Is There A Need For Is There A Need For Recombinant Collagenase In Recombinant Collagenase In Islet Isolation? Islet Isolation? Manfred Kurfuerst 1,2 , Silke Huettler 1 , Olaf Friedrich 2 , Christian Raemsch 2 1 SERVA Electrophoresis GmbH, Uetersen, Germany; 2 Nordmark Arzneimittel GmbH & Co. KG, Uetersen . ICR Islet Islet Workshop, Duarte, 2005 Workshop, Duarte, 2005 ICR Presentation ICR Islet Workshop, Duarte, July 2005 - Dr. Manfred Kurfuerst
Collagenase in Islet Isolation • Which enzymatic characteristics are essential for a good isolation? Dissociation efficacy of Collagenase Class I and Class II (rat pancreas): II+I >> II > I > Neutral Protease >> no enzyme (Vos-Scheperkeuter et al. (1997) Cell Transplant. 6 , 403-412) Presence of 125 kD band in Collagenase P as indicator for efficient lot (pig) (Chen et al. (2001) Cell Transplant. 10 , 709-716) Ratio II/I = 0.7 to 1.0 optimal with respect to yield, viability, function (rat islets) (Brandhorst et al. (2005) Transplant Proc. 37 (1), 215-6) Dosage of Neutral Protease NB (human) (Brandhorst et al. (2005) Transplant Proc. 37 (1), 241-2) Presentation ICR Islet Workshop, Duarte, July 2005 - Dr. Manfred Kurfuerst
Collagenase in Islet Isolation • Recombinant collagenase (Class I + Class II) Recombinant class I combined with “native” class II result in similar yield and digestion time as native class I and II (Wolters et al. (1995) Diabetes 44 , 227-233) Isolation results with respect to yield, viability and function comparable to Liberase HI (Brandhorst et al. (2003) Diabetes 52, 1143-1146) • Additional protease is essential Neutral Protease ( Clostridium histolyticum ) Thermolysin ( Bacillus thermoproteolyticus ) Dispase ( Bacillus polymyxa ) Presentation ICR Islet Workshop, Duarte, July 2005 - Dr. Manfred Kurfuerst
Islet Isolation Results with Collagenase NB 1 and Neutral Protease NB • Human Islet Isolation Results: Comparison Collagenase NB 1 / Neutral Protease NB and Liberase Collagenase NB 1 / Liberase P value Neutral Protease NB IEQ (10 3 ) 354.4 ± 65 271.9 ± 147 0.08 IEQ/g pancreas (10 3 ) 4.02 ± 1.20 2.36 ± 1.35 <0.05 0.83 ± 0.2 0.51 ± 0.1 IEQ/Islet number ratio <0.01 62 ± 6 53 ± 8 Purity (%) <0.05 3.8 ± 1.6 4.2 ± 2.1 Tissue volume (ml) 0.3 96 ± 1 94 ± 2 Islet Viability (%) 0.1 4.3 ± 1.5 3.8 ± 3.2 Stimulation Index 0.3 1.25 ± 0.03 7.25 ± 1.23 Apoptosis after 12h culture <0.05 Bucher, P., Berney, T. et al. (2005) Transplantation 79(1), 91-97. Presentation ICR Islet Workshop, Duarte, July 2005 - Dr. Manfred Kurfuerst
Collagenase in Islet Isolation � In principle, recombinant and non-recombinant collagenase/protease give good isolation results Problems: Lot-to-lot consistency and stability have to be assured Question: How can these problems be solved? Presentation ICR Islet Workshop, Duarte, July 2005 - Dr. Manfred Kurfuerst
Collagenase in Islet Isolation � Different approaches: • Recombinant enzyme production • Non-recombinant enzyme production according to GMP Presentation ICR Islet Workshop, Duarte, July 2005 - Dr. Manfred Kurfuerst
