MICLAB-085 Bacterial EndoToxin Testing kCA Method

1. General

1.1.         New operators must be adequately trained by a competent staff member and perform a satisfactory Operator Verification, (as per section 3.3), 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.

1.2.         Bacterial endotoxin testing is carried out in order to determine whether endotoxin has been introduced into a product via the manufacturing process, raw materials or packaging, which may cause a pyrogenic reaction to the end user.

1.3     The KCA method utilises a co-lyophilised mixture of lysate and a synthetic colour producing substrate to detect endotoxin chromogenically.  A sample is mixed with the lysate/substrate reagent, placed in the BioWhittaker KQCL reader and is automatically monitored over time for the development of a Yellow colour.  The Yellow colour is caused by the cleaving of the Yellow substrate p-nitroaniline from a colourless peptide chain by an enzyme in the Limulus Amoebocyte lysate activated by bacterial endotoxin.  The time required for the development of colour is directly proportional to the amount of endotoxin present (i.e. The more endotoxin present, the faster the colour development).  The concentration of endotoxin in unknown samples can be calculated by comparing their reaction times to the reaction time of endotoxin standards from a standard curve.

2. Materials required

1. Depyrogenated Borosilicate test tubes wrapped in foil.
2. Pyrogen free disposable pipettes 1mL, 10mL.
3. Vial opener, Parafilm, vortex, timer
4. LAL grade WFI and Magnesium Chloride for dilutions
5. Micropipettes 10-100mL, 100-1000mL with Pyrogen free micropipette tips
6. Multichannel micropipette 200mL with Pyrogen free micropipette tips
7. pH meter with 0.1M pyrogen-free HCl and NaOH for pH adjustments
8. Certified KCA reagent kit (for example the brands “Charles River Endosafe” or “Cambrex (BioWhittaker)) stored at 2-8°C.
9. Pyrogen free reagent wells
10. Pyrogen free microtitre plates (96 well)
11. BioWhittaker KQCL (brand) reader with software.

3. Verification Assays

Before any testing can be done using KCA, all reagents, equipment and operators must be verified. Results for all verification assays are to be kept in the “KCA Initial Qualification File”.  KCA Reagent qualification and equipment verification assays can be used as annual operator verification assays.  Copies should be filed in both sections of the “KCA Initial Qualification File”.

3.1.         Reagent Verification

3.1.1.     KCA Reagent Verification: for every new lot number of KCA kits from the supplier, a verification assay must be run to ensure that reagent activity conforms to specifications.  This assay consists of running 4 replicates of the 5 concentration standard range with an LAL grade WFI blank (section 5.1).

3.1.2.     Diluent Verification: for every new lot number of Water for Injection or Magnesium Chloride for dilutions, a verification assay must be run against the manufacturer’s process LAL water to ensure that the diluents are pyrogen free and are acceptable to use for KCA Bacterial endotoxin testing.  This assay consists of running duplicates of the 5 concentration standard range with a LAL grade WFI blank, (section 5.1) and duplicate samples of the new diluent and manufacturer’s LAL process water including Positive Product Controls (spike concentration of 5 EU/mL).  When entering the assay details, use the “Diluents” template.

3.2.         Equipment Verification

3.2.1     96 well Microtitre plate Verification: for every new lot number of 96 well microtitre plates, a verification assay must be run to ensure that the plates are pyrogen free and free of “hot wells”.  This assay consists of running 4 replicates of the 5 concentration standard range with an LAL grade WFI blank, (section 5.1).

3.3.         Operator Verification

3.3.1     Each new operator of the BioWhittaker KQCL reader must perform an initial qualification before performing routine testing.  Also existing operators must perform a qualification on an annual basis.  This assay consists of running 4 replicates of the 5 concentration standard range with an LAL grade WFI blank (section 5.1).

4. KCA Product Validation

Before routine testing can be performed on any product, it must be validated to ensure maximum endotoxin sensitivity and control spike recovery.  If multiple concentrations of a type of product are to be tested, all concentrations must be validated separately as different concentrations may exhibit varying levels of inhibition or enhancement.  For Validation, three (3) batches of a product must be assayed with one brand of reagent.  To cover against reagent unavailability, a second brand of reagent may be validated using one (1) batch of product.  The reagent brands to be used for KCA product validation should be available in all inclusive kit form.  To complete the validation file, three (3) batches of the product must be routinely tested.  The steps for product validation are as follows:

4.1     The Maximum Valid Dilution (MVD) must be calculated (Form 600).  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 (i.e. When a product is diluted to overcome interference, any endotoxin present is also diluted).

4.2     The MVD is equal to the endotoxin limit divided by Lambda.

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

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

4.5       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)    By corporate product specification or 4) by calculation.

See below for 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 ml)

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

4.6     Two fold serial dilutions of the product should be made from undiluted down to an appropriate dilution (eg: 1:64).  If a higher dilution is needed then dilute further however the dilution must always be less than the MVD calculated for the product.  LAL grade water for injection is commonly used as the diluent however if the product, (e.g. Heparin) is a known chelator, then another diluent should be used, (e.g. LAL grade Magnesium chloride).
pH adjustments should be made for some products as instructed.

4.7     The assay is then run as per section 7 using the 5 concentration standard range, (section 5.1) and a positive product control spike of 5 EU/mL.  If the percentage spike recovery is less than 100 then inhibition is taking place, if it is greater than 100 then Enhancement is taking place.  The dilution that gives a spike recovery closest to 100% should be used for routine product testing.

4.8     All product details and MVD calculations should be recorded on cover sheet – Form 620 and filed with the assay in the KCA Product Validation file.

5. Preparation of Standards

Reconstitute the CSE endotoxin vial with verified LAL grade Water for Injection as instructed on the KCA kit Certificate of Analysis.  This will give a 50 EU/mL standard solution.  Vortex, as per the manufacturer’s recommendations.

