1 / 38

New Approaches in Continuous BioManufacturing :

New Approaches in Continuous BioManufacturing : Continuous XD ® cell cultures (around 100 mln cells/mL) coupled to the Rhobust ® EBA integrated clarification and purification technology Gerben Zijlstra, PhD Sr. Scientist DSM Biologics, The Netherlands. Outline.

amelia
Download Presentation

New Approaches in Continuous BioManufacturing :

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. New Approaches in Continuous BioManufacturing: Continuous XD® cell cultures (around 100 mlncells/mL) coupled to the Rhobust® EBA integrated clarification and purification technology Gerben Zijlstra, PhD Sr. Scientist DSM Biologics, The Netherlands

  2. Outline • Introduction DSM Biologics • DSM XD® Technology • RHOBUST®Expanded Bed Adsorption (EBA) Technology • Continuous XD® coupled to RHOBUST®EBA • Principle • Continuous XD® examples • Rhobust® EBA on continuous XD® harvest • Concluding Remarks

  3. DSM Biologics: Who are we? • Part of DSM: A Leading Global Life Sciences & Material Sciences Company • Active in 50 countries & 5 continents at over 200 locations • 2012 revenue > € 9 billion • ~23,000 employees • DSM Biologics Manufacturing Locations • The Netherlands and Australia • DSM Biologics Services • Contract manufacturing for mammalian cell culture: • From development to commercial manufacturing • cGMP for all clinical phases & market supply • Regulatory support • Global reach • Proprietary Process Technologies: • XD® - intensifying upstream process technology • RHOBUST® - direct capture technology

  4. XD®Technology • Very high-density mammalian processes • Increased bioreactor output & yield per volume 5 – 15 fold • High & Consistent product quality • Reduced capital expenditure requirements • Lower scale-up risk • XD® is a registered trademark of DSM

  5. Cell Culture Modes Fed Batch Feed concentrate Build up Metabolites Osmo increase Changing environment Reducing cell viabilities Concentrated Harvest batch identification XD® Medium Feed Wash out Metabolites No Osmo increase Constant environment High cell viabilities Concentrated Harvest batch identification Perfusion Medium Feed Wash out Metabolites No Osmo increase Constant environment High cell viabilities Dilute harvest Large harvest XD® Process XD®: Proprietary Process Intensification Cell Culture Mode of Manufacturing Fed Batch Feed concentrate Build up Metabolites Osmo increase Changing environment Reducing cell viabilities Concentrated Harvest batch identification Fed Batch Feed concentrate Build up Metabolites Osmo increase Changing environment Reducing cell viabilities Concentrated Harvest batch identification XD® Medium Feed Wash out Metabolites No Osmo increase Constant environment High cell viabilities Concentrated Harvest batch identification XD® Medium Feed Wash out Metabolites No Osmo increase Constant environment High cell viabilities Concentrated Harvest batch identification Perfusion Medium Feed Wash out Metabolites No Osmo increase Constant environment High cell viabilities Dilute harvest Large harvest Perfusion Medium Feed Wash out Metabolites No Osmo increase Constant environment High cell viabilities Dilute harvest Large harvest XD® Process

  6. XD® : Process IntensificationScale-up - PER.C6®; Viable cell count

  7. XD® scale upScale-up - PER.C6®; Product IgG

  8. XD®: Process Intensification Bioreactor Set-up

  9. Preferred retention system: TFF Fully disposable, simple operation, robust, flexible filter choice

  10. Direct product capture • Reduced unit operations • From 3 to 1 • Higher yields • Proven scalability • Reduced labor cost & process time • Suitable for recombinant proteins, antibodies, vaccines DSM RHOBUST® Technology Tungsten carbide incorporated in the agarose bead

  11. RHOBUST®Direct Capture Technology

  12. RHOBUST® in Action with the XD® Harvest Equilibration Cell wash out XD® Harvest ~150x106 cell/mL Wash First cell breakthrough RHOBUST® 1 step! Complete cell breakthrough Elution Post-Protein A intermediate

