Preventing Cell Death
App Note / Case Study
Published: May 2, 2024
Credit: Sartorius
Water systems are crucial for biopharmaceutical laboratories, providing ultrapure water with low endotoxin levels to prevent cell death. Arium® Pro VF combines advanced purification technologies and has been rigorously tested to meet stringent regulatory standards, ensuring safe and effective pharmaceutical manufacturing.
Download this app note to discover a system that can be used for:
- The Arium® Pro VF system's ability to effectively reduce endotoxin levels in ultrapure water
- The combination of carbon adsorption, ion exchange, UV, and ultrafiltration technologies for consistent water purity
- Validation results confirming the system's compliance with US Pharmacopeia standards for endotoxin content
Preventing Cell Death
Ultrapure Water System Lowers Endotoxin Content Below Prescribed Limits
Katrin Toeppner1, Dr. Elmar Herbig2
1. Sartorius Stedim GmbH, Goettingen, Germany
2. Sartorius Lab Instruments GmbH & Co. KG, Goettingen, Germany
Introduction
The cell wall components of gram-negative bacteria, such as E. coli and pseudomonads, are designated as endotoxins. They
have a hydrophilic polysaccharide and a lipophilic lipid component and, unlike the bacteria from which they originate, are highly
heat and pH-stable. Endotoxins belong to pyrogens, i.e., they can cause fever if they come in contact with mucous membranes
or if they enter the bloodstream (with reference to Steck, 2006, [1]).
According to the prevalent pharmacopeias, the defined limit values for endotoxin content may not be exceeded during the
manufacturing process of pharmaceuticals.
In mammalian cell cultures that are used to produce biopharmaceuticals, such as immunoglobulins, the presence of endotoxins
can lead to cell death. For this reason, ultrapure media, i.e., ultrapure water, with levels proven to be below the limits must be used
to manufacture biopharmaceuticals or to propagate cell lines or cell cultures.
The objective of this study is to demonstrate that the ultrapure water produced by the Arium® Pro VF water purification system
exhibits an endotoxin content that is far below the prescribed limits and that can be used for applications of the categories
mentioned above.
June 22, 2021
Keywords or phrases:
Ultrapure water, manufacturing of pharmaceuticals,
detection of endotoxins, endotoxin concentration,
Pharmacopoeia, mammalian cell cultures, production
of immunoglobulins, gel-clot method, quantitative
chromogenic method
Find out more: www.sartorius.com
2
Endotoxin Testing
A method for detection of endotoxins is the so-called LAL
(Limulus Amebocyte Lysate) test, which uses the clotting
reaction of a lysate of amebocytes isolated from the horseshoe crab (Limulus polyphemus). This reaction involves a
primitive evolutionary coagulation cascade in which proteases are sequentially activated, thus resulting in the formation of a clot.
The coagulation cascade (also called a Limulus Enzyme
Cascade, see [2], [3]) can be triggered by endotoxins or
alternatively by ß-glucans, which are short-chain polysaccharides found in the cell walls of yeasts and molds. The
ß-glucan pathway may lead to false-positive results [1].
Based on coagulation, the liquid, colorless lysate reacts by
coagulation to form a solid, milky gel, hence the term “gelclot method.” As the sensitivity of the lysate is extremely
high, it is imperative to rule out a falsepositive reaction
caused by contamination with endotoxins or ß-glucan. For
this reason, exceptionally high purity requirements must
be placed on ultrapure water.
In addition to the gel-clot method, the endotoxin content
can also be assayed using a quantitative chromogenic
method. In this method, a synthetic chromogenic peptide,
a chromogenic substrate, is added to the lysate. Whereas
in the gel-clot method, a gel clot is formed by the clotting
enzyme, the synthetic chromogenic substrate in the chromogenic method is split by the clotting enzyme, resulting
in a color change to yellow. The intensity of the yellow coloration correlates directly with the endotoxin content of the
sample (chromogenic test). Preparing the lysate for either
the gel-clot or the chromogenic method must therefore
be done using ultrapure water.
