HYDROPONIC CULTIVATION OF NICOTIANA TABACUM FOR PRODUCTION OF RECOMBINANT PHARMACEUTICAL PROTEINS BY RHIZOSECRETION

Luisa Madeira, Pascal Drake, Tim Szeto and Julian Ma The Hotung Molecular Immunology Unit St. George’s University of London

HYDROPONIC CULTIVATION 

Well established practice in the horticultural industry. 1. Closed environment facilitates Integrated Pest Management. 2. Well suited to organic farming. 3. Not dependent on soil quality. Can be done on non-arable land as well as urban areas. 4. Stable and high yields. High product quality with little variation. 5. Water consumption is reduced by up to 90%. Major advantages for Molecular Pharming

1. Control of the cultivation process. 2. Chemically defined media as compared with soil based systems. 3. Closer to cell fermentation systems.

RHIZOSECRETION Rhizosecretion is a natural process in plants involved in: • Nodulation • Mycorrhizal colonisation • Growth inhibition of neighbouring plants • Acquisition of nutrient from soil

Molecular Milking – rhizosecretion of recombinant proteins was first described for : Green fluorescent protein (0.92mg/g.root dry weight/24h) Human placental alkaline phosphatase (5.8mg/g.root dry weight/24h) Bacterial xylanase (Borisjuk et al 1999) Monoclonal antibody (11.8mg/g.root dry weight/24h) (Drake et al., 2002)

   

Harvest over the lifetime of the plant Only harvest fully processed and secreted proteins Reduced degradation Simpler purification

SPECIFIC AIMS OF THE PROJECT 





1. Investigate strategies to optimise Mab yields from in vitro transgenic plants in hydroponic medium and develop a protocol for production by rhizosecretion. 2. Investigate the rhizosecretome and develop a simple purification strategy for recombinant Mab from hydroponic medium. 3. Determine scalability.

MATERIAL AND METHODS 



Transgenic plants: 

Transgenic plants expressing the Mab M12 were generated by Fraunhofer IME.



Mab M12 was originally raised againstthe MUC-1 epithelial cell tumour marker, but has subsequently been shown to bind to vitronectin.

Plant cultures: 

Seeds were sterilised and sown onto MS agar



Seedlings were transferred to liquid culture after 2-4 weeks (week 0)

Weeks 0-5: cultivation period Weeks 6-10: root optimisation phase Weeks 10 and beyond: harvesting period

NAA INCREASES M12 YIELDS AND ROOT MASS

NITRATE ENRICHMENT (100MM HNO3) INCREASES M12 CONCENTRATION IN HYDROPONIC MEDIUM

Max. yield 45mg/ml/wk at week 12. Average = 25mg/ml/wk over 8 weeks

250 150 100

1

2

3

4

5

1

2

3

4

5

Lane 1 – purified control M12 (40 ng) Lane 2 –WT plants in MS medium Lane 3 – M12 plants in MS medium Lane 4 –WT plants in MSN medium Lane 5 – M12 plants in MSN medium

75 50 37 75

25 20

Anti-light chain Western Blot

OSMOLARITY IS THE MAJOR REASON FOR INCREASED M12 YIELDS 

Enrichment of MS with KNO3 (MSK), NaCl (MSS). Harvesting Phase 35 30

[M12] µg/ml

25

MS

20

MSN 15

MSK

10

MSS

5 0 Week 9



Week 10

Week 11

Week 12

No differences in M12 content in leaf or root tissue were observed

POSSIBLE MECHANISMS OF ACTION OF NITRATE 

1) acted as a fertilizer and increased overall protein production and/or rhizosecretion;



2) increased mass of root tissue

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3) increased the secretion of high molecular weight proteins;

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4) increased antibody stability in hydroponic medium;

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5) decreased protease activity in hydroponic medium;

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6) acted through an as yet unknown mechanism driven by hyperosmolar conditions.

