Demonstrating Process Scalability with Robust and Turnkey Platforms
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Joe Orlando presented on Merck KGaA's platforms for robust and scalable upstream bioprocess development. Their CHOZN GS expression system provides a stable recombinant cell line, optimized media and feeds, and single-use bioreactors to efficiently develop and scale processes from shake flasks to 50L bioreactors. Using their TNFR Fc-fusion protein as an example, Merck demonstrated consistent titers, glycan profiles, and low aggregates across scales. Their turnkey solution aims to reduce development time and improve product quality and consistency throughout the upstream process.
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Demonstrating Process Scalability with Robust and Turnkey Platforms
- 1. Merck KGaA Darmstadt, Germany Demonstrating Process Scalability with Robust and Turnkey Platforms Joe Orlando, Ph.D., Principal Scientist 15th February 2018
- 2. BioProcessing Cell culture Harvest Clarification Chromatography Virus removal Tangential flow filtration Sterile filtration Formulation and Final Fill 2
- 3. BioProcessing Cell culture Harvest Clarification Chromatography Virus removal Tangential flow filtration Sterile filtration Formulation and Final fill Cell line development Media optimization Bioreactor 3
- 4. Purpose Platform Upstream Bioprocess Development • Foundation blocks Cell line, DNA vector, cell culture media & feeds, and bioreactors • Unit operation integration Turn-key solution for process development and scale up 4
- 5. Time consuming and resource intensive processes • Cell line development • Bioreactor process development Challenges to upstream process development Process consistency • Expected product titer and quality • Equivalency at small and large scale 5
- 7. Comprehensive expression platform & services Rapid and robust development CHOZN® GS-/- cell line GS auxotroph cell line with clear IP path Traceability Documentation Comprehensive cell line history documentation to support regulatory filing Expression Vector IP free GS expression vector suitable for mAbs/recombinants Process Guidance & Protocols Protocols for entire workflow Media and Feeds Fed batch & Perfusion media produced under GMP Technical Support Comprehensive product and process technical support to ensure your success CHOZN® GS Expression Platform 7
- 8. Optimized cell line development process Balancing resources and performance Transfect Selection via “minipools” Screen minipools in 96-well plates Scale-up top producing minipools Screen 7-day shake flask Fed-batch assay on top minipools (shake flask) Single Cell Cloning 200 pools 100 pools 100 pools 20 pools 3-4 weeks 1 week 2 weeks 1 week 2 weeks 10-12 weeks 8
- 9. CHOZN® GS performance Performance of 4 CHOZN® GS clones isolated from mini-pools 0 1000 2000 3000 4000 5000 Titer(mg/L) Protein Expression 0 25 50 75 100 125 CellSpecificProductivity pg/Cell/Day Cell Specific Productivity (Qp) 9
- 10. CHOZN® GS stability 75%75% High stability Studies performed on top ten producing clones from an individual project. 80% of CHOZN® clones are found to have stable productivity. Confidence in consistent production 10
- 11. Optimization of cell culture media and feed strategy 11
- 12. • Robust and sustainable supply chain • Proven manufacturability and consistency • Multisite manufacturing redundancy • Scalable GMP ready products • Animal component free • High titers with CHOZN® GS and other host cell lines EX-CELL® Advanced™ cell culture media & feeds Paired media for efficient production processes 12
- 13. EX-CELL Advanced cell culture media Optimizing feed strategy 0 25 50 75 100 125 150 7 10 12 14 %ofcontrolpeaktiter Days in culture Control 5x5% 3x10% 1x5%, 4x7.5% 1x5%, 3x10%, 1x5% 10x3% 30% improvement in peak titer 13
- 14. Scale up 14
- 15. Mobius® Single-Use Bioreactor Scalable System for Rapid Process Development and Production 15
- 16. Limited number of bioreactor runs required for efficient scale up Shake Flask Mobius® 50L Single-use bioreactor Mobius® 3L Single-use bioreactor • Duplicate shake flasks were run in each study • Cells seeded at 5x105 cells/ml • EX-CELL® Advanced™ Feed schedule: 10% days 3, 5 and 7 & 5% day 10 • Glucose maintained at 5 or 6 g/L • 3 duplicate runs evaluated • Seed density and feed strategy consistent with shake flask • Power 20 W/m3, pH 6.9, dissolved oxygen (DO) 30%, temp 36.8°C • Parameters evaluated: gas transfer, pH dead band • 2 single vessel runs evaluated • Cells seeded and fed as shake flasks • Equivalent energy dissipation or Power - primary scaling factor • Power 20 W/m3, pH 6.9, DO 30%, temp 36.8°C Scalability of the Mobius® Single-use Bioreactors https://www.emdmillipore.com/Web-US-Site/en_CA/-/USD/ShowDocument-Pronet?id=201610.040 16
- 17. Process scalability Titers similar between bioreactor scales 0 5 10 15 20 25 - 3 6 9 12 15 VCDX1E6 Days Viable Cell Density SF 50L 3L 0 0.5 1 1.5 2 2.5 - 3 6 9 12 15 Titerg/L Days Titer SF 50L 3L 17
- 18. Process predictability Glycan profiles similar across multiple scales Shake Flask Mobius® 3L Single-use bioreactor Mobius® 50L Single-use bioreactor 18
- 19. Process predictability Low aggregate profiles across multiple scales Shake Flask Mobius® 3L Single-use bioreactor Mobius® 50L Single-use bioreactor 0 0.3 0.6 0.9 1.2 50L 3L 3L Shake flask %Aggregates 19
- 20. • A stable CHOZN® GS recombinant expressing a TNFR Fc-fusion protein was subjected to scale-up • Shake flask, 3L, and 50 L bioreactor conditions were identical to those used for the benchmark recombinant cell line • Near equivalent titers were achieved at all three scales • Future parameters to potentially address • Seed density • Power and agitator speed • New feeds and strategies Process consistency – Turn key platform Scale-up of a recombinant cell line expressing a TNFR Fc-fusion protein 0 0.5 1 1.5 2 SF SF 3L 3L 50L PeakTiterg/L20
- 21. Summary and Conclusions Upstream Bioproduction • Foundation blocks Cell line, cell culture media & feeds, and bioreactors • Unit integration Turnkey solution for clone selection, process development, and scale up • Speed Platform development reduces the need for extensive optimization • Product consistency Confidence in process performance and product quality 21
- 22. Questions 22
- 23. Joe Orlando, Ph.D. Principal Scientist 80 Ashby Rd Bedford, MA 01730 USA Phone: +1 781 533-5721 Email: joe.rlando@emdmillipore.com CONtact: The vibrant M, CHOZN, Cellvento, Mobius, Advanced and EX-CELL 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. © 2017 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.