Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm
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Purification can account for majority of the manufacturing costs of most biological drugs. In the vaccine industry, purification processes are particularly complex with non-templated processes typically with low yields leading to higher than desired product costs. We are actively working with industry partners to develop purification processing platform to address these challenges using affinity chromatography technologies. Such a purification platform could be amenable to diverse heterogeneous types of vaccines such as glycoconjugates, virus like particles and viruses. In this webinar, you will learn: -Custom affinity ligand discovery, characterization, and selection strategies -Affinity ligand - base matrix immobilization strategies -Performance evaluation techniques
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Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm
- 1. Merck KGaA Darmstadt, Germany Romas Skudas Senior Scientist, Chromatography R&D 13 September 2018
- 2. Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018 The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. 2
- 3. Non-templated processes leading to long timelines for Process Development Often have complicated purification schemes leading to high COGS and low yields Current Challenges with Vaccine Downstream Purification CaptureClarificationBioreactor 1st Purification 2nd Purification Final Polishing Final Formulation Sterile Filtration Final Fill Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.20183
- 4. Current Challenges with Vaccine Downstream Purification ClarificationBioreactor Capture with target specific custom affinity ligands Final Polishing Final Formulation Sterile Filtration Final Fill Simplifying downstream purification schemes leading to reduced COGS and improved yields Potential to establish a purification template with target specific custom affinity ligands Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.20184
- 5. Divine Project Affinity ligand discovery Functionalization of chromatography resins Full process GMP-like Water recycling process development Vaccine product Industrial Process implementation DSP development pilot-scale The DiViNe consortium is a EU based commission focused on improvements to vaccine purification processes that we are actively participating in. Source: https://divineproject.eu/ The DiViNe project has received funding from the European Commission’s Horizon 2020 research and innovation programme under Grant Agreement n°635770. Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.20185
- 7. Ligand Immobilization and Support Synthesis Separation media characterization Synthesis upscaling Supply Ligand characterization and support choice Small scale immobilization GMP Mfg & Scale-up 7 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018
- 8. Ligand Selection AFFINITY RANGE 5 to 200 nM CIP CONDITIONS > 90% binding recovery 6 hours, 0.1M NaOH EXPRESSION YIELDS beyond 500 mg/L Expression yield Stability Immunogenicity Nanofitin® ligand discovery process: In silico immunogenicity prediction scores below characterized non immunogenic protein Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.20188
- 9. Nanofitin® Ligands 7kDa alternatives to antibodies… Random mutations in binding site Discovered in Yellowstone geysers Extremely stable to heat and acid (85°C and pH 2). Binding specificity redirected to the target of choice Specific, high affinity binders with conserved original features Robinson et al., Nature (1998) Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.20189 https://www.affilogic.com
- 10. …Appropriate for Affinity Separation Simple and cost-effective manufacturing by E. coli fermentation Extremly robust Affordable custom ligands Stable to T° (>80°C) and pH (0-12) Highly resistant to CIP treatments Straightforward and regio-selective conjugation to resins Tunable affinity ligands 100% in vitro selection process in 2 months Capture of macromolecules, from peptides to viruses With a level of performance comparable to Protein A Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.201810 https://www.affilogic.com
- 11. Ligand Discovery Scheme Ligand library screening Expression of leaders Affinity binding Binding kinetics at chosen conditions (e.g. BLI assay) Physical characterization (e.g. caustic stability, immunogenicity) Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.201811
- 12. Discovery Selection Process in Ribosome Display with Affilogic Propriety Libraries: •4 rounds with increasing washing pressure •Possibility of implementing desired elution parameters to orient selection towards expected specifications •Selection process optimized to reduce discovery time Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.201812
- 13. Screening Supernatants of selected Nanofitin® ligands were tested in ELISA, clones were ranked according to their binding Enzyme-Linked Immunosorbent Assay (ELISA): •Direct and indirect affinity screening modes •Automated process in 96 well plate format Ligand hits showing specific interaction with immobilized target protein 13 Direct mode – screening against epitopes opposite to biotinylation Indirect mode – screening against random epitopes 13 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018
- 14. Screen ELISA: −Biotinylated target is attached in plate bottom on streptavidin −Clones selection based on the binding signal compared to background noise −Binding signal at different ligand concentrations (1 µM, 0.1 µM, 0.01 µM) Nanofitin® Ligand Affinity Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018 Ligands showing strongest interaction with immobilized target protein CRM197 targets were coated on ELISA plates and binding of Nanofitin® ligands (1 µM) was revealed by anti-RGS HRP antibody Chosen Nanofitin® ligands present different binding profiles and affinities ranging from sub micromolar to low nanomolar 14
