International Journal of

Molecular Sciences
ArticleLow Endotoxin Recovery—Masking of Naturally
Occurring Endotoxin
Johannes Reich 1, * , Felix Alexander Weyer 1 , Hiroshi Tamura 2,3 , Isao Nagaoka 3 andHubert Motschmann 4
1
2
3
4

*

Microcoat Biotechnologie GmbH, Am Neuland 3, 82347 Bernried am Stanberger See, Germany;
f.weyer@microcoat.deLPS (Laboratory Program Support) Consulting Office, Tokyo 160-0023, Japan; htamura@lpsct.com
Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine,Tokyo 113-8431, Japan; nagaokai@juntendo.ac.jp
Institute of Physical and Theoretical Chemistry, University of Regensburg, 93040 Regensburg, Germany;
Hubert.Motschmann@chemie.uni-regensburg.de
Correspondence: j.reich@microcoat.deReceived: 10 December 2018; Accepted: 12 February 2019; Published: 15 February 2019


Abstract: Endotoxins are cell wall components of Gram-negative bacteria. A release of endotoxins
into the human blood stream results in an inflammation reaction that can lead to life-threatening
conditions like sepsis. Therefore, control for endotoxin contamination of intravenously administereddrugs is crucial. Drugs are usually tested for putative endotoxin contamination with Limulus-based
tests. However, validity of the compendial test procedures is questioned in the case of low endotoxin
recovery (LER). To assure validity, regulatory authorities request hold-time studies of endotoxin
in addition to pharmacopoeial requirements. Within these studies, endotoxin is added (spiked) toan undiluted product. The spiked product is held for a certain period of time and subsequently
diluted for endotoxin determination. Due to the known heterogeneity of endotoxin the question has
been raised as to which source represents the most adequate endotoxin spike. In the present study,
endotoxin hold-time studies were analyzed by using different sources of endotoxin. Highly purified
endotoxin, crude endotoxin extracts (Naturally Occurring Endotoxin) from different bacterial species
and varied growth conditions as well as endogenous endotoxin contaminations were investigated.
The results clearly demonstrate that endotoxin masking—an effect of LER—is dependent on theendotoxin source used. Various parameters such as bacterial strain and growth conditions lead todifferent masking susceptibilities. Due to these effects it is impossible to predict the susceptibility ofbacterial endotoxin contamination to LER. In order to determine whether a sample is prone to LER,
an endotoxin spike that is susceptible to LER is required.
Keywords: low endotoxin recovery; endotoxin; lipopolysaccharide; limulus amoebocyte lysate;naturally occurring endotoxin; bacterial endotoxin testing; masking

1. Introduction
Endotoxins play an important role in the pathogenesis and manifestation of Gram-negative
infections and in particular of septic shock. Lipopolysaccharides (LPS) are the dominating constituentsof the outer leaflet of the outer membrane of Gram-negative bacteria ([1]). Therefore, the terms
endotoxin and LPS are used interchangeably ([1–4]). Because the structures of LPS can vary significantly
in its O-antigen, core region and lipid A, not all endotoxins possess the same toxicity [5]. In order todetect endotoxins, diverse detection methods like the rabbit pyrogen test [6,7], the monocyte activation
test ([8–10], and the Limulus-based tests methods [11–14] are used. These methods have been used forInt. J. Mol. Sci. 2019, 20, 838; doi:10.3390/ijms20040838

www.mdpi.com/journal/ijms

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decades in the pharmaceutical industry to test for potential contaminations of drug products withbacterial endotoxins. Due to their simplicity and high sensitivity [15,16], the Limulus-based tests are
the most commonly used methods [15,16]. However, the effectiveness of the current test procedures isunder debate regarding Low Endotoxin Recovery (LER). In 2013, Chen and Vinther presented LER,
a masking effect of endotoxin in a matrix containing a chelator and a surfactant [17]. They showedthat endotoxin spiked into an undiluted sample could not be recovered by dilution or by magnesium
replacement, two methods commonly used to overcome test inhibition. After public recognition of the
LER phenomenon, some publicized [4,18–22], and many not publicly accessible endotoxin recovery
studies of biopharmaceutical drug products and their formulations were performed by endotoxin test
providers, contract labs, and pharmaceutical companies. LER is defined as the inability to recover
more than 50% activity over time when endotoxin is added to an undiluted product. LER cannot be
overcome by dilution. LER and the associated masking effects have been widely confirmed, especiallywhen tested drug products contained surfactants and chelators. Hold-time experiments are usually
performed using standardized endotoxins such as the reference standard endotoxin (RSE) or the control
standard endotoxin (CSE) as spike. However, since bacterial endotoxin tests are commercially used,
the source and preparations of appropriate standard endotoxin are under debate [16]. The primary
standard in bacterial endotoxin testing (BET) is RSE, which is endotoxin from E. coli O113:H10:K
negative. RSE is purified in several steps including hot phenol-extraction [23]. For better handling,lactose and polyethylene glycol are added. This standard is accepted worldwide and sets the baseline
for secondary standards. Because of limited RSE-availability, vendors of Limulus-based test systems
distribute secondary standards which are calibrated against RSE. The preparations of such secondary
standards are inspired by RSE, but the source can differ from E. coli O113. Exact production processes
and formulations are not published. Since LER hold-time studies are widely performed, the sourceof the endotoxin is controversially discussed. It is especially debated, whether LER is dependent on
the source of endotoxin [18,19,22,24,25]. Obviously, depending on source, preparation, and degree of
purification, the LPS itself and the accompanying matrix components can vary respectively [26,27].
For instance, endotoxins from different bacteria can differ in their molecular structures [28,29]. There
are differences in the lipid A (e.g., acylation), core region (e.g., substitution of sugar units) andO-antigen (e.g., distribution of sugar units). Moreover, depending on the preparation, endotoxin
suspensions may vary in their compositions. For example, crude endotoxin extracts of bacteria, also
known as Naturally Occurring Endotoxin (NOE), may contain substantial amounts of lipids andproteins, whereas phenol-extracted endotoxin preparations may only contain limited amounts ofhydrophobic matrix components. Some experiments have proposed that the detectability of selected
endotoxins in complex samples might be more robust compared to the detectability of commerciallyavailable standard endotoxins [18,19,30]. Therefore, endotoxins from different bacteria, grown undermanipulated conditions, crude and highly purified endotoxins, as well as endogenous endotoxins
were analyzed with regard to their detectability in a polysorbate/citrate matrix in the present study.
2. Results
2.1. Preparation and Characterization of Naturally Occurring Endotoxin (NOE)In order to study the masking susceptibility of endotoxins from different bacteria, crude
suspensions of bacterial endotoxin (NOE) were prepared from E. coli, E. cloacae, S. marcescens,
P. aeruginosa, B. cepacia, S. maltophilia and R. pickettii. All bacteria were grown under equal, controlled,
and defined conditions (See material and methods “prep a” for details). After 18 h of growth, theabsorbance at OD (optical density) = 600 nm of all bacterial suspensions was determined as growth
control (Table 1). While the bacterial culture of E. coli and E. cloacae showed the highest absorbancevalues at 600 nm (>1.7), lower absorbance values were obtained for S. maltophilia and B. cepacia(<0.7) (Table 1). This result indicating varying growth characteristics of different bacteria under fixed
growth conditions.

