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 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

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
Started construction 2007 validation I 2011 $750 million invested Six 20,000 liter bioreactor, one purification strain Productioin of Orencia and other biologics

Bristol Myer Squibb

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 System Development Clone Evaluation Media Flasks Development* Process Optimization** Scale Up

Identify target, isolate gene, 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, 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 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

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 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 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

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 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 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

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

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