DATA PACKAGE. Multiplex CVD I 96 96

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1 DATA PACKAGE Multiplex CVD I 96 96

2 Table of contents 1. INTRODUCTION Technology Data analysis 3 2. PERFORMANCE CHARACTERISTICS Sample types Analytical Measurement 4 Detection limit 4 Measuring ranges 4 High dose hook effect Precision Repeatability Reproducibility 2.4 Analytical Specificity Endogenous interference 2.5 Scalability 3. REFERENCES 11 TECHNICAL SUPPORT For technical support, please contact us at support@olink.com or

1. Introduction Proseek Multiplex CVD I 96 96 is a reagent kit measuring 92 cardiovascular disease related human protein biomarkers simultaneously in plasma samples.

1 TECHNOLOGY The Proseek reagents are based on PEA, a Proximity Extension Assay technology 1, where 92 oligonucleotide labeled antibody probe pairs are allowed to bind to their respective target

The assay is performed in a homogeneous 96-well format without any need for washing steps, see Figure 1.

For each data point, delta Cq (dcq) values were obtained by subtracting the value for the Extension control, thus normalizing each sample for technical variation within one run.

In the final step of the preprocessing procedure the values are set relative to a fixed background level determined by Olink.

3 1. Introduction Proseek Multiplex CVD I is a reagent kit measuring 92 cardiovascular disease related human protein biomarkers simultaneously in plasma samples. The analytical performance of the product has been carefully validated and the results are presented below. 1.1 TECHNOLOGY The Proseek reagents are based on PEA, a Proximity Extension Assay technology 1, where 92 oligonucleotide labeled antibody probe pairs are allowed to bind to their respective target present in the sample. A PCR reporter sequence is formed by a proximity dependent DNA polymerization event and is subsequently detected and quantified using real-time PCR. The assay is performed in a homogeneous 96-well format without any need for washing steps, see Figure DATA ANALYSIS Data analysis was performed by employing a preprocessing normalization procedure. For each data point, delta Cq (dcq) values were obtained by subtracting the value for the Extension control, thus normalizing each sample for technical variation within one run. Normalization between runs is then performed by subtraction of the Interplate Control (IPC) for each assay. In the final step of the preprocessing procedure the values are set relative to a fixed background level determined by Olink. The generated Normalized Protein Expression (NPX) unit is on a log2 scale where a larger number represents a higher protein level in the sample, typically with the background level at around zero, although it might differ between runs. Linearization of data is performed by the mathematical operation 2 ^NPX. Statistical analyses, e.g. coefficient of variation (CV) calculations were performed on linearized values. INCUBATION EXTENSION DETECTION Allow the 92 antibody probe pairs to bind to their respective proteins in your samples. Extend and pre-amplify 92 unique DNA reporter sequences by proximity extension. Quantify each biomarker s DNA reporter using high throughput real-time qpcr. Fig 1. Proseek Multiplex assay procedure employs three core steps: Incubation, Extension and Detection. High throughput real-time qpcr is performed by using the Fluidigm Biomark TM or Fluidigm Biomark TM HD systems. Proseek Multiplex CVD I Data Package 3

