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Technology Trends in Bioprocessing Purification

The document discusses advancements in therapeutic monoclonal antibody (mAb) processing, emphasizing market trends towards intensified capture processing and continuous operations. Key technologies introduced include continuous multi-column chromatography and in-line virus inactivation, which aim to enhance productivity, reduce costs, and streamline purification processes. The adoption of flow-through polishing techniques is highlighted for improving product yield while minimizing facility footprints and buffer requirements.

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Merck KGaA Darmstadt, Germany Takao Ito, Head of MSAT Japan & Korea BIO KOREA International Convention 18 April 2019 Considerations for Continuous Processing and Connected Polishing for mAb Technology Trends in Bioprocessing Purification
01 02 03 04 05 Agenda 2 Market Trends and the Evolution of Next Generation Processes Intensified Capture Processing Continuous Virus Inactivation Connected Flow-Through Polishing Summary
Therapeutic mAb Processing Market Trends *http://www.biophorum.com/category/resources/technology-roadmapping-resources/introduction/ 1 2 3 4
Therapeutic mAb Processing Market Trends Require Step Change in Business Driver Performance 70% 10X 90% 90%
5 Therapeutic mAb processing Factory of the Future Facility Goals FACTORY OF THE FUTURE Enablers Process Intensification Single Use Process Analytics Software & Automation
Therapeutic mAb Processing An Evolutionary Journey Across Many Disciplines 6 Today Near-term Mid-term End state Process Batch Intensified Connected Continuous Format Stainless Hybrid Single Use SU/Closed Analytics QC Manufacturing At-Line In-Line/ Real-Time Sensing Controls Standalone Control Semi- Centralized Control Full Process Control Predictive Process Control Digital Plant Pre-Digital Plant Digital Silos Connected Plant Adaptive Plant
BioContinuum™ Platform Focus Areas: (1) Cell Culture Media For Intensified Processes (2) Integrated Perfusion Bioreactor 1 & 2 3 (3) Protein A for Intensified Capture 4 (4) In-Line Viral Inactivation 5 & 6 (5) Flow Through Polishing Toolbox (6) Single-Pass UF/DF Past Current
Approach of mAb Downstream Process Intensification Low pH VI Pool CEX Pool AEX Pool VF Pool Traditional Process CEX B/E AEX FT VF with prefiltrationPA B/E VF PoolTank Less CEX B/E AEX FT VF with prefiltration PA B/E VF Pool Continuous Capture (CMC, SMB) CMC CEX B/E VF with pre-filtration AEX FT Tandem CMC PA B/E Connected Flow-through (post PA) Low pH VI Pool VF Pool AC + AEX FT CEX + VF In-line pH Pro-A+AEX: M. Shamashkin et al., Biotechnol Bioeng, 110(10) 2013 Pro-A+CEX+AEX: Neil Soice, et. al. ACS spring conference 2010 V. Warikoo et al., Biotechnol Bioeng, 109(12), 2012 R. Godawat et al., J Biotechnol. 213, 2015 AEX+HIC: J. Zhang et al., Eng Life Sci. 17(2) 2017 Depth+AC+AEX: T Yamada et al., J Chrom B 1061-1062 2017 EP 2574618 A1. 2012
01 02 03 04 05 Agenda 9 Market Trends and the Evolution of Next Generation Processes Intensified Capture Processing Continuous Virus Inactivation Connected Flow-Through Polishing Summary
Improved resin utilization Residence time operation: Higher or lower What is Continuous Multi-Column Chromatography Intensified Capture 2 column Single loading 3 column Loading in-series
Intensified Capture What is Continuous Multi-Column Chromatography ▪ Better resin utilization ▪ Higher productivity ▪ Smaller columns ▪ Lower buffer consumption 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0 10 20 30 40 50 60 70 80 1-Column (1% BT) 2-Column (1% BT) DynamicCapacity(g/L) Residence Time (min) Protein conc. in column 3 min RT 1 min RT 0,6 min RT 0,4 min RT Batch Continuous
Intensified Capture Quality and Yield of Continuous Multi-Column Chromatography 3 column continuous results Total IgG loaded : 325mg (@RT 1min) Washing : 25mg Elution : 296mg 91% Total Operation time: 8.3hr Used protein A amount: 3ml Batch results Total IgG loaded : 480mg (@RT 7.2min) Elution : 445mg 92% Total Operation time: 24hr Used protein A amount: 5ml Data generated by Chiyoda TechnoAce Co.,Ltd. ▪ Maintained performance ▪ Same robustness ▪ Comparable process yield Protein A affinity resin: Eshmuno® A
Is Multi-column Chromatography Productive? Same operation for resin: B/E • Same resin • Same buffer Batch Multi Column Changes: • Reduction of resin and buffer • Shorter residence time • Increase cycle • Interconnected operation • Complex flow path for cycling
Intensified Capture Operational Windows on Productivity Batch 2-column switching 3-column cycling Works around 20cm Bed at 250-350 cm/hr Productivity: 15-30 g/L/hr Limitation of flow rate Bed height at 5-15cm 25-40 g/L/hr Operation point is determined by 24hr run (perfusion) 3.5-4.5g/L/hr 2000L (Titer 5.4g/L) Eshmuno® A resin Least Squares Fit by response surface (Predicted by JMP software) ID 63cm ID 25cm ID 35cm Different sweet spot of resin utilization
