COMPANY PROFILE

CENTRAL RESEARCH INSTITUTE, KASAULI (H.P)

C.R.I.

Established in 1905 A scheme for the production of bacteriological department and a center of medical research in India was initiated by Sanitary Commissioner with the Government of India. This commenced on what is now known as CENTRAL RESEARCH INSTITUTE, which is located in Kasauli, Himachal Pradesh. engaged in the following activities: (i) Large scale production of Bacterial & Viral Vaccines & Sera. (ii) QC of Immunobiologicals. (iii) R & D in the field of immunology and Vaccinology (iv) Teaching & Training in the field of Microbiology

TRAINING MODULES AT CRI FROM 26-07-2010 to 4-09-2010

CENTARL DRUGS LABORATORY CENTARAL INSTRUMENTATION AND ANALYTICAL LABORATORY ANTISERA DEVISION ANIMAL HOUSE / GUINEA PIG COLONY INFLUENZA SECTION NATIONAL POLIO SURVEILLANCE LABORATOTY JAPANESE ENCEPHALITIS VACCINE DIVISION

YELLOW FEVER

INTRODUCTION
Yellow fever a viral hemorrhagic fever strikes an estimated 200000 persons worldwide each year and causes an estimated 30000 deaths Causative agent of yellow fever an arthropod borne virus from the Flavivirus genus Human sporadically become infected after being bitten by infected mosquitoes In 1927 , a strain of yellow fever vaccine , which after attenuation became known as 17D strain , was obtained from Asibi an African patient. Millions of people in many countries have since been rendered immune to the disease by vaccine prepared from this strain.

Symptoms # Acute infectious diseases # Severe headache # skin infection # pulse rate falls # Temperature declines called Fagets sign # Black vomit # Death.

Central research institute kasauli , India is the only institute in south east Asia where production of yellow fever vaccine takes place.

PRODUCTION OF YELLOW FEVER VACCINE (17D live attenuated , lyophilized)

Fertile , white leghorn chicken eggs

Incubation at 39C 0.5C in 70% humidity for 8 days

Live embryonated chicken eggs , 8 days old

Inoculation in amniotic cavity with 17D strain of ROCK FELLER FOUNDATION (SEED VIRUS)

Test on inoculum Virus titration , sterility

Normal control (2% live uninoculated fertile eggs)

AN EGG INCUBATOR

Incubation of inoculated and normal control eggs at 35C 0.5C in 70% humidity

After 4 days , harvest live and healthy embroys

33% embryos emulsion in double distilled water by homogenisation

Pooled and batch wise harvets

Tests for bulk material and content tissues

Virus titration(bulk material) , sterility , M. avium fowl pox virus , salmonellaes P.N. content , Ab toxicity test .

Embryonic extracts (harvests) containers shell frozen and stored at -70C

Embryonic suspension thawed at 30C in water bath

Centrifuged at 2000 rpm for 1 hour at 4C

Supernatant pooled and dilute with stabilizer

Test for potency and sterility

Containerization and lyophilization

Final product

Quality check testing

THE FINAL STEP

Lyophilisation of vaccine : 1.Prefreezing the drying chamber. 2.Loading of filled vials in the lyophilizer 3. cooling till -60C achieved #Primary drying #Secondary drying Temperature 24C kept for 25 hours then final packaging done for the transportation.

Diptheria Pertussis Tetanus

DIPTHERIA

Diphtheria is an upper respiratory tract illness caused by Cornibacterium diptheria, afacultative anaerobic gram positive bacteria . It is characterized by sore throat, low fever, and an adherent membrane (apseudomembrane) on the tonsils,pharynx and/or nasal cavity. Diphtheria vaccine is a vaccine used against , Cornibacterium diptheria the agent that causes dipteria. It is a component of the DPT vaccine

PERTUSIS
The first clear description of the disease was produced by Sydenham in 1679 who gave it the name pertussis (per = severe + tussis = cough) meaning a violent cough of any kind. Whole cell pertussis vaccine is prepared from one or more strain of B. pertussis

TETANUS

Tetanus, also called lockjaw, is a medical condition characterized by a prolonged contraction of skeletal musclefibers . The rough surface of rusty metal merely provides a prime habitat for a C. tetaniendospore to reside, and the nail affords a means to puncture skin and deliver endospore into the wound . An endospore is a non-metabolising survival structure that begins to metabolise and cause infection once in an adequate environment. Because C. tetani is an anaerobic bacterium, it and its endospores survive well in an environment that lacks oxygen Hence, stepping on a nail (rusty or not) may result in a tetanus infection, as the low-oxygen (anaerobic) environment is provided by the same object which causes a puncture wound, delivering endospores to a suitable environment for growth Tetanus vaccine is a vaccine used against Clostridium tetani, the agent that causes tetanus. Tetanus vaccine is required again after 10 years if the individual is exposed to possible infection.

