Endotoxin Removal

Ion Exchange Chromatography with Sartobind Membrane Adsorbers

Fig. 1 shows that endotoxin monomers in Three standard strategies are available for
solution range from molecular weight of 4 removal of endotoxin from solutions with
to 20 kDa but they can form micelles and Sartobind devices. Using the strong basic
vesicles with diameters up to 0.1 m. The ion exchanger type Q and a buffer pH lower
presence of detergents, chelators and pro- than the pl of the protein, endotoxin will
teins promotes the formation of structures be bound and protein will pass through the
like micelles (300-1000 kDa) and monomers membrane (negative chromatography).
Application Note

(10-20 kDa), while bivalent ions promote the Sartobind STIC PA is an anion exchanger and
formation of large structures like vesicles works at the same pH conditons as Q but is
(>1000 kDa)1. If the target substance in also applicable at higher salt concentrations.
the solution to be depyrogenated has a When other contaminants such as host cell
small molecular weight (e.g. buffer, salt, proteins have to bound at up to 20 mS/cm,
nucleotides, amino acids, peptides, some Sartobind STIC (Salt Tolerant Interaction
carbohydrates etc.) the endotoxins can be Chromatography) may be the membrane of
separated from the target substance by choice as these contaminants can be effec-
ultrafiltration with an appropriate cut off tively removed at such conditions. Using the
crossflow filter. However, proteins in the strongly acidic ion exchanger type S and a
same molecular weight range as endotoxins buffer pH lower than the pl of the protein,
cannot be separated by ultrafiltration. the endotoxin will pass through and the pro-
tein will bind and can be subsequently eluted.
Due to the negatively charged phosphoryl and
Introduction carboxl groups in endotoxins, ion exchange Larger volumes for production scale (gram
Endotoxins (lipopolysaccharides from chromatography is the most common depyro- kilogram) can be processed with Sartobind Q
membranes of gram negative bacteria) make genation method for proteins; however, or STIC disposable capsules with 4 mm bed
up the majority of pyrogens, which must it has several drawbacks which limit its use- height. Sartobind Q capsules with 8 mm bed
be removed from pharmaceutical products, fulness as depyrogenation step. This includes height may be applied as well.
biologicals for injection and some media handling and usage problems such as packing,
for tissue cultures. There are many factors channeling, low flow rates, long regeneration The reduction of endotoxin is expressed
to be considered when designing a depyro- times, compressibility and limited chemical with a log reduction value (LRV) which is the
genation scheme for media or solutions stability. Small flow rates and susceptibility logarithmic quotient (log10) of the sample
containing proteins and peptides: type of to fouling mean that incorporating chro- solution containing the endotoxins, divided
target substance, proteins or peptides, matography resins into process scale purifica- by the concentration of endotoxins of the
product concentration, molecular weight and tion steps can be expensive and troublesome. filtered solution.
isoelectric point (pI); electrolyte concentration; Sartorius has introduced a high capacity, EU/ml starting solution
pH and buffer system; and interactions such scaleable and ready-to-use ion exchange LRV = log
EU/ml filtrate
as interference or aggregation. In general, membrane technology, which provides excel-
these factors determine a method for an lent performance needed for depyrogenation
effective pyrogen removal. in the laboratory or for process scale.

Bivalent Bivalent
cations cations

Detergents, EDTA, Detergents EDTA

bile acids (Proteins) (Proteins)

