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 9
New Microfluidic Platform –
BioLector® Pro
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 13
pH Profile Settings
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
Pump Function
Flow diagram:1. Fill pump chamber
16
Pressure
Liquid
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 20
Applications
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 22
Examples
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 31
Automation of Microbioreactors
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
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