TOPIC DISCUSSION:

CHAPTER 12: BIOPROCESS CONSIDERATIONS IN USING ANIMAL CELLS

• ANIMAL CELL CULTURES - They are capable of making a vast array of important therapeutic
proteins. With recombinant DNA techniques, it is possible to achieve significant levels of
production of compounds made only in minute amounts in native cells. 1

This capacity is essential in some cases to the formation of a clinically useful product.

√ Inside the cytoplasm of most animal cells is an extensive network of membrane bounded
channels called the endoplasmic reticulum (ER).

√ The membranes of the ER divide the cytoplasm into two phases: the lumenal phase (inside
the endoplasmic reticulum)

√ cytosol (outside the rough endoplasmic membrane). Ribosomes are usually located on the
outer surface of the endoplasmic reticulum.

√ Some ribosomes are located in cytoplasm and may be interconnected by fine filaments. 1

√ Mitochondria are independent organelles in the cytoplasm containing DNA and are capable of
independent reproduction.

√ Each mitochondrion is surrounded by a double membrane: a smooth outer membrane and a

highly folded inner membrane called the cristae.

√ The mitochondrial matrix often contains crystallike inclusions.

√ Lysosomes are rather small cytoplasmic organelles bound by a single membrane, and they
contain various hydrolytic enzymes, such as proteases, nucleases, and esterases.

√ Lysosomes are responsible for the digestion of certain food particles ingested by the cell.

√ The Golgi body is a cytoplasmic organelle surrounded by a rather irregularly shaped

membrane called the cisternae.

√The cisternae of a Golgi body are often stacked together in parallel rows, called dictyosome.
√ The Golgi apparatus is responsible for the completion of complex glycosylation and for
collecting and secreting extracellular proteins or directing intracellular protein traffic to other
organelles.

√ Some cells contain small cytoplasmic organelles called peroxisomes and glyoxysomes.

• STRUCTURE AND BIOCHEMISTRY OF ANIMAL CELL

√ Animal cells do not have a cell wall, but are surrounded by a thin and fragile plasma
membrane that is composed of protein, lipid, and carbohydrate. This structure results in
significant shear sensitivity. 2

• Virus Vaccines - various virus vaccines (prophylactics) have been produced for animal and
human use. In some cases live virus is propagated in animal cells. Virus is then collected and
inactivated (“killed” virus) and used as a vaccine.2

• Insecticides2- Animal cell cultures have been used to produce some insect viruses that are
highly specific and safe to the environment. Several of the baculoviruses have federal approval
for use as insecticides. Genetically engineered variants with greater virulence can now be
produced. 2
CHAPTER 13: BIOPROCESS CONSIDERATIONS IN USING PLANT CELL CULTURES

• WHY PLANT CELLS?

√ The plant kingdom is very diverse. Many thousands of chemicals are produced only in plants.
3

√ The biosynthetic capabilities of plant cells have been little explored. 3

√ Only a few percent of the world’s plants have been scientifically named or described, and only
a small fraction of these have been screened for the production of novel and useful
compounds.3

EXAMPLES OF PLANT PRODUCTS OF POTENTIAL PLANT INTEREST

√ Pharmaceuticals

√ Food Colors or Dyes

√ Flavors

√ Fragrances

√ Sweeteners

√ Agricultural Chemicals

• The factory production of chemicals from plant cell tissue culture offers a number of
important advantages:3

1. Control of supply of product independent of availability of the plant itself.

2. Cultivation under controlled and optimized conditions.

3. Strain improvement with programs analogous to those used for microbial systems.

4. With the feeding of compounds analogous to natural substrates, novel compounds

not present in nature can be synthesized.3

• Potential Problems of Large-scale Immobilized Plant Cell Cultures:

1. Large-scale aseptic immobilization procedures must be developed

2. Mass transfer limitations may significantly affect cell metabolism (positively and adversely)

3. Products must be produced by nongrowing cells

4. Products must be released from the cell into the medium

5. Experience in the scale-up of immobilized-cell systems is limited4

CHAPTER 14: UTILIZING GENETICALLY ENGINEERED ORGANISMS

INTRODUCTION: we discussed how cells could be genetically engineered. The techniques of

genetic engineering are fairly straightforward; the design of the best production system is not.

