Sartobind® Ion ExchangeMembrane Adsorber capsules, 4 mm bed height

turning science into solutions

Sartobind Capsules for Economic Production of Therapeutic Proteins

High speed polishing

Sartobind SingleSep® capsules are designed

to remove charged contaminants from

therapeutic proteins at accelerated flow

rates by ion exchange membrane chro-

matography. The high throughput is a

direct result of negligible mass transfer

effects and is made possible by the >3 μm

macroporous membrane with 4 mm bed

height.

Approved technology

In 2001 Campath®* received FDA approval.

This monoclonal antibody is polished by

application of Sartobind ion exchange

Q modules in flow through mode (DNA|

endotoxin|leached protein A|virus removal).

It was the first time that a Membrane

Adsorber has been accepted in the produc-

tion of a therapeutic protein. This shows not

only Sartorius’ position as the pioneer in

adsorptive membrane chromatography but

also displays that this technology is proven

and tested.

* Campath is a trademark of Genzyme

Corporation

Efficient

– Higher throughput (g/h) for trace

impurity removal

Economical

– Saves capital

– No hardware investment & maintenance

– No column packing, testing,

regeneration

– No re-use validation

– Less unspecific binding – higher yield

– Less labor

– Buffer consumption may be

decreased 95%

Easy to use

– Disposable

– Simple and fast set up

– Handling like a filter capsule

Controlled quality

Sartobind SingleSep capsules are designed,

developed and manufactured in accor-

dance with an ISO 9001 certified Quality

Management System. They have passed

Plastic Class VI, particles and extractables

test according to current United States

Pharmacopoeia (USP) and are tested for

protein binding capacity prior to release.

Each package contains a certificate of

quality.

A validation guide is available upon

request: Order No.: 85032-536-72.

Ion Exchange Membranes

Sartobind Q

The workhorse to remove negatively

charged contaminants: DNA, host cell

proteins, viruses and endotoxins.

Sartobind S

For the removal of positively charged

contaminants such as host cell proteins.

Sartobind STIC®

In addition to conventional anion exchange

chemistries, Sartobind STIC PA (primary

amine) ligand operates at high salt condi-

tions to decrease need for dilution (e.g. of

cation exchange pools) when removing

negatively charged impurities. With a

binding capacity of 1.4 mg/cm2 the salt

tolerant adsorber is a specialist for (host

cell) protein removal. MVM virus removal

at 150 mM NaCl salt result in >4 LRV and

DNA removal in >3 LRV.

Capsule design

The Sartobind SingleSep looks like a

standard filter capsule except that the

adsorbing membrane is reeled on a core

to form a cylinder. The flow is from top

into the outside channel and then perpen-

dicular through the membrane layers to

the center of the cylinder and leaves the

capsule at the outlet.

3

l Fig. 1: Sartobind 30 inch capsule connected to a disposable bag.

Materials

Labour

Consumables

Hardware costs

IEX gel column

MaterialsLabourConsumables

�

80% less

IEX Membrane Adsorber Capsule

Flow path/4 mm bed height

Sartobind capsule

Fig. 2: Cost comparison of columns and Membrane Adsorbers for polishing application

Fig. 3: Schematic flow pattern and cross section of capsulesa) Sartobind pico 0.08 ml b) 10" capsule 180 ml

a)

b)

Application Area

In a generic downstream process for the

production of a monoclonal antibody, the

main capturing steps are followed by the

polishing step(s).

The later the anion exchanger is applied in

the process, the less contaminants have to

be removed.

The positively charged ligand of the

Q adsorbers binds negatively charged

contaminants.

Neutral or positively charged product

runs through (see Figure).

