MICLAB-080 Bacterial EndoToxin Testing (LAL) – Gel Clot Method

1. General

1.1.         The gel-clot method for bacterial endotoxin testing described in this SOP is based on the fact that Limulus Amoebocyte Lysate (LAL) will form a firm gel in the presence of bacterial endotoxin.

1.2.         New operators must be adequately trained by a competent staff member, prior to performing routine testing.  Routine testing may be performed once all training requirements are met and have been deemed to be satisfactory following review.

1.3.         The reagents referred to in the Test Method i.e. “Pyrogent” Lysate, “Pyrosperse” dispersing agent and Escherichia coli Endotoxin are manufactured by Bio Whittaker Inc. Pyrogent and Endotoxin are ideally can be purchased in a 200 test kit form “Pyrogent plus” Cat. No. N284.
Reagents of another manufacturer may not be substituted in this test method unless prior validation of their suitability has been carried out.

1.4.         Bacterial Endotoxin testing is to be conducted on all WFI samples and batches of product for injection or irrigation where required as part of product specifications.

1.4.1.     The testing regime for the gel-clot test is for the individual testing of the samples taken from the beginning, middle and end of the batch.  Therefore a total of three LAL gel-clot tests are conducted for each commercial batch.

1.5.         Documentation of LAL gel clot session should be recorded on Form 685 and stored in the “LAL Gel-Clot Test Session Results” File.  This includes the date of test session, Operator’s name, batch number, Product name, container & size, dilution, result of test, record that the information has been transferred to log book, result of standards both positive and negative and details of all Reagents used including reconstitution dates.  Make a note of any problems encountered during the test session.

1.6.         Materials required

10mL sterile disposable pipettes                       E. coli Endotoxin

1mL sterile disposable pipettes                        Pyrogent

Sterile disposable micropipette tips                   Pyrosperse

Pyrogen-free Water for Injection (WFI)               Vortex mixer

10mm x 75mm test tubes                                 Micropipettor

10mL depyrogenated test tubes                        {Heating Block (switch on 30 minutes

20mL depyrogenated test tubes                        {prior to test to gain stable temperature).

0.1N pyrogen free Hydrochloric acid	-Prepared by diluting concentrated HCl with pyrogen free WFI using pyrogen free equipment
0. 1N pyrogen free Sodium Hydroxide	-NaOH diluted to the appropriate concentration with pyrogen free WFI using pyrogen free equipment

NOTE: Depyrogenate the bottles prior to making up the HCl/NaOH solution.

2. Preparation of the Endotoxin

2.1.         Add 5mL of pyrogen free WFI to the vial of E. coli Endotoxin.  Record the date of preparation on the vial.

2.2.         Vortex the vial for 1 hour.  Check the Endotoxin concentration on the Certificate of Analysis.

2.3.         Store the reconstituted Endotoxin in this vial at 2 ‑ 8°C for up to 1 month in the fridge.

2.4.         Each day that the Endotoxin standard is to be used, vortex the vial for 1 hour.

3. Preparation of Test Solutions

3.1.         Finished Products

3.1.1.     Pool the contents from a minimum of 3 sample containers for each test required (or at least half the samples provided whichever is less) into a pyrogen free test tube.  Adjust pH if required. Carry out dilution of the samples if necessary using pyrogen‑free WFI.  The total volume of pooled sample (diluted or otherwise) must be greater than 10mL.

3.1.2.     The pH of some products must be adjusted to between 6.0 and 7.5 for testing using the Gel Clot method. Using a pH meter, measure the pH of these solutions and adjust if necessary using pyrogen free HCl (adjust down) or pyrogen free NaOH (adjust up).

3.1.3.     Transfer 10mL of the test solution (appropriately diluted if required) into a fresh pyrogen free test tube.

3.1.4.     PYROSPERSE – MUST be at room temperature before use.  Add 200mL Pyrosperse to each of the 10mL test samples to form a 2% concentration.  Vortex well.  Some precipitation of Marcain and Naropin products is to be expected.

3.1.5.    Transfer an amount of the prepared test solution into a fresh pyrogen free test tube to be used for section 4 as the PPC.  The remaining amount is the test solution.  This will depend on the Lysate sensitivity for accurate volume – see Appendix 2.

4. Preparing Positive Product control

4.1.         Prepare a positive product control for each test sample, prepared in section 3.1, by spiking with Endotoxin of concentration twice the Lysate sensitivity, e.g. When using a Lysate of sensitivity 0.06 EU/mL pipette 50mL of a 10 EU/mL Endotoxin standard into the tube containing 4mL of appropriately pH adjusted and diluted test sample.  Vortex well.  This will give an Endotoxin concentration in the Positive Product control of 0.125 EU/mL.  See Appendix 2 for calculations using other concentrations of Endotoxin standard.

5. Preparation of Raw materials

5.1         To prepare Raw Materials (Sodium Chloride, Dextrose Anhydrous & Sodium Acetate) for bacterial Endotoxin testing see Raw Material Specifications.

