Testing antivenoms - Quality control of antivenoms in Japan -
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1 Testing antivenoms - Quality control of antivenoms in Japan - Motohide TAKAHASHI, Ph.D Yoshichika ARAKAWA, MD, Ph.D Deptartment of Bacterial Pathogenesis and Infection Control National Institute of Infectious Diseases Tokyo, Japan
2 Japanese Mamushi Gloydius blomhoffii or Korean Mamushi Chinese Mamushi Gloydius blomhoffii brevicaudus Gloydius blomhoffii (Mamushi) Gloydius blomhoffii and its subspecies were proposed to be regrouped from Agkistrodon halys in (W Wüster et al., Toxicon,1997)
3 Gloydius blomhoffii and its subspecies contain at least two well characterized toxins, lethal toxin and hemorrhagic toxin. Bite injury by mamushi (outside of right foot) Two bite traces ) appear about 1.5 cm distance. Total length of snake body was 56cm (larger than middle size).
4 Supply sources of Mamushi antivenom products in each country Japan : domestically produced by KAKETSUKEN China : domestically produced by SIBP Korea : imported from China and Japan
5 Kinds of Antivenom/antitoxin (Equine) in Japan Products* Mamushi antivenom Habu antivenom Gas gangrene antitoxin Botulism antitoxin type A, B, E, & F Botulism antitoxin type E Diphtheria antitoxin Antigen for horse-immunizing Snake venom Gloydius blomhoffii Snake venom Bacterial toxin genus Clostridium Bacterial toxin genus Clostridium Bacterial toxin genus Clostridium Bacterial toxin genus Corynebacterium Protobothrops flavoviridis Marketing quantity (approximately) 3,000 vials/year 200 vials/year 2,000 vials/5 years 200 vials/5 years 300 vials/5 years 1,000 vials/5 years All the products are manufactured by KAKETSUKEN.
6 Flow sheet of Freeze-dried Mamushi Antivenom product Manufacturing process Healthy horse Venom-injection Blood collection Operating procedure Equine infectious anaemia virus et al Tetanus toxoid Whole blood collection, serum centrifugation Crude antivenom material Purification Dialysis Bulk material Addition of solution Final bulk Filling Lyophilizatuion, Sealing Final product Dilution, ph-adjustment Digestion by pepsin ph-adjustment Salting-out Dialysis, ph-adjustment Aseptic filtration Addition of stabilizer, dilution Aseptic filtration Filling Lyophilization, Sealing Packaging
7 Characterization of the Freeze-dried Mamushi Antivenom, Equine Item* Moisture content( 3%) ph ( ) Protein content(<30 mg/300u) Sterility Test for freedom from abnormal toxicity Pyrogen test( 1.3 ) Potency test Anti-lethal toxin titer( 300 U/mL) Anti-haemorrhagic titer( 300 U/mL) Freeze-dried Mamushi Antivenom pass(0.17%) pass(7.07) pass(17.3 mg/300u) pass pass pass(0.14 ) pass (400 U/mL) pass(300 U/mL) Minimum Requirement of Biological Products in Japan.
8 Gloydius blomhoffii
9 Background Mamushi snakes (Gloydius blomhoffii) that produce very similar venoms inhabit in a very wide geographical area including Japan, Korea and China continent. Antivenoms has been used for the treatment of snakebites in this region. No international or regional reference standard for mamushi antivenoms established and authorized by international organizations was available to date.
10 Materials and Methods Standard mamushi antivenom used for the assay Japanese national standard mamushi antivenom Lot C-48 (established at NIID in 1994) anti-lethal titer: 2,100 U/vial, anti-hemorrhagic titer: 3,300 U/vial, (Freeze-dried products) Candidate mamushi antivenom Produced by SIBP under the same production protocol for Chinese commercial antivenom products. (Freeze-dried products for long-term storage)
11 Potency tests Determination of anti-lethal titer mouse strains : Slc:ddY at NIID and KAKETSUKEN ICR at KFDA, Kunmin at SIBP > 3 mice/group, body weight g Determination of anti-hemorrhagic titer two rabbits (Japanese white strain), body weight approximately 3.0 kg
12 Final results of anti-lethal titer facility test potency 95% confidence interval 1 30,594 Japan 2 31,999 NIID 3 31,328 common potency 31,437 29,111-33, ,310 Japan 2 29,036 Kaketsuken 3 27,342 common potency 31,572 27,066-36, ,256 Korea 2 46,114 KFDA 3 37,638 common potency 36,391 32,832-40,335 general common potency 32,909 31,080-34,846 (U/vial)
13 Final results of anti-hemorrhagic titer facility test potency 95% C.I. 1 37,847 Japan 2 33,033 NIID 3 29,561 common potency 34,454 33,112-35,850 Japan Kaketsuken 37, ,607 Korea 2 35,402 KFDA 3 42,607 common potency 36,063 34,411-37,793 general common potency U/vial 36,226 35,440-37,030
14 Results of stability test ( stored at stored for (months) ) common 20 ( ) ( ) ( ) ( ) 37 Potency 1 ( ) ( ) ( ) ( ) ( 95% C.I. ) ( ) ( ) ( ) ( ) common ( ) ( ) ( ) ( ) 20 Regression 2 37 coefficient common Anti-lethal titers relative to the candidate stored at 4 for the same period. 2 Regression coefficient of potency.
15 Recommendations summarized in the WHO workshop on the standardization and control of antivenom meeting held in 2001 Even now still remain Alternative methods to animal test Viral validation study Refine test methods Mice test (Lethal) or Rabbit skin test (Hemorrhagic)
16 Conclusion A regional reference standard for mamushi antivenom was successfully established in cooperation with Korea, China, and Japan. The reference standard can be used for quality control of mamushi antivenom products by the currently employed protocols in each country after approval by each NRA.
17 Participated Institutions & Contributors Korea Korea Food and Drug Administration (KFDA) SH Hong, HJ Oh, SJ Hur, KH Lee People s Republic of China Shanghai Institute of Biological Products (SIBP) Z Wei National Institute for the Control of Pharmaceutical & Biological Products L Dianliang (NICPBP) Japan Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN) K Morokuma, K Ohkuma National Institute of Infectious Diseases (NIID) T Fukuda, M Iwaki, Y Nagaoka, M Takahashi
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