On-line monitoring of all key parameters in shake flasks ......combined with different feeding...

www.avt.rwth-aachen.de

Prof. Dr. Jochen Büchs

AVT - Biochemical Engineering, RWTH Aachen University

Building NGP2, D - 52074 Aachen, Germany

e-mail: [email protected]


On-line monitoring of all key parameters in

shake flasks and microtiter plates

combined with different feeding options

Bio Processing Days, Recklinghausen 19. 2. 2018


2

Successive steps of bioprocess development

Modified after Bareither and Pollard (Merck & Co. Inc.), Biotechnol. Prog. 27 (2011) 2-14

Static & shaken cultures Stirred cultures


3

Scalability between shaken and stirred microbial cultures


4


Prerequisites:

1) If sufficient oxygen supply is provided,

Bacteria and yeast may require for a medium with 20 g/L glucose

in a non-baffled 250 mL shake flask a

shaking frequency of minimum 350 rpm and a

filling volume of maximum 10 mL.

If a culture is oxygen limited, online measurement doesn’t make sense.

You will only be able to observe the level of mass transfer (low DOT, linear

increase of biomass) and not the characteristics of the cultured biology.


5


Prerequisites:


2) the mode of pH-control (use of the same buffer type and amount)

If you use a pH-buffer in shaken cultures and pH-control in stirred

cultures, you are changing two parameters at the same time:

osmotic pressure and course of pH over time


6


Prerequisites:


2) the mode of pH-control (use of the same buffer type and amount)

3) and the same inoculated medium (no separate sterilization !)

are the same in all scales, cultures can readily be scaled between

shaken and stirred cultures.

Exceptions may be cultures with filamentous microorganisms or

mammalian cells.


7

Exp

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High throughput and (!) high information contentFeeding

On-line monitoring

On-line measurement, feeding and control options


8

• Introduction to meanwhile established equipment for online

monitoring of shake flasks

(pH by optodes, DOT by optodes and suspended particles, OD by

Cell Growth Quantifier and OTR, CTR and RQ by RAMOS)

• Introduction to the BioLector and RoboLector technology (for MTPs)

• New µRAMOS technology & combination with BioLector and

well-resolved 2D-spectroscopy for online monitoring of substrates,

products and more

• Alternative experimental methodology

• Feeding by membrane-based fed-batch shake flask

• Feeding by polymer-based controlled release materials or by

microfluidic pumps in MTPs

Agenda of this talk


9








products and more





Agenda of this talk


10

pH-measurement in shake flasks applying optodes

(from a brochure of Kühner AG)


0 2 4 6 8 10 120

10

20

30

40

50

60

70

6.4

6.6

6.8

7.0

7.2

7.4

OTR

Oxy

gen

tra

nsf

er r

ate

(O

TR

)

[mm

ol/

L/h

]

Time [h]

on-line pH

On

-lin

e p

H

[-]

E. coli BL 21 pLys pRset eYFP-IL6, mineral medium, 37°C, 50 mm shaking diameter, 350 rpm; 10 mL filling volume

Combination of on-line pH and oxygen transfer rate

measurement in shake flasks

Scheidle et al. (2007), Sensors 7, 3472-3480 11


Calculated liquid distribution in shaking flasks250 ml flask, shaking diameter 25 mm, filling volume 25 ml, shaking frequency 200 rpm

0

1

2

3

4

5

6

-4

-3

-2

-1

0

1

2

3

4

y-Axis (cm)

-4 -3 -2 -1 0 1 2 3 4

x-Axis (cm)

Height (cm)

maximumliquid height

(3.07 cm)

Problem of measurement of DOT in shake flasks with optodes

14


Time [h]

DOT (suspendednanoparticles)

OTR

DOT (optode)

Comparison of DOT measurements during K. lactis cultivationsComplex YEP medium with 40 g/L glucose, 250 mL shake flask, 10 mL filling volume, 200 rpm, 50

mm shaking diameter, 30°C

15Flitsch et al., Microb Cell Fact (2016) 15:45


From a brochure of the company BlueSens

Device for on-line measurement of O2- and CO2-transfer rate (OTR, CTR, RQ)

)p(ppV

1kOTR O2outO2,

absLplugplug

Limited to ≥500 mL flasks


15Anderlei et al., Biochem. Eng. J. 17 (2004) 187-194


Respiration Activity MOnitoring System (RAMOS)


16

Commercialized by

Kühner AG Birsfelden, Swiss

and HiTec Zang GmbH,

Herzogenrath, Germany


Respiration Activity MOnitoring System (RAMOS)

Anderlei et al., Biochem. Eng. J. 17 (2004) 187-194


17

(optode)

