MICLAB-105 Gel Clot Validation Method

1. General

New operators must be adequately trained by a competent staff member and perform a satisfactory Operator Verification, (Form 590), prior to performing Gel-Clot method validations.  Gel-clot method validations may be performed once all training requirements are met and have been deemed to be satisfactory following review.

The gel clot validation method for Bacterial Endotoxin testing described in this SOP, is to determine the level of Inhibition/Enhancement of products on the LAL test for endotoxins within the allowable Maximum Valid Dilution (MVD) for each type of product.  The Gel-Clot techniques detect or quantify endotoxins based on clotting of the LAL reagent in the presence of endotoxin.

To be determined for each type of product, using the highest and lowest concentration of active.  If either concentration shows inhibition or enhancement, then each remaining concentration must be tested.  At least three (3) Production batches of each finished product should be tested for inhibition and enhancement.

1.1.             Materials required:

10ml sterile disposable pipettes

1ml sterile disposable pipettes

Sterile disposable micropipettes

Pyrogen-free water for injection (WFI)

10mm x 75mm test tubes

10ml depyrogenated test tubes

20ml depyrogenated test tubes

E.coli endotoxin

Pyrogent

Pyrospers

Vortex mixer

Micro pipettor

Heating Block.

2. Preliminary Inhibition and Enhancement Testing:

Preliminary Inhibition/Enhancement testing consists of running two (2) sets of product dilutions in parallel, one set containing product alone, the other set containing endotoxin spiked product such that the concentration of the product decreases with increasing dilution while the concentration of endotoxin remains the same.  The final concentration of the endotoxin spike should be equal to twice the sensitivity of the Lysate used.

2.1.             Method:

Preparation of Pyrogent, see MICLAB 080.

Preparation of the Endotoxin Standard, see MICLAB 080.

Preparation of the Endotoxin Working Standard, see MICLAB 080.

2.2.             Performing the Preliminary Inhibition / Enhancement Test:

a) Adjust the pH of the product (if necessary) to within the range of pH 6.0-7.5 with 0.1N pyrogen–free HCL or 0.1N pyrogen–free NaOH.

Note: Do not adjust the pH of the unbuffered saline or water.  Add Pyrosperse so that the concentration of Pyrosperse in the product is 2% i.e. 0.1ml Pyrosperse to 5ml of product.

b) Product Control Dilution:

Prepare a dilution series of the product in 2 fold increments to a dilution level of 1 in 32.  Further serial dilution of the product may be necessary, i.e.

1ml product + 1ml WFI = 2 fold dilution.

Prepare two (2) sets of six 10 x 75 mm test tubes (appropriately labelled) of the above product dilution series, i.e. from zero dilution to 1 in 32 dilutions, by pipetting 0.1ml of the appropriate dilution into 2 test tubes.

c) Product Compatibility:

Prepare a dilution series of the product in 2 fold increments with endotoxin spiked WFI +2% Pyrosperse to a product dilution level of 1 in 32 such that the concentration of endotoxin throughout the dilution series remains the same.  Further serial dilution of the product may be necessary.  The level of endotoxin spike should be equal to twice the sensitivity of Lysate used, e.g.
If the Lysate sensitivity is 0.06EU/ml the level of endotoxin spiked per dilution tube should be 0.125EU/ml.  Hence the endotoxin spike WFI must contain 0.25 EU/ml and 0.125 EU/ml

The product compatibility dilution series for a Lysate sensitivity of 0.06 EU/ml will therefore be as follows:

Tube Dilution

                   No                 Factor                     Contents of tube                                Endotoxin Conc.

                   0                    8ml product             + 0.05ml endotoxin *                          (0.20EU/ml)

                   1 – 2               1ml product             + 1ml endotoxin WFI  0.25EU/ml         0.125EU/ml

                   2 – 4              1ml product             + 1ml endotoxin WFI  0.125EU/ml       0.125EU/ml

                   3 – 8              1ml product             + 1ml endotoxin WFI  0.125EU/ml       0.125EU/ml

                   4 – 16             1ml product             + 1ml endotoxin WFI  0.125EU/ml       0.125EU/ml

                   5 – 32            1ml product             + 1ml endotoxin WFI  0.125EU/ml       0.125EU/ml

* Endotoxin diluted in WFI

Prepare two (2) sets of six 10 x 75 mm test tubes (appropriately labelled) of the above product/endotoxin dilution series, i.e. from a zero to a 1 in 32 dilution, by pipetting 100ml of the appropriate dilution into 2 test tubes.

