MONOCLONAL ANTIBODYPREPARATION AND EVALUATION

1. MONOCLONAL ANTIBODY
PREPARATION AND
EVALUATION
NIVEDITHA G
M.PHARM (2ND SEM) 1 st YEAR
NTDDS

2. Antibody
• Antibody is a glycprotein used by immune system to identify and
skill foreign objects like bacteria and viruses. Each antibody
recognizes a specific antigen unique to its target.
• Antibodies are produced by B-lymphocyte cells in responce to
antigents(foreign bodies).
• The antibdies bind with the antigen to fom an antibody/antigen
complex that is engulfed by phagocytosis and destroyed.

3. Antibody-Antigen Recognition:
An antibody recognises the part of an antigens structure.The
recognised region of antigen is called epitope.
Antigens have more than one type one type of epitope. This mens that
a number of B lymphocyte cells will recgnise the antigen and poduce
antibodies.
An immune responseresults in the prduction of a number of different
antibodies.

4. Monoclonal antibody
Monoclonal antibodies are identical antibodies which ae
produced from a single B cell clone.
They recognize single epitopes on an antigen.
In 1975, Kohler and Milstein provided the most outstanding
proof of the clonal selection theory by fusion of normal and
malignant cells (Hybridoma technology)for which they
received Novel prize in 1984.
Monoclonal antibodies are antibodies that ae identical
because they were produced by one type of immune cell, all
clones of a single parent cell.

5. Monoclonal antibodies (mAB) are single type of antibody that are
identical and are directed against a specific epitope (antigen, antigenic
determinant) and are produced by B cell clones of a single parent or a
single hybridoma cell line.
A hybidoma cell line is formed by the fusion of one B cell
lymphoocyte with a myeloma cell.
Some myeloma cell synthesize single mAB antibodies naturally.

6. Advandages of using monoclonal antibodies :
Though expensive, monoclonal antibodies are cheaper too develop
than conventional drugs because it is based on tested technology.
Side effects can be treated and reduced by using mice-human hybrid
cells or by using fractions of antibodies.
They bind to specific diseased or damaged cells needing treatment.
They treat a wide range of conditions.

7. Disadvavandages of using monoclonal antibodies
Time consuming project-any where between 6-9 months.
Very expensive and needs consideable effort to produce them
Small peptide and fragment antigens may not be good antigens-
monoclonal antibody may not recognize the original antigen
Hybridoma culture may be subject to contamination.
System is only well developed for limited animal and not for other
animals.
More than 99% of the cells do not survive during the fusion process
reducing the range of useful antibodies that can be produced against an
antigen.
It is possibility of generating immunogenicity.

8. STEPS IN THE PRODUCTION OF MONOCLONAL
ANTIBODIES:
1) PRODUCTION OF MAB BY HYBRIDOMA TECHNOLOGY: The
generation of MAB producing cells requires the use of animals, usually mice.
The procedure yields a cell line capable of producing one type of antibody
protein for a long period. A tumor from this immortal" cell line is called a
HYBRIDOMA.
2) PROPAGATION OF MAB BY:
• In vitro methods:
(a)Batch tissue culture method
(b) Semi permeable membrane method

11. • In vivo method:
(a)Mouse ascites methood
STEP 1: IMMUNIZATION OF MICE AND SELECTION OF
MOUSE DONORS FOR GENERATION OF HYBRIDOMA
CELLS:
The very first step in hybridoma technology is to immunize mice with appropriate
antigen that is prepared for injection either by emulsifying the antigen with Freund's
adjuvant or other adjuvants or by homogenizing a gel slice that contains the antigen.
The antigen can be whole cells, membrane fragment, or complex molecules. The
injections at multiple sites are repeated several times. This enables increased
stimulation of B-lymphocytes which are responding to the antigen.

