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Characterization of Assay Performance in an Electrochemiluminescence-Based Ligand-Binding Method for Detection of a Therapeutic Monoclonal Antibody Utilizing Various Detection Reagents
J. A. Lefor A. A. Wyatt D. B. Gemani R. C. Sterling A. Halford Y. Q. Xiao

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Characterization of Assay Performance in an Electrochemiluminescence-Based Ligand-Binding Method for Detection of a Therapeutic Monoclonal Antibody Utilizing Various Detection Reagents
J. A. Lefor, A. A. Wyatt, D. B. Gemani, R. C. Sterling, A. Halford, Y. Q. Xiao
Department of Immunology, MPI Research, Inc., Mattawan, MI 49071

Purpose To characterize assay performance in detection of a therapeutic humanized monoclonal antibody (mAb; drug) in nonhuman primate (NHP) serum, via assessment of various capture and detection reagents with increasing degrees of drug specificity. Methods Electrochemiluminescence (ECL) based ligand-binding assays were developed to quantify mAb utilizing a variety of capture and detection reagents. Commercially available monkey cross-adsorbed anti-human IgG (-hu IgG), drug ligand, and four clones of anti-idiotypic antibodies (-Id) to the drug were evaluated on the Meso Scale Discovery (MSD) Sector Imager 6000 platform. The following capture:detection pairs were selected, listed with increasing degree of specificity: A) -hu IgG:-hu IgG; B) ligand:-hu IgG; C) ligand:-Id, and D) -Id:-Id. Matrix interference (background) was assessed at the 1:10 minimum required dilution (MRD). Assay range was evaluated via an extended two-fold titration curve of drug in matrix, with quality controls (QCs) at concentrations ranging from 0.244 to 500ng/mL. Selectivity was tested in eight NHP serum lots. Results Background ECL signal and inter-lot variation was observed to be lowest in condition C (mean=56.2; SD=5.81; n=50), and highest in condition A (mean=1214.75; SD=1456.33; n=50). Assay range was 0.122 to 500ng/mL for condition B, extending as low as 0.061ng/mL in condition A and up to 1000ng/mL in conditions C and D. All QCs tested were within 20% mean relative error (RE). Selectivity was within 20%RE at 0.977ng/mL for 8/8, 7/8, 5/8, and 3/8 lots for conditions C, B, A, and D, respectively. Conclusion The highest-performing assay characteristics were obtained with the ligand:-Id combination, although all capture:detection conditions produced acceptable assay range and QC recovery. Assay development strategies for each mAb program should be customized, taking scope, sensitivity requirements, budget, and target species into account. While a generic approach is suitable for non-primate studies, more specific methodology utilizing ligand:-Id or -Id:-Id systems should be considered for mAb detection in NHP serum, and in support of clinical trials.

Figure 1: Assay Range The Capture and Detection pairs identified in Table 1 were titrated to determine optimal concentrations for each of the four conditions. Assay range was evaluated via an extended two-fold titration curve of drug in 10% NHP serum ranging from 0.061 to 1000ng/mL and was assayed in duplicate wells. Assay range was determined as the lowest and highest drug concentration which met the following acceptance criteria: 20% relative error (RE) and 20% coefficient of variation (CV). Assay range extends from 0.061 to 500 ng/mL for Condition A, 0.122 to 500ng/mL for Condition B, and 0.122 to 1000ng/mL for Conditions C and D. Additional points shown outside of the listed ranges were utilized as anchor points and therefore not required to meet listed criteria.

Table 2: Quality Control (QC) Samples QC samples were prepared in neat NHP serum, followed by dilution to the MRD for final assay concentrations as indicated in Table 2. QCs were assayed in duplicate wells for each of the four conditions presented in Table 1. All levels tested were within 20% relative error (%RE) when compared to nominal. QC (ng/mL) 500 125 100 10 2.5 0.977 0.244 Condition A % RE -15.2 12.7 4.2 -14.6 -16.9 -13.9 -11.2 % CV 19.9 4.1 4.4 3.6 0.8 1.1 1.9 Condition B % RE 12.0 9.3 2.6 -15.5 -12.7 -9.5 -15.6 % CV 4.4 2.1 4.4 5 3.7 2.3 8.8 Condition C % RE 1.6 10.8 4.1 -18.3 -10.2 6.0 -12.1 % CV 0.1 2.8 1.7 2.3 4.5 2.9 3 Condition D % RE 3.6 3.7 -0.6 -16.5 -17.3 -16.4 4.6 % CV 1.7 2.3 0.4 1.5 2 2.5 22

