Magdy El Ekiaby, MD Shabrawishi Hospital BTC & HTC Cairo, Egypt

Transcription

1 Magdy El Ekiaby, MD Shabrawishi Hospital BTC & HTC Cairo, Egypt

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3 Are plasma and cryoprecipitate still used? They are still largely used for treatment of hereditary bleeding disorders in many resource limited countries There are a number of clinical indications for the use of these blood components in developed and developing worlds So, increasing i safety of these products is of considerable importance

4 Definition Preparation of virally inactivated: cryoprecipitate, plasma, cryo-poor plasma Virus inactivation is done by solvent and Virus inactivation is done by solvent and detergent using a medical device

5 Objectives Easy-to-use, cost/effective, reliable, and affordable technology It can be Implemented by blood establishments or local service centers with minimum infrastructure investment

6 Project elements GMP production of plasma and its components in blood establishments Development and adaptation of SD virus inactivation procedures into blood establishments Introducing principles of quality assurance, quality control and traceability to ensure effective uniform and safe final product

7 Why SD virus inactivation technology? Solvent-Detergent virus inactivation is a gold standard and a robust technology Long clinical experience with SD-treated products Inactivation of lipid enveloped viruses (most pathogenic TTV) Less risk for transmission of nonenveloped viruses due to the mini-pool size

8 SD mini-pool: current option developed Viral inactivation step:- 1% TnBP + 1% Triton X45, 31 C Solvent-Detergent removal:- 1 oil extraction with Soya Bean Oil, followed by Filtration step to remove residual TnBP & Triton X45, Followed by 0.2 μ filtration step (bacterial filter) Performed in a sterile single use processing system with equipment that is routinely used in blood establishments

9 Equipments Laminar air flow cabinet for aseptic environment Syringe pump for SD addition under controlled flow-rate, volume, and duration Shaker incubator :- Control mixing i of SD & plasma/cryoprecipitate it t under optimal temperature Control mixing of oil and plasma/cryoprecipitate for SD removal under optimal temperature

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12 Reagents TnBP Tit Triton X45 Soya Bean oil

13 The Disposable Interconnected sterile patented bag system

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15 SD-F Cryoprecipitate bag systems Same concept as for plasma 2 main differences Pooling system for low-volume cryo units suspended in 5% glucose-salinesaline Dispensing in 7 8 bags

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17 Plasma and Cryoprecipitate Systems field tests (Cairo)

18 Materials 20 units of recovered ered plasma; 10 plasma bag systems 20 units of apheresis plasma; 10 plasma bag systems 200 units of cryoprecipitate; 5 cryo bag systems

19 Plasma: Functional assays PT (and INR) APTT Clottable fibrinogen Factors II, FV, FVII, FVIII, FIX, FX, FXI, FXIII VWF:Ag & RCo Protein C, protein S Alpha-2 antiplasmin Antithrombin

20 Plasma: Other protein assays Total protein Complement component C3 IgG IgA IgM Albumin Cholesterol Triglycerides

21 Cryo: Functional assays Clottable fibrinogen FVIII VWF:Ag, VWF:RCo, VWF:CB vwf multimers Total proteins

22 Cryo: Functional assays Clottable fibrinogen FVIII VWF:Ag, VWF:RCo, VWF:CB vwf multimers Total proteins

23 Plasma and cryo systems: Other assays TnBP Triton X-45 DEHP (phthalate) SDS-PAGE under non-reduced and reduced conditions Pre-clinical testing: general safety tests in rats

24 Viral validation study To validate the capacity of the technology to inactivate TT lipid - enveloped viruses

25 Plasma protein results

26 RECOVERED PLASMA Start S/D-F Plasma % variation Fibrinogen, mg/ml NS p Factor II, IU/mL NS Factor V, IU/mL <0.01 Factor VII,IU/mL IU/mL NS Factor VIII, IU/mL NS Factor IX, IU/mL NS Factor X, IU/mL NS Factor XI, IU/mL NS Factor XIII, IU/mL NS VWFAg, IU/mL NS VWFRCo, IU/mL NS Protein SIU/ S, IU/mL NS Protein C, IU/mL NS Cairo, 25/03/2008 CONFIDENT IAL

27 RECOVERED PLASMA Start S/D-F Plasma % variation Antithrombin, IU/mL NS Alpha 2-antiplasmin, IU/mL C3, mg/ml NS IgG, mg/ml NS IgA, mg/ml NS IgM, mg/ml NS Albumin, mg/ml NS Protein, mg/ml NS PT, sec < aptt, sec NS Cholesterol, l mg/ml 84.0 ND < Triglycerides, mg/ml <0.01 Cairo, 25/03/2008 p CONFIDENT IAL

28 Apheresis plasma Start SD-F Plasma % variation Fibrinogen, i mg/ml NS Factor II, IU/mL NS Factor V, IU/mL Factor VII, IU/mL NS Factor VIII, IU/mL NS Factor IX, IU/Ml NS Factor X, IU/mL NS Factor XI, IU/mL NS Factor XIII, IU/mL NS VWFAg, IU/mL NS VWFRCo, IU/mL NS Protein S, IU/mL NS p Protein C, IU/mL NS Cairo, 25/03/2008 CONFIDENT IAL