Mr (Monomer): 10-20 kDa Micelles Vesicles

Mr: 300-1000 kDa Mr >1000 kDa
Figure 1
Structure of endotoxin aggregates in
aqueous solutions of different composition. Lipophilic Sites
Hydrophilic sites
(Courtesy of Prof. F.B. Anspach, Hamburg Charged functional groups
University of Applied Sciences, Department Bivalent cations (e.g. Ca2+, Mg2+)
of Natural Science Technology)
1. Purification of clinical vaccine proteins2 Results
Purification of five recombinant bacterial vac-
cine proteins was investigated. The molecular Table 1: Clearance of endotoxin and DNA from protein-D
weight range was between 35 and 53 kDa.
Crude E. coli lysates were purified by a combi- Purification step Endotoxin LRV DNA LRV
nation of anionic and cationic membranes. [EU/g protein] [pg/g protein]
Membrane chromatography was able to Sterile crude 6224 180
reduce the purification process development
time. Salt gradient elution was compared to After first Q 32 2 30 1
pH elution on 15 and 100 cm2 ion exchange After second Q/C 0.02 3 0.08 3
membranes. The pH elution produced higher Sterile filtration 0.002 1 0.05
yields as well as eliminated the time necessary Total 6 4
for desalting. In order to remove very high
levels of DNA and endotoxins, two consecutive
anionic exchanger (Q) steps were utilized. Table 2: Scale-up results with protein-B
The first Q membrane was used as a product
capture step, while the second Q was a nega- Scale Purity Endotoxin DNA
tive chromatography step. The pH condition [%] [EU/g product] [pg/g product]
were optimized in order to let the product run
through the second Q and directly captured Q 100 2.8 ml (100 cm2) 99.7 0.01 0.04
on a cationic exchanger membrane (C). Disc 30 ml (1100 cm2) 98.2 0.002 0.10
Module 1.15 l (4 m2) 99.0 0.01 0.05
Membrane chromatography steps for a
vaccine protein-D purification 1 liter ion exchange membrane = 3.64 m2
1. Adsorption and elution from Q
Bind at pH 8.0
Discussion
Wash with 50 mM NaCl at pH 6.5 Process development experiments were evaluated by the ability to retain the target protein
Elute with 100 mM NaCl at pH 4.7 while removing endotoxins, DNA and contaminating proteins. Endotoxin removal was achieved
by a 3 log removal in the first anion (Q) exchange step followed by an additional 3 log reduction
2. Flow through Q and loading of C in the second anion/cation (Q/C) step. DNA removal was accomplished by a 1 log reduction in
the Q step followed by a 3 log reduction in the Q step. The process consistently gave purities of
Load onto Q and C in series at pH 4.7 > 95 % and yield of 65 % of the target protein.
The protein flows through Q and binds to C
One of the most important advantages using Membrane Adsorbers was the decreased processing
Disconnect Q from C membrane time. Small-scale purifications would yield pure product in approximately 75 minutes. When
scaled-up the processing time did not increase significantly, the entire chromatographic
3. Elution from C purification could be completed in a few hours. We took advantage of this quick equilibration
Elute at pH 5.5 time and built-in pH elution steps in nearly all of the recombinant proteins. The pH elution steps
allowed the processing from one ion-exchange step to the next with only a pH adjustment.
This purification method was adapted to This eliminated the need for time consuming buffer exchange steps. Quick binding and pH
purify four additional recombinant protein elution is not possible in bead-based columns due to the long equilibration times required.
variants. All vaccine proteins were success-
fully scaled to 30 ml (1100 cm2) Sartobind The purification process was developed using Sartobind Q 100 (2.8 ml, 100 cm2) and then scaled
disc membrane and 1.15 l (4 m2) Sartobind to 30 ml (1100 cm2) disc and 1.15 l (4 m2) Sartobind module (11- and 400-fold scale-up, respec-
modules. The 1 liter (4 m2) modules were tively). Each of the scaled-up processes provided protein of equal purity and yields compared
run at a flow rate of 7 L/min/bar. to the small-scale experiments. This data demonstrated that Membrane Adsorbers are easily
scalable.

A cleaning procedure was developed using 1 N NaOH and 1 N HCl on the anion exchangers
and 1 N NaOH on the cation exchangers. Reproducibility and regeneration was demonstrated
for 40 purification cycles by overlaying chromatograms.

Conclusion
The information gathered above demonstrated that Membrane Adsorbers are a valid option
compared to the classical resin technology. The Adsorbers have proven to achieve a higher
throughput and increased speed while maintaining the capacity, selectivity, and reproducibility
required for industrial chromatographic separations. There was an overall reduction of labor as
a result of no column packing, shorter set-up, and process times. Manufacturing capacity may
quickly be expanded by simply adding more cycles.
2. Endotoxin clearance of a immunoglobulin Table 3: Endotoxin clearance of an immunoglobulin solution with Sartobind Q 75
solution with Sartobind Q 75
Sample: Cytoglobin in 10 mM potassium Endo- Sample Total Total LRV Endo- Protein Protein Protein Bound
phosphate, pH 6.0 toxin volume endo- endo- toxin in in flow- in flow- protein
concen- [ml] toxin toxin clear- sample through through [%]
Device: one Sartobind Q 75 tration in in flow- ance [mg] [mg] [%]
[EU/ml] sample through (%)
Table 3 shows that endotoxin in a [EU] [EU]
protein solution was removed effectively
1000 10 10000 60 2.2 99.40 5 4.2 84 16
(99.4099.97 %) with Sartobind Q 75.
At the same time, the protein recovery 1000 10 10000 60 2.2 99.40 5 4.3 86 14
was 8486 %.
Control (without protein)
1000 10 10000 3 3.5 99.97

Immunoglobulins often have isoelectric points between 7.59.5 and will not bind at a pH
of 6.0. Due to some other protein impurities bound on the membrane, the endotoxin removal
decreased compared to the protein free solution (control).

3. Scale-up of endotoxin clearance from Table 4: Endotoxin removal step with Sartobind Q SingleSep 20 at cGMP runs
protease solution
A. Clutterbuck3 (Avecia Ltd., UK) successfully Run 1 Run 2
removed endotoxins and DNA from their
Process volume (Start) 50 L 47 L
GST-3C protease with Sartobind Q. He com-
pleted the process scale-up from Sartobind Q Process volume (End) 60 L 59 L
downscale units through pilot scale with 5
Process time ~1020 min ~1020 min
capsules for 100 L non-GMP demonstration
batch to the final scale with 20 capsules for Protein concentration (Bradford) 4.10 g/l 4.14 g/l
2 + 100 L cGMP production batches.
Conductivity 16.63 mS/cm 17.41 mS/cm
Sample: GST-3C protease, 46 kD (genetically Pre use endotoxin level 26,900 EU/mg 10,100 EU/mg
engineered fusion protein consisting
Post use endotoxin level 0.13 EU/mg 0.18 EU/mg
of human rhinovirus 3C protease
and GST, theoretical pI 6.8) Protein recovery over step 81.4 % 84.6 %
Buffer: 50 mM Tris, 150 mM NaCl, 3 mM
LRV endotoxins 5 5
reduced glutathione, pH 8 at 20C.