4 2. Performance characteristics 2.1 SAMPLE TYPES A) 1 GDF-15 The ability to use different sample types was evaluated with the Proseek Multiplex CVD I by collecting matched ethylenediaminetetraacetic acid (EDTA), acid citrate dextrose (ACD), and heparin plasma samples from 5 individuals. Table 1 shows signal to background values for each sample type and assay, as well as relative percentage differences compared to EDTA plasma. The results indicated that EDTA plasma is a suitable sample type for all assays. Variations observed between responses in heparin and citrate plasma, as compared to EDTA plasma, was generally small, and most of the assays will therefore function without limitation in these sample types. 2.2 ANALYTICAL MEASUREMENT DETECTION LIMIT Limit of detection (LOD) was defined as 3 standard deviations above background, and reported in pg/ ml for proteins out of 92, for which recombinant antigen was available, see Figure 2 and Table 1. B) NPX NPX LLOQ ULOQ LLOQ Concentration (pg/ml) Interleukin ULOQ LOD LOD LOD: 7.6 pg/ml LLOQ: 15.3 pg/ml ULOQ: pg/ml Hook: pg/ml Range: 3.6 log LOD: 0. pg/ml LLOQ: 0. pg/ml ULOQ: 3 9 pg/ml Hook: 3 9 pg/ml Range: 4.5 log MEASURING RANGES The analytical measuring range was defined by the lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) and reported in pg/ml. Quantification limits of LLOQ and ULOQ were calculated with the following trueness and precision criteria; relative error 30% and CV 30%, of backcalculated values, respectively. Measuring ranges were reported in order of log. See Figure 2 and Table 1. Calibrator curves were determined for protein biomarkers simultaneously in multiplex format. Two protein biomarkers lacked recombinant antigens and two were non-purified preparations. Representative assays with their analytical data are exemplified in Figure 2 and the distribution of their corresponding measuring range per assay is shown in Figure 3. Separate calibrator curves established for each assay may be viewed at HIGH DOSE HOOK EFFECT A high dose hook effect is a state of antigen excess relative to the reagent antibodies resulting in falsely lower values. If undetected, a significantly lower value will be reported which can lead to misinterpretation of results. Therefore, the high dose hook effect was determined for each analyte, here reported in pg/ml, see Figure 2 and Table 1. C) D) NPX NPX LLOQ LLOQ Concentration (pg/ml) Interleukin 1 ULOQ Concentration (pg/ml) Osteoprotegerin ULOQ Concentration (pg/ml) LOD LOD LOD: 0.24 pg/ml LLOQ: 0.24 pg/ml ULOQ: pg/ml Hook: pg/ml Range: 5.1 log LOD: 0.95 pg/ml LLOQ: 0.95 pg/ml ULOQ: pg/ml Hook: pg/ml Range: 4. log Fig 2. Calibrator curves from 4 representative assays and their corresponding analytical measurement data. 4 Proseek Multiplex CVD I Data Package

5 PAPPA CTSD mamp PRL HSP 27 MB NT-pro-BNP MMP-3 Gal-3 SPON1 FABP4 FS CHI3L1 AM IL-6RA SELE Gal-3 LEP CTSL-1 ESM-1 MPO TNF-R2 CSTB CXCL- FGF-23 ECP RETN EN-RAGE t-pa Dkk-1 PDGF Subunit B AGRP PECAM-1 PTX3 TIE2 SIRT2 ST2 IL-27 SRC ITGB1BP2 HGF VEGF-D MMP-7 CXCL-6 PSGL-1 FAS RAGE TRANCE CX3CL1 TRAIL-R2 GDF-15 REN IL- CXCL1 TM MMP- KLK6 TIM SCF LOX-1 hk11 CD40L MMP- MMP-1 TNF-R1 TRAIL CCL20 MCP-1 IL-1ra CASP- Beta-NGF TNFSF14 PIGF GH HB-EGF EGF CCL4 OPG IL-4 CD40 U-PAR CCL3 VEGF-A IL-1 CSF-1 TF IL- IL Concentration (pg/ml) Fig 3. Distribution of analytical measuring range, defined by the limits of quantification LLOQ-ULOQ, for out of 92 analytes. Proseek Multiplex CVD I Data Package 5

