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Host cells for the production of biopharmaceuticals  Many of biopharmaceuticals, especially proteins : produced by recombinant DNA technology using various.

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Presentation on theme: "Host cells for the production of biopharmaceuticals  Many of biopharmaceuticals, especially proteins : produced by recombinant DNA technology using various."— Presentation transcript:

1 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.

2  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

3 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

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5  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 Drawbacks

6  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 o C) - Expression with fusion partner : GST, Thioredoxin, GFP, - High level co-expression of molecular chaperones

7  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

8 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

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10  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,

11 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

12  Disadvantage  Produces significant quantities of extracellular proteases  Degradation of heterologous proteins  Use of mutant strain with reduced level of proteases

13 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

14 CHO cells

15 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

16  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

17 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

18  Goats and sheep : Most attractive host system  High milk production capacities : 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

19  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

20  Inefficient and time-consuming in the use of the micro- injection 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

21  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

22 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

23  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

24  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

25 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

26  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

27 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

28 Plant cell system


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