IMPACT OF HIGHER ORDER COMPLEXES ON BIOMARKER TARGET QUANTITATION

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1 IMPACT OF HIGHER ORDER COMPLEXES ON BIOMARKER TARGET QUANTITATION SURENDRAN RAJENDRAN Bristol-Myers Squibb Immunogenicity and Bioassay Summit Immunogenicity Assessment & Clinical Relevance - Assay Strategy and Risk Assessment for Safe and Efficacious Biotherapeutics 1

2 BIOMARKERS Use of Biomarkers Diagnostic Prognostic Predictive Safety Exploratory Predominant technique used for its quantitation is immunology-based Ligand Binding Assay (LBA). 2

3 Issues of Biomarker quantitation by LBA Analyte independent Interferences by hemoglobin (hemolysis), bilirubin (icterus), triglycerides (lipemia), anticoagulants, human anti-mouse antibody(hama) Analyte dependent Caused by interactions between components in sample with one or more reagent antibodies Matrix effect Drug interference is one of the matrix effects Interference may falsely increase (positive interference) or decrease (negative interference) the measured signal. 3

4 Screening for Assay Reagents Detection Reagent pab MAb 1 MAb 2 MAb 3 MAb 4 Capture Reagent pab MAb MAb MAb MAb To develop an assay for Biomarker-X, commercial & in-house antibodies were screened as both capture and detection reagents 4

5 Fluorescence F I Capture - Detection Antibody Reagents Pair Selection pab MAb 1 Y MAb 2 MAb [TA] (ng/ml) Competitor Concentration ng/ml MAb 4 Plot for Competitor=BM S (60) MAb 1: Capturing Reagent Biotinylated pab: Detection Reagent 5

6 Immunoassay on Luminex Platform Capture Biomarker Detect Biomarker Label with SAPE Read on Luminex Sample MAb 1 conjugated beads MAb 1 conjugated beads MAb 1 conjugated beads MAb 1 conjugated beads Biotinylated pab Streptavidin-SE (SAPE) Read signal Incubate Incubate Incubate 6

7 Calibration Curve Fluorescence [Biomarker-X] (pg/ml) 7 Capture: MAb 1 Detection: Biotinylated pab Range: 50 to pg/ml

8 Intra- and Inter-Assay Imprecision Intra -Assay (pg/ml) Inter -Assay (pg/ml) QC LOW MID HIGH LOW MID HIGH n Mean SD %CV

9 Analytical Recovery Biomarker-X (pg/ml) Sample Expected % Recovery

10 Dilution Linearity Serum Measured Theoretical % Dilution Scheme Conc. Conc. Recovery [Biomarker-X] (pg/ml) Assay dilution (1:2) x dilution (1:4 ) x dilution (1:8 ) x dilution (1:16 ) x dilution (1:32 ) Measured Concentration (pg/ml) Serum Dilution Linearity - 2 to 32 fold Slope: r2: Theoretical Concentration (pg/ml) 10

11 Effect of TA on Biomarker-X Quantitation Fluorescence Biomarker: 0.91 nm Biomarker: 0.18 nm [TA] (nm) Drug TA interference in biomarker-x assay by signal enhancement followed a bell shaped curve as a function of increasing [TA]. 11

12 Interference Dependence on [Biomarker-X] TA Fluorescence No TA [Biomarker-X] (pm) In the presence of TA signal enhancement increases with [Biomarker-X]

13 Detection of Higher Order Complexes 1. Size Exclusion Chromatography Multi Angle Light Scattering (SEC- MALS) - MALS detector at the SEC elution determines the mass of the size separated analytes, - Thus SEC-MALS determines both absolute molar mass and average size. 2. Dynamic Light Scattering (DLS) - DLS determines the hydrodynamic radius, - Increased particle radius, directly infers formation of higher order complexes. 13

14 Instrumental Set up for SEC - MALS Column: Silica based GFC columns - PROTEIN KW-803 Exclusion limit: 700,000 Particle size: 5 µm Size: 8.0 x 300 (mm) Buffer: 100 mm sodium phosphate buffer with 200 mm sodium chloride and 0.02% sodium azide 14

15 SEC MALS indicated Higher Molecular Mass Complexes [TA] (nm) Higher Order Complex (%) TA concentration dependence of higher order complexes formation followed a bell shaped curve similar to the fluorescence signal enhancement in the Luminex assay. 15

16 DLS Confirmed Higher Order Complexes Formation Higher Order [TA] (nm) Complex Radius (nm) Not Detectable The increase in the radius of the complex followed an immuno precipitant profile of bell shape with increase in [TA]. 16

17 Fluorescence Molar Ratio: [TA]/[Biomarker-X] No TA High levels of both TA & Biomarker-X Excessive TA [TA] Capture Ab 17 Biomarker-X Detection Ab TA

18 Conclusions An assay for Biomarker-X of broad range and good sensitivity was developed on the Luminex platform. The selected capture and detection Ab reagents do not compete with drug TA for binding to Biomarker-X. In the immunoassay there is positive interference due to complexes formation by the TA binding. SEC-MALS and DLS data indicated the presence of TAmediated higher molecular mass complexes. The interference is due to TA-mediated higher order complex structures formation leading to enhanced signals. 18

19 Thanks to Fizal Nabbie Yan Zhang Steven Piccoli Noah Ditto Paul Morin Binodh DeSilva 19