5.1.         Standards for Verification and Validation Assays (sections 3 and 4)

Five standard concentrations should be prepared: 50, 5, 0.5, 0.05 and 0.005 EU/mL.  Prepare serial dilutions as per diagram below:

Figure

5.2.         Standards for Routine Product Testing (section 6)

Four standard concentrations should be prepared: 10, 1, 0.1 and 0.01 EU/mL.  Prepare serial dilutions as per diagram below:

Figure

6. Routine Product Testing

6.1.         Sample Preparation

Samples from production labelled “Pyrogen”, as per MICLAB 095 are received in the Microlab with sterility samples.  The Product code specification will show if Bacterial endotoxin testing is to be performed and if so which kind (Gel-clot only, KCA only or Gel/KCA).  Using only KCA only and Gel/KCA designated products, prepare the samples as follows:

6.1.1.     Pool one unit of finished product solution into pyrogen free test tubes.

6.1.2.     There are no pH adjustments required for solutions tested using the KCA method as the lysate solution contains a buffer which removes any inhibitory / enhancement effect that high or low pH solutions may possess. Method validations may show that pH adjustment is unnecessary for some products tested by the KCA method.

6.1.3.     Carry out dilution of the samples if necessary using pyrogen‑free WFI.  Prepare appropriate dilutions in pyrogen free test tubes.

6.1.4     Record the assay value using a template and the 4 concentration standard range with an LAL grade WFI blank (section 3.2).  Positive Product Control spiking is at 1 EU/mL, with the exception of some product like NaCl 20% which is 5 EU/mL.

All samples are done in duplicate.

7. Biowhittaker KQCL Reader Operation

7.1.         Turn the KQCL reader on and press ‘Enter’ after it has performed a self-test.

7.2.         In the KQCL software program select ‘Template’ and the templates box will appear. This will default to the last assay of the selected type performed.

7.3.         Enter the lot number details of all reagents to be used in the assay.

7.4.         Check that the standards set-up is correct for the selected template, 4 concentrations for Routine testing and 5 concentrations for Verification and Validation assays (see section 3).  When doing Routine testing, if a stored standard curve is available for the reagents being used, this may be used for the assay.

7.5.         Select the ‘Template’ button on the screen.  This will show a representation of a microtitre plate with all test and standard positions labelled.  For Validation, Diluent Verification and Routine testing assays, the products / diluents to be assayed must be entered.  Delete existing records by double clicking on each of the entries and selecting ‘delete’.  When clear, select ‘new’, then ‘products’, scroll down until you find the desired product, highlight it then select ‘copy’.  This copies the product to the product sheet.  Complete all the required batch details ensuring that the dilution details are correct, and include the Positive Product Control (PPC) when necessary (see respective sections for details).  Select ‘OK’ to copy product details to template sheet.

7.6.         As shown on the template sheet, add 0.1mL aliquots of the Blank, Standards and Products to be assayed to the appropriate wells of the plate.  Spike the Positive Product controls as shown (10ml from 50 EU/mL standard for 5 EU/mL PPC, 10ml from 10 EU/mL standard for 1 EU/mL PPC).

7.7.         Select ‘Done’, select ‘Save’, select ‘Run’.  If only one assay is to be run on the plate select ‘OK’.  (If more than one is to be run, select ‘Add’ then select the other assays to be run.)

7.8.         Follow the instructions on screen to start the 10-minute incubation at 37°C.

7.9.         Near the end of the incubation period rehydrate the required number of chromogenic lysate vials with LAL grade WFI as instructed on the vial.  Swirl gently to fully dissolve the powder, avoiding air bubbles, and pour into reagent well.

7.10.      At the end of the incubation period follow the software instructions to add 0.1mL of lysate, with the multi channel pipette vertically from left to right, to the wells used in the microtitre plate.  Take care not to cross contaminate the pipette tips.  Follow the on screen instructions to start the assay.

7.11.      When the assay is complete, follow the instructions on screen to save and print the results.

7.12.      Interpreting results

An acceptable assay has the following:

7.12.1   A Correlation Coefficient of greater than the absolute value of 0.980.  This indicates the linearity of the points in the standard curve.  For greatest accuracy, a coefficient of above 0.997 is recommended.

7.12.2   Coefficient of variances of <10% for standards, products and positive product control spikes. This is a measure of the correlation between reaction times of replicates.  The closer to zero, the better.

7.12.3   Endotoxin standard recoveries of 100 ±25%.  (Only applicable when a standard curve stored on the Win-KQCL system is used for multiple test runs.)

7.12.4   Endotoxin recoveries from positive product control spikes must be 100 ±50%.  Less than 100% is showing inhibition, greater than 100% is showing enhancement.

7.12.5   Endotoxin content of diluents and products must conform to limits.  These limits can be found in the front of the KCA Product Validation files.  Results are printed in the product report section under “Results EU/ml”.  This is automatically calculated from the “Raw EU” result by multiplying by the dilution factor.

7.12.6   Re-Test, Out-of-Specification Result and Test Failure Response

Re-Test

A repeat test is permitted under any one or more of the following circumstances;

–          The correlation coefficient is <0.980.

–          Coefficients of variance for one or more standards are >10%.

–          PPC recoveries are >150% or <50%.

–          One product or standard well is lower and the duplicate is higher than the allowable limit.

In each case a QN is to be raised to determine the cause of the OOS result.

Out-of-Specification (OOS) Result

A Deviation Report is to be raised for an out of specification result.

Endotoxin Test Failure

As an Endotoxin test failure will result in the rejection of product and Incident Meeting will be held to investigate and report.

8. Summary of Changes

Version #	Revision History
MICLAB 085	New