BIOPHARMING

Manish Saini

Amity

Vikas Chauhan

Biotechnology , AUR

Jitendra Bhargav

B.Tech

Kuldeep Sharma

Biotechnology

Institute
/

of
M.Tech

o Introduction
o Historical Background
o Conventional
Techniques
o Procedure
o List of Therapeutic
Proteins
o Milk as a Source of
Production System
o List of Transgenic
Animals
o Conventional Vs.
Transgenics
o Current Trends
o Advantages

CONTENTS

INTRODUCTION

Biopharming is the production and use of

transgenic
plants
and
animals
genetically
engineered to produce pharmaceutical substances
for use in humans or animals (Goven, 2014).
It is also known asmolecular farming or molecular
pharming.
It is one of the most important utilization of transgenic
animals involving the target production (recombinant)
of therapeutically recognized proteins.
Therefore,
biopharming
involves
large
scale
production of recombinant proteins by using transgenic
animals
As the second wave of agricultural biotechnology, it
is quite a recent phenomenon and presents a fascinating
array of benefits and risks.
It offers the promise of revolutionizing the cost structure

Transgenic animals are the subset of genetically

modified organism (GMO) or genetically engineered
organism (GEO) whose genetic material has been altered
using genetic engineering techniques.
Since, the gene of interest inserted, is isolated from the
different species, GMO are referred to as Transgenic
animals.
The therapeutic molecules have been derived from
various other conventional biological model systems ;
Bacteria
Yeast
Insects cells
Animal (Mammalian) cell culture
Transgenic Plants
The therapeutic proteins includes the human growth

Biopharming offers several advantages over the

other conventional methods of proteins production at
higher levels.
Transgenically produced recombinant proteins have the
same

amino

acid

sequence

as

native

human

proteins because they are synthesized by the mammary

cells from a recombinant version of the native gene.
Numerous

proteins

have

been

produced

at

large

amounts in the mammary gland of transgenic sheep,

goat, cattle, pigs, fishes, chicken and rabbits and
have been advanced to clinical trials.

TORICAL BACKGROU

Proteins started being used as pharmaceuticals in the

1920s with insulin extracted from pigs pancreas.
The first human protein therapeutic derived from
recombinant DNA technology was human insulin
(Humulin) created at Genentech, developed by Eli Lilly,
and approved by the US Food and Drug Administration
(FDA) in 1982.
The commercial importance of engineered insulin is
illustrated by Lantus, which was one of the top ten
selling biopharmaceuticals in 2009.
In 1972 Paul Berg made the first recombinant DNA in
vitro by using restriction enzyme and DNA ligases.
In 1974Rudolf Jaenischcreated first genetically
modified animal by inserting a DNA virus into an earlystage mouseembryoand showing that the inserted genes
were present in every cell. However, the mice did not

In 1974, Stanley Cohen, Annie Chang and Herbert

Boyer create the first genetically modified organism,
E.coli. However, the first transgenic animal (mouse) was
made in 1980 using pronuclear microinjection technique.
The arrival of the hybridoma technology Kohler and
Milstein, 1975. brought a new level of therapeutic potential
to the use of the immune mechanisms, with monoclonal
antibodies being widely recognized as potential magic
bullets.
First transgenic Sheep and pigs were produced by
using microinjection of DNA into one pronucleus of a zygote
(Hammer et al., 1985).
Since then there has been rapid development in the use of
genetically engineered animals and constant efforts are
made to improve the efficiency of generating transgenic
livestock.
Dolly, a Finn Dorset sheep, was born on July 5th, 1996, at

NVENTIONAL TECHNIQU

All of the recombinant proteins produced and marketed

to date are produced in bacterial, yeast, and mammalian
cell culture systems.
Most of the recombinant therapeutic proteins derived
from transgenic livestock are in clinical trial phases.
Bacterial culture
Yeast culture
Insect cell culture
Mammalian cell culture

Source: D.L. Ayares,

PROCEDURE

Isolation of Gene of
Interest
Construction of
Transgene
Transfer of foreign gene
into Host
Selection of transgenic
animals
Identification of Recombinant Protein
Isolation and Purification of
Recombinant Proteins

