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VTT TECHNICAL RESEARCH CENTRE OF FINLAND LTD
In-solution antibody capture with plant-made hydrofobin-Protein A fusions
Jussi JoensuuSenior Research Scientist, VTT Ltd. PBVA 2015, Lausanne
211/06/2015 2PBVA 2015 - Lausanne
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311/06/2015 3
Hydrophobin Fusion Technology for Protein Expression and Purification
0
10
20
30
40
GF
P %
TS
P
HFB1tcup Pr1b GFP KDEL vsp 3´2x35S
Induction of protein bodies Purification by ATPS
Expression level 5.5 g / kg FW Joensuu et al. 2010, Plant Phys
Agriculture andAgri-food Canada
4411/06/2015
Antibodies – Fastly Growing Market
Monoclonal antibodies are the fastest growing area in pharmaceutical industry
Downstream processing can assign as much as 70 % overall MAb production costs
PBVA 2015 - Lausanne
Total volume 90 billion$ 2015 CAGR 13.5%
0
10
20
30
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60
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2009 2010 2015
$ Billion
Research
Diagnostic
Therapeutic
BCC Res, 2013
New inventions needed to reduce production and processing costs for emerging products and generics
3
5511/06/2015 Jussi Joensuu
Current State of Art – Column Chromatography
Sample feed
Washing
Elution
Fractionation
Waste
Multi-step process!
Antibodyproduction
medium
Purifiedantibodies
Column manufacture
Regeneration?
Equilibration• Protein production• Protein purification• Chemical coupling
to solid support• Column packing
Purification process
611/06/2015 6
The Curious Case of Hydrophobins
PBVA 2015 - Lausanne
Fungal Protein
Amphipathic
Surface active
Applications in food and material science
A purification tag for recombinant proteins
4
711/06/2015 7
Hydrophobin-Protein A
PBVA 2015 - Lausanne
Protein A
HFBProtein A is commonly used to capture antibodies
A bi-functional fusion protein for antibody handling in solutions?
811/06/2015 8
Transient Expression in N. benthamiana
PBVA 2015 - Lausanne
Kurppa et al. submitted
+
5
911/06/2015 9
Plants as Production Platform
PBVA 2105 - Lausanne
Kurppa et al. submitted
Fusion enhanced the expression up to 35-fold
1011/06/2015 10
Aqueous Two Phase Separation
PBVA 2015 - Lausanne
SURFACTANT ISOBUTANOL
HFB
TARGET
PRODUCT
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1111/06/2015 11PBVA 2015 - Lausanne
Kurppa et al. submitted
Does it work in ATPS?
1211/06/2015 12
Does it still bind IgG?
PBVA 2015 - Lausanne
Kurppa et al. submitted
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1311/06/2015 13
Concept for In-solution Antibody Fishing
PBVA 2015 - Lausanne
HFB
SURFACTANT BUFFER pH < 2.5
IgG
Protein A
IgG
RECYCLE
1411/06/2015 14
Stable expression in BY-2 suspension culture
PBVA 2015 - Lausanne
+
8
1511/06/2015 15
Does It Express? - Callus Screening
PBVA 2015 - Lausanne
Kurppa et al. submitted
1611/06/2015 16
BY-2 shake flask vs. 30l bioreactor
PBVA 2015 - Lausanne
Kurppa et al. submitted
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1711/06/2015 17
BY-2 shake flask vs. 30l bioreactor
PBVA 2015 - Lausanne
Kurppa et al. submitted
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1gHFBI-Protein A
1gHFBI-Protein A
BY-2
==
30l
T. reesei
==
10 l
Speed
Yield / Potential0% 100%0% 100%
Scalability (in house)
==
N. benthamiana
500g leaves ~40 plants
0% 100%
For Comparison (in house)
10
1911/06/2015 19
Take Home Messages
PBVA 2015 - Lausanne
Plants are a great platform for HFB-fusion proteins
Good chance for high expression levels
Low background in ATPS purification system
Antibodies can be fished with HFB- ProtA fusions
2011/06/2015 20
Tailored Proteins in Graphene BiosensorsKatri Kurppa, Markku Kainlauri, Miika Soikkeli, Sanna Arpiainen, David Gunnarsson, Päivi
Laaksonen, Mika Prunnila, Markus Linder, Jouni Ahopelto VTT Technical Research Centre of Finland: Bionanomaterials & Microsystems and Nanoelectronics
Biologicalinteractions
Graphene response
Molecularself-assembly
Proteins G-FET
DESIGN ASSEMBLY SENSING
11
2111/06/2015 21
The People
PBVA 2015 - Lausanne
Katri Kurppa
Lauri Reuter Anneli Ritala
Tuuli Teikari Markus Linder
Riitta Suihkonen
TECHNOLOGY FOR BUSINESS