Plant and Mammalian Tissue Culture

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Plant and Mammalian Tissue Culture

• Introduction to bioprocessing and pharmacutical
  biotechnology of plant and animal cell culture

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Industrial Application of Cell Culture Technology

• Large Scale-Up of cell culture
• Bioprocessing
• Pharmacutical Biotechnology
• Industrial Production
• Production of cell material, protein,
  phytochemicals and other molecules from cell
  culture
• Market 1 billion upstream processing industry
  with 5,800 employees
• Follow-on biologic or biosimilar market is
  going to grow
• Refer to products marketed after expiration of
  patents
• Product can only be made that is similar not
  identical due to complexity of biologics
• Investment and market is driven by a number of
  successful therapeutic proteins going off-patent
  between 2013 and 2017
• European and Asian guidelines and competition is
  an unknown impact

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Examples of Bioprocess

• Cell Culture and Fermentation Process
• Therapeutic Antibody Products
• Treat lymphoma, inhibit transplant rejection,
  anti-metastatic breast cancer, rheumatoid
  arthritis
• Growth Factors (HGH, PDGR, Insulin)
• Veterinarian Vaccines Diarrhea, parvovirus,
  distemper
• Many metabolites alcohols, citric acid, amino
  acids
• Antibiotics
• Blockbuster Proteins
• Remicade monoclonal antibody against TNF-a.
• Used to treat Rheumatoid arthritis and Chrons
  disease
• License approved August 1998
• Possible mechanism of action is inhibiting
  cytokine receptor activation
• 900 for a 100 mg dose! Responsible for 2.1
  billion in sales 2009
• Produced in 1,000 liter production reactors

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Examples of Manufacturing Plants

• Genentech New Vacaville
• Started construction in 2004, FDA approval 2009
• 800 million invested
• Eight 25,000 liter bioreactors
• Production of Herceptin, Avastin and Rituxan
• Bristol Myer Squibb
• Started construction 2007 validation I 2011
• 750 million invested
• Six 20,000 liter bioreactor, one purification
  strain
• Productioin of Orencia and other biologics

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Non-Mammalian Examples

• Insect Cell Culture Baculovirus
• 25 compounds in clinical trials
• Possible combitorial proteomic approach could
  lead to more effective protein therapeutics
• Yeast Pichia expression systems.
• Need to humanize the glycoprotein expression
• Immune system keys in on different sugared
  proteins
• Glycofi(Merk) is creating a multistep genetic
  engineering process to eliminate non-human
  glycosylation enzymes
• Working to batch processing of uniformly
  glycosylated products
• Plant alfalfa, barley, corn, rice and duckweek
  have been given field trials
• Edible vaccines and plant-made pharmacuticals
• No current PMP product on market first will
  likely be animal health vaccine Concert

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Production Workflow
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After discovery comes development, lots and lots
of it!
Expression SystemDevelopment
Flasks

• Screen and select the highest producing and most
  stable clone

• Develop optimal growth and production media for
  each cell line

• Optimize conditions for biomanufacturing process
  in a scale-down version

• Scale up process for use in large bioreactors for
  production of therapeutic

• Identify target, isolate gene, and develop
  expression system

• Knowing gene for the protein you want is great,
  but what cell line to use? What clone form that
  cell line is best. 100s of possibilities!
• 60 or more nutritional components in culture
  media, how many combinations? When to feed them?
  Inducers, promoters?
• What temperature? What oxygen level? CO2? pH any
  shifts? When to harvest?
• A strategy of multi-factorial design is the
  natural way to attack this type of problem, but
  is difficult to execute in cell culture because
  the parameters interact strongly-requiring a lot
  of experiments. This means models!

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Bioprocessing

• Use of biological materials to create a material
  for medical or scientific purposes
• Upstream and downstream processing

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Bioprocessing

• Use of biological materials to create a material
  for medical or scientific purposes
• Upstream processing from gene/cell to
  harvesting off cell culture media or cell biomass
• Downstream processing lysing, isolating and
  further purification of bioproduct
• All sections require validation, quality control
  and quality assurance

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Some High-Throughput Cell Culture System
Requirements

• Deliver meaningful scalable data
• Sustain cells, control temperature, O2, CO2, pH,
  agitation
• Maintain sterility
• Monitor cell density, pH, DO, metabolites,
  product titer
• Operate with accuracy and precision and provide
  control of process parameters comparable to bench
  top bioreactor systems
• Automatic operation with minimal operator
  supervision
• Integration with tools for designing experiments
  and handling data

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Cell Culture Concerns

• Mammalian cells
• Fragile and shear sensitive membranes lyse
• Suspension culture cells are needed for scale up
• Fluidized bed, hollow-fiber and packed-bed do
  provide some scale up potential
• Slow growing compared to bacteria or yeas (24
  hour doubling time)
• Low production titer
• Extended batch times facilitate potential
  contamination
• Virus removal and or inactivation is required for
  further processing
• Must start with smaller cultures then move up to
  large 10,000 and 25,000 liter cultures

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Scale up issues

• Operating issues that affect reactor design
• Heat transfer
• Foaming
• Sterility
• Oxygen transfer

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Bioreactor

• A bioreactor is a system in which a reaction or
  biological conversation is effected
• Different from fermentor
• Enzymes to produce new product (biofuels)
• Microorganisms (beer fermentor)
• Animal and Plant Cells
• Basic Design of Reactor
• Control temperature
• Maintain and analyze pH
• Measure viability of cells
• Culture composition
• Sugar, protein, carbon substrate
• Oxygen
• Product and byproduct removal
• Clean and Sanitize In Place (CIP/SIP)

