ment can be a rious source of bias. In general, the rejection of measurements solely on the basis of their relative magnitudes
is a procedure that should be ud sparingly.
Each suspected potency measurement, or outlier, may be tested against the following criterion. This criterion is bad on the variation within a single group of suppodly equivalent measurements from a normal distribution. On average, it will reject a valid obrvation once in 25 trials or once in 50 trials. Designate the measurements in order of magnitude from y
1
to y
N
, where
y
1
is the candidate outlier, and N is the number of measurements in the group. Compute the relative gap by using Table A2-1, Test for Outlier Measurements, and the formulas below:
When N = 3 to 7:
G
1
= (y
2
− y
1
)/(y
N
− y
1
)
When N = 8 to 10:
G
2
= (y
2
− y
1
)
/(y
N − 1
− y
1
)
When N = 11 to 13:
G
3
= (y
3
− y
1
)/(y
N −1
− y
1
)
If G
1
, G
2
, or G
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3
, as appropriate, exceeds the critical value in Table A2-1, Test for Outlier Measurements, for the obrved N, there is a statistical basis for omitting the outlier measurement(s).
Table A2-1. Test for Outlier Measurements
In samples from a normal population, gaps equal to or larger than the following values of G1, G2, and G3 occur with a probability P = 0.01, when outlier measurements can occur only at one end; or with P = 0.02, when they may occur at either end.
wps标尺
大一学期总结N34567
G10.9870.8890.7810.6980.637
N8910
G20.6810.6340.597
N111213
G30.6740.6430.617
EXAMPLE
Estimated potencies of sample in log scale = 1.561, 1.444, 1.517, 1.535.
Check lowest potency for outlier:
G
1
= (1.517 − 1.444)/(1.561 − 1.444) = 0.624<0.889
Therefore 1.444 is not an outlier.
Check highest potency for outlier:
G
1
= (1.561 − 1.535)/(1.561 − 1.444) = 0.222<0.889
Therefore 1.561 is not an outlier.
Outlier potencies should be marked as outlier values and excluded from the assay calculations. NMT one potency can be excluded as an outlier.
á85ñ BACTERIAL ENDOTOXINS TEST
♦Portions of this general chapter have been harmonized with the corresponding texts of the European Pharmacopoeia and/or the Japane Pharmacopoeia. Tho portions that are not harmon
ized are marked with symbols (♦
acted♦
) to specify this fact.
♦
The Bacterial Endotoxins Test (BET) is a test to detect or quantify endotoxins from Gram-negative bacteria using amoebo-cyte lysate from the horshoe crab (Limulus polyphemus or Tachypleus tridentatus).
There are three techniques for this test: the gel-clot technique, which is bad on gel formation; the turbidimetric technique, bad on the development of turbidity after cleavage of an endogenous substrate; and the chromogenic technique, bad on the development of color after cleavage of a synthetic peptide-chromogen complex. Proceed by any of the three techniques for the test. In the event of doubt or dispute, the final decision is made bad upon the gel-clot limit test unless otherwi
indicated in the monograph for the product being tested. The test is carried out in a manner that avoi
ds endotoxin contami-nation.
APPARATUS
Depyrogenate all glassware and other heat-stable materials in a hot air oven using a validated process.♦1
♦ A commonly ud minimum time and temperature is 30 min at 250°. If employing plastic apparatus, such as microplates and pipet tips for auto-matic pipetters, u apparatus that is shown to be free of detectable endotoxin and does not interfere in the test. [NOTE —In this chapter, the term “tube” includes any other receptacle such as a microtiter well.]
REAGENTS AND TEST SOLUTIONS
Amoebocyte Lysate
A lyophilized product obtained from the lysate of amoebocytes (white blood cells) from the horshoe crab (Limulus polyphe-mus or Tachypleus tridentatus ). This reagent refers only to a product manufactured in accordance with the regulations of the competent authority. [NOTE —Amoebocyte Lysate reacts to some b -glucans in addition to endotoxins. Amoebocyte Lysate prepa-
rations that do not react to glucans are available: they are prepared by removing the G factor reacting to glucans from Amoe-bocyte Lysate or by inhibiting the G factor reacting system of Amoebocyte Lysate and may be ud for endotoxin testing in the prence of glucans.]
