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Do I need a validation prior to routine testing?

As described in a previous blog (What are the common test methods for pyrogen testing and how to choose between them?), the LAL assay is an enzymatic assay. This means that substances present in the test mixture might inhibit or stimulate/enhance the proper functioning of the enzyme leading to either false negatives or false positives.  

To ensure that results are representative of the endotoxin present in a sample, a positive product control recovery validation must be performed. When using the older, qualitative gel-clot method (limit test), this validation is done prior to and separate from the routine test. When quantitative methods are used, this validation is incorporated into the assay and performed simultaneously with the routine testing.  

European and US Pharmacopoeia (EP 2.6.14, USP <85>) state that inhibition or enhancement should be ruled out. According to these guidances, an inhibition and enhancement assay consists out of the following: 

  • Unspiked sample 
  • Sample spiked with the middle concentration of the standard curve (0.5 EU/ml) 
  • At least 3 different concentrations of a standard endotoxin 
  • Unspiked water (blank) 

 Each component of testing is incorporated into our test procedures for the kinetic methods, so additional validation work is typically not required. However, as testing pharmaceuticals can often lead to inhibition or enhancement, we advise an initial assay is performed in which we will test multiple concentrations of the test item to find out the concentration that renders the lowest detection limit that does not interfere with the test enzyme. As medical device extracts do not typically inhibit nor enhance the test enzyme, a separate inhibition and enhancement test is generally not performed, but may be done upon request. When using the gel-clot method the concentration of the test item and spiked sample cannot be determined, so the spike recovery cannot be determined. Therefore, a separate inhibition assay is required for the accurate determination of the spike (PPC) recovery. 

The inhibition and enhancement assay has typically been performed on three different batches, as stated in the outdated FDA guidance of 1987. However, it is no longer required. In the most recent version of ANSI/AAMI ST72 (2019), the following is stated: 

“A minimum of one batch per product or product family shall be used to demonstrate test method suitability. For photometric techniques, a valid positive product control (PPC) fulfills the requirement for methods for test method suitability.” 

Thus, to conclude: validation is required, though a separate validation prior to a routine test is only required for the gel clot assay. 

Peter Cornelis, Ir.

Senior Expert Microbiology

Peter Cornelis graduated from the Catholic University of Leuven (Belgium) in 2000 as a Master in Applied Biological Sciences (Major Biotechnology). In 2003 he started working for Toxikon Europe (now Nelson Labs) as a study director Microbiology and in-vitro Toxicology. From 2007 until 2016, he was department supervisor for Microbiology and in-vitro Toxicology. Since 2016, he is responsible for research, validation, and development of new microbiological and in-vitro toxicological methods. Peter is a member of the ISO committee TC 194 WG5, Cytotoxicity and WG 8, Irritation and sensitization. As an expert, he was involved in the adaptation of ISO/TC 194 ISO/DTS 11796:2022(E) Biological evaluation of medical devices — Guidance for interlaboratory studies to demonstrate the applicability of validated in vitro methods to assess the skin sensitization of medical devices.

Nathan Pett

Study Director BET

Nathan Pett graduated from Utah State University in 2018 with a bachelors in Biochemistry. In 2019 he began work for Nelson Labs as a Lab Analyst and in 2021 transitioned to the role of Study Director in the BET department.

Emily Spackman, B.S., RM(NRCM)

BET Consulting Study Director

Emily Spackman has worked for Nelson Laboratories in the Bacterial Endotoxins Lab for over 16 years. She graduated from the University of Utah and holds a bachelors degree in Biology. She is a member of The National Registry of Certified Microbiologists and has experience with both medical devices and pharmaceutical products.