Comercial Rafer in Zaragoza

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1 Comercial Rafer in Zaragoza ~A novel affinity method to isolate and identify intact Exosomes~ Taku Funakoshi (Mr.) Wako Chemicals GmbH Laboratory Chemicals Division Manager

2 Research fields of Exosome Products 1% 1% 3% 6% 3% 9% 5% 12% 5% 3% 52% Medicine Dentistry Engineering Agriculture Pharmacy Science Company(contract servies) Company(diagnosis) Company(Pharmaceutical) Company(food) Public Authorities Customers in pharmaceutical companies and medicine will be main targets. Because they often focus on development of the diagnostic agent in liquid biopsy and research of specific marker in extracellular vesicles. 1

3 Publication of Exosome Isolation Kit PS PS affinity method was published at Scientific Reports on 23 September The Scientific Reports have a high impact factor than Plos One. 2

4 Publication of Exosome Isolation Kit PS A lot of the research data were obtained by Wako staff. MagCapture TM Exosome Isolation Kit PS was described in a section of Methods. 3

5 Wako s Products for microrna research Novel technology MagCapture TM Exosome Isolation Kit PS microrna and Ago-Subfamily Proteins 1. microrna Experiment Flow 2. Summary of Ago Immunoprecipitation 3. Anti-Ago Monoclonal Antibody Series 4. microrna Isolation Kit Series 4-1. MagCapture TM microrna Isolation Kit (magnetic beads) 4-2. microrna Isolation Kit (silica-based beads) 5. Total RNA Purification 5-1. microrna Extractor SP Kit etc 4

6 Description of Conventional Method Precipitation by reagent (Polymer) 10% Affinity purification by antibody 3% Ultracentrifugation 87% Fig. The percentage of isolation method reported in article Isolation Method Advantage Problem Ultracenrifugation Standard Long time and complicated protocol Difficult to use multi-sample Contamination (High background) Low reproducibility Precipitation w/ reagent (polymer) Easy-to-use Contamination (Too high background) Affinity purification w/ Magnetic beads High purity Low recovery of Exosomes Depending on a performance of monoclonal antibody and population of target antigens on Exosome surfaces. Denaturation purification (Not intact Exosome purification) 5

7 Centrifugation Problem of conventional method 1. Ultracentrifugation Good No Good 110,000xg, 70min ppt +PBS 110,000xg, 70min +PBS 110,000xg, 70min ppt:exosome Low Purity Low throughput Low reproducibility 2. Density gradient fractionation High Purity Low throughput Low reproducibility Sucrose affect exosome activity 6

8 Problem of conventional method 3. Polymer-Based Precipitant Good No Good Easy and Fast Precipitated exosome is not used for down-stream experiments. (due to low purity) Such as MS analysis. Available from some companies. 4. MAb-immobilized magnetic beads Easy operation Denaturation agent is required for elution (No Intact condition) Depending on population of exosome surface antigens Low purification efficiency 7

9 Phospholipid on the membrane surface of Exosome Scheme of molecules on/in Exosome The ratio of phospholipids in membrane (cell membrane) (exosome fraction) Phosphatidylserine PC: phosphatidylcholine SM: sphingomyelin PE: phosphatidylethanolamine PS: phosphatidylserine Mittelbrunn et al., Nature Reviews Molecular Cell Biology (2012) Trajkovic et al., SCIENCE (2009) We targeted phosphatidylserine (PS) on the membrane surface of extracellular vesicles. 8

