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High-throughput and Automated Process Development for Accelerated Biotherapeutic Manufacturing
KBI Biopharma has developed high-throughput and automated processes to accelerate biotherapeutic development. This includes establishing a high-throughput process development team utilizing automated equipment and informatics solutions. Analytical case studies demonstrate automation of a residual host cell protein ELISA using a liquid handling robot, reducing analysis time from hours to minutes per sample. A second case study outlines development of a high-throughput size exclusion chromatography method, reducing run time from 30 minutes to 6 minutes while still effectively screening for high molecular weight species. These efforts allow for real-time data generation and monitoring of process development experiments.
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High-throughput and Automated Process Development for Accelerated Biotherapeutic Manufacturing
- 1. Confidential High-throughput and automated process developmentfor accelerated biotherapeutic manufacturing Derek Ryan, Ph.D. Senior Scientist, Analytical Development KBI Biopharma, Inc.
- 2. Confidential 2 KBI Biopharma’s missionis to accelerate the development of innovative discoveries into life-changing biological products & expand global access of medicines to patients in need KBI Mission Statement
- 3. Confidential 3 Discussion Overview Overview ofKBI process development and manufacturing portfolio Development of a high-throughout process development team Analytical Case study: Automation of a residual ELISA for rapid PD support Analytical Case study: Development of a high-throughput SEC method for HMW screening Ongoing developments of high-throughput efforts at KBI
- 4. Confidential 4 PD & ManufacturingExperience by Product Type: * 9 biosimilars Recently 50+ PD programs & 12 client INDs supported per year # Mfg Batches 50 7 11 10 6 1 10 5 0 0 20 40 60 Recombinant Enzyme Enzyme Growth factor Recombinant Protein PEGylated… FC-Fusion, Fusion… Vaccine Glycoprotein Bispecific AB mAb* PD Product Types
- 5. Confidential 5 KBI Analytical Panelsby Molecule Type mAbs • Titer - Octet/ProA HPLC • Aggregate - SEC-U/HPLC • Purity - R+NR CGE • Charge - icIEF • N-Glycan - HILIC-UPLC-FLD • Binding Potency – Octet/ELISA • Biological Potency - Cell-based assay Fc-Fusions • Titer - Octet/ProA HPLC • Aggregate - SEC-U/HPLC • Purity - R+NR CGE, RP/IEX/HIC-HPLC • Charge - icIEF/IEX • N-Glycan - HILIC-UPLC-FLD • Binding Potency – Octet/ELISA • Biological Potency - Cell-based assay Bispecifics • Titer - Octet/ProA HPLC/ELISA • Aggregate - SEC-U/HPLC • Heterodimer Purity – IEX, LCMS • Purity – CGE • Charge - icIEF/IEX • N-Glycan - HILIC-UPLC-FLD • Dual/Single Binding Potency – Octet/ELISA • Biological Potency - Cell-based assays Glycoproteins (HIV env, etc.) • Titer - Octet/ELISA • Aggregate - SEC-U/HPLC • Purity - RP/IEX/HIC-HPLC, SDS-PAGE • Charge - IEX, IEF-PAGE • N-Glycan - HILIC-UPLC-FLD • Antigenicity/Binding Potency – Octet/SPR/ELISA • Biological Potency - Cell-based assay Enzymes • Titer - Octet/ELISA/Activity • Aggregate - SEC-U/HPLC • Purity - R+NR CGE, RP/IEX/HIC-HPLC • SDS-PAGE • Charge - icIEF/IEX • N-Glycan - HILIC-UPLC-FLD • Potency – Enzymatic Activity Assay
- 6. 6 5 w POOL 3 w Vector 2w SCC0 2 w ST 2 w SCC1 3 w SCC2 Early Clone Robustness Enables Integrated PD with CLD Need for monitoring US and DS process development experiments in real time KBI Process, Analytical & Formulation Development KBI GMP Manufacturing (500L to 2,000L) Month: 1 2 3 4 5 6 7 8 9
- 7. Confidential 7 Establishment of anIntegrated High-throughput Mindset for PD USPD AD DSPD
- 8. Confidential 8 History of High-throughputInitiatives at KBI Present • Aligned US AMBR platform with AD liquid handling robotics for automated sample handling • Development of high-throughput and automated analytical methods • Use of high-throughput technologies for resin evaluations or small scale capture (i.e. PreDictor Plate, Robocolumns) during DSPD • Adoption of electronic lab notebooks for data management and archival Future • Establishment of platform analytics packages for efficiency gains during development support • Sample inventory and data management in electronic lab notebooks Past • Establishment of AMBR15 and AMBR250 platforms for high- throughput USPD • Adoption of ClonePix technology for high-throughput clone selection • Adoption of Octet system for high-throughput titer determination
