from microreactor to process - clib2021 · from microreactor to process m2p-labs – the...

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


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


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


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


- 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:


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


New Microfluidic Platform –

BioLector® Pro


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


BioLector® Pro – Full Bioprocess Control at Micro-Scale

11

Scale upIn collaboration with:


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


pH Profile Settings


Feed Profile Settings

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


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


Pump Function

Flow diagram:1. Fill pump chamber

16

Pressure

Liquid


Flow diagram:1. Fill pump chamber2. Close inlet valve

Pump Function

17

Pressure

Liquid


Flow diagram:1. Fill pump chamber2. Close inlet valve3. Open outlet valve

Pump Function

18

Pressure

Liquid


Flow diagram:1. Fill pump chamber2. Close inlet valve3. Open outlet valve 4. Empty pump chamber

Pump Function

19

Pressure

Liquid


Applications


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


Examples


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


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



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


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



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




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


28


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


29


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


Automation of Microbioreactors


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


Applications of the RoboLector Platform

Media Optimization

Growth Synchronization Induction Profiling

Fed-batch Processing

Automated Sampling


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®


Thank you for your attention!

Questions?

Contact:Frank [email protected]+49-2401-805331www.m2p-labs.com

from microreactor to process - clib2021 · from microreactor to process m2p-labs – the...

Documents