sartobind® membrane...
TRANSCRIPT
Sartobind®
Membrane ChromatographyDr. Stefan Fischer-Frühholz, February
15, 2012
Senior Product Manager Membrane Chromatography
February 15, 2012 Page 2
1.
Membranes1.
Membranes
2.
Formats2.
Formats
Agenda
3.
Applications3.
Applications
February 15, 2012 Page 3
Sartobind Membrane Adsorbers are formatted already for use
Chromatographic
adsorptive
membranes
Made from
regenerated
cellulose
Surface
modified
(e.g. primary
amine
IEX)
Grafted
(e.g. quaternary
ammonium
IEX)
Conventional
ligands
O
CH2
OH
OH
O
OH
CH2
* *n
OH
OHO
O
CH2
OH
O
O
OH
OH
CH2
* *n
OH
OHOH
O
OH
hydrophilicX-linker
base
stable
cellulose
(Hydrosart®)
February 15, 2012 Page 4
Diffusion limited
gels
(time) versus
convection
limited
(flow
rate)
Average pore size 15 -
40 nm
Average pore size 3 -
5 μm
February 15, 2012 Page 5
Dynamic
binding
capacity
./. Flow
rate -> Contaminant
removal
40 x Bed
volume/min
Flow
rate
Membrane chromatographyDynamicbinding
capacity
Gel chromatography
February 15, 2012 Page 6
Dynamic
binding
capacity
./. Size
-> Capturing
large molecules
Size
exclusion
for proteins
>100 kDa when
using
gel
matrix
of microporous
30-50 nm*
100 500 kDaSize
of molecule
Membrane chromatography
Dyn.binding
capacity
Gel chromatography
*E. Karlsson, L. Rydén
and J. Brewer, Protein Purification, Principles, Jan-Christer
Janson, Lars Rydén
Eds., VCH Weinheim, pp 123, 1989
February 15, 2012 Page 7
Membranes
Ion exchange Strong: Q, S
Anion exchange Weak: D
Anion exchange Sartobind STIC®
PA
(Primary
Amine)
Salt Tolerant Interaction Chromatography
HIC Phenyl
Metal chelate Iminodiacetic
acid (IDA)
Coupling Aldehyde
Affinity Protein A
...
February 15, 2012 Page 8
Sartobind STIC®
Sartobind STIC: Salt Tolerant Interaction Chromatography
Combination of unique cellulose-based membrane structure and covalently attached salt tolerant primary amine (PA) ion exchange ligand
Primary amine (Sartobind STIC PA)
February 15, 2012 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
Hydrogel
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
February 15, 2012 Page 10
Sartobind STIC PA and Q dynamic binding capacity at 10%
0
20
40
60
80
0 100 200 300 400 500Salt conc. [mM]
10%
DB
C B
SA
[mg/
ml] Sartobind Q
Sartobind STIC PA
February 15, 2012 Page 11
Typical binding capacities
*Standard proteins: BSA (Sartobind Q and STIC, ) 20 mM TRIS/HCl pH 7.5
lysozyme (S) 10 mM KPi
pH 7,
HIC: globulin, 0.9 M (NH4
)2
SO4
Membrane area: 36.4 cm²
= 1 ml volume
Membrane Description Dynamic
binding
capacity
10 %*
Quaternary
ammonium
(Q) Strong
basic
anion
exchanger 29 mg/ml (0.8 mg/cm²)
Sartobind STIC primary amine (PA)
Weak
basic
anion
exchanger 50 mg/ml (1.4 mg/cm²) in
TRIS buffer+150 mM NaCl
Sulfonic Acid
(S) Strong
acidic
cation exchanger 26 mg/ml (0.7 mg/cm²)
Phenyl Hydrophobic
Interaction
Chromatographic
membrane
14.6 mg/ml (0.4 mg/cm²)
Selection Guide Sartobind
Membrane
Adsorber step
Q
Capture
Polishing
Low salt
High salt
Q,S
STIC
Contaminant removal:
Viruses, DNA, Host cell proteins, endotoxins, aggregates
Purification: large proteins (Factor VIII), viruses (vaccines), phages...