6 Table 1. Sample Types, Analytical Measurement; Limit of Detection, LOD, Lower Limit of Quantification, LLOQ, Upper Limit of Quantification, ULOQ, High Dose Effect, Hook, and Precision indicative of assay performance are shown for 92 analytes. Values below limit of detection were not reported (NR). Signal-to-background (2 ddcq ) Sample types Analytical measurement Precision Relative 2 ddcq to EDTA plasma Target UniProt No ACD EDTA Heparin ACD Heparin LOD LLOQ ULOQ Hook Range Intra-assay Inter-assay Inter-site Adrenomedullin P % 69% % 1% % Agouti-related protein O % 7% % % 17% Angiopoietin-1 receptor Q % 92% % % % Beta-nerve growth factor P % 1% % 25% 13% Caspase- Q % 1% % 21% 25% Cathepsin D P % % % % 14% Cathepsin L1 P % 0% % 14% % C-C motif chemokine 3 P % 79% % 1% 1% C-C motif chemokine 4 P % 5% % % % C-C motif chemokine 20 P % 72% % 9% 5% CD40 ligand P % 227% % % % Chitinase-3-like protein 1 P % 91% % 15% 11% C-X-C motife chemokine 1 P % 9% % 13% 1% C-X-C motif chemokine 6 P % 23% % % % C-X-C motif chemokine Q9H2A % 7% % 14% 14% Cystatin-B P % 74% % 13% % Dickkopf-related protein 1 O % 65% % 15% 14% Endothelial cell-specific molecule 1 Q9NQ % 57% % % 11% Eosinophil cationic protein P % 4% % 22% 24% Epidermal growth factor P % 0% % 9% 5% E-selectin P % 7% % 13% 13% Fatty acid-binding protein, adipocyte P % 2% % 14% % Fibroblast growth factor 23 Q9GZV % 5% % 21% 14% Follistatin P % 2% % 13% 14% Fractalkine P % 7% % 14% 22% Galanin peptides P % 5% % 17% 9% Galectin-3 P % 7% % % 13% Growth hormone P % 90% % 14% 15% Growth/differentiation factor 15 Q % 7% % 11% 14% Heat shock 27 kda protein P % 35% % 13% 13% Heparin-binding EGF-like growth factor Q % 4% % 14% 11% Hepatocyte growth factor P % 5% % % % Interleukin-1 receptor antagonist protein P % 0% % 14% % Interleukin-4 P051 NR NR NR NR NR % 11% 13% Interleukin-6 P % 9% % % 7% Interleukin-6 receptor subunit alpha P % 7% % 14% % Interleukin- P % 3% % % % Interleukin- Q % 0% % 11% % Interleukin-1 Q % 9% % % 14% Interleukin-27 QNEV9 Q14213 pg/ml 9 6 9% 67% % 19% 22% Kallikrein-6 Q % 2% % 22% 11% Kallikrein-11 Q9UBX % 97% % 19% 9% Lectin-like oxidized LDL receptor 1 P % 2% % % 9% Leptin P % 114% % % 21% Macrophage colony-stimulating factor 1 P % 96% % % 14% Matrix metalloproteinase-1 P % 64% % 31% 51% log 6 Proseek Multiplex CVD I Data Package

