Large scale production of recombinant proteins by using transgenic animals.

1. Manish Saini
Vikas Chauhan
Jitendra Bhargav
Kuldeep Sharma
Amity Institute of Biotechnology , AUR
B.Tech / M.Tech Biotechnology
V Semester

2. CONTENTS
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
o Disadvantages
o Ethical Issues
o Future Perspective
o References Cited

4. “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 as molecular 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 of medicine,
providing an abundant source of life-saving pharmaceuticals at much
lower prices to millions of needy patients worldwide.
Transgenic animals are nature’s cheapest ‘Factory’ or ‘Bioreactor’ for
producing the recombinant proteins.

5. 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 factors
(hormones), insulin, Factor VIII and IX, erythropoietin, antibodies etc.
that have been produced through Biopharming.

6. 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.
The recombinant proteins are isolated from the various production
systems of transgenic animals including urine, blood, milk, egg, plasma
etc.

8. Proteins started being used as pharmaceuticals in the 1920s with
insulin extracted from pig’s 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 1974 Rudolf Jaenisch created first genetically modified animal by
inserting a DNA virus into an early-stage mouse embryo and showing
that the inserted genes were present in every cell. However, the mice
did not pass the transgene to their offspring.

9. 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 the Roslin
Institute in Edinburgh, Scotland.
The first biopharming product was human serum albumin, initially
produced in 1990 in transgenic tobacco and potato plants.

11. 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
Transgenic Plants

12. Source: D.L. Ayares, 2000

14. 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

16. A typical Gene of Interest must ideally contain
-Promoter
-Enhancer
-Introns
-Transcription terminator
-Coding region

17. 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 higher levels than
cDNA transgenes as natural introns in constructs can enhance the
efficiency of transgene expression by interacting with the upstream 5′
flanking sequences in splicing events (Whitelaw et al., 1991).

19. 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 elements as well as the nature
(cDNA or genomic) and sequence of the expressed gene.

20. Example: Milk Expression Construct for Transgenic Goats

22. Source: Houdebine, 2009

23. 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;
6. DNA transfer via Somatic cell nuclear transfer (SCNT): the foreign gene is
introduced into somatic cells, the nucleus of which are introduced into the

26. 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
PCR and RT-PCR

28. 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

30. 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.

31. Serum, Milk, Egg White
Source: Genzyme Transgenics Corporation

33. Houdebine et al., 2009
Niemann and Kues, 2007

34. Mishra et al., 2014
Houdebine et al., 2009

36. 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 much higher
protein concentration – typically 10 to 1000 fold greater than tissue
culture supernatants.

37. 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 a 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 improve milk
as a food or to use it as a vehicle to provide consumers with molecules
beneficial in some way for their health.

38. Comparison of the different systems to produce recombinant
pharmaceutical proteins

40. Therapeutic proteins produced in transgenic animals currently in
commercial development

41. Contd….
Source: Goven et al., 2008

42. Comparison of the time required to obtain
recombinant proteins in different transgenic animal
species
Source: Houdebine et al., 2009

43. Comparison of the different organisms to produce recombinant
pharmaceutical proteins

45. Conventional Method Transgenic Method
Higher Cost Relatively Lower cost
Limited Post-Translational
Modifications
Almost all Post-Translational
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

47. 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.
Alternative: Transgenic approach can be used to modify the milk composition of
dairy animals to supply milk-borne human immunocompounds (rhLZ, rhLF) and
nutrients to children in a continuous manner.

49. 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
Improved quality of food based dairy products

51. 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
Large area required for set-up

53. 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

55.  Production of large scale antimicrobial peptide
 Lowering the rate of transgenic failures
 Commercialization of “improved” dairy based
food products

57. 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.
5. Houdebine, L. M. (2009). Production of pharmaceutical proteins by
transgenic animals. Comparative immunology, microbiology and
infectious diseases, 32(2), 107-121.