(T1430-02-09) Overcoming Selectivity Barriers of a Ligand Binding Assay

Purpose: Pharmacokinetic (PK) assay is critical for the determination of appropriate dosing regimens informing efficacy and patient safety. In support of a biotherapeutic program, we encountered a high background of signal in diseased population, which is attributed to non-specific binding (NSB) for a PK assay. The NSB from complex biological samples can obscure the signal from target analyte, generating false positive signals, leading to inaccurate quantitation of target analyte, compromising the selectivity of the analytical method; therefore, posing significant challenges. In addition, the target assay sensitivity could not be achieved due to this high background issue. Initial strategies focused on optimizing buffer conditions and utilizing various animal matrices to reduce NSB. Although these adjustments successfully lowered background levels, they did not achieve the required sensitivity. Ultimately, the transition from a conventional mouse hybridoma anti-idiotypic (anti-ID) antibody to a recombinant anti-ID capture reagent in combination with the optimized buffer conditions, was found to be the critical change needed to achieve the sensitive and selective assay.

Methods: The PK assay utilized a ligand binding assay (LBA) assay format to quantify MK-A in human serum electrochemiluminescence (ECL) immunoassay on the MSD Sector S 600 platform. This method utilized a 96-well streptavidin coated plate with biotinylated monoclonal antibody against MK-A as the capture reagent and sulfo-TAG labeled anti-human IgG4 antibody as the detection reagent. To address the selectivity challenge in the assay, we systematically evaluated various combinations of buffers and biological matrices during the dilution of critical reagents and sample preparations. Initially, commercially available buffers were employed to block and dilute selectivity samples, aiming to suppress non-specific binding. Subsequently, different buffers and animal matrices were evaluated for the dilution of capture and detector reagents. In an additional optimization step, the capture antibody, a hybridoma anti-ID, was replaced by a recombinant anti-ID.

Results: The initial approach to optimize the blocking and sample dilution conditions did not fully suppress the background signal. This suggests that some of NSB signal may be generated by specific binding to the capture and detector reagents. Notably, the addition of 10% mouse serum to the assay buffer used for capture and detector diluent significantly reduced background levels, indicating the interfering materials were blocked by the mouse serum (Figure 1). Alternative buffers and matrices tested either had no impact or exacerbated the issue. However, lowering the target lower limit of quantification (LLOQ) led to an increased failure rate of selectivity samples, which warranted the need for further optimization. Following extensive experiments aimed at minimizing assay background, the transition to a recombinant anti-ID capture reagent allowed all selectivity samples from the diseased populated to pass (Figure 2). The data collected included samples from healthy and diseased populated naive individuals, both in their natural state and with MK-A spiked. This approach overcame the selectivity challenges, led to an improvement of the assay sensitivity. The improved assay was successful validated with acceptable performance both in-house and at a contract research organization (CRO).

Conclusion: We developed a strategic approach to effectively address the selectivity challenge in MK-A PK assay. The incorporation of mouse matrix into the capture and detector diluent functioned as a blocking agent, potentially preventing unwanted bindings to the critical reagents such as human anti-mouse antibodies (HAMAs) etc. Additionally, by utilizing a recombinant anti-ID in place of the hybridoma, we successfully resolved the selectivity challenge with samples from disease samples. Both hybridoma and recombinant antibodies are IgG2b. One hypothesis for this improvement is that the quality of antibodies produced by hybridoma technology may differ from that of antibodies generated through recombinant DNA technology, due to variations in processes such as purification and other production steps. This systematic strategy enabled the team to efficiently develop a selective and sensitive assay and allowed for a seamless transfer to the CRO.

Figure 1

Figure 2