Host cells for the production of biopharmaceuticals
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Transcript of Host cells for the production of biopharmaceuticals
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.
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 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
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
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
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
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
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,
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 pro-
teases Degradation of heterologous proteins Use of mutant strain with reduced level of pro-teases
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
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 surro-
gate mother Transgenic animal harbors a copy of the transferred
DNA
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
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
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
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
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
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
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
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
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
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
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 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
Plant cell system