12. • Three days prior to killing of the animal, a final dose of antigen is
intravenously administered.
• The immune-stimulated cells for synthesis of antibodies have grown
maximally by this approach.
• The concentration of the desired antibodies is assayed in the serum of
the animal at frequent intervals during the course of immunization.
Mice serum's are screened using various techniques such as ELISA.

13. STEP 2: SCREENING OF MICE FOR
ANTIBODY PRODUCTION
• After immunization, blood samples are obtained from mice for measurement of
serum antibodies.
• Serum antibody titer is determined with various techniques, such as ELISA, flow
cytometer.
• If the antibody titre is high-cell fusion can be performed.
• If the titre is too low-mice can be boosted until an adequate response is achieved.
• If the titre is high enough - mice are commonly boosted by injecting antigen
without adjuvant.

14. • Then the mice are euthanized and their spleens removed for in vitro hybridoma
cell production immunization of mice. Single spleen cells from the immunized
mouse are fused with the previously prepared HGPRT defective myeloma cells.
STEP 3:PREPARATION OF MYELOMA CELL
• Myeloma cells were collected and cultured in 8-azaguinine before eight days of
fusion.
STEP 4: FUSION OF SPLEEN CELL AND MYELOMA CELLS.
• Fusion is accomplished by co-centrifuging freshly harvested spleen cells &
myeloma cells in polyethylene glycol, a substance that causes cell membranes to
fuse for a short period (a few minutes), since it is toxic.
• PEG is removed by washing and the cells are kept in a fresh medium.

15. • The cells are then distributed to 96 well plates containing feeder cells
derived from saline peritoneal washes of mice. Myeloma cells have
been genetically engineered (HGPRT) such that they cannot use
Hypoxanthine, Aminopterin, and Thymidine (HAT medium) as a
source for nucleic acid biosynthesis and will die in culture. These cells
are composed of a mixture of hybridomas (fused cells), free myeloma
cells and free lymphocytes. Only B cells that have fused with the
engineered myeloma cells will survive in culture when grown in HAT
medium.

16. • STEP-5: ALLOW UNFUSED B CELLS TO DIE. ADD
HAT CULTURE TO KILL UNFUSED MYELOMA CELLS.
• Cells are plated in hypoxanthine-aminopterin-thy midine (HAT) selection
medium- inhibitor of aminopterin which blocks nucleotide synthesis.
• Only fused cells with grow on HAT.
• Cells are distributed on feeder cells (murine bone-marrow) to promote growth of
the hybridomal cells. This happens in 7-10 days of culture.
• Selection of a single antibody producing hybrid cells is very important.
• This is possible if the hybridomas are isolated and grown individually.
• The suspension of hybridoma cells is so diluted that the individual aliquots
contain on an average one cell each.

17. • These cells, when grown in a regular culture medium, produce the
desired antibody.
• Unfused myeloma cells die because they cannot use the salvage
pathway to make nucleotides and they are poisoned with aminopterin
that blocks their pathway to nucleotide synthesis.
• Fused myeloma cells and normal cells do not die because the normal
cell partner can make nucleotides in the presence of aminopterin, it
can use the salvage pathway.

18. • STEP-6: CLONING OF HYBRIDOMA CELL LINES BY
"LIMITING DILUTION" OR EXPANSION & STABILISATION OF
CLONES BY ASCITES PRODUCTION
• A mouse is inoculated with the cell and thereby becomes a factory for producing
the MAB.
• Small clusters of hybridoma cells from the 96 well plates can be grown in tissue
culture followed by selection for antigen binding or grown by the mouse ascites
method with cloning at a later time.
• The hybridomas now are ready to be diluted and grown, thus obtaining a number
of different colonies, each producing only one type of antibody.

