1

Prof. Dr. Stefan SchillbergHead of Molecular BiologyFraunhofer IME, Aachen, Germany

Brussels, 27.06.2016

CoMoFarm Contained Molecular Farming:

Controllable Contained Systems for High Yield and Consistency

SC2 Dissemination Event - Rural Renaissance Bio-based Innovation

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Production of clinical grade material in tobacco plants

HIV-specific antibody produced in tobacco (25 mg/kg)

Permission for production of clinical grade material (two-phase process)

Tobacco cultivation and protein extraction in the greenhouse

Protein purification in the GMP facility at the IME

Pre-clinical studies in rabbits successful

Phase I clinical studies with humans successful

GMP facility at the Fraunhofer IME

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Main project ideas

The development of plant-based systems that are grown in containment

The use of controlled environments to ensure process and product consistency

Optimization to improve product yield and quality

Optimization of downstream processing

Duration: 6/2009 – 12/2012

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Consortium overview

Fraunhofer IME, Germany

Zürcher Hochschule, Switzerland

Teknologian tutkimuskeskus VTT, Finland

RWTH Aachen University, Germany

St George’s Hospital Medical School, UK

Plant Research International, The Netherlands

Forschungszentrum Jülich, Germany

Greenovation, Germany

Dow AgroSciences, US

Kühner AG, Switzerland

Protein

analytic

Plant cultivation

Genetic

engineering

Downs

tream

proce

ssing

Automation

Non-invasive

monitoring

Fermentation

technology

Medium

optimisation

Plan

t pr

oduc

tivity

Contained plant

production systems

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Contained plant production platformsTobacco plants

Tobacco MallowHairy roots

Tobacco

Suspendedplants

Moss

Suspended cells

Tobacco Arabidopsis Rice

Soil NFT LED/NFT Rhizo-secretion

Target proteins

Influenza A virus hemagglutinin (HA)

Human M12 antibody (secreted)

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Productivity of different plant systems – M12 antibody

0 50 100 150 200 250 300

Rice, medium

Arabidopsis, medium

Moss, medium

BY-2, medium

Hairy root, medium

Hairy root, tissue

Tobacco rhizosecretion

Tobacco leaves

17 mg/kg/3 weeks

6 mg/L/week

120 mg/L/week

20 mg/L/4 weeks

9 mg/L/week

0.5 mg/L/2 weeks

45 mg/L/week

270 mg/kg/8 weeks

0 5 10 15 20

Rice, medium

Moss, medium

Hairy root, tissue

Hairy root, medium

Arabidopsis, medium

Tobacco leaves

Tobacco rhizosecretion

BY-2, medium

5 mg/kg/day

1.3 mg/L/day

0.9 mg/L/day

0.9 mg/kg/day

0.7 mg/L/day

0.04 mg/L/day

17 mg/L/day

6.4 mg/L/day

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Maximizing M12 yields in culture medium

Medium optimization using fractional factorial designs (FFDs) and response surface methodology (RSM)

BY2

Hairy roots

30-fold compared to MS medium

Main factors are KNO3, NAA and PVP

BY2

Rhizosecretion

500-fold compared to MS medium

BY2

BY2

31-fold compared to MS medium

Strong interactions between several medium compounds

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Development of non-invasive monitoring systems (I)

Development of a non-invasive Respiration Activity Monitoring System (RAMOS) for measurement of the oxygen transfer rate (OTR)

BY-2 MTAD #31; Vflask = 250 mL; VL = 50 mL; d0 = 5 cm; 26 °C, 180 rpm

0

1

2

3

4

5

6

7

8

9

0 24 48 72 96 120 144 168

Time [h]

Oxy

gen

tran

sfer

rate

[m

mol

/(L*h

)] MS Medium

MSN Medium 50 mL tubesMicrotiter plates

Disposable flasks 10-200 L bioreactors

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Commercialization of CoMoFarm developments (Kühner)

Adapter for RAMOS (microtiter plates)

