5. 5
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Production systems: principle
› Genetically modified host cells are cultured on a large scale (in fermentors or bioreactors)
§ Contain gene (transfected, by engineering) that encodes for the therapeutic protein to be produced
§ Controlled conditions (clean rooms), sterile
§ Good Manufacturing Practice standards (GMP)
§ Rapid growth
§ Lysed at the correct time point
§ Isolation and purification of the therapeutic protein
§ Characterisation and quality control (spectroscopy, chromatography, calorimetry)
› Different production methods, different batches
§ Are the identical, equal, comparable?
› Choice of host organism is technological (what can be produced in the system in de desired purity?)
and economical (what are the costs?)
5
GMOs
Rules and regulations!
Upstream processing
Downstream processing
Complexity
7. 7
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Small-molecule drugs versus biologicals
Small-molecule drugs
› Exact synthesis in the laboratory relatively simple
› Structure and purity can be exactly determined
Biologicals
› Biotechnological production is complicated
› Relatively small changes in temperature, electrolytes, pH, etc. during production process can
strongly influence structure and purity of the end product, its biological activity and safety
§ Assurance and control of production process are very important
§ Final control of end product is insufficient
› Analytical methods (purity, content) 10-100x less sensitive than for small-molecule drugs
7
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Heterologous production systems
Prokaryotic, eukaryotic
› Bacteria
› Yeast cells
› Mammalian cells
› Transgenic animals
› Insect cells
› Baculovirus expression system
› Transgenic plants
§ Corn, soya, tobacco
11
The cell factory
Each system has
specific (dis)advantages
In principle, each protein can be produced
in an engineered organism, but not every protein
can be produced in every cell (post-translational
modifications!)
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Bacteria
› Suitable for production of smaller proteins (< ca. 30 kDa)
› Suitable for proteins that do not require post-translational modifications (insulin, growth
hormone)
› Simple to manipulate
§ Easy uptake of DNA from their surroundings (plasmids)
› Rapid and easy growth in big fermentors
§ Very suitable for commercial purposes
› Endotoxins (immunogenic)
› Pyrogens (cause fever)
› No post-translational modifications
› Escherichia coli (E. coli)
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Yeast cells
› Used to circumvent shortcomings of bacterial production systems
§ Also for bigger proteins (> ca. 50 kDa)
§ Post-translational modifications
- Production of authentic bioactive proteins (interferon)
› Genetic manipulation is easy
› Easy to culture on a large scale
› Rapid growth
› Protein secretion into growth medium
› Cheaper than mammalian cell systems
› No endotoxins and pathogens
› Saccharomyces cereviciae
§ Very high degree of glycosylation
› Pichia pastoris
15. 15
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Mammalian cells
› For proteins that are difficult to express in more ‘simple’ systems
› For proteins that require complete authenticity
§ Post-translational glycosylation
› Culture is expensive
› Slower growth
› May contain oncogenes and viral DNA
› Chinese hamster ovary (CHO) cells
› Baby hamster kidney (BHK) cells
› Immortalised human embryonic retinal cells
› Lymphoblastoid cells (interferon production)
› Melanoma cells (plasminogen activators)
› Hybridised tumour cells (monoclonal antibodies)
17. 17
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Transgenic animals
› For proteins that require complex post-translational modification
› Introduction of ‘foreign’ gene into animal embryo with milk-specific promoter
› Protein excreted into milk and isolated therefrom
§ For larger quantities
› It takes time to obtain production system
› Health of the animal negatively influenced
› Pharming, molecular farming
› Mouse, rabbit, pig, sheep, goat, cow
§ Recombinant antithrombin III (from transgenic goats)
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Culture systems
› Cell banks, cryopreservation
› Cells grow in a fermentor (bacteria, yeasts) or a bioreactor (mammalian cells) with liquid medium
§ Free (in suspension) or immobilised (in a matrix)
› Upstream
§ Growth and production in cells, in fermentor or bioreactor
› Downstream
§ Subsequent isolation and purification steps
› Bioreactor systems
§ Stirred tank
§ Airlift
§ Fixed bed
§ Membrane bioreactors
24. 24
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Upstream production processes
› Batch process
§ Bioreactor filled with everything necessary for grow and production
§ No additions during process
§ Waste products remain in bioreactor
§ Product harvested at end of process
§ Maximal cell density and product yield lower than for fed-batch process
› Fed-batch process
§ Substrate (growth limiting nutrients) added to bioreactor during growth and/or production phase
§ High cell density and product yield possible
§ Systems well characterised and frequently used
› Perfusion process
§ Medium constantly refreshed and waste discarded
§ Product harvested during production period
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Growth characteristics
› Lag phase
§ Adaptation to conditions in bioreactor
› Exponential phase (log phase)
§ Doubling time mammalian cells 20-40 hours
§ Temperature, pH, O2, stirring are determinants
› Stationary phase
§ Nutrients become exhausted
§ Formation of toxic waste products
› Death phase
Source: Pharmaceutical Biotechnology, 3th ed.