Comparison of DOT measurements during K. lactis cultivations

Flitsch et al.,

Microb Cell Fact (2016) 15:45


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products and more





Agenda of this talk


19

x-y manipulator shaker

MTP 1

MTP 2

MTP 3

MTP 4optical fibre bundle

gas hood for

humidified air

BioLector device for parallel quasi-continuous on-line

measurement of several parameters in micro titre plates

Samorski et al., Biotechnol. Bioeng. 92 (2005) 61-68

384 wells !

to / from

fluorescence

spectrometer


20

x-y manipulator shaker

MTP 1

MTP 2

MTP 3

MTP 4optical fibre bundle

gas hood for

humidified air

BioLector device for parallel quasi-continuous on-line

measurement of several parameters in micro titre plates

Samorski et al., Biotechnol. Bioeng. 92 (2005) 61-68

384 wells !

to / from

fluorescence

spectrometerIt is possible to measure OD, pH, DOT, tryptophan-,

NADH-, riboflavin- and GFP(derivative)-fluorescence


21

Device for parallel quasi-continuous on-line measurement of

several parameters in one micro titre plate (48 or 96 wells !)

Commercialized by

m2p-labs, Baesweiler

Kensy et al., Microbial Cell Factories 8:31 (2009)


22

Combination of pipetting robot (laminar flow) and BioLector


Huber et al., Microbial Cell Factories 8:42 (2009)

Completely automated experimentation


23

Experimental procedure using micro bioreactorsE. coli BL21 (DE3) pRhotHi-2-EcFbFP cultured on glucose & sorbitol

Oxy

gen

tra

nsf

er r

ate

(OT

R)

[m

mo

l/L/h

]

Wewetzer et al., J. Biol. Eng. 9:9 (2015)


24

Experimental procedure using micro bioreactors

Oxy

gen

tra

nsf

er r

ate

(OT

R)

[m

mo

l/L/h

]

E. coli BL21 (DE3) pRhotHi-2-EcFbFP cultured on glucose & sorbitol



25

Experimental procedure using micro bioreactors

Oxy

gen

tra

nsf

er r

ate

(OT

R)

[m

mo

l/L/h

]

E. coli BL21 (DE3) pRhotHi-2-EcFbFP cultured on glucose & sorbitol

Eight such experiments are conducted in parallel !



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products and more





Agenda of this talk


27

Transition of the RAMOS into the µRAMOS technology

8 shake flasks 48 wells of a MTP

Flitsch et al., J. Biol. Eng. 10:14 (2016)


Cultivation of E. coli BL21 (DE3) pRotHi-YFP in the new µRAMOS deviceE. coli BL21 (DE3) pRotHi-YFP, Synthetic Wilms-MOPS auto-induction medium 0.55 g/L glucose, 2 g/L lactose and 5 g/L glycerol.

RAMOS: 250 mL shake flask, VL = 10 mL, n = 350 rpm, d0 = 50 mm, 37°C.µRAMOS: 48-well round well plate, VL = 800 µL, n = 1000 rpm, d0 = 3 mm, 37°C.

Standard deviation

28Flitsch et al., J. Biol. Eng. 10:14 (2016)


29

Combination of µRAMOS and BioLector device

Ladner et al., Microbial Cell Factory (2016) 15:206

µRAMOS

BioLector


Cultivation of H. polymorpha with varying magnesium concentrations

30

µRAMOS

Cultivation conditions:Synthetic Syn-6-MES medium with 10 g L-1 glycerol and different concentrations of magnesium,48-well round Plate, 30 °C,n = 1000 rpm, VL = 0.8 mL,d0 = 3 mm


percentages refer to 3 g/L MgSO4ꞏ7H2O



31

RAMOS

BioLector

Cultivation conditions:Synthetic Syn-6-MES medium with 10 g L-1 glycerol and different concentrations of magnesium,48-well round Plate, 30 °C,n = 1000 rpm, VL = 0.8 mL,d0 = 3 mm


32




33








products and more





Agenda of this talk


34

Classical 2D-fluorescence spectroscopy in fermenters

Faassen and Hitzmann, Sensors 15 (2015) 10271-10291


35

Classical 2D-fluorescence spectroscopy in fermenters

Faassen and Hitzmann, Sensors 15 (2015) 10271-10291


Conventional procedure to obtain online information

from fermentation processes by 2D-fluorescence spectroscopy

36Faassen and Hitzmann, Sensors 15 (2015) 10271-10291

Off-line analysis

Taking samples

Off-line generation

of chemometric

models

2D-fluorescence

spectra

2D-fluorescence

spectra

Additional

fermentations

On-line

process information

Cell dry weight

Glucose

Time


Cultivation conditions:

H. polymorpha RB11

pC10-FMD (PFMD-GFP),

synthetic Syn-6-MES medium,

48-well round Plate, 30°C,

n = 1000 rpm, VL = 0.8 mL,

d0 = 3 mm

37

Well resolved evaluation of complete 2D-spectra in MTPs

Ladner et al., Biotechnology J. 11 (2016) 1605-1616


38Ladner et al., Biotechnology J. 11 (2016) 1605-1616

Cultivation of H. polymorpha on Syn6-MES medium


H. polymorpha RB11


Synthetic Syn-6-MES medium

with glycerol,


n = 1000 rpm, VL = 0.8 mL,

d0 = 3 mm

8.9 g/L glycerol

0.6 g/L CDWstart

Offline samples taken

by a pipetting robot





H. polymorpha RB11



with glycerol,


n = 1000 rpm, VL = 0.8 mL,

d0 = 3 mm

8.9 g/L glycerol

0.6 g/L CDWstart

Model development by fluorescence

data of one single well





H. polymorpha RB11



with glycerol,


n = 1000 rpm, VL = 0.8 mL,

d0 = 3 mm

8.9 g/L glycerol

0.6 g/L CDWstart

Prediction of parallel cultures

with same initial conditions




H. polymorpha RB11



with glycerol,


n = 1000 rpm, VL = 0.8 mL,

d0 = 3 mm

8.9 g/L glycerol

0.6 g/L CDWstart

Calibration Prediction

9.6 g/L glycerol

0.5 g/L CDWstart

H. polym. - prediction of cultures with different initial conditions



H. polymorpha RB11



with glycerol,


n = 1000 rpm, VL = 0.8 mL,

d0 = 3 mm

8.9 g/L glycerol

0.6 g/L CDWstart


9.6 g/L glycerol

0.5 g/L CDWstart


H. polymorpha - validation of predicted values



Cultivation of E. coli on synthetic Wilms-MOPS medium

9.2 g/L glucose


E. coli Tuner (DE3) pRhotHi-2-

LacI-eYFP.

Synthetic Wilms-MOPS medium

with glucose,


n = 1000 rpm, VL = 0.8 mL,

d0 = 3 mm

Offline samples taken

by a pipetting robot




Model development by fluorescence

data of one single well9.2 g/L glucose



LacI-eYFP.


with glucose,


n = 1000 rpm, VL = 0.8 mL,

d0 = 3 mm




Prediction of parallel cultures

with same initial conditions9.2 g/L glucose



LacI-eYFP.


with glucose,


n = 1000 rpm, VL = 0.8 mL,

d0 = 3 mm



E. coli - prediction of cultures with different initial conditions

9.2 g/L glucose


6.8 g/L glucose



LacI-eYFP.


with glucose,


n = 1000 rpm, VL = 0.8 mL,

d0 = 3 mm





LacI-eYFP.


with glucose,


n = 1000 rpm, VL = 0.8 mL,

d0 = 3 mm

9.2 g/L glucose


6.8 g/L glucose

E. coli - validation of predicted values


48

• A repertoire of online measuring techniques for shaken

bioreactors is now available that outperform those for stirred tank

bioreactors in number and quality.

• Due to the absence of bubbles optical measurement can be

conducted under much more defined conditions.

Intermediate conclusion


49








products and more





Agenda of this talk


Two-phase (diauxic) growth of Escherichia coli in culture media,

which contain glucose and sorbitol in the specified ratios

from MONOD, Recherches sur la croissance des cultures bastériennes. Hermann, Paris 1958

glucose : sorbitol1 : 1



Time [h]

Op

tica

l den

sity

[-

]

62








Time [h]

Op

tica

l den

sity

[-

]

Don‘t sample and analyse the compounds you are interested in

(too tedious).

Instead, vary the concentration of these compounds in parallel

reactors.

You will directly obtain the relevant information (response of the

culture).

62








Time [h]

Op

tica

l den

sity

[-

]

62


0 2 4 6 8 10 12 14 16

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

E. coli BL 21 pRSET eYFP-IL6; Wilms-MOPS, 200mM MOPS, 20 g/L glucose, 2 g/L sorbitolT=30°C; d

0=50mm; n=350 rpm; V

L=10 ml; OD

0=0.2

25 min rinsing, 0.9 min highflow, 5 min stopping

Time [h]

Oxy

gen

tra

nsf

er r

ate

(OT

R)

[m

ol/

L/h

]

Three-phase (triauxic) metabolism of Escherichia coli on glucose and sorbitol

Hansen et al., J. Biol. Eng. 6:11 (2012) 53


0.00

0.01

0.02

0.03

0.04

0.05

0.06

OT

R

[mo

l/L/h

]

/

0.0

0.5

1.0

1.5

2.0

2.5

Ac

eta

te

[g/L

]

So

rbit

ol

[g

/L]