- 9. Confidential 9 Keys to EstablishingEfficiency Initiatives •Investment in high-throughput or automated instrumentationEquipment •IT solutions to big data managementInfrastructure •Training •Management of dataPersonnel
- 10. Confidential 10 Equipment: High-throughput andAutomated Capabilities
- 11. Confidential 11 Infrastructure and Personnel: Establishmentof a High-throughput Core Team IT Support Construction of enterprise solutions for the organization and storage of data Automation Engineers Work in concert with IT and Scientists to get the robotics to best mimic the manual operations Scientists Provide scientific guidance on intended purpose of experiments and review results of automated assays
- 12. Confidential 12 As more developmentprograms are onboarded for upstream and downstream process development an increase in submission to AD for PQ occurs Automation of assay decreases FTE effort and data TAT Potential improvement in data quality by reducing %CV Allow for more assays to be setup in parallel by fewer FTE Case Study: Automation of residual host cell protein ELISA
- 13. Confidential 13 Capabilities of ourTECAN platform LiHa – liquid handling aspects of the assay. RoMA – manipulates objects to other locations within the deck layout. RoMALiHa PreDictor Plate ATOLL Robocolumns ELISA workspace
- 14. Confidential 14 Considerations for thedevelopment of automated HCP ELISA Fully Automated rHCP ELISA Sample logistics • Sample submissions • Storage • Stability
- 15. Confidential 15 Considerations for thedevelopment of automated HCP ELISA Fully Automated rHCP ELISA Sample logistics • Sample submissions • Storage • Stability Stability of reagents • Stability of reagents on deck • Performance of assay throughout assays
- 16. Confidential 16 Considerations for thedevelopment of automated HCP ELISA Fully Automated rHCP ELISA Sample logistics • Sample submissions • Storage • Stability Stability of reagents • Stability of reagents on deck • Performance of assay throughout assays Timing of assay events • Key steps from assay need to be taken into account • Balance of robotics capability and assay demands
- 17. Confidential 17 Considerations for thedevelopment of automated HCP ELISA Fully Automated rHCP ELISA Sample logistics • Sample submissions • Storage • Stability Stability of reagents • Stability of reagents on deck • Performance of assay throughout assays Timing of assay events • Key steps from assay need to be taken into account • Balance of robotics capability and assay demands Comparability of data to QC ELISA • Head to head testing of samples using manual and automated methods
- 18. Confidential 18 Considerations for thedevelopment of automated HCP ELISA Fully Automated rHCP ELISA Sample logistics • Sample submissions • Storage • Stability Stability of reagents • Stability of reagents on deck • Performance of assay throughout assays Timing of assay events • Key steps from assay need to be taken into account • Balance of robotics capability and assay demands Comparability of data to QC ELISA • Head to head testing of samples using manual and automated methods Data integrity, storage, and organization • Data analysis and reporting • Storage of reported results
- 19. Confidential 19 Comparability of AutomatedrHCP ELISA versus Manual rHCP ELISA 0 200 400 600 800 1000 1200 1400 Affinity Capture Eluate VIN Eluate IEX Eluate Cycle 1 IEX Eluate Cycle 2 IEX Eluate Cycle 3 IEX Pool MMC Eluate ppm(ngHCP/mgProtein) Automated ELISA Manual ELISA
- 20. Confidential 20 Automation Allowed Scalingto Meet Need without FTE Expansion 0 2 4 6 8 10 12 14 16 18 Hours Analyst touch time per 20 sample batch Automated Manual 0 1000 2000 3000 4000 5000 6000 2017 2018 2019* HCP samples tested per year *Forecasted based on submissions to date (Jan – Mar 2019)
