orla technology overview
DESCRIPTION
Orla Protein Technologies designs, engineers and manufactures proteins, peptides, antibodies and antigens for immobilisation on surfaces. Orla's elegant technologies preserve protein structure, function and bioactivity and have applications in bioanalytical surfaces, cell biology, stem cell research and assay development. See our Technology Overview to learn how Orla's innovative products and services can radically simplify and streamline your workflow.TRANSCRIPT
Technology Overview Orla Protein Technologies Ltd
March 2014
© Orla Protein Technologies Ltd 2014
1. Corporate Profile
2. Technology
3. Products and Services
Outline
© Orla Protein Technologies Ltd 2014
Orla provides a powerful bio-nanotechnology platform with wide application in the Life Sciences
• Est. 2002, spin-out from Newcastle University, UK • Founders Prof Jeremy Lakey and Dr Dale Athey • Headquarters, International Centre for Life, Newcastle upon
Tyne • Revenues from products and services • 50% shareholding in joint venture (OJ-Bio) with wireless
communications company; Japan Radio Co. Ltd for point of care diagnostics
• Validated technology platform with a wide range of current and future applications
Orla Protein Technologies Ltd
© Orla Protein Technologies Ltd 2014
• Proteins are the key functional molecules of life
• Functionality is linked to structure • Functionalities used in many ways • Often involves immobilisation • Current methods are inefficient
“Orla has developed a protein engineering platform that also overcomes problems inherent in the manufacture and immobilisation of many
proteins and has wide commercial application”
Proteins
© Orla Protein Technologies Ltd 2014
Labware
Biosensors
Drug targeting/delivery Separations
Analytical Devices Medical Devices
Surface immobilised proteins have many applications
© Orla Protein Technologies Ltd 2014
Poor Reproducibility
Not Scalable
High Cost
Non-Specific Binding
Low Functionality
Current methods are sub-optimal
© Orla Protein Technologies Ltd 2014
Reproducible
Scalable
Cost Effective
Low non-specific binding
Functional
Orla Solution
© Orla Protein Technologies Ltd 2014
To identify good natural protein ‘building blocks’ that allow:
• High level of economic expression
• Ease of protein engineering
• Easily assembled on a range of substrates
Orla Goal
© Orla Protein Technologies Ltd 2014
Orla’s patented technology allows the immobilisation of proteins in an oriented manner that retains a high level of function
Poor functionality Lack of orientation Denaturation Inaccessible active sites Poor reproducibility
Close to 100% of function retained Precise orientation Structurally intact Active sites accessible by design Excellent reproducibility
Adsorption Chemical coupling
OrlaSURF
Technology Platform
© Orla Protein Technologies Ltd 2014
OrlaSURF is a fusion protein technology. A small surface binding unit (SBU) is fused to the target protein. This acts as the anchor to the surface.
SBU Features: • Flexible for engineering • Robust, stable structure • Simple, reproducible, attachment to
surface • Applicable to many different surfaces:
plastic, gold, beads, plates • Large yield overexpression in E. coli
Orla Surface Binding Unit (SBU)
© Orla Protein Technologies Ltd 2014
Small motifs in loops • ECM motifs for cell culture surfaces • Antigenic epitopes for diagnostics • Antigenic epitopes for process monitoring • Epitopes for studying protein-protein interactions
Multiple motifs in loops • ECM motifs for cell culture surfaces • Antigenic epitopes for diagnostics • Antigenic epitopes for process monitoring • Epitopes for studying protein-protein interactions • Engineering of binding grooves for small molecules
Full length proteins / large domains • Growth factors for cell culture • Fc-binding domains of Protein A / G • Enzymes • Single chain antibodies
Flexibility of SBU for Fusion
© Orla Protein Technologies Ltd 2014
• Gold – planar biochips, nanoparticles • Plastics – well plates, TC flasks,
woven fibres, beads • Silks and other fibres • (Metal oxides, TiO2)
Homogeneous single protein surfaces
Heterogeneous multi-protein
surfaces
Protein deposited by simple, apply-and-wash process from aqueous
buffer
Surfaces
© Orla Protein Technologies Ltd 2014
ORLA88 ORLA87 ORLA89 ORLA85 ORLA86 ORLA18
ORLA18 (34 kDa) – AASurf ORLA85 (44 kDa) – GGASurf ORLA86 (44.5 kDa) – LLASurf ORLA87 (50.6 kDa) – GGAASurf ORLA88 (51.1 kDa) – LLAASurf ORLA89 (60.5 kDa) – LLGGASurf ORLA91 (50.9 kDa) – ZZ(GS4)9ZZSurf
OrlaSURF scaffold
Protein A (B domain)
Protein G (C domain)
Protein L (B domain)
OrlaSURF tools for antibody capture
© Orla Protein Technologies Ltd 2014
• Protein A binds to most human and mouse IgG subclasses (e.g., human IgG1, IgG2, IgG4; mouse IgG1, IgG2a, IgG2b, IgG3). It also binds to total IgG from cow, guinea pig, hamster, horse, pig, and rabbit.
