Graffinity: Novel affinity ligands for downstream processing of proteins

(Gr)affinity® begets affinity – a new era in downstream processing begins

Dr. Frank MOFFATTDirector of Business Development

Dec 2013

[email protected]

Graffinity clients

2

The boundaries of affinity chromatography

Capacity

Yield

SelectivityStability

Re-use

Protein A affinityIon exchange

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Moving the boundaries of affinity chromatography

Capacity

Yield

SelectivityStability

Re-useProtein A affinityGraffinity®Ion exchange

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Shrink the ligand - Shrink the cost

Capacity

Yield

Selectivity

Stability

Re-use

Cost

Protein A affinityGraffinity®Ion exchange

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?

Phases of innovation

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Commercial supply & use

Development

Discovery

Target

Milestones

Discovery

• Selection of 2-5 development candidates

Development

• Selection of ligand/system for commercialization

Commercial

• Commercial agreements & final optimization & use

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SPRscreening & imaging

1. Analog synthesis2. Linking to support3. Chromatographic

performance

Affinity LC media

Targetprotein

(Gr)affinity® begets affinity

Design

MakeTest

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Graffinty micro-arrays

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Covalently boundligands

Microstructuring SAM Formation

Spotting nL

Customized pin-tool spotter Pin tool nL drops

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SPR

sig

nal st

ren

gth

, n

m

SPR signal strength, nm

Experiment 1Exp

eri

ment

2

116,000 structurally diverse compounds

SPR imaging finds binders

Affinity starts with SPR imaging

- reproducibly

Chromatographic evaluation

• Protein A Sepharose Fast Flow (GE Healthcare)

• Immobilization to NHS-activated Sepharose 4FF

• Use of aminopropyl spacer• Amide linkage between resin & ligand• Average density of 12 - 15 µmol per mL of medium• Tested in 384 MTP format 30 µL per bed volume

NH2LIGAND

O

ON

O

O

O

NH

LIGAND+ NH2LIGAND

O

ON

O

O

O

NH

LIGAND+

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Discovery

Graffinity screening

Data evaluation

Chromatographic testing

Data evaluation

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Verification of chromatographic performance (% binding/ % purity/ selectivity versus HCP)

Selection from a non-biased library of 116,000 ligands designed to bind to proteins (typically) delivers > 50 binders

Selection of 20-40 binders for re-synthesis & linking to support media

Selection of 2-5 development candidates

DISCOVERY LEVEL DATA

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Project 1 Universal Fab binder

Graffinity® screening

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≈150 compounds Select for affinity(+) positive binding ≥ 3 antibodies

>116,000 compounds • Graffinity® SPR screening library• MW 150-600• Structurally diverse• 4 full length antibodies

Graffinity® selectivity

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≈150 compounds

≈ 60 compounds

(+) positive binders

Select for specificityNO binding to:(-) HCP(-) Fc only domain protein

Graffinity® selection

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≈60 compounds

≈30 compounds

No binding to:(-) HCP & Fc only domain protein

Select for chrom. performance evaluation Mainly neutral structures

but max. 1 positive charge Groups of common substructure

facilitates rapid SAR evolution

Graffinity® specific Fab binders

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(+) IgG 1

(+) IgG 2

(+) IgG 3

(+) IgG 4

(-) HCP

(-) Fc only

Graffinity® universal Fab binders

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Conclusions SPR screening identified universal Fab binders ✔ Primary hits identified ✔ >30 ligands selected for resynthesis & characterization ✔

Next More resynthesis More LC testing Synthesis & testing of analogues IP protection

DISCOVERY LEVEL DATA

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Project 2 HCP binder for

polishing or reverse purification

Verification of chromatographic performance

Binding of HCP & MAbs versus pH✔ Graffinity® SPR screening ✔ Ligand selection✔ Resynthesis

LC set-upImmobilization of ligands on Sepharose 4 FF30 µL packed columns in 384 well MTPs

ExperimentBinding of MAbs & HCP to resins at pH 4.0 to 7.3

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Binding of HCP & MAbs versus pH

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7.3 6.2 5.1 4.00

20406080

100Ab_151

Toc Bevac HCP

pH

Bin

din

g [%

]

7.3 6.2 5.1 4.0 0

20406080

100Ab_157 Toc Bevac HCP

pH

Bin

din

g [%

]

Proteins• tocilizumab• Bevacizumab• CHO cell HCP

Ligands• Ab_151• Ab_157

Graffinity HCP binders - first conclusions

Binding depends upon • pH • Ab• Ligand

Ab_151 is the best performing un-optimized screening hit • High HCP binding across pH range• Low MAb at lower pH <6

