Animal as Bioreactors

Dr. Asok Kr. SarkarEmail: asoksarkar09@gmail.com

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.

What is a bioreactor?

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 immobilized, is a Petri dish with agar gel. Large scale immobilized cell bioreactors are: moving media, also known as Moving Bed Biofilm ..

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 bioreactorsAnimal systems for production

Blood UrineSeminal plasmaEgg 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 low-speed, 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 from milk. The protein produced is limited to the mammary glands, hence has no adverse effect on the health of the sheep.

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 (A–H): (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 Gauge needle, embryos are loaded into an ET catheter and transferred into the lumen.

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 expression elements, could yield significant amounts of protein with a great amount of purity and recovery of the protein is non-invasive.

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 post-translational modifications that can be compared with that taking place in humans.

Impetus for Creation of Animal Bioreactors

The ability to express transgenes in milk-producing 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. This ruled out the use of standard mammalian cell culture bioreactors.

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 Pig Sheep Goat Cow • Gestation period (months) 1 4 5 5 9 • Sexual maturity (months) 5 6 8 8 15 • Time from introduction of transgene • to the beginning of lactation (months) • • Female Founder • Lactation induced in puberty • - - - - - • Natural Lactation 7 16 18 18 33 • Male Founder • Lactation induced in puberty • (daughters) - - 22 22 45 • Natural Lactation (daughters) 15 28 31 31 57 • Average progeny 8 10 1-2 1-2 1 • Annual Yield of milk production• (L/year) 4-5 300 500 800 8000• Production of the recombinant protein • /female/year (kg) 0.02 1.5 2.5 4 40

Animal bioreactorsExemplary 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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