Production of Animal Cell Products through Cell Culture

Introduction:

Cell culture is the process by which cells are grown under controlled conditions, generally
outside their natural environment. After the cells of interest have been isolated from living
tissue, they can subsequently be maintained under carefully controlled conditions.
There has been a rapid increase in the number and demand for approved
biopharmaceuticals produced from animal cell culture processes over the last few years.
In part, this has been due to the efficacy of several humanized monoclonal antibodies that
are required at large doses for therapeutic use. There have also been several identifiable
advances in animal cell technology that has enabled efficient biomanufacture of these
products

There are two major procedures:

i) Isolation of cells.

ii) Maintenance of cells in culture.

Animal products in therapeutic use:

Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and
other products of biotechnology. Culture of human stem cells is used to expand the number
of cells and differentiate the cells into various somatic cell types for transplantation. Stem
cell culture is also used to harvest the molecules and exosomes that the stem cells release
for the purposes of therapeutic development.
Biological products produced by recombinant DNA (rDNA) technology in animal cell
cultures include enzymes, synthetic hormones, immunobiologicals (monoclonal
antibodies, interleukins, lymphokines), and anticancer agents. Although many simpler
proteins can be produced using rDNA in bacterial cultures, more complex proteins that
are glycosylated (carbohydrate-modified) currently must be made in animal cells. An
important example of such a complex protein is the hormone erythropoietin. The cost of
growing mammalian cell cultures is high, so research is underway to produce such complex
proteins in insect cells or in higher plants, use of single embryonic cell
and somatic embryos as a source for direct gene transfer via particle bombardment,
transit gene expression and confocal microscopy observation is one of its applications. It
also offers to confirm single cell origin of somatic embryos and the asymmetry of the first
cell division, which starts the process.

Protein-based therapeutics are highly successful in clinic and currently enjoy

unprecedented recognition of their potential. More than 100 genuine and similar number
of modified therapeutic proteins are approved for clinical use in the European Union and
the USA with 2010 sales of US$108 bln; monoclonal antibodies (mAbs) accounted for
almost half (48%) of the sales.
 Products Applications
Erythropoietin’s

Erythropoietin-A Anemia resulting from cancer and chemotherapy.

Erythropoietin-P Anemia secondary to kidney disease.

Human Growth Hormones

hGH Human growth deficiency in children, renal cell carcinoma.

Somatotropin Chronic renal insufficiency, Turners' syndrome.

Monoclonal antibodies (Therapeutic)

Anti-lipopolysaccharide Treatment of sepsis.

Murine anti-idiotype/human B-cell

lymphoma B-cell lymphoma.

Monoclonal antibodies (Diagnostics)

Anti-Fibrin 99 Blood Clot.

99 Tcm-Fab (Breast) Blood Cancer.

PR-356CYT-356-in-lll Prostate Adenocarcinoma.

Plasminogen activator

Urokinase type plasminogen activator

Acute Myocardial Infection, Acute Stroke, Pulmonary
Tissue type plasminogen activator Embolism,

Deep Vein Thrombosis.

Recombinant plasminogen activator

Vaccines

HIV vaccines (gpl20) AIDS Prophylaxis and Treatment.

Malaria vaccine Malaria Prophylaxis.

Polio vaccines Poliomyelitis Prophylaxis.

 Table 1: Some products of therapeutic use derived from animal cell cultures (based on Feichter 1996)

There are 5 major animal cell culture products,

● Monoclonal Antibodies

● Recombinant Proteins

● Hormones

● Vaccines

● Interferons

1) Monoclonal Antibodies:

Monoclonal antibodies (mAb or moAb) are antibodies that are made by identical immune
cells that are all clones of a unique parent cell. Monoclonal antibodies can
have monovalent affinity, in that they bind to the same epitope (the part of an antigen that is
recognized by the antibody). In contrast, polyclonal antibodies bind to multiple epitopes and are
usually made by several different plasma cell (antibody secreting immune cell)
lineages. Bispecific monoclonal antibodies can also be engineered, by increasing the therapeutic
targets of one single monoclonal antibody to two epitopes.

