(M1230-09-50) Development of a Bridging ECL Method for the Detection of Anti-Drug Antibodies in Human Serum on an Automated System

Purpose: Evaluation of immunogenicity at various stages of clinical development is an essential portion of the drug development process. The first component of immunogenicity testing is detection of treatment-induced anti-drug antibodies (ADAs) using screening, specificity confirmation and titration assays.  Automated systems can be utilized to perform assays with many benefits including reduction in variability associated with pipetting and human errors, increased compliance through contemporaneous recording of all movements, and potential for increased throughput. The data presented details the incorporation of a Hamilton MicroLab Vantage (Vantage) instrument into an ADA assay traditionally completed at the bench.

Methods: A bridging ECL assay was established to assess immunogenicity of a monoclonal antibody on both the Vantage and the benchtop. The Vantage is capable of positive sample tracking, so all transfers from source to destination are captured in instrument trace logs, which can be verified against the program to show how all transfers are completed in line with “attributable, legible, contemporaneous, original and accurate” (ALCOA) data integrity principles. The Vantage method initially matches barcodes of samples to the barcode number in the LIMS worklist, and if all samples match the worklist the method is executed by Vantage using three workflows in parallel. The first workflow adds blocking buffer to the assay plate and incubates the plate with shaking. During the blocking of the assay plate, samples are diluted to the minimal required dilution (MRD) and incubated with acid dissociation solution in the second workflow. After the incubation, neutralizing and conjugate solutions are combined in a conjugate plate and the diluted samples are subsequently transferred to the conjugate plate for incubation with shaking. Towards the end of the conjugate plate incubation, the third workflow begins with removal and wash of the blocked plate from the incubator and finishes with the transfer of the samples from the conjugate plate to the blocked assay plate. The assay plate is returned to the deck after all samples have been added to all plates. Multiple parameters were evaluated to accommodate automation including incubation times and conjugate preparation. Cut point determination, sensitivity, precision, drug tolerance, and robustness tests were performed using manual and automated methods to demonstrate similarity.

Results: The automated method was successfully developed and evaluated. Screening and confirmatory cut points were established for both a healthy and a diseased population using combined data from the automated method and the benchtop. Sensitivity, drug tolerance, and precision were determined to be comparable between the two methods. Robustness was thoroughly demonstrated for both methods at or beyond the anticipated timings of each workflow.  The bridging ECL method for detection of ADAs in human serum on the automated platform was determined to be fit for purpose.

Conclusion: Automated methods are a viable platform to develop immunogenicity assays with the ability to eliminate human errors that may occur at the benchtop, increase through put, and increase compliance. Similar performance between automated and benchtop methods suggests that the methods can be used interchangeably depending on time and resources available for a project. Automation should be considered early in method development in order to design the appropriate workflow to accommodate automation. These findings demonstrate that immunogenicity assays can be successfully performed using automated systems.