19. STEP-7: SCREENING SUPERNATANT OF EACH CLONE
FOR PRESENCE OF THE DESIRED ANTIBODY (ELISA)
• The hybridomas must be screened for the secretion of the antibody of desired
specificity.
• The culture medium from each hybridoma culture is periodically tested for the
desired antibody specificity.
• The two techniques namely ELISA and RIA are commonly used for this purpose.
• In both the assays, the antibody binds to the specific antigen (usually coated to
plastic plates) and the unbound antibody and other components of the medium can
be washed off.
• Thus, the hybridoma cells producing the desired antibody can be identified by
screening.
• The antibody secreted by the hybrid cells is referred to as monoclonal antibody.

20. STEP-8: GROW THE CHOSEN CLONE OF CELLS
IN TISSUE CULTURE INDEFINITELY
• This is done in vitro on culture bottles. The single hybrid cells
producing the desired antibody are isolated and cloned. Two
techniques are commonly employed for cloning hybrid cells-limiting
dilution method and soft agar method.
• Limiting dilution method: In this procedure, the suspension of
hybridoma cells is serially diluted and the aliquots of each dilution are
put into micro culture wells. The dilutions are so made that each
aliquot in a well contains only a single hybrid cell. This ensures that
the antibody produced is monoclonal.

21. • Soft agar method: In this technique, the hybridoma cells are cultured in
soft agar. It is possible to simultaneously grow many cells in semisolid
medium to form colonies. These colonies will be monoclonal in nature.
In actual practice, both the above techniques are combined and used for
maximal production of MABS.
STEP 9:HARVEST ANTIBODY FROM THE CULTURE
SUPERRNATANT
STEP-10: CHARACTERIZATION AND STORAGE:
• The monoclonal antibody has to be subjected to biochemical and
biophysical characterization for the desired specificity.
• It is also important to elucidate the MAB for the immunoglobulin class
or sub-class, the epitope for which it is specific and the number of
binding sites it possesses.

22. • The stability of the cell lines and the MABS are important. The cells
(and MABs) must be characterized for their ability to withstand
freezing, and thawing.
• The desired cell lines are frozen in liquid nitrogen at several stages of
cloning and culture.

23. 2.IN VITRO PROPAGATION OF mAb:
A major advantage of using MAB rather than polyclonal
antiserum is the potential availability of almost infinite
quantities of a specific monoclonal antibody towad a single
epitope. In general, MAB is found either in the medium
supporting the growth of a hybridoma in vitro or in ascitic fluid
from a mouse inoculated with the hybridoma.

24. BATCH TISSUE-CULTURE METHOD:
• The simplest approach for producing MAB be in vitro is to grow the
hybridoma cultures in batches and purify the MAB from the culture
medium.
• Fetal bovine serum is used in most tissue- culture media and contains
bovine immunoglobulin at about 50µg/ml.
• In most cases, hybridoma growing in 10% fetal calf serum (FCS) can
be adapted within 8-12 days to grow in < 1% FCS or in FCS free
media. By this approach it yields concentrations that are typically
below 20µg/ml.

25. SEMI PERMEABLE-MEMBRANE-BASED SYSTEMS:
• In this method the use of a barrier, either a hollow fiber or a membrane, with a
LMW(10,000-30,000KD), has been implemented in several devices to permit
cells to grow at high densities.
• These devices are called semipermeable-membrane-based systems,
• In this method isolate the cells and MAB produced in a small chamber
separated by a barrier from a larger compartment that contains the culture
media,
• Two membrane-based systems are available:
• The mini-PREM (Hopkinton)
• The CELL Line(Integra Bioscience)

26. ADVANTAGES:
• Reduce the use of mice at the antibody-production stage.
• It is a method of choice for large scale production.
• Avoid the need to submit animal protocols to IACUC's(The Institional
Animal Care and Use Committee).
• Decrease the need for laboratory personnel.
• Using semi permeable membrane based systems produce MAB in high
concentrations.

27. DISADVANTAGES:
• Some hybridomas do not grow well in culture or are lost in culture.
The loss of proper glycosylation of the antibody might make the
antibody product unsuitable for in vivo experiments because of:
• Increased immunogenicity, reduced binding affinity.
• Changes in biologic functions, accelerated clearance in vivo.
• In vitro culture methods are generally more expensive and limited by
the amount of equipment.
• Batch culture supernatants contain less MAB per ml of medium than
the mouse ascites fluid.

