HISTORY:

In 1975, these technology developed by

Georges J.F.Kohler and Cesar Milstein.
And in 1984, they shared a Nobel prize for
this discovery.
They make a hybrid cell that will make a
numbers of monoclonal antibodies against
antigen .

PRINCIPLE:

The hybrid cell has the capacity of antibody

production derived from B-cells (spleen cell ).
At the same time it can divide continuously by
the quality derived from myeloma cell.
By combining the desired qualities of both the
cells, the technology ensures large, antibody
productionof single specificity.
Specific hybridomas(spleen cell and myeloma
cell) obtain monoclonal antibodies in artificial
media, this technology called as HYBRIDOMA
TECHNOLOGY.
MONOCLONAL ANTIBODY:

Monoclonal antibodies (mAb) are

antibodies that are identical because they
are produced by one type of immune cell, all
clones of a single parent cell.
Basically produced by white blood cell
which is called as plasma cell.
Is used for treatment of cancerous cells and
as anti-venom( anti snake venom)
PROCEDURE:

1. Immunization of specific animal which

generate hybridoma cell with spleen cell.
2. Isolation of myeloma cells.
3. Fusion between spleen cell and myeloma
cell.
4. Selection of HAT medium.
5. Isolation of hybridomacelI.
Screening of hybridoma cell.

1. Immunization of specific animal. W

An antigen immunized to an animal (like mice) via

ravenously(directlyto blood) by injection.
Where in spleen it activate B-cell which produce
plasma cell (spleen cell).
Plasma cell to produce monoclonal antibodies

Isolation of plasma cellfrom spleen of animal.

| 2. Isolation of myeloma cels.

Myeloma cells are cancerous cells which is

isolated from bone-marrow.
Myeloma cells are generally immortal in
nature (that which never dies) and has
multiplication property.

|3. Fusion of spleen cell and myeloma W

>It requires PEG (poly ethylene glycone)
medium for fusion
It can also done by electro fusion.
>Fusion between spleen cell and myeloma
cell produced five different types of cells.
Fused plasma
Fused myeloma
Hybridoma
Unfused plasma
Unfused myeloma
4. Selection of HAT medium.
(Hypoxanthine, Aminopterin, Thymidine)
Before multiplication of Anti-body, it has to
synthesize new copy of DNA and for that it
require synthesis of nucleotide.
For synthesis of nucleotide mainly two pathways
are there:
1. Salvage pathway
2. De-novo Synthesis
In 1, Salvage pathway it requires degraded part
of old nucleotide toproduce new nucleotide.
In 2, De-novo
De-novo synthesis it synthesized
completely new nucleotide by small molecules
(sugar, amino-acid).
1

So in HAT medium, Cells not synthesizeu uy

De-novo synthesis due to presence of
Aminopterin in HAT medium which blocks
Di-hydro follate enzyme which is
necessary for these synthesis.
For synthesis in salvage pathway it must
requires HGPRT enzyme (Hypoxanthine
Guanine Phospho-Ribosyl Transferase).
Where hypoxanthine and thymidine are
used as precursors.
| 5. Isolation of hybridoma cell
Spleen cell have Myeloma cell doesn't
HGPRT enzyme have HGPRT enzyme

HGPRT
1. Fused plasma present
2. Fused myeloma absent
3. Hybridoma present
4. Unfused plasma present
5. Unfused myeloma absent

Sploen cots HGPRT')

ntbody producng Myoloma ols (HGPRT)

Fusion in poyetnylene gycol

Unfused
spioen
cels

Cell death
Fused
spleen
cells
888 Hybridoma Fused
myeloma
calls

Cell death
Untused
myeioma
cels

Proliferation
 Fused myeloma and unfused myeloma
didn't have HGPRT enzyme so, can't survive
in HAT medium.
Fused plasma and unfused plasma have
HGPRT enzyme but didn't have long-life.
Hybrid cell has HGPRT enzyme from spleen
cell as well as they have the ability to
multiply repeatedly as myeloma cell.
So, isolation of hybrid cell because is only
cell which survive in HAT medium.

