INTRODUCTION

Most biopharmaceutical therapeutics (e.g. antibodies, cytokines and recombinant proteins) elicit a potential to in-duce an anti-drug antibodies (ADA) response which can, in some cases, lead to potentially serious side effects (such as allergic or anaphylactic reactions, or induction of autoimmunity) and/or loss of efficacy. This makes the im-munogenicity of therapeutic proteins a big concern for clinicians, manufacturers and regulatory agencies. There-fore, the need for appropriate detection, quantitation and characterization of ADA response is necessary.

In this project, we have implemented and fully validated a bridging Electrochemiluminescece (ECL) ImmunoAssay for quasi-quantitation of antibodies against a therapeutic compound. The intended application was to support clini-cal trials from various stages of development of the compound. Therefore, we have conducted the validation study in accordance with the principles of the following standards of Good Laboratory Practice (GLP):

•OECD Principles on Good Laboratory Practice (as revised in 1997) ENV/MC/CHEM (98) 17•European Directive 2004/10/EC

1. OBJECTIVESThe purpose of this project was to conduct a full-validation of a ECL bridging Immunoassay to meet the regulatory requirements for monitoring immuno-genicity response in clinical trials.

2. METHODSECL BRIDGING ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA):

Circulating ADA are captured and sandwiched between the biotinylated drug on one side (capture) and the sulfo-TAG-labeled drug on the other side (detection). The formed complex is subsequently bound to MSD® Streptavidin-coated plates and detected by ECL. The ECL signals were measured on a MESO SCALE DISCOVERY® 2400 SECTOR Imager (Figure 1).

VALIDATION QUALITY CONTROL SAMPLES AND KEY REAGENTS:

• Positive control ADA: Polyclonal Rabbit & monoclonal mouse anti-drug human IgGs• Negative Controls (NC): Pool of serum from drug naïve subjects• High Quality Control (HQC): NC spiked with 100 µg/ml of ADA (positive control)• Low Quality Control (LQC): NC spiked with 100 ng/ml of ADA (positive control)• Sensitive Quality Control (SensQC): NC spiked with 20 ng/ml of ADA (positive control)• Drug Interference Samples: HQC spiked with ADA at different levels• Confirmatory Cut Point (CCP) samples: A set of serum samples from drug naïve subjects and spiked or not with drug at 10 mg/ml• Titration Quality Control samples (TQC): NC spiked with ADA (positive control) followed by serial dilutions• Drug Tolerance QC (DTQC) Samples: Low DTQC (ADA at 250 ng/ml) and High DTQC (ADA at 500 ng/ml) spiked with the Drug at different concen-

trations

3. RESULTSThe following validation parameters were evaluated: screening & confirmatory cut-points, titration, precision, recovery, relative sensitivity and assay drug tolerance. Results are illustrated in the following Tables and Figures. Key results are:

SCREENING CP (TAB 1, FIG 2)

A screening CP must be determined for each screening plate as follows:CP = CPF * Plate Mean ECL signal of NCCPF = 1.33Study samples with an ECL signal > assay CP will be screened as positive and will be re-analyzed for confirmation.

CONFIRMATORY CP (FIG 3):

Confirmatory CP (CCP): 27.6% (based on false-positive rate of 0.1%). Study samples for which a signal inhibition of ≥ 27.6% was observed after spiking with the drug are considered ADA positive and re-analyzed for confirmation. If a sample is confirmed positive, the sample will undergo a titration analysis.

RELATIVE ASSAY SENSITIVITY (AVERAGE) = 24.2 ng/mL with 95% consistency (Tab 3)

TITRATION PARAMETER: TQC (Titration QC): 32 ≤ Titer ≤ 128 (Tab 2, Fig 4)

INTERFERENCE OF THE DRUG IN DETECTING ADA DEMONSTRATED FOR CONCENTRATIONS OF THE DRUG ≥ 15.7 μg/mL (Fig 5)

4. CONCLUSIONA sensitive ECL bridging immunoassay for detection of ADA was developed, then implemented and validated in human serum in our laboratory. The method showed an adequate tolerance level to free drug to be used for immunogenicity testing of clinical study samples. Validation parameters support-ed the use of the method for screening, confirmatory testing and titration of positive samples for ADA.

