［医薬品医療機器レギュラトリーサイエンス，PMDRS, 54（4），341〜351（2023）
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原著

Collaborative Study of Bacterial Endotoxins Test Using
Recombinant Factor C‑Based Procedure for Detection of
Lipopolysaccharides (Part 3)
Yutaka KIKUCHI ＊1, #, Masashi MUROI ＊2, Yukari NAKAGAWA ＊3, Akiko EBISAWA ＊3,
Mayumi HAYASHI ＊3, Honoka TAKEUCHI ＊3, Yuka KIWAMOTO ＊3, Kayoko MATSUMURA ＊4,
Rumiko YOSHIMOTO ＊5, Natsuko TSUZUKI ＊5, Nayoko OIKAWA ＊5, Mina HASHIMOTO ＊5,
Yoriko HIRAMATSU ＊5, Miki FUKAMI ＊6, Kazuya KOBAYASHI ＊6, Narumi SANDA ＊6,
Syuhei ETO ＊6, Mitsuo MORI ＊6, Olivier MARTINEZ ＊7, Masato SUZUKI ＊7,
Sachie SEKIGUCHI ＊7, Kazuyuki OUCHI ＊8, Hiroki FUKUCHI ＊9, Takeshi KITAGAWA ＊10,
Motoi KIZAWA ＊10, Tamaki MASUDA ＊11, Toshio ODA ＊12, Hikaru MIZUMURA ＊12,
Norihiko OGURA ＊12, Daizaburo IIDA ＊13, Kanako SUEOKA ＊13, Yuji TANNO ＊13
and Masakazu TSUCHIYA ＊14（Received: January 30, 2023; Accepted: May 18, 2023）

Summary
Two new recombinant cascade reagents (rCRs), PyroSmart NextGen® and PYROSTARTM Neo, have recently become
available for endotoxin testing of parenteral drugs in the Japanese market. This study investigated whether these tworCRs, as well as the current commercially available recombinant factor C reagents (rFCs) PyroGeneTM and EndoZyme® II,
can be used as alternative reagents to the amoebocyte lysate reagents currently used in the compendial Bacterial
Endotoxins Test. The two rFCs were investigated in the previous two-year study.
An Escherichia coli O113: H10: K negative culture supernatant and seven water samples (six different tap waters andone deionized water) were tested for autochthonous endotoxin, and the endotoxin levels detected with four amoebocyte

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Department of Nutrition, Faculty of Healthcare Sciences, Chiba Prefectural University of Health Sciences, 2‑10‑1 Wakaba, Mihama‑ku, Chiba, Chiba 261‑0014, Japan
Research Institute of Pharmaceutical Sciences, Musashino University, 1‑1‑20 Shinmachi, Nishitokyo, Tokyo 202‑8585, Japan
Pharmaceutical Reference Standards Center, Pharmaceutical and Medical Device Regulatory Science Society of Japan, 2‑1‑2
Hiranomachi, Chuo‑ku, Osaka 541‑0046, Japan
Section of Biological Safety Research, Chitose Laboratory, Japan Food Research Laboratories, 2‑3 Bunkyo, Chitose‑shi, Hokkaido
066‑0052, Japan
Section of Biological Science, Chitose Laboratory, Japan Food Research Laboratories, 2‑3 Bunkyo, Chitose‑shi, Hokkaido 066‑0052,
Japan
Quality Control Department, Takasaki Quality Unit, Quality Division, Kyowa Kirin Co.,Ltd., 100‑1 Hagiwara‑machi, Takasaki‑shi,
Gunma 370‑0013, Japan
Industry Business Unit, bioMérieux Japan Ltd., 2F Akasaka Tameike Tower, 2‑17‑7 Akasaka, Minato‑ku, Tokyo 107‑0052, Japan
J.K. International, Inc., 3‑1‑7 Nihonbashikayabacho, Chuo‑ku, Tokyo 103‑0025, Japan
Production Process Development Department, FUJIFILM Wako Pure Chemical Corporation, 6‑1, Takada‑cho, Amagasaki‑shi,Hyogo 661‑0963, JapanLAL Development Department, FUJIFILM Wako Pure Chemical Corporation, 1‑2, Doshomachi 3‑Chome, Chuo‑ku, Osaka 540‑8605,
Japan
Lonza K.K., 9th floor, Nihonbashi Kato Building 1‑14, Nihonbashi 2‑chome, Chuo‑ku, Tokyo 103‑0027, JapanLAL Research, Central Research Laboratory, Seikagaku Corporation, 3‑1253 Tateno, Higashiyamato‑shi, Tokyo 207‑0021, Japan
Bioscience Department, Veritas Corporation, 1‑18‑16 Hamamatsucho, Minato‑ku, Tokyo 105‑0013, JapanCharles River, 1023 Wappoo Road, Suite 43B, Charleston, SC 29407, USA
Corresponding author

