PRINCIPLE
The bacterial endotoxin test or LAL test is the most sensitive and specific means available to detect and measure bacterial endotoxins, a fever producing by-product of gram negative bacteria commonly known as Pyrogen. The gram negative bacteria causes blood coagulation in Americal Horse Shoe Crab (Limulus Polyphemus) and clotting phenomenon was an enzyme-mediated response to endotoxin by components in the intercellular fluid of the circulating cells, the amebocyte. This phenomenon is exploited for estimating the concentration of bacterial endotoxins that may be present in or on the sample. The rate of reaction depends on the concentration of endotoxin, the pH and the temperature. The reaction requires the presence of certain bivalent cations, a proclotting enzyme system and clottable protein; these are provided by the lysate.
As per BP
Before carrying out the test for endotoxins on the preparation being examined, it is necessary to verify.
1. that the equivalent used does not adsorb endotoxins,
2. the sensitivity of the lysate and
3. the absence of interfering factors.
Carry out the test in a manner that avoids microbial contamination. If necessary, treat equipment to eliminate Endotoxins.
Reagents
Limulus Amoebocyte Lysate
A lysate of amebocytes from the horse shoe crab, Limulus Polyphemus. Reconstitute the lysate as stated on the label. For each batch, confirm the stated sensitivity as prescribed under sensitivity of the lysate. The sensitivity of the lysate is defined as the lowest concentration of
Endotoxin that yields a firm gel in the test conditions and is expressed in Units per milliliter.
Water BET
Water that gives a negative result in the conditions prescribed in the test for bacterial Endotoxins on the preparation being examined. It may be prepared by distilling water three times in an apparatus fitted with an effective device to prevent the entrainment of droplets or by other means that give water of the requisite quality.
0.1M Hydrochloric Acid BET
0.1 M Hydrochloric acid that has been prepared using water BET. After adjustment to pH 6.5 to 7.5 with 0.1 M Sodium Hydroxide BET it gives a negative result in the conditions of the test.
0.1 M Sodium Hydroxide BET
0.1 M Sodium Hydroxide that has been prepared using water BET. After adjustment to pH 6.5 to 7.5 with 0.1 M Hydrochloric acid BET it gives a negative result in the conditions of the test.
Standard Endotoxin Preparation
The standard preparation is the 1st International Standard for Endotoxin , established in 1986, consisting of freeze-dried Endotoxin from Escherichia coli 0113:”H10:K negative with trehalose (supplied in ampoules containing 14000 units), or another suitable preparation the activity of which has been determined in relation to the Internaltional Standard using a gelatin method.
Procedure
Unless otherwise prescribed, prepare the solutions an dilution’s used in the test using Water BET
1. If necessary, adjust the solution being examined to pH 6.5 to 7.5 using 0.1 M Hydrochloric acid BET, 0.1 M Sodium Hydroxide BET or a suitable buffer.
2. Add a volume of the lysate appropriate to the chosen receptacle (for example a slide or a tube) to each of the requisite number of such receptacles maintained at 36 - 38 OC .
3. At intervals that will permit the examination of each receptacle and the recording of each result, add to each receptacle an equal volume of the solution being examined and immediately mix gently with the lysate.
4. Incubate, the reaction mixture without vibration and avoiding loss of water by evaporation, for a constant period that has been found suitable in the chosen experimental conditions (usually 20 to 60 min.,), examine the receptacle and record the result.
5. A positive results is indicated by the formation of a firm gel that does not disintegrate when the receptacle is gently inverted.
6. A result is negative is such gel is not formed.
Sensitivity of the Lysate
1. Prepare not fewer than four replicate series each of not fewer than three dilution’s of the Standard Preparations such that at least the final dilution in each series gives a negative result.
2. Examine the dilution’s and a negative control solution consisting of water BET, as described under procedure.
3. Calculate the average of the logarithms of the lowest concentration of Endotoxin in each series of dilutions for which a positive result is found.
4. The antilogarithm of this average gives the estimated lysate sensitivity.
5. The estimated lysate sensitivity is confirmed if it does not differ by more than a factor of 2 from the stated sensitivity.
