Large scale production of

biologicals and sources

Dr Saquiba Yesmine
 Sources of biopharmaceuticals

• The bulk of biopharmaceuticals currently on

the market are produced by genetic
engineering using various recombinant
expression systems.
• Most of the recombinant proteins that have
gained marketing approval to date are
produced either in recombinant E. coli or in
recombinant mammalian cell lines.
Sources of biopharmaceuticals
 Escherichia coli as a source of
recombinant, therapeutic proteins
• As a recombinant production system, E. coli displays a
number of advantages. These include:
• E. coli has long served as the model system for studies
relating to prokaryotic genetics. Its molecular biology is
thus well characterized.
• High levels of expression of heterologous proteins can be
achieved in recombinant E. coli. Modern, high-expression
promoters can routinely ensure that levels of expression of
the recombinant protein reach up to 30 per cent total
cellular protein.
• E. coli cells grow rapidly on relatively simple and
inexpensive media, and the appropriate fermentation
technology is well established
 Escherichia coli as a source of
recombinant, therapeutic proteins
• These advantages, particularly its ease of genetic
manipulation, rendered E. coli the primary
biopharmaceutical production system for many years.

However, E. coli also displays a number of drawbacks as a

biopharmaceutical producer. These include
• heterologous proteins accumulate intracellularly;
• inability to undertake post-translational modifications
(particularly glycosylation) of proteins;
• the presence of lipopolysaccharide (LPS) on its surface.
 Escherichia coli as a source of
recombinant, therapeutic proteins
• Intracellular protein production complicates
downstream processing (relative to extracellular
production) as:
• additional primary processing steps are required, i.e.
cellular homogenization with subsequent removal of
cell debris by centrifugation or filtration;
• more extensive chromatographic purification is
required in order to separate the protein of interest
from the several thousand additional homologous
proteins produced by the E. coli cells.
 Additional production systems
Yeast
• Attention has also focused upon a variety of additional
production systems for recombinant biopharmaceuticals.
Yeast cells (particularly Saccharomyces cerevisiae) display a
number of characteristics that make them attractive in this
regard. These characteristics include:
• their molecular biology has been studied in detail,
facilitating their genetic manipulation;
• most are GRAS-listed organisms, and they have a long
history of industrial application (e.g. in brewing and
baking);
• they grow relatively quickly in relatively inexpensive media,
and their tough outer wall protects them from physical
damage.
 Additional production systems: Yeast
• Suitable industrial-scale fermentation equipment/technology
is already available;
• they possess the ability to carry out post-translational
modifications of proteins.

However, a number of disadvantages relating to heterologous

protein production in yeast have been recognized. These include:
• Although capable of glycosylating heterologous human
proteins, the glycosylation pattern usually varies from the
pattern observed on the native glycoprotein.
• In most instances, expression levels of heterologous proteins
remain less than 5 per cent of total cellular protein.
This is significantly lower than expression levels typically
achieved in recombinant E. coli systems.
 Yeast
• Despite such potential disadvantages, several
recombinant biopharmaceuticals now approved
for general medical use are produced in yeast (S.
cerevisiae)-based systems.
• Interestingly, most such products are not
glycosylated.
• The oligosaccharide component of glycoproteins
produced in yeasts generally contains high levels
of mannose.
Recombinant therapeutic proteins approved for general
medical use that are produced in S. cerevisiae
 Fungal production systems
• In general, fungi are capable of high-level expression
of various proteins secreted into their extracellular
media.
• The extracellular production of a biopharmaceutical
is distinctly advantageous in terms of subsequent
downstream processing.
• Fungi also possess the ability to carry out post-
translational modifications. Patterns of glycosylation
achieved can, however, differ from typical patterns
obtained when a glycoprotein is expressed in a
mammalian cell line.
• This can trigger a reduction is serum half-life or
immunological complications in humans.
 Transgenic animals

