1. “Current Best Practise in
Biomanufacturing and the
Platzhalter Bild Critical Role of Innovation”
International Vellore Symposium
“Bioprocess Industry-Academia
Interaction”
July 2011
Dr. Uwe Gottschalk, VP Purification
Technologies, Sartorius Stedim Biotech

2. What are the hot Topics?
7th Annual Survey of Biopharmaceutical Manufacturing.
Eric S. Langer, BioPlan Associates Inc.

3. Existing Facilities to Meet Current Challenges

4. The USDP/DSP Interface in a World of High Titers
Data adapted from: F. Wurm Production of recombinant Protein Therapeutics
in Cultivated Mammalian Cells. Nature Biotechnology 22, 1-6 (2004)

5. DSP is Mass not Volume driven
Jim Davis, Lonza Economics of Monoclonal Antibody Production: The relationship between upstream titer and
downstream costs; IBC San Diego March 2008

6. Current Best Practise in DSP
High Titer Implications:
Increasing biomass and
contaminant levels
Protein A pool volumes
and step cost
DNA & HCP levels post
Capturing
Polishing load volumes
and conductivity
Pathogen clearance as a
moving target

7. Chromatography Technologies for DSP
Polishing (Membranes)
• Highly porous structure
• Pore size: 3 – 5μm
• Convective Flow
Capturing/IP (Resins)
• Minimal buffer use
• Bead size distribution: 15 -160 μm
• Average pore size: 15 - 40 nm
• Diffusion limited flow
• High capacity

8. Capture Costs: Why bother?
Jim Davis, Lonza Economics of Monoclonal Antibody Production: The relationship between upstream titer and
downstream costs; IBC San Diego March 2008

9. Emerging capture technologies expected to have limited market potential in
upcoming years
Technology Description Maturity Risk Relevance
• CIM, BIA Separations, methacrylate based monoliths
Monoliths • Similar to membrane adsorbers
• Purification of large biomolecules (viruses, plasmid DNA, conjugates),
good resolution
Expanded • Upfront/DSM work on single use technology for MABs
Capture and intermediate purification
bed ad- • Already used in depletion of valuable biomolecules from particle • No significant
sorption containing feedstreams at large scale (milk, juice, etc.) market in
upcoming years
expected –
• Membrane adsorber technology Potential for
Direct Capture • Own development and IP, depletion of valuable biomolecules from Niches (e.g.
MA particle containing feedstreams Vaccines, DNA)
• Currently low priority, combines cell harvest and capture chromatography
step
Ligands • instAction, Prometic (mimetic ligands), BAC, GEHC pipeline, Repligen
Protein A
Precipitation/ • Used at large scale in plasma fractionation (precipitation)
Crystallization/ and APIs (crystallization) • No immediate
Extractionn • Not developed for mABs commercialization
possible
• Potential for
Affinity • Polybatics disruptive
nano- • Disruptive technology technology
particles • Single use alternative to Protein A
• Platform character
Already used In development Start of development Low risk Moderate risk High risk
Source: Sartorius

10. Alternatives to Protein A Capture
• Product precipitation batch/continuous
• Impurity precipitation (followed by non-Protein A process)
• Alternative Capturing (Protein A Mimetics, Mixed Mode, CEX)
Issues: Selectivity, Scale up, Reproducibility, Comparability

11. addresses:
Protein A pool volumes
and step cost
D. Low BioManufacturing Paris 2007

12. Alternative Protein A Chromatography Formats:
Goal: Intensified Use/Volume Reduction
• Simulated Moving Bed (SMB) and related:
» Tarpon („single use flow path“)
» Novasep
» Chromacon
» Chromatan
» ...
______________________________________________________________________
• Expanded Bed Chromatography
» DSM/Upfront („single use flow path“)
Issues: Complexity, Scale up, Reproducibility, Comparability

13. Alternative Protein A Formats:
Goal: Low Cost – Real Single Use
2000: Oleosin Platform 2005: TMV Nanoparticles 2010: Bio Polyester Platform
Polyester
Synthase
Polyester Granule
Immunoabsorbent nanoparticles based on a 100-300 nm
tobacco mosaic virus displaying protein A
Limitation: Oleosin yields < 1kg/ha S. Werner et al. PNAS 103, 17678 - 17683
Grage, K. and Rehm, B.H.A. (2008)
Bioconj. Chemistry, 19(1):254-62.

