UNIT-V

5.1.0 PRODUCTION OF RECOMBINANT PROTEINS

Previously, mammalian therapeutic protein and peptide could be prepared
only from animal or human tissues and body fluids and were available in limited
quantities. These preparation were extremely costly to produce and had some unwanted
side effects and in certain case there were unfortunate problems with virus and prion
contamination.
Recombinant technology has allowed the production of many recombinant
proteins from various sources, including over 400 human proteins and peptides with
potential medical applications. At present, only about 10% have received approval for
one by the U.S Food and Drug Administration. The fermentation volume for industrial
scale production of human therapeutic proteins is usually no greater than 2000-5000L.
Unlike recombinant vaccines,where it is essential to retain or even enhance antigenicity.
Hence, recombinant therapeutic products must be free from antigenicity.

EXAMPLE RECOMBINANT PROTEINS FOR MEDICAL USE

1. Antibiotics
2. Cancer and Viral Diseases:
Interferons, Interleukins, tissue necrotic factor and
transforming growth factor.
3. cardio vascular Diseases:
Erythropoietin
Hirudin
Tissue plasminogen activator
4. Hormones:
Human Growth Hormone
5. Neuroloical Diseases:
Endorphins
Nerve growth factor
Neuropeptide
 6. Vaccines:
Foot-2-mouth disease
Hepatitis B
7. Wound healing and blood clotting facor:
Epidermal growth factor
Fibroblast growth factor
Clotting factor VII

5.1.1 PRODUCTION OF RECOMBINANT VACCINES

Vaccine production has several stages. First, the antigen itself is generated.
Viruses are grown either on primary cells such as chicken eggs (e.g., for influenza), or on
continuous cell lines such as cultured human cells (e.g., for hepatitis A). Bacteria are
grown in bioreactors (e.g., Haemophilus influenzae type b). Alternatively, a recombinant
protein derived from the viruses or bacteria can be generated in yeast, bacteria, or cell
cultures. After the antigen is generated, it is isolated from the cells used to generate it. A
virus may need to be inactivated, possibly with no further purification required.
Recombinant proteins need many operations involving ultrafiltration and column
chromatography. Finally, the vaccine is formulated by adding adjuvant, stabilizers, and
preservatives as needed. The adjuvant enhances the immune response of the antigen,
stabilizers increase the storage life, and preservatives allow the use of multidose vials.
Combination vaccines are harder to develop and produce, because of potential
incompatibilities and interactions among the antigens and other ingredients involved.
Vaccine production techniques are evolving. Cultured mammalian cells
are expected to become increasingly important, compared to conventional options such as
chicken eggs, due to greater productivitity and few problems with contamination.
Recombination technology that produces genetically detoxified vaccine is expected to
grow in popularity for the production of bacterial vaccines that use toxoids. Combination
vaccines are expected to reduce the quantities of antigens they contain, and thereby
decrease undesirable interactions, by using pathogen-associated molecular patterns.
 Vaccine production requires highly controlled operating conditions and
restrict to good manufacturing practices. It normally involves the growth of bacterial
cultures in sophisticated high-grade fermentation of usually not greater than 1000L
capacity.
The fermentors are designed for optimized yield of antigen and
contaminant, involving the way strict adherence to protocols that bacterial release into
environment.
The internal pressures never exceed atmospheric pressure to reduce the
risks of leakage and extract gases from fermentors must pass through sterilizing filters,
inclinators or both.
Fermentations for the production of vaccines based on whole cells aims to
maximize biomass production. For inactivated whole cell vaccines, down stream
processing usually follows cell inactivation by heat treatment or the addition of HCHO.
The microbial cells inactivated or live, are then separated from the medium
by centrifugation. All harvesting conditions or equipment incorporation absolute
microbial containment and is situated within a room maintained and a negative pressure,
so that any escape is contained.
Live attenuated vaccines are usually prepared as freeze-dried products.
For the production of vaccines based on toxins or surface antigens, the cell
growth level or conditions are aimed at producing maximum levels of these specific
cellular antigens.
Excreted toxins and any loosely bound surface antigens that are shed into
the medium are purified from the clarified culture broth and the harvested cells are safely
discarded.
Toxins are usually inactivated by treatment with heat or HCHO to become
bacterial toxoids that have no toxicity but retain their antigenicity.
Vaccine antigens for human immunization are highly purified.
Purification procedures may include conventional ammonium sulphate
precipitation techniques and various chromatographic steps.
 Affinity chromatography is usually preferably incorporated, utilizing
specific antibodies as ligands, preferably monoclonal antibodies.
For maximum effectiveness as a vaccine, the purified antigens are absorbed
onto an adjuvant that increases the immune power.
Adjuvants include, mineral salts, such as aluminium hydroxide and
potassium aluminium sulphate or water in oil estimations.
Vaccine preparations can be injected parenterally or administrated orally.
Inactivated forms are usually injected and living vaccines are mostly taken orally.
Injected vaccines stimulate antibody production in the blood stream, where as oral
vaccines stimulate local production of antibody at the mucosal surface of the intestine.