5.2.       Prepare Raw Material Solution for testing using the same method as finished product (sections 3.1.2 to 3.1.4 – test solution and section 4.1 – positive product control).

6. Testing of Process Water

6.1.       See MICLAB 055 for the method and frequency of sampling process water outlets for pyrogen testing.  The autoclaved 100mL vial of sample water will be used as the test solution.

7. Preparation of Working Endotoxin Standards

7.1.         Prepare a two fold dilution series of the vortexed Endotoxin in pyrogen free WFI to obtain final Endotoxin concentrations of 1.0, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015 EU/mL.

7.2.         When the concentration of Endotoxin is 10 EU/mL, the following dilution series will provide the necessary concentrations:

Tube 1	200mL 10 EU/mL Endotoxin + 1.8mL pyrogen free WFI	= 1 EU/mL
Tube 2	1mL of Tube 1 + 1mL pyrogen free WFI	= 0.5 EU/mL
Tube 3	1mL of Tube 2 + 1mL pyrogen free WFI	= 0.25 EU/mL
Tube 4	1mL of Tube 3 + 1mL pyrogen free WFI	= 0.125 EU mL
Tube 5	1mL of Tube 4 + 1mL pyrogen free WFI	= 0.06 EU/mL
Tube 6	1mL of Tube 5 + 1mL pyrogen free WFI	= 0.03 EU/mL
Tube 7	1mL of Tube 5 + 1mL pyrogen free WFI	= 0.015 EU/mL

See Appendix 1 for initial dilution of Tube 1 using other Endotoxin concentrations.
Tubes 2‑6 remain the same.

7.3.         Mix each dilution Tube for 30 seconds on the vortex mixer before transfer.  For routine product testing, tubes 4, 5, 6 and 7 are used for standards.  For Lysate Verification and Operator Verification, tubes 3, 4, 5, 6 and 7 should be used as standards and the test must be done in quadruplicate.

8. Performing the Qualitative LAL test

8.1.         Prepare and label the 10 x 75 mm pyrogen‑free test tubes as below.

NOTE: All solutions must be vortexed for 15 seconds prior to pipetting into tubes.

8.1.1.     Standards
For 0.06 EU/mL Lysate sensitivity use standard tubes 4,5, 6 and 7 (as prepared in Section 7).  Pipette 100mL into duplicate 10x75mm labelled test tubes. For 0.125 EU/mL Lysate sensitivity, use standard tubes 3, 4, 5 and 6.

8.1.2.     Negative control
Pipette 100mL of pyrogen‑free WFI into duplicate 10x75mm labelled test tubes.

8.1.3.     Finished Products and Raw Materials
Pipette 100mL of each test solution (as prepared in Section 3 – Finished products, Section 5 – Raw Materials) into duplicate 10x75mm labelled test tubes.

8.1.4.     Positive control
Pipette 100mL of each of the positive product control solutions (as prepared in Section 4) into single 10x75mm labelled test tubes.

8.1.5.     Water Samples

Pipette 100mL of each water sample into a single 10x75mm labelled test tube.

NOTE
If more than one heating block is used per session, a set of standards and negative controls must be used in each heating block.

9. Preparation of Pyrogent

9.1.       Prepare Pyrogent as follows.  (Each new lot number kit must be verified for its Lysate Sensitivity before use and step 7.3 for details of requirements)

9.1.1.     Remove the metal cap and grey stopper from the Pyrogent 50 test vial.  Record the date of reconstitution on the vial.

9.1.2.     Pipette 5.2 mL of WFI into the Pyrogent vial and mix gently to dissolve Pyrogent.  Avoid foaming.

9.1.3.     Pipette 100mL of the reconstituted Pyrogent to each of the above test tubes.  Swirl each tube gently to mix while transferring to heating block.  Incubate undisturbed at 37°C + 0.5 C for 60 + 2 minutes.

9.1.4.     Any remaining Pyrogent at the conclusion of the working day can be frozen for use in the next test session.  It will remain stable for up to 1 month.  Reconstituted Pyrogent can be frozen-thawed only once.

9.2.         After incubation, gently remove the tubes from the block and visually check for gel formation by inverting tubes through 180 degrees.

