QUALITY CONTROL OF SNAKE ANTIVENOM

1. QUALITY CONTROL OF SNAKE ANTIVENOM DR.M.V.KHADILKAR Technical Director, Premium Serums & Vaccines Pvt.Ltd., Narayangaon,Dist.-Pune Maharashtra

2. PRODUCTION PROCESS • Equine(Horse/Mules/Ponies) procurement • Quarantine/Complete Health screening • Regular periodic Immunization with snake venoms • Periodic Titer Testing • Plasma collection & storage • Plasma Fractionation • Batch pooling • Sterile Filtration • Containerization & Lyophilization • Labeling /Inspection • Finished Product Testing • Marketing

3. Antivenom quality and safety depends on 1.The control of source materials 2. Manufacturing procedures

4. Basic Raw Materials inAntivenom Production • Snake Venoms • Hyperimmune Plasma

5. Objectives of the purification process High safety and efficacy • No impairment of the neutralizing activity during purification • Maintenance of high purity • Free of microbial contaminants and endotoxins WHO Workshop,

6. precautions in Venom production Venom is to be obtained from snakes in a controlled and documented manner. It should be properly preserved and tested for quality Venom being the prime raw material for the production of Antivenom ,it is a key factor in the production of effective antivenom.

7. Plasma production • Animal health • Monitoring of immune response • Proper collection process are the greatest contributors to good quality plasma production

8. Collection of plasma • Maintenance of healthy & disease free herd of animals • Cleanliness of premises and complete asepsis during procedure. • Use of sterilized collection sets & containers under controlled environmental conditions • Properly designed clean room to separate plasma and operations carried out by trained personnel. • Storage in proper temperature controlled rooms. • Plasma collection vessels must be labeled with date, plasma volume, horse numbers and anti-coagulant use details. AVOIDS BIOBURDEN-HELPS IN CONTROLLING ENDOTOXINS

9. ENZYMATIC CLEAVAGE OF IMMUNOGLOBULIN MOLECULE

10. COMMON METHOD USED FOR PLASMA FRACTIONATION in India • Pepsin Digestion to get F(ab’)2 fragments from whole IgG. • Precipitation of unwanted proteins by either Ammonium Sulfate or Caprylic Acid • Concentration & Diafiltration of F(ab’)2 OTHER METHODS Use of whole IgG molecule Papain Digested Fab fragments Chromatography (Ion-Exchange/Affinity)

11. PLASMA FRACTIONATION • Use of closed system with no exposure to environment. • Least product hold time during process • Process validation-digestion, filtration, ultrafiltration • Strict supervision of critical process parameters such as Temp,pH,duration etc • Checks about cleaning & sterilization status • IPQC checks -

12. Quality Control TESTS • DESCRIPTION • pH • PROTEIN • PRESERVATIVE CONTENT • POTENCY • ABNORMAL TOXICITY • STERILITY • ENDOTOXIN • (SOLUBILITY & MOISTURE CONTENT)

13. ANTIVENOM SAFETY Depends on 1. Type of antivenom -Whole IgG/ F(ab)2 / Fab Each fragment has different pharmacokinetics Efficacy reports not supported by clinical data 2. Dose 3. Route & Speed of administration 4. Manufacturing practices Hypersensitivity skin test has limited predictability value.

14. Reasons for reactions • Contamination of plasma • Enzymatic digestion & removal of unwanted proteins • Formulation • Sterility No data from Indian clinical trials is available, however , a lot has been reported by importing countries Thought to vary from 3-80% with about 5-10% severe reactions Reports are often subjective

15. EFFICACY • MONOVALENT / POLYVALENT –Has its own advantages/disadvantages • LEVEL OF REFINEMENT-Enzyme digestion,Ion exchange chromatography/Affinity chromatography. Effect on cost???? • VENOM • QUALITY • GEOGRAPHICAL ANTIGENIC VARIATION • SPECIFICITY/PARASPECIFICITY • CLINICAL MANAGEMENT-OUTCOME DEPENDS ON • EARLY START OF TREATMENT • ADEQUATE DOSES • SUPPORT SYSTEMS

16. SNAKE ANTIVENOM I.P. • Name of Product : Snake Antivenom I.P. • (Polyvalent, Lyophilized/liquid, Equine origin Enzyme Refined Immunoglobulin) • Description of Product:Lyophilized 20.0 ml Vial • Along with 10.0 ml W.F.I. I.P. OR liquid 10 ml vial • Potency : When Reconstituted With 10.0 ml • of Sterile Water For Injection I.P. • 1.0 ml of reconstituted mixture Neutralizes • 0.60 mg. of Cobra Venom • 0.45 mg. of Krait venom • 0.60 mg. of Russell’s viper venom • 0.45 mg. of Saw scaled viper venom • Nature and concentration • of preservative : Phenol /Cresol not more than < 0.25% • Storage conditions :Store in cool dark place, avoid • exposure to sunlight for lyophilized and to store between 2-8˚C for liquid product.

17. How much antivenom is needed??( Tumbare & Khadilkar,Journal of Bombay Veterinary college:2004,12(1&2):9-11)

18. INDIAN SCENARIO • It is estimated that, India currently produces about 1.8-2.0 million Snake Antivenom vials per year • Almost 75-80% of total Snake Antivenom produced is supplied to different government institutions via tender procedure. • Current capacities can supply antivenom thatmeets about 60-70% of the requirements leading to unfortunate incidents of deaths • Many times the problem is compounded by improper distribution • This results in big demand-supply gap for this life saving drug

19. INDIAN SCENARIO • Many Indian companies have been regularly supplying this life saving medicine not only to its neighboring countries but also to countries in Africa and South Asia. • Due to the enforcement of various regulations after 2000, manufacturing of equine biological products was significantly reduced resulting in severe shortage of these life saving products. • Stringent regulatory requirements have also pushed up the production costs

20. INDIAN SCENARIO • India still has very low price as compared to many other countries • It has been observed that 2010 prices for a 10 ml vial of Indian polyvalent AVS range from about INR 300 to 500 (US$ 6.50–11.00), which is a fraction of the cost of a vial of CroFab antivenom in the USA (at over US$ 1900 per vial) or CSL antivenom in Australia (at US$ 1500 per vial) AVS is supplied by Indian antivenom producers to government hospitals at Rs 115 per vial (US$ 2.50) (Romulus Whitaker & Samir Whitaker in Current science, vol. 103, no. 6, 25 september 2012) • Most of the ASVS tenders were awarded at Rs. 190 -Rs. 210 per vial in 2011

21. My views • Dose finding studies can give guidelines for treatment protocol based on syndromic approach. • One protocol for big country like India is difficult considering regional variation in venom composition. • Use of polyvalent antivenom is more practical and economical especially considering procurement and distribution system followed. • More legal sources of venoms should be established/promoted in different regions for its availability in production process. • The Indian antivenoms are reasonably priced. • There is limit to purification considering its direct effect on price. • Collaboration between Industry & Academic institutions is needed to improve yields & reducing costs.

22. THANK YOU