(M1130-06-39) PABE: A Novel Enhanced Drug Tolerant Assay Format to Detect Anti-Drug Antibodies in the Presence of High Concentrations of rhAAT-Fc

Purpose: Detection of anti-drug antibodies (ADA) plays a key role for safety and efficacy assessment of biotherapeutics. Biotherapeutic drugs can be administered at high doses, potentially resulting in circulating therapeutic drug concentrations within mg/mL range. Therefore, developing robust ADA assays with high drug tolerance is critical. The therapeutic drug is a recombinant human Alpha-1 Antitrypsin-Fc fusion protein (rhAAT-Fc) being investigated as an augmentation therapy in patients with Alpha-1 Antitrypsin Deficiency (AATD). In clinical studies, ADA samples taken at steady state trough are expected to have ≥ 1.5 mg/mL of circulating rhAAT-Fc in addition to endogenous AAT, so development of an ADA method which could meet the needs of high drug tolerance for this program was pursued. PABE (Polyethylene glycol (PEG) Precipitation, Acid dissociation (AD), Bridging Electrochemiluminescent (ECL)) is a novel Anti-Drug Antibody assay format, developed at Company A and validated at Company B. This assay detects ADA (Free ADA, endogenous counterpart or Alpha-1-Antitrypsin [AAT]-bound ADA, and therapeutic drug bound ADA) in human serum samples. Using a combination of a bridging Master Mix format with PEG precipitation and acid dissociation resulted in a robust assay with a drastically improved drug tolerance (≥5mg/mL rhAAT-Fc) and sensitivity (≤100 ng/mL). Unlike the PandA assay format, PABE is simplified in its design and execution and does not require addition of excess drug material to samples. With this novel assay format, we developed and validated a versatile ADA method that is streamlined in execution, sensitive, robust, reproducible, and tolerant to high levels of circulating therapeutic fusion protein and its corresponding endogenous counterpart.

Methods: The method includes PEG treatment (5% final concentration of PEG-8000) to precipitate immune complexes, an AD step for complex dissociation followed by a bridging ECL with dual-labeled drug to capture and detect ADA. Controls and samples are diluted with PEG solution, then incubated overnight to precipitate immune complexes. The immune complexes are then centrifuged and washed two times in 5% PEG-8000 solution. The resulting pellet is dissociated in a weak acid solution and then neutralized in the presence of Biotinylated (Bt-rhAAT-Fc) and Ruthenylated drug (Ru-rhAAT-Fc) to allow formation of bridged drug:antibody:drug complexes. The controls and samples are then transferred to a blocked, streptavidin coated MSD plate. Following incubation and washing steps, the MSD Gold Read buffer is added. The complexes are detected using an MSD S600 plate reader. The presence of ADA is determined by comparing the signal in the samples or controls to a statistically derived threshold/assay cut point. We evaluated our method using the FDA 2014 and 2019 immunogenicity guidelines. In the validation, for the drug tolerance experiment, normal human serum was utilized which typically contains greater than 1.0 mg/mL of endogenous AAT. Drug tolerance is determined at three surrogate positive control (PC) concentrations (2000 ng/mL, 500 ng/mL and 100 ng/mL). Each PC level is assessed in presence and absence of varying concentration of rhAAT-Fc which contains two Alpha1-Antitrypsin (AAT) molecules (5000 µg/mL, 2000 µg/mL, 1000 µg/mL, 100 µg/mL, 0 µg/mL).

Results: The target acceptance criteria for validation experiments were met, including intra- and inter-assay precision at the sensitivity level of the assay, cut points, sensitivity, selectivity, free drug and endogenous AAT tolerance, prozone (Hook) effect, stability, and robustness. All PC levels assessed in the presence of varying concentration of rhAAT-Fc, screened and confirmed positive, thus, they were tolerant up to 5mg/mL of rhAAT-Fc. The PC-spiked pooled normal human serum containing endogenous levels of AAT had responses above the screening and confirmatory cut points at every level of PC tested. Therefore, established rhAAT-Fc drug tolerance is in combination with normal human levels of endogenous AAT.

Conclusion: PABE combines the advantages of both the PandA and bridging assay formats. In doing so, it provides operational ease for high throughput, low non-specific background, improved sensitivity, and superior drug tolerance. Moreover, the method possibly eliminates target interferences and enables efficient detection of immunoglobulin isotypes. This method was very effective in overcoming high concentrations of rhAAT-Fc in addition to endogenous AAT. This was because the ADA was effectively isolated and detected using the PABE format regardless of how much therapeutic drug and its endogenous counterpart may be contained in the study samples. This novel PABE assay format has potential applications in ADA assays where high levels of therapeutic fusion protein and its corresponding endogenous counterpart are expected.

References: US Food and Drug Administration. (2019). Immunogenicity testing of therapeutic protein products—developing and validating assays for anti-drug antibody detection. Guidance for industry
US Food and Drug Administration. (2014). Immunogenicity assessment for therapeutic protein products. US Food and Drug Administration.
Zoghbi, J., Xu, Y., Grabert, R., Theobald, V., & Richards, S. (2015). A breakthrough novel method to resolve the drug and target interference problem in immunogenicity assays. Journal of Immunological Methods, 426, 62–69

PABE (Precipitation, Acid dissociation, Bridging Electrochemiluminescent) Assay Format

Drug Tolerance results from Validation- Assay can detect 100ng/mL of ADA in the presence of atleast 5mg/mL of therapeutic drug