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