1. Tomo Yokomizo, Morikazu Miura
Asahi Combination Filter, Sepacell PrimaTM for Prion and Leukocyte Reduction
Asahi Kasei Medical
Tomo Yokomizo, Morikazu Miura
29th October, 2010

2. Asahi Kasei Medical One of major suppliers of LR filter
Many types of LR filter, used all over the world
Nano-filtration technology for virus removal in plasma fractionation, etc.

3. Contents - Sepacell PrimaTM - Concept and Technology
Development Status
Prion Reduction
Western blot method
Bioassay test
Leukocyte Reduction
RCC Quality
Biological Safety
Next Step

4. Concept and Technology

5. Simultaneous reduction of prions and ｌeukocytes
The Concept of Filter
One Step Filter
Simultaneous reduction of prions and ｌeukocytes
Reducing prions from RCC
Covering current LR filter functions
No Additional Operating Time
No Impact on Customer SOP
No Further RCC loss

6. Key Points of Technology
RCC
Filter
SAGM
Materials
Base fibers: Polyester
Surface: New material
Prion reduction mechanism
Adsorption: High affinity to prion
Filter design
Equivalent to the current Red Cell filter in Europe, Sepacell® Pure RC.

7. Development Status

8. Scheme of Prion Spiking Test
Scrapie infected hamster with 263k scrapie strain
Brain homogenate
LR SAG-M RCC
Microsomal fraction
(10w/v%)
Filter
Leukoreduced RCC used to eliminate phagocytic effect
Western blot

9. Prion Spiking Test Results (1)
Prion reduction confirmed at two different test sites
Sterilization tolerance to both Steam and E-Beam
Test laboratories X Y
Filter
Pool 1
Pool 2
Pool 3
Pool 4
Log10
Reduction
Factor A
> 3.0
> 4.5 B - C D
0.5
0.2
A: Sepacell PrimaTM sterilized with Steam
B: Sepacell PrimaTM sterilized with E-Beam
C: Sepacell PrimaTM sterilized with Steam twice
D: Control (Pall WBF2)
Vox Sanguinis 2008; 95 (suppl. 1): 271, P-588

10. Prion Spiking Test Results (2)
No impact observed within plasma protein variation of RCC
No negative effect induced by leukocytes observed
(at the laboratory Y)
Filter
RCC
Added
FFP
(mL)
Plasma
Protein
(mg/mL)
Log10
Reduction
Facotr
Control (Pure RC)
LR-RCC
3.2
0.92
Sepacell PrimaTM
> 4.5
9.5
7.2 22
10.8
NLR-RCC -
LR-RCC: Leukocyte reduced RCC, pooled and split before FFP addition
NLR-RCC: Non - leukocyte reduced RCC
Transfusion 2008; 48 (suppl.): 86A, SP141

11. Scheme of Bioassay Test
Filter
Microsomal fraction
(10w/v%)
Scrapie infected hamster with 263k scrapie strain
Brain homogenate
Leukoreduced RCC used to eliminate phagocytic effect
Dilution series from pre / post filtration samples
0.05 mL injected intracerebrally to Golden Syrian hamsters
Observed up to 365 days
LR SAG-M RCC

12. Bioassay Test Results
Prion reduction performance supported by Bioassay test
After 365 day observation
Log(ID50) reduction is 4.20 Log10
based on Spearman-Kärber method.
-Log10ID50 = Log10 starting dilution – ([∑p – 0.5] x Log10 dilution factor)
No abnormal or unexpected observation in the groups of;
Positive control (263k microsomal post nuclear fraction)
Sham injected control (HBSS)
Uninjected control
Vox Sanguinis 2009; 97 (suppl. 1): 101, P-092

13. LR Filter Performance (1)
Ambient Temp. Hold, Day1 Filtration
Residual Leukocytes （Log WBC/unit）
Sepacell PrimaTM
Control (Pure RC)
* No significant difference
Hemoglobin （g/unit）
Sepacell PrimaTM
Control (Pure RC)
* No significant difference

14. LR Filter Performance (2)
4 Degree Hold, Day2 Filtration
Residual Leukocytes （Log WBC/unit）
Sepacell PrimaTM
Control (Pure RC)
* No significant difference
Hemoglobin （g/unit）
Sepacell PrimaTM
Control (Pure RC)
* No significant difference
Transfusion Medicine 2008; 18 (suppl.): 30, P08

15. RCC Quality Parameters evaluated Tested at day 0, 21 and 42
Hemolysis (%)
C3a (ng/mL)
Pottasium (mmol/L) pH
Lactate (mg/dL)
2,3 – DPG (mmol/L)
ATP (micro-mol/dL)
Glucose (mg/dL)
Tested at day 0, 21 and 42
Sample (Sepacell PrimaTM, n=9)
Control (Pure RC, n=3)
The results were comparable between sample and control
in all the test

16. Hemolysis results Met CE guideline
Transfusion Medicine 2008; 18 (suppl.): 30, P08

17. Biological Safety Test Results
ISO 10993
Test Item EB
Steam
Cytotoxicity
Pass
Sensitization
Intracutaneous Reactivity
Systemic Toxicity (acute)
Haemolysis Testing
Ames Test with New Polymer
negative
Overall results show the sufficient safety of new filter.
Vox Sanguinis 2008; 95 (suppl. 1): 271, P-588

18. Summary
* C3a, Pottasium, pH, Lactate, 2,3-DPG, ATP, Glucose

19. Next step

20. Next step Sepacell PrimaTM to be CE marked in SCD kit
Ready to provide samples for in-vitro test
To be ready for clinical test soon
Asahi in-house tests are on-going
Quality control method established

21. Thank you for your attention!

22. Back up pages

23. Prion Reduction Test Spiking Study by Western Blot and Bioassay test
Facility: Laboratories in Europe and US
Materials
Agent: The hamster 263K strain of scrapie
Spiking materials: Microsomal Fraction
Medium: Leukoreduced* SAGM-RCC
* to avoid the possibility of WBC related removal of PrPSC, such as phagocytic effect and non-specific binding.

24. RCC Quality Test Results
Comparable RCC quality to control filter
Transfusion Medicine 2008; 18 (suppl.): 30, P08