2.6.32. Test for bacterial endotoxins using recombinant factor C EUROPEAN PHARMACOPOEIA 11.

The product complies with the test if colonies of the types 3. PREPARATION OF THE STANDARD ENDOTOXIN
described are not present or if the identification tests are STOCK SOLUTION
negative. The standard endotoxin stock solution is prepared from
The following section is given for information. an endotoxin reference standard that has been calibrated
against the International Standard, for example endotoxin
RECOMMENDED SOLUTIONS AND CULTURE MEDIA
standard BRP.
The solutions and culture media mentioned in this chapter
and described in general chapter 2.6.13 and the following Endotoxin is expressed in International Units (IU). The
buffered peptone medium have been found to be satisfactory equivalence in IU of the International Standard is stated by
for the purposes for which they are prescribed in this chapter. the World Health Organization.
Other media may be used provided that their suitability can NOTE : 1 International Unit (IU) of endotoxin is equal to
be demonstrated. 1 Endotoxin Unit (EU).
Buffered peptone medium Follow the specifications in the package leaflet and on the
Potassium dihydrogen phosphate 1.5 g label for preparation and storage of the standard endotoxin
stock solution.
Disodium hydrogen phosphate dodecahydrate 9.0 g
4. PREPARATION OF THE STANDARD ENDOTOXIN
Sodium chloride 5.0 g
SOLUTIONS
Peptone 10.0 g After vigorously mixing the standard endotoxin stock solution,
Purified water 1000 mL prepare appropriate serial dilutions of this solution using
water for BET.
Adjust the pH so that after sterilisation it is 7.0 ± 0.2 at 25 °C. Use the solutions as soon as possible to avoid loss of activity
Sterilise in an autoclave using a validated cycle. by adsorption.
5. PREPARATION OF THE TEST SOLUTIONS
Prepare the test solutions by dissolving or diluting active
01/2021:20632 substances or medicinal products using water for BET.
corrected 11.0 Some substances or preparations may be more appropriately
dissolved or diluted in other aqueous solutions. If necessary,
adjust the pH of the test solution (or dilution thereof) so that
the pH of the mixture of the reagent(s) and test solution falls
within the pH range specified by the test kit manufacturer,
usually 6.0 to 8.0. The pH may be adjusted by the use of
2.6.32. TEST FOR BACTERIAL acid, base or a suitable buffer, as recommended by the test
ENDOTOXINS USING RECOMBINANT kit manufacturer. Acids and bases may be prepared from
concentrates or solids with water for BET in containers free of
FACTOR C detectable endotoxin. Buffers must be validated to be free of
detectable endotoxin and interfering factors.
The test for bacterial endotoxins using recombinant
factor C (rFC) is carried out to quantify endotoxins from 6. DETERMINATION OF THE MAXIMUM VALID
gram-negative bacteria. It is performed using rFC based on DILUTION
the gene sequence of the horseshoe crab (Limulus polyphemus, The maximum valid dilution (MVD) is the maximum
Tachypleus tridentatus, Tachypleus gigas or Carcinoscorpius allowable dilution of a sample at which the endotoxin limit
rotundicauda), using a fluorimetric method. can be determined. Determine the MVD using the following
The test is carried out in a manner that avoids bacterial formula :
endotoxin contamination. endotoxin limit ´ concentration of test solution
1. EQUIPMENT λ
Endotoxin limit : the endotoxin limit for active substances
Depyrogenate all glassware and other heat-stable equipment
administered parenterally, defined on the basis of dose, is
in a dry-heat oven using a validated process. A commonly
equal to :
used minimum time and temperature is 30 min at 250 °C.
Where plastic equipment (such as microplates and pipette tips K
for automatic pipettes) is employed, it must be shown to be M
free of detectable endotoxin and not to interfere with the test.
K = threshold pyrogenic dose of endotoxin per kilogram
2. REAGENTS of body mass ;
Reagents M = maximum recommended bolus dose of product per
Recombinant factor C is based on the gene sequence of the kilogram of body mass.
horseshoe crab (Limulus polyphemus, Tachypleus tridentatus, When the product is to be injected at frequent intervals
Tachypleus gigas or Carcinoscorpius rotundicauda). All or infused continuously, M is the maximum total dose
reagents, including the fluorogenic substrate and assay buffer, administered in a single hour period.
must be free of detectable endotoxin. The endotoxin limit for active substances administered pa-
Reagent solutions renterally is specified in units such as IU/mL, IU/mg, IU/Unit
If necessary, prepare the reagents according to the test kit of biological activity, etc., in monographs.
manufacturer’s instructions. Store the reagents, refrigerated or Concentration of test solution :
frozen, as indicated by the manufacturer. – mg/mL if the endotoxin limit is specified by mass (IU/mg);
Water for BET (water for bacterial endotoxins test) – Units/mL if the endotoxin limit is specified by unit of
Water for injections R or water produced by other procedures biological activity (IU/Unit);
that shows no reaction with the reagent employed at the – mL/mL if the endotoxin limit is specified by volume
detection limit of the reagent. (IU/mL).