7 Signal-to-background (2 ddcq ) Sample types Analytical measurement Precision Relative 2 ddcq to EDTA plasma Target UniProt No ACD EDTA Heparin ACD Heparin LOD LLOQ ULOQ Hook Range Intra-assay Inter-assay Inter-site Matrix metalloproteinase-3 P % 76% % 20% % Matrix metalloproteinase-7 P % 414% % % 15% Matrix metalloproteinase- P % 4% % 15% 1% Matrix metalloproteinase- P % 1% % 39% 26% Melusin Q9UKP3 NR NR NR NR % 21% 31% Membrane-bound aminopeptidase P O % 75% % 22% % Monocyte chemotactic protein 1 P % 72% % 1% 26% Myeloperoxidase P % 1% % 1% % Myoglobin P % 5% % 17% % Natriuretic peptides B P60 NR NR NR NR NR NR NR NR NR NR NR NR NR NF-kappa-B essential modulator Q9Y6K % 52% NR NR NR NR NR % 20% 15% N-terminal pro-b-type natriuretic peptide NR NR NR NR NR NR NR NR Osteoprotegerin O % 74% % 11% 7% Ovanrian cancer-related tumor marker CA 5 QWXI % 6% NR NR NR NR NR % 20% 21% Pappalysin-1 Q NR 6% NR % 21% 5% Pentraxin-related protein PTX3 P26022 NR 2 NR NR NR % 20% 14% Placenta growth factor P % 73% % 13% 7% Platelet endothelial cell adhesion molecule P % 79% % % 13% Platelet-derived growth factor subunit B P % 56% % 14% 17% Prolactin P % 79% % 14% 9% Protein S0-A P % 206% % 22% 30% Proteinase-activated receptor 1 P % 4% NR NR NR NR NR 6% 14% 17% Proto-oncogene tyrosine-protein kinase Src P % 45% % % 15% P-selectin glycoprotein ligand 1 Q % 57% % 20% 30% Receptor for advanced glycosylation end products Q % 46% % 13% % Renin P % 0% % 13% 9% Resistin Q9HD % 75% % 1% 21% SIR2-like protein QIXJ % 27% % 20% 22% Spondin-1 Q9HCB % 31% % 13% 13% ST2 protein Q % 75% % 14% 13% Stem cell factor P % 95% % 13% % Thrombomodulin P % 92% % 14% 13% TIM-1 Q96D % 90% % 14% 13% Tissue factor P % 91% % % 17% Tissue-type plasminogen activator P % 36% % 14% 23% TNF-related activation-induced cytokine O % 5% % % 13% TNF-related apoptosis-inducing ligand P % 9% % 15% % TNF-related apoptosis-inducing ligand receptor 2 O % 2% % 15% % Tumor necrosis factor receptor 1 P % 96% % 11% 7% Tumor necrosis factor receptor 2 P % 5% % 14% 14% Tumor necrosis factor receptor superfamily member 5 P % 93% % % 14% Tumor necrosis factor receptor superfamily member 6 P % 6% % % 13% Tumor necrosis factor ligand superfamily member 14 O % 130% % 25% 17% Urokinase plasminogen activator surface receptor Q % 94% % % 9% Vascular endothelial growth factor A P % 77% % 13% % Vascular endothelial growth factor D O % 111% % 13% 11% pg/ml log Proseek Multiplex CVD I Data Package 7

8 2.3 PRECISION REPEATABILITY Intra-assay variation (within-run) was calculated as the mean coefficient of variation (% CV) for 7 individual samples, within each of the 9 separate runs during the validation studies. Inter-assay variation (between-run was calculated as the mean coefficient of variation (% CV), for the same 7 individual samples, between the 9 separate runs during the validation studies. Variation calculations were assessed on linearized values for 90 out of 92 analytes. Assays with values below limit of detection were not reported, see Table 1. Across 90 assays, the mean CV intra-assay and interassay variations were observed to be % and 15%, respectively. The distribution of both inter-assay and inter-assay variations per assay is shown in Figure 4. The mean % CV value in the first analysis ranged from 6% to 9% intra-assay. The mean % CV ranged from 13% to 17% inter-assay, and % to % inter-site, here shown in direct comparison to Olink Bioscience in Figure 5. Overall, the Proseek Multiplex CVD I showed very good reproducibility and repeatability with average intersite variation of 15%. Intra: % Inter: 15% % % % of protein biomarkers >40 % CV Fig 4. Distribution of intra-assay and inter-assay variations of Proseek Multiplex CVD I Intra Inter REPRODUCIBILITY Inter-site variation (between-site) was also investigated during the validation in a β-site study, to estimate the expected variations in values between different laboratories, with different operators and using different equipment. Seven individual samples were distributed to each site together with Proseek Multiplex CVD I reagent kits. Each site was instructed to perform the analysis of the 7 individual samples according to the same run design. Each site was also asked to perform two independent runs. The overall design of the β-site study enabled the estimation of both the intra-assay and inter-assay variations for 3 sites including Olink Bioscience, and the inter-site variation for each site, here shown in Figure 5. β 1 Intra 1: 6% Intra 2: 5% Inter: 17% β 2 Intra 1: 9% Intra 2: 5% Inter: 13% Fig 5. Validation of the Proseek Multiplex CVD I at 2 (β1-β2) different laboratories. Larger boxes shows intra-assay and interassay variations for each site and small boxes represent the inter-site run variations in direct comparison to Olink Bioscience. 2.4 ANALYTICAL SPECIFICITY ENDOGENOUS INTERFERENCE Endogenous interference from heterophilic antibodies, e.g. HAMA, and rheumatoid factor are known to cause problems in immunoassays. To evaluate the potential impact of this specific interference, a special mismatch system was designed. The only way to generate a signal here is by antibody probe pairs being brought into proximity, by cross-binding substances other than antigens, e.g. heterophilic antibodies and similarly acting rheumatoid factor. Two different mismatched probe pairs of varying antibody host species origin were designed and evaluated with a Heterophilic Assessment Panel from Scantibodies Laboratory Inc. (part no. 3KG027) and two sets of samples known to contain rheumatoid factor (< International Units/mL (IU/mL)) and rheumatoid arthritis ( arbitrary units (AU)). No interference could be detected for any of the panel samples, indicating a sufficient blocking ability in all assays in the Proseek Multiplex CVD I Proseek Multiplex CVD I Data Package

