Pharmaceutical

Biotechnology
(PCS521)
Lecture Eight
Dr. Asmaa Ramadan
Lecturer, Microbiology & Biotechnology Department – College of Pharmacy
Arab Academy for Science, Technology and Maritime Transport (AASTMT)
 Therapeutic Applications of
Pharmaceutical Biotechnology:

Monoclonal A n t i b o d i e s
Part I
- Antigens are substances that induce the production of antibodies (antibody
generators).
- Most antigens are either proteins or large polysaccharides.
- 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 its target.
Monoclonal Antibodies: Monoclonal antibodies refer to a homogeneous population
of antibodies that are produced by a single clone of plasma B cells.
Polyclonal Antibodies: Polyclonal antibodies refer to a mixture of immunoglobulin
molecules that are secreted against a particular antigen.
# Monoclonal antibodies interact with the same epitope in the antigen whereas
# Polyclonal antibodies interact with the different epitopes of the same antigen.
Preparation of Monoclonal Antibodies

• Monoclonal Antibody (mAb) are

produced by cell lines or clones
obtained from the immunized animals
with the substances to be studied.

• Cell lines are produced by fusing B-

cells from the immunized animal with
myeloma cells.
 Principle:

• The hybridoma (fused) cell has the capacity

of antibody production derived from B-cells
(spleen)

• At the same time it can divide continuously by the quality derived

from myeloma cells

• By combining the desired qualities of both cells, the technology is

able to produce large quantities of mABs with very high specificity.
Practical steps for mAbs production
• Immunize animal
• Isolate spleen cells (containing antibody-
producing B cell)
• Preparation/isolation of myeloma cells
• Fuse spleen cells with myeloma cells to form
hybridoma cells
• Hybridoma Selection (using HAT medium)
• Culture hybridoma cells (place 1 cell/well and
allow each cell to grow into a clones of cell)
• Screen supernatant of each clone for presence of
desired antibody
• Grow chosen clone of cells In Vitro or In Vivo
• Harvest antibody.
• The fusion of the B cells with myeloma cells can be done using
electrofusion. Electrofusion causes the B cells and myeloma cells to align and
fuse with the application of an electric field.
• Alternatively, the B-cells and myelomas can be made to fuse by chemical
protocols, most often using polyethylene glycol.
To understand the principle of screening, we need to
remember some notes:

• In order to multiply and produce antibodies, cells must first

synthesize new copies of DNA.

• For synthesis of DNA nucleotides, two pathways are usually

undertaken:
• De Novo Pathway
• Salvage Pathway
•
•
De
• Here, to screen for hybridomas, cells are incubated in hypoxanthine-
aminopterin-thymidine (HAT) medium for 10-14 days

• In HAT medium, cells cannot synthesize nucleotides via DeNovo

pathway, as aminopterin (folic acid analogue) blocks dihydrofolate
reductase (DHFR) enzyme, which is necessary for synthesis.

For synthesis in salvage pathway, cells require presence of:

• hypoxanthine phosphoribosyl transferase (HGPRT) enzyme
• thymidine kinase (TK) enzyme

in which hypoxanthine and thymidine are precursors for nucleotide

synthesis.
• Myeloma cells are engineered to lack HPGRT enzyme
and/or TK enzyme,
which are responsible for the synthesis of nucleotides
(salvage pathway).

• This makes myeloma cells sensitive to HAT medium

Two different pathways for nucleotide synthesis in
mammalian cells

Pyrimidine Salvage Purine Salvage

(DHFR inhibitor)
 B-cell Myeloma Cell
HGPRT (+) HGPRT (-)
TK (+) TK (-)

Unfused B-cell (+)

Unfused Myeloma cell (-)
Hybridoma cell (+)

• Unfused Myeloma don’t have the enzyme(s), so cannot survive in HAT

medium
• Unfused B-cells have the enzyme(s) and can synthesize nucleotides, but
eventually die due to short-life/limited number of replication cycles.
• Hybridoma cells have the enzyme as well as the ability to multiply repeatedly
as myeloma cell, so these are the only cells that survive HAT medium
Finally, the desired antibodies are grown either in mass
culture (in Vitro) or in Vivo using suitably prepared animals.

They are then harvested and frozen for storage.

 Step 8
To produce the desired mAB, the hybridoma
cells must be grown in either of two ways:
- In Vivo method: by injection of hybridoma cells
into the peritoneal cavity of a suitably prepared
mouse, then antibodies are isolated from the
animal.
- In Vitro mass culture: which involves culturing of
hybridoma cells in a suitable culture media and
then antibodies are isolated and purified. Once a
hybridoma colony is established, it will continually
grow in culture media and produce antibodies.
 Evolution of
Monoclonal Antibodies
1- Murine:
- Mice proteins are purified after immunization with antigen.
- Antibodies to these proteins are generated in patients
i.e. Patients treated with murine mAbs develop a human antimouse antibody
(HAMA) response

- Rapid clearance of the mAb

- Poor tumour penetration
- Hypersensitivity reactions (Anaphylactic Shock and Allergic reactions).

Recombinant DNA technology or genetic engineering used to construct hybrids

composed of human Abs regions with murine. (Chimeric , Humanized and
Human monoclonal antibodies).
2-Chimeric:

- Abs Antigen binding parts (variable region) of mouse with effector

parts (constant region) of human,
- Antibodies are approximately 65% human.
- This reduces immunogenicity and thus increases serum half-life.
3-Humanized
- Antibodies contain segments in the complementary determining
regions (CDR) or hypervariable region from non human source
interspersed among human derived segments in constant regions.

- This results in a molecule of approximately 95% human origin.

4-Human Monoclonal Antibody
• Human monoclonal antibodies are produced by transferring human
immunoglobulin genes into the murine genome, after which the
transgenic mouse is vaccinated against the desired antigen, leading
to the production of monoclonal antibodies.