• The generation of transgenic animals is most often

undertaken by directly microinjecting exogenous DNA
into an egg cell.
• In some instances, this DNA will be stably integrated
into the genetic complement of the cell.
• After fertilization, the ova may be implanted into a
surrogate mother. Each cell of the resultant transgenic
animal will harbour a copy of the transferred DNA.
• As this includes the animal’s germ cells, the novel
genetic information introduced can be passed on from
one generation to the next.
Transgenic animals
 Advantages
• Ongoing supply of product is guaranteed (by
breeding).
• Low capital investments (i.e. relatively low-
cost animals replace high-cost traditional
fermentation equipment) and low running
costs.
• High expression levels of proteins are
potentially attained.
 disadvantages
• Variability of expression levels

• Significant time lag between the generation of

a transgenic embryo and commencement of
routine product manufacture.
• Another general disadvantage of this approach
relates to the use of the microinjection technique
to introduce the desired gene into the pronucleus
of the fertilized egg. This approach is inefficient
and time consuming. There is no control over
issues such as if/where in the host genomes the
injected gene will integrate.
• Overall, only a modest proportion of manipulated
embryos will culminate in the generation of a
healthy biopharmaceutical-producing animal.
• In addition to milk, a range of recombinant
proteins have been expressed in various other
targeted tissues/fluids of transgenic animals.
Antibodies and other proteins have been
produced in the blood of transgenic pigs and
rabbits. This mode of production, however, is
unlikely to be pursued industrially for a
number of reasons:
• Only relatively low volumes of blood can be
harvested from the animal at any given time
point
• Serum is a complex fluid, containing a variety of
native proteins. This renders purification of the
recombinant product more complex.
• Many proteins are poorly stable in serum.
• The recombinant protein could have negative
physiological side effects on the producer animal.
Expression of recombinant proteins in
animal cell culture systems
• Technical advances facilitating genetic
manipulation of animal cells now allow routine
production of therapeutic proteins in such
systems.
• The major advantage of these systems is their
ability to carry out post-translational modification
of the protein product.
• As a result, many biopharmaceuticals that are
naturally glycosylated are now produced in
animal cell lines
Expression of recombinant proteins in
animal cell culture systems
• hen compared with E. coli, animal cells display
a very complex nutritional requirement, grow
more slowly and are far more susceptible to
physical damage.
• In industrial terms, this translates into
increased production costs.
 Upstream processing

• Upstream processing refers to the initial

fermentation process that results in the initial
generation of product, i.e. the product
biosynthesis phase.
• Downstream processing refers to the actual
purification of the protein product and
generation of finished product format.
• Downstream processing includes filling into its
final product containers, freeze-drying if a dried
product format is required, followed by sealing of
the final product containers.
 Cell banking systems
• Recombinant biopharmaceutical production cell lines are
most often initially constructed by the introduction into
these cells of a plasmid housing a nucleotide sequence
coding for the protein of interest.
• After culture, the resultant product-producing cell line is
generally aliquoted into small amounts, which are placed in
ampoules and subsequently immersed in liquid nitrogen.
Therefore, the content of all the ampoules is identical, and
the cells are effectively preserved for indefinite periods
when stored under liquid nitrogen.
• This batch of cryopreserved ampoules forms a ‘cell bank’
system, whereby one ampoule is thawed and the cell
therein cultured in order to seed, for example, a single
production run.
 Cell banking systems
• The cell bank’s construction design is normally
two tiered, consisting of a ‘master cell bank’
and a ‘working cell bank’.
• The master cell bank is constructed first,
directly from a culture of the newly
constructed production cell line. It can consist
of several hundred individually stored
ampoules.
 Cell banking systems

The master cell bank/working cell bank system.

 Cell banking systems

The master cell bank/working cell bank system.

 Cell banking systems
• These ampoules are not used directly to seed a production
batch. Instead, they are used, as required, to generate a
working cell bank.
• The generation of a single working cell bank normally
entails thawing a single master cell bank ampoule, culturing
of the cells therein and their subsequent aliquoting into
multiple ampoules. These ampoules are then
cryopreserved and form the working cell bank. When a
single batch of new product is required, one ampoule from
the working cell bank is thawed and used to seed that
batch.
• When all the vials that compose the first working cell bank
are exhausted, a second vial of the master cell bank is used
to generate a second working cell bank, and so on.
 Cell banking systems
• The rationale behind this master cell bank/working cell
bank system is to ensure an essentially indefinite
supply of the originally developed production cells for
manufacturing purposes.
• This is more easily understood by example. If only a
single-tier cell bank system existed, containing 250
ampoules, and 10 ampoules were used per year to
manufacture 10 batches of product, the cell bank
would be exhausted after 25 years. However, if a two-
tier system exists, where a single master cell bank
ampoule is expanded as required, to generate a further
250 ampoule working cell bank, the entire master cell
bank would not be exhausted for 6250 years.
 Cell bank and upstream processing