Figure 3: Selectivity Four female (F) and four male (M) lots of neat serum were spiked with drug and diluted to the MRD (1:10) for a final assay concentration of 0.977ng/mL. Selectivity samples were analyzed in duplicate wells under each of the four testing conditions. Percent relative error (%RE) was calculated for each lot. Selectivity results were within 20%RE for 100%, 88%, 63%, and 38% of lots for Conditions C, B, A, and D, respectively.

To develop ligand-binding assay for the quantification of a monoclonal antibody therapeutic (mAb; drug) using a variety of capture and detection reagents To characterize assay performance in terms of assay range, background, QC recovery, and selectivity of the assay to detect the mAb in nonhuman primate serum To provide data in support of drivers of efficient decision-making such as assay design, logistics, and cost effectiveness Figure 2: Matrix Background Screening Individual serum lots were diluted to the minimum required dilution (MRD; 10%) in Assay Buffer and assayed in duplicate wells to determine background ECL signal under each of the four testing conditions. Mean ECL signal, standard deviation, and inter-lot %CV were calculated across the 50 lots tested (*only 49 lots were tested for Condition B). Background ECL signal and inter-lot variation were observed to be lowest in Condition C and highest in Condition A.

Meso Scale Discovery (MSD) Multi-Array 96-well high bind plates were coated with anti-human IgG, drug target (ligand), or one of four commercially available anti-idiotypic antibody clones to the monoclonal antibody therapeutic. Plates were washed and blocked to prevent non-specific binding. Standards, QCs, and prepared samples were diluted to the minimumrequired dilution (MRD; 1:10 NHP serum) prior to plating. Following sample incubation, plates were washed, and samples were detected with either biotinylated anti-human IgG or biotinylated anti-idiotypic antibodies (n=4). Following incubation, plates were washed and were then incubated with streptavidin conjugated ruthenium chelate. Upon completion of this last incubation, a final washing step was followed by addition of MSD Read Buffer T and detection on the MSD Sector Imager 6000. Standard curves of the electrochemiluminescent signal (ECL Signal) were generated using a 4-parameter logistic fit with 1/y2 weighting. The capture:detection pairings, which were considered optimal, are presented in Table 1, and the ensuing standard curves are illustrated in Figure 1. Table 1: Assay Conditions Definition of Conditions A, B, C, and D by identification of Capture and Detection reagent pairing. Condition A Capture Detection anti-human IgG anti-human IgG Condition B ligand anti-human IgG Condition C ligand anti-idiotype (clone 1) Condition D anti-idiotype (clone 2) anti-idiotype (clone 1) n = 50 Mean ECL Signal Std Deviation Inter Lot %CV Condition A 1215 1456 120% Condition B* 95 41 43% Condition C 56 6 10% Condition D 305 41 13%

The assay range and QC performance were acceptable for all four testing conditions. However, capture via drug target (ligand), followed by detection with biotinylated anti-idiotypic antibody (Condition C), yielded the lowest and least variable background signal and showed an exceptional selectivity passing rate of 100%. Capture with drug ligand, followed by detection with biotinylated generic anti-human IgG antibody, also had an acceptable selectivity passing rate. While the cost of the assay reagents is higher for a specific assay such as Condition C than for a more generic assay such as Conditions A and B, the broad dynamic range, minimal matrix interference, and selectivity of the assay provides confidence in reproducibility, selectivity, and minimal matrix interference. Assay developed under Conditions C and D can be carried from the preclinical stages through the clinical sample analysis stage of drug development. While the generation of antiidiotypic antibodies is an expensive, difficult, and timeconsuming endeavor, their effectiveness in the clinical environment should not be understated. Assay Conditions A and B would produce satisfactory options for reduction of cost in the preclinical setting if antiidiotypic antibodies are not yet available.

1. Guidance for Industry: Bioanalytical Method Validation. U.S. Department of Health and Human Services, Food And Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM). May 2001 2. Guideline on Bioanalytical Method Validation. European Medicines Agency (EMEA), 2011. Effective February 2012.