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Introduction to bioprocessing and pharmabiotech
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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-.
• 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
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
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”
Production Workflow
After discovery comes development, lots and lots of it!
Expression SystemDevelopment
Flasks Clone Evaluation Media Development*
Process Optimization**
• 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
• 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!
Bioprocessing
Use of biological materials to create a material for medical or scientific purposes Upstream and downstream processing
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
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
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
Scale up issues
Operating issues that affect reactor designHeat transferFoamingSterilityOxygen transfer
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)
Types of Bioreactors
Internal Mechanical AgitationMost common and highly
flexibleMechanical 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 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
Packed Bed Reactors
Hollow Fiber Cell Bioreactor
Packed Bed Reactors
Hollow Fiber Cell BioreactorEnhance mass transferProvide 3D space for cells to growUsed with hepatocytes as an artificial
Liver (Bioartificial Liver – BAL)
Packed Bed Reactors
Fluidized Bed BioreactorCells 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
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 SystemsPotential 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
Regulatory Concerns
Living Production System Rather than Synthetic Importance of Cell BankVariability 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
Unique Features of Bioreactor Production
Often Complex MoleculesPost-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 bioreactorsMay use internal coils or external water jacket to control
tempCoils can pose problem for contamination but is more
effective with higher surface for potential heat transferCoils can also adversely affect mixing with additional
unwanted turbulence
Foaming
Foam is a natural byproduct – mostly protein bubbles but some lipidFoam will block and wet filters causing pressure
back-up and contaminationFoam must be controlled by chemical dispersing
agents (antifoams)Maintaining 75% volume capacity of reactor
allows for foam to be retained within the vessel
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
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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