- 15. Bio-Layer Interferometry (BLI) Assay Sensor modification with ligand Blocking of active sides Association with POI at defined conditions (Kon) Dissociation at defined conditions (Koff) Bio-Layer Interferometry (BLI) Assay: •Affinity constant estimation in defined conditions (e.g. pH, conductivity, additives, etc.) •4 stage automated process Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.201815
- 16. Binding Level Investigation Experiment: −In order to evaluate the KD of identified clones the identified ligands were fused to sensors −Affinity constant KD estimation in defined conditions (e.g. pH, conductivity, additives, etc.) Nanofitin® Ligand Affinity Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.201816 -0.5 M Glycin, pH 2.3 -0.1 M Glycin, pH 2.3 -0.25 M Glycin, pH 2.3 -10 mM KH2PO4, 0.09% Tween20, pH 7.2 (binding buffer)
- 17. Stability Nanofitin® ligands (1 µM) were incubated during 6 hours in NaOH or PBS. Affinity for CRM197 was compared by ELISA after treatment. Caustic Stability: −Expected affinity ligands should be stable enough to resist to regeneration cycle −Nanofitin® ligands were tested in cleaning-in-place conditions used in biopharmaceutical manufacturing 17 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018
- 18. Immunogenicity: −Very low immunogenicity predicted by the calculated immunogenic score for NF A, B, C and D, and low immunogenic score for E Ligand Immunogenicity The global immunogenic score represents the trend, for each Nanofitin® ligand and proportionally to its sequence length, to contain peptides with high affinity to MHC-II molecules. The higher scores are presumed to have a good chance of binding, therefore they present a potential immunogenic risk. Detailed profiles for the 8 HLA-DRB1 alleles considered are available in each Nanofitin® ligand cumulative HLA chart. 18 Immunogenicity scale low medium high Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018
- 19. Ligand Immobilization and Support Synthesis Separation media characterization Synthesis upscaling Supply Ligand characterization and support choice Small scale immobilization GMP Mfg & Scale-up 19 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018
- 20. Eshmuno® -type base bead: •Available particle sizes: 50 µm, 85 µm •Two different pore sizes •High chemical stability (NaOH CIP) •High mechanical stability •Low non-specific binding •Flexibility: Particle sizes and pore structure could be varied, if necessary For larger species (e.g. VLPs), other carrier matrices might be considered (e.g. CPG material) Selection of Carrier Matrix SEM images of Eshmuno® -type base beads 20 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018
- 21. Epoxy activation is a preferred method Surface Activation/Coupling Technologies Nf Y activation X conjugation NfOH 21 X Activation method Y Coupling method Epoxy Single or multistep reaction NH2, SH Nucleophilic substitution Aldehyde Multistep reaction NH2 Reductive amination COOH Single or multistep reaction NH2 Carbodiimide mediated condensation Maleimide Multistep reaction SH Addition Iodoacetyl Multistep reaction SH Nucleophilic substitution Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018
- 22. Immobilization: • Ligands usually carry a unique cysteine at their C-terminal Improvement of reduction protocol • pH and salt scouting for ligand immobilization in 10 ml scale Coupling yield up to 83% in the best established conditions • Comparison of binding capacities and ligand amount coupled Choice of the leading ligand to be scaled up Small Scale Ligand Immobilization Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018 Ligand reduction step Bead activation Coupling Ligand immobilization scheme 22
- 23. Influence of ligand coupling conditions on target binding capacity: −Optimum pH and salt concentration window for coupling, leading to the highest target binding capacity. −A potential to increase binding capacity through spacer selection and/or use of different supports. Ligand Immobilization Binding capacity (mg/ml) 23 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018
- 24. Support Physical Characterization Physical resin characterization: •Static binding capacity measurements using target •Bicinchoninic acid assay (BCA) for the ligand amount estimation on surface •SDS wash to remove non- covalently bound ligand BCA assay results showing immobilized ligand amount on resin 24 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018
- 25. Robotic Resin Lifetime Screening Robotic resin lifetime screening: •TECAN runs on the mini-chromatography column format (e.g. 0.2-0.6 ml) •Investigation of different regeneration solutions for resin lifetime •Binding capacity values for numerous cycles (e.g. depending on the target amount availability) Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.201825
- 26. Robotic Resin Lifetime Screening Robotic resin lifetime screening: •TECAN runs on the mini-chromatography column format (e.g. 0.2-0.6 ml) •Investigation of different regeneration solutions for resin lifetime •Binding capacity values for numerous cycles (e.g. depending on the target amount availability) 26 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018
- 27. Poll Question #2
- 28. Ligand Immobilization and Support Synthesis Separation media characterization Synthesis upscaling Supply Ligand characterization and support choice Small scale immobilization GMP Mfg & Scale-up 28 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018 o Lifetime o Optimal bind and elution conditions o Affinity range: 5–200 nM o Expression o Ability of reuse o From 10 ml scale to 1000 ml and beyond o Physical and function characterization
- 29. Affilogic Team: •Anne Chevrel •Olivier Kitten Merck KGaA, Darmstadt, Germany: •Michael M Schulte •Achim Schwaemmle •Oliver Rammo •Julia Pielok •Dominic Zorn Acknowledgements Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.201829
- 30. Romas.Skudas@emdgroup.com The vibrant M and Eshmuno are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources. © 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.