Intensified Capture Capture Column Strategy for Upstream Processing Fed-batch bioreactor 2kL, 5.4g/L Single harvest Perfusion bioreactor (30days) 150L, 2.29g/L Continuous harvest Concentrated Fed-batch 500L, 21.4g/L Single harvest UF MF BPOG Next Gen Tech Roadmap Upstream Scenarios Titers are referenced from Xu, S. et al., Biotechnol. Prog., 33(4) 2017 0% 50% 100% Intensifie dFB& Batch Intensifie dFB& CMC HD perfusion & Switching Concentra tedFB &CMC Buffer Total resin 0% 100% 200% 300% Productivity x1.7 1/4 x2.2 -97% -50% 1 2 3 4
16 Intensified Capture Impact on Long Duration Cycling Run J. Pollock et al., J. Chromatography A 2013 Continuous Batch P Perfusion Bioreactor Cell Retention device, breeding device Protein A capture Semi continuous and continuous manufacturing Centrifugation, ATF, TFF Multicolumn cycling In-line perfusate clarification Limited impurity removal Significantly more foulants Steeper loss in DBC D. Burgstaller et al., J. Chem. Technol. Biotechnol. 2017 pDADMAC Millistak® HC ProDesign with enough safety factor Improved CIP Additional perfusate clarification Cycle Number LossinDBC(%)
-5 0 5 10 15 20 -0.5 0 0.5 1 1.5 2 2.5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 pHorConductivity/4 A280(AU) Column Volumes A280 Conductivity / 4 pH wash -5 0 5 10 15 20 -0.5 0 0.5 1 1.5 2 2.5 0 5 10 15 20 25 30 35 40 45 pHorConductivity/4 A280(AU) Column Volumes A280 Conductivity / 4 pH Unconcentrated mAb Harvest 1.3 g/L titer 24 CV load SPTFF Concentrated 12.4 g/L titer 2.5 CV load equil load elute re-equil CIP Load time = 144 min Cycle time = 4.25 hour Load time = 14 min Cycle time = 2 hour SPTFF feed concentration increased capture step productivity by 115% (7.2 g/L/hr → 15.5 g/L/hr) Case Study: 1.6 L Eshmuno® A column for batch mAb capture Improve Capture Chromatography with SPTFF Intensified Capture
Flow Rate Existing resins The Drive For Higher Productivity Solutions Residence times is the key for future Higher productivity enables: ✓ Increased speed ✓ Smaller footprint → Potential for Single Use → Manufacturing flexibility ✓ Reduced COGS 3-8 min. 1-3 min. 0.5-1 min. 2-10 sec. Productivity(g/L/h) Residence Time (min) Intensified Capture 18 Conventional Column Chromatography
Multi-column cycling optimizes resin utilization and enables continuous processing: • Upstream culture strategy should be considered: Fed-batch or Perfusion • Harvest type: Single or Continuous • mAb concentration: SPTFF can boost productivity for Protein A capture • Design consideration for multi cycling operation for higher loading: Safety factor, Improved CIP, Additional perfusate clarification Summary of intensified capture
01 02 03 04 05 Agenda 20 Market Trends and the Evolution of Next Generation Processes Intensified Capture Processing Continuous Virus Inactivation Connected Flow-Through Polishing Summary
In-Line Virus Inactivation ACID → Virus inactivation kinetics → Efficient incubation chamber design PROTEIN A CAPTURE POLISHING CHROM. STEP #1 BATCH Industry standard: 60 minute hold Robust inactivation > 4 LRV 2 holding tanks required Manual process Continuous pH adjustment with dynamic hold Eliminates large holding tanks Automated Enables continuous operation 60 min HOLD BASE Applicability dependent upon: CONTINUOUS
0 0.2 0.4 0.6 0.8 1 0 0.5 1 1.5 2 2.5 CumulativeDistribution V/V50 In-Line Virus Inactivation Incubation Chamber Design – Flow Distribution Characterization Straight Tube Coiled Serpentine Early virus breakthrough → Larger safety factor Long protein exposure C. Gillespie et al., Biotechnol J. 2019 Feb;14(2)
Inactivation Kinetics Virus type PRV < XMuLV Buffer type acetate  glycine < citrate Temperature 22ºC < 16ºC mAb concentration Minimal impact In-Line Virus Inactivation Virus Inactivation Kinetics Rapid inactivation kinetics (~2-3 min) C. Gillespie et al., Biotechnol J. 2019 Feb;14(2)
• Virus inactivation at low pH is rapid and requires less than 5 minutes, implying that this process can be run in continuous mode. The kinetics of inactivation depend on pH, temperature, and buffer conditions, and are not dependent on protein concentration. • Technologies exist to move from batch to continuous virus inactivation. Efficient incubation chamber design and control offers continuous flow systems shorter processing times than traditional batch processes. • Proper implementation could result in equivalent levels of virus inactivation in batch and in-line systems. Summary of in-line virus inactivation
01 02 03 04 05 Agenda 25 Market Trends and the Evolution of Next Generation Processes Intensified Capture Processing Continuous Virus Inactivation Connected Flow-Through Polishing Summary
