Graffinity: Novel affinity ligands for downstream processing of proteins
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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 fm@novalix-pharma.com
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Graffinity: Novel affinity ligands for downstream processing of proteins
- 1. (Gr)affinity® begets affinity – a new era in downstream processing begins Dr. Frank MOFFATT Director of Business Development Dec 2013 fm@novalix-pharma.com
- 3. The boundaries of affinity chromatography Capacity Re-use Yield Protein A affinity Ion exchange Stability Selectivity 3
- 4. Moving the boundaries of affinity chromatography Capacity Re-use Yield Protein A affinity Graffinity® Ion exchange Stability Selectivity 4
- 5. Shrink the ligand - Shrink the cost Capacity Cost Yield ? Protein A affinity Graffinity® Ion exchange Re-use Selectivity Stability 5
- 6. Phases of innovation Target Discovery Development Commercial supply & use 6
- 7. Milestones Discovery • Selection of 2-5 development candidates Development • Selection of ligand/system for commercialization Commercial • Commercial agreements & final optimization & use 7
- 8. (Gr)affinity® begets affinity Target protein Affinity LC media Design SPR screening & imaging Binders 1. Analog synthesis 2. Linking to support 3. Chromatographic performance Test Make 8
- 9. Graffinty micro-arrays Microstructuring SAM Formation Covalently bound ligands Spotting nL Customized pin-tool spotter Pin tool nL drops 9
- 10. Affinity starts with SPR imaging 116,000 structurally diverse compounds - reproducibly Experiment 2 SPR signal strength, nm SPR imaging finds binders Experiment 1 SPR signal strength, nm 10
- 11. 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 O O O O + N O H2 N LIGAND LIGAND N H 11
- 12. Discovery Graffinity screening Selection from a non-biased library of 116,000 ligands designed to bind to proteins (typically) delivers > 50 binders Data evaluation Selection of 20-40 binders for re-synthesis & linking to support media Chromatographic testing Verification of chromatographic performance (% binding/ % purity/ selectivity versus HCP) Data evaluation Selection of 2-5 development candidates 12
- 13. DISCOVERY LEVEL DATA Project 1 Universal Fab binder 13
- 14. Graffinity® screening >116,000 compounds ≈150 compounds • • • • Graffinity® SPR screening library MW 150-600 Structurally diverse 4 full length antibodies Select for affinity (+) positive binding ≥ 3 antibodies 14
- 15. Graffinity® selectivity ≈150 compounds ≈ 60 compounds (+) positive binders Select for specificity NO binding to: (-) HCP (-) Fc only domain protein 15
- 16. Graffinity® selection ≈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 16
- 17. Graffinity® specific Fab binders (+) IgG 1 (+) IgG 2 (+) IgG 3 (+) IgG 4 (-) HCP (-) Fc only 17
- 18. Graffinity® universal Fab binders 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 18
- 19. DISCOVERY LEVEL DATA Project 2 HCP binder for polishing or reverse purification 19
- 20. Verification of chromatographic performance Binding of HCP & MAbs versus pH ✔ ✔ ✔ Graffinity® SPR screening Ligand selection Resynthesis LC set-up Immobilization of ligands on Sepharose 4 FF 30 µL packed columns in 384 well MTPs Experiment Binding of MAbs & HCP to resins at pH 4.0 to 7.3 20
- 21. Binding of HCP & MAbs versus pH Toc Ab_151 Bevac HCP Binding [%] 100 Proteins 50 0 7.3 6.2 pH 5.1 4.0 • • • tocilizumab Bevacizumab CHO cell HCP Ligands Ab_157 Toc Bevac HCP Binding [%] 100 • • Ab_151 Ab_157 50 0 7.3 6.2 pH 5.1 4.0 21
- 22. 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
- 23. 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
- 24. HCP & MAb (tocilizumab) Input Flow-through @ pH 6.0 24
- 25. HCP & MAb (tocilizumab) next steps Potential applications • • Reverse purification Polishing Next? • • • More resynthesis & testing Synthesis & testing of analogues More Abs & HCPs 25
- 26. Development Ligand optimization Analogue synthesis, support/linker exploration & chromatography Data evaluation Selection of 2-4 lead ligands Media optimization Tech transfer to CMO or licensee Variation of chemistry of spacer & support Data evaluation Selection of ligand/system for commercialization 26