9.3.         Test Results Interpretation, Out-of-Specification and Test Failure Response

Positive	Formation of a firm gel, that remains in place upon inversion.
Negative	Total absence of gel clot, or the formation of a viscous gel that does not maintain its integrity when inverted.
Acceptance criteria	Negative control = no gel clot
	Positive control = gel clot.
	Endotoxin Standards must gel at the labelled Lysate sensitivity ± 2-fold dilution and no negative result between 2 positive results.
Interpretation	The product under test complies when a negative result is found in the duplicate product tubes
Re-Test	If sample fails refer to guidelines in the BP and USP for permitted re-testing, e.g. testing in triplicate.  A repeat test is permitted if one product tube gels and the other gives a negative result.  If the test is positive for the product under test at a dilution less than the MVD, the test maybe repeated at a dilution not greater than the MVD. If all dilutions are positive, the endotoxin concentration is reported as equal to or greater than the greatest dilution factor x λ (the lysate sensitivity).  If the product code is also registered for the KCA method the test maybe repeated using this method. The product meets the requirements of the test if the concentration of endotoxin is less than the limits registered.
OOS Result	For all OOS results a DR is to be raised. The subsequent investigation should highlight the root cause for the OOS result and any CAPA that needs to be implemented.
Endotoxin Test Failure	An Endotoxin test failure will result an Incident Meeting to document and determine batch disposition.

10. Disposal of materials

Endotoxin spiked product solution, Endotoxin standard dilutions, used reagent bottles and contaminated disposable equipment, e.g. Test tubes and pipettes are to be disposed of into the Biohazard Bins.  Any remaining product solution and containers are to be disposed of into Security Waste bins.

11. LAL Dilutions (examples)

Product	WFI	Dilution
10mL    +	10mL    =	1/2
5mL      +	10mL    =	1/3
5mL      +	15mL    =	1/4
3mL      +	9mL      =	1/4
2mL      +	14mL    =	1/8
1ml       +	10ml     =	1/11
1mL      +	14mL    =	1/15
1mL      +	15mL    =	1/16
1mL      +	19mL    =	1/20
1mL      +	39mL    =	1/40

 

12. Appendix 1 – Initial dilution of Tube 1 using other Endotoxin concentrations

1. If Endotoxin = 10 EU/mL

            Tube 1 = 200mL + 1.8mL WFI = 1 EU/mL

2. If Endotoxin = 12 EU/mL

            Tube 1 = 200mL + 2.2mL WFI = 1 EU/mL

3. If Endotoxin = 14 EU/mL

            Tube 1 = 200mL + 2.6mL WFI = 1 EU/mL

4. If Endotoxin = 16 EU/mL

            Tube 1 = 200mL + 3.0mL WFI = 1 EU/mL

5. If Endotoxin = 18 EU/mL

            Tube 1 = 200mL + 3.4mL WFI = 1 EU/mL

6. If Endotoxin = 20 EU/mL

            Tube 1 = 200mL + 3.8mL WFI = 1 EU/mL

13. Appendix 2 – Positive Product Controls

Spike with an Endotoxin concentration of twice the Lysate sensitivity.

1. For 10 EU/mL Endotoxin
2. a)       If Lysate = 0.06 EU/mL then need to spike with 0.125 EU/mL

                      4.0mL product + 50mL of 10 EU/mL Endotoxin.

1. b)       If Lysate = 0.125 EU/mL then need to spike with 0.25 EU/mL

                      3.9mL product + 100mL of 10 EU/mL Endotoxin.

2. For 12 EU/mL Endotoxin
3. a)       If Lysate = 0.06 EU/mL then need to spike with 0.125 EU/mL

                      4.8mL product + 50mL of 12 EU/mL Endotoxin.

1. b)       If Lysate = 0.125 EU/mL then need to spike with 0.25 EU/mL

                      4.7mL product + 100mL of 12 EU/mL Endotoxin.

3. For 14 EU/mL Endotoxin
4. a)       If Lysate = 0.06 EU/mL then need to spike with 0.125 EU/mL

                      5.6mL product + 50mL of 14 EU/mL Endotoxin.

1. b)       If Lysate = 0.125 EU/mL then need to spike with 0.25 EU/mL

                      5.5mL product + 100mL of 14 EU/mL Endotoxin.

4. For 16 EU/mL Endotoxin
5. a)       If Lysate = 0.06 EU/mL then need to spike with 0.125 EU/mL

                      6.4mL product + 50mL of 16 EU/mL Endotoxin.

1. b)       If Lysate = 0.125 EU/mL then need to spike with 0.25 EU/mL

                      6.3 mL product + 100mL of 16 EU/mL Endotoxin.

5. For 18 EU/mL Endotoxin
6. a)       If Lysate = 0.06 EU/mL then need to spike with 0.125 EU/mL

                      7.5mL product + 50mL of 18 EU/mL Endotoxin.

1. b)       If Lysate = 0.125 EU/mL then need to spike with 0.25 EU/mL

                      7.4mL product + 100mL of 18 EU/mL Endotoxin.

6. For 20 EU/mL Endotoxin
7. a)       If Lysate = 0.06 EU/mL then need to spike with 0.125 EU/mL

                      8mL product + 50mL of 20 EU/mL Endotoxin.

1. b)       If Lysate = 0.125 EU/mL then need to spike with 0.25 EU/mL

                      7.9mL product + 100mL of 20 EU/mL Endotoxin.

14. Gel-Clot Procedural Flowchart
15. Summary of Changes

Version #	Revision History
MICLAB 080	New