Toxoids production

1. BACTERIAL TOXOIDS
Dr. P. Suganya
Assistant professor
Department of Biotechnology (Autonomous)
Sivakasi

2. • Are virulence factor of most of bacteria and one of the
major cause of tissue damage.
• Poisonous substances produced by microorganism
• It may be protein lipid or any tissue
Bacterial
Toxins
endotoxin exotoxin
TOXINS

3. ENDOTOXIN
• LPS of Gram negative cell has three
parts:- O- antigen , core
polysaccharide and lipid A.
• Released usuallywhen the cell is
lysed but can also be released during
vegetative growth.
• Has same chemical composition in
almost all bacteria and has same
toxic effect ( no matter which
bacteria produce it).
• Encoded by chromosomal gene.

4. PROPERTIES OF BACTERIAL ENDOTOXIN
1. Integral part of the cell wall of Gram-negative bacteria. Released
on bacterial death and in part during growth. Release is not
required for biologic activity.
2. Formed only by Gram-negative bacteria
3. Lipopolysaccharides. Lipid A portion is responsible for toxicity.
4. No specific receptor.
5. Moderately toxic. Fatal to animals in large doses.
6. Relatively heat stable. Toxicity is not destroyed above 60°C for
hours.
7. Weakly antigenic. Antibodies are protective.
8. Not converted to toxoid.
9. Synthesis directed by chromosomal genes.
10.Usually produce fever in the host by release of interleukin-1 and
other mediators..

6. EXOTOXIN
• Are most powerful and active in small quantities.
• Either secreted by organism or leak into the surrounding fluid
after lysis of bacterial cell.
• Gene for exotoxin may be present on chromosome or plasmid or
bacteriophage DNA.

7. PROPERTIES OF BACTERIAL EXOTOXIN
1.Excreted by living cells
2. Produced by Gram-positive and Gram-negative bacteria
3. Polypeptides
4. Usually bind to specific receptors on cells
5. Highly toxic. Fatal to animals in very small doses
6. Relatively heat labile. Toxicity destroyed over 60°C
7.Highly antigenic. Stimulate formation of antitoxin. Antitoxin
neutralizes the toxin
8.Converted to toxoid by formalin. Toxoid is nontoxic but antigenic.
Toxoids are used to immunize, e.g. tetanus toxoid
9. Usually controlled by extra-chromosomal genes, e.g. plasmids, phage
gene
10. Usually do not produce fever in the host.

8. TOXOID
• Toxoid is modified exotoxin. An exotoxin
has two main properties :
(1)toxicity, and
(2)antigenicity.
• In toxoid, the toxicity of the toxin is
destroyed but its antigenicity is preserved.
As such toxoids e.g. diphtheria toxoid,
tetanus toxoid can be safely used for
vaccines. Toxins can be converted to toxoid
by different methods e.g. formalin
treatment.

9. Bacteria
Responsible
Description Types of Foods Symptoms Cause
Temperture
Sensitivity
Staphylococcus
aureus
Produces a
heat-stable
toxin
Meat and seafood
salads, sandwich
spreads and high
salt foods.
Nausea, vomiting and
diarrhea within 4 to 6 hours.
No fever.
Poor personal hygiene and
subsequent temperature
abuse.
No growth below 40°
F.Bacteria are destroyed by
normal cooking but toxin is
heat-stable.
Salmonella
Producesan
intestinal
infection
High protein
foods – meat,
poultry, fish and
eggs.
Diarrhea nausea, chills,
vomiting and fever within
12 to 24 hours.
Contamination of ready-to-
eat foods, insufficient
cooking and
recontamination of cooked
foods.
No growth below 40°
F.Bacteria are destroyed by
normal cooking.
Clostridium
perfringens
Produces a spore
and prefers low
oxygen
atmosphere. Live
cells must be
ingested.
Meat and poultry
dishes, sauces and
gravies.
Cramps and diarrhea within
12 to 24 hours. No vomiting
or fever.
Improper temperature
control of hot foods, and
recontamination.
No growth below40
degrees F.Bacteria are
killed by normal cooking
but a heat- stable spore can
survive.

