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1

2 ISO

3 ISO

4 ISO(International Standardization Organization) 135 ISO

5 CD(Committee Draft) DIS(Draft for International Standard) FDIS(Final DIS) ISO ISO ANSI/AAMI RD ANSI/AAMI RD ISO/CD ISO ISO/DIS ISO/CD ISO/DIS

6 ISO/TC150/SC2/WG5 ISO/DIS CD 2nd , 13959, ISO23500 NWI 2 ISO

7 ISO/TC150/SC2/WG5 ISO/DIS CD 2nd , 13959, ISO23500 NWI 2

8 ISO/CD DIS ISO/DIS : Fluids for hemodialysis and related therapies ISO/CD : Concentrates for hemodialysis and related therapies ISO/CD : Water for hemodialysis and related therapies ISO/CD : Water equipment for hemodialysis application NWI ; RD62

9

10 CD RO 0.1mg/L ISO/CD mg/L ISO/CD mg/L 1

11 ISO/TC150/SC2/WG5 ISO/DIS CD 2nd , 13959, ISO23500 NWI 2

12 Water for hemodialysis ET cfu/ml EU/L 100 (Action Level 50) 250 ISO 1 Dialysis fluid Ultrapure dialysis fluid Online prepared substitution fluid 100 (Action Level 50) ( ) TGEA, R2A, MEA

13 ISO Dr.Ward 07 5 CD NWI ISO ( )

14

15 ISO/CD23500 Ver. 1.05

16 ET

17 /ml mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L 10 10mg/L mg/L mg/L mg/L mg/L mg/L mg/L 38 ( ) 300mg/L 14 1,4-0.05mg/L mg/L 15 1,1-0.02mg/L mg/L 16-1,2-0.04mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L 45 (TOC) 5mg/L mg/L 46 ph mg/L mg/L mg/L mg/L 50 2

18 ISO ISO/CD (mg/l) 1. 2(0.1mEq/L) 2. 4(0.3mEq/L) 3. 8(0.2mEq/L) 4. 70(3.0mEq/L)

19 Water for hemodialysis Dialysis fluid Ultrapure dialysis fluid Online prepared substitution fluid ISO ET ET cfu/ml EU/L cfu/ml EU/L 100 (Action Level 50) 100 (Action Level 50) ) ( 10) ( ( ) TGEA, R2A, MEA R2A or equivalent (agar or broth or sheet)

20

21

22 ET 68 ET ET ET 1 (1 15) 10 4 EU/ 250EU/L ( ) ( ) cfu/ml Ex). 15. PDA Journal of GMP and Validation in Japan,6,38-45,2004 Eschrichia coli (dried cell) EU/ 250EU/L E. coli 1800 /ml

23 Imidazoquinolines Flagellin Bcterial DNA (CpG DNA) Mycoplasmal Lipopeptide Peptidoglycan LPS LBP TLR 7 TLR 5 TLR 9 TLR 2 TLR 6 CD14 CD14 MD-2 TLR 4 MyD88 TRAF 6 IRAK Cytokine NF- B DNA

24 m 10nm MDP 400 -D glcan DNA fragment

25 MF

26

27 CFU MF CFU

28 Culture and Non-culture Methods Culture method : Non-culturable cells (Not colony forming VBNC or dead cells) Nucleic acid staining Non-culture method 4,6-diamidino-2-phenylindole (DAPI) Propidium iodide (PI) Intracellular enzyme activity 6-carboxyfluorescein diacetate (6CFDA) 6CF 6CFDA : Culturable cells (Colony-forming ability) Colony Microcolony CTC CTC-formazan 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) Respiratory activity

29 Culture and Non-culture Methods Culture Method Fluorescent Method 5 Fig. Culture for colony Fig. Non-culture Method - Direct Epifluorescent Filtration Technique (Escherichia coli ) Microbial cells stained with DAPI

30 Bacterial number and endotoxin level in dialysis fluids Yamaguchi, N., et al : Rapid monitoring of bacteria in dialysis fluids by fluorescent vital staining

31 Rapid and Automated Bacterial Detection Systems asu, M. (2006): JP s standpoint for chapters on rapid microbiological methods presented in New microbiology chapters of the European armacopoeia - Session II: Alternative methods for control of microbiological quality (Part 1). Strasbourg, France. 2-3 Oct

32 Water for hemodialysis ET cfu/ml EU/L 100 (Action Level 50) 250 ISO 1 Dialysis fluid Ultrapure dialysis fluid Online prepared substitution fluid 100 (Action Level 50) ( ) TGEA, R2A, MEA

33

34 (SAL:Sterility assurance level) D *D SAL *D SAL 10 6

35 ISO

36

37 (DQ Design Qualification) 5. (IQ:Installation Qualification) 6. (OQ:Operational Qualification) 7. (PQ:Performance Qualification)

38 (PQ:Performance Qualification) (a) (b) (c) Phase 1 / RO Phase 2 1 Phase 2,

39

40 Ver. 1.05

41 RO RO RO ETCF Ca

42 h 50 75%

43 ET 2 70% 50%

44

45

46

Fig. 1. Structures of NM394, NAD-358 and NAD-245 Fig. 2. Typical HPLC chromatograms of NM394 in human plasma by organic solvent extraction method (a): Blank plasma (b): Plasma spiked with NM394 and internal

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