*Figures 1A and 1B property of Meso Scale Diagnostics, LLC and used with permission.

T3348BIOANALYTICAL VALIDATION OF AN ELECTROCHEMILUMINESENCE-BASED BRIDGING IMMUNOASSAY FOR IMMUNOGENICITY TESTING OF A THERAPEUTIC ANTIBODY

FIGURE 1*: Schematic Representation of Assay Format (Fig 1a) and Principle of Detection Platform (Fig 1b)

FIGURE 1A: Indirect bridging ELISA (immunogenicity) (ELISA - Enzyme-linked Immunosorbent Assay)

FIGURE 1B: ECL Detection Schematic on MSD Sector Imager 2400 (ECL, electrochemiluminescence)

EMMANUEL GUIGNOUARD, SANDRINE MORVAN, AUDREY ARTUS, ALAIN RENOUX, RABIA HIDISGS LIFE SCIENCE SERVICES, POITIERS, FRANCE

RUN SAMPLE SIZE

MEAN ECL VALUE

95TH PERCENTILE CPF FINAL CPF

(%CV)

1 54 197 264 1.34

(9.0%)

2 55 255 319 1.25

3 51 182 241 1.32

4 56 200 305 1.53

5 53 219 256 1.17

6 55 237 320 1.35

The CP values for all validation runs (and each plate) were calculated from the mean ECL signal of NC (n=4) as follows:CP= 1.33 * Plate Mean ECL signal of NC

(CP, cut-point; CPF, cut-point factor; NC, negative control; ECL, electrochemiluminescence)

TABLE 1: Cut Point factor by assay using the empirical 95th percentile method

TABLE 2: Titration Evaluation

Titer = DF above CP + ([ECL above CP - CP] x [DF below CP - DF above CP]) / (ECL above CP - ECL below CP)

(CP, cut-point; ECL, electrochemilumunescence; TQC: Titration QC; DF: Dilution Factor)

FIGURE 2: Determination of the Confirmatory CP (CCP)

FIGURE 3: Typical Titration Curve

The relative sensitivity of the assay is defined at the intercept of the screening CP with the linear regression of the two concentrations which produce assay signals directly above and below the screening CP# No ECL nor DF below CP (however, titer calculated with null values as ECL and DF below CP)* Relative sensitivity = Conc. above CP + ([ECL above CP - CP] x [Conc. below CP - Conc. above CP]) / (ECL above CP - ECL below CP)** Assay sensitivity (95% consistency) = Mean Log + (t0.05,5 x S.D.), and antilog (10^) of the result

(ECL, electrochemiluminescence; CP, cut-point; CPF, cut-point factor; S.D., standard deviation)

TABLE 3: Relative Assay Sensitivity Evaluation

FIGURE 5: Recovery of ADA (Positive Control) Spiked in Drug Naive Sera

Recovery ADA (positive control spiked at the sensitive level (25ng/ml) in 38 drug naïve serum samples Recovery observed in 94.7 % of the samples (36 out of 38 samples).

FIGURE 1: Representative data for CPF and CP determination(ECL signals of 56 individual human sera)

TABLE 4: Intra- (tab 1a) Inter-run (tab 1b) Precision on Validation QC Samples

HQC LQC SENSQC NC

INRA-RUN MEAN ECL SIGNAL 373964 504 253 198

INTRA-RUN CV (%) 14.4 11.0 11.2 14.2

n 35 30 36 71

HQC LQC SENSQC NC

INRA-RUN MEAN ECL SIGNAL 354779 446 232 171

INTRA-RUN CV (%) 11.0 11.0 6.5 8.0

n 15 15 32 32

TABLE 1BTABLE 1A

FIGURE 4: Drug Tolerance Evaluation

HIGH POSITIVE CONTROL SAMPLE

Representative Curves from Drug Tolerance Testing in 2 Pools of Human Serum Samples spiked with ADA (Positibe control) Two Levels: Low (250 ng/ml) & High (500 ng/ml)

LOW POSITIVE CONTROL SAMPLE