HIC
February 15, 2012 Page 12
February 15, 2012 Page 13
1.
Membranes1.
Membranes
2.
Formats2.
Formats
Agenda
3.
Applications3.
Applications
February 15, 2012 Page 14
Flow
rate: 0.5 bed
volumes/min
for the column 30 bed
volumes/min
for Sartobind
Column 20 l
10 l / min
20 l
Membrane Adsorber 0.5 l (~2 m²)
14 l/min
0.5 l
15 c
m
0.4 cm
results in a flow rate of 0.5 BV / min results
in a flow
rate of 28 BV /min
Need
for Throughput
Results
in Oversizing
Columns
H.L. Knudsen
et al.,
J.
Chromatogr. A 907, 145-154, 2001
-
50 cm / 30L bed
volume
-
1000 l/h
-
BC 1500 g oversized
- 0.5 L bed volume
-
1000 l/h
-
BC 15 g sufficient
for contaminant
removal
Membrane based
system Beads
based
system
Bottleneck
at production
scale
= flow
rate
February 15, 2012 Page 15
February 15, 2012 Page 16
Sartobind membrane adsorber portfolio process scale
Q Q
S S
STIC
HICSize
ml
pico
0.08
nano
1 ml
mini
7
5”
70
10”
180
20”
360
30”
540
mega
1620
nano
3 ml
150 ml
150
Jumbo
5000
Contaminant removal:
flowthrough
mode to remove DNA, Host cell proteins, endotoxins, viruses
Singe-use
Purification: bind & elute of viruses and virus like particles, large proteins
Single-use / intra-batch use / re-use
4 mm bed height 8 mm bed height
February 15, 2012 Page 17
Sartobind 4 mm capsules
for flowthrough operation with new
size
Sartobind pico 0.08 ml membrane volume
Size
ml
pico
0.08
nano
1
mini
7
5”
70
10”
180
20”
360
30”
540
Mega
1620
nano
3 ml
150 ml
150
Jumbo 5 l
5000
Q √ √ √ √ √ √ √ √ √ √ √
S √ √ √ √ √ √ √ √ √ √
STIC √ √ √ √ √ √
HIC √* √ √ √*coming soon
February 15, 2012 Page 18
Sartobind STIC PA, Q and S for process development
Bed height 4 mm
Frontal surface area 0.19 cm²
Max pressure 6 bar
Housing material Polypropylene
Size 31 x 11 mm
Void volume 0.4 ml
*Phenyl coming soon
February 15, 2012 Page 19
Sartobind STIC PA pico for application with limited sample material and virus spiking studies
-
Smallest scaleable membrane adsorber
with 4 mm bed height
-
Decreases cost for virus spiking studies
-
Scales in binding capacity for protein,
DNA and host cell proteins as well as endotoxins. Shows same log reduction for model virus as larger capsules and throughput / pressure flow relation
31 mm
Upstream
Downstream
pico 5”
February 15, 2012 Page 20
The Sartobind pico construction realizes the smallest scaleable design with 4 mm bed height (15 layers) of membranes
February 15, 2012 Page 21
Connectivity of Sartobind pico is given by finger-tight Luer PEEK adapters (enclosed in package)
February 15, 2012 Page 22
Sartobind Q, S and STIC 10”
capsule cut-away
Vent valve
Sartobind membrane
Fleece
Sartobind membrane
Fleece
February 15, 2012 Page 23
Scale-up performance
Flushing/equilibration:
10 mM phosphate
buffer, pH 7
Loading:
2 g/l bovine serum albumin
Washing:
10 mM phosphate buffer, pH 7
Elution
1 M NaCl in equlibration
buffer
MV = membrane volume
(corrected by minus void volume, diff. membrane lots
0
0,2
0,4
0,6
0,8
1
0 5 10 15 20
MV