6. The estimated lysate sensitivity is the used in all tests performed using this lysate.
Interfering Factors
1. Operate as prescribed under sensitivity of the lysate but to prepare the dilutions of the Standard Preparation use the preparation being examined at the maximum valid dilution calculated from the expression :
The bacterial endotoxin test or LAL test is the most sensitive and specific means available to detect and measure bacterial endotoxins, a fever producing by-product of gram negative bacteria commonly known as Pyrogen. The gram negative bacteria causes blood coagulation in Americal Horse Shoe Crab (Limulus Polyphemus) and clotting phenomenon was an enzyme-mediated response to endotoxin by components in the intercellular fluid of the circulating cells, the amebocyte. This phenomenon is exploited for estimating the concentration of bacterial endotoxins that may be present in or on the sample. The rate of reaction depends on the concentration of endotoxin, the pH and the temperature. The reaction requires the presence of certain bivalent cations, a proclotting enzyme system and clottable protein; these are provided by the lysate.
As per BP
Before carrying out the test for endotoxins on the preparation being examined, it is necessary to verify.
1. that the equivalent used does not adsorb endotoxins,
2. the sensitivity of the lysate and
3. the absence of interfering factors.
Carry out the test in a manner that avoids microbial contamination. If necessary, treat equipment to eliminate Endotoxins.
Reagents
Limulus Amoebocyte Lysate
A lysate of amebocytes from the horse shoe crab, Limulus Polyphemus. Reconstitute the lysate as stated on the label. For each batch, confirm the stated sensitivity as prescribed under sensitivity of the lysate. The sensitivity of the lysate is defined as the lowest concentration of
Endotoxin that yields a firm gel in the test conditions and is expressed in Units per milliliter.
Water BET
Water that gives a negative result in the conditions prescribed in the test for bacterial Endotoxins on the preparation being examined. It may be prepared by distilling water three times in an apparatus fitted with an effective device to prevent the entrainment of droplets or by other means that give water of the requisite quality.
0.1M Hydrochloric Acid BET
0.1 M Hydrochloric acid that has been prepared using water BET. After adjustment to pH 6.5 to 7.5 with 0.1 M Sodium Hydroxide BET it gives a negative result in the conditions of the test.
0.1 M Sodium Hydroxide BET
0.1 M Sodium Hydroxide that has been prepared using water BET. After adjustment to pH 6.5 to 7.5 with 0.1 M Hydrochloric acid BET it gives a negative result in the conditions of the test.
Standard Endotoxin Preparation
The standard preparation is the 1st International Standard for Endotoxin , established in 1986, consisting of freeze-dried Endotoxin from Escherichia coli 0113:”H10:K negative with trehalose (supplied in ampoules containing 14000 units), or another suitable preparation the activity of which has been determined in relation to the Internaltional Standard using a gelatin method.
Procedure
Unless otherwise prescribed, prepare the solutions an dilution’s used in the test using Water BET
1. If necessary, adjust the solution being examined to pH 6.5 to 7.5 using 0.1 M Hydrochloric acid BET, 0.1 M Sodium Hydroxide BET or a suitable buffer.
2. Add a volume of the lysate appropriate to the chosen receptacle (for example a slide or a tube) to each of the requisite number of such receptacles maintained at 36 - 38 OC .
3. At intervals that will permit the examination of each receptacle and the recording of each result, add to each receptacle an equal volume of the solution being examined and immediately mix gently with the lysate.
4. Incubate, the reaction mixture without vibration and avoiding loss of water by evaporation, for a constant period that has been found suitable in the chosen experimental conditions (usually 20 to 60 min.,), examine the receptacle and record the result.
5. A positive results is indicated by the formation of a firm gel that does not disintegrate when the receptacle is gently inverted.
6. A result is negative is such gel is not formed.
Sensitivity of the Lysate
1. Prepare not fewer than four replicate series each of not fewer than three dilution’s of the Standard Preparations such that at least the final dilution in each series gives a negative result.
2. Examine the dilution’s and a negative control solution consisting of water BET, as described under procedure.
3. Calculate the average of the logarithms of the lowest concentration of Endotoxin in each series of dilutions for which a positive result is found.
4. The antilogarithm of this average gives the estimated lysate sensitivity.
5. The estimated lysate sensitivity is confirmed if it does not differ by more than a factor of 2 from the stated sensitivity.
6. The estimated lysate sensitivity is the used in all tests performed using this lysate.
Interfering Factors
1. Operate as prescribed under sensitivity of the lysate but to prepare the dilutions of the Standard Preparation use the preparation being examined at the maximum valid dilution calculated from the expression :
= maximum allowable endotoxin concentration/ sensitivity of lysate
2. If the sensitivity of the lysate determined in the presence of the preparation being examined does not differ by more than a factor of 2 from that determined in the absence of the preparation being examined, the latter does not contain factors that interfere in the experimental conditions and it may be examined without further treatment.
