in-solution antibody capture with plant-made hydrofobin...
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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
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Hydrophobin Fusion Technology for Protein Expression and Purification
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P %
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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
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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%
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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
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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
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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
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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?
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Transient Expression in N. benthamiana
PBVA 2015 - Lausanne
Kurppa et al. submitted
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Plants as Production Platform
PBVA 2105 - Lausanne
Kurppa et al. submitted
Fusion enhanced the expression up to 35-fold
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Aqueous Two Phase Separation
PBVA 2015 - Lausanne
SURFACTANT ISOBUTANOL
HFB
TARGET
PRODUCT
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Kurppa et al. submitted
Does it work in ATPS?
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Does it still bind IgG?
PBVA 2015 - Lausanne
Kurppa et al. submitted
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Concept for In-solution Antibody Fishing
PBVA 2015 - Lausanne
HFB
SURFACTANT BUFFER pH < 2.5
IgG
Protein A
IgG
RECYCLE
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Stable expression in BY-2 suspension culture
PBVA 2015 - Lausanne
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Does It Express? - Callus Screening
PBVA 2015 - Lausanne
Kurppa et al. submitted
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BY-2 shake flask vs. 30l bioreactor
PBVA 2015 - Lausanne
Kurppa et al. submitted
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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
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30l
T. reesei
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10 l
Speed
Yield / Potential0% 100%0% 100%
Scalability (in house)
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N. benthamiana
500g leaves ~40 plants
0% 100%
For Comparison (in house)
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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
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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
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The People
PBVA 2015 - Lausanne
Katri Kurppa
Lauri Reuter Anneli Ritala
Tuuli Teikari Markus Linder
Riitta Suihkonen
TECHNOLOGY FOR BUSINESS