P 1: ISOLATION OF GENE OF INTER

A typical Gene of Interest must ideally contain

-Promoter
-Enhancer
-Introns
-Transcription terminator
-Coding region

The gene coding for recombinant proteins should be

selected on the basis of scientific, economic and social
realities (Houdebine, 2002).
Use of expression vectors can be employed which
ideally contains the gene of interest.
Techniques that are used to isolate the gene of interest
are;
Polymerase Chain Reaction (PCR)
Reverse Transcription Polymerase Chain
Reaction (RT-PCR)
The isolation and characterisation of the gene and
associated control elements should be described as
should the process by which the final construct was
made.
However, genomic transgenes are expressed at

P 2: CONSTRUCTION OF TRANSGE

Transgenes targeting the expression of recombinant

proteins to the mammary gland are usually chimeric,
being derived from the fusion of the gene of interest
with mammary-specific regulatory sequences.
This allows the optimization of protein expression.
For example, Expression in milk is achieved successfully
with promoters from milk protein genes. Expression in egg
white is possible using the potent promoter of ovalbumin
gene.
Tissue specific expression of recombinant proteins is
more advantageous than generalized expression as it will
be easier to regulate the gene expression in a
tissue specific manner (Larrick and Thomas, 2001).
Recombinant protein expression will vary according to
the nature and size of the mammary gland regulatory

mple: Milk Expression Construct for Transgenic

3: TRANSFER OF FOREIGN GENE INTO

Source: Houdebine,

1. DNA transfer via direct microinjection into a pronucleus or

cytoplasm of embryo;
2. DNA transfer via a transposon: the gene of interest is
introduced in the transposon which is injected into a
pronucleus;
3. DNA transfer via a lentiviral vector: the gene of interest is
inserted into a lentiviral vector which is injected between zona
pellucida and membrane of oocyte or embryo;
4. DNA transfer via sperm: sperm is incubated with the foreign
gene and injected into oocyte cytoplasm for fertilization by
ICSI (Intra cytoplasmic Sperm Injection);
5. DNA transfer via pluripotent cells: DNA is introduced into
pluripotent cell lines (ES: embryonic stem cells: lines
established from early embryo, EG: embryonic germ cells: lines
established from the primordial germ cells of foetal gonads).
The pluripotent cells containing DNA are injected into early
embryos to generate chimeric animals harbouring the foreign
gene;

4: SELECTION OF TRANSGENIC AN

The first generation transgene carrier , or founder

animals, may be male or female.
If the founder is female, then the time from transgene
introduction to the first natural lactation is 18 months for
goats.
If the founder is male, then he must produce
transgenic daughters, which in turn must produce
transgenic daughters before full scale milk production can
begin.
Furthermore, the selection of transgenic animals can be
achieved by following techniques;
Probe Hybridization
Fluoroscence in situ Hybridization (FISH)
Genomic in situ Hybridization (GISH)
Southern Hybridization

5: IDENTIFICATION OF RECOMBINANT PRO

Accurate and reproducible characterization methods are

an absolute requirement to support and guide decisions
made in
developing the manufacturing process and
product formulation of protein therapeutics.
This can be, thus, achieved by following bioanalytical
techniques;
ELISA
Electrophoretic Technique
Western Blotting
Immunoelectrophoresis
Chromatography
Size Exclusion chromatography
Affinity Chromatography
Mass Spectrometry

STEP 5: ISOLATION AND PURIFICATION

OF
RECOMBINANT PROTEINS

The Source material, whether blood serum, egg white,

milk or any other tissue is manually isolated.
A close attention should be given to the purity, quality
and consistency of the product.
It contains large numbers of host derived proteins
other than the desired product, some of which may be
present in large amounts which must be removed.
A transgenic animal is unlikely to be free of pathogens
like mycoplasma, virus and bacteria. Therefore, process
should be validated for their removal, as well as limits set
for their levels in the starting material.