Pharmaceutical and Medical Device Regulatory ScienceVol. 54 No. 4（2023）

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341

［Kikuchi,: Collaborative Study of BET Using rFC‑Based Procedure（Part 3）］lysates and four recombinant protein reagents were compared. The results indicate that the four recombinant protein
reagents can detect autochthonous endotoxin in culture supernatant samples at levels comparable (within the 50%-200%
range as defined in the Pharmacopeias) to those measured with limulus amoebocyte lysate reagents. One of the fourrecombinant reagents detected autochthonous endotoxin in water at comparable levels to those obtained with lysate
reagents in all samples, whereas the other three reagents detected comparable or lower levels among different samples.
These findings suggest that there are differences in the detectability of autochthonous endotoxin in water among therecombinant protein reagents.
Key words
Bacterial endotoxin test, Amoebocyte lysate reagent, Recombinant factor C reagent, Recombinant cascade reagent

Over the last several years, rFCs comprising

1. Introduction

only recombinant factor C, an endotoxin‑sensitive

Endotoxin（also referred to as lipopolysaccharide,

serine protease zymogen, have been developedLPS）in the cell walls of Gram‑negative bacteria isand introduced to the market to aid in thea strongly pyrogenic substance that can activate

conservation of the horseshoe crab, provide a1）

innate immune responses . Trace amounts ofstable supply of reagent, and improve theendotoxin directly invading the bloodstream canconsistency of testing results by eliminating theinduce fever, and larger amounts can lead to life‑

biological lot‑to‑lot variability of amoebocyte

threatening septic shock. Therefore, methods tolysate reagents5）. Lonza and Hyglos/bioMérieux

detect endotoxin contamination in products aredeveloped and introduced the recombinant protein

critical. The Bacterial Endotoxins Test（BET）isr e a g e n t s , P y r o G e n e TM a n d E n d o Z y m e ® ,listed in chapter 4.01 of the Japanese Pharmacopoeia

respectively, to the market. Each reagent contains

2）
eighteenth edition（JP 18）
as a method to test

a single recombinant zymogen protease（factor C）

endotoxin contamination in parenteral drugs.

from horseshoe crabs. PyroGeneTM has factor C

The BET harmonized among the JP, thefrom, whereas

European Pharmacopoeia（Ph. Eur.） and the

EndoZyme® contains

factor C 6）.

United States Pharmacopeia（USP）4） is a method

Recently, a recombinant cascade reagent（rCR）,

to detect or quantitate endotoxin using

PyroSmart NextGen®, comprising rFC,

amoebocyte lysate reagent（amoebocyte

recombinant factor B and recombinant proclotting

amoebocyte lysate,

enzyme, was developed and introduced to theTAL）prepared by extraction of hemocytes frommarket by Associates of Cape Cod, Inc. andhorseshoe crabs （orSeikagaku Corporation7）. This was followed by）. The BET has threeanother rCR, PYROSTARTM Neo, from FUJIFILM

different methodologies: one is the gel‑clot assay,

Wako Pure Chemical Corporation. Both rCRs use

which involves visual observation of gel formation;

recombinant factors fromthe second is a turbidimetric assay that measures

reactions of amoebocyte lysate（panel A）, rFC

the time required to reach a threshold optical（panel B）and rCR（panel C）reagents in thedensity caused by changes in turbidity; and thepresence of endotoxin are shown in Fig. 1. These

last is a colorimetric assay, which measures optical

recombinant protein reagents cannot be used forcolor development resulting from cleavage of a

BET as described in the Pharmacopeias, since ansynthetic substrate from a peptide.

amoebocyte lysate prepared from blood corpuscle

3）

lysate, LAL or342

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. Cascade［Kikuchi,: Collaborative Study of BET Using rFC‑Based Procedure（Part 3）］

O
O

O

O

O

O

O

O

O

O

Fig. 1 Cascade Reactions of (A) Amoebocyte Lysate, (B) Recombinant Factor C and (C) Recombinant Cascade
Reagents.

extracts of horseshoe crabs（

ortesting the reactivity to LPS purified from several

2））must be used . However, the US8）different species of bacteria as well as

Food and Drug Administration（FDA） states

autochthonous endotoxin ［called naturallythat rFC‑based procedures can be used to test

occurring endotoxin（NOE）in the study reports］.