Toolbox Approach Flow-through Polishing Leached Protein A Aggregates Virus Host Cell DNA Host Cell Protein Others? (Fragments, Charge Variants) Chemistries Matrices Devices
Flow-through Polishing Existing Toolbox ▪ Eshmuno® CP-FT resin Flow through aggregate removal at high loading 5 – 10X buffer reduction Easy implementation ▪ NatriFlo® HD-Q AEX membrane Impurity clearance at high loading High velocity operation (~1 sec RT) ▪ Millistak+® Pod CR40 depth filter Activated carbon media Binding: van der Waals interactions Size-based selectivity Impurity (MW) Capacity Insulin 60 g/L Methotrexate 75 g/L Pluronic® F68 surfactant 75 g/L Antifoam C > 16 g/L
Eshmuno® CP-FT resin Benchmarking Only Eshmuno® CP-FT resin was efficient for the removal of aggregates in the flow- through mode under strong binding conditions that favor frontal chromatography M. Stone et al., J Chrom A, 1599, 2019
Batch vs. Integrated Implementation of Flow-through Technology Robust Performance with Significant Footprint Reduction Flow Through Polishing Delivers: Robust Purification across 8 molecules  High product yield (83 – 91%)  Aggregates: < 1%  Fragments: up to 1 LRV  HCP clearance: < 10 ppm Significant Facility Footprint Reduction  Lower media and buffer requirements  Eliminate intermediate hold tanks  Single-skid operation Low pH VI Pool Virus Filter Pool Carbon Depth Filter + FT-AEX FT-CEX + Virus Filtration In-line pH Low pH VI Pool Depth Filter Pool CEX Pool AEX Pool Virus Filter Pool pH Adjust & Dilution Depth Filter Bind & Elute CEX FT-AEX Virus Filtration Traditional Batch Polishing Flow Through Polishing
3-step Flow-through Polishing 100 98 96 94 92 3.0 2.5 2.0 1.5 1.0 0.5 0.0 VIA AC Feed FT-AC AEX Feed FT-AEX CEX Feed FT-CEX HMW1 HMW2 LMW1 LMW2 Monomer HMW,LMW(%) Monomer(%) 10000 1000 100 10 1 DNA DNA(pg/mg),HCP(ng/mg) HCP3985 1888 27.8 512 0.15 0 0 2.47 0.25 0.01 0.01 0 1.7 0.07 1.57 0.03 1.68 0.03 98.22 94.22 <0.76 <0.25 0.00 FT-AC FTCEXFTAEX pH5 3mS/cm Activated carbon shows 3 log10 DNA clearance. Robust clearance of HCP and DNA by FT-AEX. Eshmuno® CP-FT reduces mAb-related HMW aggregates (Dimer). mAb 10.9g/L pH7 3mS/sm T. Ichihara et al., mAbs J., 10, 2018
Feed condition: mAb-A (8.2mg/mL) Impurities: HCP = 567ng/mg IgG, DNA=7276 pg/mgIgG, HMW1 = 0.78%, HMW2= 1.65%, Monomer = 96.53%, LMW1=0.93%, LMW2=0.1%. pH6, 4mS/cm, 0.2mL/min High Loading : 200mL (1640mg mAb) Operation system : AKTAexplorer 100 Single equilibrium: 25mM sodium acetate buffer pH6 Single elution: 25mM Sodium acetate buffer pH6 w 1M NaCL Evaluation: Connected flow-through 1. AEX(1mL) – CEX(0.5mL) 2. AC(0.5mL) – AEX(1mL) –CEX(0.5mL) Connected Flow-through Purification Proof of Concept Study from DOE Optimization AC CEX T. Ichihara et al., mAbs J., 10, 2018 T. Ichihara et al., Eng. Life Sci., 19, 2019
(A) AEX - CEX(A) AEX - CEX (B) AC - AEX - CEX 5 5.5 6 6.5 20 40 60 80 100 120 140 160 180 200 220 240 pH 0 500 1000 1500 2000 2500 3000 3500 0 A280(mAU) Volume (mL) 5 5.5 6 6.5 7 0 500 1000 1500 2000 2500 3000 3500 0 20 40 60 80 100 120 140 160 180 200 220 240 pH As80(mAU) Volume (mL) 7 0 40 80 120 160 200 Conductivity(mS/sm)0 40 80 120 160 200 Conductivity(mS/sm) 0 0.2 0.4 0.6 0.8 1 MPa 0 0.2 0.4 0.6 0.8 1 MPaFigure 1. Typical chromatograms obtained from the in-series, connected flow-through polishing steps. (A) AEX-CEX, (B) AC-AEX-CEX. Load Wash Elute Load Wash Elute Evaluation of Connected Flow-through Purification Results of Connected Flow-through Polishing AEX-CEX AC-AEX-CEX Wash Load Elute Wash Load Elute 90 92 94 96 98 100 5 8 11 ○Monomer(%) ■mAbconc. (mg/mL) 0 2000 4000 6000 8000 10000 0 100 200 300 400 500 0 1000 2000 △DNA(pg/mg) ◆HCP(ng/mg) mAb loading (mg) AEX-CEX AC- AEX-CEX mAb conc. Mono- mer HCP DNA Chromatograms by in-series flow-through polishing 1/2 -1/3 reduction of processing time Extremely high pressure of elution mAb 100% breakthrough at >400mg lording AC improves HCP and DNA clearance T. Ichihara et al., Eng. Life Sci., 19, 2019
33 Value Modeling What Value Does Flow Through Polishing Deliver? Bioreactor Clarification Affinity Chromatography Virus Inactivation CEX Bind and Elute Flow Through AEX Viral Clearance Final Filtration Concentration & Diafiltration Traditional Batch (Baseline) Depth Filtration Four polishing steps run continuously without intermediate hold steps ▪ Lower buffer volume ▪ Higher loading on chromatography media and virus filter ▪ Smaller facility footprint for systems, columns, and tanks Bioreactor Clarification Affinity Chromatography Virus Inactivation Final Filtration Concentration & Diafiltration Flow Through Polishing Activated Carbon Flow Through CEX Flow Through AEX Viral Clearance Bioreactor: 2k L fed-batch Titer: 5 g/L Mab Felo, Holistic Development of Intensified Mab Processes for Higher Productivity and Improved Economics, BPI Boston, Sep 2018
