Protein arrays with 140 capture spots per well (96- wel plate) with automated, multiplex Assays: ELISAs PK/PD Assays Biomarker Assays

1. science • quality • innovation
general presentation
July 2018

2. science•quality•innovation
SQI - leadership in diagnostics
Located in Toronto, 42 employees, 4 in the U.S.
Multiplexed microarrays for protein and molecular diagnostics
Product lines:
– IVD (FDA 510k, CE, Health Canada)
– LDT for clinical diagnostic labs, pre-clinical patient screening
– RUO custom assay development, screening services for pharma
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3. science•quality•innovation
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Rheumatoid Arthritis Ig_plex™
– RF-IgA
– IgG
– IgM
– anti-CCP IgG
Celiac Ig_plex™ 4-Plex
– Anti-Tissue Transglutaminase
IgA & IgG
– Anti-Deamidated Gliadin IgA &
IgG
IVD background
Clinical dx portfolio, cleared by FDA, Health Canada
Vasculitis Ig_plex™ 3-Plex
– Anti-Myeloperoxidase IgG
– Anti-Proteinase 3 IgG
– Anti-Glomerular Basement
Membrane IgG
Crohn’s disease/IBD Ig_plex™ 9-
Plex
Pipeline, to be cleared by FDA, Health Canada
ANA Ig_plex™ 11-Plex
– dsDNA IgG
– Sm IgG
– RNP IgG
– SSA IgG
– SSB IgG
– Jo-1 IgG
– Scl-70 IgG
– PCNA IgG
– CENP IgG
– Histones IgG
– Ribo P IgG

4. science•quality•innovation
core technologies: platform, custom printed plates, screening
Ig_plex™
32 multiplexed
assays per well
in 96-well plate
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Extensive experience in custom assay development, protein & molecular
e.g. autoimmune, allergy, cardiac, sepsis, gene therapy screening
Automated platform + multiplexing = more data, efficiency at lower costs
Customer-focused with partnership model to accelerate innovation

5. science•quality•innovation
quality defined, certifications
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SQI is:
▪ ISO 13485
▪ cGMP
▪ Health Canada, FDA-audited
SQI instrument software is 21 CFR part 11 complaint
Strict QA and QC programs with manufacturing, design history files
Multiple assays cleared by FDA (IVD/510k)
Data by SQI assays submitted to FDA for drug programs

6. science • quality • innovation
dual layer multiplexing
technology

7. science•quality•innovation
level-one: multiplexing at the capture level
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Multiplexing
proteins, antibodies, oligos and more
Software reports
capture analyte level
replicate level
individual spot level
Both quantitative, qualitative
Inter plate CV’s :
IVD <10%
RUO <20%

8. science•quality•innovation
level-two: multiplexing at the detection level
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Dual layer - means one well, one sample with multiple results
▪ each well contains multiple capture spots to the ADAs
e.g. innovators, biosimilars, downstream metabolites
▪ one protein capture spot detected by multiple isotypes (level one)
▪ each isotype can be further interrogated with detectors for subclassing (level two)

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sqid analyzers are sized to meet demand
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sqidworks sqidlite sqid-X
Size
Foot Print
36" x 66"
Bench Top
20" x 48"
Bench Top
18" x 24"
Target User
High Volume Ref Labs
Top 1,000 NA / EU Labs
IVD, OEM, RUO
LDTs / R&D
cETLus, CE
Research
Non-IVD
Hands-on
time 1-plate
15 minutes 15 minutes 45 minutes
Cost $300k $160k $70k (with cabinet)

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Complete solution for microarray assays
▪ high precision pipetting unit
▪ robotic arm for plate movement
▪ barcode scanner
▪ heater/shakers for incubation,
▪ washer specialized for microarray applications
▪ proprietary drying system
▪ multi-wavelength fluorescent microarray reader
▪ automated assay development, analysis and
reporting (integrated LIS, .pdf or .csv format)
sqidlite™ system
Compact size fits onto lab bench top
Validated for IVD assays, cETLus certified, CE compatible

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• Intuitive and guided workflows with software developed and supported by SQI
• Various operator access levels controlled by user login
• Load-and-Go system
• Deck and barcode verification option
• LIS integration
• All-in-one maintenance software for all system components
quantispot® software

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Assay constructs
▪ ADA isotyping, subclassing
▪ domain, epitope mapping
▪ bridging ADA, better drug tolerance
▪ PK, drug on board testing
▪ biomarker quantitation
▪ ligand binding assays, Nab, mAb, pAb screening
Ig_plex™ performance exceeds criteria for sensitivity, drug tolerance and precision
Technology and protocols are easily transferable to CRO
Assay Capability
Analysis results, based on typical multiplexed cytokine
– LOD < 0.3 pg/mL
– LLOQ < 1 pg/mL
– Precision < 10% CV at 5 pg/mL

13. science • quality • innovation
Assay constructs:
ADA isotyping, epitope mapping, biomarkers

14. Isotyping ADA assay (BMS)
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Print adnectin FC:
one consumable for 3 pre-clinical assays
IgA IgMIgG
Mouse: anti-IgG, anti-IgM, anti-IgA
Rat: anti-IgG, anti-IgM, anti-IgA
Monkey: anti-IgG, anti-IgM, anti-IgA
Develop 3 reporter cocktails
To move assay into clinical use, Anti-hu IgG*, anti-IgM, anti-IgA
▪ reporter cocktail from SQI’s FDA approved RA Diagnostic
*Anti-hu IgG is anti-F(ab’)2 so does not bind directly to printed human Fc
ONE WELL