4）

In this study, the performance of the two newly

after validation in accordance with USP Chapter

available rCRs in Japan as well as the existing

9）<1225> Validation of Compendial Procedures .

rFCs was compared to that of four amoebocyte

The Ph. Eur. also states that rFCs can be used as

lysate reagents for the detection of autochthonouscompendial methods after validation 10）. In June

endotoxin in water, which is used as a raw

2021, the JP 18 issued Bacterial endotoxins test

material for parenteral drugs, and in culture

and alternative methods using recombinantsupernatant ofprotein‑reagents for endotoxin assay <‑ ‑which is the same bacterial strain as that used toas General Information, permitting

provide a reference standard endotoxin by the JP,

endotoxin as an alternative method to the BET>

11）

O113: H10: K negative,

alternative methods to be adopted after validation.

USP and Ph. Eur. Furthermore,

However, in Japan validation of recombinant

K negative is also used by the World Healthprotein reagents to replace amoebocyte lysate

Organization（WHO）as an international reference

reagents has so far been limited due to lengthy

standard.

O113: H10:

discussions surrounding different approaches for
implementing alternative analytical procedures by
end‑users and manufacturers 7, 12 16）.
So far, the appropriateness of using recombinant

2. Materials and Methods
Apparatus

protein reagents to test endotoxin as alternative

All glassware was washed and thenmethods to the BET has been evaluated in two

depyogenated in a hot‑air oven using a validated

studies in 201717）

and 2018

18）.

Those studies

process.

Plastic materials, such as multi‑wellconcluded that recombinant protein reagents haveplates and tips for micropipettes, were used after

lot‑to‑lot consistency and similar performance

they had been determined to be free of detectable

relative to amoebocyte lysate reagents whenendotoxin and to cause no interference with thePharmaceutical and Medical Device Regulatory ScienceVol. 54 No. 4（2023）

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or reproduction without permission is prohibited except for exception under copyright laws.

343

［Kikuchi,: Collaborative Study of BET Using rFC‑Based Procedure（Part 3）］

test.

USA）and PYROSTARTM Neo was purchased from

Endotoxin

FUJIFILM Wako Pure Chemical Corporation

JP‑Reference Standard Endotoxin（JP‑RSE）（Tokyo, Japan）. PyroSmart NextGen® was used

was purchased from the Pharmaceutical andboth in onset time assay, which measures the time

Medical Device Regulatory Science Society ofrequired to reach a threshold absorbance, and rate

Japan（PMRJ, Osaka, Japan）.

assay, which measures the mean rate of color

Water samples

development, unless otherwise mentioned.

Six tap water samples and one deionized water

Endotoxin Assay

sample were collected from the seven institutes

Endotoxin in the water samples and culturethat participated in this study. After collection,

supernatant was measured using amoebocyte

each water sample was aliquoted into tubes, andlysate and recombinant protein reagents.

the aliquots were kept frozen at ‑80℃ until use.

reagents were used according to their Instructions

Preparation of culture supernatant offor Use （IFU）. JP‑RSE was employed as a

Escherichia coli O113: H10: K negative

standard, and three two‑fold dilution series wereO113: H10: K negative was cultured
19）

All

prepared with the following concentrations: 0.125,

with some

0.0625, 0.03125, 0.015625 and 0.0078125 endotoxin

modifications. Briefly, the culture medium was aunit（EU）/mL. Autochthonous endotoxin in water

minimal microbial growth medium containing

samples and culture supernatants was measured

sodium phosphate （dibasic）, monopotassium

after dilution according to the following scheme:

phosphate, sodium chloride, ammonium chloride,

tap water #1: 50‑ and 500‑fold, tap water #2: 3‑

glucose, magnesium sulfate and calcium chloride.

and 30‑fold, deionized water #1: 5‑ and 50‑fold,

After culturing, the medium was centrifuged at

tap water #3‑#6: 5‑ and 50‑fold, culture

2,330 ×for 5 min at 4℃ to separate the culture

supernatant: 5 x 10 5‑ and 5 x 10 6‑fold. All watersupernatant. The supernatant was sterilized bysamples and the culture supernatant were tested

filtration through a membrane filter with a porewith all amoebocyte lysate and recombinantsize not exceeding 0.22μm （Merck code #protein reagents at three institutions: PMRJ, Japan

S2GPU05RE, Darmstadt, Germany） and kept

Food Research Laboratories（JFRL）and Kyowa

frozen at ‑80℃ until use.