Assumption Activated Carbon AEX FT CEX FT Virus Filtration Technology Millistak+® Carbon depth filter Eshmuno® Q resin Eshmuno® CP-FT resin Viresolve® Pro filter Yield 93% 95% 95% 98% Capacity 1500 g/m2 3000 g/L 1000 g/L 5000 g/m2 # Re-uses Single use 150 150 Single use # Buffers 1 5 4 0 Activated Carbon Flow Through CEX Flow Through AEX Viral Clearance Value Modeling Assumptions Key to Output • Single skid versus four • Yields matched to baseline process • Baseline process assumptions per BPOG costs & assumptions Bioreactor: 2k L fed-batch Titer: 5 g/L Mab 34 Felo, Holistic Development of Intensified Mab Processes for Higher Productivity and Improved Economics, BPI Boston, Sep 2018
35 Baseline Scenario vs. Flow Through Polishing • Most significant savings capital, consumables, and labor • Buffer reduction due primarily from switching CEX to flow through mode 10% COGS reduction 47% Buffer reduction 43% COGS reduction 87% Buffer reduction Polishing alone… Total process… Value Modeling COGS Savings from Reduced Buffer Use, Intermediate Storage Tanks, and Process Time/Labor Bioreactor: 2k L fed-batch Titer: 5 g/L Mab Ito BioKorea 2019 | 18- Apr-2019, Copyright Merck 2019 Felo, Holistic Development of Intensified Mab Processes for Higher Productivity and Improved Economics, BPI Boston, Sep 2018
36 Ito BioKorea 2019 | 18- Apr-2019, Copyright Merck 2019 Pre-concentrate to Boost Eshmuno® Q Resin HCP Clearance SPTFF with Flow-through Polishing 0 1 2 3 4 5 6 0 20 40 60 80 100 120 Improvementfactorabove non-SPTFFfeed mAb feed concentration (mg/mL) Improvement vs. mAb feed concentration Mass Loading (g/L resin) ▪SPTFF pre-concentration can boost Eshmuno® Q resin mass loading by a factor of 4x. ▪ Reduced loading at high feed concentrations, possibly due to mAb-HCP interactions. ▪In addition to load improvements, SPTFF concentration reduces process load time, thus increasing productivity by a factor of 5x. 0 1 2 3 4 5 6 0 20 40 60 80 100 120 Improvementfactorabove non-SPTFFfeed mAb feed concentration (mg/mL) Improvement vs. mAb feed concentration Mass Loading (g/L resin) Productivity (g/L resin/hr) Pre-Concentrate Protein SPTFF Pellicon® AEX Eshmuno® Q T Elich et al., ACS conference spring Apr 2017
Continuous diafiltration for buffer exchange Tank I Tank II Recovery Tank To next process step From previous process step UF 0 1 2 3 4 5 6 7 NormalizedtoBatch All cases: no UF, 9 diavolumes ■ Batch: 3h process, 6LMM crossflow ■ CDF low area: 19h process, 6LMM crossflow ■ CDF low pump passes: 19h process, 1LMM crossflow ■ ILDF: 19h process 30 35 40 45 0 2 4 6 8 10 12 14 16 18 20 22 24 Cycle Number Control Chart: DF Cycle Time Average = 36.9 min, σ = 1.8 min E. Goodrich et al., Recovery of biological Products, Oct 2018
Comparable performance between the batch purification process and the pool-less process configuration. • Simple operation: Single system operation and removal of the intermediate hold tank • Substantial quality: higher overall mAb yield and impurity clearance • Small processing: Higher process loading = Process compression, reduced buffer usage • Shorter process time: Single loading step • COGS reduction: ~10% across full process • SPTFF pre-concentration can boost AEX resin mass loading Summary of Flow-through Polishing
01 02 03 04 05 Agenda 39 Market Trends and the Evolution of Next Generation Processes Intensified Capture Processing Continuous Virus Inactivation Connected Flow-Through Polishing Summary
Intensified Downstream Processing Summary Industry trends are forcing bio-manufacturers to adopt a more strategic view of manufacturing − Process Intensification − Single Use − Process Analytical Technologies and controls Process design and optimization requires an “holistic view” − Upstream and Downstream − Tool box for connected impurity clearance Next generation technologies can support intensification efforts of existing batch processes while enabling future connected and continuous processing Collaborations between academics, industry, and regulatory agencies are critical for successfully moving the industry forward in next generation processing 1 2 3 4
Acknowledgements Christopher Gillespie Kevin Galipeauc Yasuhiko Kurisu Yasuhiro Kawakami Miyuki Koyama Prof. Shuichi Yamamoto (Yamaguchi univ.) Takamitsu Ichihara(Astellas) Shuhei Kondo (Chiyoda TechnoAce) Michael Phillips Romas Skudas Mikhail Kozlov Lars Peeck Ajish Potty Matthew Stone Alex Xenopoulos Renaud Jacquemart Danny Wu Thomas Elich Mochao Zhao Michael Felo Joseph Geringer Elizabeth Goodrich
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Technology Trends in Bioprocessing Purification

  • 1.
    Merck KGaA Darmstadt, Germany TakaoIto, Head of MSAT Japan & Korea BIO KOREA International Convention 18 April 2019 Considerations for Continuous Processing and Connected Polishing for mAb Technology Trends in Bioprocessing Purification
  • 2.
