Sepacell Prima TM Asahi Combination Filter, Sepacell Prima TM for Prion and Leukocyte Reduction Asahi Kasei Medical Tomo Yokomizo, Morikazu Miura 29th

Sepacell PrimaTM

Asahi Combination Filter, Asahi Combination Filter, Sepacell PrimaSepacell PrimaTMTM

for Prion and Leukocyte Reductionfor Prion and Leukocyte Reduction

Asahi Kasei MedicalTomo Yokomizo, Morikazu Miura

29th October, 2010

Sepacell PrimaTM

Asahi Kasei MedicalAsahi Kasei MedicalOne 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.

Sepacell PrimaTM

Contents

Concept and Technology Development Status

Prion Reduction Western blot method Bioassay test

Leukocyte Reduction RCC Quality Biological Safety

Next Step

- Sepacell PrimaTM -

Sepacell PrimaTM

Concept and TechnologyConcept and Technology

Sepacell PrimaTM

The Concept of Filter

Reducing prions from RCC Covering current LR filter functions

No Additional Operating Time No Impact on Customer SOP No Further RCC loss

One Step FilterOne Step FilterSimultaneous reduction of prions and Simultaneous reduction of prions and ｌｌ eukocyteseukocytes

Sepacell PrimaTM

Key Points of Technology

Materials Base fibers: Polyester Surface: New material

Prion reduction mechanism Adsorption: High affinity to prion

Filter design Equivalent to the current Red Cell fil

ter in Europe, Sepacell® Pure RC.

RCC

Filter

SAGM

Sepacell PrimaTM

Development StatusDevelopment Status

Sepacell PrimaTM

Scheme of Prion Spiking Test

FilterFilter

Microsomal fraction (10w/v%)

Scrapie infected hamster with 263k scrapie strain

Brain homogenate

Leukoreduced RCC used to eliminate phagocytic effect

Western blot

LR SAG-M RCC

Sepacell PrimaTM

Prion Spiking Test Results (1)

Test laboratories X Y

Filter Pool 1 Pool 2 Pool 3 Pool 4

Log10

Reduction

Factor

A > 3.0 > 3.0 > 3.0 > 4.5

B > 3.0 > 3.0 > 3.0 -

C - - - > 4.5

D 0.5 0.2

A: Sepacell PrimaTM sterilized with SteamB: Sepacell PrimaTM sterilized with E-BeamC: Sepacell PrimaTM sterilized with Steam twiceD: Control (Pall WBF2)

•Prion reduction confirmed at two different test sites •Sterilization tolerance to both Steam and E-Beam

Vox Sanguinis 2008; 95 (suppl. 1): 271, P-588

Sepacell PrimaTM

Prion Spiking Test Results (2)

Filter RCC

Added

FFP

(mL)

Plasma

Protein

(mg/mL)

Log10

Reduction

Facotr

Control (Pure RC)

LR-RCC

0 3.2 0.92

Sepacell PrimaTM 0 3.2 > 4.5

Sepacell PrimaTM 9.5 7.2 > 4.5

Sepacell PrimaTM 22 10.8 > 4.5

Sepacell PrimaTM NLR-RCC - - > 4.5

•No impact observed within plasma protein variation of RCC•No negative effect induced by leukocytes observed

LR-RCC: Leukocyte reduced RCC, pooled and split before FFP additionNLR-RCC: Non - leukocyte reduced RCC

Transfusion 2008; 48 (suppl.): 86A, SP141

(at the laboratory Y)

Sepacell PrimaTM

Scheme of Bioassay Test

FilterFilter

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

Sepacell PrimaTM

Bioassay Test Results

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)

•Prion reduction performance supported by Bioassay test


No abnormal or unexpected observation in the groups of;•Positive control (263k microsomal post nuclear fraction)•Sham injected control (HBSS)•Uninjected control