VIRAL VACCINES
Viral vaccines were previously available only via culture in live animals or
from animal tissue cell cultures. However, genetical engineering has allowed the
production of recombinant viral vaccines through the cloning of viral antigens into an
appropriate host microorganism.
Example: The virulence factor of hepatitis B and viral protein of foot and
mouth disease virus can be expressed in E.coli for the production of valuable vaccines.
The safe production of recombinant vaccine for dangerous bacterial
pathogens is now possible, using host organism well suited to large scale fermentation.
This has the added advantage that the host can be manipulated to amplify antigen
production.
Some lactic acid bacteria are suitable hosts and are being evaluated for use
in oral immunization. Those bacteria have generally recognized as safe state and low
immunogenicity and can be used to express antigens such as fragments of tetanus and
Diptheria toxins.

EXAMPLE VACCINES
Bacterial vaccines: chlomydia (pelvis inflammatory disease)
Tettanus
Diptheria
 Protozoa: Mabria (Plasmodium vivax)

Viral Vaccines: Hepatitis B

Herpes simplox
Influenza
Measless
Poliomylitis

DNA Vaccines: Malaria

Vaccine development has several trends

• Until recently, most vaccines were aimed at infants and children, but adolescents
and adults are increasingly being targeted.
• Combinations of vaccines are becoming more common; vaccines containing five
or more components are used in many parts of the world.
• New methods of administering vaccines are being developed, such as skin
patches, aerosols via inhalation devices, and eating genetically engineered plants.
• Vaccines are being designed to stimulate innate immune responses, as well as
adaptive.
• Attempts are being made to develop vaccines to help cure chronic infections, as
opposed to preventing disease.
• Vaccines are being developed to defend against bioterrorist attacks such as
anthrax, plague, and smallpox.

5.2.0 MONOCLONAL ANTIBODIES

Most antigens offer multiple epitopes and therefore induce proliferation and
differentiation of a variety of B-cell clones each derived from a B cell that recognizes a
particular epitope. The resulting serum antibodies are heterogenous, comprising a mixture
of antibodies, each specific for one epitope. Such a polyclonal antibody response
facilitates the localization, phagocytosis and complement-mediated lysis of antigen; it
thus has clear advantages for the organism in vivo. Unfortunately, the antibody
heterogenicity that increases immune protection in vivo often reduces the efficacy of an
antiserum for various in vitro uses. For most research, diagnostic and therapeutic
purposes, monoclonal antibodies derived from a single clone and thus specific for a
single epitope, are preferable.

HYBRIDOMA TECHNOLOGY
By fusing a normal activated, antibody producing B cell with a myeloma
cell, they were able to generate hybrid cell called a hybridoma that possess immortal
growth properties of the myeloma cell and secreted the antibody produced by B cell. The
resulting clones of hybridoma cells, which secrete large quantities of monoclonal
antibody, can be cultured indefinitely. This technique is called hybridoma technology
which is developed by Kohlar and Milstein in 1975.

