Sources of
biopharmaceuticals
DR. SHATAVARI KULSHRESTHA
Protein expression
The central dogma in molecular biology
is DNA→RNA→Protein
Most proteins are modified via an array of post-
translational modifications including protein folding,
formation of disulfide bridges, glycosylation and
acetylation to create functional, stable proteins
Protein expression refers to the second step of this process:
the synthesis of proteins from mRNA and the addition of
post-translational modifications
Source: [Link]
The following factors determine the type of expression
system used to produce recombinant proteins:
time spent in expressing the protein
ease of handling the expression system
amount of protein needed
mass of the protein
type of post-translational modifications, number of
disulfide bonds
destination of the expressed protein
E. coli as a source of recombinant,
therapeutic proteins
They can usually be cultured in large quantities,
inexpensively and in a short time, by standard
methods of fermentation. Production facilities can
be constructed in any world region, and the scale
of production can be varied as required
Heterologous protein production: The expression of
recombinant proteins in cells in which they do not
naturally occur
The first biopharmaceutical produced by genetic engineering to
gain marketing approval (in 1982) was recombinant human
insulin (trade name Humulin), produced in E. coli
An example of a more recently approved biopharmaceutical
which is produced in E. coli is that of Ecokinase, a recombinant
tissue plasminogen activator (tPA), approved for sale in the EU in
1996
Advantages
Its molecular biology is 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% total cellular protein
E. coli cells grow rapidly on relatively simple and
inexpensive media, and the appropriate
fermentation technology is well established.
Drawbacks as a biopharmaceutical
producer
1. heterologous proteins accumulate intracellularly
2. inability to undertake post-translational modifications
(particularly glycosylation) of proteins
3. the presence of lipopolysaccharide on its surface
• homologous proteins are intracellular
• Few are exported to the periplasmic space, or are released as true
extracellular proteins
• Heterologous proteins expressed in E. coli thus invariably accumulate in
the cell cytoplasm.
Intracellular protein production complicates downstream
processing (relative to extracellular production) because:
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
Inclusion bodies formation
Inclusion bodies (refractile bodies) are insoluble aggregates of
partially folded heterologous product. Because of their dense
nature, they are easily observed by dark field microscopy
Why they form?
1. when expressed at high levels, heterologous proteins overload the
normal cellular protein-folding mechanisms
2. Hydrophobic patches, normally hidden from the surrounding
aqueous phase in fully folded proteins, would remain exposed in
the partially folded product
3. Promote aggregate formation via intermolecular hydrophobic
interactions
one processing advantage of formation of inclusion bodiesis
purification by a single centrifugation step
Because of their density, inclusion bodies sediment even more
rapidly than cell debris, Low speed centrifugation thus
facilitates the easy and selective collection of inclusion bodies
directly after cellular homogenization
After collection, inclusion bodies are generally incubated with
strong denaturants, such as detergents, solvents or urea, This
promotes complete solubilization of the inclusion body (i.e.
complete denaturation of the proteins therein)
The denaturant is then removed by techniques such as dialysis
or diafiltration, This facilitates re-folding of the protein, a high
percentage of which will generally fold into its native,
biologically active, conformation.
Reference: [Link]
Reduction of Inclusion body formation
1. Reduction in the temperature of
bacterial growth (from 37℃ to
30℃)
2. Expression of the protein of interest
as a fusion partner with thioredoxin
will eliminate inclusion body
Thioredoxin is a homologous E. coli
protein (expressed at high levels)---
localized at the adhesion zones in E.