100 10-1 10-2 10-3 10-4 10-5 10-6

mean 154 213 ( > 365 )

sd 34 65 -

0% 28% 100%

mean 127 145 168 ( > 365 )

sd 8 34 40 -

0% 0% 17% 100%

Dilution series

PostFiltration

PreFiltration

Survival Rate

Survival Rate

Survival Period(Days)

Survival Period(Days)

Sepacell PrimaTM

LR Filter Performance (1)

Residual Leukocytes （ Log WBC/unit）

Hemoglobin （ g/unit）

Ambient Temp. Hold, Day1 Filtration

* No significant difference


Combination Filter PureRC

n=12 n=12

mean* 4.19 4.43

σ 0.32 0.34

Sepacell PrimaTM Control (Pure RC)


n=12 n=12

mean* 49.2 48.5

σ 4.7 6.5


Sepacell PrimaTM

LR Filter Performance (2)

Residual Leukocytes （ Log WBC/unit）

Hemoglobin （ g/unit）

4 Degree Hold, Day2 Filtration


* No significant differenceTransfusion Medicine 2008; 18 (suppl.): 30, P08


n=48 n=12

mean* 4.32 4.65

σ 0.60 0.56



n=48 n=12

mean* 50.6 51.4

σ 3.5 3.5


Sepacell PrimaTM

RCC Quality

Parameters evaluated 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 controlin all the test

Sepacell PrimaTM

Hemolysis results

Met CE guideline

Transfusion Medicine 2008; 18 (suppl.): 30, P08

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Pre filtration Post filtration 21days 42days

Hem

olys

is(%

)

samplecontrol

Sepacell PrimaTM

Biological Safety Test Results

Test Item EB Steam

Cytotoxicity Pass

Sensitization Pass

Intracutaneous Reactivity Pass

Systemic Toxicity (acute) Pass

Haemolysis Testing Pass

Overall results show the sufficient safety of new filter.

Ames Test with New Polymer negative

ISO 10993


Sepacell PrimaTM

Summary

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

Items Requirement Present Status

WB Method: > 3 Log

Exogenous: > 4 Log

Residual Leukoctyes < 1x106/unit Same as PureRC

Hemoglobin min 40g/unit Same as PureRC

Hemolysis <0.8% at 42 days ca. 0.4% at 42 days

Other RBC Quality Factors* Same as existing LRF Same as PureRC

Sterilization Steam & Irradiation Both Applicable

ISO-10993 No issue found

Ames Test Negative

Prion Reduction

Biological Safety

> 3 Log

Sepacell PrimaTM

Next stepNext step

Sepacell PrimaTM

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

Sepacell PrimaTM

Thank you for your attention!Thank you for your attention!

Sepacell PrimaTM

Back up pages

Sepacell PrimaTM

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.

Sepacell PrimaTM

RCC Quality Test Results

Hemolysis(%)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6


Hem

olys

is(%

)

samplecontrolmean, SD

C3a(ng/ mL)

0

500

1000

1500

2000


C3a

(ng/

mL)


Pottasium(mmol/ L)

0

5

10

15

20

25

30

35

40

45


Pot

tasium

(mm

ol/L

)


pH

6.4

6.6

6.8

7.0

7.2

7.4


pH


Lactate(mg/ dL)

0

50

100

150

200

250

300

350


Lact

ate(

mg/

dL)


2,3-DPG(mmol/ L)

0.0

0.5

1.0

1.5

2.0

2.5


2,3-

DPG

(mm

ol/L

)(%)


ATP(μ mol/ dL)

0

20

40

60

80

100

120


AT

P(μ

mol

/dL)


Glucose(mg/ dL)

0

200

400

600

800

1000


Glu

cose

(mg/

dL)


•Comparable RCC quality to control filter

Transfusion Medicine 2008; 18 (suppl.): 30, P08

Documents

Sepacell Prima TM Asahi Combination Filter, Sepacell Prima TM for Prion and Leukocyte Reduction Asahi Kasei Medical Tomo Yokomizo, Morikazu Miura 29th