Production of Recombinant

Therapeutic Proteins
 O utline
1 . Introduction
2. Methods used to produce recombinant proteins
3. H ow are transgenic animals produced?
4. Advantage of transgenic systems
5. L im itations of the transgenic expression system s
6. List of therapeutic proteins
 1.INTRODUCTION
Production of Recombinant Therapeutic Proteins
Recombinant DNA technology is widely used in the
production of therapeutic agents such as;

hormones, cytokines, growth factors, antibiotics,

vaccines, blood products like albumin, thrombolytics,
fibrynolytics, clotting factors such as factor VII, factor
IX, tissue plasminogen activator and many more.
• All these therapeutic agents can be produced in a large
quantity and also more economically by using rDNA
technology.

• Many proteins which may be used for medical

treatment or for research are normally expressed at
very low concentrations.

• Through rDNA technology, a large quantity of proteins

can be produced. This involves inserting the desired
protein gene into an "expression vector" which must
contain a promoter so that the protein can be
expressed.
 2. METHODS USED TO PRODUCE
RECOMBINANT PROTEINS
(i) Production of recombinant proteins in microbial bioreactors
Examples
 E.coli expression system
 Saccharomyces cerevisiae

(ii) Mammalian cell derived bioreactors

 E.g. Chinese Hamster Ovary cell (CHO) bioreactors.

 (iii) Animal Bioreactors “Pharming”

Production of Recombinant Therapeutic Proteins in the Milk of
Transgenic Animals Eg, Cows, sheep, pigs etc.
 (i) Microbial bioreactors
• The first microbial bioreactors, in particular
Escherichia coli (bacterial) and Saccharomyces
cerevisiae & Pichia pastoris (yeasts) were found to
be satisfactory for the production of simple
polypeptides such as insulin and human growth
hormone
• However, microbial bioreactors were found to be
unsuitable for proteins with complex post-
translational modifications or intricate folding
requirements, such as the coagulation factors, or
monoclonal antibodies.
• This led to the development of large-scale mammalian
cell culture, for example, the use of Chinese Hamster
Ovary (CHO) cell cuture bioreactors.

Limitations of microbial bioreactors

• Bacteria often improperly fold complex proteins,
leading to involved and expensive refolding processes
and ;
• Both bacteria and yeast lack adequate post-
translational modification machinery for mammalian-
specific N- and O-linked glycosylation, γ-
carboxylation, and proteolytic processing
 Building the Transgenes

ON/OFF Switch Makes Protein stop sign

PROMOTER INTRON CODING SEQUENCE poly A signal

Transgene

Selectable
Marker Gene

Plasmid DNA
Construct
bacterial genes
•antibiotic marker
•replication origin
An overview of the recombination process in
Escherichia coli bioreactor
 (ii) Cell culture bioreactors
• These technologies permitted the development of
numerous monoclonal antibodies, cytokines, and other
complex bioactive biomolecules.

• However, there are proteins that, due to a combination

of complex structure and large therapeutic dosing have
until now eluded (fail to be attained) recombinant
production using traditional bacterial and cell culture
bioreactors
• E.g Commercial recombinant production of complex
molecules, such as antithrombin and alpha1-antitrypsin,
has not yet been achieved in microbial or mammalian
cell derived bioreactors

• Cell culture systems require high initial capital

expenditures, lack scale-up (or down) flexibility and use
large volumes of culture media

• This led to development of transgenics technology i.e

Production of Recombinant Therapeutic Proteins in the
Milk of Transgenic Animals
 (iii) Production of Recombinant Therapeutic Proteins
in the Milk of Transgenic Animals
What is a transgenic animal?
• A transgenic animal is one which has been genetically
altered to have specific characteristics (genes) it
otherwise would not have.

• Different types of transgenic animals have been invented

to carter to specific societal needs.

• It includes; transgenic disease models, transgenic

pharmers, xenotransplanters and transgenic food
 3. How are transgenic animals produced?

The foreign DNA can be inserted in three ways:

(iii)DNA microinjection
Fusing an expression vector, comprising a gene
that is encoded for the human or humanized
target protein with mammary gland-specific
regulatory sequences, and then inserting into
the germline of the selected production
species
.
• When integrated, the milk-specific expression
construct becomes a dominant genetic
characteristic that is inherited by the progeny of the
founder animal (Figure 1).

• This general strategy makes it possible to harness

the ability of dairy animal mammary glands to
produce large quantities of complex proteins.
Fig 1.
Electrofusion: Fusion induced by electric pulse
fusion pulse fusion product

Cells brought close Heterokaryon phase:

together nuclei distinct
 Cont………
(ii) Retrovirus-Mediated Gene Transfer
• A retrovirus is a virus that carries its genetic
material in the form of RNA rather than DNA

• retroviruses used as vectors to transfer genetic

material into the host cell, resulting in a chimera

• chimeras are inbred for as many as 20 generations

until homozygous transgenic offspring are born
(iii) Embryonic Stem Cell-Mediated Gene Transfer
• This method involves isolation of totipotent stem
cells from embryos

• The desired gene is inserted into these cells

• Cells containing the desired DNA are incorporated

into the host’s embryo, resulting in a chimeric
animal
 Advantage of transgenic systems

• Transgenic livestock can be maintained and scaled-

up in relatively inexpensive facilities

• Use animal feed as raw material

• Can achieve impressive yields of recombinant

proteins.
Limitations of the transgenic expression systems

• Limitations are related to potential adverse effects

of bioactive heterologous proteins on the health of
the production animals and to a lesser extent, to
initial timelines. E.g. 12-18 months in goats

• Although transgenic expression systems are able to

perform complex post-translational modifications,
such as γ-carboxylation, β-hydroxylation or N- and
O-linked glycosylation,
-there are species-specific and tissue-specific
characteristics for these modifications that may affect
the appropriateness of a given system for the
expression of specific proteins.

This is also a challenge found with mammalian cell

culture, microbial expression systems or transgenic
plants.
•Transgenic goats
Producing anti-thrombin
(rhAT) therapeutic protein.

• It is used to treat clotting

defects as in Haemophilia
and is used to prevent
DIC and DVT also used
before surgery
 Webster and Peter
Transgenic young
Nexia Biotechnologies transferred
male goats carrying
the silk gene from Orb spiders into silk gene
goats
The resulting male goats were used
to sire silk-producing female goats
Each goat produces several grams of
silk protein in her milk
The silk is extracted, dried to a white
powder and spun into fibers
The fibers are stronger and more
flexible than steel
Tracy the sheep (1997).
The first transgenic animal to produce a recombinant
protein drug in her milk alpha-1-antitrypsin (AAT)
treatment for emphysema & cystic fibrosis. Created by
PPL Therapeutics & The Roslin Institute
 Herman the bull
-15/12/1990: first transgenic bull
carrying transgene for human
lactoferrin (Gene Pharming, The
Netherlands)

-Dec 1992: permission to

generate 50 offsprings by AI

-Oct 1993: first calf born

-March 1996: 5 cows producing

human lactoferrin in their milk
•Some Biotherapeutic companies has received approval to sell
human anti-thrombin (hAT) purified from goat’s milk in Europe

•Technology is not restricted to cows,

goats, & sheep, there is interest in
using rabbits since housing costs are
significantly less & generation time is faster

•Chickens which produce recombinant drugs in their eggs

have been produced by The Roslin Institute.
= Is it good to make transgenics from
Mr. D.O.G??

THE END