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Animal as Bioreactors

Dr. Asok Kr. Sarkar


Email:
asoksarkar09@gmail.com

What is a
bioreactor?
A bioreactor may refer to any manufactured or
engineered device or system that supports a
biologically active environment.
In one case, a bioreactor is a vessel in which a
chemical process is carried out which involves
organisms or biochemically active substances
derived from such organisms.
This process can either be aerobic or anaerobic.
These bioreactors are commonly cylindrical,
ranging in size from liters to cubic meters, and
are often made of stainless steel.
A bioreactor may also refer to a device or system
meant to grow cells or tissues in the context of
cell culture.

These devices are being developed


for use in tissue engineering or
biochemical engineering.
On the basis of mode of operation, a
bioreactor may be classified as
batch, fed batch or continuous (e.g.
a continuous stirred-tank reactor
model).
An
example
of
a
continuous
bioreactor is the chemostat.
Organisms growing in bioreactors
may be suspended or immobilized.
A simple method, where cells are

Background
The first microbial bioreactors, in
particular
Escherichia
coli
and
Saccharomyces cerevisiae, 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 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
recombinant
production
using
traditional
bacterial and cell culture bioreactors.
For
example,
commercial
recombinant
production of complex molecules, such as
antithrombin and alpha-1-antitrypsin, has not
yet been achieved in microbial or mammalian
cell derived bioreactors.

Capital investments in production plants


represent a significant portion of the
development cost of new recombinant drugs.
Also, the inherent risk associated with the
regulatory approval process is a stimulus for
the development of flexible and inexpensive
approaches
for
the
manufacture
of
therapeutic proteins.
Milk-specific production offers a way to
lessen the bite.
Thus search was made where recombinant
proteins could be manufactured on an
industrial scale.
The transgenic animals provided an answer
to it.

Animal bioreactors
Animal systems for production
Blood
Urine
Seminal plasma
Egg white
Silk worm cocoon
Milk

Transgenic Animal- A
suitable Bioreactor
The animal bioreactor refers to an animal
with
bacteria in its digestive tract. The
bacteria may be a modified bacteria.
The animal may be a cow, pig, goat, sheep,
rabbit, horse, mouse, rat or guinea pig.
The bacteria may be present in the lumen
or the rumen, in the case of a ruminant
animal.
The bacteria may comprise a plasmid with a
heterologous nucleic acid, which may be
operatively linked to a regulatory element.

An animal bioreactor is produced by


administering
a
composition
comprising bacteria to the digestive
tract of an animal.
The bacteria may be introduced to
the animal by oral, nasal, or rectal
administration and by injection.
The animal may be a germ free, a
specific pathogen free or transgenic
animal.

Preparation of an Animal
Bioreactor
Any animal with a digestive tract that is
capable
of
supporting
an
enteric
microorganism may be used as a bioreactor.
The animals may include cows, pigs, goats,
sheep, rabbits, horses, mice, rats and guinea
pigs.
The animals must be healthy and germ free.
The germ free host animals may be useful for
producing and maintaining the desired levels
of microorganisms in the digestive tract of
the animal fermentation chamber.

When germ free host animals are used,


the yield of the product of interest may be
increased by housing the animals in
sterile conditions, which may prevent
recolonization
by
other
strains
of
microorganisms.
The host animals may also be a
transgenic animal.
A transgenic host animal may contain a
foreign gene encoding a protein that
when expressed in the transgenic host
may be beneficial for the increased
viability of the microorganism in the
digestive tract of the host animal.

Administration of
Microorganisms
Animal bioreactors may be produced by
administering a microorganism to the host
animal by any method which allows for
introduction of the microorganism to the
digestive tract of the host animal including,
oral, nasal, and rectal administration.
The microorganism may be formulated in
any manner which allows for introduction
and propagation of the microorganism in
the digestive tract of the host animal
including
liquid
cultures,
lyophilized
cultures, encapsulated cultures, and agar.

Isolation of the Product of


Interest
The excreted feces may be collected in any
suitable manner.
Other methods of collecting the product of
interest include the use of tubes or catheters
sampling a particular microenvironment.
The product of interest may be collected
under any desired environment, such as
anaerobic conditions.
Other methods for collecting the product of
interest
include
the
use
of
surgical
procedures to remove the intestinal tract or
portions thereof.

In view of the bulk of the fecal matter


bearing microorganisms, the product of
interest may be isolated from the feces
using standard purification techniques .
If the product of interest is in the feces
(culture medium), the feces may be
solubilized and the product of interest is
concentrated
from
the
supernatant
followed by purification using standard
procedures including chromatography,
centrifugation and extraction.
If the product of interest is in the
cellular fraction, the microorganisms may
be lysed by a large number of chemical,
biological and physical methods.