• Protein G binds to all IgG subclasses from human, mouse and rat species. It also binds to total IgG from guinea pig, rabbit, goat, cow, sheep, and horse.
• Protein L binds to all classes of Ig (IgG, IgM, IgA, IgE, and IgD). Protein L will also bind Single Chain Variable Fragments (ScFv) and Fab Fragments. Protein L binds kappa I, III, and IV in human and kappa I on mouse.
OrlaSURF tools for antibody capture
© Orla Protein Technologies Ltd 2014
ORLA18 self-assembled monolayer vs adsorbed Protein A
Signal:noise is signal with IgG just before acid wash divided by remaining signal after acid wash i.e. the non specific binding
0 1000 2000 3000 40000
500
1000
1500Protein AORLA18
Time (s)
Res
pons
e (R
U)
Signal: Noise
IgG per unit protein
ORLA18 Au monolayer
400 1.5
ORLA18 CM5 amine coupled
340 0.5
Protein A Au adsorbed
2.5 0.06
Protein A CM5 amine coupled
260 1.6
ORLA18 has two protein A B-domains fused in tandem to the OrlaSURF scaffold N-terminus
Staphylococcus aureus protein A. Has five domains capable of binding to IgG antibody
ORLA18 performs better than native Protein A on gold Biacore chips
© Orla Protein Technologies Ltd 2014
ORLA18 good for at least 30 cycles of rabbit IgG binding and acid regeneration. Scientists at the National Physical Laboratory used an ORLA85 chip for 50 cycles of monoclonal antibody binding, antigen binding and regeneration without significant loss of signal. Highly durable protein surfaces.
Regeneration and surface durability
© Orla Protein Technologies Ltd 2014
1. Orla 18 or Protein A adsorption"2. Bind IgG"3. Anti-IgG Alk-phos conjugate"4. Chromogenic reaction (A405)"
Demonstrated by performing this assay:
Data on next
slide…
Substrate
Product Fc
1 2
Fc
3
4
ORLA18 performs better than native Protein A on polystyrene surfaces
© Orla Protein Technologies Ltd 2014
Mouse IgG No antibody0.0
0.5
1.0
1.5
2.0
2.5Hydrophobic-No ProteinHydrophilic-No ProteinHydrophobic-ORLA18Hydrophilic-ORLA18Hydrophobic-Protein AHydrophilic-Protein A
A40
5
Results of immunoassay to test for presence of functional protein on surfaces • Hydrophobic - untreated polystyrene. • Hydrophilic - polystyrene with plasma deposition of hydrophilic surface. • No antibody control - no primary antibody, only conjugate added.
ORLA18 performs better than native Protein A on polystyrene surfaces
© Orla Protein Technologies Ltd 2014
Protein L fusions bind to Fab fragments and single chain antibodies
Protein G and A fusions are used for immobilisation of Fc-tagged proteins
Fab and Fc-fusion binding
© Orla Protein Technologies Ltd 2014
Immobilisation of Fc-tagged myelin-associated glycoprotein on various Orla surfaces on Biacore. The Fc-tag bound efficiently to ORLA85 and 87. ORLA9 is a scaffold-only control that contains no IgG Fc-binding sites. ORLA85 coated plates were used for downstream assays.
