What is Endotoxin Testing?
What is Endotoxin Testing?
What is Endotoxin Testing?
LAL (Limulus amebocyte lysate) endotoxin testing is an in-vitro assay used for the detection and quantitation of bacterial endotoxins in injectable products or implantable medical devices that will make direct or indirect contact with the bloodstream or spinal fluid. This test is also used in monitoring water systems and raw materials to ensure that this does not contribute endotoxins to the finished product.<div>
This guidance provides recommendations for biological product, drug, and device firms on FDA’s current thinking concerning the testing recommendations and acceptance criteria in the United States Pharmacopeia (USP) Chapter Bacterial Endotoxins Test, USP Chapter Transfusion and Infusion Assemblies and Similar Medical Devices161>,and the Association for the Advancement of Medical Instrumentation (AAMI) ST72:2002/R2010, Bacterial Endotoxins—Test Methodologies, Routine Monitoring, and Alternatives to Batch Testing(AAMI ST72). These three documents describe the fundamental principles of the gel clot, photometric, and kinetic test methods, and recommend that appropriate components and finished products be tested for the presence of pyrogens and endotoxins. 85>
This guidance does not cover the entire subject of pyrogen and endotoxins testing. Instead, it addresses those issues that may be subject to misinterpretation and are not covered in compendial procedures or in currently available guidance documents. You should already have a thorough understanding of these documents when using this guidance.
FDA’s guidance documents, including this guidance, do not establish legally enforceable responsibilities. Instead, guidances describe the Agency’s current thinking on a topic and should be viewed only as recommendations, unless specific regulatory or statutory requirements are cited. The use of the word should in Agency guidances means that something is suggested or recommended, but not required.
<b style=”background-color: var(–bb-content-background-color); font-family: inherit; font-size: inherit; color: var(–bb-body-text-color);”>BACKGROUND
For more than 30 years, FDA has accepted the use of a Limulus Amoebocyte Lysate (LAL) test for endotoxins in lieu of the rabbit pyrogens test. In a November 4, 1977, Federal Register notice (42 FR 57749), FDA described conditions for using LAL as a finished product test. By 1983, FDA indicated in guidance that an LAL test could be used as a finished product test for endotoxins. These tests were described in a series of draft and final guidance documents. The last guidance document, Guideline on Validation of the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products, and Medical Devices, was published in 1987 (the 1987 Guidance).
FDA has found that the published USP and AAMI documents describing methods and calculation of pyrogen and endotoxins testing limits provide industry with appropriate information. We also note the continued development of USP Chapters and and FDA guidance documents. The Agency has withdrawn the 1987 Guidance because it no longer reflects the Agency’s current thinking on the topic. However, because the compendial chapters and standards do not address certain regulatory perspectives, FDA is providing supplemental information in this guidance to explain our current thinking regarding the submission and maintenance of pyrogen and endotoxins testing for FDA-regulated products. 161>85>
How do I establish a sampling plan for in-process testing and finished product release?
The current good manufacturing practice (CGMP) regulations for finished pharmaceuticals and the medical device quality system regulations require development of controls that include scientifically sound and appropriate sampling plans.
Sampling plan information is addressed in AAMI ST72, but not USP Chapter . Firms should include a sampling plan as part of their application documentation. In the sampling plan, firms should consider the potential for contamination in raw materials, in-process materials, and the finished product. Specifically, firms should take into account aspects of the manufacturing design, including consistency of a manufacturing process, impact of in-process hold times, endotoxins removal steps, and finished product endotoxins specifications. The sampling plan should be considered dynamic; firms should begin with maximum coverage and adjust their sampling plans as they gain confidence in the prevention of endotoxins in their manufacturing processes. Firms should update their regulatory filings when adjusting sampling plans. For drugs and biological products, in-process changes to sampling plans are annual reportable changes. For devices, a 30-day notice may be appropriate for in-process changes to the sampling plan.
When is retesting appropriate?
When conflicting results occur within a test run, firms should consult USP Chapter , Gel Clot Limits Test, Interpretation, for guidance on repeat testing. As specified in Chapter , if the test failure occurred at less than the maximum valid dilution (MVD), the test should be repeated using a greater dilution not exceeding the MVD. A record of this failure should be included in the laboratory results. If a test is performed at the MVD and an out-of-specification (OOS) test result occurs that cannot be attributed to testing error, the lot should be rejected. All testing procedures, including those for retesting within the above limits, should be specified in advance in written standard operating procedures approved by the firm’s quality control unit.
Is sample storage and handling important?
Yes. The ability to detect endotoxins can be affected by storage and handling. Firms should establish procedures for storing and handling (which includes product mixing) samples for bacterial endotoxins analysis using laboratory data that demonstrate the stability of assayable endotoxins content. Protocols should consider the source of endotoxins used in the study, bearing in mind that purified bacterial endotoxins might react differently from native sources of endotoxins.
Can finished product samples for analysis of bacterial endotoxins be pooled into a composite sample prior to analysis?