coli--- is a heat-stable protein
A plasmid vector has been
engineered which facilitates
expression of a fusion protein,
consisting of thioredoxin linked to
the protein of interest via a short
peptide sequence, recognized by
the protease enterokinase
The fusion protein is
invariably expressed at high
levels, while remaining in
soluble form
Congregation at adhesion
zones facilitates its selective
release into the media by
simple osmotic shock. This
can greatly simplify its
subsequent purification
After its release, the fusion
protein is incubated with
enterokinase, thus releasing
the protein of interest
3. Inclusion bodies can also be reduced by High-level co-
expression of molecular chaperones, along with the protein of
interest
Chaperones are themselves proteins which promote proper and
full folding of other proteins into their biologically active, native
three-dimensional shape
They usually achieve this by transiently binding to the target
protein during the early stages of its folding and guiding further
folding by preventing/correcting the occurrence of improper
hydrophobic associations
The inability of prokaryotes such as E. coli to carry out
post-translational modifications (particularly
glycosylation) can limit their usefulness as production
systems for some therapeutically useful proteins
Many such proteins, when produced naturally in the
body, are glycosylated
However, the lack of the carbohydrate component of
some glycoproteins has little, if any, negative influence
upon their biological activity
The unglycosylated form of interleukin-2, for example,
displays essentially identical biological activity to that
of the native glycosylated molecule. In such cases, E.
coli can serve as a satisfactory production system
The presence on its surface of lipopolysaccharide (LPS)
molecules
The pyrogenic nature of LPS renders essential its
removal from the product stream
Fortunately, several commonly employed downstream
processing procedures achieve such a separation
without any great difficulty
Expression of recombinant proteins in
animal cell culture systems
The major advantage their ability to carry out post-
translational modification of the protein product
Many biopharmaceuticals that are naturally glycosylated
are now produced in animal cell lines
Chinese hamster ovary (CHO) and baby hamster kidney
(BHK) cells have become particularly popular in this regard
In addition to recombinant biopharmaceuticals, animal
cell culture is used to produce various other biologically-
based pharmaceuticals chief amongst these are a variety
of vaccines and hybridoma cell-produced monoclonal
antibodies
Disadvantages
When compared to E. coli, animal cells display
very complex nutritional requirements, grow more
slowly and are far more susceptible to physical
damage
In industrial terms, this translates into increased
production costs
Transgenic animals (a live bioreactor)
The generation of transgenic animals
by directly microinjecting exogenous
DNA into an egg cell, introduce the
desired gene into the pro-nucleus of
the fertilized egg
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
Targeting protein production to mammary gland
Mammary-specific expression can be achieved by fusing
the gene of interest with the promoter-containing
regulatory sequence of a gene coding for a milk-specific
protein
Regulatory sequences of the whey acid protein (WAP), β-
casein and α- and β-lactoglobulin genes has been used
to promote production of various pharmaceutical
proteins in the milk of transgenic animals
the production of human
tissue plasminogen activator
(tPA) in the milk of transgenic
mice. The tPA gene was fused
to the upstream regulatory
sequence of the mouse whey
acidic protein — the most
abundant protein found in
mouse milk
subsequent production of tPA
in the milk of transgenic
goats, again using the murine
WAP gene regulatory
sequence to drive expression
Goats and sheep have proved to be the
most attractive host systems
Attractive characteristics of goats and sheep as host
system
high milk production capacities
ease of handling and breeding, coupled to well-
established animal husbandry techniques
ease of harvesting of crude product — which simply
requires the animal to be milked
pre-availability of commercial milking systems, already
designed with maximum process hygiene in mind
low capital investment (i.e. relatively low-cost
animals replace high-cost traditional fermentation
equipment) and low running costs
high expression levels of proteins are potentially
attained
at expression levels of 1 g/l, one transgenic goat would
produce a similar quantity of product in 1 day as would
likely be recoverable from a 50–100 l bioreactor system
ongoing supply of product is guaranteed (by
breeding)
Milk is biochemically well characterized, and the
physicochemical properties of the major native milk
proteins of various species are well known. This helps
rational development of appropriate downstream
processing protocols
Issues related to use of goats and sheep
as host system
Variability of expression levels (high1 g/l to less 1.0 mg/l)
characterization of the exact nature of the post-
translational modifications the mammary system is
capable of undertaking, e.g. the carbohydrate
composition of tPA produced in this system differs from the
recombinant enzyme produced in murine cell culture
systems
significant time lag between the generation of a
transgenic embryo and commencement of routine
product manufacture
Gestation period - 1 month for rabbits to 9 months for
cows
Reach sexual maturity before breeding- 5 months for
rabbits, 15 months for cows
Breed successfully and bring their offspring
Overall time lag-almost 3 years in the case of cows
or 7 months in the case of rabbits
Original transgenic embryo turns out to be male
Use of microinjection technique to
introduce the desired gene into
the pro-nucleus of the fertilized
egg
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
Approaches to overcome
such issues
Replication-defective retroviral vectors are
available which will more consistently
a) deliver a chosen gene into cells
b) ensure chromosomal integration of the gene
Application of nuclear transfer technology
Application of nuclear transfer technology
Nuclear transfer entails substituting the genetic information
present in an unfertilized egg with donor genetic information
‘Dolly’ the sheep, produced by substituting the nucleus of a
sheep egg with a nucleus obtained from an adult sheep cell
(genetically, therefore, Dolly was a clone of the original
‘donor’ sheep)
An extension of this technology applicable to
biopharmaceutical manufacture entails using a donor cell
nucleus previously genetically manipulated so as to harbour a
gene coding for the biopharmaceutical of choice
The technical viability of this approach was
proved in the late 1990s upon the birth of two
transgenic sheep, ‘Polly’ and ‘Molly’
The donor nucleus used to generate these sheep
harboured an inserted (human) blood factor IX
gene under the control of a milk protein promoter
Both now produce significant quantities of human
factor IX in their milk.
