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Plant and Mammalian Tissue Culture


... 11_Default Design Plant and Mammalian Tissue Culture Industrial Application of Cell Culture Technology Examples of Bioprocess Slide 4 Slide 5 Examples ... – PowerPoint PPT presentation

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Title: Plant and Mammalian Tissue Culture

Plant and Mammalian Tissue Culture
  • Introduction to bioprocessing and pharmacutical
    biotechnology of plant and animal cell culture

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
  • 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
  • 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

Examples of Bioprocess
  • Cell Culture and Fermentation Process
  • Therapeutic Antibody Products
  • Treat lymphoma, inhibit transplant rejection,
    anti-metastatic breast cancer, rheumatoid
  • Growth Factors (HGH, PDGR, Insulin)
  • Veterinarian Vaccines Diarrhea, parvovirus,
  • Many metabolites alcohols, citric acid, amino
  • Antibiotics
  • Blockbuster Proteins
  • Remicade monoclonal antibody against TNF-a.
  • Used to treat Rheumatoid arthritis and Chrons
  • 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
  • Productioin of Orencia and other biologics

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
  • 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

Production Workflow
After discovery comes development, lots and lots
of it!
Expression System Development
  • 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!

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

  • 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

Some High-Throughput Cell Culture System
  • Deliver meaningful scalable data
  • Sustain cells, control temperature, O2, CO2, pH,
  • 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
  • Integration with tools for designing experiments
    and handling data

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
  • 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

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

  • 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)

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

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

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

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
  • 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

Packed Bed Reactors
  • Hollow Fiber Cell Bioreactor

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)

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
  • Works best with high viscosity or gaseous
    substrates or products are used

Bioreactor Operating Modes
  • Batch Inoculate culture and allow to cultivate
    without changing media
  • Simple and allows for reduced risk of
  • 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
  • Must know very specific details on cell cultured
  • Continuous

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

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
  • 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

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
  • Impurity Profile
  • Adventitious Agents, a Host for Propagation
  • Endogenous
  • Adventitious
  • Both Theoretical and Demonstrated Concerns

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

How to get the cells?
  • Cell Isolation/Harvesting

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

  • 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

  • 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 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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