Transcript Host cells for the production of biopharmaceuticals

Host cells for the production of biopharmaceuticals
 Many of biopharmaceuticals, especially proteins :
produced by recombinant DNA technology using various
expression systems
 Expression systems : E. coli, Bacillus, Yeast(Saccharomyces
cerevisiae) , Fungi(Aspergillus), animal cells (CHO), plant
cells, insect cells
 E. coli and mammalian cells : most widely used
 Typical biopharmaceuticals produced by recombinant DNA
technology : Cytokines, therapeutic proteins, etc.
 Use of appropriate expression system for specific
biopharmaceuticals :
- Each expression system displays its own unique set of
advantages and disadvantages
- Expression level (soluble form), Glycosylation, Easy
purification, cultivation process, cell density
 Cost effectiveness feasibility
 Production system for therapeutic proteins
- Cultured in large quantity, inexpensively and in a
short time by standard cultivation methods
Eschericia coil
 Most common microbial species to produce
heterologous proteins of therapeutic interest
- Heterologous protein : protein that does not occur in host cells
ex) The first therapeutic protein produced by E. coli : Human insulin
(Humulin) in 1982, tPA (tissue plasminogen activator) in 1996
 Major advantages of E. coli
- Served as the model system for prokaryotic genetics
 Its molecular biology is well characterized
- High level expression of heterologous proteins :
- High expression promoters (~30 % of total cellular protein
- Easy and simple process : Rapid growth, simple and
inexpensive media, appropriate fermentation technology,
large scale cultivation
Drawbacks
 Intracellular accumulation of proteins in the cytoplasm
 Complicate downstream processing compared to
extracellular production
 Additional primary processing steps : cellular
homogenization, subsequent removal of cell debris by
filtration or centrifugation
 Extensive purification steps to separate the protein of
interest
 Inclusion body
- Insoluble aggregates of partially folded protein
- Formation via intermolecular hydrophobic interactions
 High level expression of heterologous proteins overloads
the normal cellular protein-folding mechanisms
Hydrophobic patch is exposed, promoting aggregate
formation via intermolecular hydrophobic interactions
 Inclusion body displays one processing advantage
- Easy and simple isolation by single step centrifugation
- Denaturation using 6 M urea
- Refolding via dialysis or diafiltration
 Prevention of inclusion body formation
- Growth at lower temperature (20 oC)
- Expression with fusion partner : GST, Thioredoxin, GFP,
- High level co-expression of molecular chaperones
 Inability to undertake post-translational modification,
especially glycosylation : limitation to the production of
glycoproteins
Cf) Unglycosylated form of glycoprotein : little effect on
the biological activity (ex : IL-2  E. coli can be used as a
good host system)
 The presence of lipopolysaccharide (LPS) on its surface :
pyrogenic nature
 More complicated purification procedure
Yeast
 Saccharomyces cerevisiae, Pichia pastoris
 Major advantages
 Their molecular biology is well characterized, facilitating
their genetic manipulation
 Regarded as GRAS-listed organisms (generally regarded as
safe) with a long history of industrial applications (e.g.,
brewing and baking)
 Fast growth in relatively inexpensive media, outer cell wall
protects them from physical damage
 Suitable industrial scale fermentation equipment/technology
is already available
 Post-translational modifications of proteins, especially
glycosylation : Highly mannosylated form
 Drawbacks
 Glycosylation pattern usually differs from the pattern
observed in the native glycoprotein : highly mannosylation
pattern
Trigger the rapid clearance from the blood stream
 Low expression level of heterologous proteins : < 5 %
 Major therapeutic proteins produced in yeast for general
medical use:
ex) Insulin, colony stimulating factor(GM-CSF) for bone
marrow transplantation, Hirudin for anticoagulation,
Fungal production systems
 Aspergillus niger
 Mainly used for production of industrial enzymes :
a-amylase, glucoamylase, cellulase, lipase, protease etc..
 Advantages
 High level expression of heterologous proteins (~ 30 g/L)
 Secretion of proteins into extracellular media
 easy and simple separation procedure
 Post-translational modifications : glycosylation
- Different glycosylation pattern compared to that in
human
 Disadvantage
 Produces significant quantities of extracellular proteases
 Degradation of heterologous proteins
 Use of mutant strain with reduced level of proteases
Animal cells
 Major advantage : Suitable for production of glycoprotein
especially glycosylation
 Chinese Hamster Ovary (CHO) and Baby Hamster Kidney
(BHK) cells
 Typical proteins produced in animal cells : EPO, tPA,
Interferons, Immunoglobulin antibodies, Blood factors etc.
