Standard Operating Procedure

Title: Gel Clot Validation Method

______________________________________________________________________________________

Department Micro Laboratory Document no MICLAB 105

Prepared by: Date: Supersedes:

Checked by: Date: Date Issued:

Approved by: Date: Review Date:

Document Owner
Micro Laboratory Manager

Affected Parties
All Microbiology Laboratory colleagues

Purpose
To describe the method of Gel-Clot validation to be used in the Micro . Lab.

Scope
The procedures outlined in this SOP are to be followed by the Micro . Lab. staff.

Definition
BET Bacterial Endotoxin Test : A test used to detect or quantify endo toxins
Endotoxin Toxic molecules originating from the outer cell wall of gram -negative bacteria
Endotoxin The maximum amount of endotoxin allowed in a sterile product or on a medical device .
Limit
Maximum A figure that shows how much a pa renteral product or raw material may be diluted without
Valid Dilution losing the ability to detect endotoxin at the limit concentration

Related Documents
MICLAB 070 Identification of Micro organisms to Genus and Species Level
MICLAB 080 Bacterial Endo Toxin Testing (LAL) - Gel Clot Method
Form 590 Verification Assay Results Sheet
Form 595 Bacterial Endotoxin Test Data
Form 600 Maximum Valid Dilution and Endotoxin Limit Calculations
Form 605 Bacterial Endotoxin Gel Clot Validation - Final inhibition and Enhancement Test
Form 610 Bacterial Endotoxin Gel Clot Validation - Preliminary inhibition and Enhancement test

EHS Statement
The reagents used in testing must be disposed of into the Biohazard Bin along with all the disposable
equipment. Safety glasses must be w orn if using IPA/solvent.

Table of Contents
1. General ................................ ................................ ................................ ................................ ............... 2

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Standard Operating Procedure
Title: Gel Clot Validation Method
______________________________________________________________________________________
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 :
ïò Prepare a dilution series of the pr oduct 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.
îò 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 sensi tivity 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
1. 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 100³l of
the appropriate dilution into 2 test tubes.
i Negative controls
Prepare 2 negative controls by pipetting 0.1ml of pyrogen free WFI into two
10 x 75mm test tubes.
i 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 Lysa te 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.
i Pipette 0.1ml of the reconstituted Pyrogent into each of the above test tubes, i.e.
i 12 tubes Product control dilution
i 12 tubes Product compatibility

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Standard Operating Procedure
Title: Gel Clot Validation Method
______________________________________________________________________________________
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 Pro duct 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 Endotoxin Limit = 5EU/Kg
2 mg/Kg 10 mL/Kg
= 250EU/mL = 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 f or 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 ,

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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

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 Form 590
Issue date:
Verification Assay Result Sheet
Verification Assay For Microbi ology Laboratory Technicians
(Ref. MICLAB 105)

Verification Assay Date:

Name of Technician:

Test Reagents

Reagents Lot No. Reconstitution Date Expiry date

Pyrogent EU/mL sensitivity

Endotoxin EU/mL potency

Pyrosperse NA 2% working concentration

Test kit NA

L.A.L, Endotoxin & Endotoxin Working Standards dilu ent.

Any sterile batch (WFI) (Tested to be L.A.L. negative) Batch No.: Expiry:

Test Session Standards - Results

Key: (+) firm gel, (-) no gel or viscous gel.

Replicate Endotoxin Concentration & Gelation Res ults (EU/mL) Endpoint

Assay
Number 1 0.5 0.25 0.125 0.06 0.03 0.015 EU/mL Log

Negative Controls
Key: (+) firm gel, (-) no gel or viscous gel.
Replicate Control
Assay No. Results
1
2

File Location: Date Printed: Page 7 of 15

 Form 590
Issue date:
Verification Assay Result Sheet
Verification Assay For Microbi ology Laboratory Technicians
(Ref. MICLAB 105)

Calculations:

GMx = 10

Where =
n
ø ; y i ÷
1
n

ø ÷
Where =

GMx =10

Standard: GM sensitivity value should fall within the range 0.03 1.25 EU/mL.
GMx = _____________ EU/mL

Has the standard been met ? YES/NO

Comments about Session :

Signature of Technician : Approved by:

Date: Date:

File Location: Date Printed: Page 9 of 15

 Form 600
Issue Date:
Maximum Valid Dilution & Endotoxin Limit Calculations
(Ref. MICLAB 105 & MICLAB 085)

Maximum Valid Dilution (MVD) and Endotoxin Limits

for
Sterile finished Products Tested by LAL Gel Clot and KCA Test methods

Product:

D = Maximum human dose /Kg mg/Kg

ml/Kg

Potency of Product mg/ml

This is not required if the dose
is expressed in ml /kg

´ = Sensitivity of Lysate EU/ml

K = 5.0 EU/Kg for parenterals except intrathecal drugs where k = 0.2

Endotoxin Li mit EU/ml

MVD = Endotoxin Limit

´

Therefore: MVD for Product = _______________

Calculation for Endotoxin Limit if required

Endotoxin Limit = K x Potency

D

Therefore: Endotoxin Limit for Product = _______________

File Location: Date Printed: Page 11 of 15

 Form 605
Issue date:
Bacterial Endotoxin Gel Clot Validation
Final Inhibition and Enhancement Test
(Ref. MICLAB 105)

Result sheet Bacterial Endotoxin test (U.S.P.) Pyrogent

Interpretation of Results :

Result Acceptance Levels

Product Endotoxin Dilution series
=______________EU/mL
A2 endpoint, Part 1 (Highest dilution
(0.5 2 x Lysate sensitivity)
positive)
Positive Controls, Part 2 (Highest =______________EU/mL
B2
dilution positive) (0.5 2 x Lysate sensitivity)
C2 Negative controls, Part 3 Pos / Neg Must be negative.
Lowest dilution giving positive Lysate in
D2 Preliminary test. (Product Compatibility Less than MVD.
Test, B1 result)
Comparison of Endotoxin
Must not differ by more than
E2 determinations in Product A 2and Water
plus or minus a 2 fold dilution.
B2. (ie Dilution level they di ffer by)

Have all the acceptance levels been met ? YES / NO

File Location : Date Printed: Page 13 of 15