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from microreactor to process
NOSPEED LIMIT
Frank Kensy, m2p-labs GmbHCLIB-Forum, 3. April 2014CREATIVE CAMPUS MONHEIM
Full Bioprocess Control in Microbioreactors –A new Option for Scale Down Models
from microreactor to process
m2p-labs – The Microbioreactor Company
Company profileCompany profile
Milestones• Spin-off from RWTH Aachen University in 2005• Market entry with first product end of 2007• >85 devices placed in the market
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
Locations & Key Facts• GmbH located in Baesweiler, Germany and Inc. in NY, USA• Ca. 500 m² office and laboratory space• Currently 17 FTE
2
from microreactor to process
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
Trends and Demands in Biotechnology
State-of-the-art: laborious and expensive systems
BioLector
3
from microreactor to process 4
Microbioreactors for better Process Understanding
Oxygen
pH
Biomass & Fluorescence
48x
Old Technology BioLector ® Technology
24x
1x
from microreactor to process 5
High parallelisation (48 reactors)
Small working volume (800µl – 2400µl)
Standard MTP format automation
Non-invasive online measurements
Defined mass transfer conditions
Temperature, humidity and gassing control
Simple handling, calibration free, no tubings
BioLector
High-Throughput Fermentation System
from microreactor to process 6
- 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
FlowerPlate®: New Horizons at Microscale
*
-> same reactor performance like industrial bioreactors
*new Geometries Patent pending In collaboration with:
from microreactor to process 7
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
Media Optimization
Huber et al., BMC Biotechnology 2011, 11:22
from microreactor to processTime [h]
0 2 4 6 8 10 12 14 16
CD
W [g
. L-1]
lip. a
ct. [
U. m
L-1]
02468
10121416
CDWlip. act.
Time [h]0 2 4 6 8 10 12 14 16
CD
W [g
. L-1]
lip. a
ct. [
U. m
L-1]
02468
10121416
CDWlip. act.
NprE YwmC YpjP Empty
spec
ific
activ
ity [U
. mg-1
]
0.00.20.40.60.81.01.21.4
NprE YwmC YpjP Empty
spec
ific
activ
ity [U
. mg-1
]
0.00.20.40.60.81.01.21.4
C.glutamicum ATCC 13032pEKEX2::SP-Cutinase T=30°C, 1200 rpm, 3 mm, media: CG XII, 0.5 mM IPTG
CDW [mg.mL-1]0 2 4 6 8 10 12 14
lipol
ytic
act
ivity
[U. m
L-1]
02468
101214
BioLector (1 mL)
Bioreactor (1 L)
1.05 +/- 0.06 U.mg-1
µ = 0.4 h-1
µ = 0.4 h-1
NprE-Cutinase
NprE-Cutinase
CDW [mg.mL-1]0 2 4 6 8 10 12 14
lipol
ytic
act
ivity
[U. m
L-1]
02468
101214
1.07 +/- 0.03 U.mg-1 spec
.lip.
act.
[U. m
g-1 ]
spec
.lip.
act.
[U. m
g-1 ]
8
Scalability: Corynebacterium glut.
Scale-up factor 1000 equal µ, YX/S, YP/X
Rohe et al.,Microbial Cell Factories 2012, 11:144
from microreactor to process
Current Practice in Bioprocess R&D
0.5 - 20L
1 experiment
Volume: Most bioprocesses are conducted as fed-batch processes!