Rat MAG
(G
ly20-Pro516) H
uman IgG
1 Pro100-Lys330
ORLA85
Binding of Fc fusions
© Orla Protein Technologies Ltd 2014
Adsorb
ed Fc-E
GF-R
ORLA 85 Fc-E
GF-R05
10152025303540455055 **
pmol
PO
4/m
inFc
Human EGF-R Met1-Ser645
ORLA85 96 well plate
Activity of kinase-Fc fusion
© Orla Protein Technologies Ltd 2014
• Plastic well plates coated with OrlaSURF protein are stable for at least 6 months at room temperature
• Gold chips coated with OrlaSURF monolayer are stable for at least 6 months when stored desiccated at 4-8°C
• Protein on surfaces is resistant to removal by detergents, DMSO, extremes of pH, high salt
Stability
© Orla Protein Technologies Ltd 2014
Assay Development Biochips Cell
Biology Design and
Manufacture
Products and Services
© Orla Protein Technologies Ltd 2014
Antibody Display Orla’s Fc protein binding plates are suitable for high throughput screening (HTS) of drug targets and drug discovery applications.
Antigen Presentation Our antigen presentation plates use OrlaSURF technology to greatly enhance the functional display of antigens e.g. HIV P24 antigen and offer improved detection of antibodies to HIV. Orla also offers custom design and manufacture.
Assay Development
© Orla Protein Technologies Ltd 2014
LGPGATLEE
LQEQI
HPVHAGPIPPGQMR
α-helical spacer
ORLA173 on 96 well plate
1 10 100 10000.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5OrlaSURF 171-controlOrlaSURF 173-p24Native p24
[anti-P24 antibody 1] ng/mL
A40
5
Native HIV P24 was adsorbed directly to wells or fused to OrlaSURF scaffold and attached to wells. Detection by two commercial mouse mAbs followed by anti-mouse-AP conjugate.
1 10 100 10000
1
2
3
[anti-P24 antibody 2] ng/mL
A40
5
Example: OrlaSURF improves detection of HIV P24 antibodies
© Orla Protein Technologies Ltd 2014
Orla’s antibody binding kits create high quality IgG-binding monolayers on gold analytical surfaces • Superior performance
compared to native proteins A, G and L
• Simple apply-and-wash protocols
• Suitable for all analytical instruments that use gold substratum
Biochips
© Orla Protein Technologies Ltd 2014
Japan Radio Co., Ltd.
Diagnostics Joint Venture:
© Orla Protein Technologies Ltd 2014
Requirement for well-controlled, reproducible cell culture conditions
Shaw, BioProcess International, Vol. 8, No. 9, October 2010, pp. 10–15
© Orla Protein Technologies Ltd 2014
Monya Baker, Stem cells in culture: defining the substrate 296 | VOL.8 NO.4 | APRIL 2011 | nature methods
“We have to stop thinking about the medium and the surface as two separate entities. We have to think about the whole environment of the cells,” Clive Glover, head of product management at STEMCELL Technologies. “In the next 2–3 years, it’s really going to be surfaces that are pulling us ahead.”
Importance of the culture surface
© Orla Protein Technologies Ltd 2014
Orla’s highly customised ECM, growth factor and cytokine surfaces reliably mimic the cellular micro-environment
Cell Biology
© Orla Protein Technologies Ltd 2014
Products for Cell Biology and Stem Cell Culture Cell Culture Plates Animal-free Fc protein binding plates for cell based assays and cell enrichment. Growth Factor Plates Surface immobilised animal-free growth factor and cytokine plates. OrlaExplorer Plates Animal-free cell culture screening plates. Orla also offers custom design and manufacture.
