1. Purpose
The purpose of this Guideline is to provide a general guideline for endotoxin testing. The guideline should aid in assuring that the products manufactured at any of the company sites as well as by a contract manufacturer meet the appropriate regulatory and company requirements and that there is a harmonized, company-wide approach to the concept of endotoxin testing.
2. Scope and Applicability
This Guideline applies specifically to endotoxin testing as it is carried out at Any manufacturing site, as well as at third party testing labs. The guideline applies, where applicable, to raw material (including water), components, in-process material and drug products, and well as to validation of dry heat sterilization procedures. In addition to the guideline, the following items should also be in place.
2.1 Standard Operation Procedures (SOPs)
SOPs shall be developed to provide clear direction for the execution of the testing procedures, material preparation, data analysis, and the development of the documentation referred to in this guideline.
2.2 Validation Protocols
Validation protocols shall be generated to verify and document that the methods used to perform endotoxin testing can reliably and consistently detect endotoxins that may be present in units being tested, and that the equipment does not interact with the endotoxin testing. These protocols, the data generated and the associated summary reports should provide documentation of the acceptability of these methods and equipment for their intended use.
3. Definitions
3.1 Endotoxin
Toxic lipopolysaccharides originating from the outer cell wall of Gram-negative bacteria.
3.2 Bacterial Endotoxin Test (BET)
A test used to detect or quantify endotoxins of Gram-negative origin.
3.3 Endotoxin Limit
The maximum amount of endotoxin allowed in raw material, sterile products or on medical devices.
3.4 Maximum Valid Dilution (MVD)
A calculation that shows how much a parenteral product or raw material may be diluted without losing the ability to detect endotoxins at the limit concentration.
3.5 Minimum Valid Concentration (MVC)
A calculation that shows the lowest concentration of a substance that can be used in a test without loosing the ability to detect endotoxins at the lowest concentration.
4. Responsibilities
4.1 It is the responsibility of the appropriate management to establish, issue, maintain and ensure compliance with this quality guideline for endotoxin testing.
4.2 It is the responsibility of every unit using third party testing labs to ensure that this Guideline is adhered to.
5. Guideline
5.1 Recognised LAL Test Methods
– Gel-clot
– Turbidimetric kinetic
– Turbidimetric endpoint
– Chromogenic kinetic
– Chromogenic endpoint
The gel-clot techniques detect or quantify endotoxins based on clotting of the LAL Reagent in the presence of endotoxin. The turbidimetric method measures increases in turbidity. Depending on the test principle used, the turbidimetric technique is classified as either end-point turbidimetric or kinetic turbidimetric. The chromogenic method measures the chromophore released from a suitable chromogenic peptide by the reaction of endotoxins with the LAL Reagent. Depending on the test principle employed, the chromogenic technique is classified as either endpoint chromogenic or kinetic chromogenic.
5.2 Endotoxin Testing
5.2.1 General Requirements
5.2.1.1 Endotoxin testing can be carried out in a general laboratory. No special environmental considerations have to be taken.
5.2.1.2 Endotoxin testing should be performed in a manner that avoids endotoxin contamination of the test equipment, supplies and materials to be tested.
5.2.1.3 Endotoxin testing should be carried out in a solution that is at a neutral pH (6.0-8.0) and has a balance of divalent cations.
5.2.1.4 The sensitivity claimed of each batch of the LAL reagent should be verified.
5.2.1.5 The absence of interference should be established for each test item by performing Inhibition/Enhancement validation studies.
5.2.1.6 Endotoxin test methods should be validated and demonstrated to be appropriate for their intended use.
5.2.1.7 Appropriate preventative maintenance procedures for critical equipment and systems should be approved and in place.
5.2.1.8 Critical instruments and equipment should be calibrated and included in the routine calibration program.
5.2.1.9 Endotoxin elimination (depyrogenation) cycles for internal processes, supplies, equipment and materials should be validated.
5.2.1.10 Endotoxin test analysts or technicians should have appropriate training and documentation of that training should be on file and available for review.
5.2.1.11 Endotoxin test methods and/or procedures should be approved, current and available for use by the analysts or technicians.
5.2.1.12 Endotoxin limits and MVD or MVC should be established for each test item.
5.2.1.13 Inhibition/Enhancement testing should be performed on each raw material, packaging component, in-process material and drug product requiring a BET specification. Ideally three separate batches or individual shipments of the item should be used for this testing.
5.2.1.14 Product test assays shall be validated before being used to release final product.
5.2.1.15 Revalidation shall be performed as prescribed by Pharmacopoeia (e.g. at product reformulation, changes in concentration, change in drug substance or drug product).
5.2.2 Methods & Procedures
The following items or issues should be addressed in approved methods and/or procedures:
5.2.2.1 Validation and Revalidation of Endotoxin Test Methods.
5.2.2.2 Sample Collection, Transport and Storage.
5.2.2.3 Endotoxin Test Methods.
5.2.2.4 Preparation, Testing, Approval and Storage of Endotoxin Test Reagent and Supplies.
5.2.2.5 Maintenance of Endotoxin Test Equipment.
5.2.2.6 Preparation and Decontamination (Depyrogenation) of Containers for Endotoxin Testing.
5.2.2.7 Out of Specification (OOS) Result Investigation and Response.
5.2.2.8 Endotoxin Test Failure Investigation and Response.
5.2.2.9 Interpretation of Endotoxin Test Results.
5.2.2.10 Retesting.
5.2.2.11 Trending of Endotoxin Test Data.
5.2.2.12 Change Control.
5.2.3 Validation
5.2.3.1 The endotoxin test methods and procedures, their associated instrumentation and equipment, depyrogenation procedures and cycles should be validated to assure the reliability of the data generated by the endotoxin test program.
5.2.3.2 The following areas should be addressed by the validation effort:
– Maximum Valid Dilution
– Minimum Valid Concentration
– Operator Proficiency
– Inhibition/Enhancement
– Endotoxin Test Methods
5.2.3.3 All proposed changes to validated systems, processes, equipment or methods shall be reviewed and approved by appropriate management prior to the change. Any changes that impact the status of a validated system, process, equipment or method may require revalidation of that system, process, equipment or method.