26. 26
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Culture medium (1)
› Composition can be varied
› Dissolved in purified water and filtered (0.2 or 0.1 μm)
› Serum was necessary; recently also ‘serum-free’ media
§ Growth factors, hormones, transport proteins (cell in), binding proteins
§ Proteins make purification difficult
§ May be contaminated with viruses, bacteria, mycoplasma, fungi
§ May contain prions (transmission spongiform encephalitis)
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Downstream processing
› Downstream processing is costly (50-80% of the total production costs) and laborous
§ Low concentrations of therapeutic protein
§ Product undergoes a number of sequential purification steps (= concentration steps)
› Design of downstream process strongly dependent of product
29. 29
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Isolation and purification
› Removal cells and cell materials from the culture broth
§ Centrifugation, depth filtration
› Removal of impurities and concentration protein
§ ‘Capture steps’
› Product in buffer for formulation (aqueous)
§ Stabilisation
› Sterilisation
§ 0.2 μm filtration
Source: Pharmaceutical Biotechnology, 4th ed.
30. 30
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Purification process
› For the design of a downstream process it is desirable to know the most important
contaminants
§ From the source of the product: bacteria, yeasts, mammalian cells
§ From the upstream process: albumin, serum, product analogues
› Physical properties product versus those of known contaminants
§ Stability against heat, isoelectric point, molecular mass, hydrophobicity, density, specific binding
properties
› Exposure of protein to high physical stress may negatively influence properties and
thus effectiveness
§ Higher temperature, extreme pH
› Production of therapeutic proteins should be save, reproducible, robust and cost-
effective
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Single-use production systems
› 3D single-use bags alternative for stainless steel bioreactors
› Used for products in development and for products on the market
› Cost-effective
§ Everything not related to the production process does not interfere
› More batches per unit of time are possible
§ No cleaning or sterilisation in between
› Flexibility in design possible
§ Changes in design not connected to validation cleaning procedures, etc.
› Speed of implementation and product to market is shortened
› Reduction costs use of water and waste water
§ Cleaning
› Reduction validation costs
§ Cleaning, sterilisation
Considerable storage room necessary
for materials (bags, tubes)
Dependency of one supplier
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Contaminants
› Purity of pharmaceutical preparations for parenteral administration should be ≥99%
› Purification process should result in the desired and well-defined
§ There is always a certain degree of variablity
› All possible contaminants must be removed
§ Originating from host cells, product-related, process-related
§ Purity of a protein product is determined by the entire purification process
› Pharmacopoeial requirements: endotoxins, pyrogens
34. 34
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Overview of possible contaminants
Source: Pharmaceutical Biotechnology, 5th ed.