3. If the sensitivity of the lysate determined in the presence of the preparation being examined differs by more than a factor of 2 from that determined in the absence of the preparation being examined, the preparation being examined acts as an inhibitor or an activator.
4. The interfering factors must be eliminated by suitable treatment such as dilution, filtration, neutralization, dialysis or addition of substances that displace absorbed Endotoxins.
5. The use of a more sensitive lysate permits the use of a greater dilution of the preparation being examined and this may contribute to the elimination of interference.
6. Ultrafiltration may be used when the interfering factor passes through a filter with a nominal separation limit corresponding to a molecular weight of 10,000 to 20,000 daltons.
7. Assymmetrical membrane filters of cellulose triacetate of polysulphone have been found to be suitable .
8. The material retained on the filter, which contains the Endotoxins, is rinsed with water BET or a suitable buffer and endotoxins are recovered in water BET or a suitable buffer.
9. The test volume and the final volume used to recover the endotoxins are determined for each preparation being examined.
10. Establish that the chosen treatment effectively eliminates interference without removing Endotoxins by repeating the test for interfering factors using the preparation being examined to which the Standard Preparation has been added and which has then been submitted to the chosen treatment.
Test for Bacterial Endotoxins in the Preparation.
1. Carry out the method described under procedure in duplicate using the maximum valid dilution of the preparation being examined which has been treated if necessary to eliminate interfering factors.
2. Examine at the same time a negative control consisting of water BET and two positive controls each of which contains the standard preparation at a concentration corresponding to twice the stated sensitivity of the lysate and one of which contains the preparation being examined (treated if necessary to eliminate interfering factors after the addition of the standard preparation) at the concentration being used in the test.
3. The test is not valid unless the negative and both positive controls give the appropriate results.
INTERPRETATION
1. The preparation being examined complies with the test if a negative result is found for both test mixtures.
2. The preparation being examined does not comply with the test if a positive result is found for both test mixtures.
3. If a positive result is found for one test mixture and a negative result for the other, repeat the test; the preparation being examined complies with the test if a negative result is found for both test mixtures.
As per USP
Reference Standard and Control Standard Endotoxins
1. The reference standard endotoxin (RSE) is the USP Endotoxin reference standard, which has defined potency of 10,000 USP Endotoxin Units (EU) per vial.
2. Constitute the entire contents of vial of the RSE with 5 mL of LAL Reagent Water, mix intermittently for 30 min. using a vortex mixer and use this concentrate for making appropriate serial dilutions.
3. Preserve the concentrate in a refrigerator, for making subsequent dilutions, for not more than 14 days.
4. Mix vigorously, using a vortex mixer for not less than 3 min. before use.
5. Mix each solution for not less than 30 sec. before proceeding to make the test solution.
6. Do not store dilutions, because of loss of activity by absorption, in the absence of supporting data to the contrary.
7. A control standard endotoxin (CSE) is an endotoxin preparation other than the RSE that has been standardized against the RSE.
8. Standardize each new lot of CSE prior to use in the test calibration of a CSE in terms of RSE must be with the specific lot of LAL reagent and the test procedure with which it is to be used.
9. Standardization of a CSE against the RSE using an LAL reagent for the gel-clot procedure may be effected by assaying a minimum of 1 vial of the CSE and 1 vial of the RSE, as directed under Test procedure, but using 4 replicate reaction tubes at each level of the dilution series of the RSE and 4 replicate reaction tubes similarly for each vial or aliquot of the CSE.
10. The antilog of the difference between the mean log10 endpoint of the RSE and the mean log10 endpoint of the CSE is the standardized potency of the CSE, which is to be converted to and expressed in Endotoxin Units per mg under stated drying conditions for the CSE, or in Endotoxin units per container, whichever is appropriate.
11. A suitable CSE has a potency of not less than 2 Endotoxin units per mg and not more than 50 Endotoxin Units per mg.
Preparatory Testing
1. Use LAL reagent of confirmed label sensitivity.
2. Treat any containers or utensils employed so as to destroy extraneous surface endotoxins that may be present, such as by heating in an oven at 250 OC or above for sufficient time.
3. The validity of test results for bacterial endotoxins requires an adequate demonstration that specimens of the article or of solutions washings, or extracts thereof to which the test is to be applied do not of themselves inhibit or enhance the reaction or otherwise interfere with the test.
