0. 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

1. 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

2. 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

3. 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

4. Cell Culture Mode of Manufacturing
XD®: Proprietary Process Intensification
Cell Culture Modes
Cell Culture Mode of Manufacturing
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
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
XD®
Process

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

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

7. XD®: Process Intensification Bioreactor Set-up

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

9. 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

10. RHOBUST® Direct Capture Technology

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

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)
23-49
(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

13. 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

14. Continuous XD® - Rhobust® principle
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

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

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

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

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

19. 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 g/L

20. 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.

21. 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.

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):
Very high Recovery: Single unit operation, Concentrated product flow
Very high Purity: Optimized Rhobust® EBA
Easy to use, no column packing, air bubbles and precipitates no problem
One step clarification and capture

23. 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

24. Thank you

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

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

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

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

29. 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

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

31. Rhobust®: In action
Elution of concentrated product

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

33. 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-scale1
(hours)
Manufacturing– scale, estimated2
Rhobust® EBA 82
99.6 67
6.8
Clarification &
ProteinA packed bed 70
99.4
118
12.5
18.5
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)

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

35. 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

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.

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