252 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 11.0 2.6.32. Test for bacterial endotoxins using recombinant factor C

λ = the lowest concentration used in the standard curve. Table 2.6.32.-1

Solution Endotoxin Solution to which Number of
concentration endotoxin is replicates
added
7. FLUORIMETRIC QUANTITATIVE TECHNIQUE A None Test solution Not less than 2
B Middle Test solution Not less than 2
This technique is used to measure the fluorescence (relative concentration
fluorescence units ; RFU) emitted by a fluorescent substrate of the standard
(reagent) after cleavage by endotoxin-activated factor C. It is curve
used as an end-point-fluorescent test. C At least 3 Water for BET Each concentra-
concentra- tion not less than 2
tions (lowest
The end-point-fluorescent test is based on the quantitative concentration
relationship between the endotoxin concentration and is designated λ)
the fluorescence of the reagent mixture at the end of the D None Water for BET Not less than 2
incubation period, expressed for example as ΔRFU :
Solution A = test solution, which may be diluted but not exceeding
the MVD.
ΔRFU = RFUt end-point - RFUt 0 Solution B (positive product control) = preparation to be examined
at the same dilution as solution A, containing added endotoxin at a
concentration equal to or near the middle of the standard curve
RFUtend-point = fluorescence of the reagent mixture at the Solution C = standard endotoxin solution at the concentrations used
end of the incubation period ; in the validation of the method as described in section 8-1.
RFUt0 = fluorescence of the reagent mixture at the Solution D (negative control) = water for BET.
start of the incubation period. The test is considered valid when the following conditions
are met :
The test is carried out at the incubation temperature – the absolute value of the correlation coefficient of the
recommended by the test kit manufacturer (usually 37 ± 1 °C). standard curve generated using solution C is greater than
or equal to 0.980 ;
– the result with solution D does not exceed the limit of
8. PREPARATORY TESTING the blank value required in the description of the reagent
mixture employed, or it is less than the endotoxin detection
Preparatory tests are conducted to ensure that the fluorometric limit of the rFC employed.
technique is valid. These tests demonstrate that the criteria for
the standard curve are satisfied (8-1) and that the test solution Calculate the mean recovery of the added endotoxin by
does not interfere with the test (8-2). subtracting the mean endotoxin concentration in the solution
(if any) (solution A, Table 2.6.32.-1) from that in the solution
Validation of the test method is required when any changes containing the added endotoxin (solution B, Table 2.6.32.-1).
are made to the experimental conditions that are likely to The test solution is considered free of interfering factors if,
influence the result of the test. under the conditions of the test, the measured concentration
8-1. ASSURANCE OF CRITERIA FOR THE STANDARD of the endotoxin added to the test solution is within 50-200 per
CURVE cent of the known added endotoxin concentration, after
subtraction of any endotoxin detected in the solution without
The test must be carried out for each lot of recombinant added endotoxin.
factor C reagent.
When the endotoxin recovery is outside the specified range,
Instrument sensitivity must be adjusted in accordance with the test solution is considered to contain interfering factors.
the recommendations of the test kit manufacturer. Repeat the test using a greater dilution, not exceeding the
MVD. Furthermore, interference of the test solution or diluted
Using the standard endotoxin solution, prepare at least 3 test solution (not exceeding the MVD) may be eliminated by
endotoxin concentrations within the range indicated by the suitable validated treatment, such as filtration, neutralisation,
test kit manufacturer to generate the standard curve. If the dialysis, heat treatment or endotoxin-specific binding steps
desired range exceeds the range indicated by the manufacturer (enrichment of endotoxin from the test solution prior to
by more than 2 log10, additional standards must be included to detection in the absence of the interfering matrix). To establish
bracket each log increase in the range. Perform the test using that the treatment chosen effectively eliminates interference
at least 3 replicates of each standard endotoxin solution as without loss of endotoxins, repeat the test for interfering
recommended by the manufacturer (volume ratios, incubation factors using the preparation being examined to which the
time, temperature, pH, etc.). standard endotoxin has been added and which has then been
submitted to the chosen treatment.
The absolute value of the correlation coefficient, | r |, must
be greater than or equal to 0.980, for the range of endotoxin 9. TEST
concentrations prepared. 9-1. PROCEDURE
8-2. INTERFERING FACTORS Follow the procedure described in section 8-2.
As factor G is absent from the test kit, false-positive results 9-2. CALCULATION
due to β-glucan activation are not expected to occur. This Calculate the endotoxin concentration of each replicate of
must be taken into account when the method is compared to solution A using the standard curve generated by the standard
other bacterial endotoxin quantification methods. endotoxin solution C.
The test is considered valid when the following 3 requirements
Select an endotoxin concentration at or near the middle of are met :
the endotoxin standard curve.
(1) the results obtained with solution C comply with the
Prepare solutions A, B, C and D as shown in Table 2.6.32.-1. requirements for validation defined in section 8-1 ;
Perform the test on at least 2 replicates of these solutions as (2) the endotoxin recovery, calculated from the endotoxin
recommended by the test kit manufacturer (volume of test concentration found in solution B after subtracting the
solution and reagent test kit mixture, volume ratio of test endotoxin concentration found in solution A, is within the
solution to reagent test kit mixture, incubation time, etc.). range of 50-200 per cent ;