4. Validation is accomplished by performing the inhibition or Enhancement Test as described below.
5. Appropriate negative controls are included.
6. Validation must be repeated if the LAL reagent source or the method of manufacture or formulation of the article is changed.
Test for Confirmation of Labeled LAL Reagent Sensitivity
1. Confirm the labeled sensitivity using at least one vial of the LAL reagent lot.
2. Prepare a series of two fold dilutions of the RSE (CSE) to give concentrations of 2l , l , 0.5 l, and 0.25 l, where l is the labeled sensitivity of the LAL reagent in Endotoxin Units per mL.
3. Perform the test on the four standard concentrations in quadruplicate and include negative controls.
4. The geometric mean endpoint concentration (see Calculation and Interpretation) must be greater than or equal to 0.5 l and less than or equal to 2.0 l.
5. Confirm the labeled sensitivity of each new lot of LAL reagent prior to use in the test.
Inhibition or Enhancement Test
1. Perform the test on aliquots of the specimen, or a dilution not to exceed the Maximum Valid Dilution, in which there is no detectable endotoxin.
2. Perform the test on the specimen without added endotoxin and with endotoxin added to give final concentration of 2 l, l , 0.5 l, 0.25 l.
3. Perform the test as directed under Test Procedure, but using not less than replicate tubes for the untreated specimen to which Endotoxin has been added.
4. In parallel with the above, test in duplicate the same endotoxin concentrations in water and untreated negative controls.
5. Calculate the geometric mean endpoint endotoxin concentration for the specimen as described under Calculation and Interpretation.
6. The test is valid for the article if the geometric mean endpoint concentration in the specimen is greater than or equal to 0.5 l and less than or equal to 2.0 l.
7. If the result obtained for the specimens to which endotoxin has been added is outside the specified limit, the article is unsuitable for the Bacterial Endotoxins Test.
8. Repeat the test for inhibition or enhancement after neutralization, inactivation, or removal of the interfering substances or after the specimen has been diluted by a factor not exceeding the Maximum Valid Dilution.
9. Use a dilution, not exceed the Maximum Valid Dilution sufficient to overcome the inhibition or enhancement of the known added endotoxin, for subsequent assays of Endotoxin in test specimens.
10. If endogeneous Endotoxin is detectable in the untreated specimens under the conditions of the test, the article is unsuitable of the Inhibition or Enhancement Test, or, it may be rendered suitable by removing the Endotoxin present by ultra-filtration, or by appropriate dilution.
11. Dilute the untreated specimen (as constituted, where applicable for administration or use), to a level not exceeding the maximum valid dilution, at which no endotoxin is detectable.
12. Repeat the test for Inhibition or Enhancement using the specimens at those dilution’s.
MAXIMUM VALID DILUTION (MVD)
1. The Maximum Valid Dilution is appropriate to Injections or to solutions for parental administration in the form constituted diluted for administration, or where applicable, to the amount of drug by weight if the volume of the dosage form for administration could be varied.
2. Where the endotoxin limit concentration s specified in the individual monograph in terms of volume (in EU per mL), divide the limit by l, which is the labeled sensitivity (in EU per mL) if the lysate employed in the assay, obtain the MVD factor.
3. Where the endotoxin limit concentration is specified in the individual monograph in terms of weight or Units of active drug (in EU per mg or in EU per Unit), multiply the limit by the concentration in mg per mL or in Units per mL of the drug in the solution tested or of the drug constituted according to the label instructions, whichever is applicable, divide the product of the multiplication by l, to obtain the MVD factor.
4. The MVD factor so obtained is the dilution limit for the preparation for the test to be valid.
Test Procedure
1. In preparing for and applying the test, observe precautions handling the specimens in order to avoid gross microbial contamination.
2. To quantify the amount of endotoxin in a specimen, an assay is performed on decreasing concentrations of specimens prepared by serial dilution.
3. Select dilution’s so that they correspond to a geometric series in which each step is greater than the next by a constant ratio.
4. Include negative and positive controls and a positive product control.
5. Use not less than 2 replicate reaction tubes at each level of the dilution series for each specimen under test.
6. A standard endotoxin dilution series involving not less than 2 replicate reaction tubes is conducted in parallel.
7. A set of standard endotoxin dilution series is included for each block of tubes, which may consist of a number of a racks for incubation together, provided environmental conditions within blocks are uniform.
8. Minimum Valid Concentration (MVC) : The lowest drug specific concentration that will permit detection of endotoxin contamination within the endotoxin limit.
