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
 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 1997.
(W Wüster et al., Toxicon,1997)
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).
 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

 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
All the products are manufactured by KAKETSUKEN.


Antigen for horse-immunizing Marketing quantity
(approximately)
Snake venom 3,000 vials/year
Gloydius blomhoffii
Snake venom 200 vials/year
Protobothrops flavoviridis
Bacterial toxin
 genus 2,000 vials/5 years
Clostridium
Bacterial toxin genus 200 vials/5 years
Clostridium
Bacterial toxin
 genus 300 vials/5 years
Clostridium
Bacterial toxin genus
 1,000 vials/5 years
Corynebacterium
Flow sheet of Freeze-dried Mamushi Antivenom product
Manufacturing process Operating procedure
Healthy horse Equine infectious anaemia virus et al
Venom-injection Tetanus toxoid
Blood collection Whole blood collection, serum centrifugation

Crude antivenom material Dilution, pH-adjustment

Purification
Digestion by pepsin
pH-adjustment
Salting-out
Dialysis Dialysis, pH-adjustment
Aseptic filtration
Bulk material
Addition of solution Addition of stabilizer, dilution
Aseptic filtration
Final bulk
Filling Filling
Lyophilizatuion, Sealing Lyophilization, Sealing

Final product Packaging

Characterization of the Freeze-dried Mamushi Antivenom, Equine

Item* Freeze-dried Mamushi Antivenom

Moisture content(  3%) pass(0.17%)

pH (6.8-7.4) pass(7.07)

Protein content(<30 mg/300U) pass(17.3 mg/300U)

Sterility pass

Test for freedom from abnormal toxicity pass

Pyrogen test( 1.3 )

 pass(0.14 )

Potency test
Anti-lethal toxin titer( 300 U/mL)
 pass (400 U/mL)
Anti-haemorrhagic titer( 300 U/mL) pass(300 U/mL)

Minimum Requirement of Biological Products in Japan.

Gloydius blomhoffii
 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.
 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)
 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 16 20 g


Determination of anti-hemorrhagic titer
two rabbits (Japanese white strain),
body weight approximately 3.0 kg
 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 - 33,949
1 36,310
Japan 2 29,036
Kaketsuken 3 27,342
common potency 31,572 27,066 - 36,827
1 31,256
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)
 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 37,543
1 42,607
Korea 2 35,402
KFDA 3 42,607
common potency 36,063 34,411 - 37,793
general common potency 36,226 35,440 - 37,030
U/vial


 Results of stability test
stored at stored for (months)
(
) 3 6 9 common

20
0.825 0.830 0.929 0.870
( 0.635 - 1.011 ) ( 0.974 - 0.974 ) ( 0.799 - 1.078 ) ( 0.792 - 0.954 )

37 0.710 0.860 0.831 0.818

Potency1 ( 0.544 - 0.866 ) ( 0.743 - 0.997 ) ( 0.715 - 0.964 ) ( 0.747 - 0.964 )
( 95% C.I. ) 0.872 0.837 0.831 0.840
45
( 0.663 - 1.098 ) ( 0.717 - 0.971 ) ( 0.715 - 0.964 ) ( 0.766 - 0.921 )

common 0.796 0.843 0.862 0.842

( 0.703 - 0.902 ) ( 0.775 - 0.916 ) ( 0.794 - 0.936 ) ( 0.799 - 0.888 )
20 10.716 14.246 12.834 12.281
Regression 2 37 11.003 17.363 12.722 12.090
coefficient 45 8.959 16.411 12.722 11.681
common 10.213 15.954 12.760 12.386
1
Anti-lethal titers relative to the candidate stored at 4
for the same period.
2
Regression coefficient of potency.
 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)
 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.
 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