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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 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

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 Chron’s 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



one purification strain Productioin of Orencia and other biologics  Bristol Myer Squibb .000 liter bioreactors Production of Herceptin. FDA approval 2009 $800 million invested Eight 25.000 liter bioreactor.Examples of Manufacturing Plants  Genentech New Vacaville         Started construction in 2004. Avastin and Rituxan Started construction 2007 – validation I 2011 $750 million invested Six 20.

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” .  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.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.

Production Workflow .

but is difficult to execute in cell culture because the parameters interact strongly-requiring a lot of experiments. how many combinations? When to feed them? Inducers. and develop expression system • Screen and select the highest producing and most stable clone • Develop optimal • Optimize growth and conditions for production media biomanufacturing for each cell line process in a “scaledown” version • Scale up process for use in large bioreactors for production of therapeutic  Knowing gene for the protein you want is great. 100s of possibilities!  60 or more nutritional components in culture media. This means models! . but what cell line to use? What clone form that cell line is best.After discovery comes development. isolate gene. lots and lots of it! Expression System Development Clone Evaluation Media Flasks Development* Process Optimization** Scale Up • Identify target. 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.

Bioprocessing  Use of biological materials to create a material for medical or scientific purposes  Upstream and downstream processing .

quality control and quality assurance . isolating and further purification of bioproduct  All sections require validation.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.

metabolites.Some High-Throughput Cell Culture System Requirements  Deliver meaningful scalable data  Sustain cells. agitation  Maintain sterility  Monitor cell density. pH. control temperature. CO2. 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 . pH. DO. O2.

000 liter cultures .Cell Culture Concerns  Mammalian cells  Fragile and shear sensitive – membranes lyse  Suspension culture cells are needed for scale up • Fluidized bed.000 and 25. 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.

Scale up issues Operating issues that affect reactor design Heat transfer Foaming Sterility Oxygen transfer .

protein. carbon substrate  Oxygen  Product and byproduct removal  Clean and Sanitize In Place (CIP/SIP) .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.

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 .

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 .Types of Bioreactors  Airlift Loop  Commonly used  Air is fed through sparger ring in center-bottom of draught tube  Air flows up the tube.

diffusion through packed bed. bed height vs. ceramic. polyester and polyurethane disks are used as a growth surface  Critical issues include: high surface to volume ration. shear and pressure effects  Reservoir of media can be external or internal .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.

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 transfer  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 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 .

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

• Not Ideal • Has Been Done for Repeated Mycoplasma Problems  Inactivation / Disposal.Equipment and Facility  Antibiotics to Prevent Microbial Contamination. Environmental Concerns • What Happens if 10.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 --.000L Catastrophic Failure • Safeguards Available to Prevent Back-flow? • Method to Inactivate Prior to Release to Environment .

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 .

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 .Unique Features of Bioreactor Production  Often Complex Molecules  Post-translational modification may / may not be important to: • • • • • • Biological activity --.

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 .

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 .

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


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 .