Monoclonal antibodies are produced using hybridoma technology which involves fusing myeloma cells with antibody producing spleen cells from immunized mice. The fused cells or hybridomas are selected in HAT medium which allows only the fused cells to survive and grow. The resulting hybridoma cells secrete a single antibody clone that can be mass produced. Key steps include immunizing mice, isolating spleen and myeloma cells, fusing the cells to generate hybridomas, screening and selecting hybridomas that secrete the desired antibody clone, and culturing the selected hybridoma cells to produce monoclonal antibodies. Monoclonal antibodies find various therapeutic applications and can be purified using techniques like affinity chromatography.

1. HYBRIDOMA
TECHNOLOGY
SUBMITTED TO-Prof. Aruna Bhatia
SUBMITTED BY-Neha Singla
MSc (hon’s)biotechnology
ROLL NO.-18011001

2. CONTENT
 Antibodies
 Monoclonal antibodies
 Production – steps
 Purification

3. WHAT ARE ANTIBODIES..??
 An antibody is a protein used by the immune
system to identify
and neutralize foreign objects
like bacteria and viruses.
Each antibody recognizes
a specific antigen unique to
the target.

4. INTRODUCTION-
 Monoclonal antibodies are the antibodies that
are made by identical immune cells that are
clones of a unique parent cell.
 Have monovalent affinity(bind to same
epitope)
 Can be used to detect or purify substance

5. Difference between monoclonal
antibody and polyclonal antibody
 Monoclonal antibodies are abs that are
identical because they were produced by one
type of immune cell .
 Polyclonal antibodies are abs that are
derieved from different cell lines. They differ in
amino acid sequence.

6. HISTORY
 Idea of magic bullets –given by PAUL
EHRLICH ,who at the beginning of 20th
century, postulated that if selective compound
against disease causing organism could be
made then it will be easy to cure disease by
injecting toxin with selective compound.
 He and Metchnikoff got noble prize (effective
syphilis treatment)

7.  In 1970’s, myeloma cell was known which are
able to grow for unlimited period.
 In 1975,KOHLER and MILSTEIN succeeded
in fusion of myeloma cell lines with B-cells to
create hybridoma cells (having capacity to
produce antibodies for number of years)
 In 1988 GREG WINTER, and his team gave
the technique to humanize monoclonal
antibodies, eliminating the reactions that other
antibodies caused

8. PRODUCTION

10. STEPS-
IMMUNIZATION OF MOUSE
ISOLATION OF SPLEEN CELLS
PRODUCTION OF HYBRIDOMA
CELLS
SCREENING OF HYBRIDOMA CELLS
CULTURING OF HYBRIDOMA CELLS
SCREENING FOR DESIRED
ANTIBODY
Selection and culture of screened
antibody

11. IMMUNIZATION OF MOUSE
 Mice are immunized every 2-3 weeks with an
antigen.

12. ISOLATION OF SPLEEN
CELLS
 Spleen cells are isolated and culture on
suitable medium.
CHARACTERISTICS OF SPLEEN CELLS-
 Limited life span i.e. die soon
 Have HPGRT enzyme for salvage pathway
 Are able to produce antibodies when exposed
to antigen.

13. Isolation of myeloma cells-
myeloma cells should be isolated from
same species.
Characteristics of myeloma cells-
 Unlimited life span
 Normal cells tend to have a low growth
fraction at saturation density whereas
neoplastic cells continue to grow faster after
reaching confluence.
 As its antibody producing ability is not
required, so it is genetically modified by two
mutations. (1. it remains immortal
2.It cannot use purine salvage pathway as the
enzyme HPGRT is deficient.)

14. PRODUCTION OF HYBRIDOMA
 Myeloma cells are grown on 8-azaguanine
(week before fusion) to ensure their
hypersensitivity to HAT medium.
 Cells are fused using PEG and even it is
possible using Sendai virus. But use of PEG
is most common these days.
 And grown on HAT medium (hypoxanthine-
aminopterin-thymidine)

15. SCREENING-
 Aminopterin present in media block De Novo
pathway then all type of cells have to utilize
salvage pathway for nucleotide synthesis in
the presence of hypoxanthine and thymidine
 But for this they require HPGRT
gene(hypoxanthine phosphoguanine ribosyl
transferase)
 Myeloma cells are lacking this gene hence
they will die of starvation

16. CONTINUE-
 B-cells are having this gene but they die off
usually because of limited life span.
 Hybridoma cells have HPGRT gene as well as
unlimited life span.
 Hence only surviving cells are Hybridoma
cells

17. CULTURING HYBRIDOMA
CELLS –PRODUCTION OF
MONOCLONAL ANTIBODIES
 Hybridoma cells are separated and culture-
one cell per well
 Cells are called clonal culture as all these are
derived from single type of cells and therefore
identical
 Screening can be done for desired antibody.

18. SCREENING FOR DESIRED
ANTIBODY
 Perform ELISA
 Antigens are immobilized in the wells and
antibodies were transferred so they can bind
to Ag.

