Dept. Of Antitoxin King Institute of Preventive Medicine Snake Venom Anti Sera against 1.Naja naja 2.Vipera russelli 3.Bungarus caeruleus 4.Echis carinatus

Snake Antivenom Sera for Naja naja, Vipera russelli, Bungarus caeruleus, Echis carinatus

Snake Venom More than 20 different constituents Mainly proteins, enzymes and polypeptide toxins Procoagulation enzymes Haemorrhagins Cytolytic or necrotic toxins Haemolytic and myolytic phopholipases A 2 Presynaptic neurotoxins -Kraits Post synaptic toxins - Cobra

Preparation of the Snake Venom Solutions

Immunization of the Equines with the Snake venom by the Standard Immunization Protocol

Bleeding and separation of Plasma Test Bleeding, Plasma separated, RBC ‘s Reinfused

A - Collection of venoms B – Horse Immunization Protocols C – Starting material of animal derived sera D – Fractionation & Purification process PRODUCTION OF ANTIVENOM SERA: Technology in the public domain (not protected by intelectual property)

Pepsin digestion of IgG. Digestion normally produces a F(ab´) 2 fragment and an extensively degraded Fc portion.

Hyperimmune plasma Acidification at pH , digestion with pepsin at 30°C for 1 hr Filtrate or supernatant F(ab̒) 2 solution Addition of 12% ammonium sulphate Heating to 56°C for 1 hr with stirring Adjust pH to 5.2 Filtration or centrifugation Discard the precipitate Addition of ammonium sulphate to reach 23% concentration; stir for 1 hr Filtration or centrifugation Discard the supernatant TYPICAL FRACTIONATION PROCESS OF F(ab’) 2 FRAGMENTS

Concentrated F(ab̒) 2 solution Final Product sterile filtration Solubilization of precipitate. Tangential flow filtration and concentration

Salient features of ASVS production Long,laborious and time consuming, process (about 1 year) Maintenance of costly and sensitive animals -Horses and mules Increasing demand for the specific life saving drug by the TN state, highly necessary to augument the production of AntiSnake Venom serum. The annual requirement of ASVS serum is about 5 lakh vials 1 vial -10 ml

King Institute is manufacturing ASVS since the year 1980 and has supplied to all the Govt. hospitals in the state of Tamil nadu The maximum number of vials was produced during year of 94,458 vials Nil Nil Nil Nil Last SUPPLIED vials during June 2012

Important issues GMP Norms License Renewal

The Govt. of Tamil nadu has sanctioned an amount of Rs crores for the construction of New Building as per c GMP Norms as Phase I allotment. The requistion for Rs crores is to be submitted as Phase II allotment for Equipmens,Horse purchase,Civil and Eletrical Modifications,Manpower and Venoms and chemicals.

Assessment of neutralizing dose of RV by monovalent antiserum by ELISA Production of IgY Antibodies against RV. Production of bivalent antibodies against RV+EV