9. Positive Product Control, (PPC) : An aliquot of test sample spiked with a known amount of endotoxin (2l). This control serves as the inhibition control for get clot assay.
10. Negative Product Control (NPC) : It is the actual test where in 0.1 mL of diluted sample is added to 0.1 mL of lysate.
Calculating the Endotoxin Limit :
Endotoxin Limit = K/M ,
3. If the sensitivity of the lysate determined in the presence of the preparation being examined differs by more than a factor of 2 from that determined in the absence of the preparation being examined, the preparation being examined acts as an inhibitor or an activator.
4. The interfering factors must be eliminated by suitable treatment such as dilution, filtration, neutralization, dialysis or addition of substances that displace absorbed Endotoxins.
5. The use of a more sensitive lysate permits the use of a greater dilution of the preparation being examined and this may contribute to the elimination of interference.
6. Ultrafiltration may be used when the interfering factor passes through a filter with a nominal separation limit corresponding to a molecular weight of 10,000 to 20,000 daltons.
7. Assymmetrical membrane filters of cellulose triacetate of polysulphone have been found to be suitable .
8. The material retained on the filter, which contains the Endotoxins, is rinsed with water BET or a suitable buffer and endotoxins are recovered in water BET or a suitable buffer.
9. The test volume and the final volume used to recover the endotoxins are determined for each preparation being examined.
10. Establish that the chosen treatment effectively eliminates interference without removing Endotoxins by repeating the test for interfering factors using the preparation being examined to which the Standard Preparation has been added and which has then been submitted to the chosen treatment.
Test for Bacterial Endotoxins in the Preparation.
1. Carry out the method described under procedure in duplicate using the maximum valid dilution of the preparation being examined which has been treated if necessary to eliminate interfering factors.
2. Examine at the same time a negative control consisting of water BET and two positive controls each of which contains the standard preparation at a concentration corresponding to twice the stated sensitivity of the lysate and one of which contains the preparation being examined (treated if necessary to eliminate interfering factors after the addition of the standard preparation) at the concentration being used in the test.
3. The test is not valid unless the negative and both positive controls give the appropriate results.
INTERPRETATION
1. The preparation being examined complies with the test if a negative result is found for both test mixtures.
2. The preparation being examined does not comply with the test if a positive result is found for both test mixtures.
3. If a positive result is found for one test mixture and a negative result for the other, repeat the test; the preparation being examined complies with the test if a negative result is found for both test mixtures.
As per USP
Reference Standard and Control Standard Endotoxins
1. The reference standard endotoxin (RSE) is the USP Endotoxin reference standard, which has defined potency of 10,000 USP Endotoxin Units (EU) per vial.
2. Constitute the entire contents of vial of the RSE with 5 mL of LAL Reagent Water, mix intermittently for 30 min. using a vortex mixer and use this concentrate for making appropriate serial dilutions.
3. Preserve the concentrate in a refrigerator, for making subsequent dilutions, for not more than 14 days.
4. Mix vigorously, using a vortex mixer for not less than 3 min. before use.
5. Mix each solution for not less than 30 sec. before proceeding to make the test solution.
6. Do not store dilutions, because of loss of activity by absorption, in the absence of supporting data to the contrary.
7. A control standard endotoxin (CSE) is an endotoxin preparation other than the RSE that has been standardized against the RSE.
8. Standardize each new lot of CSE prior to use in the test calibration of a CSE in terms of RSE must be with the specific lot of LAL reagent and the test procedure with which it is to be used.
9. Standardization of a CSE against the RSE using an LAL reagent for the gel-clot procedure may be effected by assaying a minimum of 1 vial of the CSE and 1 vial of the RSE, as directed under Test procedure, but using 4 replicate reaction tubes at each level of the dilution series of the RSE and 4 replicate reaction tubes similarly for each vial or aliquot of the CSE.
10. The antilog of the difference between the mean log10 endpoint of the RSE and the mean log10 endpoint of the CSE is the standardized potency of the CSE, which is to be converted to and expressed in Endotoxin Units per mg under stated drying conditions for the CSE, or in Endotoxin units per container, whichever is appropriate.
11. A suitable CSE has a potency of not less than 2 Endotoxin units per mg and not more than 50 Endotoxin Units per mg.
Preparatory Testing
1. Use LAL reagent of confirmed label sensitivity.
2. Treat any containers or utensils employed so as to destroy extraneous surface endotoxins that may be present, such as by heating in an oven at 250 OC or above for sufficient time.
