1. A hybridoma is a hybrid cell produced by fusing an antibody-producing cell (usually a B lymphocyte) with a myeloma cell. This allows the cell to produce monoclonal antibodies indefinitely in culture. 2. To produce monoclonal antibodies, B lymphocytes are isolated from an immunized animal and fused with myeloma cells using polyethylene glycol. The resulting hybridomas are selected in HAT medium, which allows only fused cells to survive and proliferate. 3. Once a hybridoma producing the desired antibody is identified, it can be cultured indefinitely to produce large amounts of monoclonal antibodies, which have applications in research, diagnostics and therapeutics.

1. Department of Pharmacy,
Dr. B. R. Ambedkar University, Agra
Topic- Genetic Recombination
Submitted By: Submitted To:
Krishna Kumar Dr. RS Sharma
B. Pharm VIII Sem (Asst. Professor)
Roll No- 168287365004

2. Development of hybridoma for monoclonal antibody
 A hybridoma is a hybrid cell obtained by fusing antibody –
producing cell and a multiple myeloma cell. (Multiple myeloma
is a cancer of plasma cell.)
OR
 It may be defined as a hybrid cell obtained by fusing Beta-
lymphocyte with usually a tumor cell of the antibody forming
system or of B- lympocytes ( these are called myelomas).
 The hybrid cell thus produced possess the ability to produce
antibodies due to the B-lymphocyte genome and the capacity for
indefinite growth in vitro due to the tumor (myeloma) cell
involved in the fusion.

3.  Therefore, hybridoma cells are either cultured invitro or
passaged through mouse peritoneal cavity to obtain monoclonal
antibodies. This is called hybridoma technology.
 B-lymphocytes are isolated from the spleen of an animal, e.g,
mouse, which had been immunized with the antigen against
which monoclonal antibodies are to be raised (produced).
 Myeloma cells are selected for mainly two features:
 These cells must not produce antibodies themselves, and
 They must contain a genetic marker e.g. HGPRT-trait
(hypoxanthine –guanine phospho-ribosyl transferase), which
permits an easy selection of the resulting hybrid cells.

4.  When HGPRT-cells are fused with B- lymphocytes, the
resulting cell population will consist of
 hybrid cells (hybridomes)
 myeloma cells
 B-lymphocytes
 This cell population is now cultured in HAT medium
containing the drug aminopterin.
 Similarly, the B-lymphocytes do not grow for long periods of
time in tissue culture and eventually die.
 In contrast, only the hybridoma cells proliferate on the HAT
medium since the B-lymphocyte genome makes them
HGPRT+ and they have the capability for indefinite growth
from the myeloma cell.

5.  Thus hybridomas (myeloma +B-lymphocyte hybrid cells) are
selected by using a suitable selective medium like HAT, which
allows only the hybridomas to proliferate.
 The next step consists of identification and isolation of the
hybridoma cells producing antibodies specific to the antigen
used for immunization of the animals.
 Once the desired hybrido maclone has been obtained, it is
multiplied either in vitro or in vivo to obtain monoclonal
antibodies.
 In vivo production system involves injection of hybridoma
cells into the peritoneal cavity of isogenic animals, collection
of the ascitic fluid and separation of the antibodies from it.
 In vitro production involves growing hybridoma cells are
grown in vitro in a suitable large scale culture system and the
monoclonal antibodies are purified from these cultures.

6. HAT Medium
 HAT medium is one of the several selective media used for the
selection of hybrid cells.
 This medium is supplemented with hypoxanthine,
aminopterin and thymidine, hence the name HAT medium.
 Antimetabolite aminopterin blocks the cellular biosynthesis of
purines and pyrimidines from simple sugars and aminoacid.
 However, normal human and mouse cell scan still multiply as
they can utilize hypoxanthine and thymidine present in the
medium through a salvage pathway, which ordinarily recycles
the Purines and Pyrimides produced from degradation of
nucleic acids.

7.  Hypoxanthine is converted into guanine by the enzyme
HGPRT, while thymidine is phosphorylated by thymidine
kinase (TK); both HGPRT and TK are enzymes of the salvage
pathway.
 On a HAT medium, only those cells that have active
HGPRT(HGPRT+) and TK(TK+) enzymes can proliferate,
while those deficient in these enzymes (HGPRT -and / or TK-)
cannot divide.
 Thus, one may fuse HGPRT deficient human cells (designated
as TK+HGPRT-) with TK deficient mouse cells (denoted as
TK- HGPRT+). Their fusion products (hybrid cells) will be
TK+ (due to human gene) and HGPRT+ (due to mouse gene)
and will multiply on the HAT medium, while the man and
mouse cells will fail to do so.

