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• Tissue dissociation

Islet purification Organ procurement Donor criteria Organ transport Islet characterization Islet functional analysis Cell transplant

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TISSUE DISSOCIATION

• Islet purification

Organ procurement Donor criteria Organ transport Islet characterization Islet functional analysis Cell transplant

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TISSUE DISSOCIATION

• Organ procurement

Donor criteria Organ transport

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%6 77 Low: components purified then remixed High: affected by fermentation conditions Lot to lot variation < 50 300-7000 Endotoxin U/mg 3 (C1, C2, NP) > 15 Degraded enzymes Enzyme Activities Liberase

• Crude Collagenase

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Isolation based on CI enzyme activity

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Unanswered for HI: assay used to assess collagenase activity, purity of collagenase???? Analytical anion exchange HPLC & CDA assay provides better assessment

• f stability

Product stable for 2 years at -20º Wunsch assay problematic: biased to C2 and no difference in specific activity for intact or degraded C2 VitaCyte’s recent observation confirm value of analytical anion exchange HPLC VitaCyte developed CDA assay detects C1 enzyme activity Wunsch & anion exchange HPLC assays to assess collagenase activity Unanswered: Kin’s report indicates lower human islet yield with 50:50 mixture, best with 60:40 mixture Brandhorst rat islet data show best result with 50:50 blend Optimal C1:C2 ratio is 60:40 mixture R&D confirmed prior reports in the literature Subsequent studies with islets mixed: rat islets Wolters showed C2 predominant role, Brandhorst showed C1 + C2 required, no islet recovered with C1 or C2 alone with NP Report from Lakey’s lab indicated quality of C1 critical to success of islet isolation C1 + C2 + neutral protease key enzymes for tissue dissociation

Status in 2009 Presumptions in 1994

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Page 12 S3a 98 S3b 111 S3 126 S1 676 S2 97 2 24

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C1 Col G 116 kDa

S1 670 S2a 95 S2b 89

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C2 Col H 116 kDa

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Assay specific for collagenase “active” in tissue dissociation, preferentially detects intact C1

Intact C1 collagenase specific CDA 10x > intact C2 or degraded C1 (100 kDa)

• Degraded C2 (100 kDa) inactive

Importance of “active” enzyme confirmed by incubation of purified C1 and C2 with 2% (w/w) chymotrypsin for 6 hours at 37ºC

• Led to 84% and 97% loss of C1 and C2

specific CDA, respectively, while only 7% loss of C2 specific Wunsch activity when compared to untreated purified C1 or C2

• Active collagenase must have at least 1

collagen binding & catalytic domain to be detected by the CDA assay

FITC fibrils resistant to trypsin activity

Specific CDA of rec bovine trypsin at 5.4 molar excess over substrate was < 0.1% and < 1.0 % of specific CDA of purified C1 and C2, respectively

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Assay in triplicate Detect fluorescent units (FU)

Assay Procedure

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Fractionation of 5 mg of purified 60:40 C1:C2 mixture over 1 mL Mono-Q anion exchange column, specific CDA of starting material = 37,494 CDA U/mg protein

C2 C1 C1b C1c

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100 Fx 34 100 80.1 54.6 1 102.5 19.9 45.4 100 100 2 115.5 CI 40.1 14 81.5 13.5 103.8 59.9 86 100 86.5 1 118.4 CII Fx 35 Fx 33 Fx 32 Fx 31 Fx 30 Fx 23 Fx 22 Fx 21 Fx 20 CBD Class & kDa

0.2 0.4 0.6 0.8 1 1.2 1.4 15 20 25 30 35 40 Fraction Number A280/mL 20000 40000 60000 80000 100000 120000 140000 Collagenase Specific Activity A280/mL Collagenase specific activity

% Protein per Lane on SDS-PAGE Gel

Intact C1 2 CBD

Analysis of fractions from HPLC correlated A280 to collagen degradation activity and predominant molecular species in fraction using SDS-PAGE

CBD = collagen binding domain

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Intact C1 C1b single CBD C1c single CBD Intact C2 single CBD 116 kDa C1b single CBD 100 kDa C1c single CBD 100 kDa C2 Intact C1 double CBD 116 kDa

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Supplement to accelerate digestion > 80,000 NP U/mg 003-1000 CIzyme B. Polymyxa Protease Supplement to accelerate digestion > 200,000 NP U/mg 002-1000 CIzyme Thermolysin Adipose derived cells, hepatocytes 7,000-15,000 CDA U/mg 001-3000 CIzyme Collagenase LA Porcine & monkey islets, human & porcine hepatocytes 15,000 to 30,000 CDA U/mg 001-2000 CIzyme Collagenase MA Human islets > 30,000 CDA U/mg 001-1000 CIzyme Collagenase HA Application Enzyme Specific Activity Units/mg protein Catalog # Product

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Intact C1 Single CBD C1b Single CBD C1c ")##:<G47,@2J8 @)##.:47,@2J8 @)##.4.7:G,@2J8

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")##<:.4<7<G.,@2J8 @)##<:9.4.7..,@2J8 @)##<<G.4.97:,@2J8 Intact C1 Single CBD C1b Single CBD C1c

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Blend Enzymatic Activities

10000 20000 30000 40000 50000 Roche Liberase HI Roche Liberase PI Serva NB1 Sigma Type XI VitaCyte CIzyme Collagenase CDA Specific Activity (U/mg)

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CDA 18873 U/mg Endotoxin 3.21 EU/mg

Intact C1 Single CBD C1b Single CBD C1c C2

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CDA 9024 U/mg Endotoxin 2.72 EU/mg

Intact C1 Single CBD C1b Single CBD C1c C2

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100,000 200,000 300,000 400,000 500,000 600,000 700,000 800,000

Totel IEQ

Pre-purification IEQ/organ Post-Purification IEQ/organ

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• Define categories to characterize donor and histochemical

composition of the organ

• Matching enzyme composition to organ

Tailored TDEs

• Establish ranges for C1:C2 ratios
• Identify form/dose of C1 required for different applications
• Collagenase: neutral protease ratio
• Other neutral protease?

Optimize Composition

• Recombinant collagenase in place of natural collagenase
• Cost concerns?

Improve Consistency Solution Issue

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