+

_+

4

Cell removal|Clarification

Affinity Chromatography

Cation Exchange Chromatography

Anion Exchange Adsorber Polishing Step (flowthrough):

Removal of DNA, HCP, Endotoxins

Virus Clearance

Ultra-|Diafiltration

Sartobind usage in monoclonal antibody production:

Endotoxin

DNAVirus

Host cell protein

2 7 12

pH of buffer

MAb

_ _

_

_

_

+

+

pl

Fig. 4: Isoelectric points and charges of MAbs and impurities

Binding capacity on Sartobind ion exchanger membranes

IEX ligand Type of molecule Binding capacity Binding capacity Ref.

chemistry per ml per cm2

Q BSA >29 mg >0.8 mg 1

STIC PA BSA 50 mg 1.4 mg 4

Q DNA 5.6 mg 0.16 mg 2

STIC PA DNA 10 mg 0.3 mg 4

Q Adenovirus up to 1.5+1013 up to 8+1011 Viral Particles 3

S Lysozyme >29 mg >0.8 mg 1

1 ml = 36 cm2 IEX membrane1 Sartorius quality control for minimum static

binding capacity2 J. K. Walter, (Boehringer-Ingelheim Pharma) in:

Bioseparation and Bioprocessing, G. Subramanian(ed.) Wiley VCH, 1998, Vol. II p. 447–460

3 Measured with Vivapure AdenoPack 500. Order no.:VS-AVPQ501

4 Measured in high salt buffer, 150 mM NaCl,Sartobind STIC PA manual, Order no.: 85032-536-77

5

The use of conventional chromatography

columns for flow through (FT) anion

exchange chromatography requires high

flow rates. Optimized production columns

need a certain diameter and bed height to

achieve a large throughput and therefore

have a large bed volume. That is the reason

why columns are typically oversized.

Due to the cylindrical construction of the

adsorber the high flow rate of the

membrane is combined with a large frontal

surface resulting in very high throughput

and keeping the bed volume small. Typical

process data show that operation time can

be reduced by about 80% and buffer

reduction can reach 95%. Protein loading

in FT can be two orders of magnitude

higher than with columns 6, 7.

Comparison of Q packed bed chromatography and Q Membrane Adsorber6

Q Resin Q Membrane Adsorber

Flow rate 100–150 cm/h 450–600 cm/h

Protein loading (flow through) 50–100 g/l >3,000 g/m2 or

>10.9 kg/l

Buffer used 100% 5%

Cleaning validation Yes No

6 J.X. Zhou, T. Tressel (Amgen Inc.), Membrane Chromatography as a Robust Purification System for Large-ScaleAntibody Production, BioProcess Int. 09, 2005, p. 32–37.

7 H. Knudsen et al. Membrane Ion Exchange Chromatography for Process-Scale Antibody Purification, J. Chromatography A 907, 2001, 145–154

Virus clearance

This study5 also demonstrates excellent virus log reduction values with Sartobind Q.

This enables membrane adsorber technology to replace a Q column without compromising

critical viral clearance capabilities.

Murine Pseudorabies Reo Virus Minute Virus

Leukemia Virus type 3 of Mice

Virus (MuLV) (PRV) (Reo-3) (MVM)

50 ml virus > 5.57 ± 0.25 > 5.67 ± 0.17 > 7.28 ± 0.30 > 6.77 ± 0.24

spiked protein

sample

500 ml virus 6.29 ± 0.25 > 5.67 ± 0.23 > 7.53 ± 0.29 4.41 ± 0.37

spiked protein|

buffer solution

5 R. Zhang (Abgenix Inc.) et al, Viral Clearance Feasibility Study with Sartobind Q Membrane Adsorber for HumanAntibody Purification, BioProduction 2004, Munich Germany 26–27 October 2004.

