Track: Bioanalytics - Biomolecular - Bioanalytical Innovations and Applications
Category: Late Breaking Poster Abstract
Efficient Optimization of an Affinity Capture Elution Procedure for Anti-Drug Antibody Assays by Applying a Design of Experiments Approach on the Gyrolab™
Purpose: Anti-drug antibody (ADA) assays often require the use of acid treatment and bead-based capture procedures such as affinity capture elution (ACE) to enrich ADA from complex serum matrix and improve drug tolerance. These methods take time to optimize on plate-based ligand-binding assay (LBA) platforms due to relatively long assay procedures, and acid can denature binding interactants which can negatively impact future binding events in downstream assay steps. The Gyrolab™ immunoassay platform employs microfluidic systems that promote fast assay run times and is completely automated. Using the speed and automation of Gyrolab in combination with a design of experiments (DoE) approach to improve ACE conditions, we quickly optimized ADA enrichment and recovery from serum samples containing high levels of MAb drug. We also estimated an apparent ADA concentration using a positive control (PC) anti-idiotype antibody as a surrogate standard to aid in optimizing conditions for improved recovery of spiked PC in serum matrix. The optimized ACE method may be applied to enrich ADA in clinical samples, and subsequently, samples may be analyzed on any LBA platform. Methods: A bridging ADA assay was developed on Gyrolab using a MAb PC, and a 4-factor, 2-level screening factorial design was used to optimize the ACE procedure, with a total of 16 conditions tested. Samples were initially treated with acid to dissociate bound ADA from drug, and excess biotinylated drug-bound beads were used to extract ADA. ADA bound to beads were eluted using an additional acid step, and enriched ADA stored in a suitable buffer. DoE factors and levels assessed included: bead volume (30 µL vs 60 µL), incubation time with drug-bound beads (2 hr. vs overnight), neutralization timing (before or after bead addition), and storage buffer for eluted ADA (buffer A vs B) where buffer B contained a stabilizing protein. The responses measured to determine success across the various conditions were (1) PC recovery after ACE procedure and (2) drug tolerance. To assess recovery of the spiked PC across the conditions tested, the PC itself was used as a surrogate “standard” to calculate recovered concentration after ACE procedure and compared to “no ACE” control. In addition, 100 µg/mL of spiked drug was used to assess drug tolerance by comparing to no drug control. The drug tolerance response was calculated by taking the ratio of the apparent concentration of the PC with and without 100 µg/mL drug spike for each of the different conditions. Results: The response data was analyzed using Design Expert® software (Stat-Ease, MN) with the goal of maximizing both the PC recovery and drug tolerance ratio. For the PC recovery response without drug spike, all the factors had a significant impact with drug-bound bead volume having the greatest effect followed by storage buffer, incubation time and neutralization timing. For the PC recovery response with drug spiked, the factors that impacted PC recovery in the presence of drug were (in order of magnitude of effect) incubation time, bead volume and storage buffer. The drug tolerance response showed incubation time having a substantial impact, and no other single factors having a significant effect. For all responses, a small but significant interaction between bead volume and incubation time was noted. Using the modeling power of DoE, the final optimized ACE condition which achieved the highest overall PC recovery and drug tolerance was using a combination of higher bead volume (60 µL), overnight incubation of sample with beads, neutralization after bead addition and store the enriched ADA in buffer B (with stabilizing proteins) for storage at -70⁰C. With DoE optimization, the recovery of MAb PC using the ACE procedure was improved from 5% to about 30% and drug tolerance was successfully observed at 100 µg/mL. Conclusion: Using a combination of DoE and Gyrolab technology, we quickly optimized a complex ACE procedure for enriching ADA and significantly improved serum recovery of a high affinity PC control and achieved acceptable drug tolerance at 100 µg/mL. The enriched ADA was stored at -70⁰C in an improved buffer formulation and could be subsequently tested in an ADA assay developed on any LBA platform. Our unique strategy to use the PC control in the Gyrolab “tool” ADA assay as a surrogate standard enabled the estimation of PC recovery across the different ACE conditions in the DoE. The ability to attain increased recovery of ADA, both low and high affinity antibodies, and achieve high drug tolerance are critical to enable detection of clinical ADA in patient samples.