nano lot A No.13 nano lot A No.35 5" lot B No.36 5" lot B No.30
c/c o
0
0,2
0,4
0,6
0,8
1
0 5 10 15 20MV
10''_lo t_504863_2210''_lo t_504863_0720''_lo t_504663_0320''_lo t_504663_1030''_lo t_504563_0730''_lo t_504563_04
10“
lot C No.2210“
lot C No.0720“
lot D No.0320“
lot D No.1030“
lot E No.0730“
lot E No.04
c/c o
February 15, 2012 Page 24
Sartobind Q Pico scales to larger devices
February 15, 2012 Page 25
Testing of integer and pinholed
10”
capsule by: Diffusion test and HETP -> only diffusion testing works
Comparison HET P with 5% acetone solution10" Sartobind Q SingleSep with and without hole (diameter 2mm)
0
0,1
0,2
0,3
0,4
0,5
0,6
10 10,5 11 11,5 12 12,5
V [l]
Extin
ctio
n 28
0nm
capsule 1 with hole capsule 2 with hole capsule 1 without hole capsule 2 without hole
Defect Capsule Result
HETPResultDiffusion test
None 1 0.49 PASS 1.2 ml/min
None 2 0.67 PASS 0.4 ml/min
2 mm pinhole
1 0.47 FAILNo value
2 mm pinhole
2 0.62 FAIL No value
2 mm
February 15, 2012 Page 26
Screening device 96-well plate equipped with Sartobind STIC PA (coming soon)
•
Build up from 12 x 8-strip well units
•
Screening: 0.21 cm²/well per layer (3 layers: 0.7 cm²
≙
0.017 mL)
•
Operation volumes up to 0.5 mL
•
Fractionation using 96 well deep well plates
•
Allows the implementation of all typical steps in chromatography
February 15, 2012 Page 27
Comparison Sartobind Q and STIC PA Effect of citrate buffer and salt on protein binding
•
2.7 mg/cm²
BSA were added in 2 steps of 0.5 ml, analyzing flow-through fraction
•
Polyanionic
buffers such as sodium citrate prevents binding than salt concentration
less
binding
less
binding
February 15, 2012 Page 28
Comparison of Sartobind Q and STIC PA Effect of phosphate and salt on protein and DNA binding
•
Evaluation pH 8
•
Contour plots:
-
Red: no binding
-
Green: high binding
•
Sartobind Q:
-
Sensitive to [NaCl]
•
Sartobind STIC:
-
Protein (GFP) is sensitive to [PO4
]phosphate
-
DNA is less impacted by phosphate buffers
less
binding
less
binding
less
binding
February 15, 2012 Page 29
Sartobind 8 mm capsules
for flowthrough and bind & elute applications
Size
ml
pico
0.08
nano
1
mini
7
5”
70
10”
180
20”
360
30”
540
Mega
1620
nano
3 ml
150 ml
150
Jumbo 5 l
5000
Q √ √ √ √ √ √ √ √ √ √ √
S √ √ √ √ √ √ √ √ √ √
STIC √ √ √ √ √ √
HIC √* √ √ √*coming soon
February 15, 2012 Page 30
Jumbo void
volume
optimisation
–
downscale nano 3 ml
External
channel
Membrane (8 mm)
Central
core
Internal
channel
Sartobind nano 1 ml for polishing flow
through
only
Sartobind nano 3 ml for bind & elute AND
flow
through
Upstream
flow
channel
Void volume optimized
No optimization
Top view
February 15, 2012 Page 31
Computational Fluid Dynamics: Jumbo 5 l void volume optimization
upstream
downstream
also published in: Pastor, A. and Barbe, S. (2010) Disposable Chromatography for Large-Scale Biomanufacturing, in Single-Use Technology in Biopharmaceutical Manufacture (eds
R. Eibl
and D. Eibl), John Wiley & Sons, Inc., Hoboken, NJ, USA. ch25.
