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

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

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

Basic Raw Materials in Antivenom Production Snake Venoms Hyperimmune Plasma

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,

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.

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

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

ENZYMATIC CLEAVAGE OF IMMUNOGLOBULIN MOLECULE

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)

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 -

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

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.

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

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

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.

How much antivenom is needed How much antivenom is needed?? ( Tumbare & Khadilkar,Journal of Bombay Veterinary college:2004,12(1&2):9-11) Snake Average Venom Yield in ml (Liquid) Average Venom Yield in mg (Lyophilized) Theoretical dose of antivenom required for treatment (10 ml vial) Indian Cobra 0.098-1.56 ml (0.435±0.01) 56.4-514.9mg (126.32±4.31) 21 Russell’s Viper 0.177 -1.356 ml (0.412±0.02) 20 -277.5 mg (75.98±3.66) 13 Indian Common Krait 0.03-0.25 ml (0.101±0.01) 1.25-18.89 mg (8.35±0.64) 2

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 that meets 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

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

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

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.

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