SOP for Bacterial endotoxin test by gel clot method
This Document describes (Standard Operating procedure) SOP for Bacterial endotoxin test by gel clot method
SOP for Bacterial endotoxin test by gel clot method
I. Purpose & Scope:
- The purpose of this SOP is to lay down the procedure for Bacterial endotoxin test by using Gel clot Method
- This Standard operating procedure is applicable for Bacterial endotoxin test of Raw material received from vendor and Finished product Manufactured.
II. Responsibilities:
- All Quality control personnel shall be responsible to follow and implement this SOP.
III) introduction and procedure:
Principle of Bacterial endotoxin test by gel clot method :
LAL Reagent Contains bivalent ions procloting enzyme system and clotable proteins, when reacts with endotoxin gives either gel formation, turbidity or precipitation. The formation of gel indicates the presence of endotoxin in the sample. The rate of reaction depends on the concentration of endotoxin gives more gel formation.
General Requirements for Bacterial endotoxin test by gel clot method:
- Depyrogenated pipette tips
- Depyrogenated dilution tubes and assay tubes
- Micropipettes
- Vortex mixer
- Dry Heating block
- LAL Reagent
- Controlled Standard Endotoxin (CSE)
- LAL Reagent Water (LRW)
Note: Perform the depyrogenation of assay tubes and dilution tubes at 250°C for One hour in Hot air oven.
Preparation of Reagents :
LAL Test kit contains LAL reagent, CSE and LRW. Procure this kit from recommended manufacturer ( Lonza, Charles river, ACC Cap code) along with the Certificate of Analysis. Store the Reagents as per instructions given on COA.
Reconstitution of Limulus Amoebocyte Lysate (LAL) Reagent :
- Remove aluminum seal of the Lysate vial without opening rubber stopper.
- Remove the stopper carefully, without touching the inner portion of stopper.
- Dissolve the lysate with LRW with the help of Depyrogenated pipette tips and swirl gently but thoroughly. (Do not shake & Vortex).
- Close the reconstituted vial with rubber stopper.
- Lyophilized (Un reconstituted) Limulus Amebocyte Lysate shall be stored under refrigeration at 2 to 8ºC.
- Store the reconstituted Lysate vial at Freezing condition (At -10ºC or colder) if it can used within 24 hours or according to the specification of the manufacturer.
Preparation of Control Standard Endotoxin (CSE) :
- A CSE is an endotoxin preparation other than the RSE that has been standardized against the RSE. Store the CSE at 2-8°C prior to and after reconstitution.
- Reconstitute the lyophilized CSE with LRW and vortex Reconstitute CSE for 5 to10 min or as per manufacturer’s instructions.
- Use this stock solution for making appropriate serial dilution.
- Use the reconstituted CSE vial up to 28 days or as per the Certificate of Analysis
Preparation of CSE dilutions :
- Reconstitute the CSE with LRW as per the volume recommended by the manufacturer.
- Mix vigorously the reconstituted CSE vial using a vortex mixer as per the manufacturer recommendation before making serial dilutions.
- Confirm the potency of the endotoxin ( EU/ng) from the certificate of analysis provided with each LAL kit and proceed for the dilution to get the required concentration.
For example:
If the potency of CSE is 10 EU/ng and total vial content is 10ng
= 10 EU X 10 = 100 EU/vial
If the volume recommended for reconstitution is 5 ml
= 100 / 5 = 20 EU/ml (CSE stock solution)
- Prepare standard solution having at least four concentration equivalent to 2l,l, 0.5l and 0.25l.
- Example for preparation of 0.125 EU/ml lysate sensitivity as mentioned below;
| Tube No. | CSE | LRW ( ml) | Final Concentration (EU/ml) |
| 1 | 0.1 ml of CSE stock solution | 1.9 | 1.0 |
| 2 | 0.5 ml from tube 1 | 0.5 | 0.5 (4λ) |
| 3 | 0.5 ml from tube 2 | 0.5 | 0.25(2λ) |
| 4 | 0.5 ml from tube 3 | 0.5 | 0.125 (λ) |
| 5 | 0.5 ml from tube 4 | 0.5 | 0.06 (0.5λ) |
Note: Vortex each dilution for not less than one minutes before proceeding to the next. Use dilution as soon as possible to avoid loss of activity by adsorption.
- Water for bacterial endotoxin test (LRW) :LRW is the endotoxin free water (<0.005 EU/ml) which is to be used for bacterial endotoxin test. Store the LRW between 15°C to 30°C.
Sensitivity of the Lysate:
- Confirmation of the lysate sensitivity must be carried out when a new batch of lysate is used or any change in the experimental condition which may affect the outcome of the test.
- Prepare CSE dilution from 4l solution of at least four concentration equivalent to 2λ, 1λ, 0.5λ, 0.25λ. by diluting a series of CSE solution with LRW.