• Negative controls
  Prepare 2 negative controls by pipetting 0.1ml of pyrogen–free WFI into two

10 x 75mm test tubes.

• Positive controls
  Prepare two sets of four 10 x 75mm test tubes (appropriately labelled) of the endotoxin standards dilution series to bracket the concentration of the Lysate used in the test, e.g. if the Lysate sensitivity is 0.125EU/ml the endotoxin standards to be employed are tubes No 4, 5, 6, 7, i.e. concentration of 0.25EU/ml, 0.125EU/ml, 0.06EU/ml and 0.03EU/ml.
• Pipette 0.1ml of the reconstituted Pyrogent into each of the above test tubes, i.e.
• 12 tubes Product control dilution
• 12 tubes Product compatibility
• 2 tubes Negative controls
• 8 tubes Positive controls

Immediately swirl gently to mix and incubate undisturbed at 37°C ± 0.5°C in the heating block for 60 ± 2 minutes.

After 60 minutes incubation, gently remove the tubes from the block and rotate them through 180 degrees.

Test results

Positive:  Formation of a firm gel that maintains its integrity when the test tube is inverted.

Negative:  Total absence of gel, or the formation of a viscous gel that does not maintain its integrity when inverted.

Note: The results sheet – Preliminary Inhibition/Enhancement Test is to be filled in.

3. Interpretation of results of Preliminary Inhibition/Enhancement test

The results of the Product Control Dilution series and the Product Compatibility series are to be compared.

a) The Product Control Dilution series should show no clotting (positive reaction) if the product contains no endogenous endotoxin.

b) The first tube in the Product Compatibility Dilution series that does clot, i.e. a positive reaction is the dilution of product required to overcome inhibition.  If no positive reactions are recorded, further dilution of the product is required to overcome inhibition.  Repeat the test using further dilution steps in 3(b)(1) and 3(c)(1).

3.1.             Calculations for the Maximum Valid Dilution and the Endotoxin Limits

The Maximum Valid Dilution (MVD) must be calculated.

When a product is diluted to overcome interference or inhibition, any endotoxin present is also diluted.

The MVD is the degree to which a product can be diluted before the sensitivity of the assay method to detect the diluted endotoxin concentration is exceeded.

The MVD is equal to the endotoxin limit divided by Lambda

Lambda (λ) is equal to the label claim for the gel clot lysate or the lowest standard for the KCA method.

The MVD is the maximum dilution that must not be exceeded for routine product testing.

Examples of MVD Calculations:

GEL-CLOT	KCA
Lysate sensitivity = 0.06 EU/mL	Lowest Standard = 0.005EU/mL
Endotoxin Limit for Product  = 3.0EU/mL	Endotoxin Limit for Product  = 3.0EU/mL
MVD = 3.0EU/mL 0.06EU/mL	MVD = 3.0EU/mL   0.005EU/mL
MVD = 50	MVD = 600

Please note that the higher sensitivity of the KCA method will give a larger MVD.  This, in turn, will allow a greater dilution to overcome any inhibition that may be present.

The Endotoxin Limit is determined by:
(1) specific monograph for that product (BP, EP, USP or JP),
(2) by regulatory requirement (local or export)
(3) corporate product specification or 4) by calculation.

The Endotoxin Limit = (K/D) x Potency

K = Maximum allowable endotoxin exposure

            5EU/Kg/Hour for intramuscular

            0.2 EU/Kg/Hour for intrathecal

D = Maximum human dose

Potency = drug concentration (This is not required if the dose is expressed in mLs)

An average human weight for the purpose of MVD calculation is regarded as 70kg (or 60Kg for Japan).

Examples of Calculation for Endotoxin Limits:

Endotoxin Limits
Example 1 – Product 1	Example 2 – Product 2
Dose = 2 mg/Kg	Dose = 10mL/Kg
Potency  = 100mg/mL	Potency = not applicable
Endotoxin Limit = 5EU/Kg x 100mg/mL 2mg/Kg = 250EU/mL	Endotoxin Limit = 5EU/Kg 10mL/Kg = 0.5EU/mL

Note: The product dilution level required to overcome inhibition as established by the inhibition /enhancement must be compared with the MVD calculated to ensure that the Maximum Valid Dilution for that product has not been exceeded.