70 ml Membrane volumes 150 ml
4.6 Void membrane volumes 1.3February 15, 2012 Page 32
Comparison of void volumes 4 mm, 70 ml and 8 mm, 150 ml capsules
Upstream
Membrane
Downstream
February 15, 2012 Page 33
Breakthrough curve Sartobind nano to Jumbo Flow rate 4 bed volumes/min, sample BSA (2 g/l), elution 1 M NaCl in KPI
0
0,5
1
1,5
2
2,5
3
0 10 20 30 40 50 60 70
BV
c [g
/L]
Sartobind Q nano 3 mL
Sartobind Q 150 mL
Sartobind Q Jumbo 5 L
3 ml
150 ml
5000 ml
February 15, 2012 Page 34
Pressure flow relation Sartobind Jumbo 5 l
0,00
5,00
10,00
15,00
20,00
25,00
30,00
35,00
40,00
0,00 0,10 0,20 0,30 0,40 0,50 0,60 0,70 0,80 0,90 1,00 1,10 1,20 1,30 1,40 1,50 1,60 1,70 1,80 1,90 2,00 2,10 2,20 2,30 2,40 2,50
pressure difference [bar]
flow
[l/m
in]
February 15, 2012 Page 35
Breakthrough curve (BSA) and Elution peaks with Sartobind Q Jumbo and Sartobind Q nano @ 4 BV/min
0
0,5
1
1,5
2
2,5
3
0 10 20 30 40 50 60
BV
c [g
/l]
8008483_Jumbo5L8009483_Jumbo5L8010783_Jumbo5L8032683_1_nano8032783_1_nano8032883_1_nano
2 g/l BSA break
through
normalized
to bed
volume
(BV) at 4 BV/min flow Three
Sartobind Q Jumbo 5 liter
lots vs. 3 of Sartobind Q nano 3 ml
February 15, 2012 Page 36
The Jumbo and nano are
suitable
for bind elute applications Sartobind S nano 3 ml
Pre-conditioning 2 M NaCl in equilibration
buffer 20 CVEquilibration 20 mM Tris pH 7.4, 1.8mS/cm 25 CVLoading
of protein mixture ~ 1-1.5 mg/ml for protein 1,2 and 3 500 μlWash 20 mM Tris pH 7.4, 1.8 mS/cm 4 CVElution by
linear gradient 2 M NaCl in equilibration
buffer 16 CV
nano 3 ml
February 15, 2012 Page 37
1.
Membranes1.
Membranes
2.
Formats2.
Formats
Agenda
3.
Applications
-
mAb
production-
contaminant removal
-
Virus purification
3.
Applications
-
mAb
production-
contaminant removal
-
Virus purification
February 15, 2012 Page 40
Why
use
Membrane Adsorber downstream
in mAb
production
–
major drivers
No validation
Saves 95 % buffer
10 times faster
Effective removal of DNA, viruses, Host Cell Proteins
February 15, 2012 Page 41
Gel versus Membrane when polishing of a monoclonal antibody
Q Resin Q Membrane
Flux
/ flow
rate 100 –
150 cm/h 450 –
600 cm/h
Capacity 50 –
100 g/L > 3 kg/m²
or
> 10.9 kg/L
Buffer used 100 % 5 %
Operation time 8 –
9 h 2 –
2.5 h
Cleaning
validation Yes Single use
Zhou J X, Tressel
T, AMGEN Inc.: Membrane chromatography as a robust purification
system for large-scale
antibody production. Bioprocess International, Sept. 2005, p. 32-37
February 15, 2012 Page 42
15 Reasons
why use disposables:
Reduction
of
complexity
No cleaning
validation
Reduce
time to
get
facility
running
More
convenient
Decrease
product contamination
Assure
sterilityIncrease
facility
output
Lower
maintanance
cost
Reduce
space
Reduce
water
and buffer
consumption
Product faster
to market
More
flexible
Reduce
hazardous
cleaning
solutions
Faster
optimization
Simplify
operations
February 15, 2012 Page 43
Flowthrough: Contaminant removal in Downstream Processing
• Viruses
(AEX)
• DNA (AEX)
• Host cell proteins
(AEX, CEX)
• Endotoxins (AEX)
• Leached ligand (AEX)
• Aggregates (HIC)
0.2μm Sterile
Form and FillCell-Harvest/Clarification
Purif
icat
ion
Sartobind Virus FilterDisposable UF
Buffer Exchange
2nd
column
Affinity Chrom.