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Table 1. Correlation of bacterial growth behavior and endotoxin activity. To monitor the growth ofdifferent Gram negative bacterial species (E. coli O55:B5, E. coli O113, E. cloacae, S. marcescen, P. aeruginosa,
B. cepacia, S. maltophilia and R. pickettii), the OD 600 was determined before harvesting and correlatedwith the activity of the prepared crude extract.
SourceAbsorbance(600 nm)Activity(EU/mL)E. coli O55:B5
E. coli O113
E. cloacae
S. marcescens
P. aeruginosa
B. cepacia
S. maltophilia
R. pickettii

1.9
1.7
1.7
1.4
0.9
0.7
0.3
0.8

146,174
402,789
189,103
116,175
8595
357
4557
77,815Endotoxins are usually incorporated into the bacterial cell wall. During active proliferation,
however, substantial amounts of endotoxins are released into the growth media via exocytosis. Thereby
outer membrane vesicles (OMV), LPS monomers or fragments of the bacterial outer membrane
resulting from disrupted bacterial cells can be released. All of these particles, besides the actual
bacteria, contribute to the endotoxicity (detectable endotoxin activity with bacterial endotoxin test)of the sample. In order to prepare sterile endotoxin spike material, sterile-filtrated supernatants ofbacterial suspensions were used and endotoxin activity determined. As a consequence, bacterial
cells and bigger breakdown products are removed and their activity does not contribute under given
conditions. The measured endotoxin activity of the filtrate of different bacteria suspensions spanned abroad range from approximately 400 to 400,000 EU/mL, although equal growth conditions were used
for all stains. Only a weak correlation between the measured OD 600 and endotoxin activity values
was observed.
The endotoxicity of different species varies, indicating that endotoxicity seems tobe species-dependent.
Endotoxins were applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and detected by silver staining (Figure 1). The typical ladder pattern of LPS could
be observed in most lanes. The varying intensities of the bands reflect different concentrations, whichwere approximately in agreement with the detected activities (Figure 1 and Table 1). Upon closer
examination, variations in the arrangement of bands between the different endotoxin samples could
Int.observed.
J. Mol. Sci. 2019, 20, x FOR PEER REVIEW
4 of 15beFigure 1.1. Analysis
bacterial extracts.
extracts. A
polyacrylamide gel
gel
FigureAnalysis of crude bacterial
A sodium
sodium dodecyl sulfate polyacrylamide
electrophoresis (SDS-PAGE)(SDS-PAGE) ofof thethe crude
crude extracts
extracts was
was silver-stained.
silver-stained. The
The lanes
lanes reflectreflect endotoxins
endotoxins
electrophoresis
from(1), E.
E. colicoliO113
O113(2),(2),E.E.cloacae
cloacae(3),(3),S.S.marcescens
marcescens(4),(4),
aeruginosa(5),
cepaciafrom E. coli O55:B5 (1),
P.P.aeruginosa
(5),
B.B.
cepacia(6),(6),S.
S.
maltophilia
andpickettii(8).
maltophilia(7)(7)
andR.R.
pickettii(8).

These reflect different molecular structures and again confirm the heterogeneity of endotoxins.
Taken together, crude preparations of endotoxin from different species grown at the same growth
conditions show a variable endotoxicity and are heterogeneous in their structure.

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These reflect different molecular structures and again confirm the heterogeneity of endotoxins.
Taken together, crude preparations of endotoxin from different species grown at the same growth
conditions show a variable endotoxicity and are heterogeneous in their structure.
2.2. Analysis of the Masking Ability of Different NOE
Recent publications have questioned the LER phenomenon.
They showed that a“laboratory-derived endotoxin from E. coli” was not masked after spiking it into three different
batches of a monoclonal antibody formulated in a polysorbate 80/citrate buffer [18]. Similar results
were obtained using a Natural Occurring Endotoxin from Enterobacter cloacae [25,31]. To challenge theseobservations, NOEs derived from eight different bacterial strains (E. coli O55:B5, E. coli O113, E. cloacae,
S. marcescens, P. aeruginosa, B. cepacia, S. maltophilia and R. pickettii), grown under similar conditions,as described in the chapter above, were used as an endotoxin spike for masking experiments in apolysorbate 20/citrate matrix (Table 2 and Figure 2). Crude extracts of endotoxin from E. coli, E. cloacae
and S. maltophilia resulted in low recovery already at day 0. In contrast, endotoxin from S. marcescens,
P. aeruginosa, and R. pickettii showed a gradual loss of recovery over time. The endotoxin from B. cepacia
could
be Sci.
detectedshowed no trend in reduced activity.
Int. J. Mol.
2019, 20,overx FORtime
PEERand
REVIEW
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Figure 2. Hold time study using NOEs of different
different species.
species. Endotoxin recovery over seven days at
coli
O113
(b),
E. E.
cloacae(c),(c),S.
room temperaturetemperature areare shownshown forfordifferent
differentspecies
species(E.
(E.colicoliO55:B5
O55:B5(a),(a),E.E.
coli
O113
(b),
cloacaemarcescens(d),cepacia (f),(f),S.S.maltophilia
maltophilia(g)(g)
andpickettii(h)).
spiking,a 10S.
marcescens(d),P.P.aeruginosa
aeruginosa (e),(e), B. cepacia
and
R. R.
pickettii(h)).
ForForspiking,a 10-fold
fold concentrated
solution
prepared.
concentrationof thewater
controls
corresponding
concentrated
stockstock
solution
waswasprepared.
TheTheconcentrationof thewater
controls
correspondingtoto theactivities(EU/mL)
shown
inFor(i). theForrecovery
the recovery
experiments,
the extracts
crude extracts
weretheactivities(EU/mL)are areshown
in (i).
experiments,
the crude
were spiked
into
0.05%
20 and 20
10 andmM10
sodiumcitrate. citrate.
The recoveries
were determined
after 0,spikedinto(w/v)
0.05%polysorbate(w/v) polysorbate
mM sodiumThe recoveries
were determined
1,2, 50,andrecoveries
were calculated
based basedon theoncorresponding
waterwater
control
at timeafter
1, 2,75days.
and 7Alldays.
All recoveries
were calculated
the corresponding
controlatpoint
0.
time point
0.

