(W1130-04-23) Implementation of a Semi-Automated Bead Extraction Procedure during Analyte Purification for High Throughput Immunogenicity Assays

Purpose: Therapeutic protein products have the potential of inducing immune responses that could affect both patient safety and product efficacy. Thus, monitoring anti-drug antibody responses in patients receiving protein therapeutic treatment, for drugs similar to endogenous human proteins, is an important safety assessment for regulatory agencies, drug manufacturers and clinicians. We developed an immunogenicity assay to support a clinical immunogenicity program for a recombinant protease inhibitor. Pre-treatment approaches such as solid phase extraction with acid dissociation (SPEAD), have evolved during the decade and have proven useful in improving drug tolerance and overcoming matrix interference factors from endogenous protein counterparts. The adoption of magnetic beads for analyte purification in immunogenicity assays is typically infrequent. This is primarily attributed to the time-consuming and labor-intensive procedures involved, which not only extend the assay durations but also present potential risks to the health and safety of personnel. To address these challenges and emphasize safety, we optimized a semi-automated biotin-drug bead extraction, acid dissociation (BEAD) procedure coupled with Protein AGL capture for ADA purification before detection in an ECL direct assay format.

Methods: Aiming to automate and optimize the antibody purifications steps of the BEAD Protein AGL method, we implemented the KingFisher Magnetic Particle processor platform. To detail, two steps of acid dissociation and excess biotin-drug were used to extract ADA from serum samples. The initial acid treatment was to free ADA from all non-specific and specific binding partners, such as drug and endogenous drug counterpart. Next, the free ADAs were extracted by binding to excess biotin drug on streptavidin-coated magnetic beads. After separation of biotin-drug ADA complexes on the beads, the KingFisher Flex was used for washing, and acid dissociation was applied to release ADA from the beads, which were then captured on a Protein AGL coated plate for detection using ruthenium labelled drug. Typically, immunoaffinity procedures are manual and result in lower throughput and reproducibility. We adapted, modified, and optimized the BEAD-AGL method for higher throughput and robustness by incorporating semi-automation streptavidin-biotin bead based immunoaffinity washing and elution steps on the Thermo Scientific KingFisher Flex platform. The automation of these steps was focused on fine-tuning essential parameters such as mixing speed, time, and the collection of magnetic beads. This optimization aimed to minimize bead loss during both washing and elution cycles providing additional control over those critical steps. After optimizing the parameters on the KingFisher Flex instrument, we proceeded to conduct a direct comparison between manual execution and the utilization of the KingFisher Flex platform for the immunoaffinity purification step. The evaluation covered cut-point assessment, as well as inter and intra-assay precision evaluations, establishing a thorough understanding of comparability in performance.

Results: The screening, confirmatory, and titration cut points closely align with those achieved through manual execution of the immunoaffinity elution steps and on the Thermo Scientific KingFisher Flex platform. The use of the KingFisher resulted in lower cut-points (SCP: 1.11, CCP: 29.3% and TCP: 1.28) in comparison to the manual extraction (SCP:1.25, CCP: 46.6% and TCP: 1.62) from the beads, showcasing reduced variability among all data points used for analysis. Specifically, the standard deviation for signals across all individual lots evaluated on the KingFisher Flex platform was 9%, as opposed to 18% with manual extraction. This result emphasizes the enhanced reproducibility achieved when utilizing the KingFisher Flex platform. Automating the bead washing and elution steps benefited in reducing the overall assay time by minimally an hour per operator when processing multiple plates. Which further highlights the potential of automation resulting in shorter assay turnaround times and health and safety benefits. A comparison was also conducted regarding the inter- and intra assay precision of the screening and confirmatory assays processed both manually and through the Kingfisher Flex instrument platform, revealing a percent difference within 15% for controls and comparable inter and intra assay variability where both approaches present precision below 20%. The results are detailed in Table 1.

Conclusion: Magnetic bead-based immunogenicity assays commonly encounter challenges related to robustness, user-friendliness, and prolonged assay durations, leading to inconsistent results. To overcome these limitations, we have optimized and validated a semi-automated BEAD Protein AGL assay that not only demonstrates robustness, sensitivity, and high-throughput capabilities but also ensures reproducibility through the utilization of the Thermo Scientific KingFisher Flex platform. Notably, the health and safety benefits of this platform contribute to the method's overall efficiency and high throughput. The achieved robustness, reproducibility, and enhanced throughput collectively result in shorter turnaround times and positively impacting health and safety practices during the analysis of large number of samples during long term clinical studies.

Figure 1

Table 1