    01 02 03 04 05 Agenda 2 Market Trends andthe Evolution of Next Generation Processes Intensified Capture Processing Continuous Virus Inactivation Connected Flow-Through Polishing Summary
  • 3.
    Therapeutic mAb Processing MarketTrends *http://www.biophorum.com/category/resources/technology-roadmapping-resources/introduction/ 1 2 3 4
  • 4.
    Therapeutic mAb Processing MarketTrends Require Step Change in Business Driver Performance 70% 10X 90% 90%
  • 5.
    5 Therapeutic mAb processing Factoryof the Future Facility Goals FACTORY OF THE FUTURE Enablers Process Intensification Single Use Process Analytics Software & Automation
  • 6.
    Therapeutic mAb Processing AnEvolutionary Journey Across Many Disciplines 6 Today Near-term Mid-term End state Process Batch Intensified Connected Continuous Format Stainless Hybrid Single Use SU/Closed Analytics QC Manufacturing At-Line In-Line/ Real-Time Sensing Controls Standalone Control Semi- Centralized Control Full Process Control Predictive Process Control Digital Plant Pre-Digital Plant Digital Silos Connected Plant Adaptive Plant
  • 7.
    BioContinuum™ Platform Focus Areas: (1)Cell Culture Media For Intensified Processes (2) Integrated Perfusion Bioreactor 1 & 2 3 (3) Protein A for Intensified Capture 4 (4) In-Line Viral Inactivation 5 & 6 (5) Flow Through Polishing Toolbox (6) Single-Pass UF/DF Past Current
  • 8.
    Approach of mAbDownstream Process Intensification Low pH VI Pool CEX Pool AEX Pool VF Pool Traditional Process CEX B/E AEX FT VF with prefiltrationPA B/E VF PoolTank Less CEX B/E AEX FT VF with prefiltration PA B/E VF Pool Continuous Capture (CMC, SMB) CMC CEX B/E VF with pre-filtration AEX FT Tandem CMC PA B/E Connected Flow-through (post PA) Low pH VI Pool VF Pool AC + AEX FT CEX + VF In-line pH Pro-A+AEX: M. Shamashkin et al., Biotechnol Bioeng, 110(10) 2013 Pro-A+CEX+AEX: Neil Soice, et. al. ACS spring conference 2010 V. Warikoo et al., Biotechnol Bioeng, 109(12), 2012 R. Godawat et al., J Biotechnol. 213, 2015 AEX+HIC: J. Zhang et al., Eng Life Sci. 17(2) 2017 Depth+AC+AEX: T Yamada et al., J Chrom B 1061-1062 2017 EP 2574618 A1. 2012
  • 9.
    01 02 03 04 05 Agenda 9 Market Trends andthe Evolution of Next Generation Processes Intensified Capture Processing Continuous Virus Inactivation Connected Flow-Through Polishing Summary
  • 10.
    Improved resin utilization Residencetime operation: Higher or lower What is Continuous Multi-Column Chromatography Intensified Capture 2 column Single loading 3 column Loading in-series
  • 11.
    Intensified Capture What isContinuous Multi-Column Chromatography ▪ Better resin utilization ▪ Higher productivity ▪ Smaller columns ▪ Lower buffer consumption 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0 10 20 30 40 50 60 70 80 1-Column (1% BT) 2-Column (1% BT) DynamicCapacity(g/L) Residence Time (min) Protein conc. in column 3 min RT 1 min RT 0,6 min RT 0,4 min RT Batch Continuous
  • 12.
    Intensified Capture Quality andYield of Continuous Multi-Column Chromatography 3 column continuous results Total IgG loaded : 325mg (@RT 1min) Washing : 25mg Elution : 296mg 91% Total Operation time: 8.3hr Used protein A amount: 3ml Batch results Total IgG loaded : 480mg (@RT 7.2min) Elution : 445mg 92% Total Operation time: 24hr Used protein A amount: 5ml Data generated by Chiyoda TechnoAce Co.,Ltd. ▪ Maintained performance ▪ Same robustness ▪ Comparable process yield Protein A affinity resin: Eshmuno® A
  • 13.
    Is Multi-column ChromatographyProductive? Same operation for resin: B/E • Same resin • Same buffer Batch Multi Column Changes: • Reduction of resin and buffer • Shorter residence time • Increase cycle • Interconnected operation • Complex flow path for cycling
  • 14.
    Intensified Capture Operational Windowson Productivity Batch 2-column switching 3-column cycling Works around 20cm Bed at 250-350 cm/hr Productivity: 15-30 g/L/hr Limitation of flow rate Bed height at 5-15cm 25-40 g/L/hr Operation point is determined by 24hr run (perfusion) 3.5-4.5g/L/hr 2000L (Titer 5.4g/L) Eshmuno® A resin Least Squares Fit by response surface (Predicted by JMP software) ID 63cm ID 25cm ID 35cm Different sweet spot of resin utilization
  • 15.
    Intensified Capture Capture ColumnStrategy for Upstream Processing Fed-batch bioreactor 2kL, 5.4g/L Single harvest Perfusion bioreactor (30days) 150L, 2.29g/L Continuous harvest Concentrated Fed-batch 500L, 21.4g/L Single harvest UF MF BPOG Next Gen Tech Roadmap Upstream Scenarios Titers are referenced from Xu, S. et al., Biotechnol. Prog., 33(4) 2017 0% 50% 100% Intensifie dFB& Batch Intensifie dFB& CMC HD perfusion & Switching Concentra tedFB &CMC Buffer Total resin 0% 100% 200% 300% Productivity x1.7 1/4 x2.2 -97% -50% 1 2 3 4
  • 16.
