Dept. Of Antitoxin King Institute of Preventive Medicine

Snake Venom Anti Sera against

1.Naja naja2.Vipera russelli3.Bungarus caeruleus4.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 A2• 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 ImmunizationProtocols

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 3.0-3.3, digestion with pepsin at 30°C for 1 hr

Filtrate or supernatant

F(a )b̒ 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(a )b̒ 2 solution

Final Product

sterilefiltration

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 1997-1998 of 94,458 vials

• 2000-2001 30194• 2001-2002 4304• 2002-2004 Nil• 2004-2005 2340• 2005-2006 Nil • 2006-2007 3161• 2007-2008 Nil• 2008-2009 Nil

Last SUPPLIED -1000 vials during June 2012

Important issues

GMP Norms

License Renewal

S. No. District Snake bite cases Death1 Kancheepuram 768 32 Vellore 1009 13 Thiruvannamalai 1152 04 Cuddalore 1602 05 Dharmapuri 2097 06 Salem 1499 07 Erode 1648 118 Coimbatore 1128 09 Udagamandalam 110 0

10 Trichy 210 211 Pudukottai 589 212 Dindigul 804 1713 Madurai 1039 014 Virudhunagar 1560 0

15 Sivaganga 401 316 Ramanadhapuram 508 417 Thirunelveli 848 718 Thoothukudi 482 319 Kanyakumari 20 020 Nagapattinam 589 021 Thanjavur 582 222 Villupuram 879 023 Karur 262 024 Perambalur 562 325 Thiruvallur 652 126 Thiruvarur 215 327 Theni 760 128 Namakkal 1224 729 Krishnagiri 577 6

The Govt. of Tamil nadu has sanctioned an amount of Rs. 7.36 crores for the construction of New Building as per c GMP Norms as Phase I allotment.

The requistion for Rs 12.14 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