Capturing Step
February 15, 2012 Page 44
Removal of contaminants
during
monoclonal
antibody production
Positvely
(+) charged
adsorber binds
negatively
(-) charged
contaminants
DNA Viruses
Host cell proteins
2 3 4 5 6 7 8 9 10 11 12
mAbs
Isoe
lect
ric p
oint
s
Prod
uct
Im
purit
ies
Endotoxins
pH buffer
Polishing: contaminantsare
bound
Flow
Through+-
February 15, 2012 Page 45
Anion exchange step in flowthrough polishing 2nd
step:
Sartobind Q may be sufficient, mAb
dependent –
both are options
Q or Sb.STIC
Bioreactor Centrifuge /Dept filter
Protein A AEX step Membrane
CEX Virus filter Cross Flow
Production Clarification Capturing FT Polishing Capturing Virus removal UF / DF
Reduces:
Viruses
DNA
HCP
Leached ligand
Endotoxins
February 15, 2012 Page 46
AEX as 2nd
chromatography step: higher contaminant level
Harvest
1st Protein A
2nd Anion exchange membrane adsorber step:
higher impurity load (limits load density)
3rd
Cation exchange
•Low conductivity 4-7 mS/cm allows Q usage
February 15, 2012 Page 47
Anion exchange step in flowthrough polishing 3rd
step:
Position for Sartobind STIC
Sb. STIC or Q ?
Bioreactor Centrifuge /Dept filter
Protein A CEX AEX step Membrane
Virus filter Cross Flow
Production Clarification Capturing Capturing FT Polishing Virus removal UF / DF
Reduces:
Viruses
DNA
HCP
Leached ligand
Endotoxins
February 15, 2012 Page 48
Purification bottleneck –
Facility / Tank size limitation at high mAb
titers:
5 g/L mAb
10000 Liters
200 L Protein A 30 g/L, 8 cycles, 2.5 CV Pool
4000 Liters
500 L CEX 100 g/L, 6 CV Pool, 12-15 mS/cm
3000 Liters
Q needs 4-7 mS/cm, dilution 1:1
6000 LitersAEX 10 kg/L FT
February 15, 2012 Page 49
Purification bottleneck –
Facility / Tank size limitation at high titers, e.g.:
5 g/L mAb
10000 Liters
200 L Protein A 30 g/L, 8 cycles, 2.5 CV Pool
4000 Liters
500 L CEX 100 g/L, 6 CV Pool, 12-15 mS/cm
3000 Liters
Sartobind STIC AEX 10 kg/L FT
no need for 6000 Liter tank
February 15, 2012 Page 50
Anion exchange step in flowthrough polishing 3rd
step:
Position for Sartobind STIC
Sb. STIC
Bioreactor Centrifuge /Dept filter
Protein A CEX AEX step Membrane
Virus filter Cross Flow
Production Clarification Capturing Capturing FT Polishing Virus removal UF / DF
Reduces:
Viruses
DNA
HCP
Leached ligand
Endotoxins
February 15, 2012 Page 51
Impurity
levels
after
1st
column
and 12000 g Mab product
: 1.5% aggregate 180 g
: 1000 ppm CHOP 12 g
: 200 ppm DNA 2.4 g
: 30 ppm leached ProA
0.36 g
Typical “load”
value: > 2 kg protein per
liter Sartobind Q
Gerardo
Zapata
et al., Abgenix/AMGEN,
Membrane Chromatography for Purification of Human Antibodies
in
Commercial Processes, IBC Antibody Meeting 2005
After 1st
column
February 15, 2012 Page 52
(log10
) removal for
DNA : >2 log
host cell proteins: 1.9 log endotoxin: >2.8 log
Zhang R, Bouamama
T, Tabur
P, Zapata G, AMGEN, Gottschalk U, Mora J, Reif O.
