Presentation Summer Training Zahida8

8/4/2019 Presentation Summer Training Zahida8

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COMPANY PROFILECENTRAL RESEARCH INSTITUTE, KASAULI (H.P)


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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


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TRAINING MODULES AT CRI FROM

26-07-2010 to 4-09-2010

CENTARL DRUGS LABORATORYCENTARAL INSTRUMENTATION AND ANALYTICAL LABORATORYANTISERA DEVISION

ANIMAL HOUSE / GUINEA PIG COLONYINFLUENZA SECTION

NATIONAL POLIO SURVEILLANCE LABORATOTYJAPANESE ENCEPHALITIS VACCINE DIVISION


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YELLOW FEVER


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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 virusfrom the Flavivirus genus

Human sporadically become infected after being bitten byinfected mosquitoes

In 1927 , a strain of yellow fever vaccine , which afterattenuation became known as 17D strain , was obtainedfrom Asibi an African patient.

Millions of people in many countries have since beenrendered immune to the disease by vaccine preparedfrom this strain.


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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 yellowfever vaccine takes place.


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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 ofROCK FELLER FOUNDATION (SEED VIRUS)

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

Test on inoculumVirus titration , sterility

Normal control (2% live uninoculated fertile eggs)


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Incubation of inoculated and normal control eggs at35C 0.5C in 70% humidity

After 4 days , harvest live and healthy embroys

33% embryos emulsion in double distilled water byhomogenisation

Pooled and batch wise harvets

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

Tests for bulk material and content

tissuesVirus titration(bulk material) , sterility ,

M. avium fowl pox virus , salmonellaes

P.N. content , Ab toxicity test .


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Embryonic suspension thawed at 30C in water bath

Centrifuged at 2000 rpm for 1 hour at 4C

Supernatant pooled and dilute

with stabilizerTest for potency

and sterility

Containerization and lyophilization

Final product Quality check testing


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THEFINALSTEP

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 finalpackaging done for the transportation.


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Diptheria Pertussis Tetanus


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DIPTHERIA

Diphtheria is an upper respiratory tract illness causedby Cornibacterium diptheria, afacultative anaerobic grampositive bacteria .

It is characterized by sore throat, low fever, and an

adherent membrane (apseudomembrane) onthe tonsils,pharynx and/or nasal cavity.

Diphtheria vaccine is a vaccine used against ,Cornibacterium diptheriathe agent that causes dipteria.

It is a component of the DPT vaccine
http://en.wikipedia.org/wiki/DPT_vaccinehttp://en.wikipedia.org/wiki/DPT_vaccine


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PERTUSIS

The first clear description of the disease wasproduced by Sydenham in 1679 who gave it thename pertussis (per = severe + tussis = cough)meaning a violent cough of any kind.

Whole cell pertussis vaccine is prepared from oneor more strain of B. pertussis


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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 fora C. tetaniendospore to reside, and the nail affords a means topuncture skin and deliver endospore into the wound .An endospore is a non-metabolising survival structure that begins tometabolise 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 bythe same object which causes a puncture wound, deliveringendospores to a suitable environment for growth

Tetanus vaccine is a vaccine used against Clostridium tetani, theagent that causes tetanus. Tetanus vaccine is required again after 10years if the individual is exposed to possible infection.


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

Revival on Loefflers medium at 35C for 48hours

Culture purity/morphology

Subculture in pope and Lingood medium(papain digest of meat) Shake culture (140-

160 rpm) at 35C for 24 hours

PRODUCTION OF DIPTHERIA TOXOID

Production medium: Pope and Linggood medium , papindigest of meat(maltose, salts, amino acids and vitamins)

Sterilized by filteration into fermenter followedby heat 100C for 20 minutes

Purity , Lf/MLpH


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Inoculated with seed culture growth at 35C for44-48 hours under controlled agitation and

aeration

Toxin harvested by high speed continouscentrifugation and filteration through seitz filter

pads

Crude toxoid ready for concentration andpurification

Detoxification with 0.6% formalin andincubation at 36C for 6 weeks

detoxificationLf/ML (Kf) after 6 weeks

pH , Lf/ML

Sterility testLf/ML , (Kf)

Purity , opacity

Medium sterility test


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Seed culture of Bordetella pertussis

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

Phase variation Culture purity test

3 strains are used

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

PRODUCTION OF PERTUSSIS VACCINE

Microscopic purity ,opacityPhase variation, pH

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


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Inoculated with seed culture grown for 44-48 hours at 35C 0.5Cwith 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

Vaccine is ready for mixing in DTP vaccine

Bacterial concentration adjusted to 160109organisms per ml

Harvests of different strains pooled byvibromixer

Detoxification at 56C for 10 minutes

Medium sterility

Viability , opacity

Sterility , potency


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Seed strain: cl. Tetani Harvard strainno.49205

Revived in thioglycollate medium at 35C

Purity check of culture

Grow in heart infusion glucose broth underanaerobic conditions

Production medium: Mueller and Millersmedium

Dispensed in fermenter or 20 litre stainlesssteel pots

PRODUCTION OF TETANUS TOXOID

Dispensed in fermenter or 20 litre stainlesssteel pots


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Sterilized at 115C for 20 minutes

Inoculated with seed culture

Crude toxoid ready for concentration &purification

Detoxification with 0.45% formalin. Kept at room temperature forthree days followed by incubation at 36C for 7 weeks

Harvesting of toxin by seitz filteration

Grow at 35C o.5C for 140-160 hours withproper aeration and agitation in case of

fermenter culture

Purity , lysis

Detoxification

Lf/ML

Lf/ML

MLD

Lf/ML after 7 weeks


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Purification of toxoids

# crude toxoid is concentrated & partially purifiedby 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


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ANTISERUM


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ANTISERUM

Antiserum is blood serumcontaining polyclonal antibodies.

Antiserum is used to pass onpassive immunityto many diseases

Snake venom antiserum polyvalent isprepared from purified plasma of healthyhorses, which have been immunizedagainst the venoms of most dangeroussnakes.


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1.THEANTIVENOMSHOULDNOTBEUSEDIFTURBID, EXPIRED

ORSHOWINGPRECIPITATION.2.THEANTIVENOMSHOULDBEUSEDASSOONASPOSSIBLEAFTERRECONSTITUTION.3.SHELF LIFE:THREEYEARSFORTHELIQUIDFORM.4.FIVEYEARSFORTHELYOPHILIZEDFORM STORAGE:STOREAT

2 -8C, AVOIDFREEZINGANDLIGHTEXPOSURE.5.PRESENTATION:10 MLVIAL (LIQUID)/BOX.10 MLDILUENT +VIALFORLYOPHILIZEDPOWDER/BOX.


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Quarantine of cast equines (21 days)

Inoculation with tetanus toxoid

Live embryonated chicken eggs , 8 days old

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

Immunization (primary, secondary and hyper) withrespective antigens

Bleeding of the equines @ 1.5% of body weight asper schedule

PRODUCTION OF ANTISERUM


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Enzyme refinement using pepsin

Fractionisation with ammonium sulphate at 55C

Coagulation of undigested protein andfilteration for removal of coagulated proteins & collection of

filterate

Plasma separation by

Collection of plasma

Manual method Using plasma separator

Dilution of plasma with distill water to adjust proteincontent and acidic pH


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Dialysis to remove ammonium sulphate and filteration

10-15 lots pooled to make a bulk

Distribute into final lots

Containerization and lyophilisation

Release

Quality control tests

Quality control tests on final lot

Quality control tests on bulk

Quality control tests filled lot


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THANK YOU

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Presentation Summer Training Zahida8