STEPS INVOLVED
1. A mouse is immunized with a selected antigen.
2. When immune response occurs, B-lymphocytes are harvested from the
spleen of mouse.
3. The B-lymphocytes are fused with myeloma plasma cell from mouse in
the presence of Polyethylene glycol
4. The fused cells are the hybridoma cells.
5. The fused cells are grown in a selective cell culture medium. In this
medium neither the B-lymphocyte nor the myeloma cells can survive.
6. The surviving hybrid cells are screened to obtain a cell that express
antibody of desired specificity.
7. The selected cells are cloned and recloned to produce thousands of
daughter cells.
8. The selected clones can be propagated both in vivo or in vitro to yield
monoclonal antibodies.
9. For in vivo propagation, the hybridoma cell can be injected into a mouse.
A tumour is produced in the mouse. The tumour generates large amounts of the desired
monoclonalantibody. The in vivo culture has the advantages over in vitro because it
produces large quantities of monoclonal antibodies.
10. In vitro propagation, the hybridoma is cultured outside the mouse.
PURIFICATION
Purification of monoclonal antibodies
After obtaining either a media sample of cultured hybridomas or a sample of
ascites fluid, the desired antibodies must be extracted. The contaminants in the cell
culture sample would consist primarily of media components such as growth factors,
hormones, and transferrins. In contrast, the in vivo sample is likely to have host
antibodies, proteases, nucleases, nucleic acids, and viruses. In both cases, other secretions
by the hybridomas such as cytokines may be present. There may also be bacterial
contamination and, as a result, endotoxins which are secreted by the bacteria. Depending
on the complexity of the media required in cell culture, and thus the contaminants in
question, one method (in vivo or in vitro) may be preferable to the other.
The sample is first conditioned, or prepared for purification. Cells, cell debris,
lipids, and clotted material are first removed, typically by filtration with a 0.45 µm filter.
These large particles can cause a phenomenon called membrane fouling in later
purification steps. Additionally, the concentration of product in the sample may not be
sufficient, especially in cases where the desired antibody is one produced by a low-
secreting cell line. The sample is therefore condensed by ultrafiltration or dialysis.
Most of the charged impurities are usually anions such as nucleic acids and
endotoxins. These are often separated by ion exchange chromatography. Either cation
exchange chromatography is used at a low enough pH that the desired antibody binds to
the column while anions flow through, or anion exchange chromatography is used at a
high enough pH that the desired antibody flows through the column while anions bind to
it. Various proteins can also be separated out along with the anions based on their
isoelectric point (pI). For example, albumin has a pI of 4.8, which is significantly lower
than that of most monoclonal antibodies, which have a pI of 6.1. In other words, at a
given pH, the average charge of albumin molecules is likely to be more negative.
Transferrin, on the other hand, has a pI of 5.9, so it cannot easily be separated out by this
method. A difference in pI of at least 1 is necessary for a good separation.
Transferrin can instead be removed by size exclusion chromatography. The
advantage of this purification method is that it is one of the more reliable chromatography
techniques. Since we are dealing with proteins, properties such as charge and affinity are
not consistent and vary with pH as molecules are protonated and deprotonated, while size
stays relatively constant. Nonetheless, it has drawbacks such as low resolution, low
capacity and low elution times.
A much quicker, single-step method of separation is Protein A/G affinity
chromatography. The antibody selectively binds to Protein A/G, so a high level of purity
(generally >80%) is obtained. However, this method may be problematic for antibodies
that are easily damaged, as harsh conditions are generally used. A low pH can break the
bonds to remove the antibody from the column. In addition to possibly affecting the
product, low pH can cause Protein A/G itself to leak off the column and appear in the
eluted sample. Gentle elution buffer systems that employ high salt concentrations are also
available to avoid exposing sensitive antibodies to low pH. Cost is also an important
consideration with this method because immobilized Protein A/G is a more expensive
resin.
To achieve maximum purity in a single step, affinity purification can be
performed, using the antigen to provide exquisite specificity for the antibody. In this
method, the antigen used to generate the antibody is covalently attached to an agarose
support. If the antigen is a peptide, it is commonly synthesized with a terminal cysteine
which allows selective attachment to a carrier protein, such as KLH during development
and to the support for purification. The antibody-containing media is then incubated with
the immobilized antigen, either in batch or as the antibody is passed through a column,
where it selectively binds and can be retained while impurities are washed away. An
elution with a low pH buffer or a more gentle, high salt elution buffer is then used to
recover purified antibody from the support.
To further select for antibodies, the antibodies can be precipitated out using
sodium sulfate or ammonium sulfate. Antibodies precipitate at low concentrations of the
salt, while most other proteins precipitate at higher concentrations. The appropriate level
of salt is added in order to achieve the best separation. Excess salt must then be removed
by a desalting method such as dialysis.
 The final purity can be analyzed using a chromatogram. Any impurities will
produce peaks, and the volume under the peak indicates the amount of the impurity.
Alternatively, gel electrophoresis and capillary electrophoresis can be carried out.
Impurities will produce bands of varying intensity, depending on how much of the
impurity is present.