9 Table 2. Performance characteristics. Endogenous interference was performed by addition of hemolysate, lipids and bilirubin in plasma EDTA matrix. Reported are the highest tested concentrations without impact on assay performance. Endogenous interference Endogenous interference g/l g/l Targets 1-46 Hemolysate Lipids Bilirubin Targets Hemolysate Lipids Bilirubin Adrenomedullin Matrix metalloproteinase Agouti-related protein Matrix metalloproteinase Angiopoietin-1 receptor Matrix metalloproteinase Beta-nerve growth factor Matrix metalloproteinase Caspase Melusin Cathepsin D Membrane-bound aminopeptidase P Cathepsin L Monocyte chemotactic protein C-C motif chemokine Myeloperoxidase C-C motif chemokine Myoglobin C-C motif chemokine Natriuretic peptides B CD40 ligand NF-kappa-B essential modulator Chitinase-3-like protein N-terminal pro-b-type natriuretic peptide C-X-C motife chemokine Osteoprotegerin C-X-C motif chemokine Ovanrian cancer-related tumor marker CA C-X-C motif chemokine Pappalysin Cystatin-B Pentraxin-related protein PTX Dickkopf-related protein Placenta growth factor Endothelial cell-specific molecule Platelet endothelial cell adhesion molecule Eosinophil cationic protein Platelet-derived growth factor subunit B Epidermal growth factor Prolactin E-selectin Protein S0-A Fatty acid-binding protein, adipocyte Proteinase-activated receptor Fibroblast growth factor Proto-oncogene tyrosine-protein kinase Src Follistatin P-selectin glycoprotein ligand Fractalkine Receptor for advanced glycosylation end products Galanin peptides Renin Galectin Resistin Growth hormone SIR2-like protein Growth/differentiation factor Spondin Heat shock 27 kda protein ST2 protein Heparin-binding EGF-like growth factor Stem cell factor Hepatocyte growth factor Thrombomodulin Interleukin-1 receptor antagonist protein TIM Interleukin Tissue factor Interleukin Tissue-type plasminogen activator Interleukin-6 receptor subunit alpha TNF-related activation-induced cytokine Interleukin TNF-related apoptosis-inducing ligand Interleukin TNF-related apoptosis-inducing ligand receptor Interleukin Tumor necrosis factor receptor Interleukin Tumor necrosis factor receptor Kallikrein Tumor necrosis factor receptor superfamily member Kallikrein Tumor necrosis factor receptor superfamily member Lectin-like oxidized LDL receptor Tumor necrosis factor ligand superfamily member Leptin Urokinase plasminogen activator surface receptor Macrophage colony-stimulating factor Vascular endothelial growth factor A Matrix metalloproteinase Vascular endothelial growth factor D 15g Proseek Multiplex CVD I Data Package 9

The potential impact of certain known interfering serum and plasma components was evaluated by using serial dilutions of bilirubin, hemolysate, and lipids, respectively in EDTA plasma, as shown in

No interference was detected by addition of lipids while 2 assays were observed to be affected by bilirubin and 23 assays out 92 were altered by hemolysate.