- 27. DEVELOPMENT LEVEL DATA Project 3 Specific bevacizumab binder 27
- 28. Graffinity® screen for bevacizumab Ligand Ab_12 Ligand Ab_10 Bevacizumab Specific hit series Anti RhD Tocilizumab BSA HCP Relative SPR signal strength 28
- 29. Chromatographic performance Efficient binding & high selectivity 29
- 31. Graffinity bevacizumab binder Capacity Adsorption Isotherm : Ab_12 Coverage [mg/mL] Ab_07 Ab_10 Ab_12 Protein A qm [mg/mL gel] Affinity [mg/mL] Ab_07 Ab_10 ✔ High affinity ✔ High capacity Ab_12 Protein A Kd [mg/mL] 31
- 32. Relative concentration Graffinity bevacizumab binder Ab_10 Speed of binding t [min] Protein A Ab_07 Ab_10 Ab_12 t 0.8 min 32
- 33. Moderate alkaline stability Relative concentration Half-life Ab_07 Ab_07 t 0.5 h Ab_10 Ab_12 MS SuRe t 0.5 h 33
- 34. Dynamic binding capacity c/c0 Breakthrough Dynamic binding capacity q0.1/ mg (ml gel) -1 Antibody loaded [mg/mL gel] 34
- 36. 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
- 37. Graffinity® bevacizumab binder Status ✔ Specific binders to bevacizumab ✔ The structural key features identified ✔ High static & dynamic capacity Next? • Next generation developments – e.g. purification of Lucentis 37
- 38. DEVELOPMENT LEVEL DATA Project 4 Fc binder / protein A replacement 38
- 40. Specific IgG ligands Protein Ab_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 fraction 33 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 40
- 41. High affinity & capacity Affinity Adsorption isotherm Coverage q / mg mg-1 Ab_04 Ab_182 Ab_254 Ab_314 Ab_337 Ab_352 Protein A Capacity Kd [mg/mL] Equilibrium concentration ✔ High affinity ✔ High capacity qm [mg/mL gel] 41
- 42. Relative concentration Favorable adsorption kinetics Speed of binding Time min Ab_04 Ab_182 Ab_254 Ab_314 Ab_337 Ab_352 Protein A t 0.8 / min 42
- 43. Relative concentration High alkaline stability Ab_04 Time t / h Half-life Ab_182 Ab_254 Ab_314 Ab_337 Ab_352 Ms SuRe t 0.5 h 43
- 44. Dynamic binding capacity Breakthrough curves 0.4 Protein A Ab_04 Ab_182 Ab_352 Ab_254 Ab_314 Ab_337 c/c0 0.3 0.2 0.1 Dynamic binding capacity 0 0 10 20 30 Antibody loaded [mg/mL gel] 40 Ab_04 Ab_182 Ab_254 Ab_314 Ab_337 Ab_352 Protein A q10% [mg/mL gel] 44
- 46. Specific IgG affinity ligands Ab_352 Protein A MW [g/mol] 456 - Binding IgG1 [%] 100 100 Binding HCP [%] 2 2 Kd [mg/mL] 0.152 0.005 qm [mg/mL] 45 39 t0.8 [min] 8.9 9.5 >1000 - DBC [mg/mL] 23 28 qeq [mg/mL] 35 40 purity [%] 97 98 t0.5 (0.5 M NaOH) [h] 46
- 47. Graffinity® Fc binders Protein A Graffinity® ligands Costs High Much lower Chemical stability Low-moderate Very high Leaching Problematic QC controlled Negligible Potentially no QC IgG3 binding Very Low Moderate Aggregate separation No Yes 47
- 48. 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
- 49. Commercial supply Performance optimization by CMO Rigorous process/performance testing by manufacturing partner Data evaluation Final product for testing with clients Alpha testing Feedback from client labs - performance Business cases Commercial agreements & final optimization & use 49
- 50. Timelines 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? 50
- 51. The Graffinity Pipeline Target Your choice client commissioned projects Discovery albumin, insulin, factor VII, factor VIII, alpha-1-antitrypsin, fibrinogen, plasminogen, tPA, tPA-urokinase, alkaline phosphatase, novel scaffolds or proteins Generic Fab for MAb/Ab fragments – screening hits CHO/HCP – DSP polishing / POC media Bevacuzimab specific Fab binder Development MAb Fc binder Commercial 51
- 52. OFFERS: Graffinity development projects Discovery already funded NovAliX Offered for clients to develop Exclusivity under license 1. Fab • • 2. HCP • 3. Graffinity screening done ✔ Primary hits identified ✔ Chromatographic performance demonstrated ✔ Bevacizmab • Chromatographic performance demonstrated ✔ 4. Fc 52
- 53. 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
- 54. What boundaries do you need moving? Graffinity® Capacity Cost Re-use Yield Productivity Stability Selectivity 54
- 55. (Gr)affinity® how can it work for you? Let’s talk about how to support your business projects & your personal success Dr. Frank MOFFATT Director of Business Development fm@novalix-pharma.com Mobile/handy/cell +41 7 86 49 60 50
