New Approaches in Continuous BioManufacturing:

Transcription

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 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 1

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 Page 2

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 Page 3

5 XD : Proprietary Process Intensification 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 Page 4

6 XD : Process Intensification Scale-up - PER.C6 ; Viable cell count L XD reference 50L XD run #1 50L XD run #2 VCD (*10E6 cells/ml) Time (day) Page 5

7 XD scale up Scale-up - PER.C6 ; Product IgG 100 2L XD reference 50L XD run #1 50L XD run # IgG (g/l) Time (day) Page 6

8 XD : Process Intensification Bioreactor Set-up Page 7

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

10 DSM RHOBUST Technology 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 Page 9

11 RHOBUST Direct Capture Technology Page 10

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

13 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 > (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 Page 12

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 13

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

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 Residual DNA and HCP were within normal ranges and comparable to packed bed values. Page 21 Aggregate levels and relative potency were relatively constant throughout the run.

23 22 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

24 Acknowledgements: R&D Scientist team DSM-B Gerben Zijlstra Olaf Mol Dick Smit Jurjen de Jong Piet den Boer Mark Doeven Erik kremer Henk van Urk Jaco van der Merwe R&D Director Fritjof Linz GMP Process Technologist team Imre Akkerman Maria Perlasca Mark Dressen Harriet van der Molen GMP OPS Manager Esther Heuberger All Technicians involved in this work Page 23

25 Thank you

26 25 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 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 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 14,00 12,00 200L XD Biomass corrected titer (Nephelometer) 2L P23037 Titer similar to satellite run > 10 g/l Specific productivity the same Titer (g/l) 10,00 8,00 6,00 4,00 2,00 0,00 0,00 100,00 200,00 300,00 400,00 500,00 600,00 700,00 800,00 900, ,00 IVC Page 29

31 30 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 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 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) Process Overall 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 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) 33

35 34 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

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 Page 36 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 Rhobust EBA from Cont. XD example 1 mau OD 280 ph Fractions F3- Load F4 - Wash 1 F5 Wash 2 F6 - Elution F7 - Strip F8 - Cleaning F F3 F4 F5 F6 F7 F Volume (ml)