10 Novel technology PS Affinity Method by a novel molecule patent applied for Affinity method for phosphatidylserine (PS) on the membrane surface of EVs Principle Metal ion Magnetic beads PS Chelator (metal ion removal) Magnetic beads Extracellular Vesicles Extracellular Vesicles Can purify High Purity Extracellular Vesicles Can purify Intact Extracellular Vesicles High Reproducibility Easy Operation by kit (about 3.5hours) High Yield than affinity way by antibody Easy to use multi-sample (No need of ultracentrifugation) Reference: Scientific Reports 6, A novel affinity-based method for the isolation of highly purified extracellular vesicles Wataru Nakai, Takeshi Yoshida, Diego Diez, Yuji Miyatake, Takahiro Nishibu, Naoko Imawaka, Ken Naruse, Yoshifusa Sadamura & Rikinari Hanayama 9

11 Technical Info.1 Performance Comparison with Precipitation way Electron microscopic and NanoSight analysis of obtained exosome K562 cell culture Sup. (serum-free) MagCapture TM Ultracentrifugation Polymer-base Precipitation TEM NTA Size Distribution: mean 106 ± 4.1 nm Size Distribution: mean 136 ± 3.5 nm Size Distribution: mean 183 ± 4.4 nm MagCapture purified many particles around nm in size. Numerous extracellular vesicles can be seen by electron microscopy. 10

12 Technical Info.2 Amount Comparison of Exosome isolated from Healthy Human Serum Recovered amount of Marker protein Comparison of amount of mirna (kda) M Silver stain CD CD MagCapture Ultracentrifugation Comparison to affinity method by antibody Lane 1: Control (beads only) Lane 2: MagCapture Lane 3: Ultracentrifugation CD9 CD63 Lane1 :Ultracentrifugation Lane2 :MagCapture Lane3 :anti-cd9 antibody affinity Lane4 :anti-cd63 antibody affinity Lane5 :anti-cd81 antibody affinity Lane6 :anti CD9, CD63, CD81, EpCAM antibody Mix MagCapture TM purified exosomes and micrornas more efficient than ultracentrifugation and affinity way by antibody. 11

13 Technical Info. 3 Comparison of marker protein derived from 150μl of cell culture sup. Serum-free <K562 cell culture supernatant> 10% FBS (Exosome-depleted) added Western Blotting Western Blotting (kda) Silver Staining CD63 Flotillin-2 Lamp-1 M (kda) Silver Staining CD63 Flotillin-2 Lamp-1 M * Lane 1: MagCapture (PS affinity method) Lane 2: Ultracentrifugation Lane 3: Polymer precipitation * Non-specific band Exosome recovery is higher than ultracentrifugation. 12

14 Technical Info. 4 Comparison of marker protein / 200ng of total protein Serum-free <K562 cell culture supernatant> 10% FBS (Exosome-depleted) added Western Blotting Western Blotting Silver Staining CD63 Flotillin-2 Lamp-1 Silver Staining CD63 Flotillin-2 Lamp-1 (kda) M (kda) M Lane 1: MagCapture (PS affinity method) Lane 2: Ultracentrifugation Lane 3: Polymer precipitation MagCapture showed highest purity 13

15 Technical Info. 5 Comparison of human-derived peptides identified by MASS analysis <K562 cell culture supernatant, 10% FBS(Exosome-depleted) added> The rate of human-derived peptides Detection rate of human-derived peptides 13.9% 63.6% 84.6% MASS analysis data was provided by Dr. R. Hanayama at Graduate School of Medicine, Kanazawa University and Dr. Nakai at ifrec Osaka University MagCapture is suitable for MS analysis for identification of surface antigens of exosomes. 14

16 Technical Info. 6 Amount Comparison of Exosome isolated from helthy human urine Comparision of marker proteins Comparison of particle size (NTA) (kda) M Lane 1: Polymer Precip. Lane 2:Ultracentrifugation Lane 3:MagCapture MagCapture (PS affinity) Size Distribution: mean 107 ± 1.2 nm Polymer precip. Ultracentrifugation CD9 Size Distribution: mean 176 ± 1.6 nm Size Distribution: mean 191 ± 9.4 nm MagCapture also recovered high-purity exosome from urine sample 15