0

5

10

15

20

Glu

co

se

[g

/L]

0.00

0.01

0.02

0.03

0.04

0.05

0.06

OT

R

[mo

l/L/h

]

/

0.0

0.5

1.0

1.5

2.0

2.5

Ac

eta

te

[g/L

]

So

rbit

ol

[g

/L]

0

5

10

15

20

Glu

co

se

[g

/L]

0.00

0.01

0.02

0.03

0.04

0.05

0.06

OT

R

[mo

l/L/h

]

/

0.0

0.5

1.0

1.5

2.0

2.5

Ac

eta

te

[g/L

]

So

rbit

ol

[g

/L]

0

5

10

15

20

Glu

co

se

[g

/L]

0 2 4 6 8 10 12 140.00

0.01

0.02

0.03

0.04

0.05

0.06

Time [h]

OT

R

[mo

l/L/h

]

/

0.0

0.5

1.0

1.5

2.0

2.5

Ac

eta

te

[g/L

]

So

rbit

ol

[g

/L]

0

5

10

15

20

Glu

cose

[g

/L]

Oxygen transfer rates (OTR) of E.

coli BL21 pRSET eYFP-IL6 in

mineral medium with different

initial sorbitol concentrations

Experimental conditions:

Wilms-MOPS medium with 20 g/L glucose

and different sorbitol concentrations

30°C, initial pH = 7.5

250 mL flask, 10 mL filling volume

50 mm shaking diameter

350 rpm shaking frequency

0.5 g/L sorbitol

1 g/L sorbitol

1.5 g/L sorbitol

2 g/L sorbitol

54

Hansen et al., J. Biol. Eng. 6:11 (2012)


55

Exp

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High throughput and (!) high information contentFeeding

On-line monitoring

On-line measurement, feeding and control options


56








products and more





Agenda of this talk


Offline fed-batch shake flask

Online fed-batch shake flask

Any compound (even liquids like glycerol) and also

mixtures can be fed at virtually any rate.

Flexible membrane-based feeding for shake flasks

57Philip et al., Microbial Cell Factories 16:122 (2017)


Offline fed-batch shake flask

Online fed-batch shake flask

Any compound (even liquids like glycerol) and also

mixtures can be fed at virtually any rate.


58Philip et al., Microbial Cell Factories 16:122 (2017)

Must be functional at the required operating conditions

avoiding oxygen limitation (e.g. 350 rpm, VL = 10 mL) !


59

E.coli BL21 (DE3) pRhotHi-2-EcFbFP, Wilms-MOPS-mineral medium (0.2 M MOPS), 37 °C, 350 rpm,

50 mm shaking diameter, 10 mL filling volume, inoculation OD600 0.5, 2 mL reservoir filling volume, Reichelt

dialysis membrane, 18.1 mm2 membrane area

Philip et al., Microbial Cell Factories 16:122 (2017)



60

Flexible membrane-based feeding for shake flasksParallel cultivations with membrane-based fed-batch shake flasks and 3 L-laboratory scale reactors in

batch and fed-batch operation mode

E.coli BL21 (DE3) pRhotHi-

2-EcFbFP, Wilms-MOPS-

mineral medium (0.2 M

MOPS), 37 °C

Philip et al.

Microbial Cell Factories

16:122 (2017)


61








products and more





Agenda of this talk


62

MTP feeding glucose from controlled release system

glucose

feeding

polymer based controlled release systems

Commercialized

by Kühner Deutschland GmbH, Kohlscheid


BioLector Pro – Microfluidic Bioprocess Control

32 parallel microbioreactors with

active pH control and continuous substrate feeding

63

Commercialized

by m2p-labs, Baesweiler


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Scale-up from MTP to fermenterMicrotiter plate

Sartorius BIOSTAT Bplus

culture volume: 1 L

kLa determination with

micro-RAMOS device

kLa determination with

online exhaust gas analyser

Flowerplate, m2p-labs

culture volume: 500 µL

Stirred tank reactor

Scaling Factor:

2000

matched kLa-values

kLa ≈ 450 1/h

Funke et al., Microbial Cell Factories 2010, 9:86


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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

DO

T

[%]



66

Scale-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


Scale up from our microfluidic MTP’s into lab

scale fermentors is possible without restrictions.


67

Successive steps of bioprocess development

Small scale

systems

n 96

n 48

Modified after Bareither and Pollard (Merck & Co. Inc.), Biotechnol. Prog. 27 (2011) 2-14


68

Front view of the major’s house Cathedral of Aachen

Thank you for your attention !

I am looking forward to answering

your questions.

On-line monitoring of all key parameters in shake flasks ......combined with different feeding...

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Transcript of On-line monitoring of all key parameters in shake flasks ......combined with different feeding...