Water for Bacterial Endotoxins Test (BET)
U Water for Injection or water produced by other procedures that shows no reaction with the lysate employed, at the de-
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tection limit of the reagent.
Lysate TS
Dissolve Amoebocyte Lysate in Water for BET , or in a buffer recommended by the lysate manufacturer, by gentle stirring.Store the reconstituted lysate, refrigerated or frozen, according to the specifications of the manufacturer.
PREPARATION OF SOLUTIONS
Standard Endotoxin Stock Solution
A Standard Endotoxin Stock Solution is prepared from a USP Endotoxin Reference Standard that has been calibrated to the current WHO International Standard for Endotoxin. Follow the specifications in the package leaflet and on the label for prepa-ration and storage of the Standard Endotoxin Stock Solution . Endotoxin is expresd in Endotoxin Units (EU). [N OTE —One USP Endotoxin Unit (EU) is equal to one International Unit (IU) of endotoxin.]
Standard Endotoxin Solutions
After mixing the Standard Endotoxin Stock Solution vigorously, prepare appropriate rial dilutions o
f Standard Endotoxin Solu-tion , using Water for BET . U dilutions as soon as possible to avoid loss of activity by adsorption.
Sample Solutions
Prepare the Sample Solutions by dissolving or diluting drugs using Water for BET . Some substances or preparations may be more appropriately dissolved, or diluted in other aqueous solutions. If necessary, adjust the pH of the solution to be examined (or dilution thereof) so that the pH of the mixture of the lysate and Sample Solution falls within the pH range specified by the lysate manufacturer, usually 6.0–8.0. The pH may be adjusted by u of an acid, ba, or suitable buffer as recommended by the lysate manufacturer. Acids and bas may be prepared from concentrates or solids with Water for BET in containers free of detectable endotoxin. Buffers must be validated to be free of detectable endotoxin and interfering factors.
♦1 For a validity test of the procedure for inactivating endotoxins, e Dry-Heat Sterilization under Sterilization and Sterility Assurance of Compendial Articles á1211ñ.U Lysate TS having a nsitivity of not less than 0.15 Endotoxin Unit per mL.
♦
DETERMINATION OF MAXIMUM VALID DILUTION (MVD)
The maximum valid dilution is the maximum allowable dilution of a specimen at which the endotoxin limit can be deter-mined. Determine the MVD from the following equation:
MVD = (endotoxin limit × concentration of Sample Solution)/(l)
Endotoxin Limit
The endotoxin limit for parenteral drugs, defined on the basis of do, equals K/M♦2
♦
, where K is a threshold pyrogenic do of endotoxin per kg of body weight, and M is equal to the maximum recommended bolus do of product per kg of body weight. When the product is to be injected at frequent intervals or infud continuously, M is the maximum total do admin-istered in a single hour period. The endotoxin limit for parenteral drugs is specified in the individual monograph in units such as EU/mL, EU/mg, EU/Unit of biological activity, etc.
Concentration of Sample Solution
mg/mL: in the ca of endotoxin limit specified by weight (EU/mg);
Units/mL: in the ca of endotoxin limit specified by unit of biological activity (EU/Unit);
mL/mL: when the endotoxin limit is specified by volume (EU/mL).
l: the labeled nsitivity in the Gel-Clot Technique (EU/mL) or the lowest concentration ud in the standard curve for the Turbidimetric Technique or Chromogenic Technique.
GEL-CLOT TECHNIQUE
The gel-clot technique is ud for detecting or quantifying endotoxins bad on clotting of the lysate reagent in the pres-ence of endotoxin. The minimum concentration of endotoxin required to cau the lysate to clot under standard conditions is the labeled nsitivity of the lysate reagent. To ensure both the precision and validity of the test, perform the tests for confirm-ing the labeled lysate nsitivity and for interfering factors as described in Preparatory Testing, immediately below.
Preparatory Testing
TEST FOR CONFIRMATION OF LABELED LYSATE SENSITIVITY
Confirm in four replicates the labeled nsitivity, l, expresd in EU/mL of the lysate prior to u in the test. The test for confirmation of lysate nsitivity is to be carried out when a new batch of lysate is ud or when there is any change in the test conditions that may affect the outcome of the test. Prepare standard solutions having at least four concentrations equivalent to 2l, l, 0.5l, and 0.25l by diluting the USP Endotoxin RS with Water for BET.