PRODUCTION OF DIPTHERIA TOXOID

Seed strain C. diphtheria PW8 strain (sub strain CN 2000)

Revival on Loefflers medium at 35C for 48 hours

Culture purity/morphology

Subculture in pope and Lingood medium (papain digest of meat) Shake culture (140160 rpm) at 35C for 24 hours
pH Purity , Lf/ML

Production medium: Pope and Linggood medium , papin digest of meat(maltose, salts, amino acids and vitamins) Sterilized by filteration into fermenter followed by heat 100C for 20 minutes

Medium sterility test

Inoculated with seed culture growth at 35C for 44-48 hours under controlled agitation and aeration
Purity , opacity pH , Lf/ML

Toxin harvested by high speed continous centrifugation and filteration through seitz filter pads
Lf/ML , (Kf) Sterility test

Detoxification with 0.6% formalin and incubation at 36C for 6 weeks

Lf/ML (Kf) after 6 weeks

detoxification

Crude toxoid ready for concentration and purification

PRODUCTION OF PERTUSSIS VACCINE

Seed culture of Bordetella pertussis

3 strains are used

Revived on Bordet Gengou medium, incubation at 35C for 72 hours

Phase variation Culture purity test

Subculture on fresh Bordet Gengou medium , incubation at 35C for 48 hours

Subculture in verway medium , shake culture (140-160 rpm) at 35C for 48 hours
Phase variation, pH Microscopic purity ,opacity

Production medium: semi-synthetic B-2 medium sterilized in fermentor at 121C for 20 minutes

Medium sterility

Inoculated with seed culture grown for 44-48 hours at 35C 0.5C with proper agitation and aeration till pH 8.0 to 8.2 is attained

Bacterial cells harvested high speed centrifuges; bacterial mass suspended in methiolated saline

Detoxification at 56C for 10 minutes

Viability , opacity

Harvests of different strains pooled by vibromixer

Bacterial concentration adjusted to 160109 organisms per ml

Sterility , potency

Vaccine is ready for mixing in DTP vaccine

PRODUCTION OF TETANUS TOXOID

Seed strain: cl. Tetani Harvard strain no.49205

Revived in thioglycollate medium at 35C

Purity check of culture

Grow in heart infusion glucose broth under anaerobic conditions Dispensed in fermenter or 20 litre stainless steel pots Production medium: Mueller and Millers medium Dispensed in fermenter or 20 litre stainless steel pots

Sterilized at 115C for 20 minutes

Inoculated with seed culture

Grow at 35C o.5C for 140-160 hours with proper aeration and agitation in case of fermenter culture
Lf/ML Purity , lysis

Harvesting of toxin by seitz filteration

MLD Lf/ML

Detoxification with 0.45% formalin. Kept at room temperature for three days followed by incubation at 36C for 7 weeks
Lf/ML after 7 weeks Detoxification

Crude toxoid ready for concentration &purification

Purification of toxoids # crude toxoid is concentrated & partially purified by ultrafilteration # fractional precipitation by adjoint # sterilization by filteration # ready for final mixing as DPT vaccine

Mixing of bulk vaccine # tested pools of the vaccines taken # thorough mixing in fermenter fitted with vibromixer # distributed in 20L bottles # stored at 4C to 8C for 3 months for maturation

ANTISERUM

ANTISERUM
Antiserum

is blood serum containing polyclonal antibodies. Antiserum is used to pass on passive immunityto many diseases Snake venom antiserum polyvalent is prepared from purified plasma of healthy horses, which have been immunized against the venoms of most dangerous snakes.

1.THE ANTI VENOM SHOULD NOT BE USED IF TURBID, EXPIRED OR SHOWING PRECIPITATION. 2.THE ANTIVENOM SHOULD BE USED AS SOON AS POSSIBLE AFTER RECONSTITUTION. 3.SHELF LIFE:THREE YEARS FOR THE LIQUID FORM. 4.FIVE YEARS FOR THE LYOPHILIZED FORM STORAGE:STORE AT 2 -8C, AVOID FREEZING AND LIGHT EXPOSURE. 5.PRESENTATION:10 ML VIAL (LIQUID)/ BOX.10 ML DILUENT + VIAL FOR LYOPHILIZED POWDER/ BOX.

PRODUCTION OF ANTISERUM
Quarantine of cast equines (21 days)

Inoculation with tetanus toxoid

Live embryonated chicken eggs , 8 days old

Immunization (primary, secondary and hyper) with respective antigens

Allocation of the equines to carious products (ASVS, ARS, DATS, etc)

Bleeding of the equines @ 1.5% of body weight as per schedule

Plasma separation by

Manual method

Using plasma separator

Collection of plasma

Dilution of plasma with distill water to adjust protein content and acidic pH

Enzyme refinement using pepsin

Fractionisation with ammonium sulphate at 55C

Coagulation of undigested protein and filteration for removal of coagulated proteins & collection of filterate

Dialysis to remove ammonium sulphate and filteration

Quality control tests 10-15 lots pooled to make a bulk Quality control tests on bulk Distribute into final lots Quality control tests on final lot

Containerization and lyophilisation

Quality control tests filled lot Release

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