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Implementing a Fully Single-Use, Integrated mAb Biosimilars Purification Platform for Next Generation Manufacturing
Merck KGaA, Darmstadt, Germany, is implementing a fully single-use, integrated monoclonal antibody biosimilars purification platform under the NextBiopharmDSP project, aimed at creating a more efficient and environmentally friendly downstream process for biopharmaceutical manufacturing. The project involves multiple industry and academic partners, and focuses on innovations such as continuous capture, viral inactivation, and flow-through downstream processing at a 1000L scale. Funded by the EU's Horizon 2020 program, the initiative seeks to enhance productivity, reduce costs, and increase accessibility of biopharmaceuticals to patients.
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Implementing a Fully Single-Use, Integrated mAb Biosimilars Purification Platform for Next Generation Manufacturing
- 1. Merck KGaA Darmstadt, Germany Implementinga fully single-use, integrated mAb biosimilars purification platform for next generation manufacturing Josselyn Haas Durr European Biomanufacturing Engineer Manager
- 2. 2 The life sciencebusiness of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada.
- 3. 3 0 0 0 0 0 0 0Agenda Project overview 1 Primaryclarification 2 Continuous capture 3 Continuous viral inactivation 4 Full flow-through DSP 5 Implementation at 1000L scale 6 Conclusions 7
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- 5. Title Next-generation biopharmaceuticaldownstream process Acronym: nextBioPharmDSP Webpage: www.nextbiopharmdsp.eu General 4 year project (March 1, 2015 – February 28, 2019) Partners Industry: Lek Pharmaceuticals d.d. (Slovenia), Sandoz GmbH (Austria), Millipore SAS (France) Academia: University of Natural Resources and Applied Life Sciences (Austria), Karlsruhe Institute of Technology (Germany), National Institute of Chemistry (Slovenia) SME: National Systems srl (Italy) Objective Develop and implement a more efficient, cost-effective and environmentally friendly downstream process for the manufacture of monoclonal antibodies and biosimilars. Horizon 2020: EU-funded Program for Research & Innovation Snapshot 5 Budget Total budget: 10,6 mio € EU funding: 8,4 mio € 70 % for industry 100 % for academia
- 6. 1000L Fed-Batch Bioreactor Optimizing BiopharmaceuticalDownstream Processing Establish fully connected disposable DSP platform for biologics production Bioreactor Centrifuge Filtration Protein A VI CEX b/e AEX f/t VF UF/DF Bioreactor + pre-treatment Filtration Continuous VI Continuous capture Carbon & AEX f/t CEX & VF f/t UF/DF Continuous DSP - 3 days - 4kg Capture Intermediate / Polishing VF UF/DF Primary Separation 6 Batch Process Continuous Stainless Steel Format Single-Use
- 7. Primary clarification 20 Flocculation followed bydepth filtration outperforms traditional clarification strategies
- 8. Screening more than70 combinations ! Screening 3 mAbs 5 CHO harvest techniques High process window 5 pre- treatment agents 3 depth filter families IgG1(B), IgG2(A), IgG1(C) Fed-batch un-treated Fed-batch centrifuge Fed-batch pre-treated harvest Perfusion ATF Perfusion TFF Caprilic Acid Calcium Chloride PEG pDADMAC Clarisolve® mPAA Millistak+® HC depth filter (3) Millistak+® HC Pro synthetic depth filter (2) Clarisolve® depth filter (3) 8 Primary clarification Cell density: 4,9M – 10M cells/mL Viability: 72 – 98% Turbidity: 1040 – 2600 NTU 2 strategies for flocculant addition Batch Continuous
- 9. 5.2 1.9 3.4 0 1 2 3 4 5 6 7 8 9 10 Untreated + Millistak+® HCD0HC/F0HC depth filter pDADMAC + Clarisolve® 40MS synthetic depth filter Area(m²) Minimum Depth Filtration Area (Fed-Batch, 1000L, 2h) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0 100 200 300 400 500 600 FilterResistance(psi/L/m².h) Filtrate Volume (L/m²) Untreated - Millistak+® HC D0HC/F0HC Caprilic Acid 0.25% - Clarisolve® 20MS 100mM CaCl2/10mM PO4 - Clarisolve® 20MS Untreated - Clarisolve® 20MS/Millistak+® HC F0HC Untreated - Millistak+® HC Pro D0SP/X0SP PEG 4% - Clarisolve® 40MS Clarisolve® mPAA 0.05% - Clarisolve® 40MS pDADMAC 0.0375% - Clarisolve® 40MS pDADMAC pre-treatment followed by Clarisolve® 40MS depth filter outperformed traditional depth filtration 4,5x 9 Primary clarification