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Purification of tocilizumab

Experiment

• Ligand Ab_151• Chromatography on 30 µL columns packed in MTPs• Reverse purification of tocilizumab from HCP @ pH 6.0• Initital IgG purity 50 % (protein level)• SDS-PAGE with silver staining of input & flow-through

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HCP & MAb (tocilizumab)

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HCPTo

cHCP +

TocHCP

Toc

HCP + Toc

Input Flow-through@ pH 6.0

HCP & MAb (tocilizumab) next steps

Potential applications• Reverse purification• Polishing

Next?• More resynthesis & testing• Synthesis & testing of analogues• More Abs & HCPs

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Development

Ligand optimizatio

n

Data evaluation

Media optimization

Tech transfer to CMO or licensee

Data evaluation

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Variation of chemistry of spacer & support

Analogue synthesis, support/linker exploration & chromatography

Selection of 2-4 lead ligands

Selection of ligand/system forcommercialization

DEVELOPMENT LEVEL DATA

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Project 3 Specific bevacizumab binder

Relative SPR signal strength

Specifichit series

Ligand Ab_12 Ligand Ab_10

Bevacizumab

Anti RhD

Tocilizumab

BSA

HCP

Graffinity® screen for bevacizumab

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Chromatographic performance

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Efficient binding & high selectivity

Bevacizumab Fab specificity

Ab_07

Ab_10

Ab_12

Protein A

Binding [%]0 20 40 60 80 100

IgGOther IgGFcFab

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Graffinity bevacizumab binder C

over

age

[m

g/m

L]

Adsorption Isotherm : Ab_12

[mg/mL]

0 10 20 30 40 50 60 70

Protein A

Ab_07

Ab_10

Ab_12

Affinity

Kd [mg/mL]

0.001 0.01 0.1 11

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✔ High affinity ✔ High capacity

qm [mg/mL gel]

Capacity

Protein A

Ab_07

Ab_10

Ab_12

Graffinity bevacizumab binder

Ab_10

Rel

ativ

e co

nce

ntr

atio

n

t [min]Protein A

t 0.8 min

Ab_07

Ab_10

Ab_12

Speed of binding

0 1 2 3 4 5 6 7 8 9 10

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Moderate alkaline stability

Half-life Ab_07

t 0.5 h

Rel

ativ

e co

nce

ntr

atio

n

Ab_07

Ab_10

Ab_12

MS SuRe

t 0.5 h

0 100 200 300 400 500 600

0.1 M NaOH 0.5 M NaOH

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Dynamic binding capacity

0

10

20

30

40

50

Ab_10 Protein A

Dynamic binding capacityq 0

.1/

mg

(ml g

el)

-1

0

0.1

0.2

0.3

0.4

0.5

0 20 40 60

Protein AAb_10

Antibody loaded [mg/mL gel]

c/c 0

Breakthrough

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Preparative chromatography

Ab_10

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Ab_10

ProtA

ProtA

HC

LC

EluateAb_

10

Graffinity® bevacizumab binders

Ab_10 Ab_130 Protein A

MW [g/mol] 348 328 35 kDa

Binding IgG [%] 100 100 100

Binding HCP [%] 8 3 2

Yield [%] 96 98 96

Purity [%] 96 84 100

DBC [mg/mL] 36 37 28

Kd [mg/mL] 0.072 0.079 0.005

qm [mg/mL] 63 68 39

t0.8 [min] 5.8 5.3 9.5

t0.5 (0.1 M NaOH) [h] 130 516 -

t0.5 (0.5 M NaOH) [h]

11 60 -

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Status

✔ Specific binders to bevacizumab

✔ The structural key features identified

✔ High static & dynamic capacity

Next?

• Next generation developments – e.g. purification of Lucentis

Graffinity® bevacizumab binder

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DEVELOPMENT LEVEL DATA

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Project 4 Fc binder / protein A replacement

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Graffinity® Fc binders

Ab_04

Specific IgG ligands

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ProteinAb_04

(%)Ab_182

(%)Ab_254

(%)Ab_314

(%)Ab_337

(%)Ab_352

(%)

Protein A

(%)

Bevacizumab (IgG1)

100 100 100 100 100 100 100

Palivizumab (IgG1)

100 92 100 100 100 100 100

Tocilizumab (IgG1)

100 99 100 100 100 100 100

Cetuximab (IgG1)

100 100 100 100 100 100 100

Denosumab (IgG2)

100 86 100 98 100 100 100

hu IgG3

fraction33 16 63 34 56 48 6

hu IgG4 fraction

n.d. n.d. 100 100 100 98 99

hu total IgG fraction

100 94 95 100 100 100 95

HCP binding 30 24 18 12 7 2 2

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High affinity & capacity

0.001 0.01 0.1 11

Ab_04

Ab_182

Ab_352

Protein A

Ab_254

Ab_314

Ab_337

Affinity

Kd [mg/mL]

qm [mg/mL gel]

