The SQI Ig_PLEX Technology

Transcription

1 The SQI Ig_PLEX Technology

2 Introduction to SQI SQI Diagnostics is a diagnostics systems company that enables antibody testing with proprietary technology in multiplexing, miniaturization and automation. Life sciences instruments and diagnostics company founded in 1999 Providing automated multiplexed assay technology to clinical diagnostics and pharma ADA & biomarker markets First assays are FDA approved autoimmune diagnostic tests Microarray printing, multiplexed assays and walk-away automation speak to characterizing the immune response to therapeutic proteins including ADA isotype and subclass. Collaborative assay development tools and processes Headquartered in Toronto, Canada 2

3 EARLY DAYS: SQI ASSAYS FOR AUTOIMMUNE DISEASES Multiplexing at two levels: Planar arrays of capture spots Fluorescent reporter cocktails for isotyping Multiplexing IgG, IgA, IgM IgM IgA IgG Antigen 1 IgG IgA Antigen 2 A Rheumatoid Arthritis Differentially labelled a-human 2o antibodies, combined with a multi-wavelength (3x) scanner permits simultaneous measurement of patient immunoglobulin classes 3 Printing for unique control of target epitope presentation

4 MULTIPLEXED ANTIBODY DETECTION Multiplexed Antibody Detection For Immunogenicity Multiplexing Isotypes and IgG subclasses IgG2 IgG4 IgG3 IgG1 IgA IgM IgE One Spot Therapeutic proteins printed One well, one sample, multiple results Each well includes multiple capture spots ADAs to innovators and biosimilars, metabolites or subunits Each spot can be interrogated for multiple isotypes Within well replicates: interrogated independently, integrated for results reporting 4

5 What Exactly Are We Doing In One Workflow Neat Serum Ig_ consumable Start up Routine ADA Specificity Incubation On Deck Automated Dilution Acid Dissociation & Neutralize Serum & Reporter Diluents Primary Incubation Clean Up Shut Down Results Reporting Algorithmic Analysis Assay Scan 1st Wash / Rinse Assay Dry Secondary Incubation Final Rinse Flexible automation enables multiplexing of ADA s or biomarkers with or without specificity and acid dissociation Assay incubation times as in ELISA 5 2nd Wash / Rinse

6 SQiD Systems Sized to Meet Demand SQiDworks 6 SQiDlite SQiD-X Throughput results / hour 330+ results / hour 200+ results / hour Size Foot Print 36" x 66" Bench Top 20" x 48" Bench Top 18" x 24" Target Customer High Volume Ref Labs Top 1,000 NA / EU Labs IVD, OEM, RUO (LDTs NonIVD), Top 5,000 Labs Research / Non-IVD Hands-on Time for 1 Plate 15 minutes 15 minutes 45 minutes

7 SQiDlite Bench-Top System Load & Go capability for significant reduction in labor and supplies Flexible, configurable, automated workflow Bench-Top Size Full LIS Integration (up & down) CFR Part 11 compliant <3 hours for 1 plate User friendly software to report out what is required 7

8 Ample Deck Room for Additional Fluidic Processes Intermediate Dilution Plate Scanner Input Bay Drying Station 6 Fluid Troughs Multiple Tip Racks Biotek Input Bay 8 Ig_PLEX Plate 96 Sample Racks

9 Assay Development Toolkit Control and configure multiple aspects of your run Dilutions, Incubations, Washing, Drying Scanning and Reporting LIS reporting and integration Assay Development tool for in house automation optimization Ideal for LDT User friendly interface Multiple configurations SQI can optimize automation and provide locked specs Ideal for RUO and OEM Seamless integration from development Co-developed specs Rely on our expertise 9

10 Why Ig_PLEX for bioanalytics? Automated analysis of ADAs including isotype & subclass using differentially labeled secondary antibodies Array multiple captures: Tx protein innovators, biosimilars, drug metabolites or recombinant protein subunits Automate sample/drug pre-incubation for specificity confirmation or dissociation/neutralization if required Protein or antibody captures for custom biomarkers or drug on board assays Non-cell based ligand binding assays for neutralizing antibodies IVD proven technology with precision, sensitivity and method concordance 10

11 We Understand The Implications Of FDA Guidance Some of the suggestions Serum dilutions should be the same for screening and quantitation Should include H, M, L and negative controls Use cut off normalization techniques Assay should be robust and reproducible Comparisons made using the same testing technology Be aware of the impact of Fc interference Screening assays should detect all isotypes (including IgE and IgG4) 11

12 Results from Clinical and Laboratory Defined Samples

13 SQI s HIT Assay Multiplexing Isotypes on anti PF4/heparin (IgG, IgA,IgM) Generic ADA assay: LMW heparins and biosimilars Assay parallelism and standard curve Detection limit for serum antibodies Assay performance on lab defined and clinical samples Heparin inhibition for specificity 13