    16 Intensified Capture Impact onLong Duration Cycling Run J. Pollock et al., J. Chromatography A 2013 Continuous Batch P Perfusion Bioreactor Cell Retention device, breeding device Protein A capture Semi continuous and continuous manufacturing Centrifugation, ATF, TFF Multicolumn cycling In-line perfusate clarification Limited impurity removal Significantly more foulants Steeper loss in DBC D. Burgstaller et al., J. Chem. Technol. Biotechnol. 2017 pDADMAC Millistak® HC ProDesign with enough safety factor Improved CIP Additional perfusate clarification Cycle Number LossinDBC(%)
  • 17.
    -5 0 5 10 15 20 -0.5 0 0.5 1 1.5 2 2.5 0 5 1015 20 25 30 35 40 45 50 55 60 65 pHorConductivity/4 A280(AU) Column Volumes A280 Conductivity / 4 pH wash -5 0 5 10 15 20 -0.5 0 0.5 1 1.5 2 2.5 0 5 10 15 20 25 30 35 40 45 pHorConductivity/4 A280(AU) Column Volumes A280 Conductivity / 4 pH Unconcentrated mAb Harvest 1.3 g/L titer 24 CV load SPTFF Concentrated 12.4 g/L titer 2.5 CV load equil load elute re-equil CIP Load time = 144 min Cycle time = 4.25 hour Load time = 14 min Cycle time = 2 hour SPTFF feed concentration increased capture step productivity by 115% (7.2 g/L/hr → 15.5 g/L/hr) Case Study: 1.6 L Eshmuno® A column for batch mAb capture Improve Capture Chromatography with SPTFF Intensified Capture
  • 18.
    Flow Rate Existing resins TheDrive For Higher Productivity Solutions Residence times is the key for future Higher productivity enables: ✓ Increased speed ✓ Smaller footprint → Potential for Single Use → Manufacturing flexibility ✓ Reduced COGS 3-8 min. 1-3 min. 0.5-1 min. 2-10 sec. Productivity(g/L/h) Residence Time (min) Intensified Capture 18 Conventional Column Chromatography
  • 19.
    Multi-column cycling optimizesresin utilization and enables continuous processing: • Upstream culture strategy should be considered: Fed-batch or Perfusion • Harvest type: Single or Continuous • mAb concentration: SPTFF can boost productivity for Protein A capture • Design consideration for multi cycling operation for higher loading: Safety factor, Improved CIP, Additional perfusate clarification Summary of intensified capture
  • 20.
    01 02 03 04 05 Agenda 20 Market Trends andthe Evolution of Next Generation Processes Intensified Capture Processing Continuous Virus Inactivation Connected Flow-Through Polishing Summary
  • 21.
    In-Line Virus Inactivation ACID →Virus inactivation kinetics → Efficient incubation chamber design PROTEIN A CAPTURE POLISHING CHROM. STEP #1 BATCH Industry standard: 60 minute hold Robust inactivation > 4 LRV 2 holding tanks required Manual process Continuous pH adjustment with dynamic hold Eliminates large holding tanks Automated Enables continuous operation 60 min HOLD BASE Applicability dependent upon: CONTINUOUS
  • 22.
    0 0.2 0.4 0.6 0.8 1 0 0.5 11.5 2 2.5 CumulativeDistribution V/V50 In-Line Virus Inactivation Incubation Chamber Design – Flow Distribution Characterization Straight Tube Coiled Serpentine Early virus breakthrough → Larger safety factor Long protein exposure C. Gillespie et al., Biotechnol J. 2019 Feb;14(2)
  • 23.
    Inactivation Kinetics Virus type PRV <XMuLV Buffer type acetate  glycine < citrate Temperature 22ºC < 16ºC mAb concentration Minimal impact In-Line Virus Inactivation Virus Inactivation Kinetics Rapid inactivation kinetics (~2-3 min) C. Gillespie et al., Biotechnol J. 2019 Feb;14(2)
  • 24.
    • Virus inactivationat low pH is rapid and requires less than 5 minutes, implying that this process can be run in continuous mode. The kinetics of inactivation depend on pH, temperature, and buffer conditions, and are not dependent on protein concentration. • Technologies exist to move from batch to continuous virus inactivation. Efficient incubation chamber design and control offers continuous flow systems shorter processing times than traditional batch processes. • Proper implementation could result in equivalent levels of virus inactivation in batch and in-line systems. Summary of in-line virus inactivation
  • 25.
    01 02 03 04 05 Agenda 25 Market Trends andthe Evolution of Next Generation Processes Intensified Capture Processing Continuous Virus Inactivation Connected Flow-Through Polishing Summary
  • 26.
    Toolbox Approach Flow-through Polishing Leached ProteinA Aggregates Virus Host Cell DNA Host Cell Protein Others? (Fragments, Charge Variants) Chemistries Matrices Devices
  • 27.
    Flow-through Polishing Existing Toolbox ▪Eshmuno® CP-FT resin Flow through aggregate removal at high loading 5 – 10X buffer reduction Easy implementation ▪ NatriFlo® HD-Q AEX membrane Impurity clearance at high loading High velocity operation (~1 sec RT) ▪ Millistak+® Pod CR40 depth filter Activated carbon media Binding: van der Waals interactions Size-based selectivity Impurity (MW) Capacity Insulin 60 g/L Methotrexate 75 g/L Pluronic® F68 surfactant 75 g/L Antifoam C > 16 g/L
  • 28.