- 10. Surface installed 15m²3,85m² Clarification train Two-step clarification: D0HC X0HC One-step clarification: pDADMAC 40 MS Consumable cost Equivalent (including Flexware® assemblies and flocculant) Water for flush 1500 L 385 L Hold up volume 150 L 100 L Operation time 1,5 day 1 day pDADMAC pre-treatment followed by Clarisolve® 40MS depth filter exhibit benefits compared to traditional set up 5–10M TC/mL 72–98% Viability 1040–2600 NTU harvest turbidity 50% safety factor € 3 mAb 1000L CHO - 75% - 75% - 33% - 33% 2:1 = 10 Primary clarification
- 11. Primary clarification Large scaleimplementation 11 Bioreactor Flocculation in the bioreactor pDADMAC 10 % stock solution Water Buffer Bioburden reduction Clarisolve® 40 MS depth filter Clarified harvest
- 12. Continuous capture 30 Continuous Protein-A captureenhances productivity and provides a constant quality eluate
- 13. Continuous Multi-Column (CMC)Capture Batch Operation 10% BT Batch loading (~70% resin utilization) Loading time Proteinconcentration • Load • Wash • Elute • Clean • Regen 1 Protein conc. Continuous Operation 60% BT Continuous loading (~90% resin utilization) Loading time Proteinconcentration 1 2 3 • Wash • Elute • Clean • Regen • Load Speed and Cost benefits •Process Intensification •Reduced COGs •Higher binding capacity •Maintained performances and robustness 13 Load Wash- Regen STEP 1 3(ProtA) 2(ProtA) 1(ProtA) Optimizes resin utilization and enhances productivity
- 14. Small beads offerbetter performances for continuous capture Particle size: 35 – 49 μm Particle size: ~ 85 μm Particle size: 35 – 49 μm Particle size: ~ 85 μm Batch chromatography Continuous chromatography 10 Protein A resins evaluated • DBC for several residence times (RT) • BT curve shape & BT point • Removal of HCP, aggregates and step yield no significant differences • DNA removal some differences - not critical 14 Protein A resins screening ® ® Resin Resin
- 15. Continuous Chromatography Continuous captureon 3 columns: a robust and consistent operation 15 Processes developed on several resins – optimized step lengths 50 – 60 cycles performed – consistent performance Method duration: 65 – 75 min / cycle RT 1.5 min, high loadings (60 – 70 mg/mL) High productivity and resin utilization
- 16. Development of adisposable 3-columns system • Fully disposable flowpaths (incl. flowmeters, pump heads and UV) • 8 to 120 l/h per line • 30 days of continuous operation • Fit for columns from 7 to 25 cm i.d. • 3 inlets product line, 6 inlets buffer line 16 Continuous Multi-Column (CMC) Capture
- 17. From concept toreality 17 Continuous Multi-Column (CMC) Capture
- 18. Continuous viral inactivation 40 Fulfill adouble challenge: make it single use and continuous!
- 19. Viral inactivation isdependant on pH, buffer type and temperature, but NOT on mAB concentration 19 Virus inactivation at low pH is rapid and requires less than 5 minutes. The kinetics depend on pH, temperature, and buffer conditions, and are not dependent on protein concentration. The incubation chamber that can achieve a target inactivation time with an appropriate safety factor must be designed appropriately.
- 20. Semi-continuous virus inactivation: Concept 20 Ensure homogeneity and correct incubation time Continuous feed 20
- 21. From 3D conceptto reality 21 Semi-continuous virus inactivation Pumps for pH regulation C2 WYE inlet Flexware® and F1/F2 valve C1 Acid/Base bags holder C1 to C1B pump C1B
- 22. Full flow-through DSP polishing 50 Newtechnologies
- 23. pH 5 Protein Aelution material after VI AEX FT-CEXCarbon AEX FT-CEX FT-CEX pH 7 MAb acidic pI basic LowMWhigh Larger acidic HCP, DNA, and viruses Anion Exchangers HCP Cation Exchangers Smaller HCP’s and cell culture components Activated Carbon Functionality Media 23 Toolbox Approach Flow-Through Polishing
- 24. Flow-Through Polishing Technologies investigated 24 Virusfiltration Eshmuno® Q resin, (2) AEX resin prototypes Natriflo® HD-Q Membrane Adsorber, Sartobind® Q, Sartobind® STIC, Mustang® Q, QyuSpeed® D (1) AEX fiber prototype Eshmuno® CP-FT resin, Eshmuno® CPX resin Sartobind® S, Mustang® S, Natriflo® HD-Sb hydrogel membrane Millistak+® Carbon Filter Viresolve® Pro solution Activated Carbon Cation Exchange Anion Exchange