APPLICATIONS OF HYBRIDOMA TECHNOLOGY

Hybridoma technology helps to produce monoclonal antibodies in large
quantities. The monoclonal antibodies are used in the following fields,
1. Hybridoma cells produce blood group antibodies on a large scale
2. The phenotypic differences between B-lymphocytes and T-lymphocytes
can be identified.
3. The origin of tumour cells can be traced.
4. Hybridoma technology helps to locate cancerous tissues.
5. Monoclonal antibodies can be utilized as biologically guided missile for
therapeutic approaches in cancer and many other diseases.
6. The function of the immune system can be studied.
7. The antigen can be purified.
8. Antigenic determinant on various infective agents can be identified.
9. Monoclonal antibodies are used in the diagnosis of infectious diseases
such as gonorrhoea.
10. The monoclonal antibodies can be used as immunoprophylaxis against
various infectious agents.
11. Monoclonal antibodies help to establish embryological relationship
between animals.
12. Monoclonal antibodies are used in fluorescent immunoassay of
gentamycin.
 5.2.0 PRODUCTS OF CELL CULTURE

5.2.1 PLANT CELL CULTURE PRODUCTS

In recent years, plant protoplast, cell and organ cultures have become an
important tool for crop improvement, commercial production of natural compounds and
many more in the development of forestry. The major applications of in vitro cultured
plant protoplast/cells/tissues as below;
1. Tissue culture applications in order to capitalise upon the totipotency of cells.
2. Cell and protoplast cultures coupled with DNA vectors to overcome problems
caused by barriers to gene transfer through sexual means.
3. Culture of plant cells for the production of useful compounds
4. Extension and increase of efficiency of biological nitrogen fixation
5. Transfer of genes for nitrogen fixation ability to non fixing species

LARGE SCALE CULTIVATION OF PLANT CELLS

The chemical compounds produced by plants are collectively referred to as
phtochemicals. Biotechnologists have special interest in plant tissue culture for the large
scale production of commercially important compounds. These include pharmaceuticals,
flavours, fragrances, cosmetics, food additives, feed stocks and antimicrobials.
Most of these products are secondary metabolites that i.e, which do not take
part in the metabolism of plants. Thus secondary metabolites are not needed directly by
plants as they do not have any significant function. Although the native plants are capable
of producing the secondary metabolites of commercial interest, tissue culture systems are
preferred.
In order to achieve industrial production of the desired metabolite, large
scale cultivation of plant cells is required. Plant cells are generally 10-100 times larger
than bacterial or fungal cells. When cultured, plant cells exhibit changes in volumes and
thus variable shapes and sizes. Further, cultured cells have low growth rate and genetic
instability. All these aspects have to be considered for mass cultivation of cells.
The following four different culture systems are widely used
1. Free cell suspension culture
2. Immobilized cell culture
3. Two phase system culture
 4. Hairy root culture


Modern plant tissue culture is performed under aseptic conditions under

filtered air. Living plant materials from the environment are naturally contaminated on
their surfaces (and sometimes interiors) with microorganisms, so surface sterilization of
starting materials (explants) in chemical solutions (usually alcohol or bleach) is required.
Mercuric chloride is seldom used as a plant sterilant today, as it is dangerous to use, and
is difficult to dispose of. Explants are then usually placed on the surface of a solid culture
medium, but are sometimes placed directly into a liquid medium, particularly when cell
suspension cultures are desired. Solid and liquid media are generally composed of
inorganic salts plus a few organic nutrients, vitamins and plant hormones. Solid media are
prepared from liquid media with the addition of a gelling agent, usually purified agar.

In-vitro tissue culture potato explants

The composition of the medium, particularly the plant hormones and the
nitrogen source (nitrate versus ammonium salts or amino acids) have profound effects on
the morphology of the tissues that grow from the initial explant. For example, an excess
of auxin will often result in a proliferation of roots, while an excess of cytokinin may
yield shoots. A balance of both auxin and cytokinin will often produce an unorganised
growth of cells, or callus, but the morphology of the outgrowth will depend on the plant
species as well as the medium composition. As cultures grow, pieces are typically sliced
off and transferred to new media (subcultured) to allow for growth or to alter the
morphology of the culture. The skill and experience of the tissue culturist are important in
judging which pieces to culture and which to discard.
 As shoots emerge from a culture, they may be sliced off and rooted with
auxin to produce plantlets which, when mature, can be transferred to potting soil for
further growth in the greenhouse as normal plants.