  13. Results Fed-batch and classical Protein-A packed bed vs. XD® and RHOBUST® Protein-A: RHOBUST® experiments with XD® Harvest With high cell viabilities ~10% higher yield HCP, DNA & res.Protein A After column in normal range

  14. Outline • Introduction DSM Biologics • DSM XD® Technology • RHOBUST®Expanded Bed Adsorption (EBA) Technology • Continuous XD® coupled to RHOBUST®EBA • Principle • Continuous XD ® examples • Rhobust® EBA on continuous XD® harvest • Concluding Remarks

  15. Continuous XD® - Rhobust® principle Diluted Waste Stream Concentrated Product Bleed Medium Product eluate Low pH treatment Waste Waste Elution buffer Tungsten carbide incorporated in the agarose bead 2-8°C Product/Cell Bleed XD® Bioreactor Cell/Product Bleed Rhobust®EBA+Low pH Low pH treated EBA bulk

  16. Continuous XD® example 1: Myeloma - IgG Continuous XD® cultures with Myeloma cells producing highly potent IgG at around 60 mln cells/mL.

  17. Continuous XD® example 1: Myeloma - IgG The Qp (slope of cumulative titer) was constant at maximum Qp.

  18. Continuous XD® example 2: CHO - IgG Continuous XD® cultures with CHO cells producing BiosimilarIgGat around 100 mln cells/mL.

  19. Continuous XD® example 2: CHO - IgG The IgG titer in the product was bleed around 2.5 g/L in production phase.

  20. Continuous XD® example 4: PER.C6® – IgG Continuous XD® with IgG producing PER.C6® cells at around 100 mln cells/mL. IgG titer in the bleed 4.5 - 5 g/L

  21. Rhobust® EBA example 2: IgG An continuous XD® bioreactor processed with eight Rhobust® EBA runs (6 cell bleeds and 2 final bioreactor harvest loads). Product recovery, averaging at 93% was substantially higher compared to the combination of dead-end filtration and fixed bed Protein-A.

  22. Rhobust® EBA example 2: IgG Residual DNA and HCP were within normal ranges and comparable to packed bed values. Aggregate levels and relative potency were relatively constant throughout the run.

  23. Concluding remarksAdvantages Continuous XD® technology coupled to Rhobust® EBA • USP (Continuous XD®cell culture): • Functional advantages: • Robust, stable performance: Stable growth rate by Cell bleed • Very high Productivity: Very high cell density x Maximum Qp No product loss: Bleed = Product! • Constant Quality: High viability & Constant environment • Operational advantages: • Concentrated product flow: Harvest holding point possible • DSP (Rhobust® EBA Clarification / Capture): • Functional advantages: • Very high Recovery: Single unit operation, Concentrated product flow • Very high Purity: Optimized Rhobust® EBA • Operational advantages: • Easy to use, no column packing, air bubbles and precipitates no problem • One step clarification and capture

  24. Acknowledgements: R&D Scientist team DSM-B GMP Process Technologist team Gerben Zijlstra Imre Akkerman Olaf Mol Maria Perlasca Dick Smit Mark Dressen Jurjen de Jong Harriet van der Molen Piet den Boer Mark Doeven Erik kremer Henk van Urk Jaco van der Merwe R&D Director GMP OPS Manager Fritjof Linz Esther Heuberger All Technicians involved in this work

  25. Thank you Gerben.Zijlstra@DSM.com

  26. Outline • Introduction DSM Biologics • DSM XD® Technology • RHOBUST®Expanded Bed Adsorption (EBA) Technology • Continuous XD® coupled to RHOBUST®EBA • Principle • Continuous XD ® examples • Rhobust® EBA on continuous XD® harvest • Concluding Remarks

  27. Proprietary Technologies vs. Classical Concept Optimize individual Unit Operations Process Intensification & Process Integration