Figure 2: Production of Monoclonal Antibodies by hybridoma. (Source: Protocol developed by

ABGENEX®)
2) Recombinant Proteins:

Recombinant proteins are novel proteins, which are often used to study functions and structure
of nature proteins. There are basically two methods for producing recombinant proteins. One is
molecular cloning, a laboratory method used to make recombinant DNA. The other method is the
polymerase chain reaction which is used for the replication of the specific DNA sequence. Between
the two methods, PCR is mainly used in the test tube, without living cells. The recombinant DNA
is need to integrated to a cloning vector for expression. Generally, plasmids or viruses can be used
as cloning vector. The option of vector for molecular cloning relies on the option of host organism,
the size of the DNA to be cloned, and the way in which the foreign DNA is to be expressed.

Cell culture technology enables manufacturing of such recombinant proteins:

cytokines(Interleukin, Interferon), chemokines(Eoxtain, MIP, GRO), growth factors(EGF,
GMCSF, MIP), viral antigens(preS1, PreS2), enzymes(PP, UC), hormones(PTH, exedin),
neurotrophins(NGF, BDNF, NT-4) and CD antigens. The genes of these products come from
different species: mouse, rat, human, rhesus macaque and canine. In order to make the gene express
efficiently, different expression systems: mammalian system, and insect system is already
launched.

Chemokines CD Antigen Growth Factors Cytokines

 Viral Antigen Enzymes Hormones Neurotrophins

3) Hormones:

A hormone is a specific molecule that act as a chemical messenger and produced by endocrine
glands in multicellular organisms. They travel by blood the circulatory system to target organs
with a view to regulate different physiological function of the body and to regulate behavior.
Different types of animal and human cell lines can be used to produce different types of hormones
such as growth hormone, parathyroid hormone, thyrocalcitonin, and adrenocortical steroid
hormones. Hormones and other valuable cellular products can be produced by cell culture based
methods which are difficult to obtain in other ways.

4) Vaccines:

Vaccines are antigenic substances that prepare the immune system to fight a disease-causing germ
or from special detrimental antigens by imitating an infection and provides active acquired
immunity to a particular disease. Generally, a vaccine contains an agent that assimilate a disease-
causing microorganism or any pathogen and is often made from weakened or killed forms of the
microbe, its toxins or one of its surface proteins. The immune system is tricked into making a
“memory” of that germ without fighting the real germ because they stimulate the body's immune
system to identify the agent as a threat, destroy it, so that the immune system can recognize and
destroy any of these microorganisms that it later encounters.

The production of virus-based vaccines in large-scale cultures of animal cells in the early 1950s
was the first industrial application of animal cell culture technology. Examples of human vaccines
produced in animal cells include the ones against measles, mumps and rubella. Other examples
include vaccines for polio, rabies, chicken pox, hepatitis B.

5) Interferons:

Interferons are group of signaling proteins that are produced by the body’s cells as a defensive
response to several pathogens. They act as one of the most important modulators of the immune
response. Production of various types’ interferons is mainly induced in the presence of microbes
and their toxic products. Interferons can also fight bacterial and parasitic infections, induce
inhibition of cell division and promote the differentiation of cells. They are produced by all
vertebrate animals and possibly by some invertebrates as well. Three forms of interferon, alpha
(α), beta (β), and gamma (γ) have been recognized. These interferons can be divided into two
different groups: type I includes the alpha and beta forms and type II consists of the gamma form.
Three types of interferons are produced and secreted from specific cell lines:

Interferons (IFN) Cell Lines

IFNα B Lymphocytes

IFNβ Fibroblasts

IFNγ T Lymphocytes

Reference:

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proteins by mammalian cells in suspension culture. Therapeutic Proteins: Methods and
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Milián, E. and Kamen, A.A., 2015. Current and emerging cell culture manufacturing technologies
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