These results show, in clear contrast to the aforementioned data, that NOEs from different
bacteria, but grown and prepared under equivalent conditions, exhibit different masking
susceptibilities and can be masked in a polysorbate 20/citrate matrix.
2.3. Analysis of the Masking Ability of Different NOEs Depending on Their Growth Conditions
Every preparation of LPS is a heterologous mixture of various LPS molecules [32,33]. While all
LPS molecules contain the lipid A moiety and polysaccharides, the degree of acylation, substituents

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Table 2. Hold time study using naturally occurring endotoxins (NOEs) from different species. The holdtime study was performed over 7 days using the prepared NOEs for different species. All recoveries inpercentage were calculated based on the water control at t(0).
Masking Time in Days
E. coli O55:B5
E. coli O113
E. cloacae
S. marcescens
P. aeruginosa
B. cepacia
S. maltophilia
R. pickettii

Endotoxin Recoveries Based on the Water Control t(0) (%)
0

1

2

3

7

32
42
20
106
135
248
20
108

7
0
4
46
29
81
1
73

8
0
1
29
25
141
1
80

2
0
0
12
23
92
0
71

0
0
0
8
16
113
0
55

Activity of the Water Control (EU/mL)
40.9
64.7
50.9
60.5
79.6
48.5
37.9
68.4

These results show, in clear contrast to the aforementioned data, that NOEs from different bacteria,
but grown and prepared under equivalent conditions, exhibit different masking susceptibilities andcan be masked in a polysorbate 20/citrate matrix.
2.3. Analysis of the Masking Ability of Different NOEs Depending on Their Growth Conditions
Every preparation of LPS is a heterologous mixture of various LPS molecules [32,33]. While all
LPS molecules contain the lipid A moiety and polysaccharides, the degree of acylation, substituents(e.g., aminoethanol) as well as the sugars (e.g., O-antigen) varies. Additionally, bacteria might adapt
to the environmental growth conditions, which results in modifications of the LPS molecule. One
potential change due to environmental conditions is the modification of an LPS-phosphate group with
an amino-arabinose group [34]. In order to exclude that the observed masking effects of NOEs in apolysorbate 20/citrate matrix were caused by growth conditions, we performed a similar experimentwith endotoxin derived from two different growth conditions. Three bacterial strains, E. coli O113,
P. aeruginosa and B. cepacia were grown under conditions including either rich-nutrition media (100% LB)
at elevated temperatures (37 ◦ C) or low-nutrition (1% LB) at room temperatures (prep b). NOEs wereprepared as described before. A buffer containing 10 mM sodium citrate and 0.05% (w/v) polysorbate
20 was spiked with 100 EU/mL from both preparations and held for up to 7 days, respectively(Figure 3a). The endotoxin from E. coli O113 was not recovered independent of growth conditions after
one day of masking. In contrast, the recoveries of crude extract from B. capecia showed no significant
decline (Recovery >50%), independent of growth condition (Figure 3b). Recovery of endotoxin from
P. aeruginosa depend on growth conditions (Figure 3c). For both P. aeruginosa preparations the same
recovery trends are observed, but the recovery dropped below 50% already within one day (bacteria
grown at 37 ◦ C using 100% LB media). For the second preparation, the recovery dropped below 50%
only after 2 days. Thus, modified growth conditions resulted in diverging masking kinetics.

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aeruginosa preparations the same recovery trends are observed, but the recovery dropped below 50%
already within one day (bacteria grown at 37 °C using 100% LB media). For the second preparation,thedroppedInt. J.recovery
Mol. Sci. 2019,20, 838 below 50% only after 2 days. Thus, modified growth conditions resulted
6 of in15
diverging masking kinetics.

Figure 3. Masking ability of different NOEs depending on the growth condition. Endotoxin recovery
was plotted as function
endotoxin fromfrom different
different bacteria
bacteria ((a) E.
E. colifunction ofof incubation
incubation time.
time. 100 EU/mL
EU/mL endotoxincoli
O113:H21,(b)
B.
cepaciaand(c)P.
aeruginosa)
were
spikedintosamples
containing
10mM
sodium
O113:H21, (b) B. cepacia and (c) P. aeruginosa) were spiked into samples containing 10 mM sodium
citrate andand 0.05%
polysorbate20
20andand incubatedincubated atat room
room temperaturetemperature (RT).
(RT). The
The used
used endotoxin
endotoxin
citrate0.05% (w/v)(w/v) polysorbate
extracts were
were derived
Squares reflectreflect recovery
recovery of
extracts
derived fromfrom two
two different
different bacterial
bacterial growth
growth conditions.
conditions. Squaresof◦ C using 100% LB media. Triangles reflect recovery of endotoxin
endotoxinfrombacteria
grown
at
37
endotoxin from bacteria grown at 37 °C using 100% LB media. Triangles reflect recovery of endotoxin
from bacteria
LB media.
media. Each
Each endotoxin
endotoxin was
was preparedprepared ininfrombacteria grown
grown at
at room
room temperaturetemperature using
using 1%1% (v/v)(v/v) LB
triplicate.
Thecorresponding
endotoxin
measurementsofthreerepetitions
were
analyzed
on
the
same
triplicate. The corresponding endotoxin measurements of three repetitions were analyzed on the same
microtiter plate.
For calculation
calculation ofof thethe data
data points
points thethe meanmean value
value ofof thethe three
three individual
individual preparations
preparations
microtiterplate. For
was
used.
The
error
bars
represent
thestandard
deviation
of
the
three
replicates.
was used. The error bars represent the standard deviation of the three replicates.