6. Screening of hybridoma cell.

ELISA screening method which done by
incubating hybridoma culture in which
secondary enzyme gets conjugate and
formation of colored product shows positive
hybridoma.
Used for multiplying the hybridoma cells
o In-vivo
o In-vitro
 In vivo

ro
e

In-vivo procedure involves introductio..

hybridoma cells into the peritoneal cavity of
the animal , then from ascetic fluid
antibodies are isolated.
In-vitro method involves culturing of
hybridoma cells in suitable culture media
and then antibodies are isolated and
purified.
Once a hybridoma colony is established, it
will continually grow in culture medium
like RPMI-1640 and produce antibodies.
Storage: liquid nitrogen.
APPLICATION OF HYBRIDOMA TECHNOWY
º Serological;
. Identification of ABO blood group
Diagnosis:
Detection of pregnancy by assaying of hormones with
monoclonal.
Separation of one substance from a mixture of very similar
molecules.
> Immunopurification:
. Purification of individual interferon using monoclonal.
Inactivation of T-lymphocytes responsible for rejection of organ
transplants.
Therapy:
Removal of tumor cell from bone marrow.
Treatment of acuterenal failure.
Treatment malignant leukemic cells, B cell lymphomas, and a
variety of allograft rejections after transplantation.
 EQUIPMENTS

Fermenter:
" A Fermenter is a device that accomplishes the fermentation process by ti..y f
certain microorganisms, and thus it also refers as "Biofermenter or Bioreactor". it
is equipped with all the elements that are necessary to carry out the commercial
production of substances like antibiotics, enzymes, beverages etc. in many
industries. -MPELLER SHAT
ASEPTIC
NOCULATION
PPE
STIRRER
SHAFT SEAL
wORONG LEVEL
BASIC DESIGN OF FERMENTER (OF BROTH
BAFFLE H

SAMPLING
POINT

MPELLER
STERILE AR
ORAIN POINT
AIR SPARGER

Dagam of a tmeter wih one mutstladd impeler. H, lermentr height Lgd heigt D. ank
dametr P.impeler dameter.
 BASIC sTEPS OF FERMENTATION PROCESS AND GENERAL REQUIREME

The basic steps for fermentation process are:

1. Formulation of fermentation media: it is chemically a cell culture medi.
a mixture of molecules used for cellular metabolism and growth. e.g. Carbon
source, energy source, oxygen, nitrogen etc.
2. Sterilisation: sterilisation of fermentation media, Fermenters and other
equipments.
3. Inoculum formation: production of an active, pure culture In sufficient quantity
to inoculate into the production vessel.
4. Fermentation: growth of the micro-organism in the production fermenter under
optimum conditions for formation of by-products.
5. Extraction and purification of the product.
Procuenert of

Carton Nbogen, Phogrorua, Suphur

mnor and ace imes, rowth tactons

Pparaton of medum Producton strin

For proocton

Figure shows the upstream

fermentation process

BASIC STEPS OF FERMENTATION PROCESS AND GENERAL REQUIREMEP

The general requirements for fermentation process are:

1. Fermentation medium
2. Inoculum
3. Fermenter
4. Sterilisation
5. Growth of organism
6. Extraction and purification
 METHODS OF FERMENTATION PROCESS

Batch Continuous
Fermentation Fermentation
process process
Three Methods
Of
Fermentation process

Solid state
Fermentation
process

Batch fermentation: Batch culture is a closed culture system which contains

limited amount of nutrients.
Continuous fermentation: it is open culture system where nutrients are
continuously added to fermenter and product is continuously removed at same
rate.
Solid state fermentation: the fermentation medium is in the solid state, here the
micro-organisms are grown on solid substratum in the absence or near absence of
free water.

