(M1030-06-31) Utilizing LC-MS to Support of PABAD-Based Detection of Neutralizing Antibodies

Purpose: Detecting neutralizing antibodies (NAbs) effectively is crucial because they can reduce drug effectiveness, interfere with drug binding to their target, and potentially lead to serious adverse effects. It's particularly challenging in conditions like cancer, where high drug concentrations in patients can complicate detection by conventional methods. To address this, our team developed a novel approach called PABAD (PEG precipitation, Acid dissociation and Biotin-drug as an Assay Drug). This method offers several advantages over traditional industry methods. It eliminates the need for cumbersome magnetic beads, reduces acid incubation steps from two to one, and minimizes the use of Biotin-drug. Importantly, PABAD is capable of handling high drug concentrations in patient samples, up to 400 µg/mL. In the PABAD method, polyethylene glycol (PEG) is used to selectively precipitate NAbs bound to the drug, effectively excluding them from the solvent. This innovation enhances our ability to accurately detect NAbs and mitigate the interference caused by high drug levels, thereby improving the reliability of our assays. This project focused on exploring why some NAbs positive controls (PC) work better with the PABAD method than others. We used LC-MS to fine-tune the method, investigate the PEG precipitation process, and measure how well the method recovered drug and PCs in the PEG-precipitated pellet. Our aim was to improve our ability to detect a wider variety of NAbs from patient samples in conditions where drug levels are high.

Methods: In a competitive ligand binding assay format, we evaluated the inhibition differences of two potential PC NAbs using the PABAD method. Optimization of the PABAD method involved screening for NAbs, testing different acid dissociation conditions, and determining the optimal percentages of PEG-NaCl for precipitation. LC/MS was used to directly measure the recovery of NAbs, drugs, and serum IgGs after PEG precipitation across the tested conditions. Immunoaffinity enrichments followed by LC-MS analysis with isotype controls was used to selectively semi-quantify the amounts of PC, drug and non-specific IgG carryover in the pellet.

Results: Among the potential neutralizing antibodies tested as positive controls, two showed effective inhibition of the drug, sharing similar binding kinetics and humanized backbones. Both were stable under acidic conditions and effectively separated the positive control (PC) and drug complex upon treatment. However, PC 1 was not compatible with PEG precipitation, whereas PC 2 exhibited robust tolerance and sensitivity to the drug. LC/MS assays were developed to better understand the recovery of PC, drug, and IgG carryover after PEG-NaCl precipitation, particularly focusing on why PC 1 was incompatible with the PABAD assay. Using unique signature peptides targeting PC isotypes and the drug's complementarity-determining region (CDR), LC/MS confirmed the NAb assay results, showing robust recovery of PC 2 compared to PC 1. The LC/MS data also illustrated the precipitation of free drug and serum IgG, dependent on the percentage of PEG-NaCl, with optimal recovery of the PC and drug complex observed at 5% PEG-NaCl. Competitive ligand binding assay data validated that the interaction between PC and the drug, even without PEG, influenced the measured percent inhibition differences between these NAbs. Additionally, reducing the drug levels for PC 1 enhanced recovery after PEG precipitation, suggesting that further removal of free drug could improve the recovery of this particular PC.

Conclusion: We are actively refining PABAD conditions to enhance the precipitation of PC 1 and other NAbs that do not work well with PEG. Our focus is on identifying the specific characteristics of these NAbs that affect their precipitation efficiency. By expanding our understanding and identifying more NAbs, we aim to cover a broader spectrum in patient samples, thereby improving the detection and reporting of ADA (anti-drug antibody) responses.

Acknowledgements: The authors declare no conflicts of interest.