design and specifications of bioreactors
TRANSCRIPT
Bioreactor Specification and DesignBy Gary Wirt
Jacobs
Topics to Discuss
• Introduction: History and definition
• Process Functions
– Aeration
– Agitation
– Temperature Control
– PH control
• Hardware
• Materials of Construction
• Disposables
Some Basic Definitions
• Bioreactor: A system used for the growth and
maintenance of a population of mammalian or insect cells
• Fermentor: A system used for the growth and
maintenance of a population of bacterial or fungal cells
• Stirred Tank: Mechanically agitated pressure vessel
History
• 1954 Cell culturing techniques developed
• 1986
– Ortho Biotech's Orthoclone OKT3
• kidney transplant rejection
• first monoclonal antibody treatment
– Biogen's Intron A and Genentech's Roferon AF
• First biotech-derived interferon drugs for the treatment of cancer
• Kaposi's sarcoma, a complication of AIDS.
– Chiron's Recombivax HB
• First genetically engineered human vaccine
• Approved for the prevention of hepatitis B.
Source: Biotechnology Institute Web Site www.biotechinstitute.org
Differences between
mammalian cells and bacteria• Mammalian Cells
– Fragile & shear sensitive cell membrane
– Slow growing (24 hour doubling time)
– Low product titer
– Low oxygen demand
– Extended batch times
– Virus removal / inactivation required
• Bacteria
– Robust with strong cell wall
– Fast growing (20 minute doubling time)
– High product titer
– High oxygen demand
– Fast batch cycle time
– No viral threat
More differences between
mammalian cells and bacteria• Mammalian Cells
– Products are usually extracellular
– Can produce very large molecules (Enbrel has 934 amino acids and MW = 150 kDA)
– Can produce glycosylated proteins
– Can produce fully humanized antibodies
• Bacteria
– Products are often intracellular
– Limited in ability to produce really large molecules (upper limit is probably calcitonin: 45 kDa. Insulin is 6 kDa)
– Can not produce glycosylated proteins.
– Can not produce fully humanized antibodies
Mammalian Cells & Bacteria
• Hybridoma • E. Coli
Functional Inputs and Outputs
T-1
Aeration System
Temperature Control
System
Process Vent
System
Sterile Addition
Systems
CIP
Agitation System
Sampling System
Harvest System
Process Drain
System
Media Feed
Vessel
Assembly
Operating Modes
• Batch
• Fed Batch
• Perfusion (continuous feed)
– Spin filter
– Centrifuge
– Settling
Media comparison
L-Arginine 211 mg Biotin 0.024 mg
L-Histidine 21 mg
Calcium
pantothenate 0.7 mg
L-Lysine 29.3 mg Choline chloride 0.69 mg
L-Methionine 4.48 mg i-inositol 0.54 mg
L-Phenylalanine 4.96 mg Niacinamide 0.6 mg
L-Tryptophan 0.6 mg
Pyridoxine
hydrochloride 0.2 mg
L-Tyrosine 1.81 mg Riboflavin 0.37 mg
L-Alanine 8.91 mg Thymidine 0.7 mg
Glycine 7.51 mg Cyanocobalamin 1.3 mg
L-Serine 10.5 mg Sodium pyruvate 110 mg
L-Threonine 3.57 mg Lipoic acid 0.2 mg
L-Aspartic acid 13.3 mg CaCl2 44 mg
L-Glutamic acid 14.7 mg MgSO4.7H2O 153 mg
L-Asparagine 15 mg Glucose 1.1 g
L-Glutamine 146.2 mg NaCl 7.4 g
L-Isoleucine 2.6 mg KCl 285 mg
L-Leucine 13.1 mg Na2HPO4 290 mg
L-Proline 11.5 mg KH2PO4 83 mg
L-Valine 3.5 mg Phenol red 1.2 mg
L-Cysteine 31.5 mg FeSO4 0.83 mg
Thiamine
hydrochloride 1 mg CuSO4.5H2O 0.0025 mg
Hypoxanthine 4 mg ZnSO4.7H2O 0.028 mg
Folic acid 1.3 mg NaHCO3 1.2 g
Ham's Tissue Culture Medium for Mammalian Cells
(amounts dissolved in 1 liter of triple distilled water)
Glucose 5 g
Na2HPO4 6 g
KH2PO4 3 g
NH4Cl 1 g
NaCl 0.5 g
MgSO4 0.12 g
CaCl2 0.01 g
Minimal Medium for E. coli
(grams/ liter)
Media Sterilization
• Fermentation media can usually be thermally
sterilized in the fermentor.