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
Problems related to use of tissue fluid of transgenic animals
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
Therapeutic proteins have also been successfully
expressed in the urine and seminal fluid of various
transgenic animals
Issues of sample collection, volume of collected fluid and
the appropriateness of these systems renders unlikely their
industrial-scale adoption
targeted production of recombinant proteins in the egg
white of transgenic birds
Targeted production is achieved by choice of an
appropriate egg white protein promoter sequence. Large
quantities of recombinant product can potentially
accumulate in the egg, which can then be collected
and processed with relative ease.
Transgenic Plants
Agrobacterium-based vector-mediated gene transfer
Agrobacterium tumefaciens and A. rhizogenes are
soil-based plant pathogens
Upon infection, a portion of Agrobacterium Ti
plasmid is translocated to the plant cell and is
integrated into the plant cell genome
Depending upon the specific promoters used,
expression can be achieved uniformly throughout
the whole plant or can be limited, e.g. to expression
in plant seeds
Virulence Region: The virulence region codes for virulence genes
that are responsible for the transfer of T-DNA to the plant cells and
also recruiting various effector proteins for infecting the plant cells
T-DNA: The T-DNA region is the crucial region that gets transferred
to the plant cell for infection. It is approximately 15-20 kbp in length
and is transferred to the plant cell via means of genetic
recombination
Opine Catabolism: The opine catabolism region is the region from
where the bacteria sources its nutrients for the whole process.
Opines are derivatives of amino acid or sugar phosphates that can
be catabolized to use in the form of nutrients. The types of opines
found in Ti-plasmid are nopaline and octopine types
Origin of Replication: The origin of replication is the region where
replication of the plasmid is initiated
Advantages
cost of plant cultivation is low
harvest equipment/methodologies are
inexpensive and well established
ease of scale-up
proteins expressed in seeds are generally stable
in the seed for prolonged periods of time (often
years)
plant-based systems are free of human
pathogens (e.g. HIV)
Disadvantages
variable/low expression levels sometimes
achieved
potential occurrence of post-translational gene
silencing (a sequence-specific mRNA
degradation mechanism)
glycosylation patterns achieved differ
significantly from native human protein
glycosylation patterns
seasonal/geographical nature of plant growth
edible vaccines
oral vaccines in edible plants or fruit, such as
tomatoes and bananas
Animal studies have clearly shown that ingestion
of transgenic plant tissue expressing recombinant
sub-unit vaccines (induces the production of
antigen-specific antibody responses, not only in
mucosal secretions but also in the serum
The approach is elegant in that direct
consumption of the plant material provides an
inexpensive, efficient and technically
straightforward mode of large-scale vaccine
delivery, particularly in poorer world regions
Problems
the immunogenicity of orally administered
vaccines can vary widely
the stability of antigens in the digestive tract varies
widely
the genetics of many potential systems remain
poorly characterized, leading to inefficient
transformation systems and low expression levels