Fig. 5: No size exclusion effects for viruses

Fig. 6: Oversized column compared to Sartobind capsule for polishing application

Capsules vs. Columns

6

Dimensions and connections

Capsule with 4 mm pico nano mini 5” 10”, 20”, 30” mega

bed height

Connector Luer Lock Luer Lock Sanitary 3’’ Sanitary 11’’ Sanitary 11’’ Sanitary 1’’

Capsule 31+ 11 mm 37+ 33 mm 87+45 mm 270+70 mm 365+100 mm (10”) 910+190 mm

(height+diameter) 620+100 mm (20”)

870+100 mm (30”)

Inlet connector female female 25 mm outer d 50.5 mm outer d 50.5 mm outer d 50.5 mm outer d10 mm inner d 15 mm inner d 36 mm inner d 36 mm inner d

Outlet connector as above as above as above 50.5 mm outer d as above as above

19 mm inner d

Hose barb version

Capsule (height+width) n. a. n. a. 103+45 mm 190+70 mm n. a. n. a.

Hose barb connectors n. a. n. a. 6–12 mm 14.5 mm n. a. n. a.

Recommended internal n. a. n. a. 6–10 mm 12 mm n. a. n. a.

diameter of flexible tube

n.a. = not availableRelated products are Sartobind MA units for laboratory use and the Sartobind System Jumbo line with 8 mm bed height (Sartobind nano 3 ml, 150 ml and Jumbo 5 l) with void volume optimization for bind & elute and polishing.

Technical data

Membrane

Base material Stabilized reinforced cellulose

Membrane thickness 275 μm

Pore size (nominal) >3 μm

Ion exchanger types – strong anion Q (quaternary ammonium)

R-CH2-N+(CH3)3

– strong cation S (sulfonic acid) R-CH2-SO3-

– anion exchanger (primary amine) -NH2

Capsule materials

Outer cage, inner core, Polypropylene

end caps, capsule housing,

nonwovens

O-ring in vent valve* Silicone

Bed height 4 mm

* only with 10”, 20”, 30” and mega capsules

Operation

Depyrogenation 1 N NaOH 30–60 min at 20°C

Autoclaving 121°C for 30 minutes for one cycle only

Maximum operating pressure 4 bar, 0.4 MPa, 58 psi

5 bar, 0.5 MPa, 72.5 psi (Sartobind STIC PA 5")

Fig. 7: Sartobind pico 0.08 ml for processdevelopment and virus studies

Fig. 8: Sartobind Q SingleSep nano 1 ml

Ordering Information

Sartobind Q capsules

Order no. Chemistry Description Quantity Bed Bed Nominal Frontal Protein Recom- Approx.

adapter inlet volume height membrane surface* binding mended Capsule

and outlet [ml] [mm] area area capacity** flow rate weight

[cm2] [g] [l/min]

Sartobind pico

92IEXQ42DD-11--D S Luer female 10 0.08 4 2.9 cm2 0.19 0.002 0.002 1.5

Sartobind nano

92IEXQ42DN-11 Q Luer female 1 1 4 36 cm2 2.4 0.029 0.03 10 g

92IEXQ42DN-11--A Q Luer female 4 1 4 36 cm2 2.4 0.029 0.03 10 g

Sartobind mini

92IEXQ42D4-OO--A Q hose barb 4 7 4 250 cm2 20 0.2 0.2 30 g

92IEXQ42D4-SS--A Q sanitary flange 4 7 4 250 cm2 20 0.2 0.2 30 g

Sartobind 5 inch

92IEXQ42D9-OO--A Q hose barb 4 70 4 2,500 cm2 160 2 1.9 220 g

92IEXQ42D9-SS--A Q sanitary flange 4 70 4 2,500 cm2 160 2 1.9 230 g

Sartobind 10 inch

92IEXQ42D1-SS Q sanitary flange 1 180 4 6,600 cm2 450 5.3 5.0 800 g

Sartobind 20 inch

92IEXQ42D2-SS Q sanitary flange 1 360 4 1.3 m2 900 10.5 10 1.4 kg

Sartobind 30 inch

92IEXQ42D3-SS Q sanitary flange 1 540 4 2 m2 1,350 15.8 15 2 kg

Sartobind mega

92IEXQ42DC3SS Q sanitary flange 1 1620 4 6 m2 4,050 48 50 5.6 kg

* Frontal surface area = average cylinder surface

** Typical dynamic binding capacity at 10% breakthrough was measured with BSA for Q | PA and lysozyme for S chemistry.

For Sartobind STIC PA 150 mM NaCl was added to the buffer.

