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
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
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, PhDSr. ScientistDSM Biologics, The Netherlands
• 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
2
Page 3
• 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?
Page 4
• 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
Page 5
Perfusion Medium FeedWash out MetabolitesNo Osmo increaseConstant environmentHigh cell viabilitiesDilute harvestLarge harvest
Continuous XD® cultures with Myeloma cells producing highly potent IgG at around 60 mln cells/mL.
Continuous XD® example 1: Myeloma - IgG
Page 17
The Qp (slope of cumulative titer) was constant at maximum Qp.
Continuous XD® example 2: CHO - IgG
Page 18
Continuous XD® cultures with CHO cells producing Biosimilar IgG at around 100 mln cells/mL.
Continuous XD® example 2: CHO - IgG
Page 19
The IgG titer in the product was bleed around 2.5 g/L in production phase.
Continuous XD® example 4: PER.C6® – IgG
Page 20
Continuous XD® with IgG producing PER.C6® cells at around 100 mln cells/mL.IgG titer in the bleed 4.5 - 5 g/L
Rhobust® EBA example 2: IgG
Page 21
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.
Rhobust® EBA example 2: IgG
Page 22
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.
– 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
23
Page 24
R&D Scientist team DSM-B GMP Process Technologist teamGerben Zijlstra Imre AkkermanOlaf Mol Maria PerlascaDick Smit Mark DressenJurjen de Jong Harriet van der MolenPiet den BoerMark DoevenErik kremerHenk van UrkJaco van der Merwe
R&D Director GMP OPS ManagerFritjof Linz Esther Heuberger
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
Rhobust® EBA on Cont. XD® example 1
34
Process Overall yield
(%)
PurityHP-SEC
(%)
Buffer use
(CV)
Process timelab-scale1
(hours)
Process timeManufacturing–
scale, estimated2
(hours)Rhobust® EBA 82 99.6 67 6.8 6.8
Clarification &ProteinA packed bed
70 99.4 118 12.5 18.5
Cell density: 60-90 million cells/mL (viability >80%)Antibody titer: 1.3 g/LComparison: MabDirect proteinA EBA vs. clarification & protein A
packed bed chromatographyLoad 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)
• 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
35
Continuous XD® example 3: PER.C6® – Rec.
Page 36
Continuous XD® culture with PER.C6® cells producing recombinant protein (> 300 kD). Discrete intermittent product bleeds were taken for subsequent DSP
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.