NO SPEED LIMIT. Full Bioprocess Control in Microbioreactors A new Option for Scale Down Models

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1 NO SPEED LIMIT Full Bioprocess Control in Microbioreactors A new Option for Scale Down Models Frank Kensy, m2p-labs GmbH CLIB-Forum, 3. April 2014 CREATIVE CAMPUS MONHEIM

2 m2p-labs The Microbioreactor Company Company profile Business Areas & Technology Enabling Technology for Life Science Market Intelligent Bioprocessing Tools to reduce time to market 5 Technology Patents in major markets Established worldwide customer base Milestones Spin-off from RWTH Aachen University in 2005 Market entry with first product end of 2007 >85 devices placed in the market Locations & Key Facts GmbH located in Baesweiler, Germany and Inc. in NY, USA Ca. 500 m² office and laboratory space Currently 17 FTE 2

3 Trends and Demands in Biotechnology Trends in biotechnology: Genetic engineering diversity Chemical synthesis biotechnological steps Time-to-market faster development Demands in early bioprocess development: Characterisation of genetic elements, growth and expression Selection of most productive strains Media and parameter optimization State-of-the-art: laborious and expensive systems BioLector 3

4 Microbioreactors for better Process Understanding Old Technology 1x BioLector Technology Biomass & Fluorescence Oxygen ph 24x 48x 4

5 High-Throughput Fermentation System High parallelisation (48 reactors) Small working volume (800µl 2400µl) Standard MTP format automation BioLector Non-invasive online measurements Defined mass transfer conditions Temperature, humidity and gassing control Simple handling, calibration free, no tubings 5

6 FlowerPlate : New Horizons at Microscale - high mass transfer OTR (> 0.11 mol/l/h) - broad volume range ( ml) - reduced spilling - no optical cross talk - effective mixing * - no foaming - continuous contact of liquids to optodes - multiparameter reading possible -> same reactor performance like industrial bioreactors *new Geometries Patent pending In collaboration with: 6

7 Media Optimization E. coli BL21(DE3) prhothi-2-ecfbfp, modified WR-medium with 7.5 g/l Glucose conditions: T = 37 C, VL = 200 μl, n = 950 rpm, do= 3 mm, no induction Huber et al., BMC Biotechnology 2011, 11:22 7

8 Scalability: Corynebacterium glut. Bioreactor (1 L) CDW [g. L -1 ] lip. act. [U. ml -1 ] CDW [g. L -1 ] lip. act. [U. ml -1 ] CDW lip. act. µ = 0.4 h Time [h] BioLector (1 ml) µ = 0.4 h -1 CDW lip. act. NprE-Cutinase NprE-Cutinase Time [h] lipolytic activity [U. ml -1 ] lipolytic activity [U. ml -1 ] CDW [mg. ml ] / U. mg CDW [mg. ml -1 ] Scale-up factor 1000 equal µ, Y X/S, Y P/X / U. mg -1 specific activity [U. mg -1 ] spec.lip.act. [U. mg -1 ] specific spec.lip.act. activity [U [U. mg -1. mg -1 ] ] NprE YwmC YpjP NprE YwmC YpjP Empty C.glutamicum ATCC pekex2::sp-cutinase T=30 C, 1200 rpm, 3 mm, media: CG XII, 0.5 mm IPTG Empty Rohe et al., Microbial Cell Factories 2012, 11:144 8

9 New Microfluidic Platform BioLector Pro 9

10 Current Practice in Bioprocess R&D Volume: L Most bioprocesses are conducted as fed-batch processes! batch fed-batch biomass, feed biomass feed 1 experiment time Advantages: controlled process no overflow high productivity 10

11 BioLector Pro Full Bioprocess Control at Micro-Scale In collaboration with: Scale up 11

12 Design of the Microfluidic Control Chip 2 Reservoir Wells 4 Cultivation Wells ph channels Feeding channels - In total 32 active bioreactors in a 48 well microplate - 2 Reservoir wells per 4 culture wells - Feed control via microvalves and/or pump chambers - Flexible use of the 2 channels: - ph control (acid, base) - Feed + ph control (one direction) - 2x Feed 12

13 ph Profile Settings 13

14 Feed Profile Settings - Feeding profile (constant, linear, exponential) - Signal triggered feeding (e.g. DO-controlled) 14

15 Microfluidic Pump Scheme for Fed-Batch Process control (ph-control, fed-batch) reservoir with pressure connection reaction well microtiter plate optode fluidîc layer membrane pneumatic layer Inlet valve pump chamber Outlet valvel 15