Taken together, this result demonstrates that masking can be dependent on the growth conditions
Taken together, this result demonstrates that masking can be dependent on the growth
and the nutrition content in the media.
conditions and the nutrition content in the media.
2.4. Comparison of the Masking Behavior of Purified Lipopolysaccharides (LPS) and NOE
2.4. Comparison of the Masking Behavior of Purified Lipopolysaccharides (LPS) and NOE
For this study we analyzed NOEs from different species. However, in the pharmaceutical industry
For this study we analyzed NOEs from different species. However, in the pharmaceuticalpurified LPS preparations are used as reference standard endotoxin and as control standard endotoxin.
industry purified LPS preparations are used as reference standard endotoxin and as control standard
These standards are purified by phenol-chloroform extraction and further steps (e.g., size exclusion
endotoxin. These standards are purified by phenol-chloroform extraction and further steps (e.g., size
chromatography). They are the endotoxin of choice when masking is analyzed in hold-time studies.
exclusion chromatography). They are the endotoxin of choice when masking is analyzed in hold-time
As most of the studies that are used to analyze masking and its mechanism are performed using purifiedstudies. As most of the studies that are used to analyze masking and its mechanism are performed
LPS preparations [20,21,35], masking for purified LPS preparations is well established. Purification ofusing purified LPS preparations [20,21,35], masking for purified LPS preparations is well established.
spike material is the basis for standardization because of its required identity and known purity [36].
Purification of spike material is the basis for standardization because of its required identity andTo further evaluate the impact of endotoxin purification, endotoxins from one bacterial species (E. coli
known purity [36]. To further evaluate the impact of endotoxin purification, endotoxins from one
O55:B5) were prepared by different methods and subsequently analyzed. Phenol-extracted endotoxin
bacterial species (E. coli O55:B5) were prepared by different methods and subsequently analyzed.
and crude supernatants of bacterial suspension were spiked into a polysorbate 20/citrate matrix andPhenol-extracted endotoxin and crude supernatants of bacterial suspension were spiked into a
incubated for up to six days at room temperature (Figure 4a). Both preparations showed no recovery
polysorbate 20/citrate matrix and incubated for up to six days at room temperature (Figure 4a). Bothafter one day of incubation, which confirms the pronounced masking susceptibility of endotoxin frompreparations showed no recovery after one day of incubation, which confirms the pronounced
E. coli.
masking susceptibility of endotoxin from E. coli.
Due to the fast kinetics of masking, the experiment was also performed at decreased incubation
temperature 2 to 8 °C (Figure 4b). The reduced incubation temperature was chosen, as endotoxin
masking can be decelerated, allowing a better resolution of slight differences in masking
susceptibilities (Figure 4b). After 2 days of incubation, 50% and 14% after 14 days of the initialendotoxin content could be detected within the NOE at 2 °C to 8 °C. In comparison, the recovery ofphenol-extracted endotoxin was already low after one day of incubation (27%) and no significant
endotoxin content was detectable after three days of incubation. At lower incubation temperature theInt. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW

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detectabilityof NOE
Int. J. Mol. Sci. 2019,20, 838 decreased slower, compared to phenol-extracted endotoxin. However, 7bothof 15
preparations of endotoxin were affected by masking.

thethemaskingbehaviorof purified
endotoxins
and NOEs.
of endotoxin
Figure 4.4. Differences
Differencesin inmaskingbehaviorof purified
endotoxins
andRecovery
NOEs. Recovery
offrom E. colifrom
O55:B5is shown
overincubation
100 EU/mLof gel-filtrated
endotoxin (blackbars)
endotoxin
E. coliO55:B5is shown
over time.
incubation
time. 100
EU/mL of gel-filtrated
endotoxinand sterile
bacterial(grey bars)(greywerebars)
spikedsamples
containing
10 mM(blackbars) filtrated
and sterile
filtratedsuspension
bacterial suspension
wereinto
spikedinto samples
containing
sodiumcitrate
and
0.05%
(w/v)
Polysorbate
20.
The
sampleswereincubated
up
to
14
days
at
RT (a)
10 mM sodium citrate and 0.05% (w/v) Polysorbate 20. The samples were incubated up to 14 daysat◦and(a)
4 andC (b).
RT
4 °C (b).