Yes. With some exceptions (see below), finished drug product units may be pooled into a composite sample and assayed for bacterial endotoxins. The composite sample may be represented by the entire unit or partial aliquots (equal volumes) of finished product containers from one manufactured lot of aqueous-based pharmaceuticals. Pooling would generally be accepted for small-volume parenterals (those with volumes of 100 mL or less) as long as the MVD is adjusted to a proportional, lower value because of the potential for diluting a unit containing harmful levels of endotoxins with other units containing lower, less harmful, levels of endotoxins. This “adjusted MVD” is obtained by dividing the MVD computed for an individual sample by the total number of samples to be pooled. FDA suggests pooling no more than three units per composite in keeping with the concept of testing representative beginning, middle, and end finished product containers. If this reduction in MVD results in an inability to overcome product-related assay interference because of an insufficient dilution, then the samples should be tested individually.
Finished medical devices may also be pooled into a composite sample and assayed for bacterial endotoxins. Testing for medical devices should be conducted using rinsing/eluting and sampling techniques as described in ISO 10993-1 and ISO 10993-12, as also used for inhibition/enhancement. Sampling can be adjusted for special situations. After a suitable eluate/extract pool is obtained from a finished production lot, this pooled extract should be kept under conditions appropriate for stability until it is tested in duplicate.
FDA recommends that pooled samples be a composite of aseptically removed aliquots (after at least 30 seconds of vigorous mixing) from each of the product containers. In this way, the original, individual containers will be available for possible retesting in the event the pooled sample displays an OOS result.
Some product types should not be pooled. Two examples are drug products that have an initial low MVD (see discussion above of “adjusted MVD”) and products that are manufactured as a suspension, because sample aliquot homogeneity may present significant interference issues. FDA also does not recommend pooling in-process samples from different in-process stages of the manufacturing process because it may be difficult to ensure the homogeneity of these materials.
May a firm use alternative assays to those in the USP for a compendial article?
Yes, firms may use alternative methods and/or procedures if they provide advantages in terms of accuracy, sensitivity, precision, selectivity, or adaptability to automation or computerized data reduction, and in other special circumstances. Such alternative procedures and methods should be validated as described in the USP General Chapter , Validation of Compendial Procedures, and should be shown to achieve equivalent or better results. When a difference appears or in the event of a dispute, the final decision is made based upon the USP compendial gel clot method unless otherwise indicated in the monograph for the product being tested.
What is the best process for transitioning from one alternate bacterial endotoxins test (BET) method to another?
The transition between tests that measure the same entity (e.g., LAL cascade) can be made by comparing the two tests to verify the equivalence of the new method. The comparison of the limit of detection and inhibition/enhancement is fundamental. The sensitivity of the new method can be evaluated on spiked product samples. In addition to using spiked samples, a battery of field samples of product found to be positive may be a good source for comparing results from the methods. The method validation should also attempt to address the variability found in the normal use of the method and the manufacturing environment (e.g., source materials or seasonal changes).
For drug, animal drug, and biological products, the transition to a new method should be submitted in a prior approval supplement (PAS). Alternatively, once a firm has established a general method for making the transition between tests, it may submit the method for review in a PAS—comparability protocol (CP). The CP should describe, in detail, the methods used to transition between assays and the acceptance criteria used to establish the equivalence of the new method. After approval of the CP, results of implementation of the CP may be directed to be reported in a reduced reporting category (Supplement—Changes Being Effected or Annual Report or Special Report (21 CFR 314.80)). The firm should maintain the study protocol, final report, and all data at the facility for FDA review. The firm should confirm the filing process with the appropriate review division before submitting a CP. For Class III devices, the transition to a new assay requires a 30-day notice filed under 21 CFR 814.39(e). See FDA’s guidance, Modifications to Devices Subject to Premarket Approval (PMA) – The PMA Supplement Decision-Making Process, Manufacturing changes for Class I and II devices should be in accordance with the quality system regulation, 21 CFR part 820. Design control, production and process control requirements can be found at 21 CFR 820.30, 21 CFR 820.70, 21 CFR 820.72, and 21 CFR 820.75.
For devices, a 30-day notice may be appropriate for changes to quality control testing used on incoming components, raw materials, the in-process device, or the finished device, including performing end-product pyrogen testing on non sterile samples prior to sterilization. Manufactures of medical devices should demonstrate a sensitivity that is consistent with the route of administration for the device and the type of body contact. Manufacturers may use another endotoxin test after demonstrating a reproducible correlation between methods and the USP reference standard.
What happened to the endotoxins limit table in Appendix E of the 1987 Guidance?
The endotoxins limit table is out of date due to the increase in numbers of dosage (regimes) and drug strengths since the publication of the 1987 Guidance. The appropriate way to establish the endotoxins limit is to use the calculation methods provided in the USP or AAMI standards. Monograph limits may also not account for current product strengths or dosage regimes; these should also be checked using the calculations recommended in the standards. If there are several components in a finished product, then the overall endotoxins limit for parenterally-administered products should not exceed the overall threshold limit specified in the USP Bacterial Endotoxins Test, regardless of an individual component endotoxins limit. Intrathecally-administered products, ophthalmics, or devices (see question 11 for devices) may have endotoxins limit requirements that are not based on the calculation for parenterally administered products. FDA encourages firms to check with the appropriate office or review division about these products.
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