Outline of the upstream processing stages involved in the production of a single batch of product.
Initially, the contents of a single ampoule of the working cell bank (a) are used to inoculate a few
hundred millilitres of media (b). After growth, this laboratory-scale starter culture is used to
inoculate several litres/tens of litres of media present in a small bioreactor (c). This production-
scale starter culture is used to inoculate the production-scale bioreactor (d), which often contains
several thousands/tens of thousands litres of media. This process is equally applicable to
prokaryotic or eukaryotic-based producer cell lines, although the bioreactor design, conditions of
growth, etc., will differ in these two instances
 Downstream processing
• Downstream processing serves to (:
a) recover the therapeutic protein from its
producer cell source upon completion of the
upstream processing phase,
b) purify the protein and
c) formulate the protein into final product
format
 Downstream processing
• Downstream processing is undertaken under clean-
room conditions in order to protect the product
stream from environmental contamination.
• In addition, the water used as solvent during
downstream processing (and, indeed, often during
upstream processing) is highly purified ‘water for
injections’ (WFI).
• Standard potable (drinkable) water contains
contaminants (e.g. microorganisms, dissolved organic
and particulate matter, etc.) that could either react
with the protein directly or that would have an
adverse effect upon patient health if present in the
final product.
 Insulin
• Insulin is a hormone central regulating carbohydrate
and fat metabolism in the body.

• Insulin is secreted by the Islets of Langerhans of

pancreas which catabolizes glucose in blood.

• Insulin causes liver cells, muscle cells and fat tissue

to take up glucose from the blood and store it as
glycogen in the liver and muscle.
Insulin Production
 Structure
• Insulin consists of two polypeptide chains,
Chain A ( 21 amino acid long) and B ( 30 amino
acid long). Its precursor is proinsulin which
also contains two polypeptide chains, A and B,
and is connected with a third peptide chain –C
(35 amino acid long).
 Production of Insulin

 In the Islets of Langerhans, insulin accumulates in secretary

vesicles as a single polypeptide chain called proinsulin.

 Before secretion into the bloodstream the third C chain of the

proinsulin molecule is excised, leaving the A and B chains
joined by disulphide bridges as the active insulin.

 E. coli is not capable of removing the C chain.

 There are several strategies for producing insulin from

bacteria, but the most successful is to synthesize the A and B
separately and then join them together.
 Production of Insulin

• The gene sequence of determining the A chain has been fused

to the ß-galactosidase gene (lac Z) of E.coli. The whole lac-Z-
A chain fusion is cloned into pBR322. Bacteria with this
plasmid synthesize ß-galactosidase with the insulin A chain.

• The B chain is produced in an identical manner.

• After purification of the two chains they are mixed , oxidized

and then reduced which allows the disulphide bridges to form
and active insulin to be produced.
Strategy for insulin production
Production of Recombinant Insulin
 Production of Recombinant Insulin

preproinsulin proinsulin insulin

Insulin
Inserting the vector into the required organism (E. coli).
 Inserting the vector into the required organism (E. coli).

• The recombinant plasmid is inserted

into the bacteria by the process of
transformation.

• The recombinant bacteria are sorted

by growing them in the presence of
an antibiotic. The bacteria which
survive are the ones which have taken
up the plasmid.

• They are said to be transformed.

Human Insulin Production by Bacteria
Human Insulin Production by Bacteria
 Human Insulin Production by Bacteria

Mix the recombinant plasmid

with bacteria.
Human Insulin
Production by
Bacteria
Regulation of blood glucose level
 Human Insulin Production by Bacteria

One cell with the

recombinant plasmid

A fermentor used to grow

recombinant bacteria.
 Human Insulin Production by Bacteria

The final steps are to collect the bacteria, break open the cells, and purify the
insulin protein expressed from the recombinant human insulin gene.
Production of Growth Hormone