FlowCon gas mixing station

LED-unit for shaken incubators

Adapter for RAMOS (disposable flasks)

SB200-X pH and DO control

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Costs for M12 production in tobacco plants (greenhouse) DSP of 200 kg tobacco leaves containing 400 mg ER-retarded M12 per kg

Position ValueDuration 9-10 weeksTotal costs € 87,550.-M12 production yields ~400 µg/g FLWFinal M12 yield 77 g (~ 88% recovery)DSP 84% of total costsCosts per g M12 € 1,137.-

Consumables DSP 62%

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Novel automated vertical farmfor production of malaria vaccines

Multilayer plant growth levels

Continuous plant production process

Soilless cultivation

Flexible range of plant species

Automated plant scanning

GMP-compliant production + DSP

Funded by the Fraunhofer Zukunfsstiftung

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Additional impact / exploitation

Establishment of plant suspension cells from fruits

Cell line optimization using statistical experimental designs (DoE)

From process to product

Pear

Service tree

More intense cooperation with industrial partners

- Direct funding from industrial partners

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Meeting agenda

www.comofarm.org

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Backup slides

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National

Applied Ecology Division Schmallenberg (1959)

Molecular Biology Division Aachen (2000)

Bioresources Gießen (2009)

TMP Frank-furt (2012)

Biopolymers Münster (2010)

International

Center for Molecular Biotechnology Newark, USA (2001)

Center for Systems Biotechnology Santiago, Chile (2010)

Fraunhofer Institute for Molecular Biology and Applied Ecology IME - R&D facilities

ScreeningPort Hamburg (2014)

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Production of clinical grade material in tobacco plants

HIV-specific antibody produced in tobacco (25 mg/kg)

Permission for production of clinical grade material (two-phase process)

Tobacco cultivation and protein extraction in the greenhouse

Protein purification in the GMP facility at the IME

Pre-clinical studies in rabbits successful

Phase I clinical studies with humans successful

GMP facility at the Fraunhofer IME

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Increased M12 in tobacco plants through breeding

T0

T1

T2

T3

77180

600

268

791

75

0

200

400

600

800

1000

Secreted M12 ER-retarded M12

M12

[mg/

kg F

LW]

9555

M12 levels increased 5-fold and 8-fold, respectively, in subsequent generations

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Maximizing M12 yields in culture medium (II)

Co-expression of secreted M12 antibody and DsRed

Single high fluorescent cells were isolated 5.6-fold improved M12 levels in the culture medium

16.9 17.4

60.165.1

1st FACS rd.M12

[µg/

mL

cultu

re v

olum

e]

DsR

ed fluorescence cells [%]

0

10

20

30

40

50

60

70

80

0

10

20

30

40

50

60

70

80

3.06

53.9

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FACS-based cell line optimisation – ER-retarded M12

Co-expression of M12 antibody and DsRed

Determination of DsRed fluorescence intensity by flow cytometry of 10,000 cells

Optimisation of antibody yield and production stability through FACS-based selection of elite single cells

14-fold improved M12 levels in the ER

0

20

40

60

80

100

120

140

160

180

200

0

50

100

150

200

250

300

350

400

450

M12

ant

ibod

y [µ

g/g

fresh

wei

ght] D

sRed fluorescence intensity [G

eometric m

ean]

13

10

145

105

117

172

1st FACS rd.

182 171

255

448

2nd FACS rd.

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Costs for M12 production in BY2 cells (200 L bioreactor) Transgenic BY2 cell line secreting 20 mg M12 per L

Position Value single run Value 2 refillsDuration 2.5 weeks 4.5 weeksTotal costs € 10,400.- € 16,800.-M12 production yields ~ 20 µg/g FLW ~ 20 µg/g FLW

Final M12 yield 0.67 – 0.88 g (75 – 85% recovery)

2.0-2.6 g (75 – 85% recovery)

DSP 77% of total costs 77% of total costsCosts per g M12 € 12,722.- € 6,224.-

Consumables DSP 68.4%