Conclusion
Demonstration batch
The final product from cGMP production had great purity, quantity and activity, low
Endotoxin level of affinity column eluate:
endotoxin and low DNA as well. The Sartobind Q capsule was extremely easy to use and
42,900 EU/ml
saved up to 100 production plant hours per batch and enabled significant cost saving.
Flowthrough after Sartobind Q was taken
for endotoxin every 5 L (50 L total, initial
100 L was reduced to the half before the
endotoxin removal step).
Endotoxin breakthrough was detected
after 10 L, therefore, Sartobind Q 20, 4 mm
bed height (5 times larger membrane volume
than 5) was chosen for the final cGMP runs.
4. Purification of recombinant Helicobacter pylori urease for immunization References
against H. pylori 1. Petsch, D. and Anspach, F.B.,
Helicobacter pylori infects the gastric epithelium of approx. half of the worlds population and Endotoxin removal from protein solutions.
causes a histologic gastritis. An estimated 20 % of those infected will go on to develop either J. Biotechnol. 76, 97-119 (2000)
peptic ulcer or gastric adenocarcinoma, which is the second-most-common cause of cancer
mortality worldwide2. 2. McMaster, R. et al.,
Purification of clinical vaccine poteins by
Recombinant H. pylori urease was purified, which was used in immunoassays and for immu- high speed membrane chromatography.
nization of mice,4,5 or of rhesus monkeys 6. Using Sartobind Q, endotoxin contamination was ISPPP 98 Vienna, Austria, Abstract pp 29
reduced to <1.5 ng/mg of urease 4,5, 6. (1998)

3. Clutterbuck, A. et al.,
5. Endotoxin removal at high conductivity with Sartobind STIC Endotoxin reduction using disposable
In this experiment, the ability of Sartobind STIC PA anion exchanger for endotoxin removal membrane adsorption technology in cGMP
at high salt concentration was tested. manufacturing. BioPharm International 20 (5),
24-31(2007)
Materials
Endotoxin: LONZA N 185 LPS E. coli 055:B5 4. Weltzin, R. et al.,
Buffer: 20 mM TRIS +150 mM NaCl, pH 7.5, Parentral adjuvant activities of Escherichia
conductivity 16 mS/cm coli heat-labile toxin and its B subunit for
Sartobind STIC PA nano 1 ml (salt tolerant anion immunization of mice against gastric
exchange membrane adsorber, order number Helicobacter pylori infection. Infect. Immun.
92STPA42DN-11--A), 3 lots, one device from 68(5), 2775-2782 (2000)
each lot.
5. Londono-Arcila, P. et al.,
Methods Attenuated Salmonella enterica Serovar Typhi
The Sartobind STIC PA nano 1 ml was sanitised expressing urease effectively immunizes
with 1 N NaOH for 30 minutes at room mice against Helicobacter pylori challenge
temperature. To avoid false negative results as part of a heterologous mucosal priming-
(NaOH in high concentration results in a parenteral boosting vaccination regimen.
detectable inhibition), the unit was then Infect. Immun. 70(9), 5096-5106 (2002)
thoroughly washed with 250 ml arium water
to reach a conductivity of <2 mS/cm 6. Solnick, J.V. et al.,
(no inhibition of the test). The sample was Immunization with recombinant Helicobacter
rebuffered with 50 ml 20 mM Tris with 150 mM pylori urease in specificpathogen-free rhesus
NaCl, pH 7.5, 16 mS/cm. monkeys (Macaca mulatta). Infect. Immun.
Altogether 4000 ml of endotoxin sample with a 68(5), 2560-2565 (2000)
concentration of 1000 EU/ml (in total 4 Mio EU)
were applied to the capsule at 10 ml/min flow
rate.

Results
The endotoxin concentration after the Sartobind STIC treatment was lower than the detection
limit (< 0.0125 EU/ml) using all three devices.

Table 5: Endotoxin removal with Sartobind STIC PA nano 1 ml Sartorius Stedim Biotech GmbH
August-Spindler-Strasse 11
before after LRV 37079 Goettingen, Germany
Endotoxin concentration 1000 EU/ml < 0.05 EU/ml > 4.3 Phone +49.551.308.0
Fax +49.551.308.3289
www.sartorius-stedim.com
This results shows that the Sartobind STIC PA can reduce endotoxins from high levels down USA Toll-Free +1.800.368.7178
to below detection limit at higher conductivity levels compared to endotoxin removal with Q UK +44.1372.737159
chemistry. France +33.442.845600
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Ver. 11 | 2010