3. The validity of test results for bacterial endotoxins requires an adequate demonstration that specimens of the article or of solutions washings, or extracts thereof to which the test is to be applied do not of themselves inhibit or enhance the reaction or otherwise interfere with the test.
4. Validation is accomplished by performing the inhibition or Enhancement Test as described below.
5. Appropriate negative controls are included.
6. Validation must be repeated if the LAL reagent source or the method of manufacture or formulation of the article is changed.
Test for Confirmation of Labeled LAL Reagent Sensitivity
1. Confirm the labeled sensitivity using at least one vial of the LAL reagent lot.
2. Prepare a series of two fold dilutions of the RSE (CSE) to give concentrations of 2l , l , 0.5 l, and 0.25 l, where l is the labeled sensitivity of the LAL reagent in Endotoxin Units per mL.
3. Perform the test on the four standard concentrations in quadruplicate and include negative controls.
4. The geometric mean endpoint concentration (see Calculation and Interpretation) must be greater than or equal to 0.5 l and less than or equal to 2.0 l.
5. Confirm the labeled sensitivity of each new lot of LAL reagent prior to use in the test.
Inhibition or Enhancement Test
1. Perform the test on aliquots of the specimen, or a dilution not to exceed the Maximum Valid Dilution, in which there is no detectable endotoxin.
2. Perform the test on the specimen without added endotoxin and with endotoxin added to give final concentration of 2 l, l , 0.5 l, 0.25 l.
3. Perform the test as directed under Test Procedure, but using not less than replicate tubes for the untreated specimen to which Endotoxin has been added.
4. In parallel with the above, test in duplicate the same endotoxin concentrations in water and untreated negative controls.
5. Calculate the geometric mean endpoint endotoxin concentration for the specimen as described under Calculation and Interpretation.
6. The test is valid for the article if the geometric mean endpoint concentration in the specimen is greater than or equal to 0.5 l and less than or equal to 2.0 l.
7. If the result obtained for the specimens to which endotoxin has been added is outside the specified limit, the article is unsuitable for the Bacterial Endotoxins Test.
8. Repeat the test for inhibition or enhancement after neutralization, inactivation, or removal of the interfering substances or after the specimen has been diluted by a factor not exceeding the Maximum Valid Dilution.
9. Use a dilution, not exceed the Maximum Valid Dilution sufficient to overcome the inhibition or enhancement of the known added endotoxin, for subsequent assays of Endotoxin in test specimens.
10. If endogeneous Endotoxin is detectable in the untreated specimens under the conditions of the test, the article is unsuitable of the Inhibition or Enhancement Test, or, it may be rendered suitable by removing the Endotoxin present by ultra-filtration, or by appropriate dilution.
11. Dilute the untreated specimen (as constituted, where applicable for administration or use), to a level not exceeding the maximum valid dilution, at which no endotoxin is detectable.
12. Repeat the test for Inhibition or Enhancement using the specimens at those dilution’s.
MAXIMUM VALID DILUTION (MVD)
1. The Maximum Valid Dilution is appropriate to Injections or to solutions for parental administration in the form constituted diluted for administration, or where applicable, to the amount of drug by weight if the volume of the dosage form for administration could be varied.
2. Where the endotoxin limit concentration s specified in the individual monograph in terms of volume (in EU per mL), divide the limit by l, which is the labeled sensitivity (in EU per mL) if the lysate employed in the assay, obtain the MVD factor.
3. Where the endotoxin limit concentration is specified in the individual monograph in terms of weight or Units of active drug (in EU per mg or in EU per Unit), multiply the limit by the concentration in mg per mL or in Units per mL of the drug in the solution tested or of the drug constituted according to the label instructions, whichever is applicable, divide the product of the multiplication by l, to obtain the MVD factor.
4. The MVD factor so obtained is the dilution limit for the preparation for the test to be valid.
Test Procedure
1. In preparing for and applying the test, observe precautions handling the specimens in order to avoid gross microbial contamination.
2. To quantify the amount of endotoxin in a specimen, an assay is performed on decreasing concentrations of specimens prepared by serial dilution.
3. Select dilution’s so that they correspond to a geometric series in which each step is greater than the next by a constant ratio.
4. Include negative and positive controls and a positive product control.
5. Use not less than 2 replicate reaction tubes at each level of the dilution series for each specimen under test.
6. A standard endotoxin dilution series involving not less than 2 replicate reaction tubes is conducted in parallel.
7. A set of standard endotoxin dilution series is included for each block of tubes, which may consist of a number of a racks for incubation together, provided environmental conditions within blocks are uniform.
