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monoclonal antibody : production & application.pptx
1. Monoclonal Antibodies;
Preparation and Application
Guided by :
Mr. Arya. S. Vyas
Presented by :
Harshid Kukadiya
M.Pharm (Sem II)
Department of Pharmaceutics
L. M. College of Pharmacy, Ahmedabad
2. TABLE OF CONTENTS
Antigen, Antibody,
Monoclonal Antibody;
structure & type
Introduction Production
major steps of mAb
production
Application
Different application of
in research & industry
Approved mAb
Approved mAb for
treatment by authority
01 02
03 04
4. Antigen-Antibody
An antigen is a foreign substance that
enters your body.
This can include bacteria, viruses,
fungi, allergens, venom and other
various toxins.
An antibody is a protein produced by
our immune system to attack and
fight off these antigens.
Antigen Antibody
5. Antibody structure
● Also known as an immunoglobulin (Ig)
● They are glycoproteins comprising of 82–
96% polypeptides and 4–18%
carbohydrates.
● They are large, Y-shaped protein having
four peptide chains, produced by plasma
cells
● Two of these fragments are identical and
retain the antigen binding capacity
associated with an intact antibody.
● The two identical long chains are called
heavy chains and the two identical short
chains are called light chains.
6.
7. They are the antibodies that are identical because they were
produced by one type of immune cell, all clones of a single
parent cell.
Monoclonal antibody
8. Monoclonal antibodies (mAb) are a single type of antibody that are
identical and directed against a specific epitope (antigen, antigenic
determinant).
They are produced by B-cell clones of a single parent or a single hybridoma
cell line. A hybridoma cell line is formed by the fusion of a one B-cell
lymphocyte with a myeloma cell.
Monoclonal antibodies can have monovalent affinity, in that they bind to
the same epitope.
The idea of "magic bullets" was first proposed by Paul Ehrlich, who, at the
beginning of the 20th century, postulated that, if a compound could be made
that selectively targeted a disease-causing organism, then a toxin for that
organism could be delivered along with the agent of selectivity.
13. TABLE OF CONTENTS
Antigen, Antibody,
Monoclonal Antibody;
structure & type
Introduction Production
major steps of mAb
production
Application
Different application of
in research & industry
Approved mAb
Approved mAb for
treatment by authority
01 02
03 04
15. Production steps
Immunization of mouse
Cell fusion
Isolation of hybridomas of single specificity
Characterization and storage
Screening of products
Selection of Hybridomas
Cloning and propagation
16. Immunization of mouse
Mouse is immunized by injecting with an appropriate antigen along with
adjuvant.
Injection of antigens at multiple sites are repeated several times for
increased stimulation of antibodies.
3 days prior to killing of an animal a final dose is given intravenously.
Spleen is aseptically removed and disrupted to release the cells.
By centrifugation lymphocytes are separated from rest of the cells.
17. Cell fusion
Lymphocytes are mixed with myeloma cells and is exposed to PEG
for a short period.
The mixture is then washed and kept in fresh medium.
The mixture contains hybridomas, free myeloma cells, and free
lymphocytes.
18. Selection of Hybridomas
HAT selection
H = Hypoxanthine
A = Aminopterin Blocks De nova pathway
T = Thymidine
Only cells capable of nucleotide synthesis by the salvage pathway survive in
HAT medium.
HAT are metabolites for the salvage pathway.
( Key enzyme : HGPRT )
19. Isolation of hybridomas of single specificity
This is done by method known as limited dilution.
The cells are distributed in multi well culture plates at very low
density
20. Screening, cloning and propogation of products
The two techniques namely ELISA and RIA are commonly used for the
screening purpose.
In next step desired antibody are identified and they are isolated and
cloned.
Separate clones of activated B cells, each producing antibodies of a
single specificity known as monoclonal antibody.
21. Characterization and storage
In the final step, these monoclonal antibodies are characterized and
stored.
Mostly they are stored in liquid nitrogen.
Now this monoclonal antibodies are ready to use in treating and
diagnosis diseases.
22.
23. TABLE OF CONTENTS
Antigen, Antibody,
Monoclonal Antibody;
structure & type
Introduction Production
major steps of mAb
production
Application
Different application of
in research & industry
Approved mAb
Approved mAb for
treatment by authority
01 02
03 04
25. Therapeutic application :
Immune‐mediated diseases
Antibody Type Medical uses
Alemtuzumab Humanized, mAb, IgG1 Multiple sclerosis
Brodalumab IgG2Human, mAb, IgG2 Plaque psoriasis
Canakinumab Human, mAb, IgG1
Cryopyrin‐associated
periodic syndrome
Monoclonal antibodies have revolutionized the treatment of
autoimmune diseases.
It can target different components of the immune system .
26. Oncology
Antibody Type Medical uses
Trastuzumab Humanized, mAb, IgG1 Metastatic breast cancer
Rituximab Chimeric, mAb, IgG1
non‐Hodgkin lymphoma
anfd eukemia
Avelumab Human, mAb, IgG1 Merkel‐cell carcinoma
treatment of various neoplasias, including both hematologic ma‐
lignancies and solid tumors
To target tumor antigens and kill cancer cells
27. Infectious diseases
Antibody Type Medical uses
Bezlotoxumab Human, mAb, IgG1
Clostridium difficile
colitis
Raxibacumab Human, mAb, IgG1
Anthrax (prophylaxis
and treatment)
There are advantages of mAbs for the treatment of infections, over
immune sera-derived preparations, such as low lot-to-lot variability,
low risk of pathogen transmission, and no immunological
complications associated with the use of heterologous sera.