Viral Clearance Feasibility Study With Sartobind Q Membrane Adsorber for Human Antibody Purification. IBC’s 3rd European Event BioProduction
2004.
Polishing Step: hMAB
after
capture
Virus Size
(nm) Enveloped LRV Run 1 LRV Run 2
MVM: Minute Virus of Mice 16 –
15 No ≥6.77 ±
0.24 4.41 ±
0.37
Reo-3: Reovirus
Type III 75 –
80 No ≥7.28 ±
0.30 ≥7.53 ±
0.29
MuLV: Murine
Leukemia
Virus 80 –
110 Yes ≥5.57 ±
0.25 6.29 ±
0.32
PRV: Pseudorabies
virus 150 -
250 Yes ≥5.67 ±
0.17 ≥5.76 ±
0.23
February 15, 2012 Page 53
Impurity
Levels after
2nd column:
: << 100 ppm CHO protein
: << 20 ppm DNA
Typical “load”
value:
10.9 kg antibody/l membrane(180 times more than Q resin)
Feng
Li et al., Amgen, J. BioProcessing,
09/10, 23-30, 2005
Polishing Step after
Intermediate Purification
After 2nd
column
pH: 7.2 / conductivity: 4 mS/cm / flow rate: 450 cm/h / 1 % spikeprotein concentration: 4.3 g/l Mab load: 10.9 kg/l membrane (3 kg/m²) /*LRV = 5.59 at 600 cm/hr
Zhou J and Tressel
T, Amgen: Basic Concepts in Q Membrane Chromatography for Large-Scale Antibody Production. Biotechnol
Prog., 22 (2) 341 –
349, 2006.
February 15, 2012 Page 54
Virus removal study
for MAb
Virus Enveloped LRV Run 1 LRV Run 2 Virus recovery (%)
MVM: Minute Virus of Mice no ≥
6.03 ±
0.21 ≥
6.03 ±
0.20 100
Reo-3: Reovirus
Type III no ≥
7.00 ±
0.31 ≥
6.94 ±
0.24 100
MuLV*: Murine
Leukemia
Virus yes ≥
5.35 ±
0.23 ≥
5.52 ±
0.27 > 70
PRV: Pseudorabies
virus yes ≥
5.58 ±
0.28 ≥
5.58 ±
0.22 100
February 15, 2012 Page 55
HCP binding on Sartobind Q vs. STIC PA at different salt concentrations: Salt tolerant STIC less dependent on salt->large window of operation
Binding of HCP Sartobind Q
0102030405060708090
100
6,0 6,5 7,0 7,5 8,0 8,5 9,0
pH
B re
lativ
e to
the
max
[%]
Binding of HCP Sartobind ST IC PA
0102030405060708090
100
6,0 6,5 7,0 7,5 8,0 8,5 9,0
pH
Bind
ing
rela
tive
to t
he m
ax [%
]
Sartobind Q
Sartobind STIC PA
0mM NaCl
100mM NaCl
200mM NaCl
300mM NaCl
400mM NaCl
500mM NaCl
February 15, 2012 Page 56
HCP breakthrough at 10 membrane volumes per min (resins 1 MV/min)
0
5
10
15
20
25
30
35
40
45
1 10 100 1000
Membrane volume [ml]
UV
(280
nm) [
mA
U]
Sartobind Q Sartobind STIC PA AEX resin 1AEX resin 2 Mixed mode resin
Sartobind STIC PA binds HCP most effectively
Buffer: 20 mM Tris pH 7.5, 150 mM NaCl
February 15, 2012 Page 57
DNA binding capacity
0
2
4
6
Sartobind Q Q-Sepharose FF
mg / m
l
DNA removal from
a therapeutic
antibody.
Campath 1-H
•
...clears
the DNA
•
below
detection
limit
•
„superior pressure/flow relation
•
...the process time is reduced 23-fold, excluding the benefit in handling for set-up.
