H₂SO₄ and the plate was read at 450 nm via an absorbance microplate reader. For the hybrid µLC-MS/MS analysis, SMART Digest IA streptavidin magnetic beads were incubated with CaptureSelect Human IgG-FC biotin conjugate for 1 hr at RT. Diluted standards, QCs, and samples were then added to a well plate with the coated magnetic beads and internal standard. Sample capture was conducted for 1.5 hr at RT with shaking, and subsequently the plate was washed using a Thermo Fisher KingFisher Apex with beads being transferred to a plate containing digestion buffer. Samples were digested for 2 hr at 70 °C and the magnetic beads were removed post-digestion. The samples were then split for further ADC analysis as well as for further cleanup by solid-phase extraction. µLC-MS/MS analyses were conducted using an Eksigent M5 micro LC system coupled with a Sciex 6500+ QTrap mass spectrometer.

Results: The hybrid µLC-MS/MS workflow produced linear calibration curves over the range of 1 to 5000 ng/mL of antibody measurement and 0.5 to 2000 ng/mL of ADC measurement, both with high correlation (r² > 0.99). No matrix interference was found for either measurement and retention time shift was within acceptable range (CV < 0.6%). Using small sample volumes (1 µL of plasma), the workflow was successfully applied to a preclinical murine PK study, in which a proprietary DAR 8 ADC was dosed. Total antibody as well as total ADC plasma concentrations were quantitated through successive measurements from the hybrid µLC-MS/MS approach. The total antibody plasma exposure was correlated with data obtained from an ELISA assay using the same set of samples and it was found that there was high agreement between the two techniques (R² = 0.970). Additionally, throughput for the ELISA was much greater than the hybrid µLC-MS/MS workflow; however, the hybrid µLC-MS/MS workflow enabled measurements of additional metrics (i.e. total ADC, free payload, etc.) that would otherwise require specialized reagents for an ELISA.

Conclusion: With increasingly intricate ADCs that include lower-dosed, highly cytotoxic payloads, multiple cytotoxic payloads, and more, there is greater emphasis for sensitive and information-rich bioanalytical methodologies. Herein, a cost-effective hybrid µLC-MS/MS workflow accurately measures total antibody and total ADC using only 1 µL of mouse plasma. Moreover, this approach provides reliable plasma exposure concentrations when compared to traditional ELISA approaches, but also offers additional data when compared to such methods.

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General workflow for quantitative analysis of antibody–drug conjugates (ADCs) by the implementation of solid-phase extraction in micro-flow liquid chromatography – tandem mass spectrometry (µLC-MS/MS). ADC with a DAR of 8 was conjugated stochastically at all potential interchain cysteines.

Comparison of total antibody concentration analyzed by ELISA and µLC-MS.

Correlation plots of total antibody between ELISA and µLC-MS/MS