14 Anti-PF4/Heparin Single well, 3 Color Scan IgG IgA Total Ig IgG IgA IgM IgM PF4/Heparin complex printed at 50, 125 and 400 mg/ml Sample Positive for IgG, IgA and IgM Signal (MFI) on 400 mg/ml PF4/Hep IgG IgA IgM (55.38 AU/ml) (56.89 AU/ml) (21.67 AU/ml)

15 HIT Assay: Parallelism of Multiplexed response Parallelism of Anti-PF4/Hep Antibody Response G+A+M, G+M and G+A Samples Warkentin 2 IgG Warkentin 2 IgA Warkentin 2 IgM Akers 1 IgG Akers 1 IgM Proteogenex 30.5 IgG Intensity Proteogenex 30.5 IgA :50 1:100 Sample Dilution 15 1:200 1:400

16 HIT Assay: Semi-Quantitative Standard Curve X (Units) Y (INT) Semi-Quant. Cutoff AU/ml IgG IgA IgM Fitting Parameter for formula: A B C D F(X) = (A-D)/(1+(X/C)^B)+D R^299.99%

17 LLOD : HIT Assay Sensitivity Objective: Determine detection limit for HIT antibodies GAM-DY-488 KKO HIT MAb Heparin/PF4 Precision : 9-15 % CV Detection limit of 2 ng/ml HemosIL Acustar LOD 6.5 ng/ml 17

18 Multiplexed HIT (ANTI-PF4/HEPARIN) ASSAY Sample Category Lab-Defined Clinically-Defined IVD Predicate IgG Positive A/M frequencies, not agreements Confirmed HIT, heparin-dependent, RUO Predicate G/A/M IgG IgA IgM IgG IgA IgM % Positive Agreement (Sensitivity) 86.4 (38/44) 72.7 (32/44) 52.3 (23/44) 100 (8/8) 100 (6/6) 83.3 (5/6) % Negative Agreement (Specificity) 86.4 (57/66) 95.5 (63/66) 98.5 (65/66) 100 (0/8) 100 (2/2) 100 (2/2) % Overall Agreement 86.4 NA NA Lab-defined samples predicated on Aniara Zymutest HIA IgG PVS/PF4 Generates less specific neo-epitope Negatives: healthy patient samples (unscreened for HIT) 18 RUO Assay PF4/Heparin Warkentin TE, et alblood, 2009;113: 4963

19 Multiplexed Inhibition: Heparin Dependent Specificity IgG, A & M from sample in 2 wells, +/- high dose heparin HIT specificity defined as ~40% inhibition by high dose heparin. Average inhibition: IgG 48% IgA 65% IgM 45% Pre-screened lab-defined (LD) and clinically confirmed HIT samples Incubated in the presence of 125 U/ml unfractionated heparin (UFH) 19

20 MULTIPLEXED ANTIBODY DETECTION Multiplexed Antibody Detection For Immunogenicity Multiplexing Isotypes and IgG subclasses IgA IgG1 IgG2 IgG4 IgG3 PF4/Heparin printed 2 x 3 color cocktails 20 IgM

21 Multiplexed HIT ASSAY : IGG SUBCLASS DETERMINATION OF LAB-DEFINED SAMPLES IgG1, 2, 3, 4, A & M from sample in 2 wells : Representative subset of 40 samples assayed [39/40 a-pf4/heparin +, 33/40 IgG +] Antibody IgG1 IgG2 IgG4 IgA IgM Res ul t MFI AU/mL MFI AU/mL MFI AU/mL MFI AU/mL MFI AU/mL MFI AU/mL PR30.46 PR30.47 PR30.48 PR30.49 PR30.50 PR30.52 PR30.54 PR30.55 PR30.56 PR30.57 PR30.58 AK4 AK5 Control Cutoff Frequency 72.5% (29/40) 21 IgG3 5% (2/40) 37.5% (15/40) 2.5% (1/40) 57.5% (23/40) 37.5% (15/40)

22 MULTIPLEXED ANTIBODY DETECTION Multiplexed Antibody Detection For Immunogenicity Multiplexing Isotypes and IgG subclasses IgA IgG1 IgG2 IgG3 IgM IgG4 One Spot Therapeutic protein printed 1 x 6 color cocktail 22

23 Elimination of Fluorescent Crosstalk With 3 channel scanner, 2 wells for 6 Ig types, 6 color scanning for single well Excitation and emission filters enable discrete wavelengths preventing cross talk of detectors 23

24 Multiplexed Antibody Detection: Screen Isotypes and Subclasses in a Single Well 6-plex Reactivity of a Sample* Positive for All Isotypes and Subclasses MFI signal for IgA, IgM and each IgG subclass Detected by 3 color cocktail +/- 3 unlabeled 2os RPE Signal Additivity Using 2 subclass 2os in a single well *Mixed 2 samples for 6 Ig types: (S1): A,M,G1,G2,G3, (S 2): A,M,G2,G3,G4 24 G2-RPE G3-RPE Calculated Actual G1-RPE G4-RPE Calculated Actual