•
...diminish
a loss of product
•
...installed as in-line filters and can be disposed after use*.
Average of three 12,500 liter batches
with 15-50 m odule
050100150200
After protein
A
After
Sartobind Q
pg DNA pro m
g
Protein
Galliher P, Fowler E, Millennium Pharmaceuticals Inc.: Validation of Impurity Removal by the CAMPATH-1H Biomanufacturing
Process. IBC’s Biopharmaceutical Production Week, Paradise Point Resort –
San Diego, CA, November 12-15, 2001
J. K. Walter, Boehringer
Ingelheim
Pharma,
Bioseparation
and
Bioprocessing, G. Subramanian
(ed.) Wiley VCH, Vol. II p.
447-460, 1998
FDA approval
March
2001
February 15, 2012 Page 56
DNA binding capacity at 10 % breakthrough and 16.8 mS/cm
February 15, 2012 Page 59
400 440 480 500mM (NH4 )2 SO4 concentration
HIC phenyl
membrane works
at flow
through
conditions between
400 – 500 mM ammonium
sulfate
February 15, 2012 Page 60
Aggregate removal from
a recombinant
protein by
Sartobind Phenyl
Ebert,S., Rentschler Biotechnologie, Fischer-Frühholz, S., SSB, Efficient
Aggregate
Removal from
Impure
Pharmaceutical
Active
Antibodies, BioProcess International, Vol.
9, No. 2, Feb 2011, pp. 36-42.
February 15, 2012 Page 61
Endotoxins can
be
removed
5 LRV with Sartobind Q
Endotoxins were removed from GST-protease from levels 26.900 EU/mg to 0.13 EU/mg
protein solved in TRIS / 150 mM NaCl buffer pH 8 with Sartobind Q SingleSep 20“
at cGMP
runs.*A. Clutterbuck et al., Avecia, Endotoxin reduction using disposable membrane adsorption technology in cGMP manufacturing, BioPharm
International 20 (5), 24-31 (2007)
Run 1 Run 2 Process volume (Start) 50 L 47 L Process volume (End) 60 L 59 L Process time ~10 – 20 min ~10 – 20 min Protein concentration (Bradford) 4.10 g/l 4.14 g/l Conductivity 16.63 mS/cm 17.41 mS/cm Pre use endotoxin level 26,900 EU/mg 10,100 EU/mg Post use endotoxin level 0.13 EU/mg 0.18 EU/mg Protein recovery over step 81.4 % 84.6 % LRV endotoxins 5 5