17 Summary of MagCapture TM Exosome Isolation kit PS Comparison with other purification method Methods Exosome Purity Exosome Recovery Recovery Intact vesicles MagCapture TM ( PS Affinity) Yes Ultracentrifugation Polymer-based Precipitation Affinity Method using antibody Yes Yes No Can purify High Purity Extracellular Vesicles Can purify Intact Extracellular Vesicles High Reproducibility Easy Operation by kit (about 3.5hours) High Yield than affinity way by antibody Easy to use multi-sample (No need of ultracentrifugation) Concentrating cell culture media beforehand, compatible with large scale (Data is not shown, please see reference data. ) 16

18 Product Information MagCapture TM Exosome Isolation Kit PS Code No. Product package tests MagCapture TM Exosome Isolation Kit PS tests Exosome isolation magnetic beads are reusable up to 4 times. (Details is written in manual) The kit components are included enough as considering recycling case. Kit composition of MagCapture Exosome Isolation Kit PS(10tests) 1Streptavidin Magnetic Beads 600μL 1 tube 2Biotin-labeled Exosome Capture 100μL 1 tube 3Exosome Capture Immobilizing Buffer 35mL 1 tube 4Exosome Binding Enhancer(500 ) 500μL 1 tube 5Washing Buffer 75mL 2 tubes 6Exosome Elution Buffer 5mL 1 tube 7Reaction Tubes 22 tubes Sample: cell culture supernatant, serum, urine This kit is NOT suitable for plasma treated with chelate agents such as EDTA and citric acid. Storage:

19 Our Goal Exosome isolation method (Gold Standard) Ultracentrifugation Switch! MagCapture TM Exosome Isolation Kit PS (New Gold Standard) 18

20 Exosomes + Large EVs Exosomes Large EVs Sample pretreatment (condition of centrifugation) Sample(cell culture sup.) Exosome + Large EVs microvesicles nm Pellet Sup. 300xg, 5min Size Distribution: mean 150 ± 2.3 nm exosomes nm 1,200xg, 20min Pellet Sup.1 (1,200xg sup.) Purification step Exosome van der Grein SG et al., Front Immunol.(2014) 10,000xg, 30min Sup.2 (10,000xg sup.) Pellet (10,000xg pellet) Purification step Purification step Pretreatment condition is optional according to purpose. Large EVs Size Distribution: mean 119 ± 0.7 nm Size Distribution: mean 219 ± 14.7 nm (kda) M Lamp-1 19

21 1 st purification 2 nd purification 3 rd purification Optional sample pretreatment (concentration) Supernatant (Capacity:~50mL) 1,200xg sup. or 10,000 g sup. Concentrated by ultrafiltration (cut-off: 100KDa) 1mL (~50-fold concentrated) 50mL 5mL (10-fold concentrated) 50mL 1mL (50-fold concentrated) Purification step 3 Flotillin-2 2 nd purification mean that using sup. after 1 st purification. 3rd purification mean that using sup. after 2 nd purification. In the case of large volume (-50ml), concentration make it possible to collect exosome efficiently. 20

22 Product Information Analyzing RNAs after Extracellular Vesicle Purification microrna Extractor SP Kit Code No. Product package microrna Extractor SP Kit 50 tests Exosome observation NanoSight nanoparticle analyzing system 21

23 Scheme of PS affinity ELISA Anti-exosome surface protein Antibody (Primary antibody) Exosome HRP-conjugated Anti-mouse IgG (Secondaty antibody) Absorbance detection Tim4-coated plate 22

24 Comparison of conventional ELISA (using antibodies) with PS affinity ELISA Sample:K562 cells-derived exosomes(ultracentrifuge Purification) Primary antibody:cd63 Ab 23