• Cell culture media is usually filter sterilized into the
bioreactor.
• Thus, fermentors are designed to be sterilized full,
and bioreactors are designed to be sterilized empty.
PROCESS FUNCTIONS
• Aeration
• pH
• Agitation
• Temperature Control
Aeration Systems
• Bioreactors:
– Low gas flow rates typically on the order of 0.01 VVM
– Inlet gas is a mixture of Air (for DO control and CO2 stripping), Oxygen (for DO control without excessive gas flow rates), and CO2 (for pH control.
– Foaming usually not a problem
• Fermentors:
– High gas flow rates,
typically on the order of 1-
1.5 VVM.
– Inlet gas is primarily air.
Occasional applications
require oxygen enrichment.
– Foaming is frequently a
problem.
– Oxygen transfer rate is
usually the limitation to
productivity.
Oxygen Requirements
• 0.05 - 0.5 mMoles / L / Hr
• 150 – 1500 mg / L/ Hr
• 0.1 VVM
Cell Specific O2 Demands
Fleischaker and Sinskey, 19610.05FS-4
Danes et al, 19630.064Skin fibroblast
Danes et al., 19630.37HLM (liver)
Phillips and McCarthy 1956
Phillips and Andrews 1960
0.097
0.10
HeLa
referencemMol O2 / L-hour @ 109 cells / ml
human
pH Control
• Gas mixtures
– Bicarbonate buffer
– Bicarbonate addition
• Addition of dilute acid or caustic
Gas Manifold System
Agitation
• Cells are shear sensitive
• Mixing to prevent gradients in dissolved oxygen and temperature
• Scale-up based on shear and mixing
Agitator features
• Large axial flow impellers
• Angle mount to eliminate baffles
• Low RPM / Shear
Scale-up Criteria
• Maintain mixing time with scale
tML/ tMs = (Ns4DiL/ NL
4Dis)
• Constant Shear
Ss = SL(Dis/ DiL)1/3
Temperature Control
• Closed or semi-closed re-circulating temperature
control
• Minimize difference between jacket temperature
and bioreactor contents temperature (∆T<18C)
• Cascade temperature control
Temperature Control Module
HARDWARE
• Seed to Production
• Tanks
• Agitators
• Valves
• Traps
• The Specification
Bioreactors Travel in Packs
• Expanding a cell population to production scale
requires a series of systems of successively larger size.
• Starting with a frozen vial of cells, a typical train sequence is:
– Mammalian: Spinners/5 liter Wave/20 liter stirred
tank/100 liter/500 liter/ 2,500 liter/Multiple 10,000 to 15,000 liter units.
Small Scale Applications
• Laboratory – Bench top equipment for discovery or
process development, typically under 30 liters
Pilot Scale Applications
• Pilot – Skid mounted equipment for scale up
studies, process optimization, or small volume
production. Typically under 2000 liters
Production Scale Applications
• Skid or module based systems. Ranging up to
25,000 liters for cell culture
• GMP Bioreactors - for regulated industries such as
pharmaceuticals or biotechnology
Large Mammalian Cell Culture
Reactors
20,000 Liter Cell Culture
20,000 Liter Cell Culture
The Tank• Polished 316L SS pressure vessel.
• Fully drainable
• Above 100 liters, designed for complete CIP
• Custom fittings to minimize dead legs in ports, eliminate hold-up, and enable insertion of probes and sensors.
• Primary difference between bioreactor and fermentor vessels is geometry – taller vessels (H/D = 2.5 –3.0) are used for bacterial processes to improve oxygen mass transfer. Shorter vessels for mammalian cell culture (H/D = 1.5) improve mixing.
The Agitator• Bioreactor Agitator:
– Low shear
– High mixing capacity
– Power input typically <1kw/1,000 liters
– Primary scaling criteria is mixing time.