Mix a volume of the Lysate TS with an equal volume (such as 0.1-mL aliquots) of one of the Standard Endotoxin Solutions in each test tube. When single test vials or ampuls containing lyophilized lysate are ud, add solutions directly to the vial or am-pul. Incubate the reaction mixture for a constant period according to the directions of the lysate manufacturer (usually at
37±1° for 60±2 min), avoiding vibration. To test the integrity of the gel, take each tube in turn directly from the incubator, and invert it through about 180° in one smooth motion. If a firm gel has formed that remains in place upon inversion, record the result as positive. A result is negative if an intact gel is not formed. The test is considered valid when the lowest concentra-tion of the standard solutions shows a negative result in all replicate tests.
The endpoint is the smallest concentration in the ries of decreasing concentrations of standard endotoxin that clots the lysate. Determine the geometric mean endpoint by calculating the mean of the logarithms of the endpoint concentrations of the four replicate ries and then taking the antilogarithm of the mean value, as indicated in the following formula:
geometric mean endpoint concentration = antilog (S e/f)
where S e is the sum of the log endpoint concentrations of the dilution ries ud, and f is the number of replicate test tubes. The geometric mean endpoint concentration is the measured nsitivity of the lysate (in EU/mL). If this is not less than 0.5l and not more than 2l, the labeled nsitivity is confirmed and is ud in tests performed with this lysate.
♦2K is 5 USP-EU/kg of body weight for any route of administration other than intrathecal (for which K is 0.2 USP-EU/kg of body weight). For radiopharmaceutical products not administered intrathecally, the endotoxin limit is calculated as 175 EU/V, where V is the maximum recommended do in mL. For intrathecally ad-ministered radiopharmaceuticals, the endotoxin limit is obtained by the formula 14 EU/V. For formulations (usually anticancer products) administered on a per square meter of body surface, the formula is K/M, where K = 100 EU/m2 and M is the maximum do/m2.
震撼的意思是什么♦
TEST FOR INTERFERING FACTORS
Usually prepare solutions (A–D) as shown in Table 1, and perform the inhibition/enhancement test on the Sample Solutions at a dilution less than the MVD, not containing any detectable endotoxins, operating as described for Test for Confirmation of Labeled Lysate Sensitivity . The geometric mean endpoint concentrations of Solutions B and C are determined using the formula described in the Test for Confirmation of Labeled Lysate Sensitivity . The test for interfering factors must be repeated when any condition changes that is likely to influence the result of the test.Table 1. Preparation of Solutions for the Inhibition/Enhancement Test for Gel-Clot Techniques
Solution Endotoxin Concentration/Solution to Which Endotoxin Is Added Diluent Dilution Factor Endotoxin Concentration Number
of
Replicates A a None/Sample Solution ———4B b 2l /Sample Solution Sample Solution 12l 4 21l 4 40.5l 4 80.25l 4C c 2l /Water for BET Water for BET 12l 2 21l 2
40.5l 2
80.25l 2
D d None/Water for BET ———2a Solution A : A Sample Solution of the preparation under test that is free of detectable endotoxins.
b
Solution B : Test for interference.c Solution C : Control for labeled lysate nsitivity.
d
Solution D : Negative control of Water for BET
.
The test is considered valid when all replicates of Solutions A and D show no reaction and the result of Solution C confirms
the labeled nsitivity.
If the nsitivity of the lysate determined in the prence of Solution B is not less than 0.5l and not greater than 2l , the
Sample Solution does not contain factors that interfere under the experimental conditions ud. Otherwi, the Sample Solution to be examined interferes with the test.
If the sample under test does not comply with the test at a dilution less than the MVD, repeat the test using a greater dilu-tion, not exceeding the MVD. The u of a more nsitive lysate permits a greater dilution of the sample to be examined, and this may contribute to the elimination of interference.
Interference may be overcome by suitable treatment such as filtration, neutralization, dialysis, or heating. To establish that the chon treatment effectively eliminates interference without loss of endotoxins, perform the assay described above using the preparation to be examined to which Standard Endotoxin has been added and which has then been submitted to the chon treatment.