8. Minimum Valid Concentration (MVC) : The lowest drug specific concentration that will permit detection of endotoxin contamination within the endotoxin limit.
9. Positive Product Control, (PPC) : An aliquot of test sample spiked with a known amount of endotoxin (2l). This control serves as the inhibition control for get clot assay.
10. Negative Product Control (NPC) : It is the actual test where in 0.1 mL of diluted sample is added to 0.1 mL of lysate.
Calculating the Endotoxin Limit :
Endotoxin Limit = K/M ,
where K = 0.5 EU/Kg/hr. for all parentals
M = Max. dose of product/Kg/hr.
Calculating MVD & MVC :
Lambda = Lysate Label Claim Sensitivity
Potency of the product = Concentration of the product in Units / mL
MVC = Lambda x M (dosage)/ K (constant)
Lambda = Lysate Label Claim Sensitivity
Potency of the product = Concentration of the product in Units / mL
MVC = Lambda x M (dosage)/ K (constant)
OR
MVC = Lambda / Endotoxin Limit
Eg. Fluconozole MVC = 0.125 EU/mL x 200 mg/60 Kg/ 5.0 EU/Kg == 0.083 mg/mL
MVD = (potency of product) x Endotoxin Limit / Lambda
or
MVD = Potency/ MVC
Eg. Flucanozole MVD = [2 mg/mL] / [0.83 mg/mL] = 24
I.e., the final formulation of Zolstan infusion can be diluted 24 (1 + 23) times for endotoxin test, when lysate of 0.125 EU/mL sensitivity is used.
Preparation And Storage Of Reagents
1. Since the form and amount per container of standard endotoxin and of LAL reagent may vary, constitution and / or dilution of contents should be as directed in the labeling.
For Lysate of 0.125 EU/mL sensitivity : If CSE contains 100 EU/vial, which can reconstituted with 5 mL of LRW, each 1 mL will have 20 EU. To prepare 4 lambda solution, mix 1.95 mL of LRW and 0.05 mL of reconstituted CSE. Dilute this four times (1+3) to get a solution of lambda strength (0.125 EU/mL).
2. The pH of the test mixture of the specimen and the LAL reagent is in the range 6.0 to 8.0 unless specifically directed otherwise in the individual monograph.
3. The pH may be adjusted by the addition of endotoxin - free Sodium Hydroxide or Hydrochloric acid or suitable buffers to the specimen prior to testing.
4. Collect LAL powder into the bottle of the vial. Rehydrate with LAL by gentle mixing. Do not vortex. Discord the vial if the vacuum is absent or if color or opacity is present. Lyophilized LAL reagent is stored at 2 - 8 OC . Rehydrated LAL can be frozen (below -20 OC ) and used upto four weeks after reconstitution. LAL may be frozen and thawed only once. While in use LAL should be kept at 2 - 8 OC.
Procedure For Testing The Samples
1. Preparation of control curve / positive & water controls :
Prepare a control standard endotoxin series to get 2 l, 0.5 l , and 0.25 l as per the directions provided in the Certificate of Analysis for the specified lot of Lysate. A control series must be run with first set of tests each day. Otherwise test only a 2l endotoxin level, in there is no change in test parameters. Prepare a series of endotoxin control in the following way in 10 x 75 mm test tube
I.e., the final formulation of Zolstan infusion can be diluted 24 (1 + 23) times for endotoxin test, when lysate of 0.125 EU/mL sensitivity is used.
Preparation And Storage Of Reagents
1. Since the form and amount per container of standard endotoxin and of LAL reagent may vary, constitution and / or dilution of contents should be as directed in the labeling.
For Lysate of 0.125 EU/mL sensitivity : If CSE contains 100 EU/vial, which can reconstituted with 5 mL of LRW, each 1 mL will have 20 EU. To prepare 4 lambda solution, mix 1.95 mL of LRW and 0.05 mL of reconstituted CSE. Dilute this four times (1+3) to get a solution of lambda strength (0.125 EU/mL).
2. The pH of the test mixture of the specimen and the LAL reagent is in the range 6.0 to 8.0 unless specifically directed otherwise in the individual monograph.
3. The pH may be adjusted by the addition of endotoxin - free Sodium Hydroxide or Hydrochloric acid or suitable buffers to the specimen prior to testing.
