Purpose: Immunogenicity assessment of biotherapeutics is monitored in most preclinical and clinical studies via the detection and characterization of anti-drug antibodies (ADA) with a three-tiered strategy involving screening, confirmation, and semi-quantitative titer assessment. Various analytical methods have been used to detect or monitor ADAs including ELISA, surface Plasmon resonance, and electrochemiluminescence-based platforms. The bridging method has become the preferred assay as it can detect all isotypes of ADAs and usually has high drug tolerance. However, it requires preparation and labeling of unique capture and detection reagents specific for each drug, thus could be laborious and resource intensive and may not be suitable for preclinical ADA testing where multiple lead candidates are typically evaluated. Here we describe the development and implementation of a novel and fast approach to detect preclinical ADA against therapeutic proteins and monoclonal antibodies with Capillary Electrophoresis/immunodetection using the “Peggy Sue” system from ProteinSimple.
Methods: Peggy Sue is a capillary-based western/immunoassay platform that can separate proteins by size or charge. We adopted this system to ADA testing by separating a drug of interest in the capillary by charge (pI) followed by immobilization to the capillary wall via a proprietary, photoactivated capture chemistry. When ADA containing samples are passed through the capillary, ADAs are expected to bind to the immobilized drug and can be detected using a HRP-conjugated secondary antibody resulting in a chemiluminescent signal that can be detected and quantitated.
Results: As a proof of concept we tested a positive control mAb against an engineered protein and found the method to be specific to the immobilized drug. Next a set of monkey serum samples that have been dosed with this drug and tested as ADA positive by a bridging assay was tested on Peggy Sue using anti-monkey IgG-HRP as detection. The result shows good correlation between these two methods: all day 14 ADA positive samples tested by the bridging method were also tested positive by Peggy Sue. The Peggy Sue method used only 5-10 ul of samples and is fully automated with sensitivity comparable to the bridging method. It can also be used to detect ADA against mAb drugs, thus having a broad application. In addition to charge separation, Peggy Sue can also be used for size separation. However, since size separation requires denaturing a protein, this may not be ideal to detect ADA against native form of a drug.
Conclusion: The main advantages of the Peggy Sue ADA assay are: 1) no requirement for preparation and labeling of capture and detection reagents, thus making the approach ideal at the preclinical stage when multiple lead candidates are evaluated, allowing for rapid analysis without the need for tailored assay optimization; 2) reduced sample volume and automation; 3) valuable charge/size characterization of the immunogenic agent and is ideal for further characterization of ADA specificity against complex biologics such as bispecific or multi-specific biotherapeutics where individual modules/domains can be separated and tested easily.
Shuli Zhang– Principal Scientist, Merck Research Laboratories