Host cells for the production of biopharmaceuticals

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(Saccharomycescerevisiae) , 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

5. 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

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 oC) - 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, Pichiapastoris • 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

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 • Aspergillusniger • 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  Easyrecovery 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-lactoglobulingenes

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 : 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

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 - Agarobacteriumtumefaciens 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 Bombyxmorinuclear polyhedrovirus(BmNPV) in conjunction with cultured silkworm cells • Virus Autographacalifornicanuclear 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, VibragenOwega - Use of silkworm for the production of feline interferon ω

28. Plant cell system