16 Pump Function Flow diagram: 1. Fill pump chamber Pressure Liquid 16

17 Pump Function Flow diagram: 1. Fill pump chamber 2. Close inlet valve Pressure Liquid 17

18 Pump Function Flow diagram: 1. Fill pump chamber 2. Close inlet valve 3. Open outlet valve Pressure Liquid 18

19 Pump Function Flow diagram: 1. Fill pump chamber 2. Close inlet valve 3. Open outlet valve 4. Empty pump chamber Pressure Liquid 19

20 Applications 20

21 Applications Clone screening under different process conditions Media optimization at different ph values Fermentation parameter optimization Optimization of feed profiles in Fed-Batch Scale down model Bioprocess characterization Tool for PAT and QbD 21

22 Examples 22

23 Microfluidic Fed-Batch Cultivation in MTP E. coli K12 fed-batch fermentation with constant feed 6g/L/h Wilms-MOPS minimal medium 10 g/l Glucose, OD start =0.12, V start =500 µl, S F =500 g/l Glucose, n=1000 rpm Funke et al., Microbial Cell Factories 2010, 9:86 23

24 Microfluidic Fed-Batch Cultivation in MTP E. coli K12 fed-batch fermentation with exponential feed (µ=0.2 1/h) Wilms-MOPS minimal medium 10 g/l Glucose, OD start =0.12, V start =500 µl, S F =500 g/l Glucose, n=1000 rpm Funke et al., Microbial Cell Factories 2010, 9:86 24

25 Scale-Up from MTP to Fermenter Microtiter plate Stirred tank reactor Flowerplate, m2p-labs culture volume: 500µl k L a determination with micro-ramos device Scale-up by matched k L a-values k L a 450 1/h Scaling Factor: 2000 Sartorius BIOSTAT B plus culture volume: 1L k L a determination with online exhaust gas analyses 25

26 Scale-Up of ph-control from MTP to Fermenter E.coli K12 in minimal medium (10g/L glucose) acid: 1M H 3 PO 4 ; base: 2M NH 4 ; V start = 500 µl; T = 37 C; OD start = 0.1; BioLector: Ø 3 mm; n=1000 rpm Funke et al., Microbial Cell Factories 2010, 9:86 26

27 Scale-Up of ph-control from MTP to Fermenter E.coli K12 in minimal medium (10g/L glucose) acid: 1 M H 3 PO 4 ; base: 2 M NH 4 MTP: V start = 500 µl; T = 37 C; OD start = 0.1; BioLector: Ø 3 mm; n=1000 rpm fermenter: V start = 1 L; T = 37 C; OD start = 0.1; stirrer speed: 950 rpm Funke et al., Microbial Cell Factories 2010, 9:86 27

28 Scale-Up of ph-control from MTP to Fermenter E.coli K12 in minimal medium (10g/L glucose) acid: 1 M H 3 PO 4 ; base: 2 M NH 4 MTP: V start = 500 µl; T = 37 C; OD start = 0.1; BioLector: Ø 3 mm; n=1000 rpm fermenter: V start = 1 L; T = 37 C; OD start = 0.1; stirrer speed: 950 rpm 28

29 Scale-Up of ph-control from MTP to Fermenter E.coli K12 in minimal medium (10g/L glucose) acid: 1M H 3 PO 4 ; base: 2M NH4 MTP: V start = 500 µl; T = 37 C; OD start = 0.1; BioLector: Ø 3 mm; n=1000 rpm fermenter: V start = 1L; T = 37 C; OD start = 0.1; stirrer speed: 950rpm 29

30 Conclusion BioLector Pro with Microfluidics Real Fed-batch cultivation in micro-scale ( µl) No liquid handling system required Accurate ph control with acid and/or base (max. 2 lines) Dosing with less than 50 nl 32 individual controlled fermentations Results scalable to standard stirred tank bioreactor 30

31 Automation of Microbioreactors 31

32 Flexible Automation of the BioLector Robot + BioLector = RoboLector Freedom Evo, Tecan Microlab Star, Hamilton Huber et al., Microbial Cell Factories 2009, 8:42 RoboLector, m2p-labs + Combination with HT Downstream Processing RoboColumns, Atoll GmbH 32

33 Fermentation in the RoboLector with online Multiparameter Monitoring x Riboflavins (488/520 nm) [a.u.] Scattered light [a.u.] Cal. ph [ ] NADH (365/450 nm) [a.u.] Cal. po 2 [% a.s.] Actual volume [µl] Time [h]

34 Applications of the RoboLector Platform Media Optimization Fed-batch Processing Automated Sampling Growth Synchronization Induction Profiling 34

35 Summary BioLector High-Throughput Fermentation Online Monitoring Scalability BioLector Pro + individual ph Control + Fed-batch Processing RoboLector + Automated Sampling + Automated Induction + Automated Feeding Fully Controlled and Automated Bioprocessing Plattform 35

36 Thank you for your attention! Questions? Contact: Frank Kensy from microreactor to process