3.2.             Final Inhibition/Enhancement Test

The Inhibition/Enhancement test is then to be repeated using the diluted product concentration in 4(b) containing varying concentrations of endotoxin that bracket the lysate sensitivity and comparing this product series with a series of the same endotoxin concentration in water alone.

Method:

Preparation of Pyrogent, see MICLAB 080.

Preparation of the Endotoxin Standard,

Preparation of the Endotoxin Working Standard,

4. Performing the Final Inhibition/Enhancement Test:

a)Adjust the pH of the product (if necessary) to within the range of pH 6.0 – 7.5 with 0.1N pyrogen-free HCL or 0.1N pyrogen-free NaOH.

Note: Do not adjust the pH of the unbuffered saline or water.  Add Pyrosperse so that the concentration of Pyrosperse in the product is 2%, i.e. 0.1ml Pyrosperse to 5ml of product.

Product endotoxin dilutions:

i) Prepare 20ml of Product diluted with pyrogen-free WFI.

ii) Pipette 10ml of this diluted product into a pyrogen-free test tube and spike with endotoxin to give the most concentrated endotoxin level required for an endotoxin series to bracket the Lysate sensitivity, e.g. if the Lysate sensitivity is 0.125 EU/ml the endotoxin series will be 0.25 EU/ml, 0.125 EU/ml, 0.06 EU/ml and 0.03 EU/ml.

Therefore to spike the diluted product with a endotoxin concentration of 0.25 EU/ml use:

10ml diluted product +0.1ml (25 EU/ml) endotoxin    = Tube No.1.
= Endotoxin conc. 0.25EU/ml

iii) Prepare a 2 fold dilution series of the endotoxin spiked diluted product using the additional 10ml of diluted product prepared in (i) as the diluent such that the product concentration remains constant throughout the dilution series whereas the endotoxin concentration decreased with increasing dilutions, eg. for Lysate sensitivity 0.125 EU/ml:

Tube                                                                                   Endotoxin

No.                                                                                            Conc.

1     10ml diluted product +0.1ml (25 EU/ml) endotoxin

2     1ml diluted product/endotoxin spiked @0.25 EU/ml + 1ml diluted product=0.125EU/ml, (i.e. Tube No.1)

3           1ml Tube No. 2 + 1ml diluted product

4           1ml Tube No. 3 + 1ml diluted product

iv) Prepare 4 sets of four 10 x 75 mm test tubes (appropriately labelled) endotoxin standards dilution series to bracket the concentration of the Lysate used in the test, i.e. used the same endotoxin concentration diluted in the water as are used in product Endotoxin Dilutions.

b) Positive controls

Prepare 4 sets of four 10 x 75mm test tubes (appropriately labelled) of the endotoxin standards dilution series to bracket the concentration of the Lysate used in the test, i.e. use the same endotoxin concentration diluted in water as are used in Product Endotoxin Dilutions.

c) Negative controls

Prepare 2 negative controls by pipetting 0.1ml of pyrogen-free WFI + 2% Pyrosperse into two 10 x 75mm test tubes.

d)Pipette 0.1ml of the reconstituted Pyrogent into each of the above rest tubes, i.e.

• 16 tubes Product Endotoxin Dilution
• 2 tubes Negative controls
• 16 tubes Positive controls.

Immediately swirl gently mix and incubate undisturbed at 37°C ± 0.5°C in the Heating block for 60 ± 2 minutes.

e) After 60 minutes incubation, gently remove the tubes from the block and rotate them through 180 degrees.

f) Test results

Positive:  Formation of a firm gel that maintains its integrity when the test tube is inverted.

Negative:  Total absence of gel, or the formation of a viscous gel that does not maintain its integrity when inverted.

g) The results sheet – Final Inhibition/Enhancement test – is to be filled out.

5. Summary of Changes:

Version #	Revision History
MICLAB 105	New

Associated Forms

Form 590

Form 595

Form 600

Form 605

Form 610

End of Procedure