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2-64 Multi-Fermentation POD For High Density Recombinant Protein Expression and Production From 80 ml Up to 10L Scale ‘‘More than a Product, the bioPOD is a Concept with Innovation as a Continuous Improvement”
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2-64 Multi-Fermentation POD For High Density Recombinant
Protein Expression and Production
From 80 ml Up to 10L Scale
‘‘More than a Product, the bioPOD is a Concept with Innovation as a Continuous Improvement”
www.fogalebiotech.com September 2011
Bioprocessing Goals Of The Platform
1/ BACTERIAL CULTURE (0.1 1/ BACTERIAL CULTURE (0.1 –– 300 L)300 L)
2/ FERMENTATION OPTIMIZATION FOR RECOMBINANT PROTEIN PROCESSES:2/ FERMENTATION OPTIMIZATION FOR RECOMBINANT PROTEIN PROCESSES:
-- PRODUCTIONPRODUCTION-- PROTOCOLSPROTOCOLS
3/ LABELLING OF PROTEINS FOR 3/ LABELLING OF PROTEINS FOR STRUCTURAL BIOLOGYSTRUCTURAL BIOLOGY
4/ PURIFICATION4/ PURIFICATION
The Concept of the BioPOD has Started at Pasteur Institute (Paris)
Courtesy of Pasteur Institute Paris
Gap of Tool for optimization ProcessGap of Tool for optimization Process
Fermentation Classical Scale-Up
10-mL micro plates
1-L Fernbach flask
2-L fermentor
15-L fermentor300-L fermentor
Courtesy of Pasteur Institute Paris
The project :The project :Production of a large collection Production of a large collection
of recombinant proteinsof recombinant proteins
The goal :The goal :Reduction of volume and size vessel keeping Reduction of volume and size vessel keeping
the same protein yieldsthe same protein yields
The solutions :The solutions :Miniaturization of the reactors Miniaturization of the reactors Use of an High Density mediaUse of an High Density media
Courtesy of Pasteur Institute Paris
Protein Requirements @ 100 ml scaleDepending of Final Applications
Applications Purified Protein
Functional Activity Studies 100 ng to 1 mg
Antibody Preparation 2 to 5 mg
Structural Studies 10 to 50 mg
Industrial Projects, Diagnostics, Drug Discovery > 500 mg
Parameters AND Multiple Influence to be Considered for Production of Soluble Target Proteins in E. coli
§ Host Strains (DE3 pLysS, AI , KRX)§ Media (High Density, Auto Inducible)§ Growth and temperatures of Induction *§ Optical Density for Induction*§ Inducer Concentration§ Growth Rate*§ Co-expression of Chaperones§ tRNA Complementation Plasmids§ Fusion Proteins§ Construction of Gene Fragments
Needs for a Parallel Approach to Test Many Variables for a Given Experiment
Taking always in consideration:
BIOLOGICAL ASPECTS + TECHNOLOGICAL ASPECTS (*)
Op
tica
l D
en
sity
/ T
em
p.°
C
Culture Time
Growth
Induction
Biomass ProductionProtein Synthesis
Recombinant Protein
Original Design
Courtesy of Pasteur Institute Paris
80 ml FERMENTOR
T°C controlpH probeOxygen probe
Sparger
Gas Outlet
Inlet air/O2 Injection septum
Feeding or sampling
MINI-BIOREACTORS DESIGN
Design of the bioPOD vessel
2 X 80 ml FERMENTOR VERSION
4 X 80 ml FERMENTOR VERSION
8 X 80 ml FERMENTOR VERSION
16 X 80 ml FERMENTOR VERSION
Scale-up of the bioPOD DesignLaunch Starting 4th Quarter 2011
3
1
2
CONTROL UNIT
IT’S MODULAR!
