2-64 Multi-Fermentation POD         For High Density Recombinant       Protein Expression and Production              From...
The Concept of the BioPOD has Started at Pasteur Institute (Paris)               Bioprocessing Goals Of The Platform   1/ ...
Fermentation Classical Scale-Up           10-mL micro plates                 1-L Fernbach flask          Gap of Tool for o...
The project :  Production of a large collection     of recombinant proteins                                  The goal :   ...
Protein Requirements @ 100 ml scale        Depending of Final Applications      Applications            Purified ProteinFu...
Parameters AND Multiple Influence to be Considered for        Production of Soluble Target Proteins in E. coli§ Host Strai...
Original DesignCourtesy of Pasteur Institute Paris
MINI-BIOREACTORS DESIGN         80 ml FERMENTOR                                             Feeding                 Gas Ou...
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
CONTROL UNIT123
IT’S MODULAR!
LOCAL CONTROL PANEL
PUMP MODULE
PUMP MODULE
BioPOD VESSELMATERIALSSENSORSSET UPCLEANINGPRACTICALADVICES
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: Si...
PRINCIPLE OF A RECIPE• THE BIOMASS GROWTH IS CONTROLING  THE FERMENTATION• AUTOMATION OF A PROCESS AS MUCH  AS POSSIBLE• M...
Optical Density / Temp.°C                                 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  Cultivation Recipesare Already In...
BOSSVIEW RECIPE STARTING WINDOW                                  33
Select Recipe     Select Reactor
The Selected Recipe is Loaded to Selected Fermentor                          Recipe Diagram                          Biolo...
Creation of a New Recipe by Adding New Boxes Phases                                              Edit a the new           ...
B.O.S.S view PROCESS DASHBOARD WINDOWProcess Dashboard folder
B.O.S.S view GRAPH WINDOW                                   On line Optical Density and Temperature Shift                 ...
B.O.S.S view Fed-batch Process Control                        F:     Feeding Flow rate (mL/h)                        µ:   ...
Main Features                 Fully automated cultivation system                        for microbial cultures- Feedback C...
WHY bioPOD IS UNIQUE?§ Rapid Parallel Investigation of Multiple Cultures for Process Optimization§ Production at Small Sca...
BIOLOGICAL PERFORMANCES          ACHIEVED / PUBLISHED•   BATCH & FED BATCH WITH E. COLI•   FED BATCH WITH PICHIA PASTORIS•...
Courtesy of Pasteur Institute Paris
Courtesy of Pasteur Institute Paris
Courtesy of Pasteur Institute Paris
TEST OF DIFFERENT AUTO INDUCIBLE MEDIA WITH E. coli                                        (1) Auto Inductible            ...
E.coli Batch and Fed-Batch for the Production - Same Recombinant Protein               Complex high density medium        ...
Pichia Pastoris Fed-Batch - Production of Secreted Proteins             F=18 mL/h/L                  F=3,6 mL/h/L         ...
Production of recombinant Proteins  using bioPOD & 2 L Fermentor                      Induction   Bioreactor   Volume     ...
2011 – 2012 Fermentation Extension of the PortfolioMODEL           WORKING VOLUME TOTAL VOLUME      AVAILABLEF1           ...
2011 – 2012 Cell Culture Extension of the PortfolioMODEL           WORKING VOLUME TOTAL VOLUME        AVAILABLEC1         ...
THE SCIENTIFIC ARGUMENTS•   BIOLOGICAL PERFORMANCES•   MORE AUTOMATION Vs. COMPETITION•   BIOLOGICAL SUPPORT•   POSSIBLE C...
THE TECHNICAL ARGUMENTS•   NO EQUIVALENT TO THE BioPOD•   MODULAR•   SCALABLE•   NO SPECIFIC SOFTWARE LANGUAGE•   NO NEED ...
THE SALES ARGUMENTS• PRICE COMPETITIVE• WE ARE SELLING A PERFORMANCE• WE ARE SELLING A SOLUTION• WE ARE NOT ONLY SELLING A...
Scientific References•   Pasteur Institute - Paris (32 fermentors)•   CNRS - Paris - (4 fermentors)•   CNRS - Montpellier ...