Kirin Co., Ltd. All water samples and the culture

Amoebocyte lysate reagentssupernatant were subjected to testing withaccording to the reported method

Endochrome‑K™ with endotoxin‑specific

PyroSmart NextGen® in onset time assay and withreconstitution buffer was purchased from Charles

PYROSTARTM Neo at Musashino University. All

River Laboratories（MA, USA）. Endospecy® ES‑

water samples and culture supernatant were

50M was purchased from Seikagaku Corporation

tested at participating institutions other than those（Tokyo, Japan）and Kinetic‑QCL™ was purchased

mentioned above using their own amoebocyte

TMfrom Lonza（MD, USA）. PYROSTAR

ES‑F waspurchased from FUJIFILM Wako Pure Chemicallysate and recombinant protein reagents.

Data analysis‑test was conducted using Excel 2019

Corporation（Tokyo, Japan）.

（Microsoft, Tokyo, Japan） with the software

Recombinant protein reagents
As rFCs, EndoZyme® II was purchased from

OriginPro 2022（Lightstone, Tokyo, Japan）.

Hyglos / bioMérieux （Munich, Germany） andPyroGene™ was purchased from Lonza （MD,USA）. As rCRs, PyroSmart NextGen ® waspurchased from Associates of Cape Cod, Inc.（MA,344

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3. Results
Water and culture supernatant samples wereVol. 54 No. 4（2023）

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［Kikuchi,

Table 1

: Collaborative Study of BET Using rFC‑Based Procedure（Part 3）］

Evaluation of Endotoxin Panel Using Amoebocyte Lysate Reagent
Endochrome‑KNo.

sample1

EU/mLaEndospecy ES‑50M

RSD (%)b 50%Min ‑ 200%Maxc

45880 ± 2386

10.40

20790 ‑ 100100

d

EU/mL

4

46800 ± 2815

RSD (%) 50%Min ‑ 200%Max

12.03

20030 ‑ 107300

4

4

O113:H10:K negative2

Tap water #1

16.27 ± 1.763

21.67

5.773 ‑ 39.93

4

15.50 ± 2.458

31.71

5.219 ‑ 43.92

3

Tap water #2

0.1845 ± 0.008788

9.524

0.07930 ‑ 0.3940

40.1935 ± 0.01555

16.08

0.07930 ‑ 0.4680

44Deionized water #1

3.692 ± 0.3117

16.88

1.473 ‑ 8.826

42.926 ± 0.2375

16.24

1.156 ‑ 6.918

45

Tap water #3

3.129 ± 0.2702

17.27

1.228 ‑ 7.555

4

2.489 ± 0.3091

24.84

0.8084 ‑ 6.066

4

6

Tap water #4

3.057 ± 0.2476

16.20

1.161 ‑ 6.792

42.507 ± 0.07016

5.596

1.150 ‑ 5.192

4

7

Tap water #5

1.380 ± 0.1206

17.47

0.5832 ‑ 3.437

40.9623 ± 0.05614

11.67

0.3998 ‑ 2.079

48

Tap water #6

0.7609 ± 0.06782

17.83

0.3198 ‑ 1.761

40.8142 ± 0.06737

16.55

0.3365 ‑ 1.980

4Kinetic‑QCL
No.

sample1

PYROSTAR ES‑F

EU/mL

RSD (%)