AGITATION OF PLANTS TO INCREASE MAB RELEASE Agitation started

Week 0

1

Seedling transferred to hydroponic culture

2

3

4

5

6 Fresh medium

7

8 NAA

9

10 Fresh medium NAA

11 Fresh medium NAA

... 13 Sample

AGITATION INCREASES PROTEIN RELEASE BUT ALTERS ROOT PHENOTYPE

Shaking M

Ct

Sh

250

Week 8

a

150 100 75 50 37 25 20 15 10

SDS-PAGE of week 8 pooled samples from the Control (Ct) and Shaking (Sh) groups.

Week 10

b

Control

ASSESSMENT OF DIFFERENT NAA TREATMENT PROTOCOLS

Group 2

Group 3

Week 4

Week 5

Fresh MSN/ NAA

NAA

Week 6 Week 7 Week 8 Fresh Fresh MSNg/ NAA MSNg/ NAA NAA Sample Fresh MSN Fresh MSN/ NAA

NAA

NAA

NAA

Week 9 Week 10 Week 11 Fresh Fresh Fresh MSNg/ MSNg/ MSNg/ NAA NAA NAA Sample Sample Sample Fresh Fresh MSNg/ MSNg/ NAA NAA Sample Fresh Fresh MSNg/ MSNg/ NAA NAA Sample

Week 12 Week 13

Fresh MSNg/ NAA Sample Fresh MSNg/ NAA Sample

30 25 20 Group 1 15

Group 2 Group 3

10 5 0 8

9

10

11 Week

12

13

14

Week 14

Sample

35

[M12] µg/ml

Group 1

Week 0 Set up hydroponic cultures in MS Set up hydroponic cultures in MS Set up hydroponic cultures in MS

Fresh MSNg/ NAA Sample Fresh MSNg/ NAA Sample

Sample

Sample

CHARACTERISATION OF THE TOBACCO RHIZOSECRETOME 1

2

3

4

Lane 1: Markers 2: Transgenic (CV-N) plant in MS medium 3: Transgenic (CV-N) plant in MS medium + NAA 4: Transgenic (M12) plant in MSN medium + NAA

150

100 75

* 50

Gamma

• LC/MS/MS analysis of trypsin digested gel sections

37

Kappa

25 20

• Abundance of proteins was calculated using emPAI (exponentially modified protein abundance index).

15

10

•CV-N sample – 74 proteins, CV-NAA – 96 proteins, M12 – 104 proteins

CV-N

Coomassie stained reducing gel

• Recombinant proteins were the most abundant in all samples • * Major band in lane 3 is a subtilisin-like protease –related to treatment with NAA, and associated with lateral root emergence

THE RHIZOSECRETOME

DIRECT PURIFICATION OF M12 FROM HYDROPONIC MEDIUM 

a

Hydroponic medium from plants at Weeks 8, 12 and 15 were pH adjusted to 7.5 and pass through a 0.22mm filter.



Samples were loaded onto a Protein A agarose column.



Bound antibody was eluted with 100mM glycine pH2.5 1

b

2

3

150

100 75

50 37

25

10

SDS-PAGE under reducing conditions, stained with Coomassie Blue

1. Eluted Mab from Week 8 2. Eluted Mab from Week 12 3. Eluted Mab from Week 15

PRODUCTIVITY OF M12 

3 batches of M12 were prepared from groups of 10 plants.



Hydroponic medium was sampled weekly from Weeks 8-15 of culture



Average purified antibody yield was 13.3mg/10 plants after Protein A affinity purification. A scalable system?

510 plants occupy 0.7m2

SUCROSE IS THE PRIMARY CARBON SOURCE – LIGHT IS NOT REQUIRED.

07

Av [protein]/level/week 06

S1X

Rec. protein ug/ml)

05

S2X

04

S3X

03

02

01

00 Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Week 16

Week 17

Week 18

HANDLING LARGE NUMBERS OF PLANTS CAN BE SIMPLIFIED BY AUTOMATION. Off the shelf robotics.