Dueconsequence,
to the fast kinetics
of masking, thekinetics
experimentalso performed
decreased method
incubation
In
endotoxin-masking
can wasbe affectedby theatextractionof◦temperature2
to8
C(Figure4b).
The
reducedincubation
temperaturewaschosen,asendotoxin
endotoxin, but the kinetics rather depends on the source of bacteria than on the purification state ofmasking
can be decelerated, allowing a better resolution of slight differences in masking susceptibilities
theendotoxin.
(Figure 4b). After 2 days of incubation, 50% and 14% after 14 days of the initial endotoxin content
could
be detected
withinNOE at Endotoxin
2 ◦ C to 8 ◦Contamination
C. In comparison, the recovery of phenol-extracted
2.5.
Masking
Behaviorof an theEndogenous
endotoxin was already low after one day of incubation (27%) and no significant endotoxin content was
For all of the examples above, the source of endotoxin was known. Endotoxins were consciously
detectable after three days of incubation. At lower incubation temperature the detectability of NOE
added to samples containing surfactants and chelators. In order to examine a real endotoxin
decreased slower, compared to phenol-extracted endotoxin. However, both preparations of endotoxin
contamination, the detectability of an endogenous contaminated monoclonal antibody was analyzed.
were affected by masking.
The lyophilized antibody was solubilized in four different chelator-containing buffer systems (A: 25
In consequence, endotoxin-masking kinetics can be affected by the extraction method of endotoxin,mM sodium citrate pH 6.5, B: 10 mM sodium citrate pH 7.5, C: 160 mM trehalose and 10 mM sodium
but the kinetics rather depends on the source of bacteria than on the purification state of the endotoxin.
phosphate pH 6.2 and D: 10 mM sodium phosphate pH 7.5). An average endotoxin activity of 135
EU/mL
and 114
EU/mL
determined
before Contamination
and after sterile filtration, respectively (Figure 5a).
2.5. Masking
Behaviorof anwas
Endogenous
Endotoxin
The different buffer systems as well as the filtration had no major effects on the detectability of the
For all of the examples above, the source of endotoxin was known. Endotoxins were consciously
endogenous endotoxin contamination of the antibody. Only small differences between the activity
added to samples containing surfactants and chelators. In order to examine a real endotoxin
before and after the filtration step were observed. As expected, no masking was observed in the buffer
contamination, the detectability of an endogenous contaminated monoclonal antibody was analyzed.
systems containing only the chelating agent. As is known from previous studies on the mechanism
The lyophilized antibody was solubilized in four different chelator-containing buffer systemsof LER [20,21], masking is driven by a simultaneous presence of a surfactant (e.g., Polysorbate 20 or(A: 25 mM sodium citrate pH 6.5, B: 10 mM sodium citrate pH 7.5, C: 160 mM trehalose and 10 mM
Polysorbate 80) and a chelator (e.g., sodium citrate). Therefore, 0.07% (w/v) of the surfactant
sodium phosphate pH 6.2 and D: 10 mM sodium phosphate pH 7.5). An average endotoxin activity ofPolysorbate 80 was added to the antibody in buffer A, 0.05% (w/v) Polysorbate 80 to the antibody in
135 EU/mL and 114 EU/mL was determined before and after sterile filtration, respectively (Figure 5a).
buffer B, 0.04% (w/v) Polysorbate 20 to the antibody in buffer C and 0.05% (w/v) Polysorbate 20 to
The different buffer systems as well as the filtration had no major effects on the detectability of thethe antibody in buffer D, respectively. The endotoxin concentration was directly determined after
endogenous endotoxin contamination of the antibody. Only small differences between the activity

Int. J. Mol. Sci. 2019, 20, 838

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before and after the filtration step were observed. As expected, no masking was observed in the buffer
systems containing only the chelating agent. As is known from previous studies on the mechanismof LER [20,21], masking is driven by a simultaneous presence of a surfactant (e.g., Polysorbate 20
or Polysorbate 80) and a chelator (e.g., sodium citrate). Therefore, 0.07% (w/v) of the surfactant
Polysorbate 80 was added to the antibody in buffer A, 0.05% (w/v) Polysorbate 80 to the antibody inbufferB,Sci.
0.04%
Polysorbate
20 to the antibody in buffer C and 0.05% (w/v) Polysorbate 208toofthe
Int.
J. Mol.
2019,(w/v)
20, x FOR
PEER REVIEW
15
antibody in buffer D, respectively. The endotoxin concentration was directly determined after adding
addingthe detergents0). Comparable
as determined
directly
after filtrationwere
the detergents(day 0). (dayComparable
activitiesactivities
as determined
directly after
filtration
were observed
observed(FigureAfter an incubation
ofroom
3 daysat room temperature,
the content
endotoxin
was(Figure 5a).
After 5a).
an incubationof 3 days attemperature,
the endotoxin
wascontent
determined
determinedagain. A significant
decrease(90%oftothe
98%)of the endotoxin
activity
be observedinagain. A significant
decrease (90%
to 98%)
endotoxin
activity could
becould
observed
in all fourall
four samples
5b).
samples(Figure (Figure
5b).

Figure 5.
5. Masking
Masking ofof anan endogenous
endogenous endotoxin
endotoxin contamination.
contamination. Endotoxin
Endotoxinactivity
activityofof an
an unknown
unknown
Figureendotoxin contamination
contamination under
under different
different buffer conditions is shown. 1010 mg/mL
mg/mLmonoclonalmonoclonalantibody
antibody
endotoxin(MAK33)weresolubilizedin25
mM
sodiumcitrate,pH
6.5(bufferA);
10mM
sodiumcitrate,pH(MAK33) were solubilized in 25 mM sodium citrate, pH 6.5 (buffer A); 10 mM sodium citrate, pH 7.5
7.5(bufferB);
160
mM
trehalose,50mM
sodiumphosphatepH
6.2(buffer
C)
and
10
mM
sodium(buffer B); 160 mM trehalose, 50 mM sodium phosphate pH 6.2 (buffer C) and 10 mM sodium
phosphate,pH
pH7.5(buffer
7.5(bufferD).
D).Endotoxin
Endotoxin
content
wasdetermined
beforesterilefiltration
phosphate,content
wasdetermined
beforeandandafteraftersterilefiltration(0.2® was®used.
(0.2 µm)(a).endotoxin
For endotoxin
detection
EndoZyme
was used.
filtrationand (w/v)
0.05%µm)(a). Fordetection
EndoZyme
After After
filtration
0.07%0.07%(w/v) (w/v)
and 0.05%(w/v) polysorbate
were added
to the sample
in A
bufferB, respectively.
(w/v)
and (w/v)
0.05%
polysorbate
80 were80added
to the sample
in buffer
and A
B, andrespectively.
0.04% 0.04%(w/v) and
0.05%
(w/v)
polysorbate
20
were
added
to
the
sampleinbufferC
andbufferD,
respectively.
Endotoxin
polysorbate 20 were added to the sample in buffer C and buffer D, respectively. Endotoxin activity
activity
was determined
immediately
after additionof polysorbate
andincubation
after incubationof daysthree
wasdetermined
immediately
after additionof polysorbate(day 0)(dayand 0)
afterof three® was used.
® was used.
days3)
(day
3) at
(b).
For endotoxin
detection
the EndoLISA
(day
at RT
(b).RTFor
endotoxin
detection
the EndoLISA

Endotoxin masking
masking was
was observed
observed inin allall fourfour antibody
antibody preparations,preparations, independent
independent ofof thetheEndotoxin
compositionofthemasking
buffer.
Accordingtoprevious
publications,
all
of
these
buffershave
composition of the masking buffer. According to previous publications, all of these buffers havemasking capabilities
capabilities [20,21].
[20,21]. Thus,
Thus,thetheresults
resultsare
are consistentconsistent with
with thethe above
above mentioned
mentioned data
data andandmaskingliterature, nono further
further replicates
replicates oror statisticalstatistical analysis
analysis ofof thethe data
data were
were performed.
performed. The
The present
present data
data
literature,

show a series of experiments, in which NOEs can be masked under LER-conditions, but with various
masking kinetics. Individual masking kinetics are consistent when reproduced (Figure 3) orcompared to other experiments (Figures 2–4). Finally, the data clearly demonstrates that an
endogenous endotoxin contamination, which reflects a real naturally occurring endotoxin, is masked
in a commonly occurring formulation buffer of biologicals.