-
Sample: 20 mM Tris pH 7.5, 150 mM NaCl
spiked with 500 EU/ml
-
Device: Sartobind STIC PA nano 1 ml
-
Flow rate 10 ml/min
-
Sample load volume: 8000 ml
-
Total load 4,000,000 EU
-
Detection limit of test: 0.05 EU/ml
February 15, 2012 Page 62
Endotoxin removal on Sartobind STIC nano 1 ml LRV >4.3
4 Million Endotoxin Units (1000 EU/ml)
Complete removal
Before
EU/ml
After
EU/ml
LRV
Concentration 1000 <0.05 >4.3
February 15, 2012 Page 63
Bakteriophage
Phi X 174 is considered as surrogate for viruses
Microviridae
ssDNA
27-33 nm diameter
pI = 6.4-6.7
K. Brorson, H. Shen, S. Lute, J.S. Pérez, D.D. Frey, Journal of Chromatography A 1207 (2008) 110-121.
M. Phillips, J. Cornier, J. Ferrence, C. Dowd, R. Kiss, H. Lutz, J. Carter, J. Chromatogr. A 1078 (2005) 74.
February 15, 2012 Page 64
Applications
flowthrough polishing
on anion
exchanger, typical
spiked viruses
to prove
virus
safety
of Mabs
Positvely
(+) charged
anion
exchange
adsorber binds
negatively
(-) charged
viruses
Viruses
4 5 6 7
8 9
Isoe
lect
ric p
oint
s
Prod
uct
Im
purit
ies
pH bufferViruses
are
bound-
Source
for pI of viruses: Daniel M. Strauss et al., Biotechnol. Bioeng. 2009, 104, 371–380
Phage pI
ΦX174 6.4-6.7
February 15, 2012 Page 65
LRV of ΦX174 (pI 6.4-6.7) of Sartobind Q and Sartobind STIC low and high conductivity 25 mM Tris pH 7.5, 0, 50, 150 mM NaCl
Load 4*107 pfu/ml ΦX174, LP15, flow rate 20ml/min
00,5
11,5
22,5
33,5
44,5
5
25mMTRIS/HCl
25mMTRIS/HCl
25mMTRIS/HCl
25mMTRIS/HCl
20mMKPi
20mMKPi
20mMKPi
20mMKPi
7,5 7,5 8 8 7,5 7,5 8 8
0 150 0 150 0 150 0 150
LRV
Sartobind Q
Sartobind STIC
Buffer
pH
NaCl [mM]
February 15, 2012 Page 66
Phosphate and other multivalent anions inhibit the binding of ΦX174
February 15, 2012 Page 67
Phosphate inhibits phage binding but DNA contaminant is bound Sartobind STIC to be used at binding conditions of viruses
Ortho phosphate
mM
Phage in flowthrough
% of start material
DNA in flowthrough
% of start material
0 <0.00001 <1
2 <0.0001 <1
10 0.001 <1
30 75 -
83 <1
0.41 ml (15 cm², 3 layers) Sartobind STIC PA were loaded with 150 ml ΦX 174, 1 x 107
PFU/ml, salmon sperm DNA 200 ng/ml at 10 ml/min, buffer 20 mM Tris/HCl pH 7.5, 150 mM NaCl plus phosphate.
February 15, 2012 Page 68
Sartobind Q Sartobind STIC
Protein Binding [g/l]
BSA in 200 mM NaCl (20 mS/cm) 3.6 36
DNA Binding [g/l]
DNA in 50 mM NaCl (7 mS/cm) 7.3 22
LRV with Mouse Minute Virus (MMV)in 150 mM NaCl 1.81 > 4.96
Salt tolerant anion exchanger Sartobind STIC Primary Amine
•
Provides higher binding capacity compared to Q anion exchanger at higher conductivity
• Enables the polishing at broader operating conditions (high salt)
February 15, 2012 Page 69
February 15, 2012 Page 70
Sartobind / AEX column
Polishing
February 15, 2012 Page 71
Sartobind in Virus Purification
• Influenza virus
• Adenovirus
• Lentivirus
• Adenoassociated
virus
• Densonucleosis
virus
• Baculovirus
• Foot
and mouth
desease
virus
• Pseudrabies
virus
• Bovine
herpesvirus
• Rotavirus
like
particles
• Yellow
fever
virus
• Bacteriophages
February 15, 2012 Page 72
Membrane Adsorbers vs. Density Gradient –
Adenovirus purification
Membrane Adsorbers Density gradient ultra centrifugation
2 hours 36 hours
Up to 1013 VP/ml 106 VP/ml
No carryover, disposable, no validation, simple
Carryover validation
No contaminants Toxic CsCl2
, sucrose removal from finished product
February 15, 2012 Page 73
Membrane Adsorbers vs. Columns –
Adenovirus purification
• Capacity membranes > 10 fold higher than beads
• 20 L module instead of 200 L column
• 25-fold higher volumetric flow rate
February 15, 2012 Page 74
Purification of PPV virus spike
PDA Technical Report No. 47: Preparation of virus Spikes used for Virus Clearance Studies 2010, p19-20
February 15, 2012 Page 75
Sartobind in virus
purification –
targets
Capture
Influenza vaccine
Rabies
vaccine
Adenovirus-vectored
vaccine
Polio vaccine
Conjugated
vaccine
VLP
Polishing
DNA removal
HCP removal
February 15, 2012 Page 76
Thank
you!
www.sartorius-stedim.com/sartobind
www.sartorius-stedim.com/sartobind-stic