28. Diagnostic application
Monoclonal antibodies have become key components in a vast array of
clinical laboratory diagnostic tests. Their wide application in detecting
and identifying serum analytes, cell markers, and pathogenic agents
has largely arisen through the exquisite specificity of these unique
reagents.
Anti β HCG antibody can be used to detect pregnancy only 14 days
after conception.
Other monoclonal antibodies allow rapid diagnosis of hepatitis,
influenza, herpes, streptococcal infections.
29. Diagnostic application
Monoclonal antibodies have become key components in a vast array of
clinical laboratory diagnostic tests. Their wide application in detecting
and identifying serum analytes, cell markers, and pathogenic agents
has largely arisen through the exquisite specificity of these unique
reagents.
Anti β HCG antibody can be used to detect pregnancy only 14 days
after conception.
Other monoclonal antibodies allow rapid diagnosis of hepatitis,
influenza, herpes, streptococcal infections.
30. As therapeutic agent
In destroying disease causing organisms : mAb promote efficient
opsonization of pathogenic organisms (by coating with antibody) and
enhance phagocytosis.
In the immunosuppression of organ transplantation : in the normal
medical practice, immunosuppressive drugs such as cyclosporin and
prednisone are administered to overcome the rejection of organ
transplantation. In recent years, mAbs specific to T-lymphocyte
surface antigens are being used for this purpose.
31. Protein purification
Monoclonal antibodies can be produced for any protein and can be
used conventionally for the purification of the protein against which it
was raised.
These include the specificity of the mAb to bind to the desired protein,
very efficient elution from the chromatographic column and high
degree of purification.
32. Blood group Typing
Before blood transfusion, blood group typing is essential.
Blood group typing is carried out to find out compatibility between
donor and recipient blood groups. in all cross-match tests, a specific
chemical reaction of antibodies with erythrocyte antigens is carried out
to monitor agglutination
Recently at the National Institute of Immunology monoclonal
antibodies against blood group antigens A and B have also been
developed.
33. As a vehicle for drug delivery
An antibody can be used as an attached drug (magic bullets) or toxin
(immunotoxin) at cancer cells. By this approach, higher concentration
of the drug can be built up locally, minimizing systemic toxicity.
Indeed a far better survival rate is observed in experimental mice given
Daunamycin loaded on mAb raised against mouse lymphomas as
compared to those receiving the drug alone.
mAb covalently bound to a toxin or toxin-subunit knowm as immuno-
toxin have been observed to be effective in therapy of some cancers
34. TABLE OF CONTENTS
Antigen, Antibody,
Monoclonal Antibody;
structure & type
Introduction Production
major steps of mAb
production
Application
Different application of
in research & industry
Approved mAb
Approved mAb for
treatment by authority
01 02
03 04
37. GTU Questions :
1. Explain in detail the method of producing monoclonal antibodies by hybridoma
technology. ( winter 2018 )
2. Describe in brief the applications of monoclonal antibodies.
( winter 2018 )
3. Discuss method of preparing monoclonal antibodies. ( winter 2019 )
4. Discuss preparation and applications of Monoclonal Antibodies.
( winter 2021 )
5. Write a short note on monoclonal antibodies. ( winter 2022 )
6. Describe the pharmaceutical applications of monoclonal antibodies.’
( summer 2022 )
7. Describe the method of producing monoclonal antibodies by hybridoma
technology. ( winter 2018 )
8. State the applications of monoclonal antibodies in drug targeting giving
Examples. ( summer 2021 )
38. Reference :
[1] Bayer, V. An Overview of Monoclonal Antibodies. In Seminars in oncology
nursing; Elsevier, 2019; Vol. 35, p 150927.
[2] Fesseha, H. Monoclonal Antibody and Its Diagnostic Application- Review. BJSTR,
2020, 30 (4). https://doi.org/10.26717/BJSTR.2020.30.004997
[3] Berger, M.; Shankar, V.; Vafai, A. Therapeutic Applications of Monoclonal
Antibodies. The American Journal of the Medical Sciences, 2002, 324 (1), 14–30.
https://doi.org/10.1097/00000441-200207000-00004
[4] Payne, W. J.; Marshall, D. L.; Shockley, R. K.; Martin, W. J. Clinical Laboratory
Applications of Monoclonal Antibodies. Clinical Microbiology Reviews, 1988, 1 (3),
313–329. https://doi.org/10.1128/cmr.1.3.313
39. [5] Production of Monoclonal Antibodies - Yokoyama - 2013 - Current Protocols in
Immunology-WileyOnlineLibrary
https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/0471142735.im0205
s102 (accessed Mar 16, 2024)
[6] Wang, S. S.; Yan, Y. (Susie); Ho, K. US FDA-Approved Therapeutic Antibodies
with High-Concentration Formulation: Summaries and Perspectives. Antib Ther,
2021, 4 (4), 262–272. https://doi.org/10.1093/abt/tbab027