STUDY OF MEDIUM

Fermentation medium:
Medium with nutritional and hormonal requirements of cells in optimum
concentration.
Small modifications in the medium could change cell line stability, product quality,
and yield.
Composition of media:
=>Carbon source-dextrin, glucose, starch etc
=>Nitrogen source-yeast extract, soybean meal, cotton seed meal etc.
=>lnorganic salts-ammonium salts, ammonium nitrate etc.
=>Vegetable/Animal fat-soybean oil, linseed oil etc.
=>Growth regulators - Vitamin B12
=>Trace elements-zinc, copper etc.
=>Buffers-to maintain pH eg calcium carbonate etc
=>Oxygen etc
Inoculum:
" It is the pure bacterial cell culture that is introduced to the medium .
" The cell then grows in the medium, conducting metabolism.
 EQUIPMENTS

Fermenter:
" AFermenter is a device that accomplishes the fermentation process by ti. . f
certain microorganisms, and thus it also refers as "Biofermenter or Bioreactor". it
is equipped with all the elements that are necessary to carry out the commercial
production of substances like antibiotics, enzymes, beverages etc. in many
industries. MPELLER SHAFT

ASEPTIC
INOCULATION
PPE

STIRRER
SHAFT SEAL
wORONG LEVEL
BASIC DESIGN OF FERMENTER (OF BROTH)
BAFFLE

4-P
SAMPUING
POINT

MPELLER
STERILE AR
LINE RDRAIN POINT

AR SPARGER

Dagram of a fermenter wih one ms-tladed inpeler. H. fermenter heigt L lquld height: D, tank
dameter, P, impeller dameter.

EQUIPMENTS

S.No. Part Purpose

1 Top plate cover (made of stecl)
2 Clamp top plate conpressed onto vessel using clamp
3 Seal separates top plate from vessel (glass) to prevent air leakage
Vessel glass, jacketed, stel with ports for various outputs, inputs, probes
cte
Drive motor used to drive mixing shaft
6 Drive shaft mixes the medium evenly with its impeller
7 Marine impeller for plant tissue culture
8 Baffles prevert sedimentation on sides and proper mixing
9 Sparger air supplier / after filtration via membranes ensures cficient
dispersal -by attachedto impeller
10 Exit gas cooler like condenser remove as much moisture as possible from exhaust
11 Ino culation port to add inoculum
needle
12 Fecd pumps regulates the flow rates of additives (medium, nutrients) variable
specd
13 Peristaltie fixed speed pumps - used for cortinuous sampling
pumps
14Singe pump using a syinge- mo stly used in batch
15 Exit gas analysis CO; analyzer, O2 analyzer, mass spectrometer
16 Sample pipe through which samples are drawn
17 3 wayinlet to insert different probes
VARIOUS COMPONENTS OF AN IDEAL FERMENTER WITH THEIR PURPOSES
 EQUIPMENTS

Specific parts of fermenters are:

1-Body: It is important to select the material that can with stand repeate
sterilisetion cycles.
e.g. Glass : advantages
gives smooth surface
" Non toxic
" Corrosion proof
" Easy to examine the interior part of vessel during fermentation process.
"Steel: As per AlSI(American Iron and Steel Institute) -Steel alloys are those
counting less than 4% chromium while Stainless steel are those contacting more
than 4% chromium.
Fro making of fermenter body mostly stainless steels are preferred
" Steel coated with glass/phenolic epoxy can also be used.

2- Seal: to seal top plate and vessel to prevent air leakage.

Types are:
" Gasket seal- can be used to seal glass and metal
"Lip seal- for joining of glass to metal
" O-ring seal-for joining of metal to metal or glass to metal.

EQUIPMENTS

3-Agitator: (Impellers)
The agitator is required for mixing of bulk fluid , gas phase mixing , air d p f ,
Oxygen transfer etc to maintain uniform environment.
Types of agitator are as follows:

a) Mounted to a shaft (al

through
the lid a bearing in

b) Driven by an external
power source or direct
drive

c) Direct drive - action

varied by using
different
blades
impeller (d)

d) Spining of medium in
circular direction k mie
 EQUIPMENTS

4-Baffles: There are normally 4 baffles incorporated into of vessel of ferme

sized to prevent vortex (swril effect) or to improve proper aeration efficiency.
Baffles are metal strips which are radially attached to the fermenter wall.

Four baffles in the fermentation vessel

5-Spargers: The air introducing device in the fermenter is the Spargers. The different
types of sparers are:
. Orifice spargers Spwger ind

Porous spargers Cs iet

. Nozzle spargers

Rnador el

Quá Cree Spgr

Sparger giving out air forming bubbles in the liquid Sparger device