8. Application of monoclonal antibodies (Mabs)
 Diagnostic application:
 When Mabs are used to detect the presence of aspecific antigen or of
antibodies specific to an antigen in a sample or samples, this constitutes
a diagnostic application. Some examples of diagnostic applications are
as follows:
1. Mabs are available for the un equivocal classification of blood
groups e.g. ABO, Rh, etc.
2. Mabs are applied for a clear and decisive detection of
pathogens involved in disease (disease diagnosis).
3. Mabs can be used for the accurate detection of specific
chromosomes of a given species.

9.  Therapeutic application:
1. Antibodies specific to a cell type, say, tumor cells, can be
linked with a toxin polypeptide to yield a conjugate molecule
called immunotoxin. The antibody component of immunotoxin
will ensure its binding specifically and only to the target cells
and the attached toxin will kill such cells.
2. Mabs can be administered to provide passive immunity against
diseases.
3. Mabs are very useful in the purification of antigens specific to
pathogens; these purified antigens are used as vaccines.
 Immunopurification:
1. The highly specific interaction of an antibody to the antigen is
used to purify antigens present in small quantities as a mixture
with several types of molecules; this is known as
immunopurification.

10. Drugs produced by biotechnology
 The European Federation of Biotechnology (FEB) considers
‘biotechnology ’as— ‘the integration of natural sciences and
organisms, cells, parts there of, and molecular analogues for
products and services.’
 New Biotechnological processes essentially embrace almost
all methods of genetic modification by recombinant DNA and
cell fusion techniques, together with the ‘magic touch’ of the
modern developments of these so called ‘traditional-
biotechnological processes’.
 Interestingly, these processes will, in many instances, function
at relatively low temperature, will consume little energy, and
will mainly on inexpensive substrates for biosynthesis.

11.  Although there are a large number of ‘drugs’ that have
been evolved via the biotechnological processes. Few of
which are listed below:
a) Altepase [Activase®],
b) Human Insulin [Humulin (R)],
c) Humatrope: Growth Hormone, and
d) HepatitisB [Recombinant HB (Merck)— a Hepatitis B vaccine]

12. Alteplase [ Activase®]
 Activase® (Alteplase) is FDA approved for treatment of myocardial
infarction (heartattack), acute ischemic stroke (blood clot in the brain)
& acute massive pulmonary embolism (blood clots in the lungs).
 Activase is one of the recombinant tissue plasminogen activator, or r-
tPA and is produced by recombinant DNA technology.
 For certain patients, Activase may improve the chances of recovery
from stroke with little or no disability. Patients can receive Activase
only if they begin treatment within 3hours after their stroke symptoms
start and only after they have had as can to rule out bleeding in the
brain.
 Tissue plasminogen activator (abbreviated tPA or PLAT) is a protein
involved in the break down of blood clots. It is a serine protease found
on endothelial cells, the cells that line the blood vessels.

13.  As an enzyme, it catalyzes the conversion of plasminogen to
plasmin, the major enzyme responsible for clot breakdown.
Because it works on the clotting system, tPA is used in clinical
medicine to treat only embolic or thrombotic stroke. Use is
contraindicated (not advisable) in hemorrhagic stroke and
head trauma.
 tPA may be manufactured using recombinant biotechnology
techniques. tPA created this way may be referred to as
recombinant tissue plasminogen activator (rtPA).
Recombinant tissue plasminogen activators (r-tPAs) include
alteplase, reteplase, and tenecteplase (TNKase).
 Storage: Alteplase need to be stored preferably at –20°C or
even below in perfectly sealed containers.

14.  Units: The activity of alteplase may be measured in terms of
International Units (IU) by employing the 2nd
International
Standard for the Tissue Plasminogen Activator established in
1987, although it is an usual practice to express the doses by
weight. The Specific Activity of alteplase is 580000IUs. mg–1
 Pharmacokinetics: It has been duly observed that alteplase
gets cleared from the plasma, chiefly via metabolism in the
liver.
 Note: IU is the amount of an enzyme that catalyses the
transformation of 1 micromole of substrate per minute
(under defined conditions of pH, concentration, and
temperature)

15. Uses and Mechanism of Action :
 The various applications and possible mechanism of action of
‘alteplase’ are as follows:
 It is a thrombolytic agent, which is a predominant representative of a
single-chain form of the endogenous enzyme tissue plasminogen
activator meticulously produced by there combinant DNA
technology. Very much similar to the endogenous tissue
plasminogen activator, it converts fibrin-bound plasminogen to the
corresponding active form of plasmin, there by causing in marked
and pronounced fibrinolysis and dissolution of clots.
 Alteplase is employed very much akin (similar in quality and
character) to steptokinase both in the management and treatment of
thrombo-embolic disorders, specifically the myocardial infarction
and venous thrombo-embolism.

