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Bioreactor Specification and Design

By 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


Media Feed CIP Process Vent System

Aeration System

Sterile Addition Systems

Vessel Assembly Temperature Control System


T-1

Sampling System

Agitation System Process Drain System

Harvest System

Operating Modes

Batch Fed Batch Perfusion (continuous feed) Spin filter Centrifuge Settling

Media comparison
Ham's Tissue Culture Medium for Mammalian Cells (amounts dissolved in 1 liter of triple distilled water) L-Arginine L-Histidine L-Lysine L-Methionine L-Phenylalanine L-Tryptophan L-Tyrosine L-Alanine Glycine L-Serine L-Threonine L-Aspartic acid L-Glutamic acid L-Asparagine L-Glutamine L-Isoleucine L-Leucine L-Proline L-Valine L-Cysteine Thiamine hydrochloride Hypoxanthine Folic acid 211 mg 21 mg 29.3 mg 4.48 mg 4.96 mg 0.6 mg 1.81 mg 8.91 mg 7.51 mg 10.5 mg 3.57 mg 13.3 mg 14.7 mg 15 mg 146.2 mg 2.6 mg 13.1 mg 11.5 mg 3.5 mg 31.5 mg 1 mg 4 mg 1.3 mg Biotin Calcium pantothenate Choline chloride i-inositol Niacinamide Pyridoxine hydrochloride Riboflavin Thymidine Cyanocobalamin Sodium pyruvate Lipoic acid CaCl2 MgSO4.7H2O Glucose NaCl KCl Na2HPO4 KH2PO4 Phenol red FeSO4 CuSO4.5H2O ZnSO4.7H2O NaHCO3 0.024 mg 0.7 mg 0.69 mg 0.54 mg 0.6 mg 0.2 mg 0.37 mg 0.7 mg 1.3 mg 110 mg 0.2 mg 44 mg 153 mg 1.1 g 7.4 g 285 mg 290 mg 83 mg 1.2 mg 0.83 mg 0.0025 mg 0.028 mg 1.2 g
(grams/ liter) Minimal Medium for E. coli

Glucose

5g

Na2HPO4

6g

KH2PO4 NH4Cl NaCl MgSO4 CaCl2

3g 1g 0.5 g 0.12 g 0.01 g

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 11.5 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


human HeLa HLM (liver) Skin fibroblast FS-4 mMol O2 / L-hour @ 109 cells / ml 0.097 0.10 0.37 0.064 0.05 reference
Phillips and McCarthy 1956 Phillips and Andrews 1960 Danes et al., 1963

Danes et al, 1963

Fleischaker and Sinskey, 1961

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/ NL4Dis)

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-toback. 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 Valves
Source: 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.pbmvalve.com

Reference: www.jordanvalve.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

Xcellerex Up to 2,000L WV Rocking Up to 500L WV

HyClone 50L to 1000L WV

Connectors

Sterile Tube Fuse, GE

Millipore Colder

Pall Pall Kleanpak Colder Steam-Thru

Conventional LSCC

Typical 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!
Working Project A B C D E F G H I J K Volume Liters 20,000 20,000 15,000 7,500 3,000 12,000 12,500 5,000 15,000 12,000 500 Batch Batch Batch Batch Perfusion Batch Batch Batch Batch Batch Perfusion* Type Auto On/Off Valves 316 156 137 122 101 85 84 72 45 24 2 RTDs 55 38 42 32 31 27 29 24 15 5 0 Traps 63 30 43 40 35 33 32 20 19 10 0 Feed Vessels 3 2 1 3 1 0 0 0 0 2 0

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 Doesnt 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