50%Min ‑ 200%Max

EU/mL

60250 ± 4732

15.71

27000 ‑ 148000

447980 ± 5148

21.46

20120 ‑ 126300

4

32.95

4.500 ‑ 40.20

4

11.44 ± 1.224

21.41

4.358 ‑ 28.90

4

RSD (%) 50%Min ‑ 200%Max

O113:H10:K negative2

Tap water #1

14.99 ± 2.470

3

Tap water #2

0.2326 ± 0.03955

34.01

0.08055 ‑ 0.6600

40.1700 ± 0.01445

17.00

0.07680 ‑ 0.4265

4

4Deionized water #1

4.155 ± 0.6370

30.66

1.505 ‑ 11.00

4

3.044 ± 0.1522

9.999

1.422 ‑ 6.986

45

Tap water #3

2.809 ± 0.4239

30.18

1.0280 ‑ 7.980

43.072 ± 0.2619

17.05

1.339 ‑ 7.600

4

6

Tap water #4

3.644 ± 0.3677

20.18

1.386 ‑ 8.980

42.920 ± 0.2633

18.04

1.170 ‑ 6.987

4

7

Tap water #5

1.233 ± 0.04630

7.513

0.5525 ‑ 2.640

4

1.119 ± 0.02161

3.863

0.5360 ‑ 2.350

48

Tap water #6

1.078 ± 0.1326

24.61

0.4675 ‑ 2.950

40.5857 ± 0.05223

17.84

0.2304 ‑ 1.433

4aValues shown are mean ± standard error of results from participating independent institutions.
Relative standard deviation.
cRange between 50% of minimum and 200% of the maximum the levels detected with amoebocyte lysate reagent.
dNumber of institutions that carried out measurements.
btested for autochthonous endotoxin with fouramoebocyte lysate reagents and four recombinant

4. Discussion

protein reagents. The mean and standard error

In this study and the two previous studies 17, 18）,

for each reagent were calculated, and the results

the appropriateness of using recombinant protein

are shown in Tables 1 and 2, and Fig. 2 and 3.

reagents to test endotoxin as an alternative

Autochthonous endotoxin was detected in all

method to the BET was evaluated by ten

water samples and the culture supernatant withinstitutions.

both amoebocyte lysate and recombinant protein

Autochthonous endotoxin in both culture‑test analysis,

supernatant and water samples was detected with

autochthonous endotoxin levels detected with both

both amoebocyte lysate and recombinant protein

amoebocyte lysate and recombinant protein

reagents with acceptable repeatability andreagents showed equal reagent‑to‑reagent

reproducibility.

variance among all samples, with the exception of

measured at three or more institutions, inter‑

culture supernatant（Table 3）.

institutional differences between recombinant andreagents.

As shown byMoreover, for each sample

amoebocyte lysate reagents were similar,
suggesting that there was no difference inPharmaceutical and Medical Device Regulatory ScienceVol. 54 No. 4（2023）

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345

［Kikuchi,

Table 2

: Collaborative Study of BET Using rFC‑Based Procedure（Part 3）］

Evaluation of Endotoxin Panel Using Recombinant Protein Reagent
EndoZyme II

No.

sample1aPyroGeneb

EU/mL

RSD (%)

c

113500 ± 1189

2.095

27.42

PyroSmart NextGen‑onset time

EU/mL

RSD (%)

EU/mL

447640 ± 5043

21.17

4

54700 ± 3176

12.99

5

4

4.205 ± 0.7796

37.08

4

15.40 ± 1.444

20.97

5

RSD (%)

O113:H10:K negative2

Tap water #1

2.386 ± 0.3271

3

Tap water #2

0.02861 ± 0.0009355

6.539

4

0.03048 ± 0.001705

11.19

4

0.1890 ± 0.01822

21.56

5

4 Deionized water #1

1.647 ± 0.03545

4.304

4

1.759 ± 0.1825

20.75

4

3.230 ± 0.3885

26.89

5

5

Tap water #3

0.4557 ± 0.1028

45.11

4

0.7513 ± 0.05651

15.04

4

2.600 ± 0.2012

17.31

5

6

Tap water #40.7006 ± 0.05401

15.42

4

0.8350 ± 0.08132

19.48

4

2.940 ± 0.1654

12.58

5

7

Tap water #5

0.3460 ± 0.02350

13.59

4

0.2966 ± 0.01731

11.67

4

0.8130 ± 0.05953

16.37

5

8

Tap water #6

0.1462 ± 0.02364

32.35

4

0.06563 ± 0.007883

24.02

4

0.7900 ± 0.05788

16.38

5

PyroSmart NextGen‑rateNo.

sample1

EU/mL

PYROSTAR Neo

RSD (%)EU/mL

RSD (%)