29 Apheresis plasma Start S/D-F plasma % p Antithrombin, IU/mL Alpha 2-antiplasmin,U/mL NS C3, mg/ml NS IgG, mg/ml NS IgA, mg/ml NS IgM, mg/ml NS Albumin, mg/ml NS Protein, mg/ml NS PT, sec <0.05 aptt, sec <0.05 Cholesterol, mg/ml < Triglycerides, mg/ml Cairo, 25/03/2008 CONFIDENT IAL

30 Cryoprecipitate protein results

31 Cryoprecipitate Start cryo S/D-F cryo % p Factor VIII, IU/mL NS Fibrinogen, mg/ml NS Factor XIII, IU/mL < VWF :Ag, IU/mL NS VWF :Rco, IU/mL NS RCo/Ag NS VWF/CB, IU/mL NS CB/Ag < 0.05 %> 15 mers NS > 15 mers / control plasma ratio NS %> 10 mers NS > 10 mers / control plasma ratio NS Anti-A, Anti-B titer 0 0 Cairo, 25/03/2008 CONFIDENT IAL

32 VWF multimers pattern 15 mers 10 mers 5 mers

33 SDS-PAGE results

34 SDS-PAGE Non-reduced Plasma Cryo

35 SDS-PAGE Reduced β γ Plasma Cryo

36 TnBP and Triton X-45 results

37 10000 Solvent/Detergent Treatment After oil extraction Final Product Res sidue of TnB BP (in ppm) Aphersis Type of Plasma Recovered Ti b t l h h t (T BP) id ( ) i h i d Tri-n-butyl phosphate (TnBP) residues (ppm) in apheresis and recovered plasma after solvent / detergent treatment, oil extraction and in final products.

38 Solvent/Detergent Treatment After oil extraction Final Product Resid due of Tr riton X45 (in ppm m) Aphersis Type of Plasma Recovered Fi 2 Tt X45 id ( ) i h i d d l Fig. 2. Trton X45 residues (ppm) in apheresis and recovered plasma after solvent / detergent treatment, oil extraction and in final products.

39 DEHP results

40 residue e in ppm Start After S/D treatment After oil extraction Final product DEHP Apheresis Plasma type Recovered Fi 3 Di(2 th lh l) hth l t (DEHP) id ( ) i h i d Fig. 3. Di(2-ethylhexyl) phthalate (DEHP)residues (ppm) in apheresis and recovered plasma after solvent / detergent treatment, oil extraction and in final products.

41 General safety tests

42 General safety test Toxicity study in rats 4 rats per group (including saline, start plasma and cryo, SD-F plasma and cryo) 6.5 ml/kg body weight* Control of survival, behavior, gain weight, food consumption, water consumption (14 days) * Equivalent to about 50 iu FVIII/kg (cryo)

43 Gain weight - Rat general safety test No significant differences in Body weig ght % Increasing Salin Start plasma Start cryo S/D plasma / oil ext. S/D cryo / oil ext. Plasma / AKS6 Cryo / Cuno Sp32 Plasma / MKP Cryo / MKP 10/100, TnBP/Triton 150/300, TnBP/Triton Days post Treatment Fig. 6. Percent increasing in body weight after single dose injection of start plasma and cryo, S/D treated plasma and cryo after oilextraction, S/D plasma filtered on AKS6, S/D Cryo filtered on Cuno SP32, S/D plasma and cryo filtered on MKP filter (final product).

44 Gain weight - Rat general safety test Cryoprecipitate p Percent increase e in body weight 60 A B C Control SD-F cryo Start cryo Days post-infusion

45 Viral validation study of SD in the disposable system Performed at Texcell/Pasteur Institute (miniature small oval bags) Spiking studies using:- HIV1, relevant virus Pseudo Rabies Virus (PRV) as a model of Herpes Virus Bovine Viral Diarrhea Virus (BVDV) as a recognized model for HCV >4 log reduction of the 3 viruses in two minutes

46 Overall quality of SD-F plasma Excellent recovery: coagulation factors Anticoagulant and protease inhibitors (AT, alpha 2-AP, Protein S, Protein C) Clear, cell free, sterile-filtered plasma TnBP < 10 ppm Triton X-45 < 50 ppm Low DEHP content

47 Cairo, 25/03/2008 CONFIDENT IAL

48 Overall quality of SD-F cryoprecipitate p (1) Concentrated virally-inactivated cryo FVIII = 8 10 iu/ml Fibrinogen = mg/ml VWF:Rco, :CB, :Ag = IU/m multimers > 15 mers and > 10 mers = same as in starting cryo Reduced or negative ABO iso-agglutinins titers

49 Overall quality of SD-F cryoprecipitate p (2) Clear, cell free, sterile-filtered cryo TnBP < 10 ppm Triton X-45 < 50 ppm Low DEHP content

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51 Clinical Use The product can be used in the treatment of the following conditions:- Hemophilia A vwd Fibrinogen deficiency Severe post partum hemorrhage DIC FXIII deficiency i???

52 Next step: Multicentric field tests

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55 In press

56 In press

57 Groups involved in Prototype validations *E Egypt: *F France: Shabrawishi Hospital HPPS Cairo University Hematology * Switzerland laboratory, University VIPS Hospital, Lille * Brazil DTS INSERM Unit 837, Lille