Serum, Milk, Egg

White

Source: Genzyme Transgenics

OF THERAPEUTIC PROTE

Niemann and Kues, 2007

Houdebine et al., 2009

Houdebine et al., 2009

Mishra et al., 2014

MILK AS THE SOURCE

OF
PRODUCTION SYSEM

The mammary gland has generally been considered the

tissue of choice to express valuable recombinant proteins
in transgenic animal bioreactors because milk is easily
collected in large volumes and the ease of
production and purification (Meade et al. 1999;
Rudolph 1999)
As a result, a great deal of effort has been made to
produce transgenic bioreactors with the traditional dairy
species, such as sheep, goats, pigs and cows.
Foreign proteins are commonly reported to be expressed
into transgenic milk at rates of several grams per litre.
Milk as a source material of recombinant protein offers a

Furthermore, milk does not contain active protease

that can break down the protein of interest.
The

concentration

of

protein

in

milk

remains

consistent over the production cycle.

By

Contrast,

milk

provides

relatively

clean

feedstream , consisting of about 87% water, 4% fat and

9% non fat solids.
Recombinant proteins usually accumulate in the soluble
whey fraction of milk , thereby facilitating their
recovery.
It allows to produce pharmaceutical proteins in milk to

Comparison of the different systems to produce

recombinant pharmaceutical proteins

LIST OF TRANSGENIC
ANIMALS

Therapeutic proteins produced in transgenic

animals currently in commercial development

Contd.

Source: Goven et al.,

Comparison of the time required to

obtain recombinant proteins in
different transgenic animal species

Source: Houdebine et al., 2009

Comparison of the different organisms to produce

recombinant pharmaceutical proteins

VENTIONAL VS. TRANSGEN

Conventional
Method

Transgenic
Method

Higher Cost

Relatively Lower cost

Limited Post-Translational
Modifications

Almost all PostTranslational Modifications

Low yield of recombinant

proteins

Higher yield of
recombinant proteins

Scale up is difficult

Scale up is highly efficient

Isolation complexity

Relative ease in isolation

Aseptic environment
required

Requires normal care

CURRENT TRENDS

Source: Bertolini, L., Bertolini, M., Murray, J., & Maga, E.

(2014, October). Transgenic animal models for the
production of human immunocompounds in milk to
prevent diarrhea, malnourishment and child mortality:
perspectives for the Brazilian Semi-Arid region. In BMC
Proceedings (Vol. 8, No. Suppl 4, p. O30). BioMed
Central Ltd.
Diarrhea is major cause of high child mortality rate in brazil
Usually happens to malnutrition children
ORS treats the consequences and not the root cause of
disease
Fast recovery time for breast feeded children : attributed to
the antimicrobial actions of human milk proteins, such as
lysozyme and lactoferrin that can enhance intestinal and systemic
immunological functions.
But not an option always present for poor families.

ADVANTAGES
OF
BIOPHARMING

Lower cost
Production
Scale up
Set up
Infrastructure and operating cost
More efficient system
Stability of proteins
Higher production
Post-Translational Modifications
Potential for new and better drugs
Reduce allergenicity or
immunogenicity
Economic advantage of dairy
species
No or reduced aseptic conditions

DISADVANTAGES
OF
BIOPHARMING

Low rate of success

Health related complications in transgenic
animals
Labor intensive
Prior Extensive knowledge required
Potential pathogenic infection
Male variety can not be use directly
Relatively time consuming
Gestation period
Sexual maturity age
Defined Mating

ETHICAL ISSUES

Concerns about escape of transgene

Should patents be allowed on transgenic
animals?
Limits data and animal sharing
Risk of escape of transgene viral vector
Risk from drug resistance gene markers
Welfare of other life forms?
Ecological concerns

FUTURE
PERSPECTIVE

 Production

of

large

scale

antimicrobial peptide
Lowering the rate of transgenic
failures
Commercialization of improved
dairy based food products

REFERENCES
CITED

1. Ayares, D. L. (2000). Transgenic protein production:

Achievements using microinjection technology and the
promise of nuclear transfer. Journal of Animal Science,
78(suppl_3), 8-18.
2. Goven, J. (2014). Biopharming. Encyclopedia of Food
and Agricultural Ethics, 217-226
3. Whitelaw, C. B. A., Archibald, A. L., Harris, S.,
McClenaghan, M., Simons, J. P., & Clark, A. J. (1991).
Targeting expression to the mammary gland: intronic
sequences can enhance the efficiency of gene
expression in transgenic mice. Transgenic research,
1(1), 3-13.
4. Larrick, J. W., & Thomas, D. W. (2001). Producing
proteins in transgenic plants and animals. Current
opinion in biotechnology, 12(4), 411-418.