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New Approaches in Continuous BioManufacturing: Continuous XD ® cell cultures (around 100 mln cells/mL) coupled to the Rhobust ® EBA integrated clarification.

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Presentation on theme: "New Approaches in Continuous BioManufacturing: Continuous XD ® cell cultures (around 100 mln cells/mL) coupled to the Rhobust ® EBA integrated clarification."— Presentation transcript:

1 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

2 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 Outline 1

3 Page 2 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 DSM Biologics: Who are we?

4 Page 3 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 XD ® Technology

5 Page 4 Perfusion Medium Feed Wash out Metabolites No Osmo increase Constant environment High cell viabilities Dilute harvest Large harvest 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 Cell Culture Modes XD ® Process XD ® : Proprietary Process Intensification

6 Page 5 XD ® : Process Intensification Scale-up - PER.C6 ® ; Viable cell count

7 Page 6 XD ® scale up Scale-up - PER.C6 ® ; Product IgG

8 Page 7 XD ® : Process Intensification Bioreactor Set-up

9 Preferred retention system: TFF Page 8 Fully disposable, simple operation, robust, flexible filter choice

10 Page 9 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 Tungsten carbide incorporated in the agarose bead DSM RHOBUST ® Technology

11 Page 10 RHOBUST ® Direct Capture Technology

12 XD ® Harvest ~150x10 6 cell/mL Post-Protein A intermediate Equilibration First cell breakthrough Elution Wash Cell wash out Complete cell breakthrough RHOBUST ® 1 step! RHOBUST ® in Action with the XD ® Harvest Page 11

13 Page 12 RHOBUST ® experiments with XD ® Harvest Results Fed-batch and classical Protein-A packed bed vs. XD ® and RHOBUST ® Protein-A: Yield (%)Purity (%) HCP (ug/mg Mab) DNA (ng/mg Mab) Protein A (ppm) Fed-batch, clarification, Protein-A Packed Bed > 85> 95 1 – 15 (n=15) (n=2) 9-12 XD ®, Protein-A RHOBUST ® > 90> (n=19) (n=6) 4-14 With high cell viabilities ~10% higher yield HCP, DNA & res.Protein A After column in normal range

14 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 Outline 13

15 Continuous XD ® - Rhobust ® principle Page 14 Tungsten carbide incorporated in the agarose bead 2-8°C Product/Cell Bleed Elution buffer Product eluate Low pH treatment Waste Medium XD ® Bioreactor Cell/Product Bleed Rhobust ® EBA+Low pH Low pH treated EBA bulk Concentrated Product Bleed Diluted Waste Stream

16 Continuous XD ® example 1: Myeloma - IgG Page 15 Continuous XD ® cultures with Myeloma cells producing highly potent IgG at around 60 mln cells/mL.

17 Continuous XD ® example 1: Myeloma - IgG Page 16 The Qp (slope of cumulative titer) was constant at maximum Qp.

18 Continuous XD ® example 2: CHO - IgG Page 17 Continuous XD ® cultures with CHO cells producing Biosimilar IgG at around 100 mln cells/mL.

19 Continuous XD ® example 2: CHO - IgG Page 18 The IgG titer in the product was bleed around 2.5 g/L in production phase.

20 Continuous XD ® example 4: PER.C6 ® – IgG Page 19 Continuous XD ® with IgG producing PER.C6 ® cells at around 100 mln cells/mL. IgG titer in the bleed g/L

21 Rhobust ® EBA example 2: IgG Page 20 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 Page 21 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 remarks Advantages 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 22

24 Page 23 R&D Scientist team DSM-BGMP Process Technologist team Gerben ZijlstraImre Akkerman Olaf MolMaria Perlasca Dick SmitMark Dressen Jurjen de JongHarriet van der Molen Piet den Boer Mark Doeven Erik kremer Henk van Urk Jaco van der Merwe R&D DirectorGMP OPS Manager Fritjof LinzEsther Heuberger All Technicians involved in this work Acknowledgements:

25 Thank you

26 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 Outline 25

27 Page 26 Proprietary Technologies vs. Classical Concept Optimize individual Unit Operations Process Intensification & Process Integration

28 Page 27 XD ® : Process Intensification Bioreactor Set-up Concentrated Product Bleed

29 Page 28 XD ® : Process Intensification Scaled-up in different 50 L Single Use Bioreactors

30 Scale-up: 200 L XD ® Results Page 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 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 Outline 30

32 Page 31 Rhobust ® : In action Elution of concentrated product

33 32 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 33 ProcessOverall yield (%) Purity HP-SEC (%) Buffer use (CV) Process time lab-scale 1 (hours) Process time Manufacturing– scale, estimated 2 (hours) Rhobust ® EBA Clarification & ProteinA packed bed Cell density: 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 remarks Advantages 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: MabDirect ProteinA, MabDirect MIMO, FastLine SP –Large ligand library (Incl):IMAC, Q, DEAE 34

36 Continuous XD ® example 3: PER.C6 ® – Rec. Page 35 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. Page 36

38 Rhobust ® EBA from Cont. XD ® example 1 37 OD 280 pH Fractions –F3- Load –F4 - Wash 1 –F5 – Wash 2 –F6 - Elution –F7 - Strip –F8 - Cleaning mAU Volume (mL) F F3 F4 F5 F6 F7 F8


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