Is There A Need For Is There A Need For Recombinant Collagenase In - - PowerPoint PPT Presentation

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Is There A Need For Is There A Need For Recombinant Collagenase In - - PowerPoint PPT Presentation

Nov 08, 2022 886 likes •1.06k views

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,

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SLIDE 1

Presentation ICR Islet Workshop, Duarte, July 2005 -

  • Dr. Manfred Kurfuerst

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 Huettler1, Olaf Friedrich2, Christian Raemsch2

1SERVA Electrophoresis GmbH, Uetersen, Germany; 2Nordmark Arzneimittel GmbH & Co. KG, Uetersen.

ICR ICR Islet Islet Workshop, Duarte, 2005 Workshop, Duarte, 2005

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SLIDE 2

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)

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SLIDE 3

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)

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SLIDE 4

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 / Neutral Protease NB Liberase P value IEQ (103) 354.4 ± 65 271.9 ± 147 0.08 IEQ/g pancreas (103) 4.02 ± 1.20 2.36 ± 1.35 <0.05 IEQ/Islet number ratio 0.83 ± 0.2 0.51 ± 0.1 <0.01 Purity (%) 62 ± 6 53 ± 8 <0.05 Tissue volume (ml) 3.8 ± 1.6 4.2 ± 2.1 0.3 Islet Viability (%) 96 ± 1 94 ± 2 0.1 Stimulation Index 4.3 ± 1.5 3.8 ± 3.2 0.3 Apoptosis after 12h culture 1.25 ± 0.03 7.25 ± 1.23 <0.05

Bucher, P., Berney, T. et al. (2005) Transplantation 79(1), 91-97.

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SLIDE 5

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?

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SLIDE 6

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
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SLIDE 7

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)
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SLIDE 8

Presentation ICR Islet Workshop, Duarte, July 2005 -

  • Dr. Manfred Kurfuerst

Production of Collagenase N: Fermentation

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SLIDE 9

Presentation ICR Islet Workshop, Duarte, July 2005 -

  • Dr. Manfred Kurfuerst

Production of Collagenase NB: Chromatography System

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SLIDE 10

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

21.5 31 36.5 55.4 66.3 97.4 116.3 [kDa]

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SLIDE 11

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

  • 50

50 100 150 200 250 1 2 3 4 5 6 7 8 9 10

time [min] Abs [280 nm]

NB 1 GMP #1-1 NB 1 GMP #1-2 NB 1 GMP #2-1 NB 1 GMP #2-2 NB 1 GMP #3-1 NB 1 GMP #3-2 NB 1 GMP #4-1 NB 1 GMP #4-2 Reference-1 Reference-2

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SLIDE 12

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: Final results after 12 months for solubilized enzymes in PE cups: 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 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

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SLIDE 13

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)

0,0 20,0 40,0 60,0 80,0 100,0 120,0 25 °C 5 °C

Storage conditions PZ activity [%]

Starting 3 months 6 months 9 months 12 months 18 months

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SLIDE 14

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)

0,0 20,0 40,0 60,0 80,0 100,0 120,0 140,0 +25 °C / 60 % r.h. +5 °C

  • 25 °C
  • 85 °C

Storage conditions DMC activity [%]

Starting 1 month 6 months 12 months 18 months

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SLIDE 15

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