Chemical
methods
for
disrupting
microbial cell walls include treatment
with
alkali,
organic
solvents
or
detergents.
If the product of interest is stable at
about pH 10.5-12.5, lysis may be carried
out on a large scale at low cost.
Lysis may also be performed using
enzymatic treatments which may be
highly specific and which may be
performed under mild conditions.
After cell disruption, cell debris is
removed by methods including lowspeed, high-capacity centrifugation or
membrane microfiltration.

The microorganisms may also be physically


disrupted by nonmechanical methods including
osmotic shock and repeated cycles of freezing
and thawing.
Lysis may also be performed by mechanical
procedures such as sonication, wet milling,
high-pressure
homogenization
and
impingement.
A number of proteinaceous products of
interest may be present as insoluble particles
present in inclusion bodies within the
microorganism.
After cell disruption, such inclusion bodies may
be separated from the bulk of the remaining
cell components.

The product of interest may be renatured using


standard techniques including, but not limited,
dissolving in 6 M guanidinium chloride followed by
renaturing in an appropriate buffer.
The required degree of purity of the final product
of interest depends on its end use.
Crude preparations may be satisfactory in some
instances, but additional purification steps may be
required for other products of interest using
standard separation techniques such as
chromatography, centrifugation and extraction.

Production Of Bovine Growth Hormone Using A Porcine Bioreactor

Ten 4-week old germ-free pigs, which test


negative for bacteria by rectal swabs, are fed a
24
hour
culture
of
bacteria
previously
transformed with pBGH33-4 , which expresses
high levels of bovine growth hormone.
Rectal swabs from each pig are collected on days
2, 4, 7, 9, 11, 14, 17, 19, 21 and 24 and plated on
LB supplemented with tetracycline to measure
the growth of the modified bacteria in the large
intestines.
The rectal swabs are also plated on LB to ensure
that other bacteria have not colonized the large
intestines.

Feces are collected from the pigs


beginning 4 days after induction.
The feces are dissolved in Tris buffer and
sonicated to lyse the modified bacteria.
Bovine growth hormone in the resulting
supernatant is then precipitated with 30%
to 70% ammonium sulfate and then
resuspended and dialyzed in Tris buffer.
The dialyzed sample is then loaded on an
SDS-polyacrylamide gel.
Alpha-BGH antibodies used to probe the
gel to verify the presence of BGH.

Large-Scale Production Of Threonine Using Bovine Bioreactor

3000 dairy cows are colonized with a bacterial


strain that over-expresses L-threonine .
The cows are housed in a dairy facility under
comparative microbial isolation.
The cows are born in the facility and leave the
facility only to be milked.
The food rations for the cows is controlled to
minimize toxic substances excreted in the feces.

The feces are collected via drains in


the floor of the facility and transported
to a collector on a lower level.
Feces are collected from the cows
beginning 4 days after induction.
The feces are dissolved in Tris buffer
and sonicated to lyse the modified
bacteria.
L-threonine
in
the
resulting
supernatant is then purified by batch
chromatography
as
described
by
Jansen et al.

Transgenic Sheep as
Bioreactor
The research in producing transgenic sheep
is now focused on producing sheep with
better
growth,
increased
meat
and
developing the mammary gland of this
mammal as a bioreactor.
The pharmaceutically important proteins are
made to secrete into the milk generated by
the sheep.
Though the amount of milk compared to
cattle is
less, yet lactation in sheep can produce
significant amount of milk on annum basis.

There are several advantages associated


with sheep in using them as bioreactor:
Sheep are mammals and therefore the
protein produced in them would be more
like the one generated in the human
being.
Biologically they mature more quickly
compared to cattle and therefore more
economical.
Large amounts of the pharmaceutically
important proteins can be produced as it
is easy to maintain a flock of sheep.
Milk
can
be
easily
extracted
and
downstream processing is simpler and
therefore proteins can be easily purified

Applications of Transgenic
Sheep
1) Human growth hormone has been
successfully introduced into sheep in order
to increase their
development, growth and meat
production. Such
transgenic sheep has shown
considerable improvement in their body
weight, feed efficiency, meat/fat ratio and
fat composition. The gene for ovine growth
hormone is usually placed under control of
metallothionein promoter1

2) Transgenic sheep have been produced with the


idea of increased wool production and improved
wool quality, and this is being one of the major
research efforts. However, these initial works
have not been that promising and much further
work is needed to improve wool production and
its quality.
3) Clotting factor IX is an important protein
produced
naturally in the body. It helps the
blood to form clots
and stops bleeding. Injections of factor IX are
used to
treat hemophilia B, a condition which is
sometimes
called Christmas disease. If one
does not have enough factor IX and become
injured, the blood will not form clots as it should
and may result in damage of muscles and joints.