    Eshmuno® CP-FT resin Benchmarking OnlyEshmuno® CP-FT resin was efficient for the removal of aggregates in the flow- through mode under strong binding conditions that favor frontal chromatography M. Stone et al., J Chrom A, 1599, 2019
  • 29.
    Batch vs. IntegratedImplementation of Flow-through Technology Robust Performance with Significant Footprint Reduction Flow Through Polishing Delivers: Robust Purification across 8 molecules  High product yield (83 – 91%)  Aggregates: < 1%  Fragments: up to 1 LRV  HCP clearance: < 10 ppm Significant Facility Footprint Reduction  Lower media and buffer requirements  Eliminate intermediate hold tanks  Single-skid operation Low pH VI Pool Virus Filter Pool Carbon Depth Filter + FT-AEX FT-CEX + Virus Filtration In-line pH Low pH VI Pool Depth Filter Pool CEX Pool AEX Pool Virus Filter Pool pH Adjust & Dilution Depth Filter Bind & Elute CEX FT-AEX Virus Filtration Traditional Batch Polishing Flow Through Polishing
  • 30.
    3-step Flow-through Polishing 100 98 96 94 92 3.0 2.5 2.0 1.5 1.0 0.5 0.0 VIAAC Feed FT-AC AEX Feed FT-AEX CEX Feed FT-CEX HMW1 HMW2 LMW1 LMW2 Monomer HMW,LMW(%) Monomer(%) 10000 1000 100 10 1 DNA DNA(pg/mg),HCP(ng/mg) HCP3985 1888 27.8 512 0.15 0 0 2.47 0.25 0.01 0.01 0 1.7 0.07 1.57 0.03 1.68 0.03 98.22 94.22 <0.76 <0.25 0.00 FT-AC FTCEXFTAEX pH5 3mS/cm Activated carbon shows 3 log10 DNA clearance. Robust clearance of HCP and DNA by FT-AEX. Eshmuno® CP-FT reduces mAb-related HMW aggregates (Dimer). mAb 10.9g/L pH7 3mS/sm T. Ichihara et al., mAbs J., 10, 2018
  • 31.
    Feed condition: mAb-A (8.2mg/mL) Impurities:HCP = 567ng/mg IgG, DNA=7276 pg/mgIgG, HMW1 = 0.78%, HMW2= 1.65%, Monomer = 96.53%, LMW1=0.93%, LMW2=0.1%. pH6, 4mS/cm, 0.2mL/min High Loading : 200mL (1640mg mAb) Operation system : AKTAexplorer 100 Single equilibrium: 25mM sodium acetate buffer pH6 Single elution: 25mM Sodium acetate buffer pH6 w 1M NaCL Evaluation: Connected flow-through 1. AEX(1mL) – CEX(0.5mL) 2. AC(0.5mL) – AEX(1mL) –CEX(0.5mL) Connected Flow-through Purification Proof of Concept Study from DOE Optimization AC CEX T. Ichihara et al., mAbs J., 10, 2018 T. Ichihara et al., Eng. Life Sci., 19, 2019
  • 32.
    (A) AEX -CEX(A) AEX - CEX (B) AC - AEX - CEX 5 5.5 6 6.5 20 40 60 80 100 120 140 160 180 200 220 240 pH 0 500 1000 1500 2000 2500 3000 3500 0 A280(mAU) Volume (mL) 5 5.5 6 6.5 7 0 500 1000 1500 2000 2500 3000 3500 0 20 40 60 80 100 120 140 160 180 200 220 240 pH As80(mAU) Volume (mL) 7 0 40 80 120 160 200 Conductivity(mS/sm)0 40 80 120 160 200 Conductivity(mS/sm) 0 0.2 0.4 0.6 0.8 1 MPa 0 0.2 0.4 0.6 0.8 1 MPaFigure 1. Typical chromatograms obtained from the in-series, connected flow-through polishing steps. (A) AEX-CEX, (B) AC-AEX-CEX. Load Wash Elute Load Wash Elute Evaluation of Connected Flow-through Purification Results of Connected Flow-through Polishing AEX-CEX AC-AEX-CEX Wash Load Elute Wash Load Elute 90 92 94 96 98 100 5 8 11 ○Monomer(%) ■mAbconc. (mg/mL) 0 2000 4000 6000 8000 10000 0 100 200 300 400 500 0 1000 2000 △DNA(pg/mg) ◆HCP(ng/mg) mAb loading (mg) AEX-CEX AC- AEX-CEX mAb conc. Mono- mer HCP DNA Chromatograms by in-series flow-through polishing 1/2 -1/3 reduction of processing time Extremely high pressure of elution mAb 100% breakthrough at >400mg lording AC improves HCP and DNA clearance T. Ichihara et al., Eng. Life Sci., 19, 2019
  • 33.
    33 Value Modeling What ValueDoes Flow Through Polishing Deliver? Bioreactor Clarification Affinity Chromatography Virus Inactivation CEX Bind and Elute Flow Through AEX Viral Clearance Final Filtration Concentration & Diafiltration Traditional Batch (Baseline) Depth Filtration Four polishing steps run continuously without intermediate hold steps ▪ Lower buffer volume ▪ Higher loading on chromatography media and virus filter ▪ Smaller facility footprint for systems, columns, and tanks Bioreactor Clarification Affinity Chromatography Virus Inactivation Final Filtration Concentration & Diafiltration Flow Through Polishing Activated Carbon Flow Through CEX Flow Through AEX Viral Clearance Bioreactor: 2k L fed-batch Titer: 5 g/L Mab Felo, Holistic Development of Intensified Mab Processes for Higher Productivity and Improved Economics, BPI Boston, Sep 2018
  • 34.