time bi
omas
s, fe
ed
batch fed-batch
Advantages:• controlled process• no overflow• high productivity
10
biomass
feed
from microreactor to process
BioLector® Pro – Full Bioprocess Control at Micro-Scale
11
Scale upIn collaboration with:
from microreactor to process 12
Design of the Microfluidic Control Chip
2 Reservoir Wells
- 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
4 Cultivation Wells
pH channels
Feeding channels
from microreactor to process 14
Feed Profile Settings
- Feeding profile (constant, linear, exponential)- Signal triggered feeding (e.g. DO-controlled)
from microreactor to process
Microfluidic Pump Scheme for Fed-Batch
fluidîc layer
membrane
pneumatic layer
microtiter plate
reservoir with pressure connection reaction well
pump chamber
optode
Inlet valve Outlet valvel
Process control(pH-control, fed-batch)
15
from microreactor to process
Flow diagram:1. Fill pump chamber2. Close inlet valve
Pump Function
17
Pressure
Liquid
from microreactor to process
Flow diagram:1. Fill pump chamber2. Close inlet valve3. Open outlet valve
Pump Function
18
Pressure
Liquid
from microreactor to process
Flow diagram:1. Fill pump chamber2. Close inlet valve3. Open outlet valve 4. Empty pump chamber
Pump Function
19
Pressure
Liquid
from microreactor to process 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
from microreactor to process
Microfluidic Fed-Batch Cultivation in MTP
E. coli K12 fed-batch fermentation with constant feed 6g/L/hWilms-MOPS minimal medium 10 g/L Glucose, ODstart=0.12, Vstart =500 µL, SF=500 g/L Glucose, n=1000 rpm
23Funke et al.,Microbial Cell Factories 2010, 9:86
from microreactor to process
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, ODstart=0.12, Vstart =500 µL, SF=500 g/L Glucose, n=1000 rpm
24Funke et al.,Microbial Cell Factories 2010, 9:86
from microreactor to process
Scale-Up from MTP to Fermenter
Microtiter plate
Sartorius BIOSTAT Bplusculture volume: 1L
kLa determination with micro-RAMOS device
kLa determination withonline exhaust gas analyses
Flowerplate, m2p-labsculture volume: 500µl
Stirred tank reactor
Scale-up by matched kLa-values
kLa ≈ 450 1/h
Scaling Factor:2000
25
from microreactor to process
Scale-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
26Funke et al.,Microbial Cell Factories 2010, 9:86
from microreactor to process
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
Scale-Up of pH-Control from MTP to Fermenter
27Funke et al.,Microbial Cell Factories 2010, 9:86
from microreactor to process
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
Scale-Up of pH-Control from MTP to Fermenter
28
from microreactor to process
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
Scale-Up of pH-Control from MTP to Fermenter
29
from microreactor to process 30
Conclusion 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 32
Flexible Automation of the BioLector
Huber et al., Microbial Cell Factories 2009, 8:42
Freedom Evo,Tecan
Microlab Star, Hamilton
RoboLector,m2p-labs
Robot + BioLector = RoboLector
+ Combination with HT Downstream Processing
RoboColumns, Atoll GmbH
from microreactor to process 33Time [h]
0 12 24 36 48 60 72
Scattered light [a.u.]
‐800
‐600
‐400
‐200
0
200
400
600
Ribo
flavins (4
88/520
nm) [a
.u.]
‐2
‐1
0
1
2
3
4
5
6
7
NAD
H (3
65/450
nm) [a
.u.]
‐10
‐5
0
5
10
15
20
25
Cal. pH
[‐]
‐5
‐4
‐3
‐2
‐1
0
1
2
3
4
5
6
7Ca
l. pO
2 [% a.s.]
0
50
100
150
200
250
300
350
400
Actual volum
e [µL]
200
400
600
800
1000
1200
140048 x
Fermentation in the RoboLectorwith online Multiparameter Monitoring
from microreactor to process 34
Applications of the RoboLector Platform
Media Optimization
Growth Synchronization Induction Profiling
Fed-batch Processing
Automated Sampling
from microreactor to process 35
Summary
BioLector® • High-Throughput Fermentation
• Online Monitoring
• Scalability
+ individual pH Control
+ Fed-batch Processing
+ Automated Sampling
+ Automated Induction
+ Automated Feeding
Fully Controlled and Automated Bioprocessing Plattform
BioLector® Pro
RoboLector®
from microreactor to process
Thank you for your attention!
Questions?
Contact:Frank [email protected]+49-2401-805331www.m2p-labs.com