- 25. Millistak+® CR40 depthfilter is the main contributor to HCP reduction level 25 Activated Carbon Binding: van der Waals interactions Size-based selectivity Protects AEX CAP.E. AFTER AC AFTER AEC AFTER CECCAP.E. AFTER AC AFTER AEC AFTER CEC
- 26. Eshmuno® CP-FT resinoutperforms CEX membrane adsorbers for aggregates removal in Frontal chromatography mode 26 FT cation exchange
- 27. Unit Operation TechnologySelection Flow-Through Polishing – Implementation Strategy for 1000L Reactor Protein A Eluate after VI Millistak+® CR40 Activated Carbon Depth Filter (1.2 m2) Eshmuno® Q AEX FT Resin (2 x 1.1L) Eshmuno® CP-FT CEX FT Resin (2 x 1.1L) Viresolve® Pro Virus Filtration (0.22 m2) adjust pH=5 pH = 7 Conductivity < 5 ms/cm 27
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- 29. Technical runs 1000L ScaleSetup CAP VIN AC-AEX CEX-VF 29
- 30. DSP continuous processcontrol: system handshaking Bioreactor + pre-treatment Clarification Continuous Virus Inactivation Continuous capture f/t Carbon f/t AEX f/t CEX & VF UF/DF pH adjustment Switch of VIN collection tank Full VIN tanks stop CAP VIN ready starts Carbon/AEX pH adj. starts CEX/VF Breakthrough detection Peak detection Flow VPE VIN = Viral Inactivation30
- 31. Continuous capture duringthe 4 runs Data Run 1 Run 2 Run 3 Run 4 Number of cycles Total 90 92 94 139 col 1 30 30 32 46 col 2 30 31 31 46 col 3 30 31 31 47 Loading speed (cm) 180 240 210 210 Elution speed (cm) 180 280 280 280 31
- 32. 32 Capture – CMCskid Trends (run 4) demonstrate the consistency of the capture process 32
- 33. 4 technical runs at1000L scale 2,5 kg mAb purified in 2,5 days > 80% yield all runs and steps considered 9–12 g/l mAb concentration after Viresolve® Pro solution CONSISTENT purity and quality over runs • No bioburden • RPC and CZE profiles comparable to reference33
- 34. 34 HCP removal consistently< 40 ppm (Target < 100 ppm) 0 50 100 150 200 250 300 350 400 450 500 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 50 52 54 56 58 60 62 HCP(ppm) Time (h) Run 3 AC AEX.E CEX.E VIN 0 50 100 150 200 250 300 350 400 450 500 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 HCP(ppm) Time (h) Run 4 AC AEX.E CEX.E VIN Technical runs detailed results
- 35. 35 Aggregates removal <0.2% (target < 1%) 0 1 2 3 4 5 6 7 8 9 10 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 52 54 56 58 Aggregatecontent(%) Time (h) Run 4 AC AEX.E CEX.E VIN 0 1 2 3 4 5 6 7 8 9 10 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 50 52 54 56 58 60 62Aggregatescontent(%) Time (h) Run 3 AC AEX.E CEX.E VIN Technical runs detailed results Aggregates content in pool Run 2: < LOQ Run 3: 0.2% Run 4: 0.1 %
- 36. 36 Protein A removalbelow LOD (target < 5 ppm) 0 5 10 15 20 25 30 35 40 45 50 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 50 52 54 56 58 60 62 ProteinA(ppm) Time (h) Run 3 AC AEX.E CEX.E VIN 0 5 10 15 20 25 30 35 40 45 50 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 ProteinA(ppm) Time (h) Run 4 AC AEX.E CEX.E VIN Technical runs detailed results Activated carbon and Eshmuno® CP-FT resin are the main contributors
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- 38. Main benefits 38 € • Facilityinvestment: 35% • Running costs: 30% • Cost of materials: 50% • CO2 emissions: 25% • Reduced water, buffer, resins consumption (water 10x) Productivity Footprint Economics Single-Use Environmental • Reduce bioburden risk • No carry-over issues • No cleaning, regeneration, steaming • Rapid change-over • Complete DSP in 30 m2 (10-15x smaller) • Elimination of large intermediate hold tanks • Flexibility (mobile equipment) • Capable of processing up to 3000L harvest in 24 hours (up 12 kg of mAb) nextBioPharmDSP project helps to make highly efficient biopharmaceuticals more accessible to patient
- 39. You are invited! NextGeneration Bioprocessing Forum October 8-9, 2019 M Lab™ Collaboration Center Molsheim, France
- 40. Trademarks and logosused in this presentation are the property of companies collaborating in the nextBioPharmDSP project. Thank you! www.nextbiopharmdsp.eu This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 635557
- 41. josselyn.haas@milliporesigma.com Josselyn Haas The vibrantM, Eshmuno, Viresolve, Millistak+, Flexware, NatriFlo and BioContinuum 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. © 2019 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.