host cells for the production of biopharmaceuticals
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Host cells for the production of biopharmaceuticals . Many of biopharmaceuticals, especially proteins : produced by recombinant DNA technology using various expression systems - PowerPoint PPT PresentationTRANSCRIPT
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Host cells for the production of biopharma-ceuticals
Many of biopharmaceuticals, especially proteins : produced by recombinant DNA technology using var-ious expression systems
Expression systems : E. coli, Bacillus, Yeast(Saccharomyces cerevisiae) , Fungi(Aspergillus), animal cells (CHO), plant cells, in-sect cells
E. coli and mammalian cells : most widely used
Typical biopharmaceuticals produced by recombinant DNA technology : Cytokines, therapeutic proteins, etc.
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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
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Eschericia coil
Most common microbial species to produce het-erologous proteins of therapeutic interest
- Heterologous protein : protein that does not occur in host cells ex) The first therapeutic protein produced by E. coli : Human in-
sulin (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 in-
expensive media, appropriate fermentation technol-ogy, large scale cultivation
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Intracellular accumulation of proteins in the cy-toplasm
Complicate downstream processing compared to ex-tracellular production
Additional primary processing steps : cellular ho-mogenization, 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 interac-
tions
Draw-backs
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High level expression of heterologous proteins over-loads the normal cellular protein-folding mecha-nisms
Hydrophobic patch is exposed, promoting aggre-gate formation via intermolecular hydrophobic interac-tions
Inclusion body displays one processing advantage - Easy and simple isolation by single step centrifu-gation - 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
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Inability to undertake post-translational modifica-tion, especially glycosylation : limitation to the pro-duction 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 sur-
face : pyrogenic nature More complicated purification procedure
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Yeast Saccharomyces cerevisiae, Pichia pastoris
Major advantages Their molecular biology is well characterized, facilitat-
ing their genetic manipulation Regarded as GRAS-listed organisms (generally re-
garded as safe) with a long history of industrial appli-cations (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
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Drawbacks Glycosylation pattern usually differs from the pattern
observed in the native glycoprotein : highly mannosy-lation pattern
Trigger the rapid clearance from the blood stream
Low expression level of heterologous proteins : < 5 %
Major therapeutic proteins produced in yeast for gen-eral medical use:
ex) Insulin, colony stimulating factor(GM-CSF) for bone marrow transplantation, Hirudin for anticoagu-lation,
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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
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Disadvantage Produces significant quantities of extracellular pro-
teases Degradation of heterologous proteins Use of mutant strain with reduced level of pro-teases
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Animal cells Major advantage : Suitable for production of glycopro-
tein 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 fac-
tors expensive complicate the purification procedure Slow growth rate: long cultivation time Far more susceptible to physical damage Increased production cost
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CHO cells
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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 surro-
gate mother Transgenic animal harbors a copy of the transferred
DNA
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In order for the transgenic animal system to be practi-cally 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 mam-mary 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
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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 ani-mals : 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 at-tained :
> 1 g protein/L milk
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On-going supply of product is guaranteed by breeding Ease downstream processing due to well-character-
ized 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 mi-cro-injection technique to introduce the desired gene into the egg
Other approaches than microinjection Use of replication-defective retroviral vectors : consis-
tent 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
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No therapeutic proteins produced in the milk of transgenic animals had been approved for general medical use
Alternative approach : production of therapeutic pro-teins 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
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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 inte-
grated into the plant cell genome
Expression of therapeutic proteins in plant tissue : Table 3.16
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Potentially attractive recombinant protein pro-ducer
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 silenc-
ing (a sequence specific mRNA degradation mechanism) Different glycosylation pattern from that in human Seasonal/geographical nature of plant growth
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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 re-
combinant sub-unit vaccines induces the production of antigen-specific antibody responses
Direct consumption of plant material provides an inexpensive, efficient and technically straightfor-ward 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 character-
ized Inefficient transformation systems and low expression levels
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Insect cell-based system Laboratory scale production of proteins Infection of cultured insect cells with an engineered
baculovirus (a viral family that naturally infects in-sects) 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
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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
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Alternative insect cell-based sys-tem
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
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Plant cell system