14. Two Birds – one Stone: Contaminant Precipitation at Pfizer and Medarex
addresses:
Fig 7a. precipitation based process Fig 7b. TFF based process
Contaminant
precipitation
Protein A pool volumes
TFF
and step cost
DNA & HCP levels post
HCP < 1000 ng/mg
CEX HCP < 10ng/mg CEX
Dilution
Capturing
Dilution
Q Membrane Q Membrane HCP < 1000 ng/mg
HCP BDL 2 g/ml
20 g/ml
Dilution
VF
Mix Mode
VF
Process Scale Precipitation of Precipitation of Process-Derived
Impurities in Mammalian Cell Culture Impurities in Non-Protein A
Broth; J. Glynn et al. In: Gottschalk U Purification Schemes for MAb; J. Wang
(ed) Process-scale Purification of et al. BioPharm Intl. 10/2009, 2-9
Antibodies. Wiley, NY.

15. Chromatography Technologies for DSP
Polishing (Membranes)
• Highly porous structure
• Pore size: 3 – 5μm
• Convective Flow
Capturing/IP (Resins)
• Minimal buffer use
• Bead size distribution: 15 -160 μm
• Average pore size: 15 - 40 nm
• Diffusion limited flow
• High capacity

16. Pete Gagnon 2007

17. Selection Guide Convective Media
Q,S Low salt Polishing in
flowthrough:
STIC viruses, DNA, Host
ng
is hi cell proteins,
Pol High salt endotoxins,
HIC aggregates
Convective Media
Cap Purification: large
t ure
proteins (Factor
Q,S VIII), viruses
(vaccines), phages...

18. Sartobind STIC® - Next Generation of Membrane Adsorbers
Shares same cellulose base membrane as Q: >3 μm pore size
0.5 μm
Here is the
binding
capacity Sartobind Q Grafted
Quaternary ammonium
Sartobind STIC Direct derivatisation
Binding capacity + ligand density
is distributed
more evenly + pore accessibility
Primary amine (Sartobind STIC PA)
I. Tatárova, I., R. Fáber, R. Denoyel, M. Polakovic, J. Chromatography A 1216 (2009) 941
March 7, 2011 Page 9

19. Host cell protein removal from up 10 kg mAb per L (pH 8, 500 ppm HCP load, 10
MV/min
Application Note Sartorius-Stedim Biotech: 85032-540-18, 05/2011

20. Sartobind STIC
Sartobind Q Sartobind STIC
Binding Capacity (g/m²)
BSA 2 mS/cm @ 0 mM NaCl 9 19
BSA 20 mS/cm @ 200 mM NaCl 1 10
DNA 7 mS/cm @ 50 mM NaCl 2 6
Removal of ΦX174 (LRV)
LRV 1.4 mS/cm @ 0 mM NaCl 3,7 5,1
LRV 6.7 mS/cm @ 50 mM NaCl 0,1 5,1
LRV 16.8 mS/cm @ 150 mM NaCl 0,1 4,8
Removal of MVM (LRV) at Wuxi Apptec
Trial 1 16.8 mS/cm @ 150 mM NaCl 2.10 3,82
Trial 2 16.8 mS/cm @ 150 mM NaCl 1.81 >4,96

21. Source: 2nd Annual Survey of the Bioprocessing Market
for Single-Use Solutions
Aspen Brook Consulting, 2010

22. Current Challenge in UF
• Process efficiency should be HIGH
• Membrane cleaning should be EASY
• Final Mab concentration may reach 20%
• Maximum system pressure is LIMITED
E ECO
Pumping Requirements 8 L/m2/min 2 L/m2/min
Viscosity High Low
MAb Concentration >15% <10%

23. Select the Right Products
Flux vs. Concentration for Mab
Sartorius ECO & E cassette
Crossflow Rate: 360 L/m2-Hr
100
90
ECO
80
eate FLux (LMH)
70
60
50
40
E
Perm
30
20
10
0
10 100 1000
Concentration (g/l)

24. Yes we can!

25. Upcoming Alternatives
Przybycien, Pujar, Steele: Current Opinion in Biotechnology 2004, 15 469-478

26. Disruptive Technologies from Inception to Maturation
Konstantinov, K. Towards fully continuous bioprocessing: What can we learn from Pharma? Cell Culture Engineering
XII, Banff, Canada (2010)

27. Trend in New Drug Production Scales

28. Flexible and Disposable

29. New Facilities to meet Future Challenges

30. Thank you!
Uwe.Gottschalk@sartorius-
stedim.com