(M1230-11-64) Implementation of Solid Phase Extraction Acid Dissociation (SPEAD) Methodology in Reconciling Drug Tolerance Demands of Immunogenicity

Purpose: Humanized monoclonal antibodies have seen a rise in usage in the pharmaceutical industry and has integral part in the targeted therapy approach. One of the many complex facets of this approach is in testing for any potential in vivo immunological response. Although these antibodies offer a high specificity, there remains some safety concerns that must be considered. The immunogenicity of anti-drug antibody (ADA) response requires testing to make sure the therapeutic is safe. High dosages of such drugs can induce an immune response which can interfere with efficacy of the monoclonal antibody drug. Common methods such direct, bridging, and sandwich ligand binding assay offer simple methodology to such an approach, but can have potential short comings when analyzing drug tolerance. Somewhat more complex approaches can thus be utilized to help satisfy the demand, such as Solid Phase Extraction Acid Dissociation (SPEAD) assays and Affinity Capture Elution (ACE) assays. The focus of this current experiment is on the utilization of SPEAD to improve the ability to detect drug tolerance in the 400-500 ug range of free circulatory drug. In using this method to satisfy all drug tolerance demands and bolster the assay, special care must be taken to ensure no other necessary criteria, such as selectivity or sensitivity is negatively impacted and the amount of non-specific bonding potential in early steps of the method kept to a minimum.

Methods: Solid Phase Extraction Acid Dissociation is a two-day, multi-step assay. The initial day involves diluting the samples in a biotin-conjugated solution. A low concentration of a biotin-conjugated antibody solution is prepared by spiking the biotin-conjugated drug into the assay buffer. From there samples are diluted in the prepared low concentration solution and incubated overnight in a refrigerated environment. The following day these samples were placed ona shaker briefly then transferred to a Pierce High-Binding Capacity (HBC) Streptavidin coated plate, followed by incubation.  The HBC plate is then washed three times using a standard wash buffer. Acetic acid is then added to the washed HBC plate and allowed to incubate on a shaker for 30-60 minutes at room temperature. The acid dissociated samples are then transferred to a fresh plate and diluted with the conjugated antibody mixture, treated with base to neutralize the acid, and incubated for one hour. This final solution is then transferred to a blocked Streptavidin coated MSD plate and incubated for one more hour. The final step in the process involves washing the sample coated MSD plate, adding the MSD read buffer and reading the plate on the MSD Meso Sector plate reader

Results: While the traditional bridging method for this assay were initially successful in meeting the required assay parameters, it was summarily unable to meet the required demands of drug tolerance. Multiple acids, buffers, concentrations, sample dilutions and plate variations were tested, all of which results in no discernable drug tolerance of free drug in matrix at a positive control of 250 ng/mL. Implementing the SPEAD method yielded immediate results. At a low concentration of biotin pre-loading on Day 1, the drug tolerance was able to be acquired at a range between 400-500 ug/mL of free circulatory drug at a positive control of 250 ng/mL. Further testing shows that the SPEAD method had a similar sensitivity of the initial bridging method of 12.4 ng/mL, no exhibition of the prozone effect at high PC concentrations and passed all subsequent testing criteria. The initial success was reached using the same acid, bases and buffer that are commonly found in traditional bridging methods.

Conclusion: Utilization of the SPEAD method improved drug tolerance four-to-five-fold compared to traditional bridging assay. Although optimization of traditional bridging method achieved most assay validation requirements, the required drug tolerance level could not be met. The final drug tolerance level of 400-500 ug/mL of free drug at a positive control level of 250 ng/mL was achieved using traditional assay buffer and acid. Subsequent usage of this SPEAD method can theoretically be utilized to overcome similar hurdles for other humanized monoclonal antibody assays.

Graphic of ADA complex with free drug interference



Step wise SPEAD format with graphic and optimal condiitions



SPEAD results vs traditional bridging method