16.  Alteplase has almost negligible effect upon the circulating,
unbound plasminogen; and hence, may be termed as a fibrin-
specificagent.
It was perhaps thought that fibrin specificity could bean
absolute necessity for minimising the prevailing risk of
ensuing haemorrhage intimately associated with the
application of thrombolytics; although the latest fibrin
specific drugs usually give rise to appreciable bleeding
incomparison to the non-specific thrombolytics.

17. Humulin : Humulin®
 Humulin: Humulin® is the branded product of the famous pharmaceutical
manufacturer, Lilly, containing human-insulin and its host of variants,
being produced by it indifferent countries across the globe.
Description of Insulin:
 Insulin is a pancreatic-hormone essentially involved in the regulation of
blood- glucose concentrations and also having a specific role in the protein
and lipid metabolism.
 In usual practice, the human, porcine, bovine or mixed porcine-bovine
insulin is administered to such patients having insulin- dependent diabetes
mellitus in the management and control of their blood-glucose
concentrations. It may also be used necessarily in certain non-insulin-
dependent diabetics. Insulin is also an essential component of the
emergency management and control of diabetic ketoacidosis.
Insulin is ‘a hormone produced by the β-cells of the islets of Langerthans of
the pancreas and essentially comprise of two separate chains of amino
acids, the A and B chains, joined together by two disulphide bridges’.

19. Human Insulin
 Human insulin is a dimer comprising one chain of 21
amino acid (A chain) and the other 30 amino acids (B
chain).
 Both the chains A and B are derived from a single poly
peptide chain, and are held together by two disulphide
bridges.
 Generally, the insulin gene codes for preproinsulin, a
polypeptide containing the A and B polypeptides of the
active insulin, and a connecting polypeptide that is
absent from mature insulin.
 Insulin is processed from preproinsulin via proinsulin by
enzymatic cleavage of the connecting polypeptide from
the A and B chains.

20. Production of human insulin

21.  These plasmids were transformed into E.coli strains.
 The transformed bacteria thus produces the fusion protein of A
chain and B chain separately.
 The A and B chains were separated by amethionine residue from the
β-galactosidase sequence encoded by lacZα.
 Therefore, the insulin sequences were separated from the β-
galactosidase sequences by treating the fusion proteins with
cyanogen bromide.
 The purified chains A and B were then attached to each other by
disulphide bonds to produce insulin.
 This method how ever turned out to be an inefficient reaction.
 Therefore, a gene representing B, C and A Chains was synthesized
and expressed in E.coli; in this case, the intervening chain is
removed proteolytically following spontaneous folding of the
proinsulin molecule.

22. Humatrope® [Growth Hormone]
 Growth hormone is an anabolic hormone secreted by the anterior
pituitary which stimulates tissue growth and anabolism. It is found
to affect fat, carbohydrate and mineral metabolism.
 Humatrope (somatropin): It is a polypeptide hormone of rDNA
origin.
Manufactured by Eli Lilly and Company, it is used to stimulate
linear growth in pediatric patients who lack adequate normal human
growth hormone. It has 191 amino acid residues and a molecular
weight of 22,125 daltons.
 Its amino acid sequence is identical to that of human growth
hormone of pituitary origin (anteriorlobe). Humatrope is synthesized
in a strain of E. coli modified by the addition of a gene for human
growth hormone.

23.  Other human growth hormone produced from rDNAs include;
Omnitrope (Sandoz), Nutropin, Norditropin, Genotropin
(Pfizer).
 Units: One Ampoule of the First International Standard
(1987): 4.4 units of the human growth hormone (somatropin)
are contained in 1.75 mg of freeze-dried purified human
growth hormone, with 20 mg of glycine, 2 mg of mannitol, 2
mg of lactose, and 2mg of sodiumbicarbonate.

24. Pharmacokinetics
 Somatropin is well-absorbed after subcutaneous or IM
injection.
 After IV injection it has a half-life of about 20-30 minutes;
however, after subcutaneous or IM administration the
prevailing serum concentrations usually decline having a half-
life of 3-5 hours, on account of the relatively more prolonged
release from the site of injection. It is found to be metabolised
in the liver and excreted in bile.

25. Uses and Mechanism of Action
 Somatropin is a synthetic human growth hormone; and Somatrem is
its corresponding methionyl analogue. The ‘drug’ promotes the
growth of muscular, skeletal, and other tissues, stimulates protein
anabolism; and also affects fat and mineral metabolism.
 The hormone exhibits adiabetogenic action upon the carbohydrate
metabolism specifically.
 These cretion is observed to be pulsatile and solely depends upon
the neural and hormonal influences, such as:
 Hypothalamic release-inhibiting hormone e.g.,somatostatin, and
 Hypothalamic releasing hormone e.g.,somatorelin. In fact, there are certain
physical and physiological factors that largely influence an enhanced secretion
of the growth hormone, such as : sleep, emotional stress, and hypoglycaemia