25 Abs.(450nm-620nm) Abs.(450nm-620nm) Performance of PS affinity-exosome ELISA 1. Evaluation of detection limit We evaluated detection limit of each sample. Sample is purified exosome derived from culture supernatant of K562 and COLO201 with MagCapture TM Exosome Isolation Kit PS. <Exosom ELISA> Purified K562 exosomes (Anti-CD63 MAb) Purified COLO201 exosomes (Anti-CD63 MAb) Exosomal protein (pg) Exosomal protein (pg) Detection limit(a SD):50pg Quantitative limit(a 0 +10SD) :134pg Detection limit(a SD):11pg Quantitative limit(a 0 +10SD) :34pg Detection limit of Exosome ELISA K562 exosomes 50pg, COLO201 exosomes 11pg Wako ELISA detected exosomes thousand times as sensitive as WB. Confidential 24

26 Sensitivity of exosome detection Sample:COLO201 and K562 cells-derived exosomes (PS affinity purification) Primary antibody:cd63 Ab Detection limit(blank+3.3sd) Determination limit(blank+10sd) COLO201 cell culture supernate 0.11ng/mL(11pg) 0.34ng/mL(34pg) K562 cell culture supernate 0.50ng/mL(50pg) 1.34ng/mL(134pg) COLO201 cellsderived exosomes (kda) CD

27 Linearity of exosomes in culture media Standard:K562 and COLO201 cells-derived exosomes (PS affinity purification) Sample:K562 and COLO201 cell culture supernate Primary antibody:cd63 Ab K562 Dilution Assay value Expected value % of expected Ratio Factor ( ) (ng/ml) (ng/ml) 1: : : : : COLO201 Dilution Assay value Expected value % of expected Ratio Factor ( ) (ng/ml) (ng/ml) 1: : : : :

28 Detection of individual normal human serum Normal human serum (1/100) Sample:Normal human serum(1/100) Primary antibody:cd63 Ab or BLANK(TBS- T) Normal human serum-derived exosomes 40ng 20ng 10ng Sample:Normal human serum-derived exosomes (PS affinity purification) 40, 20, 10ng Primary antibody:cd63 Ab 27

29 Abs.(450nm-620nm) Comparison of ExoTEST (HansaBioMed) with PS affinity ELISA Plate:Tim4-coated plate or ExoTEST plate Sample:COLO201 cells-derived exosomes (PS affinity purification) Primary antibody:cd9 Ab(BD Biosciences) (our recomendation) Secondaty antibody: Secondary Antibody HRP-conjugated Anti-mouse IgG(100 ) 10 Purified COLO201 exosomes (Anti-CD9 MAb) TIm4-coated 和光プレート plate Hansa ExoTEST プレート plate Exosomal protein (ng) Wako Plate detected exosomes hundred times as sensitive as ExoTEST. 28

30 Comparison of ExoELISA(SBI) with PS affinity ELISA Sample:COLO201 cells-derived exosomes (PS affinity purification) Primary antibody:cd63 Ab 29

31 Expression analysis of exosome marker protein by WB and ELISA Sample:Each cell-derived exosomes(ps affinity purification) Primary antibody:cd9 Ab, CD63 Ab and CD81 Ab Purified Exosome 150ng CD9 Purified Exosome 1ng CD63 CD81 30

32 Product information Code No. Name of product Size PS Capture TM Exosome ELISA Kit (Anti-mouse IgG POD) 96 tests PS Capture TM Exosome ELISA Kit (Anti-mouse IgG POD) (96 tests) 1Exosome Capture 96 well plate 8well 12strip 2Reaction / Washing Buffer(10 ) 50mL 2 vials 3Exosome Binding Enhancer(100 ) 10mL 1 vial 4Control Primary Antibody Anti CD63(100 ) 120μL 1 vial 5Secondary Antibody HRP-conjugated Anti-mouse IgG(100 ) 120μL 1 vial 6TMB Solution 12mL 1 vial 7Stop Solution 12mL 1 vial 8Plate Seal 4 sheets Measurable sample:cell culture supernatant, serum and EDTA-treated Plasma Storage:2-8 31

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