Int. J. Mol. Sci. 2019, 20, 838

9 of 15

show a series of experiments, in which NOEs can be masked under LER-conditions, but with various
masking kinetics. Individual masking kinetics are consistent when reproduced (Figure 3) or comparedto other experiments (Figures 2–4). Finally, the data clearly demonstrates that an endogenous endotoxin
contamination, which reflects a real naturally occurring endotoxin, is masked in a commonly occurringformulation buffer of biologicals.
3. Discussion
Endotoxins are complex molecules originating from the outer membrane of Gram-negative
bacteria containing a hydrophobic lipid moiety and a polysaccharide moiety. The polysaccharide
part of the endotoxin is highly diverse and varies in number of sugars within a unit, the nature of
the linkages of the sugars, as well as the number of repetitive units. O-antigen sugars appear to
be most variable, core structures appear to be less variable. In contrast, lipid moieties of LPS areconsidered highly conserved within a genus [1,37]. Gram-negative bacteria exhibit only minimal
growth requirements and can thus be found in clean water, processed water, or buffers [38], required
for the production of biopharmaceuticals. As endotoxins are strong stimulators of the human immune
system mediated by the Toll-like receptor 4 complex, monitoring of endotoxins is a crucial step in the
production of biopharmaceuticals. While the Limulus-based tests are the method of choice to test for apotential endotoxin contamination, these have been challenged under the light of the low endotoxin
recovery phenomenon. To evaluate whether a sample is affected by LER, regulatory authorities request
hold-time studies to evaluate detection of an endotoxin spike in an undiluted drug product over time,
but the kind of endotoxin used as spike was under debate. Recent studies suggested the use of NOE
as standards for hold-time studies instead of purified endotoxin. Moreover, it is hypothesized thatmasking of endotoxin is mainly induced by the use of purified endotoxin [39,40]. The authors claimthat the use of NOEs is closer to contaminations observed during biopharmaceutical drug production.
Purified endotoxins are not observed in nature and exhibit different properties due to the extraction
process and missing pieces like proteins or lipids. Hence, to investigate whether the phenomenon of
LER is limited to standardized endotoxins from E. coli, detectability of NOEs from different bacterial
species was studied in a typical biopharmaceutical drug product matrix containing polysorbate andsodium citrate. Such a matrix has been shown to induce LER. In this work, it could be shown thatsome NOEs were masked as well. However, masking of NOEs was shown to depend on species andgrowth conditions. While no masking was observed over a 7-day period for NOEs from B. cepacia andR. pickettii, masking was observed for crude extracts of E. coli, E. cloacae, S. marcescens, P. aeruginosaand S. maltophilia. These results show endotoxins which are rapidly affected by LER (e.g., E. coli O113)
and endotoxins which are less affected by LER. Endotoxin from B. cepacia was not affected by masking
under given conditions. Regarding B. cepacia, it is described in the literature that the LPS possess
an unusual structure. The bacteria lower the anionic charge of the cell surface by the substitution of
4-amino-4-deoxyarabinose residues bound to phosphates of the lipid A backbone [41–43]. This might
explain the observed limited masking susceptibility. These results would prove NOEs unsuitable forendotoxin hold-time studies. The absence of masking in a drug product could be due to the analyzed
species and its respective growth conditions. However, masking would be observed in this exact
drug product, if a different species had been used. It has to be pointed out that not all endotoxins are
similarly affected when growth conditions are modified. A change of masking susceptibility cannot
automatically be observed when comparing recovery kinetics of endotoxins from the same source, butderived from different growth conditions.
These results can be explained with an adaptation of the bacteria to an unfavorable environment toensure viability. It is known that bacteria are able to modify their primary LPS structure under certain
growth conditions. Thereby they can reinforce the external membrane to assure optimal protection
against the environment [34]. Recent publications have claimed that the endotoxin extraction method is
the reason for LER, as the endotoxin is extracted from its native environment and can adopt non-natural
aggregation states. The present findings, using highly purified endotoxin and NOE from the same