    Assumption Activated Carbon AEX FTCEX FT Virus Filtration Technology Millistak+® Carbon depth filter Eshmuno® Q resin Eshmuno® CP-FT resin Viresolve® Pro filter Yield 93% 95% 95% 98% Capacity 1500 g/m2 3000 g/L 1000 g/L 5000 g/m2 # Re-uses Single use 150 150 Single use # Buffers 1 5 4 0 Activated Carbon Flow Through CEX Flow Through AEX Viral Clearance Value Modeling Assumptions Key to Output • Single skid versus four • Yields matched to baseline process • Baseline process assumptions per BPOG costs & assumptions Bioreactor: 2k L fed-batch Titer: 5 g/L Mab 34 Felo, Holistic Development of Intensified Mab Processes for Higher Productivity and Improved Economics, BPI Boston, Sep 2018
  • 35.
    35 Baseline Scenario vs.Flow Through Polishing • Most significant savings capital, consumables, and labor • Buffer reduction due primarily from switching CEX to flow through mode 10% COGS reduction 47% Buffer reduction 43% COGS reduction 87% Buffer reduction Polishing alone… Total process… Value Modeling COGS Savings from Reduced Buffer Use, Intermediate Storage Tanks, and Process Time/Labor Bioreactor: 2k L fed-batch Titer: 5 g/L Mab Ito BioKorea 2019 | 18- Apr-2019, Copyright Merck 2019 Felo, Holistic Development of Intensified Mab Processes for Higher Productivity and Improved Economics, BPI Boston, Sep 2018
  • 36.
    36 Ito BioKorea 2019 |18- Apr-2019, Copyright Merck 2019 Pre-concentrate to Boost Eshmuno® Q Resin HCP Clearance SPTFF with Flow-through Polishing 0 1 2 3 4 5 6 0 20 40 60 80 100 120 Improvementfactorabove non-SPTFFfeed mAb feed concentration (mg/mL) Improvement vs. mAb feed concentration Mass Loading (g/L resin) ▪SPTFF pre-concentration can boost Eshmuno® Q resin mass loading by a factor of 4x. ▪ Reduced loading at high feed concentrations, possibly due to mAb-HCP interactions. ▪In addition to load improvements, SPTFF concentration reduces process load time, thus increasing productivity by a factor of 5x. 0 1 2 3 4 5 6 0 20 40 60 80 100 120 Improvementfactorabove non-SPTFFfeed mAb feed concentration (mg/mL) Improvement vs. mAb feed concentration Mass Loading (g/L resin) Productivity (g/L resin/hr) Pre-Concentrate Protein SPTFF Pellicon® AEX Eshmuno® Q T Elich et al., ACS conference spring Apr 2017
  • 37.
    Continuous diafiltration forbuffer exchange Tank I Tank II Recovery Tank To next process step From previous process step UF 0 1 2 3 4 5 6 7 NormalizedtoBatch All cases: no UF, 9 diavolumes ■ Batch: 3h process, 6LMM crossflow ■ CDF low area: 19h process, 6LMM crossflow ■ CDF low pump passes: 19h process, 1LMM crossflow ■ ILDF: 19h process 30 35 40 45 0 2 4 6 8 10 12 14 16 18 20 22 24 Cycle Number Control Chart: DF Cycle Time Average = 36.9 min, σ = 1.8 min E. Goodrich et al., Recovery of biological Products, Oct 2018
  • 38.
    Comparable performance betweenthe batch purification process and the pool-less process configuration. • Simple operation: Single system operation and removal of the intermediate hold tank • Substantial quality: higher overall mAb yield and impurity clearance • Small processing: Higher process loading = Process compression, reduced buffer usage • Shorter process time: Single loading step • COGS reduction: ~10% across full process • SPTFF pre-concentration can boost AEX resin mass loading Summary of Flow-through Polishing
  • 39.
    01 02 03 04 05 Agenda 39 Market Trends andthe Evolution of Next Generation Processes Intensified Capture Processing Continuous Virus Inactivation Connected Flow-Through Polishing Summary
  • 40.
    Intensified Downstream Processing Summary Industrytrends are forcing bio-manufacturers to adopt a more strategic view of manufacturing − Process Intensification − Single Use − Process Analytical Technologies and controls Process design and optimization requires an “holistic view” − Upstream and Downstream − Tool box for connected impurity clearance Next generation technologies can support intensification efforts of existing batch processes while enabling future connected and continuous processing Collaborations between academics, industry, and regulatory agencies are critical for successfully moving the industry forward in next generation processing 1 2 3 4
  • 41.
    Acknowledgements Christopher Gillespie Kevin Galipeauc YasuhikoKurisu Yasuhiro Kawakami Miyuki Koyama Prof. Shuichi Yamamoto (Yamaguchi univ.) Takamitsu Ichihara(Astellas) Shuhei Kondo (Chiyoda TechnoAce) Michael Phillips Romas Skudas Mikhail Kozlov Lars Peeck Ajish Potty Matthew Stone Alex Xenopoulos Renaud Jacquemart Danny Wu Thomas Elich Mochao Zhao Michael Felo Joseph Geringer Elizabeth Goodrich
  • 42.
    Schedule an in-personor remote visit today. EMDMillipore.com/mlab
  • 43.
    The vibrant M,M Lab, BioContinuum, Eshmuno, Millistak+, NatriFlo 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. © 2020 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.