50820 ± 2981

10.16

3

74920 ± 5946

17.75

5

26.20

3

10.47 ± 1.774

37.89

5

O113:H10:K negative2

Tap water #1

14.31 ± 2.165

3

Tap water #2

0.1491 ± 0.008495

9.867

3

0.04996 ± 0.002622

11.74

5

4

Deionized water #1

3.929 ± 0.09098

4.011

3

3.198 ± 0.2781

19.45

5

5

Tap water #3

2.846 ± 0.3591

21.86

3

1.289 ± 0.1465

25.42

5

6

Tap water #4

2.772 ± 0.1125

7.026

3

1.626 ± 0.2486

34.19

5

7

Tap water #5

0.7927 ± 0.01538

3.360

3

0.4279 ± 0.03375

17.64

5

8

Tap water #6

0.6314 ± 0.1053

28.87

3

0.3122 ± 0.02964

21.23

5

a

Values shown are mean ± standard error of results from participating independent institutions.
Relative standard deviation.
c
Number of institutions that carried out measurements.
b

In this study, we adopted the BET assay

reproducibility.
The reagent‑to‑reagent variability among thevariability of 50%‑200% as defined in thefour amoebocyte lysate reagents in detecting thePharmacopeias to investigate the comparability of

autochthonous endotoxin level in each water

endotoxin levels in samples detected bysample andculture supernatant was 1.26‑

recombinant protein reagents and by thefold at minimum（Endochrome‑K/Endospecy ES‑amoebocyte lysate reagents. Endotoxin levels

50M in tap water #3）and 1.84‑fold at maximum

detected by recombinant protein reagents falling（Kinetic‑QCL / PYROSTAR ES‑F in tap water

within the 50%‑200% range of the respective#6）. Among the four recombinant protein

lysate reagents were considered comparable forreagents, the reagent‑to‑reagent variability was

the present purpose.

2.38‑fold at minimum（EndoZyme II/PyroGene fordetected autochthonous endotoxin in all seventhe culture supernatant） and 12.04‑fold atwater samples and the culture supernatant sample

maximum （PyroSmart NextGen‑onset time /at comparable levels. PyroGeneTM and EndoZyme®

PyroGene for tap water #6）. Some recombinant

II detected autochthonous endotoxin in two

protein reagents exhibited lower detectability ofsamples, deionized water #1 and the culture

autochthonous endotoxin in water samples.

supernatant, at levels comparable to the lysate346

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PyroSmart NextGen ®

［Kikuchi,: Collaborative Study of BET Using rFC‑Based Procedure（Part 3）］

endotoxin in water between rFCs and rCRs is notclear. However, one possibility is that it might bedepend upon whether or not factor B is includedin the recombinant protein reagents. Factor B isreported to participate in the recognition ofendotoxin as well as in signal transduction within
the cascade 20）. The slight differences in endotoxin
d e t e c t i o n w i t h P y r o S m a r t N e x t G e n® a n d
PYROSTARTM Neo, which are both rCRs, might beascribed to differences of other formulation
components in those reagents. Nevertheless, thedetectability of autochthonous endotoxin in theculture supernatant of

O113: H10: K

negative and most purified LPS from several typesof bacteria in the previous two‑year study 17, 18） byrecombinant protein reagents was comparable tothat with the four amoebocyte lysate reagents.
The same tendency, including low measured
potency of LPS such as

LPS, isalso reported by another group

7）. These findings

may suggest that autochthonous endotoxin in tap
and deionized water is less able to associate withor activate factor C, compared to standard
endotoxin. Further studies may be necessary to
Fig. 2 Comparison of Amoebocyte Lysate andRecombinant Protein Reagents for Detectionof Autochthonous Endotoxin
Culture supernatant of
O113:H10: K negative
was measured with various amoebocyte lysate（■）andrecombinant protein reagents（□）. The relative endotoxin
activities（EU/mL）against RSE were calculated to compare thespecificity of each reagent. Values are the mean ± standard
error shown in Tables 1 and 2.

elucidate why the relative detectability ofendotoxin by recombinant protein reagents and byamoebocyte lysate reagents varies depending on
the origin of the endotoxin.
When selecting a commercially available
recombinant protein reagent to be used for testing
endotoxin, it is recommended that end‑users
consider aspects of product quality anddevelopment, in addition to the performance of

Neo detectedrFC 12） or rCR 16）. Most amoebocyte lysate reagents

autochthonous endotoxin in six samples （tapare currently licensed by the FDA, meaning thatwater #1, 3, 4, 5 and 6, deionized water #1）andthe development and product processes comply

culture supernatant at comparable levels. This

with regulatory guidelines and current Goodtendency of rFCs to detect lower levels of

Manufacturing Practice （cGMP） and/or

autochthonous endotoxin in tap and deionizedInternational Organization for Standardizationwater than those measured with lysate reagents（ISO）standards. Similar quality controls for thereagents levels.