28. INVIVO MOUSE ASCITES METHOD
In vivo propagation involves ascites production from rodent hybridomas. During this
process, the monoclonal cells are injected into the peritoneal cavity of a mouse and
allowed to grow. After 2 weeks, there is a build-up of ascitic fluid, which contains
the antibodies. These antibodies can then be removed from the peritoneal cavity
using a needle syringe.
Advantages:This method usually produces very high concentrations that often do not
require further conc. Procedures that can denature antibody and decrease
effectiveness. The high conc. of the desired MAB in this method avoids the effects
of contaminants. Relatively inexpensive and easy
Disadvantages:This method involves the continued use of mice requiring daily
observation. MAB produced by this method can contain various mouse proteins and
contaminants. This method can cause significant pain or distress in mice. There are
ethical concerns with using animals.

29. Major Applications:
• 1) Diagnostic Applications
• Biochemical analysis
• Diagnostic Imaging
• (2) Therapeutic Applications
• . Direct use of MABS as therapeutic agents
• MABS as targeting agents.
• (3) Protein Purification
• 1) Diagnostic Application

30. la. Biochemical analysis
• Routinely used in radioimmunoassay (RIA) and enzyme-linked immune
sorbent assays (ELISA) in the laboratory.
• These assays measure the circulating concentrations of hormones (insulin,
human chorionic gonadotropin, growth hormone, progesterone, thyroxine,
triiodothyronine, thyroid stimulating hormone) and several other tissue and
cell products (blood group antigens, blood clotting factors, interferon's,
interleukins, tumor markers).
• Eg. Pregnancy by detecting the urinary levels of human chorionic
gonadotropin.
• Hormonal disorders analysis of thyroxine, triiodothyronine
• Cancers estimation of plasma carcino embryonic antigen in colorectal cancer,
and prostate specific antigen for prostate cancer

31. Ib. Diagnostic imaging
• Radiolabeled-MABs are used in the diagnostic imaging of diseases, and this
technique is referred to as immune scintigraphy. The radioisotopes commonly
used for labeling MAb are iodine-131 and technetium-99. The MAB tagged with
radioisotope are injected intravenously into the patients.
• These MABS localize at specific sites (say a tumor) which can be detected by
imaging the radioactivity. In recent years, single photon emission computed
tomography (SPECT) cameras are used to give a more sensitive three
dimensional appearance of the spots localized by radiolabeled-MABS.
• Myocardial infarction, DVT(Deep vein thrombosis), atherosclerosis etc.

32. 2. Therapeutic Application:
• 2a.Direct use of MABS as therapeutic agents
• In destroying disease-causing organisms: MABS 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 cyclosporine 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
• In the treatment of cancer:
• MABS, against the antigens on the surface of cancer cells, are useful for the treatment of
cancer. The antibodies bind to the cancer cells and destroy them via different pathways.

33. • Liposomes are sacs or vesicles formed spontaneously when certain
lipid molecules are exposed to aqueous environment.
✓Drug entrapped in liposomes that are coated with MABS directed
against
• Tissue specific antigens are being tried for drug delivery.
✔Unfortunately, the progress in this approach has been limited, since
such liposomes do not reach the target cells.
• They are retained mostly in the liver and spleen (reticulo endothelial
cells), and degraded.

34. 3. Protein Purification:
• Monoclonal antibodies can be produced for any protein. And the so produced
MAb can be conveniently used for the purification of the protein against which
it was raised.
• MABS columns can be prepared by coupling them to cyanogen bromide
activated Sepharose (chromatographic matrix). The immobilized MABs in this
manner are very useful for the purification of proteins by immune affinity
method.
• There are certain advantages of using MABS for protein purification. 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.