4. Collect LAL powder into the bottle of the vial. Rehydrate with LAL by gentle mixing. Do not vortex. Discord the vial if the vacuum is absent or if color or opacity is present. Lyophilized LAL reagent is stored at 2 - 8 OC . Rehydrated LAL can be frozen (below -20 OC ) and used upto four weeks after reconstitution. LAL may be frozen and thawed only once. While in use LAL should be kept at 2 - 8 OC.
Procedure For Testing The Samples
1. Preparation of control curve / positive & water controls :
Prepare a control standard endotoxin series to get 2 l, 0.5 l , and 0.25 l as per the directions provided in the Certificate of Analysis for the specified lot of Lysate. A control series must be run with first set of tests each day. Otherwise test only a 2l endotoxin level, in there is no change in test parameters. Prepare a series of endotoxin control in the following way in 10 x 75 mm test tube
2. Preparation of test solution and controls :
i. Asepticaly withdraw sample solution. Calculate MVD for the sample and dilute to get a dilution which is equal to half MVD. Vortex for 15 seconds. Label as ‘STOCK’.
ii. Negative Product Control : Take 50 µl of half MVD solution in 10 x 75 mm test tube.
iii. Positive Product Control : Take 50 µl of half MVD solution in 10 x 75 mm test tube and add 50 µl of 4 l CSE solution to give a concentration of 2 l.
iv. Negative Water Control : Take 100 µl of LAL reagent water in 10 x 75 mm test tube (blank, NWC).
Incubation
Immediately prior to testing, rehydrate or thaw out the LAL frozen under validated conditions for storage and rehydration.
Inoculate all the test specimens and controls, beginning with the Negative Control and ending with Positive Control (with the greatest endotoxin concentration), with 100 µl of LAL reagent and mix. Promptly incubate the reaction tubes at 37 OC for 60 ±2 minutes. Observe and record the results.
Interpretation Of Results :
1. A positive reaction is characterized by the formation of a firm gel that remains when inverted through 180 OC.
2. Record such a result as positive (+).
3. A negative result is characterized by the absence of such a gel or by the formation of a viscous gel that does not maintain its integrity.
4. Record such a result as negative ( - ) .
5. Handle the tubes with care, and avoid subjecting them to unwanted vibrations, or false negative observation may result.
6. A valid test yields :
I. A positive gel clot in the positive product control and in 2 l and l CSE control.
ii. A negative gel (no gel) reaction in the negative controls of product (NPC) and water (NWC).
iii. A negative gel is half l and in 0.25 l (one forth lambda conc.,)
iv. the following results are also accepted
Test Tube No. Result
1,2 - -
3,4 + +
5,6 + +
7,8 + -
9,10 - -
v. A negative gel with the negative product control, means that there is less than the USP limit of endotoxin.
vi. If the PPC does not gel, then there is product inhibition. In this case product must be diluted and the test should be repeated.
vii. If the Ppc gels and the product and pyrogen free water do not, then the product is considered to have less than the endotoxin than the labeled Lysate sensitivity.
7. The test is invalid if the positive product control is negative or the endotoxin standard does not show the endpoint concentration to be within ±1 two-fold dilutions from the label claim sensitivity of the LAL reagent or if any negative control is positive.
8. Proceed to Endotoxin content Calculation to determine the amount of endotoxin present in the test specimen.
VALIDATION OF LAL TEST / Geometric Mean Calculation
Prepare serial two fold idltuions of endotoxin bracketing lysate sensitivity using the 1 EU/mL dilution and pyrogen free water. After each dilution vortex tube for one full minute.
Label test tubes in a quadruplicate and add 0.1 mL of each dilution and a PFW negative contro Add 0.1 mL of LAL to each tube in incubator block and incubate at 37 OC for one hour and observe for the clot formation. Calculate the Geometric Mean End Point to determine lysate sensitivity. Acceptable variation is one half to two times the labeled lysate sensitivity.
Eg. Table for Assay Results of GEL CLOT method.
Each end point value is converted to Log10 . The individual log10 values are averaged and lysate sensitivity is taken as the antilog10 of this average log value.
Eg. In the above case the mean Log10 end point is -0.996
Antilog10 means = 0.10 EU/mL
if the label claim of endotoxin is 0.125 EU/mL the results met the validation test.
INTERPRETATION
The article meets the requirements of the test if the concentration of endotoxin is not more than that specified in the individual monograph.
NOTE: Use the method as per the Pharmacopoeial status (grade) of the material. In case of In-house specifications, follow the method as per IP or as specified .
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