PYROSTARTM

was observed in our first study in 2017previously described

7）

.

17）

and wasdevelopment, manufacturing, and master cell

The reason for thebanking of recombinant protein reagents aredifference in detectability of autochthonous

critical from an end‑user perspective.

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［Kikuchi,: Collaborative Study of BET Using rFC‑Based Procedure（Part 3）］

Fig. 3 Comparison of Results of Amoebocyte Lysate and Recombinant Protein Reagents for
Endotoxin Panel Testing
Water samples containing endotoxin, collected from independent institutions, were tested with various
amoebocyte lysate（■）and recombinant protein reagents（□）. The relative endotoxin activities（EU/mL）
against RSE were calculated to compare the specificity of each reagent. Values are the mean ± standard error
shown in Tables 1 and 2.

348

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［Kikuchi,: Collaborative Study of BET Using rFC‑Based Procedure（Part 3）］

Fig. 3 Continued

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［Kikuchi,: Collaborative Study of BET Using rFC‑Based Procedure（Part 3）］

Table 3 Equality of Variacne of Endotoxin Level by Amoebocyte Lysate andRecombinant Protein Reagents
No.

sample12

O113:H10:K negative

Tap water #1

valueacritical valueb

valuecdistributiond

16.55

15.10

0.04396 not equal variance

7.509

15.10

0.1294 equal valiance0.1243 equal valiance3

Tap water #2

7.742

15.10

4

Deionized water #1

3.030

15.10

0.3893 equal valiance5

Tap water #3

13.67

15.10

0.05746 equal valiance6

Tap water #4

4.988

15.10

0.2178 equal valiance7

Tap water #5

1.982

15.10

0.6008 equal valiance8

Tap water #6

2.342

15.10

0.5104 equal valianceavalue was calculated from the ratio of variance from the two groups
critical value is the value where the area is 0.05 when the degree of freedom is (4, 3) in a two‑sided test in the
distribution table.
cvalue is the probability that the null hypothesis of equal variances holds.
d
The difference between amoebocyte lysate and recombinant protein reagent groups was considered significant
when <0.05 in a two‑sided test.
b

2016, 8.0, p.194‑198.

A further study to compare the ability ofamoebocyte lysate and recombinant protein
reagents to detect endotoxin fromis in4）United States Pharmacopeia. <85> Bacterial endotoxins
test. 2019, 42, p.6434‑6438.
5）Ding, J.L.; Chai, C.; Pui, A.W.M.; Ho, B. Expression of fulllength and deletion homologues ofprogress.

Factor C in:

immunoreactivity and endotoxin binding

Acknowledgments. 1997, 4, p.33‑43.

This study was supported by a Research Grant on Testing
Methods of the Japanese Pharmacopoeia from the6）Loverock, B.; Simon, B.; Burgenson, A.; Baines, A. A

Pharmaceutical and Medical Device Regulatory Science

recombinant factor C procedure for the detection ofSociety of Japan.

gram‑negative bacterial endotoxin.
. 2010, 36, p.321‑329.
7）Stevens, I.; Ogura, N.; Kelley, M.; DʼOrdine, R. L.;

Conflicts of Interest
O.M., M.S. and S.S. are employees of Hyglos/bioMérieux

Mizumura, H.; Oda, T.; Akiyoshi, J.; Jahngen, E. G.

Japan, Ltd.; K.O. is an employee of J.K. International, Inc.;

Advanced Recombinant Cascade Reagent PyroSmart

T.K., H.F. and M.K. are employees of FUJIFILM Wako

NextGen® for Bacterial Endotoxins Test as Described

Pure Chemical Corporation; T.M. is an employee of Lonza;

in the Pharmacopeias.

2022, 5, p.105‑114.

T.O., H.M. and N.O. are employees of Seikagaku

8）U.S. Department of Health and Human Services, U.S.

Corporation; D.I., H.S. and Y.T. are employees of Veritas

Food and Drug Administration. Guidance for industry‑

Corporation; and M.T. is an employee of Charles River. The

pyrogen and endotoxins testing: questions andother authors declare that they have no conflict of interest.

answers. 2012. http://www.fda.gov/drugs/guidanceco
mplianceregulatoryinformation/guidances/ucm314718.
htm.

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351