  28. XD®: Process Intensification Bioreactor Set-up Concentrated Product Bleed

  29. XD®: Process IntensificationScaled-up in different 50 L Single Use Bioreactors

  30. Scale-up: 200 L XD®Results • 200 L XD® run with CHO cell line performed in PD with 2L satellite run under equal conditions • 200 L XD® run performed in standard Sartorius STR bioreactor (only increased size side ports) • Successful scale-up to equal cell densities (130 mln cells/mL in 200 L) • No oxygen or other limitation observed • Titer similar to satellite run > 10 g/L • Specific productivity the same

  31. Outline • Introduction DSM Biologics • DSM XD® Technology • RHOBUST®Expanded Bed Adsorption (EBA) Technology • Continuous XD® coupled to RHOBUST®EBA • Principle • Continuous XD ® examples • Rhobust® EBA on continuous XD® harvest • Concluding Remarks

  32. Rhobust®: In action Elution of concentrated product

  33. RHOBUST® GMP column (30 cm diameter) and MabDirect ProteinA adsorbent • GMP EBA column • 30cm diameter • 20cm - 60cm settled bed • EBA level monitoring and control (ultrasound) • Low pressure system • RFD (variable speed) • Disposable flow path • UV, flow, conductivity, pressure, temperature, pH using AktaReady • Operational • MabDirect ProteinA • RSF available

  34. Rhobust® EBA on Cont. XD® example 1 Cell density: 60-90 million cells/mL (viability >80%) Antibody titer: 1.3 g/L Comparison: MabDirect proteinA EBA vs. clarification & protein A packed bed chromatography Load ratio: Approx. 22 mg IgG/mL settled bed (both protein A resins) 1 Clarification and chromatographic process (pH treatment, filtration and filling not included) 2 Clarification manufacturing scale will take 6-8 hours (includes dilution, pre-rinse, filtration, post-rinse)

  35. Concluding remarksAdvantages RHOBUST® technology • Operational: • Easy to use, no column packing • Can deal with air bubbles and precipitated material • One step clarification and capture • No separate clarification  8-hour time reduction of process time • Suitable for other high viscosity feed streams (incl. microbial and yeast). • Development and Scale-up • Reduced-scale model available (1 cm and 2 cm column + AKTA Explorer) • Scalable concept: • Pilot scale unit (10 – 60 cm columns + Rhobust Flex or AKTA Ready) • Fully automated GMP unit (10 – 60 cm colums + AKTA Ready) • 30cm-diameter EBA column with floating piston • EBA level monitoring and control (ultrasound) • Disposable flow path and in process monitoring in place (AKTA Ready) • Resins and ligands: • Available Resins: MabDirectProteinA, MabDirect MIMO, FastLineSP • Large ligand library (Incl): IMAC, Q, DEAE

  36. Continuous XD® example 3: PER.C6® – Rec. Continuous XD® culture with PER.C6® cells producing recombinant protein (> 300 kD). Discrete intermittent product bleeds were taken for subsequent DSP

  37. Principle of the Kremer Method • A one step flow-through intermediate purification and polishing procedure, which can optionally be followed by virus filtration. • Benefits: • One unit operation for 2 chromatography steps • Standard dual pump chromatography systems required • Product in flow-through, impurities bind ( small resin volumes) • No intermediate storage • Good removal of aggregates and HCPs • The Kremer method™ has successfully been applied to post Rhobust® intermediate yielding very similar purity as classical DSP.

  38. mAU 3500 10.0 3000 2500 8.0 2000 1500 6.0 1000 4.0 500 0 F4 F6 F5 F7 F3 F 2.0 500 1000 1500 Volume (mL) Rhobust® EBA from Cont. XD® example 1 • OD 280 • pH • Fractions • F3- Load • F4 - Wash 1 • F5 – Wash 2 • F6 - Elution • F7 - Strip • F8 - Cleaning F8

More Related