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Types of Bioreactors

• Internal Mechanical Agitation
• Most common and highly flexible
• Mechanical agitation paddles
• Disperses gas bubbles
• Increases times of bubbles (oxygen transfer)

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Types of Bioreactors

• Internal Mechanical Agitation
• Bubble-Column Reactor
• Disperse gas through reactor with plates to
  enhance dispersion and mixing
• Low-Sheer but air / liquid interface produces
  denaturation and cell lysis
• Energy efficient low power required

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Types of Bioreactors

• Airlift Loop
• Commonly used
• Air is fed through sparger ring in center-bottom
  of draught tube
• Air flows up the tube, forming bubbles and
  exhausts at top
• Degassed liquid (now more dense) flows down
  creating a circulation flow
• Larger fermentors and reactors use this style to
  meet oxygen and cooling needs

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Packed Bed Reactors

• Used for monolayer (adherent) cell cultures
• Initially used glass beads to grow cells then
  flow media through beads to change media and
  oxygen
• Glass is still used but also macroporus glass
  beads, ceramic, polyester and polyurethane disks
  are used as a growth surface
• Critical issues include high surface to volume
  ration, diffusion through packed bed, bed height
  vs. shear and pressure effects
• Reservoir of media can be external or internal

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Packed Bed Reactors

• Hollow Fiber Cell Bioreactor

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Packed Bed Reactors

• Hollow Fiber Cell Bioreactor
• Enhance mass transfer
• Provide 3D space for cells to grow
• Used with hepatocytes as an artificial
• Liver (Bioartificial Liver BAL)

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Packed Bed Reactors

• Fluidized Bed Bioreactor
• Cells are immobized cultured, on small
  particles which move with the fluid
• Large numbers of particles create a large surface
  area for high rate of heat, nutrient and oxygen
  transfer
• Works best with high viscosity or gaseous
  substrates or products are used

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Bioreactor Operating Modes

• Batch Inoculate culture and allow to cultivate
  without changing media
• Simple and allows for reduced risk of
  contamination
• Lower capital investment and greater flexibility
  with media adjustments
• Slower must prepare one batch at a time
• Small amounts of product are produced
• Fed Batch allows cells to grow to high density.
  
• Use concentrated feedstock
• Add in growth limiting nutrient/substrate not a
  change in media
• Allows for high cell density with higher working
  time
• Must know very specific details on cell cultured
  used
• Continuous

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Bioreactor Operating Modes

• Batch Inoculate culture and allow to cultivate
  without changing media
• Fed Batch allows cells to grow to high density.
  
• Continuous- perpetual feeding process
• Culture medium is fed to cells constantly
• May be automated and thus less expensive
• Less non-productive time spent emptying, filling
  and sterilizing reactor
• Higher risk of contamination
• Greater processing costs more media
• Used in high volume production

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Regulatory Concerns

• Mammalian Production Systems
• Potential for Adventitious Virus
• Indicate Breach in cGMP Practices Even if Virus
  Has No Pathogenic Effect in Humans
• Likely Source is Raw Material
• Potentially Costly Impact --- Equipment and
  Facility
• Antibiotics to Prevent Microbial Contamination,
• Not Ideal
• Has Been Done for Repeated Mycoplasma Problems
• Inactivation / Disposal, Environmental Concerns
• What Happens if 10,000L Catastrophic Failure
• Safeguards Available to Prevent Back-flow?
• Method to Inactivate Prior to Release to
  Environment

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Regulatory Concerns

• Living Production System Rather than Synthetic
• Importance of Cell Bank
• Variability of Living Organisms
• Complex Physiology
• Balancing Growth vs Production
• Spent Culture Medium is Full of Enzymatic
  Activity
• Impurity Profile
• Adventitious Agents, a Host for Propagation
• Endogenous
• Adventitious
• Both Theoretical and Demonstrated Concerns

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Unique Features of Bioreactor Production

• Often Complex Molecules
• Post-translational modification may / may not be
  important to
• Biological activity --- increase or decrease
• Purity Profile
• Serum Half Life
• Immunogenic Nature of the Molecule(s)
• Stability
• Subsequent Chemical Modification
• Family of molecules rather than single entity
• Differential Toxicity or Clinically Relevant
  Activity Differences

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How to get the cells?

• Cell Isolation/Harvesting

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Heat Transfer

• Large masses of cells actively respiration will
  produce heat
• Control of heat by transfer is one of the two
  main limitations on size of bioreactors
• May use internal coils or external water jacket
  to control temp
• Coils can pose problem for contamination but is
  more effective with higher surface for potential
  heat transfer
• Coils can also adversely affect mixing with
  additional unwanted turbulence

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Foaming

• Foam is a natural byproduct mostly protein
  bubbles but some lipid
• Foam will block and wet filters causing pressure
  back-up and contamination
• Foam must be controlled by chemical dispersing
  agents (antifoams)
• Maintaining 75 volume capacity of reactor allows
  for foam to be retained within the vessel

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Sterility

• Sterilization in place (SIP) cleaning of reactor
  and bed without dismantling reactor or feed tubes
• Pressurized steam is used for in-place
  sterilization of probes, valves and seals
• All crooks, crevices and surfaces are potential
  contaminants and must be sterilized
• Sterilization must be verified and validated

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Cleaning

• Cleaning in place (CIP) is performed after each
  run and before a new run is initiated
• Highly alkaline detergents, bases and acids are
  used with copious amounts of water
• Cleaning solutions are often plumbed into system
  for automation

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