graffinity: novel affinity ligands for downstream processing of proteins
DESCRIPTION
Graffinity is a potentially revolutionary technology for the discovery of novel media for the purification of protein biologics. Proof of concept is sound. One example is a ligand that works as well as & better than protein A. We are open to suggestions or proposals for collaboration. CONTACT [email protected]TRANSCRIPT
(Gr)affinity® begets affinity – a new era in downstream processing begins
Dr. Frank MOFFATTDirector of Business Development
Dec 2013
The boundaries of affinity chromatography
Capacity
Yield
SelectivityStability
Re-use
Protein A affinityIon exchange
3
Moving the boundaries of affinity chromatography
Capacity
Yield
SelectivityStability
Re-useProtein A affinityGraffinity®Ion exchange
4
Shrink the ligand - Shrink the cost
Capacity
Yield
Selectivity
Stability
Re-use
Cost
Protein A affinityGraffinity®Ion exchange
5
?
Phases of innovation
6
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
7
SPRscreening & imaging
1. Analog synthesis2. Linking to support3. Chromatographic
performance
Affinity LC media
Targetprotein
(Gr)affinity® begets affinity
Design
MakeTest
8
Graffinty micro-arrays
9
Covalently boundligands
Microstructuring SAM Formation
Spotting nL
Customized pin-tool spotter Pin tool nL drops
10
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+
11
Discovery
Graffinity screening
Data evaluation
Chromatographic testing
Data evaluation
12
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
13
Project 1 Universal Fab binder
Graffinity® screening
14
≈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
17
(+) IgG 1
(+) IgG 2
(+) IgG 3
(+) IgG 4
(-) HCP
(-) Fc only
Graffinity® universal Fab binders
18
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
19
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
21
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
22
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
23
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
25
Development
Ligand optimizatio
n
Data evaluation
Media optimization
Tech transfer to CMO or licensee
Data evaluation
26
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
27
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
29
Efficient binding & high selectivity
Bevacizumab Fab specificity
Ab_07
Ab_10
Ab_12
Protein A
Binding [%]0 20 40 60 80 100
IgGOther IgGFcFab
30
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
31
✔ 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
32
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
33
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
34
Preparative chromatography
Ab_10
35
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 -
36
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
37
DEVELOPMENT LEVEL DATA
38
Project 4 Fc binder / protein A replacement
39
Graffinity® Fc binders
Ab_04
Specific IgG ligands
40
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
41
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
42
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
43
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
44
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
45
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
52
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
46
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
47
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
48
Commercial supply
Performance
optimization
by CMO
Data evaluation
Alpha testing
Business cases
49
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
50
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
51
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
52
OFFERS: Research projects
Client funded Access to a proprietary & powerful technology Exclusive solutions IP protectable performance advantages
• Discovery of novel ligands for any target
53
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