GMP compliance / documentation • Production compliant to international GMP guidelines � Highly reproducible batches � Validation of production process � Complete production documentation available (Traceability) � Dedicated production facility (no cross contamination possible) � Qualified personnel � Validated and calibrated equipment � Stability Study (compliant to ICH guidelines Q1A for drug substances) Safety issues • � Test for Abnormal Toxicity according to Pharm. Eur. � Virus validation studies � Certificate of Suitability (CoS-TSE) from European Authorities EDQM for fermentation ingredients Filing of Drug Master File (US) , PTA (FR) • Presentation ICR Islet Workshop, Duarte, July 2005 - Dr. Manfred Kurfuerst
Production of Collagenase N: Fermentation Presentation ICR Islet Workshop, Duarte, July 2005 - Dr. Manfred Kurfuerst
Production of Collagenase NB: Chromatography System Presentation ICR Islet Workshop, Duarte, July 2005 - Dr. Manfred Kurfuerst
Lot-to-lot consistency: Collagenase NB 1 GMP Grade Gelatin Zymogram of Collagenase NB 1 GMP Grade [kDa] 116.3 97.4 66.3 55.4 36.5 31 21.5 Presentation ICR Islet Workshop, Duarte, July 2005 - Dr. Manfred Kurfuerst
Lot-to-lot consistency: Collagenase NB 1 GMP Grade RP-HPLC of Collagenase NB 1 GMP Grade 250 200 NB 1 GMP #1-1 NB 1 GMP #1-2 150 NB 1 GMP #2-1 NB 1 GMP #2-2 Abs [280 nm] NB 1 GMP #3-1 100 NB 1 GMP #3-2 NB 1 GMP #4-1 NB 1 GMP #4-2 Reference-1 50 Reference-2 0 0 1 2 3 4 5 6 7 8 9 10 -50 time [min] Presentation ICR Islet Workshop, Duarte, July 2005 - Dr. Manfred Kurfuerst
Stability Data of Collagenase NB 1 GMP Grade and Neutral Protease NB GMP Grade • Stability study according to ICH guidelines (Int. Conference on Harmonisation) Preliminary results after 18 months for lyophilized enzymes in glass vials: Storage temperature Storage duration Collagenase NB 1 +5 °C 18 months Neutral Protease NB -25 °C and -85 °C 18 months +5 °C 6 months Final results after 12 months for solubilized enzymes in PE cups: Storage temperature Storage duration Collagenase NB 1 -25 °C and -85 °C 12 months 50 Pz U/mL in Tris Neutral Protease NB -25 °C and -85 °C 12 months 10 mg/mL in water Presentation ICR Islet Workshop, Duarte, July 2005 - Dr. Manfred Kurfuerst
Stability Data: Collagenase NB 1 GMP Grade Stability of Collagenase NB 1 GMP Grade (Lyophilisate) 120,0 100,0 PZ activity [%] 80,0 Starting 3 months 6 months 60,0 9 months 12 months 40,0 18 months 20,0 0,0 25 °C 5 °C Storage conditions Presentation ICR Islet Workshop, Duarte, July 2005 - Dr. Manfred Kurfuerst
Stability Data: Neutral Protease NB GMP Grade Stability of Neutral Protease NB GMP Grade (Lyophilisate) 140,0 120,0 DMC activity [%] 100,0 Starting 80,0 1 month 6 months 12 months 60,0 18 months 40,0 20,0 0,0 +25 °C / 60 % r.h. +5 °C -25 °C -85 °C Storage conditions Presentation ICR Islet Workshop, Duarte, July 2005 - Dr. Manfred Kurfuerst
Summary • Collagenase NB 1/Neutral Protease NB, Liberase HI and recombinant collagenase show comparable efficacy in islet isolation • Recombinant enzyme production for islet isolation: Three enzymes have to be produced separately (collagenase class I, collagenase class II, neutral protease) in complex expression systems. Lot-to-lot consistency is difficult to achieve • GMP compliant production only for Collagenase NB 1/Neutral Protease NB: - Highly reproducible lots - Stability data available - Safe product - Fulfills all requirements of the authorities Mit freundlicher Genehmigung von Dr. Brandhorst, Uni Gießen Presentation ICR Islet Workshop, Duarte, July 2005 - Dr. Manfred Kurfuerst
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