An Ultrapure Water System
The Arium® Pro VF system (Fig. 1) is designed to produce
ultrapure water from pretreated drinking water and removes any contaminants still present in this potable water.
Ultrapure water production requires continuous recirculation and a constant water flow rate, which is achieved using
a pump system with controlled pressure. The conductivity
of the water is measured at the feed water inlet and at the
downstream port, or product water outlet.
The system used in the tests described in this paper operates with two different cartridges. These are filled with
a special active carbon adsorber and mixed-bed ion exchange resins to deliver ultrapure water with a low TOC
content. Moreover, the unit has an integrated UV lamp
that has an oxidizing effect at wavelengths of 185nm and
254nm, respectively.
Furthermore, the system has a built-in ultrafilter module
used as a crossflow filter. The ultrafilter membrane utilized
in this filter retains colloids, microorganisms, endotoxins,
RNA and DNA. A 0.2μm final filter installed at the water
outlet serves to remove particulates and bacteria during
dispensing of the ultrapure water stream. The process that
the unit employs to produce ultrapure water is shown in
Figure 2.
Figure 2: Schematic flow diagram
Test Methods
In the tests performed to assay the endotoxin content in
ultrapure water obtained from the system, both the gel-clot
method and the chromogenic method were conducted.
Gel-Clot method
The lyophilized lysate (Charles River Endosafe R 15006) was
dissolved in ultrapure water obtained from the system and
frozen by portions in pyrogen-free test tubes. An endotoxin
standard series consisting of LPS E. coli 055:B5 (Lonza
N 185) was prepared in concentrations of 0.001 EU/mL to
25 EU/mL using ultrapure water from the Arium® Pro VF
system. The dissolved lysate was tested using the endotoxin
standard prepared in order to verify that it had the sensitivity indicated (0.06 EU/mL). A sample of 100μL was pipetted into 100μL of the lysate and incubated in a water bath
at 37 °C. After one hour, each tube was individually removed
from the water bath, turned by 180° and assessed for gelclot formation.
UV lamp (185|254nm)
Conductivity
measurement
(inlet water)
Inlet water
input
Pump
Cartridge 1 Cartridge 2
Reject water
outlet
Conductivity
measurement
(product water)
Final
filter
0.2µm
Sanitization port
Ultrafilter
Product water
3
Sample Endotoxin
Standard Prepared
[EU/mL]*
GelClot
Chromogenic
Assay [EU/mL]*
Arium® Pro VF
Ultrapure Water
0 No 0**
Endotoxin Standard 0.001 No 0**
Endotoxin Standard 0.005 No 0**
Endotoxin Standard 0.01 No 0.009
Endotoxin Standard 0.05 No 0.055
Endotoxin Standard 0.1 Yes 0.083
Endotoxin Standard 0.5 Yes 0.63
Endotoxin Standard 1.0 Yes 1.2
Endotoxin Standard 5.0 Yes 4.47
Endotoxin Standard 10 Yes 8.4
Endotoxin Standard 25 Yes 25
* EU/mL, endotoxin unit; 1 EU approximately corresponds to 100 picograms of endotoxin
(as a rule of thumb)
** Below the quantifiable detection limit
Table 1: Assay of the endotoxin content in Arium® Pro VF ultrapure water
and endotoxin standard samples using the gel-clot method and the chromogenic method
Figure 4 shows the data collected on endotoxin concentrations in tap water, deionized water (DI water) and Arium®
water in comparison with two selected endotoxin standards
(0.05 EU/mL and 0.005 EU/mL).
Chromogenic method
The chromogenic substrate (Charles River Endochrome K
R1710K) was dissolved in ultrapure water produced by the
water purification system. This chromogenic substrate and
the endotoxin standard samples were pipetted into 96
endotoxinfree well plates in a ratio of 1:1. The absorption of
each sample was measured continuously at one-minute
intervals over one hour at 405nm using a TECAN Safire
plate reader at 37 °C.
