(M1530-02-06) Improving Drug Tolerance in Anti-Drug Antibody (ADA) Assay using PandA Method – Case Studies

Purpose: One of the significant technical challenges in Anti-Drug Antibody (ADA) assays is achieving drug tolerance (DT). High levels of therapeutic drugs can mask ADAs, making them undetectable. This interference occurs because the drug can bind to the antibodies, forming complexes that the assay cannot detect. To overcome the drug tolerance in ADA assays, some type of sample pretreatment is needed. The pretreatment procedure could be acid dissociation, Solid Phase Extraction and Acid Dissociation (SPEAD), Affinity Capture Elution (ACE), or Precipitation and Acid Dissociation (PandA).

Methods: The ADA sample is treated with an excess of the drug to enable the formation of drug-ADA complexes. The complex is then precipitated using polyethylene glycol (PEG). Following precipitation, the pellet is washed several times with a wash solution containing PEG. At the end of the wash steps, the pellet is resuspended using an acid solution. This pretreatment helps remove excess drug, thereby improving drug tolerance. The acidified sample is directly coated onto a Meso Scale Discovery (MSD) plate and detected using a sulfotag-labeled drug as the detection reagent.

Results: The PandA method was able to achieve drug tolerance by upto100-fold compared to other pretreatment methods such as acid dissociation or SPEAD. This method was proven to be successful for different drugs, including a fusion protein, a naked antibody, and an antibody-drug conjugate. The results clearly indicate that the PandA method significantly enhances drug tolerance across various drug types. For instance, while other methods failed to achieve any drug tolerance for the fusion protein, the PandA method achieved a tolerance of 50 mg/mL. Similarly, for naked antibody in this case study, the PandA method improved drug tolerance from 50 µg/mL to 1000 µg/mL. For antibody-drug conjugate, the improvement was from 10 µg/mL to 1000 µg/mL.

Conclusion: The PandA method was instrumental in achieving the desired drug tolerance for three different drugs. Like any sample pretreatment process, the PandA method also involves the optimization of some critical steps to achieve consistent results. Overall, the PandA method proved to be consistent in improving drug tolerance, while other methods were not as successful. This consistency and effectiveness make the PandA method a valuable tool in ADA assays, ensuring that ADAs can be detected even in the presence of high levels of therapeutic drugs. In summary, the PandA method stands out as a robust and reliable approach to overcoming drug tolerance in ADA assays. Its ability to significantly enhance drug tolerance across various drug types highlights its potential for broader application in the field of immunogenicity testing. Future studies could further optimize and validate this method, potentially extending its use to other therapeutic drugs and improving the accuracy and reliability of ADA detection.

References: Zoghbi J, Xu Y, Grabert R, Theobald V, Richards S. A breakthrough novel method to resolve the drug and target interference problem in immunogenicity assays. J Immunol Methods. 2015 Nov;426:62-9. doi: 10.1016/j.jim.2015.08.002. Epub 2015 Aug 6. PMID: 26255760.

The table below summarizes the drug tolerance achieved with different methods: