NO SPEED LIMIT Full Bioprocess Control in Microbioreactors - A new Option for Scale Down Models - CLIB2021
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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
from microreactor to process
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
from microreactor to process 2
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
from microreactor to process 3
Microbioreactors for better Process Understanding
Old Technology BioLector ® Technology
Biomass &
1x Fluorescence
Oxygen
pH
24x
48x
from microreactor to process 4
High-Throughput Fermentation System
High parallelisation (48 reactors)
Small working volume (800µl – 2400µl) BioLector
Standard MTP format automation
Non-invasive online measurements
Defined mass transfer conditions
Temperature, humidity and gassing control
Simple handling, calibration free, no tubings
from microreactor to process 5
FlowerPlate®: New Horizons at Microscale
- high mass transfer OTR (> 0.11 mol/L/h)
- broad volume range (0.8 – 1.5 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:
from microreactor to process 6
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.,
from microreactor to process BMC Biotechnology 2011, 11:22 7
Scalability: Corynebacterium glut.
Bioreactor (1 L) Scale-up factor 1000
NprE-Cutinase
16 14
equal µ,1.4YX/S, YP/X
lipolytic activity [U mL ]
. -1
]
. -1
14 CDW 12
spec.lip.act. [U mg-1]
specific activity [U. mg
1.2
lip. act. [U mL ]
-1
12 lip. act. 10 1.0
CDW [g L ]
. -1
10 8 0.8
.
8 µ = 0.4 h-1 0.6
6
6 0.4
4 4 0.2
2 2 1.07 +/- 0.03 U.mg-1 0.0
0 NprE YwmC YpjP Empty
0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14
Time [h] CDW [mg.mL-1]
C.glutamicum ATCC 13032
BioLector (1 mL) pEKEX2::SP-Cutinase
T=30°C, 1200 rpm, 3 mm,
media: CG XII, 0.5 mM IPTG
NprE-Cutinase
16 14
lipolytic activity [U mL ]
. -1
14 12
CDW
lip. act. [U mL ]
-1
12 1.4
-1] ]
. -1
10
CDW [g L ]
lip. act.
. -1
mg
10 1.2
[U.mg
8
.
8 1.0
activity[U
6 µ = 0.4 h-1 6 0.8
spec.lip.act.
4 0.6
4
0.4
2 2 1.05 +/- 0.06 U.mg-1
specific
0.2
0 0 0.0
0 2 4 6 8 10 12 14 16 0 2 4 6
8 10 12 14 NprE YwmC YpjP Empty
Time [h] -1
CDW [mg.mL ] Rohe et al.,
from microreactor to process Microbial Cell Factories 2012, 11:144 8
New Microfluidic Platform –
BioLector® Pro
from microreactor to process 9
Current Practice in Bioprocess R&D
Volume: 0.5 - 20L Most bioprocesses are conducted
as fed-batch processes!
batch fed-batch
biomass, feed
biomass
feed
time
Advantages:
• controlled process
• no overflow
1 experiment • high productivity
from microreactor to process 10BioLector® Pro – Full Bioprocess Control at Micro-Scale In collaboration Scale up with: from microreactor to process 11
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
from microreactor to process 12pH Profile Settings from microreactor to process 13
Feed Profile Settings
- Feeding profile (constant, linear, exponential)
- Signal triggered feeding (e.g. DO-controlled)
from microreactor to process 14Microfluidic 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
from microreactor to process 15Pump Function
Flow diagram:
1. Fill pump chamber Pressure
Liquid
from microreactor to process 16Pump Function
Flow diagram:
1. Fill pump chamber Pressure
2. Close inlet valve
Liquid
from microreactor to process 17Pump Function
Flow diagram:
1. Fill pump chamber Pressure
2. Close inlet valve
Liquid
3. Open outlet valve
from microreactor to process 18Pump Function
Flow diagram:
1. Fill pump chamber Pressure
2. Close inlet valve
Liquid
3. Open outlet valve
4. Empty pump chamber
from microreactor to process 19Applications from microreactor to process 20
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
from microreactor to process 21Examples from microreactor to process 22
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, ODstart=0.12, Vstart =500 µL, SF=500 g/L Glucose, n=1000 rpm
Funke et al.,
from microreactor to process Microbial Cell Factories 2010, 9:86 23Microfluidic 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, ODstart=0.12, Vstart =500 µL, SF=500 g/L Glucose, n=1000 rpm
Funke et al.,
from microreactor to process Microbial Cell Factories 2010, 9:86 24Scale-Up from MTP to Fermenter
Microtiter plate Stirred tank reactor
Scale-up by
matched kLa-values
kLa ≈ 450 1/h
Flowerplate, m2p-labs Sartorius BIOSTAT Bplus
culture volume: 500µl culture volume: 1L
kLa determination with
Scaling Factor: kLa determination with
micro-RAMOS device 2000 online exhaust gas analyses
from microreactor to process 25Scale-Up of pH-Control from MTP to Fermenter
E.coli K12 in minimal medium (10g/L glucose)
acid: 1M H3PO4; base: 2M NH4; Vstart = 500 µL; T = 37 °C; ODstart = 0.1; BioLector: Ø 3 mm; n=1000 rpm
Funke et al.,
from microreactor to process Microbial Cell Factories 2010, 9:86 26Scale-Up of pH-Control from MTP to Fermenter
E.coli K12 in minimal medium (10g/L glucose)
acid: 1 M H3PO4; base: 2 M NH4 MTP: Vstart = 500 µL; T = 37 °C; ODstart = 0.1; BioLector: Ø 3 mm; n=1000 rpm
fermenter: Vstart = 1 L; T = 37 °C; ODstart = 0.1; stirrer speed: 950 rpm
Funke et al.,
from microreactor to process Microbial Cell Factories 2010, 9:86 27Scale-Up of pH-Control from MTP to Fermenter
E.coli K12 in minimal medium (10g/L glucose)
acid: 1 M H3PO4; base: 2 M NH4 MTP: Vstart = 500 µL; T = 37 °C; ODstart = 0.1; BioLector: Ø 3 mm; n=1000 rpm
fermenter: Vstart = 1 L; T = 37 °C; ODstart = 0.1; stirrer speed: 950 rpm
from microreactor to process 28Scale-Up of pH-Control from MTP to Fermenter
E.coli K12 in minimal medium (10g/L glucose)
acid: 1M H3PO4; base: 2M NH4 MTP: Vstart = 500 µL; T = 37°C; ODstart = 0.1; BioLector: Ø 3 mm; n=1000 rpm
fermenter: Vstart = 1L; T = 37°C; ODstart = 0.1; stirrer speed: 950rpm
from microreactor to process 29Conclusion BioLector® Pro with Microfluidics
• Real Fed-batch cultivation in micro-scale (800–2400 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
from microreactor to process 30Automation of
Microbioreactors
from microreactor to process 31Flexible Automation of the BioLector
Robot + BioLector = RoboLector
Freedom Evo, Microlab Star, RoboLector,
Tecan Hamilton m2p-labs
Huber et al.,
Microbial Cell Factories 2009, 8:42
+ Combination with HT Downstream Processing
RoboColumns,
from microreactor to process Atoll GmbH 32Fermentation in the RoboLector
with online Multiparameter Monitoring
1400 400 7 25 7 600
48 x
6
350 6
20 400
1200 5
5
300 4
15 200
Riboflavins (488/520 nm) [a.u.]
1000 3 4
NADH (365/450 nm) [a.u.]
Scattered light [a.u.]
Actual volume [µL]
250
Cal. pO2 [% a.s.]
2 10 0
Cal. pH [‐]
3
800 200 1
2
0 5 ‐200
150
600 ‐1 1
0 ‐400
100 ‐2
0
400 ‐3
‐5 ‐600
50 ‐1
‐4
200 0 ‐5 ‐10 ‐2 ‐800
0 12 24 36 48 60 72
from microreactor to process Time [h] 33Applications of the RoboLector Platform
Media Optimization
Fed-batch Processing
Automated Sampling
Growth Synchronization Induction Profiling
from microreactor to process 34Summary
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
from microreactor to process 35Thank you for your attention!
Questions?
Contact:
Frank Kensy
kensy@m2p-labs.com
+49-2401-805331
from microreactor www.m2p-labs.com
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