10. Bacteria
Responsible
Description
TYPES OF
FOODS
Symptoms Cause Temperture Sensitivity
Clostridium
botulinum
Produces a spore
and requires a low
oxygen
atmosphere.
Produces a heat-
sensitive toxin
Home-
canned
foods.
Blurred vision,
respiratory
distress and
possible DEATH.
Improper
methods of
foods.
Type E and Type B can grow
at 38° F. Bacteria destroyed by
cooking and the toxin is
home-processing destroyed by boiling for 5 to 10
minutes. Heat-resistant spore
can survive.
Vibrio Requires salt for
parahaemolyticus growth.
Diarrhea, cramps,
Raw and vomiting,
cooked headache and
seafood. fever within 12 to
24 hours.
Recontamination
of cooked foods
or eating raw
seafood.
No growth below 40° F.
Bacteria killed by normal
cooking.
Bacillus cereus
Produces a spore
and grows in
normal oxygen
atmosphere.
Starchy
food.
Mild case of
diarrhea and
some nausea
within 12 to 24
hours.
Improper holding
and storage
temperatures
after cooking.
No growth below 40° F.
Bacteria killed by normal
cooking, but heat-resistant
spore can survive.
E. coli
Can produce
toxins that are
heat stable
Meat
and
Diarrhea,
abdominal
cheeses. cramps, nofever.
Inadequate
cooking.
Recontamination
of product.
Organisms can be controlled by
heating. Can grow at refrigeration
temperatures.

11. PRODUCTION OF TOXINS

12. SELECTING THE STRAIN FOR VACCINE
PRODUCTION
GROWING THE MICRO-ORGANISM
ISOLATION & PURIFICATION OF
MICROORGANISM
INACTIVATION OF ORGANISM
FORMULATION OFVACCINE
QUALITY CONTROLAND LOTRELEASE
Upstream
processing
Downstream
processing

13. 1.Clarification:
ULTAPrime GFcapsule
Filtration(0.6µm)
2.Diafiltration(0.4µm) in buffer[20mM
Tris+0.5M NaCl, pH7.5]
3.DNA binding Anion Exchange
Chromatography: Toremove gDNA
4.Sterile Filtration(0.2µm): Stainless steel
filter precoatedwith buffer.
DOWNSTREAM
PROCESSING

14. CAUSATIVE AGENT
• Clostridiumtetani
Left.Stainedpusfrom amixedanaerobicinfection.Atleast
three different clostridiaareapparent.
Right.Electronmicrographof vegetativeClostridiumtetani
cells.
9

15. Tetanus is an acute and highly fatal disease caused by exotoxins
produced by the bacterium Clostridium.tetani.
Cl. tetani produces two exotoxins- tetanolysin and
tetanospasmin.
Tetanus toxoid is one of the most immunogenic antigens available for the
protection against an infectious disease in the world.
Tetanus Vaccine produced by anaerobic fermentation.

16.  The production of tetanus toxoid in a large culture vessel was described
by R. 0. Thomson By employing a semicontinuous procedure, 70 liters
of culture every 48 hrs could be produced. When fully continuous
methods were attempted, the organisms tended to lose toxigenicity.
 J. R. Hepple was able to obtain large volumes of high potency
material by batch cultivation in tanks, but did not report any yields
PRODUCTION OF TETANUS TOXOID

17. The production medium was prepared by following
the procedure
•in a 100-gal, steam-jacketed, glass-lined fermentor, equipped with
temperature control and a two-bladed gear-driven agitator, and with
proper connections for introducing inoculation and withdrawing
samples.
•The medium was sterilized at 118 to 122 C for 1 hr, cooled rapidly to
36 to 39 C, and inoculated immediately.

18. SEED CULTURES OF CLOSTRIDIUM TETANI
• Seed cultures of Clostridium tetani (Massachusetts strain) were maintained at -70 C in
1.0-ml amounts in glass ampoules.
• One or four of these ampoules were thawed and inoculated into 200 to 300 ml of Brewer's
thioglycolate broth contained in 500-ml bottles
• . After incubation at 37 C for 24 to 30 hrs, these cultures were used to inoculate the tank.
• Tank cultures were incubated for 7 to 8 days at 36 to 39 C.
• Either nitrogen or air was introduced to the culture surface within the
tank through a sterile filter stack packed with glass wool.
• Satisfactory toxin yields were obtained in both 200- and 300- liter volumes with gas
atmospheres of either nitrogen or air.

19. CULTIVATION (ANAEROBICALLY)
CELL LYSIS
CENTRIFUGATION
SUPERNATANT
DETOXIFICATION
PROCESS INVOLVED INTETANUS
TOXOID PRODUCTION

20. PURIFICATION BY ULTRAFILTERATION(O.2 um)
FRACTIONAL SALT PRECIPITATION
DIALYSIS/GEL FILTRATION
(REDUCE NH4S04 )
ABSORBED ON ADJUVANTS+ ADDITION OF PRESERVATIVES
AUTOMATED FILLING OF TOXOID IN AMPOULES AND
VIAL

21. Alum
12
Productionstrain Seedculture Fermentation Purification
Adjuvant
Filling
Labeling Packaging



Inoculation
Inspection


Ultrafilteration

Glycine
soln
Formaldehyde solnMonosodium
glutamate
Inactivation
Thimersol


Stabilizer
Preservative
Dilutant