Professional Documents
Culture Documents
Introduction to bioprocessing and pharmacutical biotechnology of plant and animal cell culture
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 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
Started construction in 2004, FDA approval 2009 $800 million invested Eight 25,000 liter bioreactors Production of Herceptin, Avastin and Rituxan
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
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
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
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
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 design
Heat transfer Foaming Sterility Oxygen 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
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
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
Continuous
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
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
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
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, 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