USES OF PHYTOCHEMICALS
According to a WHO survey, nearly 70-80% of the world population
depends on herbal drugs. It is a fact that many chemicals with complex structures that
cannot be chemically synthesized can be conveniently produced in plants.
The production of speciality chemicals in a plant is a multibillion industry.
The plant cell cultures provide laboratory managed sources for the supply of useful plant
products. Although hundreds of new compounds are identified every year in plants, only
a few of them are commercial importance. Attempts are made to produce them in cell
culture systems.
Shikonine is the dye produced by the cells Lythospermum erythrorhizon on
a commercial scale. The other products successfully produced in plant culture include
analgistics, antimalarial, muscle relaxants, drugs to control cardiovascular diseases,
hypotensives, perfumes, insecticides, food sweeteners and anticancer agents.
Sometimes the cost of the plant product is unimaginably high. For 1Kg of
vincristine and vinblastine respectively cost $3,50,000 and $1,00,000.
Phtoalexins
Plants are capable of defending themselves when attacked by
microorganisms by producing antimicrobial compounds collectively referred to as
Phytoalexins. These are the chemical weapons of defense against pathogenic
microorganism. Some of the phytoalexins that induce the production of secondary
metabolites are regarded as elicitors. Some chemicals can also act as elicitors.
Ex., actinomycin-D, sodium salt of arachidonic acid, ribonuclease A, chitosan, etc.

BIOTRANSFORMATION USING PTC

The conversion of one chemical into another using biological sysytems as
biocatalyst is regarded as biotransformation or bioconversion. The biocatalyst may be
free or immobilized and the process of biotransformation may involve one or more
enzymes.
A good example of biotransformation by plant cell cultures is the large
scale production of cardiovascular drug digoxin from digitoxin by digitali lanata. Digoxin
production is carried out by immobilized cells of D.lanat in airlift bioreactors. Cell
cultures of D.purpurea can convert steviol into steviobiocide and steviocide which are
100 times sweeter than cane sugar

5.2.2 ANIMAL CELL CULTURE PRODUCTS

Animal cell culture (ACC) is the process of culture of animal cells outside
the tissue (in vitro) from which they were obtained. The process of ACC is carried out
under strict laboratory conditions of asepsis, sterility and controlled environment
involving temperature, gases and pressure. It should mimic the in vivo environment
successfully such that the cells are capable of survival and proliferation in a controlled
manner.
Culturable cells

Theoretically, cells of any type can be cultured upon procurement in a viable

state from any organ or tissue. However, not all types of cells are capable of strangeness
in such an artificial environment because of many reasons on which the artificial
environment may fail to mimic the biochemical parameters of the source environment.
Some good examples include the absence of growth regulators, cell to cell signal
molecules, etc. Under optimal conditions of maintenance, the cell culture established can
be sub-cultured (passaging) until a pure-culture of specific cell type is obtained. This can
repeatedly sub-cultured to maintain as a cell-line. As a matter of fact, cell lines from
cancerous tissues have also been established. The presence of excess growth regulators or
other factors may often render the cells to undergo rapid uncontrolled proliferation
resulting in a cancerous state.
Media
The artificial environment is generally known as media. A media comprises
an appropriate source of energy for the cells which they can easily utilize and compounds
which regulate the cell cycle. A typical media may or may not contain serum. The latter
is called a serum-free media. Some of the common sources of serum can be fetal bovine
serum, equine serum, and calf serum. Both types of media have their own sets of
advantages and disadvantages.
Applications
Consider drug testing in the process of discovery of a new drug. The test drug
must pass through many phases after which it gets approved and marketed. Among the
preliminary phases, one such involves the testing of the test drug on animals for toxicity
or efficacy and efficiency. Now this can also be harmful and/or fatal to the animal on
which it is being tested. This can be minimized if the drug is tested on a cell line it is
targeted against as a cure, thereby assessing the toxicity on an initial scale thus reducing
the probability of death on the test animal.
Production of therapeutically significant biological compounds like hormones and
proteins on an industrial scale has been made simpler, faster and more efficient by the use
of cell lines in the place of the living animal themselves.
Vaccines effective against many viral infections and diseases require the
cultivation and mass production of the virus followed by its attenuation. The drawback in
this is that virus requires a living medium to replicate and multiply. Rather than the
traditional concept- “Sacrifice one life to save many”, ACC can be employed to mass
produce the virus. Passively, ACC can be employed to reduce the virulence of particular
virus strains by cultivating them on cells other than target cells which the virus infects
followed by repeated passaging.
Studies on regenerative medicine can be understood on deeper concepts if ACC is
fully exploited as cells behave in a spectrum of patterns under various environments
which can be simulated in an ACC laboratory and followed in vitro.
Active research on stem cell culture, proliferation leading to organogenesis is at a
slow phase due to the non-availability of research materials. This can however be
overcome if ACC is fully utilized. The fruits of such a study can be more than
overwhelming towards the betterment of human life.
ACC also finds application in the preservation of highly valuable cord blood cells
which are nothing but stem cells specific to an individual.
ACC is a potential tool in Assisted Conception which requires the maintenance of
sperm and the egg from the donors viable under laboratory conditions after which they
are allowed to fertilize (in vitro) followed by re-implantation.