Table 2 shows the highest concentrations without impact on assay performance for each component. A) Hemolysate 24-plex were plotted against the 4-plex, 72-plex and 96-plex for each analyte.

10 The potential impact of certain known interfering serum and plasma components was evaluated by using serial dilutions of bilirubin, hemolysate, and lipids, respectively in EDTA plasma, as shown in Figure 6. These additions represent different patient health conditions and/or sample collection irregularities. No interference was detected by addition of lipids while 2 assays were observed to be affected by bilirubin and 23 assays out 92 were altered by hemolysate. The latter is probably due to actual analyte leaking out from the disrupted blood cells rather than disturbance of the assay mechanism. Table 2 shows the highest concentrations without impact on assay performance for each component. A) Hemolysate 24-plex were plotted against the 4-plex, 72-plex and 96-plex for each analyte. The correlation coefficient R 2 value generated by linear regression analysis reflects the correlation between the multiplex assays. The R 2 values were >0.99 for the different multiplex blocks, as shown in Figure 7, demonstrating the scalability of the system. A) 24-plex (dcq) y = x R² = g/l 7.5 g/l 3.75 g/l 1. g/l 0.94 g/l 0.47 g/l 0.23 g/l Serum plex (dcq) B) Lipids B) y = 1.017x R² = Serum 24-plex (dcq) C) Bilirubin plex (dcq) Serum C) y = 1,025x - 0,427 R² = 0,9971 Fig 6. Endogenous interference. Levels tested for hemolysate were g/l hemoglobin, lipids and bilirubin The highest hemolysate concentration translates to about % hemolysis. 24-plex (dcq) 2.5 SCALABILITY Assay performance was further evaluated with regard to scalability, meaning the capability of the Proseek Multiplex technology to maintain the same quality of performance irrespective of multiplex grade. A stepwise increase of multiplex grade (24, 4, 72 and 96) was performed and the observed dcq values for the plex (dcq) Fig 7. Scalability of the Proseek Mutliplex technology platform. This experiment was performed using the Proseek Multiplex Oncology I panel. Human serum samples were analyzed with a 24-plex, 4-plex and 72-plex assay and the complete Proseek Mutliplex Oncology I panel. The observed dcq (log2) values were plotted, and the correlation coefficient R 2 value was generated by linear regression. Proseek Multiplex CVD I Data Package

11 3. References 1. Lundberg M, Eriksson A, Tran B, Assarsson E and Fredriksson S. Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of owabundant proteins in human blood. Nucleic Acid Res 6 June (2011). doi:.93/nar/gkr424 Proseek Multiplex CVD I Data Package 11

12 This product is for research use only. Not for use in human diagnostic or therapeutic procedures. This product includes a license for non-commercial use of Proseek products. Commercial users may require additional licenses. Please contact Olink Proteomics AB for details. There are no warranties, expressed or implied, which extend beyond this description. Olink Proteomics AB is not liable for property damage, personal injury, or economic loss caused by this product. The following trademarks are owned by Olink AB: Olink, Olink Bioscience and Proseek. This product is covered by several patents and patent applications including US 6,511,09, US 7,306,904, and related US and foreign patents. This product is sold under license from PHRI Properties, Inc. and may be used under PHRI Properties patent rights outside the field of human in vitro diagnostics. Components in the Proseek Multiplex Probe Kit utilise Lightning-Link technology and are provided under license from Innova Biosciences. 20 Olink Proteomics AB. All third party trademarks are the property of their respective owners. 0969, v1.3, Olink Proteomics Dag Hammarskjölds v. 52B SE Uppsala, Sweden

Data Package. Multiplex Oncology I 96 96

Data Package. Multiplex Oncology I 96 96 Data Package Multiplex Oncology I 96 96 Table of contents 1. Introduction 3 1.1 Technology 3 1.2 Data analysis 3 2. Performance characteristics 4 2.1 Sample types 4 2.2 Analytical Measurement 4 Detection