BioPod General Presentation

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

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p.°

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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

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p.°

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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)