√ The choice of host cell, the details of the construction of the vector, and the choice of
promoter must all fit into a processing strategy. 5
• That strategy includes plans not only for efficient production but also for how a product is to
be recovered and purified. 6

• The development of processes for making products from genetically engineered organisms
requires that many choices be made. 6

• Another important consideration is whether the product will be used in foods. For example,
some yeasts (e.g., S. cerevisiae) are on the FDA GRAS list (generally regarded as safe), which
would greatly simplify obtaining regulatory approval for a given product.6

• MAMMALIAN CELLS : Mammalian cell culture is chosen when the virtual authenticity of the
product protein must be complete.

√ 6But for bioreactor processes, mammalian cell tissue culture will provide the product closest
to its natural counterpart.

√ Another advantage is that most proteins of commercial interest are readily excreted. Slow
growth, expensive media, and low protein expression levels all make mammalian cell tissue
culture very expensive.6

• TRANSGENIC PLANTS AND PLANT CELL CULTURES:

√ Proteins, including many complex protein assemblies, such as antibodies and virus-like
particles (as vaccines), can be made inexpensively in plants. 6

√ Transgenic plants offer many potential advantages in addition to cost. Since plant viruses are
not infective for humans, there are no safety concerns with respect to endogenous viruses or
prions.

√ While inexpensive, witheasy scale-up, it takes 30 months to test and produce sufficient seed
for unlimited commercial use.

√ Such long lead times are undesirable. Further, environmental control on fieldgrown crops is
difficult, so the amount (and possibly quality) of the product can vary from time to time and
place to place.

• Host Cell Mutations6:

√ Mutations in host cells can also occur that make them far less useful as production systems for
a given product. 7

√ The key feature of this category of genetic instability is that a host cell mutation imparts a
growth advantage to the mutant, so that it will eventually dominate the culture. In this case the
mutant cell will contain unaltered plasmids but will make very little of the target, plasmid-
encoded protein.7

• METABOLIC ENGINEERING

√ The principle motivations for metabolic engineering are the production of specialty chemicals
(e.g., indigo, biotin, and amino acids), utilization of alternative substrates (e.g., pentose sugars
from hemicellulose)7

√ A goal of the engineering approach has been to develop rational design techniques for
metabolical engineering.

√ Due to the highly nonlinear, dynamic nature of a cell and its metabolism, uninformed changes
intended to improve production of a specific compound often fail to give the desired result.

√ Real progress has been made toward the industrial use of metabolically engineered cells. 7

• IMPORTANT TERMINOLOGIES

√ Genetic instability is the loss of the genetic information to make the target protein. 7

√ Segregational instability arises when a plasmid-free cell is formed during cell division. 7

√ Structured instability results when the cell loses the capacity to make the target protein in
significant quantities due to changes in plasmid structure.7

√ Host-cell derived instability occurs when chromosomal mutations decrease effective plasmid-
encoded protein production while the cell retains the plasmid.

√ Growth-rate-dependent instability is a function of the growth-rate differential between

plasmid-free and plasmid-containing cells;7

√ E. coli greatly facilitates sophisticated genetic manipulations, but process or product

considerations may suggest alternative hosts.7
√ P. pichia can produce very high concentrations of proteins, but the use of methanol presents
challenges in reactor control and safety.

√ Bacillus
and the lower
fungi may
have well-
developed
secretion
systems
that would be
attractive
if they can
be

harnessed.8

The use of this approach is illustrate in the following.

Example 14.4.
Estimate the fraction of plasmid-containing cells in a batch culture under the following
circumstances. Cells are maintained at constant, maximal growth rate of 0.693 h-1 during scale-
up from shake flask through seed fermenters into production fermenters. The total time for this
process is 25 h. Assume that the inoculum for the shake flask was 100% plasmidcontaining cells.
It is known that the growth rate for a plasmid-free cell is 0.97 h-1. The value of P is 0.001.

Solution

First we calculate a and ng.9