0 10 20 30 40 50

Capacity

Cov

erag

e q

/ m

g m

g-1

Equilibrium concentration

Adsorption isotherm

✔ High affinity ✔ High capacity

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Favorable adsorption kineticsR

elat

ive

con

cen

tra

tion

Time min

0 1 2 3 4 5 6 7 8 9 10

t 0.8 / min

Speed of binding

Ab_04Ab_182

Ab_352Protein A

Ab_254Ab_314Ab_337

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High alkaline stabilityR

elat

ive

con

cen

tra

tion

Time t / h

0 200 400 600 800 1000

t 0.5 h

Half-lifeAb_04

Ab_182

Ms SuRe

Ab_254

Ab_314

Ab_337

Ab_352

Dynamic binding capacity

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0 5 10 15 20 25 30

Dynamic binding capacity

q10% [mg/mL gel]

Ab_04

Ab_182

Ab_352Protein A

Ab_254Ab_314Ab_337

0

0.1

0.2

0.3

0.4

0 10 20 30 40

Protein A

Ab_04

Ab_182

Ab_352

Ab_254

Ab_314

Ab_337

Antibody loaded [mg/mL gel]

c/c 0

Breakthrough curves

Preparative chromatography

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feed

Ab_04

Ab_18

2Ab_

254

Ab_31

4Ab_

337

feed

Ab_35

2

Prote

in A

[ %]

0

20

60

100Yield Purity

Ab_0

4

Ab_1

82

Ab_2

54

Ab_3

14

Ab_3

37

Prote

in A

Ab_3

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Specific IgG affinity ligands

  Ab_04Ab_18

2Ab_25

4Ab_31

4Ab_33

7Ab_352

Protein A

MW [g/mol] 397 404 368 442 382 456 -

Binding IgG1 [%] 100 100 100 100 100 100 100

Binding HCP [%] 30 24 18 12 7 2 2

Kd [mg/mL] 0.076 0.063 0.076 0.224 0.081 0.152 0.005

qm [mg/mL] 39 36 47 42 49 45 39

t0.8 [min] 9.8 6.4 9.8 9.4 7.8 8.9 9.5

t0.5 (0.5 M NaOH) [h]

400 390 >1000 >1000 >1000 >1000 -

DBC [mg/mL] 20 22 25 18 25 23 28

qeq [mg/mL] 25 30 39 28 41 35 40

purity [%] 81 89 86 91 91 97 98

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Graffinity® Fc binders

  Protein A Graffinity® ligands

Costs High Much lower

Chemical stability Low-moderate Very high

Leaching ProblematicQC controlled

NegligiblePotentially no

QC

IgG3 binding Very Low Moderate

Aggregate separation No Yes

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Graffinity® Fc binders

✔✔✔ Chromatographic performance

“as good as or better than Protein A”

Next

• Find partner(s) for product development & commercialization

• Offer for beta testing (from our “lead” partner

• Next generation developments

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Commercial supply

Performance

optimization

by CMO

Data evaluation

Alpha testing

Business cases

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Feedback from client labs - performance

Rigorous process/performance testing by manufacturing partner

Final product for testingwith clients

Commercialagreements &final optimization& use

Timelines

Aims Discover

3-6 months

Develop3-12 months

Produce

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Total timescale6 -18 months

Depends on:-1. Aims – what is good enough? “quick & dirty” or fully optimized performance?

2. Results - how challenging is the target? How many DMT cycles? Chemistry?

The Graffinity Pipeline

Target

Discovery

Development

Commercial

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MAb Fc binder

Your choiceclient commissioned projects

Generic Fab for MAb/Ab fragments – screening hitsCHO/HCP – DSP polishing / POC mediaBevacuzimab specific Fab binder

albumin, insulin, factor VII, factor VIII, alpha-1-antitrypsin, fibrinogen, plasminogen, tPA, tPA-urokinase, alkaline phosphatase, novel scaffolds or proteins

OFFERS: Graffinity development projects

Discovery already funded NovAliX Offered for clients to develop Exclusivity under license

1. Fab• Graffinity screening done ✔• Primary hits identified ✔

2. HCP• Chromatographic performance demonstrated ✔

3. Bevacizmab• Chromatographic performance demonstrated ✔

4. Fc

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OFFERS: Research projects

Client funded Access to a proprietary & powerful technology Exclusive solutions IP protectable performance advantages

• Discovery of novel ligands for any target

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What boundaries do you need moving?

Capacity

Yield

Productivity

SelectivityStability

Re-use

Cost

Graffinity®

54

(Gr)affinity® how can it work for you?

Dr. Frank MOFFATTDirector of Business Development

[email protected]/handy/cell+41 7 86 49 60 50

Let’s talk about how to support your business projects

& your personal success