Embryo Transfer in sheep and


goat. Embryo Transfer in
sheep and goats (AH): (A),
preparation of anesthetized
ewe on a cradle; (B) and (C),
two skin incisions (each 1cm)
are made on either side of the
mid ventral line, 5 cm away
from the udder; (D, E and F),
insertion
of
trocar
and
cannula, cannula is left on the
abdomen
through
which
endoscope is inserted into the
abdomen; (G), insertion of the
endoscope
through
the
cannula
to
visualize
the
ovaries, corpora lutea and
reproductive tract; (H), a
uterine horn is punctured
close to the utero-tubular
junction using a blunt-end 18

Transgenic Chickens as
Bioreactors
The hen since from long time has been a
potential
candidate
to
produce
human
biopharmaceuticals at low-cost with high-yield.
The reason for this is simple as:
The yolk and white of the egg are sterile.
The technology for fractionating egg yolk and
egg white proteins is available.
Highly
automated
systems
for
efficiently
producing and collecting thousands of eggs per
day are well established.
The egg white contains ~4 g of protein, more
than half of which comes from the expression of
a single gene i.e. ovalbumin gene (OV).
Hence, the OV promoter, combined with its other

Added to this typically a hen lays


>300 eggs per year.
Therefore a single hen could
potentially produce 300 g of raw
product annually.

Above
all
they
show
posttranslational modifications that can
be compared with that taking place
in humans.

Impetus for Creation of Animal Bioreactors

The ability to express transgenes in milkproducing animals has resulted in the creation
of bioreactors.
These are animals that produce large amounts
of a given recombinant protein in their milk.
These recombinant proteins are produced in
fully biologically active form through proper
posttranslational
modification
(PTM),
for
purification and therapeutic use.
This approach has been used to produce
recombinant tissue plasminogen activator ,
granulocyte colony-stimulating factor, Ig , and
lactoferrin in the milk of goats and cows.

A recombinant form of human


antithrombin III (ATryn) produced
in goats is currently approved in
Europe.
It is used for the prevention of
clotting in surgical patients with
hereditary antithrombin deficiency.
This is the first instance of a
transgenically produced drug being
approved for use in humans.

Atryn: The First Transgenically


Produced Biopharmaceutical
The recombinant production of AT
presented numerous challenges.
Antithrombin is a complex glycoprotein
carrying 4 N-linked glycosylation sites and
3 disulfide bonds.
These characteristics, which are crucial
for the functions of AT, precluded the use
of
microbial
bioreactors
for
its
recombinant production.
In addition, the therapeutic use of AT
calls for large amounts, often grams, of
purified protein per course of treatment.

Thus the expression in the milk of transgenic


dairy goats was employed.
The promoter region of the goat beta-casein
gene was linked to hAT cDNA.
This transgene was introduced into the
chromosomes of goat embryos, which were
then transferred to surrogate mothers.
The resulting goats carrying this transgene
produce the gene product, rhAT, in their milk.
Transgenic offspring from the line selected
for commercial development consistently
express rhAT in their milk at approximately 2
g/L.
Expression levels of up to 10 g/L were
observed in other lines that were not
developed further because of timing issues.

The human AT purified from transgenic goat's


milk is structurally indistinguishable from
human plasma-derived AT (hpAT) with the
exception of the carbohydrates.
The main glycosylation differences observed for
rhAT were the presence of fucose and GalNAc, a
higher level of mannose, and a lower level of
galactose and sialic acid.
Several
independent
laboratories
have
determined that differences in glycosylation of
AT do not affect the intrinsic rate constant of
the uncatalyzed or heparin catalyzed inhibition
of thrombin.
Thus, glycosylation does not impact the major
biological
activity
of
AT.

Table 2. Chronology of recombinant protein production in the mammary gland


of transgenic strains from different species [14]
Rabbit

Cow
Gestation period (months)
1
9
Sexual maturity (months)
5
15
Time from introduction of transgene
to the beginning of lactation (months)

Pig

Sheep

Goat

Female Founder
Lactation induced in puberty
Natural Lactation
7
33
Male Founder
Lactation induced in puberty
(daughters)
45
Natural Lactation (daughters)
15
57
Average progeny
8
1
Annual Yield of milk production
(L/year)
4-5
8000
Production of the recombinant protein
/female/year (kg)
0.02
40

16

18

28
10

18

22
31
1-2

300

500

1.5

2.5

22
31
1-2

800

Animal bioreactors
Exemplary categories of polypeptides

Growth factors
Hormones
Antiviral proteins
Lipocortins
Lipotropins
Interleukins
Interferons
Stimulating factors
Kinases
Transmembrane
regulators
Immunoglobulins
Milk lipases
Cell surface proteins
Human pancreatic enzymes
Enkephalins
Silk proteins
Spider silk proteins

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