orla technology overview

Technology Overview Orla Protein Technologies Ltd

March 2014

© Orla Protein Technologies Ltd 2014

1. Corporate Profile

2. Technology

3. Products and Services

Outline


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


•  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


Labware

Biosensors

Drug targeting/delivery Separations

Analytical Devices Medical Devices

Surface immobilised proteins have many applications


Poor Reproducibility

Not Scalable

High Cost

Non-Specific Binding

Low Functionality

Current methods are sub-optimal


Reproducible

Scalable

Cost Effective

Low non-specific binding

Functional

Orla Solution


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’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


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)


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


•  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


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


•  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


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


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


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


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


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


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


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


•  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


Assay Development Biochips Cell

Biology Design and

Manufacture

Products and Services


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


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’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


Japan Radio Co., Ltd.

Diagnostics Joint Venture:


Requirement for well-controlled, reproducible cell culture conditions

Shaw, BioProcess International, Vol. 8, No. 9, October 2010, pp. 10–15


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’s highly customised ECM, growth factor and cytokine surfaces reliably mimic the cellular micro-environment

Cell Biology


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.


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


•  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


ORLA1 – RGDS motif ORLA0 – OrlaSURF scaffold control

Micro-patterned surfaces for cell adhesion


•  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


•  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


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


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