LOCAL CONTROL PANEL
PUMP MODULE
PUMP MODULE
BioPOD VESSELMATERIALS
SENSORS
SET UP
CLEANING
PRACTICAL ADVICES
AERATION MODULE
BASE SUPPORT WITH FERMENTORS
9 Built-In Recipes are Pre-Defined to Run Easily Main FermentationCulture Protocols For Protein Production :
Ø Recipe 1: Single chemical induction without temperature shift
Ø Recipe 2: Single chemical induction with one temperature shift
Ø Recipe 3: Single chemical induction with two temperature shifts
Ø Recipe 4: Periodic chemical induction without temperature shift
Ø Recipe 5: Periodic chemical induction with one temperature shift
Ø Recipe 6: Dual chemical inductions without temperature shift
Ø Recipe 7: Dual chemical inductions with two temperature shifts
Ø Recipe 8: Thermal induction with two temperature shifts
Ø Recipe 9: Single chemical induction in fed-batch process culture
PRINCIPLE OF A RECIPE
• THE BIOMASS GROWTH IS CONTROLING THE FERMENTATION
• AUTOMATION OF A PROCESS AS MUCH AS POSSIBLE
• MODELISATION USING MATHEMATICAL ALGORITHMS AVAILABLE FROM LITTÉRATURE
• SPECIFIC MATHEMATICAL MODELS
Growth
Induction
Recipe 2
Op
tica
l D
en
sity
/ T
em
p.°
C
InductionGrowth
Recipe 1
Periodic Induction
Growth
Recipe 3
Opt
ical
Den
sity
/ Tem
p.°C
Growth
Periodic Induction
Recipe 4
Induction 1
Growth
Induction 2
Recipe 5
Fed-BatchGrowth
Induction
Recipe 6
Example of Some Pre-Filled Recipes Built-In BO.S.S view Software
Recipe NR 2
Recipe NR 6
AUTOMATION OF A FERMENTATION
B.O.S.S view
SOFTWARE CONTROL
OF FERMENTATION PROCESSES
Control of Parameters Programmed Set Points
§ Better Reproducibility
§ Various Steps of Culture Protocols can be Performed at any time
§ Culture Parameters can be Stored and Retrieved from a Data Base
§ Open System Adapted to Develop new Specific Cultivation Protocols without Computer Skills
Fully AutomatedProcesses
Cultivation Recipes are Already Integrated
Easy Writing of New Recipes
33
BOSSVIEW RECIPE STARTING WINDOW
Select Reactor
Select Recipe
35
The Selected Recipe is Loaded to Selected Fermentor
Recipe Boxes Phases
Recipe Parameters
Biological Data
Recipe Diagram
36
Creation of a New Recipe by Adding New Boxes Phases
Edit a the newphase
Process Dashboard folder
B.O.S.S view PROCESS DASHBOARD WINDOW
On-line O.D
Temperature
Optical Density Calibration for E.
y = 1,0029xR2 = 0,946
0
20
40
60
80
100
120
140
0 20 40 60 80 100 120 140
Online Optical Density
On line Optical Density and Temperature Shift of Selected Cultures
B.O.S.S view GRAPH WINDOW
B.O.S.S view Fed-batch Process Control
F : Feeding Flow rate (mL/h)µ : Growth rate µ (h-1)Yx/s : Conversion ratio (gDCW/g) V : Volume (L)S : Concentration of the feeding substrate (g/L)
Main FeaturesFully automated cultivation system
for microbial cultures
- Feedback Control and Programming of Automated Biological RecipesSequences during Fermentation
- Temperature, pH, dissolved oxygen (DO) and biomass are modeled/monitored on-line
- Events, such as Temperature Shifts and Addition of Inducer, as a Function of Cell Density are built in.
- At-Line/On-Line Cell Density from 0.05 to 500 OD600 nm
- Independent Heating/Cooling Peltier devices (4-65°C)
WHY bioPOD IS UNIQUE?
§ Rapid Parallel Investigation of Multiple Cultures for Process Optimization
§ Production at Small Scale and True Scale-Up to Larger Volumes
§ Simplified Manipulations for Culture Runs
§ Reduced Cost and Time Saving Using Reduced Culture Volume Reaching High Density
§ At-line /On-line Monitoring of Cell Density with O.D and Capacitance
§ Pre-Programmed Recipes (Batch, Fed-Batch…) with Different Levels of Automation
§ Intuitive, Flexible and Versatile Software Interface Allows to Edit New Recipes
§ No Computer Skills Needed
§ Data Base for Systematic Data Retrieval and Powerful Search Functions for Rapid comparison of Different Cultivation Logs and Graphs
BIOLOGICAL PERFORMANCESACHIEVED / PUBLISHED
• BATCH & FED BATCH WITH E. COLI• FED BATCH WITH PICHIA PASTORIS• FED BATCH WITH SACCAROMYCES• pH STAT MODE • CONTINUOUS MODE• CHEMOSTAT MODE
Courtesy of Pasteur Institute Paris
Courtesy of Pasteur Institute Paris
Courtesy of Pasteur Institute Paris
SDS-PAGE of Soluble and Insoluble, Total Protein Extracts after Expression of a Recombinant Protein in BL21 DE3 E.coli Host Strain Grown in Different Auto-Inducible Media.