Editor's Notes
- ExperimentChromatography on 30 µL columnsInjection: 3.3 cv IgG or flowthrough from protein A chromatography (HCP)Elution: 5 CV glycine-Cl, 50 mM, pH 2.5ResultsQuantitative binding by all ligandsNo binding to negative control (CEA)Promising selectivity
- ExperimentPapain cleavage of IgGPurification of Fab & Fc fragments by protein A chromatography & SECChromatography of Fc & Fab fragments on µL-scaleChromatography of a second control IgG (“other IgG”) on µL-scaleResultSpecific binding to Fab region of bevacizumabConclusionNot a generic Fab binder
- ExperimentEquilibrium batch adsorption in MTPs0.5 - 5 mg/mL (3.3 – 33 µM) IgG in PBS5 µL gel, 110 µL reaction volume25 °C, 1000 rpm, ≥ 3 hQuantification by BradfordFitting with Langmuir isotherm equationResultsAffinity of ligands sufficient for typical IgG titers in feed streamsCapacity comparable to protein A sepharose FF or better
- Favorable adsorption kineticsBinding faster than for Protein A Rapid adsorption allows for high dynamic capacities at short contact timesExperimentDynamic batch adsorption in MTPs0.75 mg/mL (5 µM) IgG in PBS5 µL gel, 110 µL reaction volume25 °C, 1000 rpm, 2.5 – 80 minConcentration measurements by Bradford assayDetermination of t0.8ResultsBinding faster than for Protein A Rapid adsorption allows for high dynamic capacities at short contact times
- ExperimentTreatment of ligands with sodium hydroxide 1 - 2 mM ligand in 20% DMSO0.1 / 0.5 M NaOH, room temperatureAnalysis by LC-MSResultsAb_07 very stable in sodium hydroxideConclusion Potential to improve alkaline stability through chemical structural optimization
- ExperimentFrontal analysis with pure IgG on short columnsColumns: Omnifit 25 mm x 3 mm (0.177 mL)Sample: 1 mg/mL IgG in PBSDetermination of dynamic binding capacity at 10% breakthroughFlow rate: 50 cm/h (3 min column residence time)ResultHigher dynamic binding capacity with Ab_10 SepharoseConclusion Potential of further gains in capacity by matrix / linker optimization
- ExperimentSimulation of feed by spiking IgG into HCPInitial IgG purity: 20 % Injection: 100 cv IgG + HCPWash: 20 cv PBS, pH 7.3Elution: 20 cv glycine, 50 mM, pH 3.0Online monitoring at 280 nmAnalysis of eluate fractions by SDS-PAGEResultAb_10 96% Recovery & high purityConclusionComparable to protein A but specific for bevacizumab
- * Ab_130 :next generation with improved alkaline stability
- ExperimentChromatography on 30 µL columnsInjection: 3.3 cv IgG or HCPElution: 5 cv Glycine-Cl, 50 mM, pH 2.5ResultsComplete binding of IgG1, IgG2 and IgG4Only moderate binding of IgG3Similar binding pattern than for Protein A
- ExperimentEquilibrium batch adsorption in MTPs0.5 - 5 mg/mL (3.3 – 33 µM) IgG in PBS5 µL gel, 110 µL reaction volume25 °C, 1000 rpm, ≥ 3 hQuantification by BradfordFitting with Langmuir isotherm equationResultsAffinities are in the submicromolar rangeAffinity is sufficient for typical IgG titers in feed streamsCapacity comparable to Protein A sepharose FF or better
- ExperimentDynamic batch adsorption in MTPs0.75 mg/mL (5 µM) IgG in PBS5 µL gel, 110 µL reaction volume25 °C, 1000 rpm, 2.5 – 80 minConcentration measurements by Bradford assayDetermination of t0.8ResultsBinding kinetics in the range of Protein A or fasterRapid adsorption allows for high dynamic capacities at short contact times
- ExperimentTreatment of ligands with 0.5 M sodium hydroxide1 - 2 mM ligand in 20% DMSORoom temperatureAnalysis at various time points by LC-MSCalculation of t0.5ConclusionLigands have excellent alkaline stability
- ExperimentFrontal analysis with pure IgG on short columnsColumns: Omnifit 25 mm x 3 mm (0.177 mL)Sample: 1 mg/mL IgG in PBSDetermination of dynamic binding capacity at 10% breakthroughFlow rate: 50 cm/h (3 min column residence time) ResultDynamic binding capacity 20 - 25 mg/mL ConclusionPotential for higher capacity by matrix / linker optimization
- ExperimentSpiking of IgG into HCPInitial IgG purity: 20 % Injection: 100 CV IgG + HCPWash: 20 CV PBS, pH7.3Elution: 20 CV glycine, 50 mM, pH 3.0ResultsRecovery comparable to Protein APurity >95 % ConclusionFurther ligand optimization is still possible