• Fermentor Agitator
– High power input
– Radial impellers (Rushton turbines) are common – high speed
– Power input up to 10 kw/1,000 liters typical
– Primary scaling criteria is oxygen transfer rate
Top v. Bottom Drive
• Top Drive
– Seal is not exposed to direct contact with culture media
– Shaft is longer
– Drive occupies valuable top head real estate
– Need to remove agitator or provide headlift mechanism on smaller vessels
• Bottom Drive
– Seal is exposed to direct contact with culture media
– Shorter shaft
– Top head is left free for pipe, ports and probes
– Can open top head on small vessels without removing agitator
Seal Design
• Cartridge type double
mechanical seal
• Seals are arranged back-to-
back. Increasing sealant
pressure increases sealing force
• Sealant is clean steam condensate
• Seal can be sterilized with vessel, separately, or both.
• Seal assembly can be pressure
tested before installation
Diaphragm ValvesSource: ITT, Pure-Flo, Integrated Block Valve CD, IBV-07(C)
Avoid Dead Legs
Source: ITT, Pure-Flo, Integrated Block Valve CD, IBV-07(C)
Radial Diaphragm Valves
Reference: www.asepco.com
Other Valve Types
Reference: www.jordanvalve.com
Reference: www.pbmvalve.com
Traps
• Fast acting
• Sanitary
• Thermostatic
• Allow adequate drip leg
The Specification
– Scope of Work
– Mechanical
– Electrical
– Instrumentation and Controls
– Testing Requirements
– Quality Assurance - At Site
– Options
– Reference Specifications
Data Sheets
• Process and General Data
• Electrical Requirements
• Major Equipment Data
• Piping
• Control System
• Instrument Listings
Scope of Work
• List all equipment to be included
• Include items such as tagging, skidding and wiring
Mechanical
• Details of equipment
• Include references to any standard specifications
• Piping
• Materials
Electrical
• Wire to a common point
• Reference specifications
Instrumentation and Controls
• Refer to P&IDs if provided
• Reference specifications
• CRTs, printers
• Refer to instrument list
• Who provides automation
Testing requirements
• Factory Acceptance Test (FAT)
• Site Acceptance Test (SAT)
Reference Specifications
• Piping
– Materials
– Finishes
• Specifications for Skidded Equipment
• Electrical
– VFDs
– Motors
• Control Systems
Single Use Bioreactors
RockingUp to 500L WV
XcellerexUp to 2,000L WV
HyClone50L to 1000L
WV
Connectors
Colder
Pall
Sterile Tube Fuse, GEMillipore
Colder Steam-Thru®Pall Kleanpak™
Typical LSCC
Conventional LSCC
LSCC Employing Single Use
Simplified LSCC
Simplify the Remaining
Stainless Steel• A Comparison of production bioreactors built in the last five
years shows an order of magnitude difference in complexity, yet they all do the same thing!
13531101Perfusion3,000E
0202472Batch5,000H
0002Perfusion*500K
210524Batch12,000J
0191545Batch15,000I
0322984Batch12,500G
0332785Batch12,000F
34032122Batch7,500D
14342137Batch15,000C
23038156Batch20,000B
36355316Batch20,000A
Feed VesselsTrapsRTD’sAuto On/Off
ValvesType
Working
Volume
Liters
Project
Simplify the Remaining
Stainless Steel• Over a third of the piping and automation in a conventional
LSCC facility can be eliminated.
Single Use Concerns
• Operating Costs Depend on Location
• Compatibility Testing Required
• Animal Derived Component Free (ADCF) Film
• Difficult to Pipe Long Distances
• Limited in Size
• Doesn’t Support “Lights Out” Manufacturing
• Containment
Where Are We Headed?
• Single Use
– All Biotech Facilities Incorporate Some Single Use
– Companies Are Developing Processes Using Only Single Use Systems
• Bioreactor Size Will Decrease
– Higher Titers
– Improved Pharmacology
– Alternative Expression Systems
Conclusion
• Bioreactors need to be specified to meet the
specific needs of the cell culture process
• Conditions of temperature, dissolved oxygen and
pH are important to cell growth
• Hardware and materials need to be chosen that are
cleanable and sterilizable
• The specification should include hardware as well
as testing and documentation requirements