- Take depyrogenated assay tubes and lable the tubes by numbering arrange quadruplicate in stand and proceed as per following table;
| Sample | CSE dilution used | LRW | Lysate (µl) | No.of replicates |
| 2λ | 100 µl of 2λ | – | 100 µl | 4 |
| 1 λ | 100 µl of 1 λ | – | 100 µl | 4 |
| 0.5λ | 100 µl of 0.5λ | – | 100 µl | 4 |
| 0.25λ | 100 µl of 0.25λ | – | 100 µl | 4 |
| NWC | – | 100 µl | 100 µl | 1 |
- Calculate the sensitivity of the lysate by finding out end point log for all sets.
- From these logs determine mean log and antilog of mean log is the sensitivity of the lysate.
- Lysate sensitivity = Antilog of Mean log;
Where, mean log = ∑e/f,
∑e = Sum of log end point concentration of the series of dilution used and
f = Number of replicate tubes.
- Sensitivity of the lysate should be in range of 0.5λ to 2λ. Where λ is label claim lysate sensitivity.
- If same lot of Lysate is received up to one year, no need to perform Lysate sensitivity of that Received Lot.
- Record the result in Annexure I.
Testing of sample :
- Make the entry of the Sample in Annexure V.
- Specified quantity of sample shall be weighed in depyrogenated vial/dilution tube using analytical balance. Prepare the sample solution by dissolving or diluting drug substance using LRW.
- If necessary adjust the pH of the solution to be examined in between 6.0 to 8.0.The pH may be adjusted by use of an acid, base or suitable buffer as recommended by manufacturer.
- For bacterial endotoxin test of raw material and finish product calculate the MVD (Maximum valid dilution ) using following formula;
M.V.D. = Endotoxin Limit (EU/mg) X Potency of product (mg/ml)
Lysate Sensitivity (EU/ml)
- Take out lysate and standard endotoxin from the refrigerator, allow it to attain room temperature before employing in the test.
- Take clean depyrogenated tubes from oven and allow it to attain room temperature.
- Put “ON” heating block, set and monitor for constant temperature of 37.0oC ± 1.0o
- Arrange sample, CSE, LAL reagent water and depyrogenated tubes into BET room of the microbiological testing area.
- Mark the depyrogenated tubes in duplicate as positive control (PC), product positive control (PPC), Test Sample and Negative water control (NWC).
- Prepare 1 EU/ml of CSE, by making appropriate dilution of standard endotoxin with LAL reagent water (Standard A). From this solution prepare the CSE upto 4λ.
- Prepare positive control (PC) by diluting 50 µl of 4l CSE in 50 µl of LAL reagent water will finally gives 2λconcentrations in the sample.
- Prepare product positive control (PPC) by mixing 50 µl of sample of appropriate test dilution with 50 µl of 4λ CSE which will finally gives 2lconcentrations in the sample.
- Prepare Test sample by taking appropriate test dilution of 100 µl sample.
- Prepare the Negative water control (NWC) by taking 100 µl of LRW.
- Add 100 µl Lysate in each of positive control, product positive control, Test sample, Negative water control test tubes with the help of depyrogenated pipette tips.
- Gently shake these tubes for uniform mixing and incubate in the heating block immediately for 60 ± 2 minutes at 37oC ± 1o
- Records the time of incubation and after incubation the tubes shall be observe for the presence of gel.
- Prepare the dilution of the sample and record the result in Annexure III.
- Record the consumption of lysate in Annexure II and consumption of CSE in Annexure IV.
Observations for Bacterial endotoxin test by gel clot method :
- Remove the tube gently and visually observe for gel formation without disturbing the tubes.
- For confirmation of gel formation, gently invert the tubes through 180°.
- If gel formation takes place after invert the tubes through 180°,record such result as positive (++).A negative result is characterized by the absence of such a gel. Record such results as Negative (- -).
Precautions while Bacterial endotoxin test by gel clot method :
- Use depyrogenated glassware’s only.
- Only depyrogenated micropipette tips shall be used and discarded after use.
- The dilution of CSE shall not be stored because of loss of activity by adsorption.
- During usage, CSE solution shall be mixed vigorously for given time period by manufacturer.
- Reconstituted control standard endotoxin shall be stored at about 2-8oC for not more than 28 days.
- Do not vortex Lysate.
- Follow temperature and time of incubation strictly.
Failure investigation:
- When Positive result observed on both the tubes of test preparation, investigate the cause of its failure by checking following parameters.
- Check product dilution, CSE dilution, and lysate dilution.
- Whether glassware are cleaned and depyrogenated as per given procedure.
- Check sensitivity report of lysate lot and matched CSE.
- Check Heating block temperature and calibration.
- Check Micropipette Calibration.
- Check any source of contamination occurs due to Microbiologist.