Results
In the range of 0.001 EU/mL to 0.05 EU/mL, the gel-clot
method showed a negative result; i.e. no gel-clot formation
(no endotoxins detected; Table 1). A concentration of
≥ 0.1 EU/mL resulted in the formation of a gel-clot. This
result shows that within the detection sensitivity limits of
the gel-clot method, no endotoxins were present in the
ultrapure water filtered by this particular system.
The 60 absorption readings at 405nm (Figure 3) in the
chromogenic method yielded curves with different slopes
depending on the specific endotoxin content of the standard samples. The times that were needed to reach a specific absorption were determined using a special computer
program and used as the basis for extrapolating the samples with an unknown endotoxin content (for values, see
Table 1 – chromogenic assay). As the standard samples
with the lowest concentrations (0.005 and 0.001 EU/mL)
and the Arium® water did not attain the extinction value
of 1 required to calculate the endotoxin content, they were
assessed as below the detection limit.
The values attained with Arium® Pro VF ultrapure water
were considerably lower than those plotted for the curve
of 0.001 EU/mL.
25 EU/mL
10 EU/mL
5 EU/mL
1 EU/mL
0,5 EU/mL
0,1 EU/mL
0,05 EU/mL
0,01 EU/mL
0,005 EU/mL
0,001 EU/mL
Arium® water
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0 600 1200 1800 2400 3000 3600
time [sec]
absorption [405nm]
Endotoxin standards 25 EU/mL–0,001 EU/mL
Figure 3: Absorption readings for endotoxin standard samples over a
60-minute period using the chromogenic method.
0,05 EU/mL
0,005 EU/mL
Arium® water
tap water
DI water
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0 600 1200 1800 2400 3000 3600
time [sec]
absorption [405nm]
Figure 4: Endotoxin assay of water samples.
The endotoxin content in water samples was calculated
from the values measured using the endotoxin standards
and is shown in Table 2.
Sample Endotoxin Concentration [EU/mL]*
Arium® Pro VF Ultrapure Water <0.001
DI Water 0.02
Tap Water 25
Table 2: Table showing the endotoxin content in different water samples
Germany
Sartorius Lab Instruments GmbH & Co. KG
Otto-Brenner-Strasse 20
37079 Goettingen
Phone +49 551 308 0
USA
Sartorius Corporation
565 Johnson Avenue
Bohemia, NY 11716
Phone +1 631 254 4249
Toll-free +1 800 635 2906
For further information, visit
www.sartorius.com
Specifications subject to change without notice.
Copyright Sartorius Lab Instruments GmbH & Co. KG.
Status: 06 | 2021
In the DI water sample, endotoxins of 0.02 EU/mL were
detected, which was below the currently valid limits for WFI
(water for injection). In tap water, with 25 EU/mL an exceptionally high endotoxin load was found. This finding was not
further analyzed. By contrast, an exceptionally low value of
<0.001 EU/mL was measured in ultrapure water obtained from
the system, which is far below the usual limits encountered.
Discussion
The test results show that ultrapure water produced by the
Arium® Pro VF system can be readily used as an affordable
alternative for preparing samples to be assayed in endotoxin tests as the detectable concentrations of endotoxin
in the ultrapure water produced are exceptionally low
(<0.001 EU/mL). The results obtained confirm earlier tests
in which an endotoxin load of <0.001 EU/mL was found in
Arium® ultrapure water [4].
The endotoxin concentration is far below the limits required
by the United States Pharmacopeia, which therefore makes
this water theoretically suitable for use in the manufacture/
endotoxin monitoring of pharmaceutical products. Examples
of such uses are product formulations, diafiltration solutions,
chromatography buffers and water for extraction, rinsing and
sanitization steps as well as for cell culture solutions.
Precisely within the scope of cell culture applications one has
to pay attention to contaminations in the various steps of
these processes. To maintain control over the effects of endotoxins on such cultures and on the sensitivity of cells, particularly with respect to such endotoxins, the media for cell
growth must be demonstrably free of detectable endotoxins
(see also [5]).
Acknowledgements
Our special thanks go to Dr. Stephanie Steck of Rentschler
Biotechnologie GmbH
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