Sartobind® Membrane...

Sartobind®

Membrane ChromatographyDr. Stefan Fischer-Frühholz, February

15, 2012

Senior Product Manager Membrane Chromatography

February 15, 2012

1.

Membranes1.

Membranes

2.

Formats2.

Formats

Agenda

3.

Applications3.

Applications

February 15, 2012

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

Diffusion limited

gels

(time) versus

convection

limited

(flow

rate)

Average pore size 15 -

40 nm

Average pore size 3 -

5 μm

February 15, 2012

Dynamic

binding

capacity

./. Flow

rate -> Contaminant

removal

40 x Bed

volume/min

Flow

rate

Membrane chromatographyDynamicbinding

capacity

Gel chromatography

February 15, 2012

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

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

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

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

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

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

February 15, 2012

1.

Membranes1.

Membranes

2.

Formats2.

Formats

Agenda

3.

Applications3.

Applications

February 15, 2012

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

February 15, 2012

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

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

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

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

The Sartobind pico construction realizes the smallest scaleable design with 4 mm bed height (15 layers) of membranes

February 15, 2012

Connectivity of Sartobind pico is given by finger-tight Luer PEEK adapters (enclosed in package)

February 15, 2012

Sartobind Q, S and STIC 10”

capsule cut-away

Vent valve

Sartobind membrane

Fleece

Sartobind membrane

Fleece

February 15, 2012

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

Sartobind Q Pico scales to larger devices

February 15, 2012

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

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

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

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

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

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

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

Comparison of void volumes 4 mm, 70 ml and 8 mm, 150 ml capsules

Upstream

Membrane

Downstream

February 15, 2012

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

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

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

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

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


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


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


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


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+-


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


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


Anion exchange step in flowthrough polishing 3rd

step:

Position for Sartobind STIC

Sb. STIC or Q ?


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


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


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


Anion exchange step in flowthrough polishing 3rd

step:

Position for Sartobind STIC

Sb. STIC


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


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


(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


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.


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


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


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


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

DNA binding capacity at 10 % breakthrough and 16.8 mS/cm


400 440 480 500mM (NH4 )2 SO4 concentration

HIC phenyl

membrane works

at flow

through

conditions between

400 – 500 mM ammonium

sulfate


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.


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


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


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.


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


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]


Phosphate and other multivalent anions inhibit the binding of ΦX174


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.


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)


Sartobind / AEX column

Polishing


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


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


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


Purification of PPV virus spike

PDA Technical Report No. 47: Preparation of virus Spikes used for Virus Clearance Studies 2010, p19-20


Sartobind in virus

purification –

targets

Capture

Influenza vaccine

Rabies

vaccine

Adenovirus-vectored

vaccine

Polio vaccine

Conjugated

vaccine

VLP

Polishing

DNA removal

HCP removal

http://de.wikipedia.org/w/index.php?title=Datei:Influenza_virus.png&filetimestamp=20050216000608

http://de.wikipedia.org/w/index.php?title=Datei:Poliovirus.png&filetimestamp=20061105173605

http://de.wikipedia.org/w/index.php?title=Datei:Rabies_Virus_EM_PHIL_1876.JPG&filetimestamp=20060530175609


Thank

you!

[email protected]

www.sartorius-stedim.com/sartobind

www.sartorius-stedim.com/sartobind-stic

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