BioPod General Presentation
BioPod General Presentation
BioPod General Presentation
BioPod General Presentation
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BioPod General Presentation

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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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BioPod General Presentation

  1. 1. 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
  2. 2. The Concept of the BioPOD has Started at Pasteur Institute (Paris) 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/ PURIFICATIONCourtesy of Pasteur Institute Paris
  3. 3. Fermentation Classical Scale-Up 10-mL micro plates 1-L Fernbach flask Gap of Tool for optimization Process 2-L fermentor 15-L fermentor 300-L fermentorCourtesy of Pasteur Institute Paris
  4. 4. The project : Production of a large collection of recombinant proteins The goal : Reduction of volume and size vessel keeping the same protein yields The solutions : Miniaturization of the reactors Use of an High Density mediaCourtesy of Pasteur Institute Paris
  5. 5. Protein Requirements @ 100 ml scale Depending of Final Applications Applications Purified ProteinFunctional Activity Studies 100 ng to 1 mg Antibody Preparation 2 to 5 mg Structural Studies 10 to 50 mg Industrial Projects, > 500 mgDiagnostics, Drug Discovery
  6. 6. Parameters AND Multiple Influence to be Considered for Production of Soluble Target Proteins in E. coli§ Host Strains (DE3 pLysS, AI , KRX) Biomass Production Protein Synthesis§ Media (High Density, Auto Inducible) Optical Density / Temp.°C§ Growth and temperatures of Induction *§ Optical Density for Induction* Growth§ Inducer Concentration§ Growth Rate* Induction§ Co-expression of Chaperones§ tRNA Complementation Plasmids§ Fusion Proteins Recombinant Protein§ Construction of Gene Fragments Culture Time Needs for a Parallel Approach to Test Many Variables for a Given Experiment Taking always in consideration: BIOLOGICAL ASPECTS + TECHNOLOGICAL ASPECTS (*)
  7. 7. Original DesignCourtesy of Pasteur Institute Paris
  8. 8. MINI-BIOREACTORS DESIGN 80 ml FERMENTOR Feeding Gas Outlet or sampling Inlet air/O2 Injection septum T°C control pH probe Oxygen probe Sparger
  9. 9. Design of the bioPOD vessel
  10. 10. 2 X 80 ml FERMENTOR VERSION
  11. 11. 4 X 80 ml FERMENTOR VERSION
  12. 12. 8 X 80 ml FERMENTOR VERSION
  13. 13. 16 X 80 ml FERMENTOR VERSION
  14. 14. Scale-up of the bioPOD DesignLaunch Starting 4th Quarter 2011
  15. 15. CONTROL UNIT123
  16. 16. IT’S MODULAR!
  17. 17. LOCAL CONTROL PANEL
  18. 18. PUMP MODULE
  19. 19. PUMP MODULE
  20. 20. BioPOD VESSELMATERIALSSENSORSSET UPCLEANINGPRACTICALADVICES
  21. 21. AERATION MODULE
  22. 22. BASE SUPPORT WITH FERMENTORS
  23. 23. 9 Built-In Recipes are Pre-Defined to Run Easily Main FermentationCulture Protocols For Protein Production :Ø Recipe 1: Single chemical induction without temperature shiftØ Recipe 2: Single chemical induction with one temperature shiftØ Recipe 3: Single chemical induction with two temperature shiftsØ Recipe 4: Periodic chemical induction without temperature shiftØ Recipe 5: Periodic chemical induction with one temperature shiftØ Recipe 6: Dual chemical inductions without temperature shiftØ Recipe 7: Dual chemical inductions with two temperature shiftsØ Recipe 8: Thermal induction with two temperature shiftsØ Recipe 9: Single chemical induction in fed-batch process culture
  24. 24. 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
  25. 25. Optical Density / Temp.°C Example of Some Pre-Filled Recipes Built-In BO.S.S view Software Periodic Induction Induction Growth Growth Growth Induction Recipe 1 Recipe 2 Recipe 3 Induction 1 Induction 2 InductionOptical Density / Temp.°C Growth Growth Fed-Batch Growth Periodic Induction Recipe 4 Recipe 5 Recipe 6
  26. 26. Recipe NR 2
  27. 27. Recipe NR 6
  28. 28. AUTOMATION OF A FERMENTATION
  29. 29. B.O.S.S view SOFTWARE CONTROL OF FERMENTATION PROCESSES Cultivation Recipesare Already Integrated § Better Reproducibility Control of Parameters § Various Steps of Culture Protocols canProgrammed Set Points be Performed at any time § Culture Parameters can be Stored and Retrieved from a Data Base Fully Automated Processes § Open System Adapted to Develop new Specific Cultivation Protocols Easy Writing without Computer Skills of New Recipes
  30. 30. BOSSVIEW RECIPE STARTING WINDOW 33
  31. 31. Select Recipe Select Reactor
  32. 32. The Selected Recipe is Loaded to Selected Fermentor Recipe Diagram Biological Data Recipe Parameters Recipe Boxes Phases 35
  33. 33. Creation of a New Recipe by Adding New Boxes Phases Edit a the new phase 36
  34. 34. B.O.S.S view PROCESS DASHBOARD WINDOWProcess Dashboard folder
  35. 35. B.O.S.S view GRAPH WINDOW On line Optical Density and Temperature Shift of Selected Cultures Optical Density Calibration for E. 140 y = 1,0029x 120 R2 = 0,946 100 80 On-line O.D 60 40 Temperature 20 0 0 20 40 60 80 100 120 140 Online Optical Density
  36. 36. 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)
  37. 37. Main Features Fully automated cultivation system for microbial cultures- Feedback Control and Programming of Automated Biological RecipesSequences during Fermentation- Temperature, pH, dissolved oxygen (DO) and biomass aremodeled/monitored on-line- Events, such as Temperature Shifts and Addition of Inducer, as a Function ofCell 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)
  38. 38. 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 Rapidcomparison of Different Cultivation Logs and Graphs
  39. 39. BIOLOGICAL PERFORMANCES ACHIEVED / PUBLISHED• BATCH & FED BATCH WITH E. COLI• FED BATCH WITH PICHIA PASTORIS• FED BATCH WITH SACCAROMYCES• pH STAT MODE• CONTINUOUS MODE• CHEMOSTAT MODE
  40. 40. Courtesy of Pasteur Institute Paris
  41. 41. Courtesy of Pasteur Institute Paris
  42. 42. Courtesy of Pasteur Institute Paris
  43. 43. TEST OF DIFFERENT AUTO INDUCIBLE MEDIA WITH E. coli (1) Auto Inductible Medium Designed (2) Invitrogen MagicMedia (3) Novagen Overnight Express Instant TBSDS-PAGE of Soluble and Insoluble, TotalProtein Extracts after Expression of aRecombinant Protein in BL21 DE3 E.coli HostStrain Grown in DifferentAuto-Inducible Media.
