procedure for conducting the Bacterial Endotoxin Test of Purified Water and Operation of Cyclomixer

1.0       Purpose:

To lay down the procedure for conducting the Bacterial Endotoxin Test of Purified Water and Operation of Cyclomixer. 

2.0       Scope:

This procedure applies to testing of bacterial endotoxins for purified water by gel clot method and also applies to operation of cyclomixer.           

3.0       Responsibility:

3.1       The responsibility of performing the bacterial endotoxin test for purified water and operation of cyclomixer lies with Microbiologist/Analyst.

4.0       Definitions/Abbreviations:

4.1       Endotoxins: Endotoxins are lipopolysaccherides, which form an integral part of gram-negative bacteria.

4.2       LAL: Limulus Amebocyte Lysate. 

4.3       CSE: Control Standard Endotoxin         

4.4       LAL Water: Limulus Amebocyte Lysate Water

5.0       Procedure:

5.1       Reagents:

5.1.1    Control Standard Endotoxin (CSE)

5.1.2    LAL Reagent (Limulus Amebocyte Lysate)

5.1.3    LAL Reagent Water (LRW)

5.2       Calculation of Endotoxin Limit:

5.2.1    Endotoxin Limit:  K/M 

Where,

K = Threshold human pyrogenic dose of Endotoxin per Kg of body weight (5 EU/Kg)

M = Maximum human dose per Kg of body weight in a single hour period.

5.3       Calculation of Maximum valid dilution (MVD):

5.3.1    MVD is the maximum allowable dilution of a specimen at which the Endotoxin limit can be determined.

MVD = Endotoxin Limit x Concentration of sample / l          

Where, l = Labeled sensitivity of Lysate

5.4       Preparation of Standard Endotoxin Solution:          

Reconstitute the standard Endotoxin vial with (10 ng) with LAL reagent Water (as per manufacturer recommendation) and vortex on cyclomixer for 5 minutes (stock solution).

Prepare the 1.0 EU/ml solution from stock solution.

            1.0ml of solution (1.0 EU/ml)    +  1.0 ml of LRW      ---- (0.5 EU/ml)      ------  4l

            1.0 ml of solution (0.5 EU/ml)   +  1.0 ml of LRW      ---- (0.25 EU/ml)    ------  2l.

            1.0ml of solution (0.25 EU/ml)   +  1.0 ml of LRW      ---- (0.125 EU/ml)  ------  l

            1.0 ml of solution (0.125EU/ml) +  1.0 ml of LRW     ---- (0.06 EU/ml)    ------  l/2 

            1.0 ml of solution (0.06 EU/ml)  + 1.0 ml of LRW     ---- (0.03 EU/ml)    ------  l/4

            Reconstituted Standard Endotoxin stock solution can be stored as per manufacturer recommendations (shall be stored at 2° - 8° C for 28 days).

5.5       Preparation of Lysate solution:

5.5.1Reconstitute the lysate vial with LAL reagent water as per manufacturer recommendations (Example: with 1.2 ml of LAL reagent water) and swirl gently for 10Sec.

5.5.2The reconstituted lysate solution shall be stored as per manufacturer recommendations (Example: at 2 – 8 C for 24 hours and below -20 C for 28 days).

5.6       Test for confirmation of Labeled Sensitivity of Lysate:

5.6.1    Pipette out 100 μl of Standard Endotoxin Solutions of 2λ, λ, λ/2 and λ/4 into each test tube in quadruplicate.

5.6.2Add 100 μl of Lysate solution into each above standard solution and mix gently for 10sec.

5.6.3For Negative control, take 100 μl of LAL reagent water in a test tube and add 100 μl of Lysate solution.

5.6.4Incubate each test tube at 37° ± 1° C for a period of 60 min ± 2 minutes, avoiding vibration of tubes. Take each tube after completion of 60 min. ± 2 min. and observe the contents carefully.

5.6.5The positive result is characterized by formation of firm gel that remains its integrity when inverted through 180°.

5.6.6The negative result is characterized by the absence of intact gel.

5.6.7The end point is the last positive test results in the series of decreasing concentrations of standard endotoxin.

5.6.8Calculate the mean value of the logarithms of the end point concentration and then the antilogarithm of the mean value using the following formula.

            Geometric mean endpoint concentration = antilog (å e/f)

            Where, åe is the sum of the log end point concentrations of the dilution

            f is the number of replicate test tubes.

5.6.9The geometric mean endpoint concentration is the measured sensitivity of LAL reagent.

Acceptance criteria: It should be within 2 fold variation.

5.6.10The test for confirmation of Lysate sensitivity shall be carried out for one vial of new batch / Lot of LAL reagent used.

5.7Preparation of sample solution:

5.7.1Sampling of purified water for BET test should be done in depyrogenated test tubes. The sample pH should be 6.0 to 8.0, If required adjust pH of the sample by adding Tris buffers or endotoxin free equivalent buffers.

5.7.2Prepare the sample solution with LAL reagent water in 1: 1 dilution i.e, 50 l of sample and 50l of LAL reagent water.

5.8       Testing Procedure:

5.8.1Depyrogenated glassware shall be used in the test.

5.8.2Pipette out 50 l of LAL reagent water in a test tube and add 50 l of sample (MVD/2).

5.8.3For Negative control, pipette out 100 l of LAL regent water in a test tube.

5.8.4For Positive control, pipette out 50 l of LAL reagent water in a test tube and add 50l of standard solution of 4.

5.8.5For product positive control, pipette out 50 l of sample (MVD/2) and 50 l of standard solution of 4 in a test tube.

5.8.6Add 100 l of LAL reagent (Lysate) to all the above test tubes.

5.8.7Test each dilution in duplicate.

5.8.8Incubate each test tube at 37° ± 1° C for a period of 60 min ± 2 minutes, avoiding vibration of tubes. Take each tube after completion of 60 min. ± 2 min. and observe the contents carefully.

5.8.9The positive result is characterized by formation of firm gel that remains its integrity when inverted through 180°.

5.8.10The negative result is characterized by the absence of intact gel.

5.8.11Recording of Results:  Record the Positive result as ‘+’ and Negative result as ‘– ’.

5.9       Interpretation of results:

5.9.1The test is valid, if the positive controls are positive and Negative controls are negative.

5.9.2    The test is invalid, if the positive control and product positive controls are negative or negative controls are positive and endotoxin standard does not show the end point concentration with in the two fold dilution of the label claim sensitivity of Lysate.

5.9.3    Repeat the test, if a positive result is found for one of the test duplicates and negative for the other.

5.10     Testing Frequency: Frequency of samples from purified water storage tank and return loop shall be done only as and when need basis either as per customer requirement or as per the requirement of other manufacturing sites with in the group of Mylan.  

5.11     Operation of Cyclomixer:

5.11.1  Switch ‘ON’ the main.

5.11.2  Place and hold the test tube with the sample to be vortexed on the rubber cavity of the cyclomixer.

5.11.3  Adjust the speed of the mixer with the control knob.

5.11.4  After mixing, switch OFF the instrument.

5.11.5  Clean the instrument with sterile cloth after work is done.

6.0       Related Documents: Nil

Microbiology, conducting, bacterial endotoxin, purified water, cyclomixer