Using Ligang-Binding Assay Sensitivity for Improved Matrix Tolerance and Related Parameters by Tailored Sample Dilution.

Transcription

1 Using Ligang-Binding Assay Sensitivity for Improved Matrix Tolerance and Related Parameters by Tailored Sample Dilution. Dr. Mark Spengler, Chimera Biotec

2 Chimera Biotec Company Overview Bioanalytical CRO with proprietary platform Focus: Ultra-sensitive LBA s Founded 2000 Marketing Imperacer since 2004 Collaboration with PRA International for GLP Studies Revenues: 50/50 (EU/US)

3 Basic Principle improved performance Antibody-DNA Conjugate replaces Antibody-enzyme Conjugate Processing & Read-out by real-time PCR 3

4 ELISA vs. Imperacer Biosource TNFa ELISA kit: Chimera Biotec TNFa Imperacer kit: improved performance LOD: 10 pg/ml (40,000,000 molecules absolute) LOD: 0.01 pg/ml (12,000 molecules absolute) Using the same set of antibodies 4

5 Comparison of Platforms RIA Western ELISA Luminex Gyros ECL, MSD Singulex Imperacer Assay Sensitivity basic good excellent Choose the appropriate platform for your assay

6 Imperacer Assay Development & Bioanalysis Conjugation Assay set-up Conjugation maximization of sensitivity assay engineering pre-study validation GLP Non-GLP Option 1: Preclinical Chimera Biotec (non-glp) Option 2: Clinical PRA-International (GLP) Option 3: BA in-house (non-glp / GLP): Imperacer Instrumentation 6

7 Saliva Cells Biological Sample: Matrix compounds ( ) interfere with target ( ) detection Brain Faeces Food Serum Sensitive IPCR target detection after sample dillution Minimizing Matrix Effects :1000 1:10e6 1:10e9 NC :1000 1:10e6 1:10e9 NC... through simple sample dilution 7

8 Imperacer [delta Ct] Minimizing Matrix Effects pure 1:10 1: ng/ml 10ng/ml 1ng/ml NC Serum Buffer Serum 1:10 Buffer 1:10 Serum 1:100 Buffer 1:100 Minimizing Matrix Effects through Dilution 8

9 Examples from Clinical assays 1. Anti-cytokine Drug 2. Replacement Therapy 3. Immunogenicity 4. Clinical Study enrollment

10 Minimizing Matrix Effects: Background Anti-Cytokine Drug Detection of a Cytokine-Drug complex in the presence of interfering endogenous antibody Target: Cytokine -Drug complex Detection conjugate directed against IgG Cytokine-specific therapeutic IgG Interfering unspecific IgG in serum Cytokine Cytokine-specific Capture antibody For more details please refer to poster presentation: A23 10

11 Imperacer [delta [delta Ct] Ct] Minimizing Matrix Effects: Background Anti-Cytokine Drug NC 500 pg/ml target 5 ng/ml target 50 ng/ml target 15 Serum (pure) Pure 1+1 SDB SDB6100 Dilution Buffer A 1+9 SDB SDB2100 Dilution Ratio Dilution Buffer B Tailored sample dilution enables massive background reduction. Maximizing Performance by appropriate sample dilution buffer Current LOD: 1 pg/ml; aiming at sub pg/ml levels of the Cytokine 11

12 Minimizing Interfering Effects: Endogenous Counterpart Replacement Therapy Endogenous Protein Imperacer detection conjugate directed against drug specific moiety Replacement Drug (endogenous protein + specific domain) The Challenge: Endogenous moiety specific capture antibody Sandwich Imperacer in Replacement Therapy Study At Standard dilution ratio either endogenous protein or unspecific antibodies saturate the capture plate 12

13 Imperacer [delta Ct] Minimizing Interfering Effects: Endogenous Counterpart Replacement Therapy µg/ml 400ng/ml 160ng/ml 64ng/ml 25.6ng/ml 10.24ng/ml 4.10ng/ml NC 25 Buffer Non-Depleted Matrix (individual) Depleted Matrix Non-Depleted Matrix (pool) Buffer Non-Depleted Matrix (individual) Depleted Matrix Non-Depleted Matrix (pool) 1+49 Sample Dilution Ratio 1+99 Sample Dilution Ratio 1 : 50 1 : 100 Dilution Ratio High sample dilution in appropriate sample dilution buffer neutralizes interference by endogenous counterpart. 13

14 Minimizing Interfering Effects: Endogenous Counterpart Replacement Therapy Samples with unknown conc. of endogenous protein can be quantified against STD curve in depleted matrix or native matrix pool. 14

15 Minimizing Drug Interaction Immunogenicity / ADA detection Source: Spengler et al., BBRC (2009) 15

16 Minimizing Drug Interaction Immunogenicity / ADA detection Comparison of Assay Platforms ELISA MSD IMPERACER Sample Volume 100 µl 100 µl 30 µl Sensitivity Using mab Drug Tolerance (mab : Drug) Sample Pre- Treatment (Acid Dissociation) 70 ng/ml 20 ng/ml 40 pg/ml 1 : 1 1 : 40 1: 1000 Increased Background Increased Background Matrix Interference NA Sponsor in-house ELISA & ECL assays compared to Imperacer Same Antibodies & Reagents used No sample pre-treatment was used in Imperacer procedure NA From ELISA to a novel Immuno-PCR (i-pcr) based immunogenicity assay in quest to improve the drug tolerance Poster presentation at the AAPS NBC 2010, Darshana Jani 1, Joyce Sobolowski 1, Jeannine Keefe 1, Michael Adler 2, and Jaya Goyal 1 Clinical Science and Technology, Preclinical and Clinical Development Sciences, Biogen Idec Inc. Cambridge, MA 1 and Chimera Biotec, Dortmund Germany 2 16

17 Ligand-Binding Assay Sensitivity Study Group Profiling 6 Biomarker detection common technology Biomarker detection Imperacer technology Biomarker screening using classical LBA (e.g. ELISA) Biomarker screening using Imperacer Concentrations below detection limit 2 1 Critical biomarker level 0 s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 s11 s12 s13 s14 s15 s16 s17 s18 s19 s20 s21 s22 s23 s24 s25 number of analysed individual samples 0 s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 s11 s12 s13 s14 s15 s16 s17 s18 s19 s20 s21 s22 s23 s24 s25 number of analysed individual samples - Most concentrations are below detection limit - Detection of base level concentrations - Identification of patients with critical biomarker level Ultra-sensitive patient screening enables dramatic increase in drug responder rates Option: Companion diagnostics

18 Imperacer Dive deeper into the Proteome Thank You! Contact: Chimera Biotec GmbH Mark Spengler Director of Project Management PRA International Martin Nemansky Scientific Director