Annexure:
Annexure I : Report for Lysate Sensitivity
Annexure II : LAL Reagent Stock/Consumption Record
Annexure III : Bacterial endotoxin test report
Annexure IV : Control standard endotoxin stock/consumption record
Annexure V : Bacterial Endotoxin Test Inward Register
Annexure- I
Report for Lysate Sensitivity
Date of Testing :———————– Heating block I.D.:————————
Incubation Start time:—————— Incubation End time:———————
Start Temperature:——————— End Temperature:————————-
| Make : | Supplier : | ||||
| Details | LAL Reagent | Control Standard Endotoxin | LAL Reagent Water | ||
| Batch No. | |||||
| Sensitivity/Potency | |||||
| Opened On | |||||
| Use Before | |||||
| Expiry Date | |||||
I) CSE Dilution Details : The reconstituted CSE Vial contain_____ EU/ml and dilutions are as per below table;
Preparation of Standard CSE 1 EU/ml (A): ________________________________________
1 ml of A +________ ml of LRW = 0.5 EU/ml (B)
1 ml of B +________ ml of LRW = 0.25 EU/ml (C)
1 ml of C +________ ml of LRW = 0.125 EU/ml (D)
1 ml of D +________ ml of LRW = 0.06 EU/ml (E)
1 ml of E +________ ml of LRW = 0.03 EU/ml (F)
II) Negative Control:
100µl of LRW + 100 µl Lysate.
III) Test :
| Sample | CSE dilution used | LRW | Lysate (µl) | No.of replicates |
| 2λ | 100 µl of 2λ | – | 100 µl | 4 |
| λ | 100 µl of λ | – | 100 µl | 4 |
| 0.5λ | 100 µl of 0.5λ | – | 100 µl | 4 |
| 0.25λ | 100 µl of 0.25λ | – | 100 µl | 4 |
| NWC | – | 100 µl | 100 µl | 2 |
Observed the gel formation after keeping the tubes at 37°C± 1°C for 60± 2 min.
IV) Observation :
| Set No. | Concentration | End point | Log | Mean Log | |||
| 1 | |||||||
| 2 | |||||||
| 3 | |||||||
| 4 | |||||||
Note : + ve : Gel formation – ve : No Gel formation
Negative Control:
Lysate Sensitivity = Antilog of Mean log
=
Acceptance limit: Lysate sensitivity must be within two fold dilution of lable claim i.e.within 0.5λ to 2λ,where λ is sensitivity of lysate.
Conclusion: The end point of Lysate sensitivity is found within/ not within two fold dilution as mentioned in lable claim sensitivity by manufacturer.
Analysed By: Checked By:
Date: Date:
Annexure II
LAL Reagent Stock/Consumption Record
Date of Receipt :———————- Quantity Received : ———————-
Lot.No./Batch No. : ———————- Sensitivity of Lysate : ——————–
Mfg.Date : —————————— Expiry Date : ——————————
| Date | Reconstituted On | Reconstituted Volume (ml) | Used for | Balance Reconstituted Volume (ml) | Stock vial | Done By Sign/Date | Checked By
Sign/Date |
Verified By :———————
( Department Head)
Annexure- III
Bacterial endotoxin test report
I) Sample Details :
| Name of Sample | Date of Analysis | ||
| Batch No. | A.R.Number | ||
| Specification Number | Type of Product |
II) Equipment and Incubation Details :
| Analytical Balance I.D | Heating Block I.D. | ||
| Vortex mixure I.D | Micropipette code | ||
| Incubation temp. | Incubation time | ||
| Incubation Start at | Incubation end at |
III) Test Reagent Details :
| CSE Batch No. : | Lysate Lot No.: | LRW Batch No.: |
| Exp. Date: | Exp. Date: | Exp. Date: |
| Potency of CSE: | Sensitivity of lysate: | Mfg Date: |
| Reconstituted On: | Reconstituted On: | Opend On: |
| Make : | Make : | Make : |
IV) CSE Dilution : CSE Concentration______ EU/ml (A) (After rehydration as per COA)
| Volume of CSE added
(µL) |
Volume of LRW added
(µL) |
CSE Concentration
(EU/ml) |
V) Sample Preparation :
Weight of product taken :—————- Volume of LRW added :—————–
Potency/Concentration : —————— Endotoxin Limit :————————-
M.V.D. = Endotoxin Limit(EU/mg) X Potency of product(mg/ml)
Lysate Sensitivity (EU/ml)
=
=
VI) Product Dilution :
| Dilution Factor | Volume of Sample (µL) | Volume of LRW (µL) |
VII) Test Results :
| Sample Details | LRW (µL) | CSE (µL) | Sample (µL) | Lysate (µL) | Result | |
| Tube 1 | Tube 1 | |||||
| Positive Control | ||||||
| Product Positive Control | ||||||
| Negative water control | ||||||
| Test Sample | ||||||
Note : + ve : Gel formation – ve : No Gel formation
Conclusion : The Product Complies/does not comply with the endotoxin limit____________.
Analysed By : Checked By :
Date : Date :
Annexure- IV
Control standard endotoxin stock/consumption Record
Date of Receipt :———————- Quantity Received : ———————-
Lot.No./Batch No. : ———————- Sensitivity of CSE : ——————–
Mfg.Date : —————————— Expiry Date : ——————————
| Date | Reconstituted On | Reconstituted Volume (ml) | Used for | Balance Reconstituted Volume (ml) | Stock vial | Done By
Sign/Date |
Checked By
Sign/Date |
Verified By :————————-
( Department Head)
Annexure V
Bacterial Endotoxin Test Inward Register
| Name of Product | Batch.No | A.R.No | CSE Reconstituted on | Lysate Reconstituted on | Analysis done on | Done By | Checked By |
