2-64 Multi-Fermentation POD for High Density Recombinant Protein Production at

100 ml Scale

THE PROTEIN MACHINE ON THE BENCH

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

tical

Den

sit

y/

Tem

p.°

C

Culture Time

Growth

Induction

Biomass ProductionProtein Synthesis

Recombinant Protein

THE SCIENTIFIC ARGUMENTS

• BIOLOGICAL PERFORMANCES

• MORE AUTOMATION

• 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 PERFORMANCES

• WE ARE SELLING A SOLUTION

• WE ARE NOT ONLY SELLING AN EQUIPMENT

• OUR SUPPORT IS ON THE PROCESS

WHERE THE bioPOD WAS CREATED?

Pasteur Institute – Paris (France)Platform Nr 5 – Dr. BELLALOU

Bioprocessing Goals Of The Platform

1/ BACTERIAL CULTURE (0.1 – 300 L)

2/ FERMENTATION OPTIMIZATION FOR RECOMBINANT PROTEIN PROCESSES:

- PRODUCTION- PROTOCOLS

3/ LABELLING OF PROTEINS FOR STRUCTURAL BIOLOGY

4/ PURIFICATION

80-mL biorecator

T°C controlpH probeOxygen probe

Sparger

Gas Outlet

Inlet air/O2Injection septum

Feeding or sampling

MINI-BIOREACTORS CONCEPTION

2011 Design

2 X 100 ml FERMENTATION VERSION

4 X 100 ml FERMENTATION VERSION

8 X 100 ml FERMENTATION VERSION

16 X 100 ml FERMENTATION VERSION

OVERVIEW OF THE SPECIFICATIONS

MAIN FEATURES

CONTROL UNIT

FERMENTOR

RECIPES

SUPERVISION SOFTWARE

bioPOD IS UNIQUE!

3

1

2

CONTROL UNIT

IT’S MODULAR!

LOCAL CONTROL PANEL

PUMP MODULE

PUMP MODULE

AERATION MODULE

BASE SUPPORT

BASE SUPPORT

BASE SUPPORT WITH FERMENTORS

Design of the bioPOD Vessel

BioPOD VESSEL IS SIMPLE!

BioPOD VESSEL

MATERIALS

SENSORS

SET UP

CLEANING

PRACTICAL ADVICES

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

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

Nine Built-In Recipes are Pre-Defined to Run EASILY Main Fermentation Protocols for protein production

Recipe 1: Single chemical induction without temperature shift

Recipe 2: Single chemical induction with one temperature shift

Recipe 3: Single chemical induction with two temperature shifts

Recipe 4: Periodic chemical induction without temperature shift

Recipe 5: Periodic chemical induction with one temperature shift

Recipe 6: Dual chemical inductions without temperature shift

Recipe 7: Dual chemical inductions with two temperature shifts

Recipe 8: Thermal induction with two temperature shifts

Recipe 9: Single chemical induction in fed-batch process culture

Growth

Induction

Recipe 2

Op

tical

Den

sit

y/

Tem

p.°

C

InductionGrowth

Recipe 1

Periodic Induction

Growth

Recipe 3

Op

tica

l D

en

sity

/ Te

mp

.°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

32

BOSSVIEW RECIPE STARTING WINDOW

Select Reactor

Select Recipe

34

The Selected Recipe is Loaded to Selected Fermentor

Recipe Boxes Phases

Recipe Parameters

Biological Data

Recipe illustration

35

Creation of a New Recipe by Adding New Boxes Phases

Edit a the new

phase

Process Dashboard folder

B.O.S.S view PROCESS DASHBOARD WINDOW

On-line O.D

Temperature

Optical Density Calibration for E.

y = 1,0029x

R2 = 0,946

0

20

40

60

80

100

120

140

0 20 40 60 80 100 120 140

Online Optical Density

Optical D

ensity a

t 600nm

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 Features

Fully automated cultivation system for microbial cultures at 100 ml scale

- 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 Optical Density from 0.05 to 350 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 Monitoring of O.D

Pre-Programmed Recipes (Batch and Fed-Batch) with Different Levels of Lab 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 PERFORMANCESPUBLISHED

• BATCH & FED BATCH WITH E. COLI

• FED BATCH WITH PICHIA PASTORIS

• FED BATCH WITH SACCAROMYCES

HDM Design

A high-density medium formulation was designed, which enabled to grow E. coliup to an OD600 of 100 in batch cultivation with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 80 ml culture were at least equivalent to those obtained in a Fernbach flask with 1 liter of conventional medium.

80-mL reactors Batch Cultures

(37°C, 0,5 VVM)

0

10

20

30

40

50

60

70

80

90

Optical D

ensity 6

00nm

LB media

Hyper Broth

TurboBroth

HDM media

5-mL

micro-plate80-mL

fermentor10-mL

tube

1-L Shaker

flask

1-6 to 16-L

fermentor

DVT. HIGH DENSITY CULTURE MEDIA

SDS-PAGE of Soluble and Insoluble, Total

Protein Extracts after Expression of aRecombinant Protein in BL21 DE3 E.coli HostStrain Grown in DifferentAuto-Inducible Media.

(1) Auto Inductible Medium Designed

(2) Invitrogen MagicMedia

(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

Before

Induction

End

Culture

End

Culture

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 16

Culture Time (h)

On

lin

e O

ptica

l D

en

sity

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 36

Culture 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 70

Culture Time (h)

Optical D

ensity ;

D.O

. (%

)

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 on

supernatants samples

Time (h) after methanol induction

scFv fragment

Meth

anol

transitio

n

Glycerol

Batch

Gly

cero

l fe

d-b

atc

h

MethanolFed-Batch

F=18 mL/h/L F=3,6 mL/h/L

F=7,3 mL/h/L

F=10,9 mL/h/L

SCIENTIFIC REFERENCES

• Pasteur Institute (Paris)

• GLYCODE (France)

• CNRS (Paris)

• CNRS (Montpellier)

• Unicamp (Brazil)

• KRIBB (Korea)

• HKIB (China )

• MIT (USA)