(1) Auto InductibleMedium Designed
(2) InvitrogenMagicMedia
(3) Novagen Overnight Express Instant TB
TEST OF DIFFERENT AUTO INDUCIBLE MEDIA WITH E. coli
E.coli Batch and Fed-Batch for the Production - Same Recombinant Protein
Complex high density medium
With Fed-Batch
§ Higher Bacterial Biomass
§ Control of the Growth Rate µ
§ Higher Amount of Target Soluble Protein
SDS-Page on crude bacterial extracts
BeforeInduction
EndCulture
EndCulture
Sol Insol Sol Insol Sol Insol
Batch Process Fed-Batch Process
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12 14 16Culture Time (h)
IPTG induction
Batch Process
Growth and Induction: 24°C
0
20
40
60
80
100
120
0 4 8 12 16 20 24 28 32 36Culture Time (h)
Chemically Defined Medium
Batch Process Fed-Batch Process
IPTG induction
µ : 0,15 (h-1)
µ : 0,05 (h-1)
Growth and Induction: 24°C
0
50
100
150
200
250
300
350
400
450
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70Culture Time (h)
4,4
4,8
5,2
5,6
6,0
pH
Pichia Pastoris Fed-Batch - Production of Secreted Proteins
Successful Scale-Down of the Invitrogen fed-batch Pichia protocol in BSM Media Described for 20-L fermentor
M.W 0 16 20 24 40 44h
SDS-Page onsupernatants samples
Time (h) after methanol induction
scFv fragmentM
ethan
ol
transi
tion
Glycerol
Batch
Gly
cero
l fe
d-b
atc
h Methanol
Fed-Batch
F=18 mL/h/L F=3,6 mL/h/LF=7,3 mL/h/L
F=10,9 mL/h/L
Production of recombinant Proteins using bioPOD & 2 L Fermentor
13.412.6
1.30.91
9772
1 liter70
FermentorbioPOD
14°C, ON1
14°C, ON1Rv2256c
7.013.8
0.360.29
5121
1 liter62 ml
FermentorbioPOD
14°C, ON1
14°C, ON1Rv1827
6.011
0.490.63
8257
1 liter66 ml
FermentorbioPOD
14°C, ON1
14°C, ON1Rv2238c
5.45.7
0.260.33
4858
1.6 liter60 ml
FermentorbioPOD
20°C, 3 h 20°C, 4.5 h
T. cruziracemase
2632.7
2.12.3
8070
1.6 liter80
FermentorbioPOD
37°C, 2 h37°C, 2h
Rv2543
Purified protein
µg/OD600
Purified protein mg/ml culture
Final OD600
VolumeBioreactorInduction protocolStrain
1 ON, over night
2011 – 2012 Fermentation Extension of the Portfolio
MODEL WORKING VOLUME TOTAL VOLUME AVAILABLE
F1 0.2 - 1 L 1,2 L SEP. 2011
F3 0.4 - 2.9 L 3.6 L SEP. 2011
F5 1 - 6 L 7.5 L 1st Q. 2012
F10 3,5 – 11 L 14 L 1st Q. 2012
2011 – 2012 Cell Culture Extension of the Portfolio
MODEL WORKING VOLUME TOTAL VOLUME AVAILABLE
C1 0,2 – 0,75 L 1,2 L 1st Q. 2012
C3 0,4 – 2,5 L 3.6 L 2nd Q. 2012
C5 1 - 5 L 7.5 L 3rd Q. 2012
C10 3,5 – 10 L 14 L 4th Q. 2012
THE SCIENTIFIC ARGUMENTS
• BIOLOGICAL PERFORMANCES• MORE AUTOMATION Vs. COMPETITION• BIOLOGICAL SUPPORT • POSSIBLE COLLABORATIVE WORK• OPEN TO IDEAS AND INNOVATION
THE TECHNICAL ARGUMENTS
• NO EQUIVALENT TO THE BioPOD• MODULAR• SCALABLE• NO SPECIFIC SOFTWARE LANGUAGE• NO NEED TO BE A BIOPROCESS EXPERT• QUICK RESULTS OBTAINED
THE SALES ARGUMENTS
• PRICE COMPETITIVE• WE ARE SELLING A PERFORMANCE• WE ARE SELLING A SOLUTION • WE ARE NOT ONLY SELLING AN EQUIPMENT• OUR SUPPORT IS ON THE PROCESS AND
EQUIPMENT• SCIENTIFIC SUPPORT IN ASIA
Scientific References
• Pasteur Institute - Paris (32 fermentors)• CNRS - Paris - (4 fermentors)• CNRS - Montpellier ( 8 fermentors)• Unicamp - Brazil (8 fermentors )• KRIBB - Korea (2 fermentors)• HKIB - China (8 fermentors)• MIT - USA (2 fermentors)