This document discusses monoclonal antibodies, including their structure, types, methods of preparation, applications, and marketed products. Monoclonal antibodies are homogeneous antibodies produced by identical immune cells that are clones of a single parent cell. They are prepared using the hybridoma technology which involves immunizing an animal, fusing immune cells with myeloma cells to form hybridomas, screening and selecting hybridomas that produce the desired monoclonal antibody. Monoclonal antibodies have various therapeutic applications in areas such as cancer, autoimmune diseases, organ transplants, and infections. They are also used for diagnostic purposes.

1. Presented by : SHIKHA SINGH
Under the guidance of : Dr. Srinivas Mutalik
Department of Pharmaceutics
Registration No. 160617009
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MONOCLONAL ANTIBODIES

2. CONTENTS
• Introduction
• Structure of monoclonal antiboby
• Types of monoclonal antibodies
• Methods for preparation of monoclonal antibodies
• Applications of monoclonal antibodies
• Marketed preparations
• Conclusion
• References
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3. INTRODUCTION
• Antibodies are glycoprotiens(globulins) present in the serum, also known as
immunoglobulins(Ig’s) & are produced by B-lymphocytes
• Monoclonal antibodies are the monospecific antibodies & are made by identical immune cells
that are all clone of a parent cell
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4. • Antibodies function as markers, binding to the antigen so that the antigen molecules can be
recognized & destroyed by phagocytes
• Naked mAbs are antibodies that work by themselves which are non-cytotoxic, drug or
radioactive
• Conjugated mAbs are linked to a chemotherapeutic drug, radioactive particle, or a cytotoxin,
work by take these substances directly to the cells
Types of mAbs
Naked mAbs
Conjugated
mAbs
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5. STRUCTURE OF ANTIBODY
• Y-shaped structure heterodimeric molecules
• Heavy chain and light chain
• Fab Fragment and Fc Fragment
• Paratope and Epitope Light chain
Heavy chain
FAB region
FC region
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6. TYPES OF ANTIBODIES
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7. Advantages
 Homogeneity: mAbs represents single antibody molecule that binds to antigens with the same
affinity & promote the same effector functions
 Specificity: The product of a single hybridoma reacts with the same epitope on antigens
 Selection: It is possible to select for specific epitope specificities
 Antibody Production: Unlimited quantities of a single well-defined mono-specific antibody can
be generated
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8. Disadvantages
 Affinity: Average affinity of monoclonal antibodies is generally lower than polyclonal
antibodies.
 Cross-reactions: Antibodies sometimes display unexpected cross-reactions with unrelated
antigens.
 Time and effort commitment: Very large.
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9. PREPARATION METHODS OF mAbs
Preparation
methods of
mAbs
Main methods
Hybridoma
technology
Large scale
production
Encapsulated
hybridoma cells
Alternative
methods
Using Bacteria
as precursor
Polymerisation
Chain Reaction
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10. Hybridoma technology
• Production of mAbs involved:
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Immunization
Cell fusion
Selection of
hybridomas
Screening of
hybridomas
Cloning &
propagation
Characterization
& storage
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11. Immunization
Step 1: - Immunization Of Mice & Selection Of Mouse Donor For
Generation Of Hybridoma cells
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ANTIGEN + ADJUVANT
SPLEEN REMOVED
(source of cells)
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12. Cell fusion
Step 2: - Fusion of Myeloma Cells with Immune Spleen Cells &
Selection of Hybridoma Cells
FUSION
HGPRT
MYELOMA CELLSSPLEEN CELLS
Fused cells, Free
myeloma & spleen cells
PEG added &
washed (3mins)
HAT
Medium
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13. Selection of hybridoma
 Myeloma cell- HGPRT- &
cannot survive in HAT
medium
 B cell (spleen)- HGPRT+ &
survive in HAT medium but
after some division undergoes
normal cell death
 Hybrid cell (fused)- survive in
HAT medium
Step 3: - Formation & selection of Hybridoma Cells is based on 2
pathways De-novo pathway & salvage pathway
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14. Screening of hybridoma
Step 4: - Screening of product
ELISA RIA
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15. Cloning and propagation of hybridoma
Step 5: - Cloning & propagation of product
Two techniques are commonly employed for cloning hybrid cells
1. Limiting dilution method or expansion
2. Soft agar method
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16. Characterization and storage of hybridoma
Step 6: - Characterization & storage of product
 The monoclonal antibody has to be subjected to biochemical & biophysical characterization for
the desired specificity
 The stability of the cell lines & the MABs are important
 The cells (and MABs) must be characterized for their ability to withstand freezing & thawing
 The desired cell lines are frozen in liquid nitrogen at several stages of cloning & culture
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18. Large scale production
 Production of mabs in culture bottles is rather low- 5-10g/ml
 Yield can be increased by growing the hybrid cells as ascites in the peritoneal cavity of mice
which yields about 5-20 mg of mabs/ml
 It is superior than the in vitro cultivation techniques
 Ascitic fluid in mice may have high risk of contamination by pathogenic organism of animal & in
addition, several animals have to be sacrificed to produce mabs
 Hence, many workers prefer in vitro techniques rather than the use of animals
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19. Encapsulated hybridoma cells