 Drawbacks
 Very complex nutritional requirements : growth factors
 expensive  complicate the purification procedure
 Slow growth rate: long cultivation time
 Far more susceptible to physical damage
 Increased production cost
CHO cells
Transgenic animals
 Transgenic animals : live bioreactor
 Generation of transgenic animals :
 Direct microinjection of exogenous DNA into an egg cell
 Stable integration of the target DNA into the genetic
complement of the cell
 After fertilization, the ova are implanted into a surrogate
mother
 Transgenic animal harbors a copy of the transferred DNA
 In order for the transgenic animal system to be practically
useful, the target protein must be easily and simply
separable from the animal without any injury
: Simple way : to produce a target protein in a mammary
gland
 Easy recovery of a target protein from milk
 Mammary-specific expression : Fusion of a target gene with
the promoter-containing regulatory sequence of a gene
coding for a milk-specific protein
ex) Regulatory sequences of the whey acid protein (WAP,
the most abundant protein in mouse milk), β-casein,
α- and β-lactoglobulin genes
ex) Production of tPA in the milk of transgenic mice
- Fusion of the tPA gene to the upstream regulatory
sequence of the mouse whey acid protein
 More practical approach : production of tPA in the milk of
transgenic goats
 Production of proteins in the milk of transgenic animals :
ex) tPA (goat) : 6 g/L,
Growth hormone (Rabbit) : 50 mg/L
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Goats and sheep : Most attractive host system
High milk production capacities : 700-800 L/year for goat
Ease of handling and breeding
Ease of harvesting of crude product : simply requires the
animal to be milked
 Pre-availability of commercial milking systems with
maximum process hygiene
 Low capital investment : relatively low-cost animals replace
high-cost traditional cultivation equipment, and low running
costs
 High expression levels of proteins are potentially attained :
> 1 g protein/L milk
 On-going supply of product is guaranteed by breeding
 Ease downstream processing due to well-characterized
properties of major native milk proteins
 Issues to be addressed for practical use
 Variability of expression levels (1 mg /L ~ 1 g/L)
 Different post-translational modifications, especially
glycosylation, from that in human
 Significant time lag between the generation of a transgenic
embryo and commencement of routine product recovery:
- Gestation period ranging from 1 month to 9 months
- Requires successful breeding before beginning to lactate
- Overall time lag : 3 years in the case of cows, 7 months in
the case of rabbits
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Inefficient and time-consuming in the use of the microinjection technique to introduce the desired gene into the
egg
 Other approaches than microinjection
 Use of replication-defective retroviral vectors : consistent
delivery of a gene into cells and chromosomal integration
 Use of nuclear transfer technology
 Manipulation of donor cell nucleus so as to harbor a
gene coding for a target protein
 Substitution of genetic information in un unfertilized
egg with donor genetic information
 Transgenic sheep, Polly and Molly, producing human
blood factor IX, in 1990s
 No therapeutic proteins produced in the milk of transgenic
animals had been approved for general medical use
 Alternative approach : production of therapeutic proteins
in the blood of transgenic pigs and rabbits
 Drawbacks
- Relatively low volumes of blood can be harvested
- Complicate downstream processing because of
complex serum
- Low stability of proteins in serum
Transgenic plants
 Expression of heterologous proteins in plant :
 Introduction of foreign genes into the plant species :
Agrobacterium-based vector-mediated gene transfer
- Agarobacterium tumefaciens
A. rhizogenes : soil-based plant pathogens
 When infected, a proportion of Agarobacterium Ti
plasmid is trans-located to the plant cell and integrated
into the plant cell genome
 Expression of therapeutic proteins in plant tissue :
Table 3.16
 Potentially attractive recombinant protein producer
 Low cost of plant cultivation
 Harvest equipment/methodologies are inexpensive
and well established
 Ease of scale-up
 Proteins expressed in seeds are generally stable
 Plant-based systems are free of human pathogens(eg., HIV)
 Disadvantages
 Variable/low expression levels of proteins
 Potential occurrence of post-translational gene silencing
(a sequence specific mRNA degradation mechanism)
 Different glycosylation pattern from that in human
 Seasonal/geographical nature of plant growth
 Most likely focus of future transgenic plants :
 Production of oral vaccines in edible plants or fruit, such
as tomatoes and bananas
- Ingestion of transgenic plant tissue expressing
recombinant sub-unit vaccines induces the production of
antigen-specific antibody responses
 Direct consumption of plant material provides an
inexpensive, efficient and technically straightforward
mode of large-scale vaccine delivery
 Several hurdles
 Immunogenicity of orally administered vaccines vary widely
 Stability of antigens in the digestive tract varies widely
 Genetics of many potential systems remain poorly characterized
 Inefficient transformation systems and low expression levels
Insect cell-based system
 Laboratory scale production of proteins
 Infection of cultured insect cells with an engineered
baculovirus (a viral family that naturally infects insects)
carrying the gene coding for a target protein
 Most commonly used systems
 Silkworm virus Bombyx mori nuclear
polyhedrovirus(BmNPV) in conjunction with cultured
silkworm cells
 Virus Autographa californica nuclear polyhedrovirus(AcNPV)
in conjunction with cultured armyworm cells
 Advantages
 High level intracellular protein expression
- Use of strong promoter derived from the viral
polyhedrin : ~30-50 % of total intracellular protein
- Cultivation at high growth rate and less expensive media
than animal cell lines
- No infection of human pathogens, e.g., HIV
 Drawbacks
- Low level of extracellular secreted target protein
-Glycosylation patterns : incomplete and different
 No therapeutic protein approved for human use
Alternative insect cell-based system
 Use of live insects
- Live caterpillars or silkworms
 Infection with the engineered baculovirus vector
Ex) Veterinary pharmaceutical company, Vibragen Owega
- Use of silkworm for the production of feline
interferon ω
Plant cell system