19. SELECTION OF
ANTIBODIES
 Desired antibodies are grown in mass culture
after looking for positive results

20. PRODUCTION OF
MONOCLONAL ANTIBODY –
IN VIVO
 In this procedure mice or rats are used.
 Initially , the immune system of the
experimental animals are suppressed (1-
2weeks) before the injection of hybridoma cells
intraperitoneally.
 Suppression is done by injecting a primer, such
as pristane (2,6,10,14-tetramethylpentadecane)
or Freund’s incomplete adjuvant.
 The hybridoma cells then multiply in the
peritoneal cavity

21. CONT….
 The ascitic fluid which will be formed is a very rich
source of the secreted antibody.
 When an adequate amount of ascites has formed ,
the animal is killed and ascitic fluid Is collected.
 Sometimes ascitic fluid is tapped or drained from
the peritoneal cavity while the animal is under
anaesthetic, with a second and final harvest being
taken once the ascites has reformed. The mAb
product can be harvested 5-21 days after the
injection of hybridoma cells.

22. CONT…
 Approximately 5ml of ascites can be obtained from a
mouse, and 10-40ml from a rat. Thus, for the
production of a mAb with a given specificity, it may be
necessary to use one or more mice, depending on the
amount of antibody required.
 Main advantage of ascites method is the extremely
high yield of antibody, which generally lies in the range
of 1-20mg/ml.

23. DISADVANTAGES OF IN VIVO
MAB PRODUCTION-
 The main disadvantage of the ascites method is that it
is extremely painful for the animals used, due to the
following:
 a) the injection of primer;
 b) the resulting peritonitis caused by the primer;
 c) abdominal tension
 d) the invasive tumours which result (4-6). Proper
animal husbandry facilities are mandatory. The mAbs
produced generally show a reduced immunoreactivity of
60-70%, as opposed to an immunoreactivity of 90-95%
for antibodies produced in vitro, due to contamination
by biochemically identical immunoglobulins.

24. CONT….
 . The mAbs produced generally show a reduced
immunoreactivity of 60-70%, as opposed to an
immunoreactivity of 90-95% for antibodies produced in
vitro, due to contamination by biochemically identical
immunoglobulins.
 There is also a potential risk of product contamination
by viruses which are pathogenic to humans. A further
disadvantage is that the individual batches of
harvested ascitic mAb are of variable quality, and they
are contaminated with bioreactive cytokines.

25. IN VITRO MAB PRODUCTION-
 The antibodies produced generally express an
immunoreactivity of 90-95%, irrespective of the
system used. Three categories of in vitro production
system can be identified according to the principle
underlying the culture system:
 a) static and agitated suspension cultures
 b) membrane-based and matrix-based culture
systems
 c) high cell density bioreactors.

26. SCALE UP-
 Plastic bags can be used to culture
suspension cells.
 They are gas permeable and can be agitated
by rocking on trays on a flat rocking platform

27. AIR LIFT FERMENTORS-
 Large-scale fermentors frequently use the air
lift principle
 5% CO2 in air is pumped into a porous steel
ring at the base of the central cylinder, and
bubbles stream up the center, carrying a flow
of liquid with them, and are released at the
top, while the medium is recycled to the
bottom of the cylinder.
 Used extensively in the biotechnology
industry, up to capacities of 20,000 L.

29. BELLO CELL AERATOR
CULTURE
 This device has a bellows medium
compartment that alternately forces medium
over cells anchored in porous matrices and
withdraws it again,
in a ‘‘breathing’’ motion
of the bellows.
 Optimum mixing and
aeration is claimed
with minimum shear.

30. STIRRER CULTURE-
 The stirring speed should be between30 and
100 rpm, sufficient to prevent cell
sedimentation, but not so fast as to create
shear forces that would damage the cells.
 Antifoam (Dow Chemical Co.) or Pluronic F68
(Sigma), 0.01–0.1%, should be included when
the serum concentration is above 2%,
particularly if the medium is sparged.
 In the absence of serum, it may be necessary
to increase the viscosity of the medium with
carboxymethyl cellulose (1–2%) (molecular
weight ∼105).

32. EXAMPLES-
 In 1986, mab named OKT-3 developed by
ortho pharmaceutical was the first murine
mab to be licensed for therapeutic use. OKT-3
recognizes a surface antigen on T –
lymphocyte and is one of the most effective
agents in preventing immunological rejection
of transplanted kidneys.
 REO –PRO--- FOR coronary angioplasty
 Anifrolumab - systemic lupus erythematosus

33.  Adecatumumab-prostate and breast cancer
 Herceptin – breast cancer
 Anrukinzumab-asthma
 Brodalumab-Plaque psoriasis
 Certolizumab pegol-rheumatoid arthritis

34. PURIFICATION-
 Sample is first conditioned ,or prepared for
purification
 Cells, cell debris, lipids and clotted material
are first removed by centrifugation followed by
filtration with a 0.45 micrometer.
 In cases where desired antibody is produced
by a low secreting cell line , the sample is
concentrated by ultrafiltration.

35.  Most of the charged impurities are removed by ion
exchange chromatography . Either cation exchange
chromatography is used at a low enough pH that the
desired antibody binds to column while anions flow
through. Even in some cases size exclusion
chromatography is also used, like transferrin. But has
some drawbacks of low elution.
 To achieve max. purity in single step, affinity
chromatography can be performed, in this Ag is
attached to sepharose 4B with which Ab is having
affinity and elution is generally done using low pH
buffer or more gentle, is then used to purify antibody.

36. REFERENCES
 culture of Animal Cells – R. IAN Freshney
 Essentials of immunology-Dr. S.K. GUPTA
 Kuby
 https://pdfs.semanticscholar.org/0e88/97a118
cc684a02fe0cca7a00aea2f04d2fd9.pdf
 www.gvax.org/history.htm

37. Thank
You