EMA Q&A - When is Testing for Endotoxin Masking Effects necessary for biological Products?

In EudraLex Volume 4: EU Guidelines on Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use. Part 1, Chapter 6: Quality control states e.g. under 6.3 “Finished product assessment should embrace all relevant factors, including production conditions, results of in-process testing, a review of manufacturing (including packaging) documentation, compliance with Finished Product Specification and examination of the final finished pack."

Low Endotoxin Recovery/Maskierung

This provides the basis for the necessity of low endotoxin recovery studies for certain products. The EMA has now answered the question “When should low endotoxin recovery (LER, also known as ‘endotoxin masking’) be investigated?” as part of an update to its question and answer catalog on biologics. EMA states: 

"LER (low endotoxin recovery) refers to the reduced ability to detect spiked endotoxin in products tested using the compendial Limulus Amebocyte Lysate (LAL) assay. This phenomenon may be due to endotoxin masking, and its causes are not completely understood. Current knowledge suggests that LER is inherent to certain product formulations. Thus, for product formulations that contain a combination of a surfactant (e.g. polysorbate) and a chelator (e.g. EDTA, citrate, phosphate, histidine), LER studies should be submitted in the MAA dossier.

LER studies, requested in a MAA, should be performed by spiking undiluted samples with known amounts of endotoxin and by monitoring the recovery over specific time intervals. LER studies should reflect conditions relevant for the manufacturing process, including relevant temperatures, and should reflect potential hold times during manufacture.

Based on current scientific state of the art, a minimum of four time-points should be evaluated to ensure valid and accurate results. Two consecutive data points falling below 50% recovery (lower compendial limit), should be considered as LER.

If LER is detected, the Applicant should propose an adequate mitigation strategy (e.g. the compendial method should be optimized or an alternative method developed). For LER exhibiting products, it is strongly recommended to set the finished product specification limit for bacterial endotoxin as low as reasonably achievable, according to actual manufacturer`s data. This approach would help to ensure that a larger safety margin is built into the finished product endotoxin specification limit. The recommended location of the LER studies is CTD section 3.2.P.5.3 (validation of analytical procedures) or CTD 3.2.P.2.3 (manufacturing process development) with a cross-reference in 3.2.P.5.3, as appropriate.

Of note, quality control (QC) sample hold-times and storage conditions are distinct from hold time applied during LER studies. Indeed, QC samples may be diluted and/or frozen prior to testing, while this is not the case for LER studies.  The appropriateness of the QC samples respective hold time i.e. maximum allowable time between sampling and testing, is a general GMP requirement1.

LER investigations should focus on the finished product (FP) as it is administered to the patient. LER studies may not be necessary for the active substance (AS) if the matrix is similar for both AS and FP. However, if there are differences in the matrix, a risk assessment should be performed to evaluate whether LER testing on the FP is sufficiently representative for the AS.

PDA Technical Report No. 82 on LER is recognised as a relevant standard for designing LER studies and could therefore be considered by Applicants when designing LER studies. Although the technical report primarily focuses on protein-based products, in the absence of specific guidance, it can also be consulted for other products such as vaccines and gene therapy."

More detailed information

The original text and further questions and answers can be found directly in the document "Questions and answers for biological medicinal products".

In this context, please note the ECA Academy's special practical laboratory course on Low Endotoxin Recovery on March 18/19 in Bernried, Germay  which covers the handling of LER both in theory and in practical laboratory training.