  44. 44. E.coli Batch and Fed-Batch for the Production - Same Recombinant Protein Complex high density medium Chemically Defined Medium 70 Batch Process Batch Process Fed-Batch Process 60 120 Growth and Induction: 24°C 50 Growth and Induction: 24°C 100 µ : 0,05 (h-1) 40 IPTG 80 induction 30 IPTG 60 induction µ : 0,15 (h-1) 20 40 10 20 0 0 0 2 4 6 8 10 12 14 16 Culture Time (h) 0 4 8 12 16 20 24 28 32 36 Culture Time (h) SDS-Page on crude bacterial extracts With Fed-Batch Batch Process Fed-Batch Process § Higher Bacterial Biomass Sol Insol Sol Insol Sol Insol § Control of the Growth Rate µ § Higher Amount of Before End End Target Soluble Protein Induction Culture Culture
  45. 45. Pichia Pastoris Fed-Batch - Production of Secreted Proteins F=18 mL/h/L F=3,6 mL/h/L F=10,9 mL/h/L F=7,3 mL/h/L450 6,0 Time (h) after methanol induction Glycerol Methanol Glycerol fed-batch transition Fed-Batch Methanol400 M.W 0 16 20 24 40 44h Batch350 5,6300250 5,2200 scFv fragment pH150 4,810050 0 4,4 SDS-Page on 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 supernatants samples Culture Time (h) Successful Scale-Down of the Invitrogen fed-batch Pichia protocol in BSM Media Described for 20-L fermentor
  46. 46. Production of recombinant Proteins using bioPOD & 2 L Fermentor Induction Bioreactor Volume Final Purified Purified Strain protocol OD600 protein protein mg/ml µg/OD600 culture Rv2543 37°C, 2 h Fermentor 1.6 liter 80 2.1 26 37°C, 2h bioPOD 80 70 2.3 32.7 T. cruzi 20°C, 3 h Fermentor 1.6 liter 48 0.26 5.4 racemase 20°C, 4.5 h bioPOD 60 ml 58 0.33 5.7 Rv2238c 14°C, ON1 Fermentor 1 liter 82 0.49 6.0 14°C, ON1 bioPOD 66 ml 57 0.63 11 Rv1827 14°C, ON1 Fermentor 1 liter 51 0.36 7.0 14°C, ON1 bioPOD 62 ml 21 0.29 13.8 Rv2256c 14°C, ON1 Fermentor 1 liter 97 1.3 13.4 14°C, ON1 bioPOD 70 72 0.91 12.6 1 ON, over night
  47. 47. 2011 – 2012 Fermentation Extension of the PortfolioMODEL WORKING VOLUME TOTAL VOLUME AVAILABLEF1 0.2 - 1 L 1,2 L SEP. 2011F3 0.4 - 2.9 L 3.6 L SEP. 2011F5 1-6L 7.5 L 1st Q. 2012F10 3,5 – 11 L 14 L 1st Q. 2012
  48. 48. 2011 – 2012 Cell Culture Extension of the PortfolioMODEL WORKING VOLUME TOTAL VOLUME AVAILABLEC1 0,2 – 0,75 L 1,2 L 1st Q. 2012C3 0,4 – 2,5 L 3.6 L 2nd Q. 2012C5 1-5L 7.5 L 3rd Q. 2012C10 3,5 – 10 L 14 L 4th Q. 2012
  49. 49. THE SCIENTIFIC ARGUMENTS• BIOLOGICAL PERFORMANCES• MORE AUTOMATION Vs. COMPETITION• BIOLOGICAL SUPPORT• POSSIBLE COLLABORATIVE WORK• OPEN TO IDEAS AND INNOVATION
  50. 50. THE TECHNICAL ARGUMENTS• NO EQUIVALENT TO THE BioPOD• MODULAR• SCALABLE• NO SPECIFIC SOFTWARE LANGUAGE• NO NEED TO BE A BIOPROCESS EXPERT• QUICK RESULTS OBTAINED
  51. 51. 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
  52. 52. 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)

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