7

8

Sartobind STIC capsules

Order no. Chemistry Description Quantity Bed Bed Nominal Frontal Protein Recom- Approx.

adapter inlet volume height membrane surface* binding mended Capsule

and outlet [ml] [mm] area area capacity** flow rate weight

[cm2] [g] [l/min]

Sartobind pico

92STPA42DD-11--D STIC PA Luer female 10 0.08 4 2.9 cm2 0.19 0.004 0.002 1.5 g

Sartobind nano

92STPA42DN-11--A STIC PA Luer female 4 1 4 36 cm2 2.4 0.050 0.03 10 g

Sartobind 5 inch

92STPA42D9-FF--A STIC PA sanitary flange 4 70 4 2500 cm2 160 3.5 1.9 230 g

Sartobind 10 inch

92STPA42D1-SS STIC PA sanitary flange 1 180 4 6600 cm2 450 9 5 800 g

Sartobind 30 inch

92STPA42D3-SS STIC PA sanitary flange 1 540 4 2 m2 1,350 27 15 2 kg

Sartobind mega

92STPA42DC3SS STIC PA sanitary flange 1 1620 4 6 m2 4,050 81 50 5,6 kg

Sartobind S capsules

Sartobind pico

92IEXS42DD-11--D S Luer female 10 0.08 4 2.9 cmÇ 0.19 0.002 0.002 1.5

Sartobind nano

92IEXS42DN-11 S Luer female 1 1 4 36 cm2 2.4 0.025 0.03 10 g

92IEXS42DN-11--A S Luer female 4 1 4 36 cm2 2.4 0.025 0.03 10 g

Sartobind mini

92IEXS42D4-OO--A S hose barb 4 7 4 250 cm2 20 0.175 0.2 30 g

92IEXS42D4-SS--A S sanitary flange 4 7 4 250 cm2 20 0.175 0.2 30 g

Sartobind 5 inch

92IEXS42D9-OO--A S hose barb 4 70 4 2,500 cm2 160 1.75 1.9 220 g

92IEXS42D9-SS--A S sanitary flange 4 70 4 2,500 cm2 160 1.75 1.9 230 g

Sartobind 10 inch

92IEXS42D1-SS S sanitary flange 1 180 4 6,600 cm2 450 4.6 5.0 800 g

Sartobind 30 inch

92IEXS42D3-SS S sanitary flange 1 540 4 2 m2 1,350 15.8 15 2 kg

* Frontal surface area = average cylinder surface

** Typical dynamic binding capacity at 10% breakthrough was measured with BSA for Q | PA and lysozyme for S chemistry.

For Sartobind STIC PA 150 mM NaCl was added to the buffer.

9

Accessories

Order no. Description

5ZGI--0001 Stainless steel holder for one 10, 20 or 30 inch capsule, 3 legs

5ZGLG-0004 Stainless steel holder for three 10, 20 or 30 inch capsules, 3 legs

5ZALB-0002 Stainless steel distribution adapter for three 10, 20 or 30 inch capsules

9ZAIAM0001 Stainless steel legs for mega, 3 items

1ZAOGV0003 Adapter Sanitary 25 mm 1–3“ to UNF10–32 to connect mini to LC systems, 2 items

1ZA---0004 Adapter Luer male to UNF – 10–32 female, PEEK, 1 item

Fig. 10: A Sartobind mega capsuleon stainless steel legs

Fig. 9: Adapter to connect mini with sanitaryconnection to liquid chromatography systems

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