Limit Test
PROCEDURE
Prepare Solutions A, B, C, and D as shown in Table 2, and perform the test on the solutions following the procedure above for Preparatory Testing , Test for Confirmation of Labeled Lysate Sensitivity .
Table 2. Preparation of Solutions for the Gel-Clot Limit Test
Solution *Endotoxin Concentration/
Solution to Which
Endotoxin Is Added Number of Replicates
A None/Diluted Sample Solution 2
B 2l /Diluted Sample Solution 2
C 2l /Water for BET 2
D None/Water for BET 2
* Prepare Solution A and the positive product control Solution B using a dilution not greater than the
MVD and treatments as described for the Test for Interfering Factors in Preparatory Testing . The positive control Solutions B and C contain the Standard Endotoxin Solution at a concentration corresponding to twice the la-beled lysate nsitivity. The negative control Solution D consists of Water for BET.
INTERPRETATION
The test is considered valid when both replicates of Solutions B and C are positive and tho of Solution D are negative. When a negative result is found for both replicates of Solution A, the preparation under test complies with the test. When a positive result is found for both replicates of Solution A, the preparation under test does not comply with the test.
When a positive result is found for one replicate of Solution A and a negative result is found for the other, repeat the test. In the repeat test, the preparation under test complies with the test if a negative result is found for both replicates of Solution A. The preparation does not comply with the test if a positive result is found for one or both replicates of Solution A. However, if the preparation do
es not comply with the test at a dilution less than the MVD, the test may be repeated using a greater dilu-tion, not exceeding the MVD.
Quantitative Test
PROCEDURE
The test quantifies bacterial endotoxins in Sample Solutions by titration to an endpoint. Prepare Solutions A, B, C, and D as shown in Table 3, and test the solutions by following the procedure in Preparatory Testing, Test for Confirmation of Labeled Lysate Sensitivity.
Table 3. Preparation of Solutions for the Gel-Clot Assay
Solution
Endotoxin Concentration/春天歌
Solution to Which Endotoxin
Is Added Diluent
Dilution
Factor
Endotoxin
Concentration
Number
of
Replicates
A a None/Sample Solution Water for BET1—2
2—2
4—2
8—2
B b2l/Sample Solution—1
2l2
C c2l/Water for BET Water for BET12l2
21l2
40.5l2
80.25l2
D d None/Water for BET———2
a Solution A: Sample Solution under test at the dilution, not to exceed the MVD, with which the Test for Interfering Factors was completed. Subquent dilution of the Sample Solution must not exceed the MVD. U Water for BET to make a dilution ries of four tubes containing the Sample Solution under test at concentra-tions of 1, 1/2, 1/4, and 1/8 relative to the concentration ud in the Test for Interfering Factors. Other dilutions up to the MVD may be ud as appropriate.
b Solution B: Solution A containing standard endotoxin at a concentration of 2l (positive product control).
c Solution C: Two replicates of four tubes of Water for BET containing the standar
d endotoxin at concentrations of 2l, l, 0.5l, and 0.25l, respectively.
d Solution D: Water for BET (negativ
e control).
CALCULATION AND INTERPRETATION
The test is considered valid when the following three conditions are met: (1) Both replicates of negati
ve control Solution D are negative; (2) Both replicates of positive product control Solution B are positive; and (3) The geometric mean endpoint con-centration of Solution C is in the range of 0.5l to 2l.
To determine the endotoxin concentration of Solution A, calculate the endpoint concentration for each replicate by multiply-ing each endpoint dilution factor by l. The endotoxin concentration in the Sample Solution is the endpoint concentration of the replicates. If the test is conducted with a diluted Sample Solution, calculate the concentration of endotoxin in the original Sample Solution by multiplying by the dilution factor. If none of the dilutions of the Sample Solution is positive in a valid assay, report the endotoxin concentration as less than l (if the diluted sample was tested, report as less than l times the lowest dilu-tion factor of the sample). If all dilutions are positive, the endotoxin concentration is reported as equal to or greater than the greatest dilution factor multiplied by l (e.g., initial dilution factor times eight times l in Table 3).农村小额信贷
The preparation under test meets the requirements of the test if the concentration of endotoxin in both replicates is less than that specified in the individual monograph.
