(T0930-06-35) Analysis of Drug-Antibody Ratio (DAR) of ADC (M9346A-SPDB-DM4) in Rabbit Cornea and K₂EDTA Plasma Using SEC-MS

Purpose: M9346A-SPDB-DM4 is an ADC consisting of an FRα-targeting antibody, a cleavable linker and a potent tubulin-targeting agent (maytansinoid payload DM4). Each antibody molecule is conjugated to an average of three to four molecules of DM4 molecules, which acts as a potent antimitotic agent by inhibiting microtubule dynamics. DAR value determines the potency and toxicity of the ADC. Low DAR significantly limits the potency of the therapeutics and high DAR may hinder the pharmacokinetic properties, making it crucial to analyze the DAR throughout drug metabolism. This study developed a sensitive and robust assay to analyze the DAR of ADC, using M9346A-SPDB-DM4 as an ADC research tool, in rabbit cornea and plasma using size exclusion chromatography (SEC) with high resolution mass spectrometry (HRMS) detection.

Methods: Rabbit cornea samples were homogenized using a Covaris CP02 cryoPREP Dry Pulverizer. ADC in rabbit cornea homogenate and plasma were extracted by immunocapture using biotinylated goat anti-human IgG, followed by cleanup using a Kingfisher or DynaMag™-2 Magnet. ADC was eluted in 0.1% TFA in MeCN/H₂O (20:80, v/v) and injected onto an ACQUITY UPLC Protein BEH column with a mobile phase composed of FA, TFA, water, and acetonitrile. A Vanquish UHPLC coupled with Orbitrap Exploris 240 system was used to separate ADC and record the Total Ion Chromatogram (TIC). Chromatograms were deconvoluted using Thermo BioPharma Finder 2.0 software. The average DAR was calculated from the deconvoluted spectra.

Results: During method development, various chromatographic columns, including reverse phase and SEC, were assessed to optimize the separation and detection of M9346A-SPDB-DM4 components. For cornea tissue homogenization, both bead-based and bead-free methods were evaluated, with cryopulverization identified as the optimal approach for consistent matrix preparation. Our experiments confirmed that the expected masses aligned consistently with the observed masses of unconjugated antibody (DAR0) and various conjugated species (e.g., DAR1, DAR2) within the DM4 distribution profile. For the cornea assay, positive control samples were prepared by spiking the analyte into blank rabbit cornea homogenate. Using 200 µL of tissue homogenate, we achieved a sensitivity of 1.00 μg/mL, with eight rabbit corneas pooled for sample analysis. For the plasma assay, positive control samples were prepared by spiking the analyte into blank rabbit plasma. Using 200 µL of matrix, we achieved a sensitivity of 0.5 μg/mL. Cornea samples from Day 14 and plasma samples from Day 1 and Day 14 were analyzed. Plasma samples showed a higher overall DAR of 1.9 (up to DAR5 species detected) on Day 1, which decreased to 0.63 (up to DAR2 species detected) by Day 14. The DAR profiles were similar between plasma and cornea after 14 days.

Conclusion: This study successfully developed a sensitive assay to quantify M9346A-SPDB-DM4 DAR in rabbit cornea and plasma, highlighting its potential for broader application in pharmacokinetic studies and clinical research. The method can be easily adapted for other tissues or matrices.