 Production of mAb can be substantially
increased by increasing the hybridoma cell
density in suspension culture
 By this approach, a much higher
concentration of Monoclonal antibody
production (10-100g/ml) can be achieved
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20. Alternative methods
Using Bacteria as a precursor:
Bacteria Antibodies
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21.  Polymerase chain reaction:
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22. APPLICATION
Applications of mAbs:
1. mAbs as Target drug delivery
2. Therapeutic uses of mAbs
3. Diagnostic use
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23. mAbs as Target drug delivery
1. Dissolution clot
2. Immunoliposomes
3. Immunomicrospheres
4. Radio immunotheraphy
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24. Dissolution clot
Mechanism
mAb is coupled with tPA & used for degradation of blood clots
mAb-tPA complex due to a high affinity gets attached to fibrin
conversion of plasminogen to plasmin which is in turn dissolves
blood clot (fibrin)
Clot lysis has been reported by using mAbs-tPA complex in
animal
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25. Immunoliposomes
Antigens expressed by tumor cells are present in higher ratio than
the normal cells
Immunoliposomes are expected to bind more to high antigen density
tumor cells than to low antigen density normal cells
Low valency immune-liposomes were found to allow better
discrimination between target and normal cells
High drug loading potential that a small number of antibody
molecules conjugated to the surface of an immune-liposome can
deliver many more drug molecules to the target
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26. Immunomicrospheres
• Biocompatible & biodegradable polymers, antibodies
have been conjugated to polymeric microparticles for
controlling their in vivo deposition
• Another study has evaluated the in vivo drug delivery
potential of albumin immune-microspheres in mice,
microspheres bearing Lewis lung carcinoma MABs
demonstrated slightly higher localization in lung
carcinoma at 24 h after its administration
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27. Radio immunotheraphy
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28. Therapeutic uses of mAbs
• Pregnancy
• HIV
• Cancer
• Autoimmuno diseases
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29. Pregnancy
 A monoclonal antibody can be used to detect pregnancy only 14 days after conception
 A pregnant woman has the hormone human chorionic gonadotrophin (HCG) in her urine
 mAb have been produced against HCG to which enzymes is attached, which can later interact
with a dye & produce a colour change
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30. HIV
 The test of HIV infection is based on detecting the presence of HIV antibody in the patient’s
blood serum
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31. Cancer
 Cancer cells carry specific tumour-associated
antigens (TAA) on their plasma membrane.
 Monoclonal anti-TAA antibodies have been
produced
 Drugs which kill tumour cells or inhibit key
proteins in tumour cells are attached to
monoclonal anti-TAA antibodies
 Cancer cells are specifically targeted, avoiding
damage to healthy host cells
 mAbs are being used to track cancer antigens &
alone or linked to anticancer agents, to attack
cancer metastases
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32. Autoimmuno diseases
 Monoclonal antibodies used for autoimmune diseases include infliximab and adalimumab,
which are effective in rheumatoid arthritis, Crohn's disease & ulcerative Colitis
 Basiliximab & daclizumab activated T cells & thereby help preventing acute rejection of kidney
transplants
 Omalizumab inhibits IgE which is useful in moderate-to-severe allergic asthma
 The monoclonal antibody known as OKT3 is saving organ transplants threatened with rejection
& preventing bone marrow transplants from setting off graft-versus-host disease
 mAbs neutrilize the action of lymphocytes responsible for the rejection of grafts & destroy the
auto-antibodies produced in auto-immune disease.
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33. Diagnostic use
 mAbs can be used to detect for the presence & quantity of this substance, for instance in a
Western blot test (to detect a substance in a solution) or an immunofluorescence test
 mAbs can also be used to purify a substance with techniques called immunoprecipitation &
affinity chromatography
 mAbs allow rapid diagnosis of hepatitis, influenza, herpes, streptococcal & Chlamydia infections
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34. Antibody Brand name Type Indication
Palivizumab Synagis humanized Respiratory Syncytial Virus
Panitumumab Vectibix human Colorectal cancer
Ranibizumab Lucentis humanized Macular degeneration
Rituximab Rituxan, Mabthera chimeric Non-Hodgkin lymphoma
Tocilizumab Actemra Humanised Rheumatoid arthritis
Tositumomab Bexxar murine Non-Hodgkin lymphoma
Trastuzumab Herceptin humanized Breast cancer

35. Conclusion
 Monoclonal antibodies are new biological agents that have good clinical effects & an
extended choice in the treatment spectrum to the patients who were not responding to the
existent treatments
 New therapeutic approaches are rapidly emerging and further studies may help in designing
more specific Mabs that would spare the normal tissue, have less adverse effects and
improve the patient’s quality of life
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36. References
1. AM Carpbell, monoclonal antibody and immunosensor, edited by P.C. van der VLIET -
Department for Physiological Chemistry, University of Utrecht, Utrecht, Volume 23, 1991
Elsevier
2. http://www.biotecharticles.com/Others-Article/Hybridoma-Technology-A-Biotechnology-
Technique-378.html,25th Jan 2017
3. http://pathology.wustl.edu/research/hybridoma.php?page=advantages, 30th Jan 2017
4. http://en.wikipedia.org/wiki/Monoclonal_antibodies,27th Jan 2017
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