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Isotyping assay: IgG, IgE, IgM response
+ Pepsin
Protein G
Print full length mAb for Drug Tolerance
(rabbit polyclonal control)
Generate F(ab’)2 for human isotyping
IgA IgM IgG
F(ab’)2
Rabbit-a- mAb
mAb
Goat-a-rabbit
ADA assay: polyclonal positive control
IgG1 Hu mAb ADA

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ADA assay drug tolerance
▪ anti-HUmAb rabbit polyclonal PC (1:25 MRD)
▪ indirect assay format, no dissociation
Drug Tolerance of Rabbit Pab @ 500 ng/ml ≥ 400 ug/ml (forecast 598.5 ug/ml)
250 ng/ml ≥ 400 ug/ml (forecast 446.4 ug/ml)
125 ng/ml = 170 ug/ml drug (forecast)
62.5 ng/ml = 90 ug/ml drug (forecast)

17. science•quality•innovation
a-drug1 mAb
Biotin
a-drug1 mAb
drug1
a-drug2 mAb a-drug3 mAb
Biotin
a-drug3 mAb
Biotin
a-drug2 mAb
drug3drug2
multiplex PK
Potentially co-administered hu mAb based drugs
Standard ranges, bias , recovery and selectivity meet customer spec
*no hook effect detected

18. Epitope (Domain) Mapping
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Domain mapping: Identifying portion of drug responsible for immunogenicity
ONE WELL
scaffold/linker/hu Fc (IgG1)
Intact drug
21-plex: 7 captures x 3 isotypes
7 captures 3 detectors:
IgG, IgE, IgM1. Intact drug
2. Null binder
3. Alt linker
4. Drug-scaffold only
5. Null binder-scaffold only
6. Fc alone
7. Endogenous scaffold source
5-plex multispecies reporter
4 positive control Abs: a-drug, a-framework, a-ID, a-scaffold source

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ADA domain mapping: method development
1. Print optimization for 7 captures
2. Ag Cross-reactivity with positive controls
3. Reporter cocktail development for detection of primate ADA, mouse and rabbit PCs
and normalization of response for IgG, IgE and IgM detection
4. Limit of detection, matrix effects and MRD confirmation for 4 positive controls (PCs)
5. Specificity of PC responses by soluble antigen competition
6. Drug tolerance for three anti-drug positive controls
7. Cut point for each positive control and isotype on each of the captures
▪ Screening cut points using 150 individual monkey and human, naive serum
▪ Plate-specific floating cut points for mouse and Hu sera
▪ Specificity (confirmation) cut points in monkey serum for drug, Fc and
endogenous
8. Assay precision (inter-spot, inter-well, inter-plate and inter-print lot) for 4 PCs at high
and low positive levels
9. Domain mapping and isotyping of monkey samples from tox study

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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 and
compared with results from Milliplex Bead Array for 20 samples
Cytokine IL-2 Il1-b IL-4 IL-6 IL-8 IL-10 IFNg TNFa
% Positive
Agreement
(Sensitivity)
25
(1/4)
82
(9/11)
75
(12/16)
78
(7/9)
100
(16/16)
80
(8/10)
71
(5/7)
83
(5/6)
% Negative
Agreement
(Specificity)
100
(16/16)
100
(9/9)
100
(4/4)
100
(11/11)
100
(4/4)
100
(10/10)
92
(12/13)
79
(11/14)
% Overall
Agreement
85 90 80 90 100 90 85 80

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a-TNF
TNF
BIOTIN
a-TNF
SQI 8-PLEX CYTOKINE MICROARRAY
IL-2 IL-4 IL-6 IL-8 IL-10 IL-1Beta TNF-Alpha IFN-Gamma
LLOD (pg/mL) 1.15 0.32 0.53 0.6 0.77 0.24 0.77 5.0
LLOQ (pg/mL) 1.25 0.63 0.63 1.25 1.25 0.63 1.25 7.5
ULOD (ng/mL) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
%CV (25pg/mL) 5.3 8.2 9.7 4.7 6.2 5.8 7.9 5.3
% Recovery at
25pg/mL (in
serum/plasma)
96/91 90/104 86/84 80/70 87/85 78/84 71/67 72/74
8-plex cytokine microarray

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summary
SQI’s Ig_plex system multiplexes at two levels
Microarray printing of multiple captures
Differentially labeled detectors
SQI platform has demonstrated success with
Isotyping ADA assays
Domain mapping of ADA response
Bridging assays
Pharmacokinetics, including multiplexed PK
Multiplexed Biomarker Quantitation
Xplex™ streptavidin plates allow researchers to
Develop their own assays to detect and isotype/subclass anti-drug with SQI
species and isotype specific reporter cocktail development
Develop other bioanalytical assays including bridging & 1-3 color sandwich assays
Choice of scaled automation platforms and assay development toolkit
to configure and run the assay

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Contacts:
Eric Brouwer, CSO, w: 416.674.9500 x242
Betty Cosgrove, Business Manager (East), m: 203.435.598
Jeff Terryberry, Discovery Scientist, w: 416.674.9500 x289
thank you