Int. J. Mol. Sci. 2019, 20, 838

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species (E. coli O55:B5), show that masking is not caused by the purification process itself, as bothpreparations were similarly masked. At decreased temperature, the masking process of the NOE
was slower than observed for the purified endotoxin. These results indicate a change in the masking
kinetics, probably due to the additional elements like proteins and lipids within the crude extract.
However, masking characteristics remained unchanged, independent of the purification process. Taken
together, masking characteristics of a given endotoxin preparation are mainly influenced by growth
conditions, not by the purification process. With the exception of Schwarz et al. [4], all publishedstudies either used a purified endotoxin standard or a lab-derived NOE to analyze masking andto simulate a possible contamination. Within this study, the masking behavior of an endogenous
endotoxin contamination affecting a commercial antibody was analyzed, as this represents a realistic
case. The contamination is observed in the final product and the bacterial species and source of thecontamination is unknown. This represents a scenario that pharmaceutical quality control departments
face during product release. The masking behavior of this contamination was analyzed using fourcommonly used buffers for drug product formulation. Within all four buffers, rapid masking wasobserved in the tested product. Less than 10% of the initial endotoxin activity was measured after
three days. These results clearly show that endotoxin masking is not linked to the purification process,but rather to the molecular structure. This emphasized that NOEs are no solution to overcome LER,
as masking was also observed for an endogenous endotoxin contamination. The use of NOEs in
hold-time studies could instead be misleading, as masking would not be observed with the analyzed
product, but might indeed be observed with an endogenous contamination.
The origin of LPS is inevitably connected to the respective bacteria. However, an intrinsic
heterogeneity is certain. It is impossible to predict source and cause of potential bacterial endotoxin
contaminations of a sample. As a consequence, the masking susceptibility of a potential endotoxin
contamination is unknown, too. In order to ensure reliable detection of endotoxins, the masking
capability of a sample has to be evaluated. To analyze the masking capability of a sample, endotoxin
hold-time studies have to be performed with endotoxin spikes, that are susceptible to masking. The
results above have shown that standardized endotoxins from E. coli exhibit a pronounced susceptibility
to endotoxin masking and represent an appropriate source for endotoxin hold-time studies. With
regard to the heterogeneity of endotoxins and the diversity of sample compositions, a panel of different
endotoxins might be the safest way to determine the masking capability of a sample and to ensure
detectability of a potential contamination. A reference standard and an additional control standard
from different species could be used as such a panel. Lastly, as soon as a sample is identified withthe capability of endotoxin masking, a suitable detection method needs to be developed in order toproperly detect endotoxins and to avoid the underestimation of a contamination.
4. Material and Methods
4.1. Chemicals and Materials
Polysorbate 20, polysorbate 80, sodium chloride, sodium azide, trisodium citrate, phosphoricacid, sodium dihydrogenphosphate, potasium dihydrogenphosphate, disodium hydrogen
phosphate-heptahydrate, 2-amino-2-(hydroxymethyl)-1,3-propanediol (tris), 2-mercaptoethanol,
isopropanol, D(+)-glucose, sodium chloride, calcium dichloride and magnesium dichloride wereobtained from Sigma-Aldrich Chemie GmbH, Steinheim, Germany. Ammonium hydroxide,
formaldehyde and D(+)-trehalose-dihydrate were obtained from AppliChem GmbH, Darmstadt,
Germany. Acetic acid, glycerol, periodic acid, sodium hydroxide, silver nitrate, sodium dodecylsulfate(SDS) and yeast extract (powdered) were obtained from Carl Roth GmbH & Co. KG, Karlsruhe,
Germany. Bromophenol blue sodium salt was obtained from Merck Chemicals GmbH, Darmstadt,
Germany. 20× Tris-tricine/SDS electrophoresis buffer were obtained from Serva Electrophoresis
GmbH, Heidelberg, Germany. The mouse monoclonal antibody (MAK33) was obtained from Roche
Diagnostics Deutschland GmbH, Mannheim, Germany. Tryptone Bacto TM was obtained from Becton

Int. J. Mol. Sci. 2019, 20, 838

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Dickinson GmbH, Heidelberg, Germany. Depyrogenated water, depyrogenated borosilicate glass tubes
and recombinant Factor C tests, EndoZyme® and EndoLISA® were obtained from Hyglos GmbH,
Bernried, Germany. Prior to the experiments, all relevant materials were tested for endotoxins andproven to contain less than 0.05 EU/mL.
4.2. Endotoxins and Bacteria
Endotoxin from Escherichia coli O55:B5 (gel-filtrate) was obtained from Sigma-Aldrich Chemie,
Steinheim, Germany. Freeze-dried bacteria from E. coli O55:B5 (DSM 4779), Enterobacter cloacae (DSM
30054) and Pseudomonas aeruginosa (DSM 500 71) were obtained from Leibniz Institute DSMZ-German
Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. Freeze-dried bacteria from
E.coli O113 (Ecor 30) was obtained from Escherichia coli Reference Collection, East Lansing, USA.
Freeze-dried bacteria from Burkholderia cepacia (2008 B02-12.20.164) and Stenotrophomonas maltophilia(DSMZ 50 170) were obtained from Robert Koch-Institute, Wernigerode, Germany. Bacteria from
Ralstonia pickettii (isolate) were a kind gift from Hyglos GmbH, Bernried, Germany.
4.3. Preparation of Crude Endotoxin Extracts (Naturally Occurring Endotoxins, NOEs) (Prep a)
For bacterial growth, 5 mL lysogeny broth (LB) media (10 g/L sodium chloride, 5 g/L yeast
extract and 10 g/L tryptone) were inoculated with the desired bacterial strain, followed by incubation
overnight at 37 ◦ C in a shaking incubator (Platform shaker: Innova 2300, New Brunswick Scientific
Co, Enfield, USA; Incubator: Wärmeschrank für Plattformschüttler, Mytron Bio- und Solartechnik
GmbH, Heilbad Heiligenstadt, Germany). Afterwards, 200 µL of the preparatory culture weretransferred into 500 mL of media (12.8 g/L disodium hydrogenphosphat-heptahydrat, 3 g/L potassium
dihydrogenphosphat, 0.5 g/L sodium chlorid, 1 g/L ammonium chloride, 0.01 g/L calcium dichloride
and 0.4 wt% glucose) and incubated for 24 h at 37 ◦ C. Growth of bacteria was monitored by lightabsorption at 600 nm using a spectro photometer (V550 Jasco Germany GmbH, Gross-Umstadt,
Germany). Bacterial growth was stopped by temperature reduction to 4 ◦ C. Crude bacterial extracts(Natural occurring endotoxins) were prepared by centrifugation of the culture at 4500 rpm (Heraeus
Multifuge 3 S-R) and sterile filtration (0.2 µm, Pall GmbH, Dreieich, Germany) of the supernatant. For
conservation 0.05% (v/v) sodium azide were added. Required endotoxin concentrations for endotoxin
recovery studies were adjusted by dilution with depyrogenated water.
4.4. Preparation of NOEs from Bacteria Grown under Low-Nutrition Conditions (Prep b)
Five mL LB media (10 g/L sodium chloride, 5 g/L yeast extract and 10 g/L tryptone) were
inoculated with the desired bacterial strain, followed by incubation overnight at 37 ◦ C in a shaking
incubator (Platform shaker: Innova 2300, New Brunswick Scientific Co, Enfield, USA; Incubator:
Wärmeschrank für Plattformschüttler, Mytron Bio- und Solartechnik GmbH, Heilbad Heiligenstadt,
Germany). Afterwards, the preparatory culture was transferred into 20 mL of LB media or a minimal
media (1% LB media in depyrogenated LRW) and incubated for 18 h at room temperature. Bacterial
growth was stopped by temperature reduction to 4 ◦ C and sterile filtration (0.2 µm) of the bacterial
suspension. For conservation 0.05% (v/v) sodium azide was added. Required endotoxin concentrations
for endotoxin recovery studies were adjusted by dilution with depyrogenated water.
4.5. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Silver Staining
The NOEs of all different species were vortexed for 30 s. 40 µL of each sample were mixed
with 10 µL sodium dodecyl sulfate (SDS) loading buffer and boiled at 100 ◦ C for 10 min. 18 µLof these samples were loaded on a 20% gradient Tris-Tricin gel (Anamed Elektrophorese GmbH,
Rodau, Germany). The sodiumdodecylsulfatepolyacrylamid gelelectrophoresis was performed in
Tris-Tricine/SDS buffer at 130 V for 90 min. The gel was fixed overnight in fixation solution (25%(v/v) isopropanol and 7% acetic acid) and oxidized for 5 min using an oxidation solution (0.06 g/mL
periodic acid, 0.33% (v/v) Isopropanol and 0.09% (v/v) acetic acid. The gel was washed 5 times inInt. J. Mol. Sci. 2019, 20, 838

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depyrogenated water and silver stained for 10 min in staining solution (350 µL sodium hydroxide(8 M), 1 mL concentrated ammonium hydroxide (28% (w/v), 2 mL silver nitrate (20% w/v) and
75 mL depyrogenated water). The stained gel was washed 5 times with depyrogenated water anddeveloped for 10 min with the development solution (1 mg/mL citric acid, 0.037% formaldehyde). The
development was stopped using acetic acid (10% v/v).
4.6. Sample Masking Preparation for Masking Kinetics
Samples were prepared in 5 mL glass tubes with sample volumes of 1 mL per sample. Unlessotherwise described, samples were spiked with a 10-fold or concentrated stock solution of the respective
endotoxin. The pH of buffer components was adjusted to 7.5 if not otherwise described. Before adding
the spikes to the sample, the stock solution was shaken at 1400 rpm for 10 min. After spiking, thesamples were incubated at defined temperatures and for defined periods of time. Immediately after
incubation, the samples were mixed at 1400 rpm for 2 min again, and diluted in depyrogenated waterin order to avoid test interference. Necessary dilutions of the particular sample compositions were
determined prior to the actual experiment.
Samples with different incubation periods were measured on the same microtiter plate, to avoid
variation from test to test. Therefore, endotoxin recovery kinetics was performed in a reverse manner.
The particular sample was aliquoted and all aliquots were stored under equal conditions over time.
The aliquot with the longest endotoxin incubation period was spiked first (e.g., 7 days prior to themeasurement). Further aliquots with shorter incubation periods were spiked later in accordancewith the respective incubation period. The zero-time point aliquot was spiked immediately before
measurement. Resulting kinetics consists of various time points and reflects one experiment, if nototherwise indicated.
The validity of the actual measurement was controlled by spiking the defined endotoxin amounts
into the diluted samples (Positive Product Control (PPC)). Endotoxin determination in a diluted sample
was considered valid, if 50% to 200% of the spiked endotoxin (PPC) was recovered. To control theaccuracy of the endotoxin spiked into the undiluted samples, equal amounts of endotoxin were spiked
into depyrogenated water (water control), mixed and identically incubated as the actual sample. The
spike into the undiluted sample was considered valid if the water control was in the range of 50% to200% of the theoretical expected spike concentration.
For calculation of the recovery, the determined endotoxin concentrations in the tested samples
were compared to the endotoxin concentrations at time zero in water controls and stated as percent.
4.7. Sample Preparation using an Endogenous Endotoxin Contamination
The lyophilized monoclonal antibody (MAK33) was solubilized at a concentration of 10 mg/mL
in buffer A (25 mM sodium citrate, pH 6.5), buffer B (10 mM sodium citrate, pH 7.5), buffer C (160 mM
Trehalose, 50 mM sodium phosphate pH 6.2) and buffer D (10 mM sodium phosphate, pH 7.5). The
endotoxin concentration of the natural contamination was determined before and after sterile filtration
using the EndoZyme® assay. After filtration, 0.07% (v/v) polysorbate 80 were added to the sample inbuffer A, 0.05% (v/v) polysorbate 80 were added to the sample in buffer B, 0.04% (v/v) polysorbate
20 were added to the sample in buffer C and 0.05% (v/v) polysorbate 20 were added to the sample
in buffer D, respectively. The endotoxin content was determined immediately after the addition ofpolysorbate (day 0) and after incubation at room temperature (19–25 ◦ C) after 3 days (day 3) using the
EndoLISA® assay. For calculation of endotoxin recovery, the determined endotoxin concentrationsin the actual sample was compared to the determined endotoxin concentration in the water control
stated as percentage.
4.8. Endotoxin Detection
For bacterial endotoxin detection, Limulus-based tests (rFC) were used according to
manufacturer’s instructions. The amount of fluorescence substrate (amino-methylcoumarin) released

Int. J. Mol. Sci. 2019, 20, 838

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was measured fluorometrically at 440 nm (Excitation: 380 nm) with a FLx800 fluorescence microplate
reader (BioTek Instruments GmbH, Bad Friedrichshall, Germany). All samples were measured in
duplicate and average values were used for further calculations. Standard curves were fitted using afour-parameter logistic non-linear regression model. The detection limit of the assay was 0.005 EU/mL(EndoZyme® ) and 0.05 EU/mL (EndoLISA® ). If not otherwise indicated, EndoZyme was used forendotoxin detection.
Microsoft Excel 2010, Version 14.0.7015.1000 was used to calculate endotoxin recovery, plot
graphs and to simulate endotoxin recovery kinetics. Gen5 Data Analysis Software Version 2.05 from
BioTek Instruments GmbH, Bad Friedrichshall, Germany was used to calculate standard curves fordetermination of endotoxin concentrations.
Author Contributions: J.R. was responsible for measurements, reading and writing. F.A.W. was responsible fordata curation and writing. H.T. and I.N. were responsible for details of the study concept, reading and writing.
H.M. was responsible for the general study concept and supervision.
Funding: This research received no external funding.
Acknowledgments: The authors wish to express their gratitude to the participating organizations, which enableda charitable and comprehensive, collaborative study between academia and industry.
Conflicts of Interest: The authors declare no conflict of interest.

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