© Orla Protein Technologies Ltd 2014
Motifs from ECM proteins: Fibronectin Laminin Collagen Vitronectin E.g. RGDS, IKVAV, YIGSR and many more…
Growth Factors: FGF-1, 2, 4 – fibroblast growth factors IL-3, IL-4 – interleukins EGF – epidermal growth factor SHH – sonic hedge hog SCF – stem cell factor and many more…
Customised protein surfaces for cell culture
© Orla Protein Technologies Ltd 2014
• Concentration of soluble FGF-1 is at least 10x greater than ORLA90. • No need to replenish ORLA90 at every medium change - significant
cost saving per culture cycle. • ORLA90 - animal free and defined. • Can make mixed surfaces of ORLA90 with OrlaSURF proteins
displaying adhesion motifs, growth factors or Fc-binding proteins.
Comparison of soluble and surface-attached FGF-1
© Orla Protein Technologies Ltd 2014
ORLA1 – RGDS motif ORLA0 – OrlaSURF scaffold control
Micro-patterned surfaces for cell adhesion
© Orla Protein Technologies Ltd 2014
• Client X – antibody engineering
• Client Y – specific surface proteins
• Client Z – cell binding proteins
‘Made-to-measure’ and ‘bespoke’ protein design and production
Contract Design and Manufacture
© Orla Protein Technologies Ltd 2014
• Patented technology and know-how, developed for commercial utility
• Revenues from contract R&D manufacturing, services, tools and products
• Technology licensing
• Strategic partnering
Business Model
© Orla Protein Technologies Ltd 2014
1. A. P. Le Brun, S. A. Holt, D. S. H. Shah, C. F. Majkrzak, J. H. Lakey, The structural orientation of antibody layers bound to engineered biosensor surfaces. Biomaterials 32, 3303 (2011).
2. A. P. Le Brun, S. A. Holt, D. Athey, D. S. Shah, J. H. Lakey, Self-Assembly of Protein Monolayers Engineered for Improved Monoclonal Immunoglobulin G Binding. International Journal of Molecular Sciences 12, 5157 (2011).
3. M. J. Cooke et al., Neural differentiation regulated by biomimetic surfaces presenting motifs of extracellular matrix proteins. Journal of Biomedical Materials Research Part A 93A, 824 (2010).
4. S. A. Holt et al., An ion channel containing model membrane: structural determination by magnetic contrast neutron reflectometry Soft Matter 5 2576 (2009).
5. A. P. Le Brun, S. A. Holt, D. S. Shah, C. F. Majkrzak, J. H. Lakey, Monitoring the assembly of antibody-binding membrane protein arrays using polarised neutron reflection. European Biophysics Journal with Biophysics Letters 37, 639 (2008).
6. M. J. Cooke et al., Enhanced cell attachment using a novel cell culture surface presenting functional domains from extracellular matrix proteins. Cytotechnology 56, 71 (2008).
7. M. J. Cooke et al., Presentation of extracellular matrix motifs by biomimetic substrates to control cellular attachment and differentiation. Journal of Anatomy 212, 89 (2008).
8. D. S. Shah et al., Self-assembling layers created by membrane proteins on gold. Biochemical Society Transactions 35, 522 (June 1, 2007, 2007).
9. D. A. Cisneros, D. J. Muller, S. M. Daud, J. H. Lakey, An approach to prepare membrane proteins for single-molecule imaging. Angewandte Chemie-International Edition 45, 3252 (2006).
10. S. Terrettaz et al., Stable self-assembly of a protein engineering scaffold on gold surfaces. Protein Science 11, 1917 (2002).
11. Q. Hong, S. Terrettaz, W. P. Ulrich, H. Vogel, J. H. Lakey, Assembly of pore proteins on gold electrodes. Biochem Soc Trans 29, 578 (2001).
Selected Publications
© Orla Protein Technologies Ltd 2014
Biomedicine West Wing International Centre for Life Newcastle upon Tyne United Kingdom NE1 4EP T: +44 (0) 191 2313127 E: [email protected] W: www.orlaproteins.com
Contact Orla