Transcript
Platzhalter Bild
“Current Best Practise
in Biomanufacturing
and the
Critical Role of Innovation”
International Vellore Symposium “Bioprocess Industry-Academia
Interaction”
July 2011
Dr. Uwe Gottschalk, VP Purification Technologies, Sartorius Stedim
Biotech
What
are
the
hot Topics?
7th Annual Survey of Biopharmaceutical Manufacturing. Eric S. Langer, BioPlan
Associates Inc.
Data adapted from: F. Wurm
Production of recombinant Protein Therapeuticsin Cultivated Mammalian Cells. Nature Biotechnology 22, 1-6 (2004)
The
USDP/DSP Interface in a World of High Titers
Jim Davis, Lonza
Economics of Monoclonal Antibody Production: The relationship between upstream titer and downstream costs; IBC San Diego March 2008
DSP is
Mass
not
Volume
driven
Current
Best Practise
in DSP
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
High Titer
Implications:
Chromatography Technologies for DSP
Polishing
(Membranes)
• Highly porous structure
• Pore size: 3 –
5μm
• Convective Flow
• Minimal buffer useCapturing/IP
(Resins)
• Bead size distribution: 15 -160 μm
• Average pore size: 15 -
40 nm
• Diffusion limited flow
• High capacity
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
Emerging capture
technologies expected to have limited market potential in upcoming years
Source: Sartorius
MaturityMaturity RiskRiskDescriptionDescription
•
CIM, BIA Separations, methacrylate
based monoliths•
Similar to membrane adsorbers•
Purification of large biomolecules
(viruses, plasmid DNA, conjugates), good resolution
•
Upfront/DSM work on single use technology for MABs•
Already used in depletion of valuable biomolecules
from particle containing feedstreams
at large scale (milk, juice, etc.)
•
Membrane adsorber
technology•
Own development and IP, depletion of valuable biomolecules
from particle containing feedstreams
•
Currently low priority, combines cell harvest and capture chromatography step
•
instAction, Prometic
(mimetic ligands), BAC, GEHC pipeline, Repligen
Protein A
•
Used at large scale in plasma fractionation (precipitation)
and APIs (crystallization)•
Not developed for mABs
•
Polybatics•
Disruptive technology•
Single use alternative to Protein A•
Platform character
TechnologyTechnology
Monoliths
Expanded bed ad-
sorption
Direct Capture MA
Ligands
Precipitation/Crystallization/
Extractionn
Affinity
nano-
particles
RelevanceRelevance
•
No significant market in upcoming years expected –
Potential for Niches (e.g. Vaccines, DNA)
•
No immediate commercialization possible
•
Potential for disruptive technology
Capt
ure
and
inte
rmed
iate
pur
ific
atio
n
Already used In development Start of development Low risk Moderate risk High risk
• 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
Alternatives to Protein A Capture
• 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
Alternative Protein A Chromatography
Formats:Goal: Intensified
Use/Volume
Reduction
Limitation: Oleosin yields < 1kg/ha
2000: Oleosin
Platform 2005: TMV Nanoparticles
Immunoabsorbent
nanoparticles
based on a tobacco mosaic virus displaying protein AS. Werner et al. PNAS 103, 17678 -
17683
Polyester Granule100-300 nm
Grage, K. and Rehm, B.H.A. (2008) Bioconj. Chemistry, 19(1):254-62.
Polyester Synthase
2010: Bio Polyester Platform
Alternative Protein A Formats:Goal: Low Cost
–
Real Single Use
CEX
TFF
Q Membrane2 g/ml
Dilution
HCP < 1000 ng/mg
VF
Mix Mode
Fig 7b. TFF based process
Dilution
HCP < 1000 ng/mg
CEX HCP < 10ng/mg
Contaminant precipitation
Q Membrane20 g/ml
Fig 7a. precipitation based process
VF
HCP BDL
Dilution
CEX
TFF
Q Membrane2 g/ml
Dilution
HCP < 1000 ng/mg
VF
Mix Mode
Fig 7b. TFF based process
Dilution
HCP < 1000 ng/mg
CEX
TFF
Q Membrane2 g/ml
Dilution
HCP < 1000 ng/mg
VF
Mix Mode
Fig 7b. TFF based process
Dilution
HCP < 1000 ng/mg
CEX
TFF
Q Membrane2 g/ml
Dilution
HCP < 1000 ng/mg
VFVF
Mix Mode
Fig 7b. TFF based process
Dilution
HCP < 1000 ng/mg
CEX HCP < 10ng/mg
Contaminant precipitation
Q Membrane20 g/ml
Fig 7a. precipitation based process
VF
HCP BDL
Dilution
CEX HCP < 10ng/mg
Contaminant precipitation
Q Membrane20 g/ml
Fig 7a. precipitation based process
VFVF
HCP BDL
Dilution
Two Birds –
one Stone: Contaminant Precipitation at Pfizer and Medarex
Protein A pool volumes and step cost
DNA & HCP levels post Capturing
addresses:
Precipitation of Process-Derived Impurities in Non-Protein APurification Schemes for MAb; J. Wang et al. BioPharm
Intl. 10/2009, 2-9
Process Scale Precipitation of Impurities in Mammalian Cell Culture Broth; J. Glynn
et al. In: Gottschalk U (ed) Process-scale Purification of Antibodies. Wiley, NY.
Chromatography Technologies for DSP
Polishing
(Membranes)
• Highly porous structure
• Pore size: 3 –
5μm
• Convective Flow
• Minimal buffer useCapturing/IP
(Resins)
• Bead size distribution: 15 -160 μm
• Average pore size: 15 -
40 nm
• Diffusion limited flow
• High capacity
Convective Media
Q,S
Capture
Polishing
Low salt
High salt
Q,S
STIC
Polishing in flowthrough:
viruses, DNA, Host cell proteins, endotoxins, aggregates
Purification: large proteins (Factor VIII), viruses (vaccines), phages...
HIC
Selection
Guide Convective
Media
March 7, 2011 Page 9
Sartobind STIC®
-
Next Generation of Membrane Adsorbers Shares same cellulose base membrane as Q: >3 μm pore size
0.5 μmHere is the binding capacity
Binding capacity is distributed more evenly
Sartobind Q
Grafted
Quaternary ammonium
Sartobind STIC
Direct derivatisation
+ ligand density
+ pore accessibility
Primary amine (Sartobind STIC PA)
I. Tatárova, I., R. Fáber, R. Denoyel, M. Polakovic, J. Chromatography A 1216 (2009) 941
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
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 ApptecTrial 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
Source: 2nd Annual Survey of the Bioprocessing Market for Single-Use SolutionsAspen Brook Consulting, 2010
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%
Flux vs. Concentration for MabSartorius ECO & E cassetteCrossflow Rate: 360 L/m2-Hr
0
10
20
30
40
50
60
70
80
90
100
10 100 1000Concentration (g/l)
Per
mea
te F
Lux
(LM
H)
ECO
E
Select the Right Products
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)
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