QC of biologicals is very complex
35. 35
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Biosimilars (1)
› Definition: ‘Biological medicinal product, equivalent to biological medicinal product
that has previously obtained marketing authorisation (‘biological reference medicinal
product’)’
§ Similar / equivalent but not identical
§ EU Guideline on similar biological products (23.10.2014)
› Compare: small-molecule drugs
› Generic drugs, identical to brand-name (proprietary) drugs, can be produced easily and brought to
the market
› Testing of bioequivalence (and proof of)
36. 36
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Biosimilars (2)
› Biologicals are complex products
§ A product of another manufacturer may not be fully identical to that of the original manufacturer
(patent holder, owner of the production process)
- Different cell lines, differences in production process
- Differences in biological effect (action, safety) are possible
§ Impurities (other than in reference product) may be present
- Safety aspects
› Proof of ‘comparability’ required for registration
› If a patient switches from an originator to a biosimilar, good instructions (different
administration aids, storage, preparation) and monitoring (therapeutic and adverse effects)
are necessary
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Viruses (1)
› Parenterals should be free from viruses
§ Risk of viral contamination not always clear
› Potential contaminants of cultured mammalian cells
§ Virus needs living cell to replicate
§ Good screening of cell banks
§ Nutrients (serum!), production cell line, human handling
› Concentration low: difficult to detect
§ Electron microscopy, RT-PCR, in vitro assays
§ Detection methods not available for all viruses
› Inactivation and removal by physical and chemical methods
§ Heat, radiation, sonification, extreme pH, detergents, solvents, disinfectants
- Be careful for damage to the product
§ Nanofiltration (15 nm)
§ Ion exchange chromatography
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Bacteria
› Limit bioburden
§ Sterilise raw materials, utensils, equipment
› Work under strictly aseptic condition
§ Clean rooms, HEPA-filters for air treatment, procedures
› Possible addition of antibiotics to culture medium
§ No beta-lactam antibiotics (hypersensitivity)
§ Preference for antibiotics-free culturing (residues are persistent)
› Filtration over 0.2 μm filter is sufficient
› Pyrogens (endotoxins of gram-negative bacteria)
§ People are sensitive for picogram quantities
§ Make materials pyrogen free before use
§ Removal (ultrafiltration, ion exchange chromatography)
§ Sensitive tests available
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Cellular DNA
› Production of recombinant proteins in mammalian cells may result in the presence of
DNA fragments carrying oncogenes
› Purification procedure should reduce cellular DNA fragments in the material to a safe
level
§ Validation of the methods applied
› WHO, Ph. Eur.: contamination of nucleic acids not more than 100 pg of 10 ng per
dose, dependent of the cell type used for the production
43. 43
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Undesired proteins
› Biotech products always contain small quantities of host-, process- and product-
related proteins
§ Aggregates of the protein, product with heterogenicity in disulfide bridges, N- en C-terminus
variants, variants in glycosylation, desamidated products, parts of the protein (proteolysis)
- E. coli: protein synthesis start with methylmethione; desired: NH2-terminus
› Potential antigens for the patient
§ Immune reaction in case of repeated treatment
§ Discriminate between immunological reaction against the therapeutic protein or against impurities
› Impurities often strongly resemble the desired protein
§ Difficult to remove
§ Sensitive and specific immunoassays necessary for in-process control
44. 44
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Concluding remarks
› Biologicals broaden the pharmacotherapeutic horizon
§ Monoclonal antibodies (mAb) form nowadays a large segment
§ Improved therapies, new possibilities to treat diseases
› Many biotech products are in the pipeline
§ Each year new registrations
§ High costs
› Biosimilars (when patent terminates) may reduce costs
› Pharmaceutical production processes first set up in the 1980s
§ Now: bioreactors of ten thousands of litres, >70% product recovery
45. 45
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Assignments and what to know
› What are the principles of a production system for therapeutic proteins?
› What is a biosimilar and what is the clinical impact of the use of biosimilars?
› Compare the various heterologous production systems for therapeutic proteins
§ Main characteristics, applications
› What is the difference between upstream and downstream processes?
› Why are downstream processes so costly?
› What does the Indonesian Pharmacopoeia contain about therapeutic proteins?
