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