Capacity: 66 jars Price: 36K Euros

GILSON GX-271/281 LIQUID HANDLER

INEXPENSIVE, LOW-TECH OPTIONS ALSO AVAILABLE

Vacuum

Reservoir

Filtered air

Labman Automation UK

CONCLUSIONS 

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

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A Mab production platform was developed that combines hydroponic cultivation of transgenic plants with rhizosecretion of the target recombinant protein. An optimised production system for a human monoclonal antibody was identified, which enabled high yields and consistency between batches. The rhizosecretome contains a complicated mixture of plant proteins, but the target recombinant protein is the major component, and is readily purified by affinity chromatography.

This simple, low-tech and inexpensive system could be used for gram quantities of antibody at lab scale, or could readily be scaled up for production of 10’s of grams. Further improvements will be targeted to maintaining the initial high yields of recombinant protein rhizosecretion and avoiding the decline in productivity towards the end of the harvesting phase.

ACKNOWLEDGEMENTS



The Hotung Endowment



EU FP7 – CoMoFarm project – Stefan Schillberg (Co-ordinator)



National Institutes of Health – Microbicide Innovation Program (III)



Dr. Helen Byers – St. George’s Biomics Unit

CONTROL ROOTS 

Roots of tobacco plants grown in soil or MS agar without NAA

ep

A

B

ct en+st

C

C

D

D

Figure 6.3 Control roots from plants grown in soil (A and B) or MS agar (C and D). A and B) detail of the root tip; C) root branching into a lateral root; D) detail of the root hairs mat. Bar = 250 µm (A and B) and 400 µm (C and D).

Figure 6.5 Confocal images of control roots showing A) the different cell layers in a tobacco root meristem: epepidermis, ct-cortex, en-endodermis and st-stele; B) root apical meristem; C) root hair initiations (arrowed); D) elongated root hairs (arrowed). Bar = 100 µm.

NAA TREATED ROOTS 

More root hairs, lateral roots and nodules; epidermis missing in cases A

B lp

cl

lp

C

D

lp cl

ct

E

F

ap

Figure 6.6 Roots from plants treated with NAA. A) Root tip with dense mat of root hairs; B) detail of extensive root branching with nodules visible; C) Detail of root nodules outside epidermis; D) Detail of root nodules inside epidermis; E and F) Root tips with thickened apices. Bar = 200 µm (A, C, D, E and F), 600 µm (B).

Figure 6.8 Confocal image of a root tip with two lateral primordial emerging from the same site (lp) causing extensive cortex proliferation (ct) and undifferentiated cells detaching from the tissue around them (cl). Ap-root apex. Bar = 200 µm

NAA TREATED ROOTS 

Confocal microscopy revealed amount of root hairs, tissue disintegration and confirmed nodules as lateral root primordia. B

A

ct

ep

rh rh

D

C

ct lp lp lp

ct rh

Figure 6.7 Confocal images of NAA treated roots showing A) meristem lacking parts of the epidermis, nonviable endodermis cells (arrowed) and a mass of long root hairs (rh); B) detail of the meristem with loose cortex cells (ct) and root hairs emerging from every epidermis cell (ep); C) root lacking epidermis and two lateral root primordia (lp); D) detail of lateral root primordium emerging from pericycle and loose endodermis cells. Bar = 25 µm in B and D, 100 µm in A and C.

NAA TREATED AND AGITATED ROOTS 

Short and thick roots with browning tissue resembling callus



Dense mat of root hairs

A

B

cl

C

D

C

D

Figure 6.9 Roots from NAA treated and agitated plants. A and B) Macro images, C) Detail of a single root tip, D) Mat of short and thick root structures. (cl) callus. Bar = 0.5 cm in A, 0.25 cm in B and 0.2 cm in C and D.

NAA TREATED AND AGITATED ROOTS 

No lateral root primordia, high density of root hairs, callus A

B

A

B

C

D

C

E E

D

F F

Figure 6.10 Confocal images of roots from NAA treated and agitated plants. A) Intact root apex, B) Root with numerous hairs emerging, C) Detail of root hairs emerging from every cell on the epidermis, D) Mass of undifferentiated cells, E) Older root with callus initiation, F) Callus mass. Bar = 50 µm in A and C, 100 µm in B, and 25 µm in D, E and F.