25 Applications of the Ig_PLEX Platform

26 Applications of the of the Ig_PLEX platform Screening Assays for Immunogenicity Assessment Monoclonal antibodies, therapeutic proteins, fusion proteins, peptides Compare subunits, metabolites, epitopes and/or biosimilars in a single well Screen pre-existing Biotherapeutic reactive antibodies Single printed array combines with species-specific reporter cocktails to address preclinical and clinical studies ( rat, mouse, rabbit, monkey, human) Gold standard comparable or better sensitivity and drug tolerance combined with immune response characterization and flexible automation Multiplexed determination of Biomarkers Print sandwich assay capture antibodies for protein quantitation Directly labeled or biotin/ SA-fluor Multiplexed cytokine panels : 26

27 SQI 8-PLEX CYTOKINE M SQI 8PLEX CYTOKINE MICROARRAY ICROARRAY Full Range Standard Curves for SQI 8-plex Cytokine Assay IL-1beta IL-6 IL-4 IL-10 IFN-gamma BIOTIN α-tnf TNF-alpha IL RIU TNF IL α-tnf ng/ml Cytokine IL-2 IL-4 IL-6 IL-8 IL-10 TNF Alpha INF Gamma IL-1 Beta LLOD (pg/ml) LLOQ* (pg/ml) ULQ* (ng/ml) ULD (ng/ml) CV% % Recovery (Serum/ Plasma) 25pg/ml 85/95 102/89 82/88 116/ /120 95/82 88/87 85/84 *precision (CV) <15%, accuracy (bias) <20% 27

28 SQI 8-PLEX CYTOKINE PANEL: ASSAY PERFORMANCE Recovery and CV of 8 cytokines spiked into normal heparanized plasma Cytokine spike IL2 IL4 IL6 NP 10pg NP 50pg NP 100pg % Recovery NP 10pg 76% 81% NP 50pg 94% 88% NP 100pg 99% 94% CV (n >6) NP 10pg 5.0% 8.1% NP 50pg 8.2% 8.9% NP 100pg 7.5% 9.0% IL8 IL10 TNFa % 115% 106% 76% 88% 97% 106% 98% 100% 86% 82% 89% 74% 90% 96% 61% 82% 84% 9.1% 11.1% 12.2% 11.6% 14.1% 6.5% 15.6% 8.8% 11.9% 18.1% 9.6% 6.7% 7.8% 9.7% 5.5% 8.2% 10.9% 7.3% Average Recovery and CV from 50 pg/ml spiked into normal plasma 52 18% 28 IL % IL % IL % IFNg Assay Reproducibility: 6 plates from 2 lots (n=126) IL2 IL1b IL % TNFa 50 14% IL1b 52 13% IFNg 44 18%

29 SQI 8-PLEX CYTOKINE PANEL: MULTISITE AGREEMENT ANALYSIS ON AUTOIMMUNE DISEASE SERUM AND PLASMA SQI screened in-house Autoimmune Disease samples for 8 plex cytokine expression Compared with results from Milliplex Bead Array for 20 samples Cytokine IL-2 Il1-b IL-4 IL-6 IL-8 IL-10 % Positive Agreement (Sensitivity) (1/4) (9/11) (12/16) (7/9) (16/16) (8/10) (5/7) (5/6) (16/16) (9/9) (4/4) (11/11) (4/4) (10/10) (12/13) (11/14) % Negative Agreement (Specificity) % Overall Agreement 29 IFNg TNFa

30 Applications of the of the Ig_PLEX platform Multiplexed determination of Biomarkers incorporating multicolor labeling Improve assay specificity with differentially labeled detector antibodies Differentiate non specific interactions with directly labeled reporters Where Biomarker modification or complex formation is under investigation, a single capture can be combined with differentially labeled detectors to measure multiple states in a single well TP htau 30 Phospho-Tau

31 Summary: Isotyping antibodies, ADA and measuring Biomarkers are key tools for enhancing our knowledge of the biological and therapeutic properties of Biologics as well as some aspects of the etiology and progression of diseases. The SQI automated platform multiplexes at two levels : Microarray printing of multiple captures Differentially labeled detectors Assay kinetics, fluidics and time to result equivalent to ELISA A technology borne from IVD that provides: Multiplexing isotypes with IgG subclasses (IgE and IgG4) Multiplexing ADA specificity confirmation Nanogram detection of serum antibodies Multiplexed Biomarker quantitation 31 Acknowledgements: Jeff Terryberry Stuart Carmichael Subo Perampalam T.E. Warkentin

32 Questions? Robert Massé Vice President, Large Molecule Bioanalysis Algorithme Pharma (450) x 2305 rmasse@algopharm.com Jaymie R. Sawyer, Ph.D. Vice President, Research and Development SQI Diagnostics Systems Inc. (416) x 242 jsawyer@sqidiagnostics.com