General Notices (1) apply to all monographs and other texts 253
2.6.33. Residual pertussis toxin EUROPEAN PHARMACOPOEIA 11.0

(3) the result obtained with solution D (negative control) L-Histidine hydrochloride 45.8 mg
does not exceed the limit of the blank value required in the monohydrate
description of the reagent mixture employed, or it is less than
the endotoxin detection limit of the rFC employed. L-Isoleucine 7.88 mg

9-3. INTERPRETATION L-Leucine 26.2 mg

The preparation being examined complies with the test if the
mean endotoxin concentration of the replicates of solution A, L-Lysine hydrochloride 73.0 mg
after correction for dilution and concentration, is less than the
L-Methionine 8.96 mg
endotoxin limit for the product.
Guidelines on the test for bacterial endotoxins are given in L-Phenylalanine 9.92 mg
general chapter 5.1.10.
L-Proline 69.0 mg

L-Serine 21.0 mg
01/2020:20633
L-Threonine 23.0 mg

L-Tryptophan 4.1 mg

L-Tyrosine disodium salt 13.5 mg

dihydrate
2.6.33. RESIDUAL PERTUSSIS TOXIN
L-Valine 23.0 mg
The test for residual pertussis toxin is performed in vitro,
using a Chinese Hamster Ovary (CHO) cell-based assay, and Vitamins
is intended for the assay of non-adsorbed purified pertussis
components. Biotin 70 μg
Pertussis toxin BRP is suitable as a reference pertussis toxin D-Calcium pantothenate 0.5 mg
preparation.
The CHO cell-clustering assay (CHO assay) is based on the Choline chloride 14.0 mg
induction of clusters in non-confluent CHO cell cultures by
active pertussis toxin. The cultures are then examined under a Folic acid 1.3 mg
microscope and any clusters present are counted. The assay 18.0 mg
myo-Inositol
may be used as a quantitative test or as a limit test with the
sensitivity determined within each assay using dilutions of a Nicotinamide 37 μg
reference pertussis toxin preparation.
The sensitivity of the CHO assay to pertussis toxin is verified Pyridoxine hydrochloride 60 μg
with pertussis toxin BRP or a suitable reference preparation
Riboflavin 40 μg
calibrated in International Units using a suitable CHO assay.
The assay sensitivity is defined as the lowest concentration in Thiamine hydrochloride 0.3 mg
the reference preparation dilution series to produce a positive
response, i.e. at least 10 CHO cell clusters. A suitable assay Vitamin B12 1.4 mg
has a sensitivity of at least 5 mIU/mL.
Inorganic salts
The following method is given as an example.
Reagents and equipment Calcium chloride, anhydrous 0.1020 g
– Pertussis toxin BRP. Cupric sulfate pentahydrate 2 μg
– CHO-K1 cell line (ATCC No. CCL-61 or ECACC
No. 85051005). Disodium hydrogen phosphate, 0.1155 g
anhydrous
– Kaighn’s modified Ham’s F-12K medium.
Media that have a slightly different composition from that Ferric sulfate heptahydrate 0.8 mg
in Table 2.6.33.-1 may also be used. Proline is an essential
component of the medium. Adjust to pH 7.2 ± 0.2 if Magnesium chloride, anhydrous 49.7 mg
necessary.
Magnesium sulfate, anhydrous 0.1920 g
Table 2.6.33.-1. – Kaighn’s modified Ham’s F-12K medium
Potassium chloride 0.2850 g
Amino acids
Sodium bicarbonate 2.5000 g
L-Alanine 18.0 mg
Sodium chloride 7.5300 g
L-Arginine hydrochloride 0.4220 g
Zinc sulfate heptahydrate 0.144 mg
L-Asparagine monohydrate 30.0 mg
Other components
L-Aspartic acid 26.6 mg
D-Glucose 1.2600 g
L-Cysteine
hydrochloride 70.0 mg
monohydrate Hypoxanthine sodium salt 4.0 mg

L-Glutamic acid 29.0 mg Lipoic acid 0.21 mg

L-Glutamine 0.2920 g Phenol red 3.0 mg

Glycine 15.0 mg Putrescine dihydrochloride 0.32 mg

254 See the information section on general monographs (cover pages)