- 21. Confidential 21 Process development iscapable of generating 100’s of samples for aggregate testing Management of a sample inventory of this size outside of 96 well plates not realistic Adopted HPLC autosamplers that can hold up to 16, 96 well plates in a temperature controlled chamber Method run time can significantly affect data turnaround time Example: 200 injections (samples, ref. brackets, etc.) using our platform “QC” SEC- HPLC method would take nearly a week of nonstop run time to collect the data. 200 samples * 30 min per sample = 100 hour run time (~4 days), not including data workup Development of a High-throughput SEC Method: How can Analytics Provide Real Time Aggregate Data? Prompted development of new high throughput SEC methods to reduce run time during critical process development evaluations
- 22. Confidential 22 Acq Method Set:UPSW3000 3_INS3624 AU 0.00 0.05 0.10 0.15 0.20 0.25 0.30 Acq Method Set: UPSW3000 4_INS3624 AU 0.00 0.05 0.10 0.15 0.20 0.25 0.30 Acq Method Set: UPSW3000_INS3624 AU 0.00 0.05 0.10 0.15 0.20 0.25 0.30 Minutes 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80 10.00 Evaluation of High-throughput SEC Method: Optimizing Run Time 0.3 mL/min 0.4 mL/min 0.5 mL/min TOSOH, UP-SW3000 (2 µm, 15 cm x 4.6 mm) Mobile Phase: 100 mM phosphate, 150 mM NaCl pH 7.3
- 23. Confidential 23 3.037 Main-3.420 4.178 4.982 AU 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 Minutes 1.80 2.00 2.202.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 12.855 13.584 Main-15.695 19.549 AU -0.002 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.016 0.018 0.020 Minutes 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 HT Method Shows Comparable HMW in the Context of Screening “QC” method: 5 µm particle size 30 min run time High-throughput Method: 2 µm particle size 6 min run time Method %HMW %Main %LMW QC SEC-HPLC 1.95 97.91 0.15 HT SEC-HPLC 2.19 97.16 0.65 NIST IgG standard: unstressed 12.855 13.584 Main-15.695 19.549 AU 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00 3.037 Main-3.420 4.178 4.982 AU 0.00 0.10 0.20 0.30 0.40 0.50 0.60 Minutes 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00
- 24. Confidential 24 3.037 Main-3.420 4.178 4.982 AU 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 Minutes 1.80 2.00 2.202.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 12.855 13.584 Main-15.695 19.549 AU -0.002 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.016 0.018 0.020 Minutes 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 “QC” method: 5 µm particle size 30 min run time High-throughput Method: 2 µm particle size 6 min run time Method %HMW %Main %LMW QC SEC-HPLC 2.72 96.39 0.9 HT SEC-HPLC 3.20 96.44 0.35 NIST IgG standard: Heat Stress1 12.855 13.584 Main-15.695 19.549 AU 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00 3.037 Main-3.420 4.178 4.982 AU 0.00 0.10 0.20 0.30 0.40 0.50 0.60 Minutes 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 1held at 37 oC for 24 hours HT Method Shows Comparable HMW in the Context of Screening
- 25. Confidential 25 Typical QC SECmethod is 30 - 40 minutes per sample plus HPLC mechanical movements • 1rack of 96 samples is about 48 - 72 hrs of HPLC time (excluding reference brackets) HT SEC method is 6 minutes per sample plus HPLC mechanical movements • 1plate of 96 samples is about 11hrs of HPLC time • Represents an at least 5x decrease in duration of TAT for data Metrics of QC versus HT SEC-HPLC Methods Method considered fit for purpose of HT screening testing for PD troubleshooting Additional comparability library of other molecules being assembled
- 26. Confidential 26 Ongoing Goals forthe High-throughput team in 2019 and beyond Analytical Development •Continued automation of plate based ELISA •Automation of small scale purification •Utilization of liquid handling robotics for sample preparation Upstream Process Development •Continued implementation of high-throughput process development on the AMBR15 and AMBR250 platforms •Utilization of liquid handling robotics for automated sampling and retain aliquoting Downstream Process Development •Utilization of liquid handling robotics for rapid resin screening experiments •Utilization of liquid handling robotics for automated sample and retain aliquoting
- 27. Confidential 27 Analytical HT Team: KirstenDysinger John Russell Phoebe Lee Sean Johnson Acknowledgements Tim Kelly, Ph.D. Jimmy Smedley, Ph.D. Sigma Mostafa, Ph.D. Upstream HT Team: Niket Bubna David Chang Downstream HT Team: Leslie Wolfe, Ph.D. Thomas Lindsey
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