Question and Answers to Endotoxin Masking and Low Endotoxin Recovery (LER)

Masking of Endotoxins and LOW Endotoxin Recovery is a currently often discussed topic related to the quality of pharmaceutical products. As part of the webinar on Endotoxin Masking and Low Endotoxin Recovery (LER) end of September the participants had a number of questions. The speaker, Johannes Reich, answered these questions at the end of the webinar. Following you will find a summary of these questions and answers.

"The following questions and answers are a summary of the points raised in the webinar. The provided answers are not binding and shall only serve for guidance.

• Do studies for time-dependent endotoxin recovery have to be conducted for all formulations?
  Yes, it is not sufficient to merely test single substance groups or formulation classes, since endotoxin masking depends on numerous different factors. For instance, different pH settings may significantly influence masking under otherwise identical conditions.
  
• What is the difference between "test interference" and "endotoxin masking"?
  Both phenomena have different underlying mechanisms. In case of "test interference", the detection system/ the enzymatic reaction is directly disturbed by the ambient conditions. For example, protease inhibitors can impair the detection enzyme and thereby cause a false-negative result.
  On the other hand, if "endotoxin masking" occurs, the endotoxin to be detected itself is influenced by the ambient conditions. This means that the active form (supramolecular structure) of the endotoxin has changed, leading to lower activities that are detected. The enzymatic detection reaction is however not inhibited in this case. In practice, both phenomena may occur simultaneously.
  
• How can you differentiate between interference and masking?
  As interference is time-independent and masking is time-dependent, time-dependent experiments are required in addition to the positive control. This means that the recovery rate decreases over time, if masking occurs. To differentiate between the phenomena, a sufficiently high spike concentration has to be chosen for the undiluted samples to later dilute for in turn excluding test interferences.
  
• How long and at which incubation temperature shall a masking experiment be conducted?
  The length of incubation is highly dependent on the respective sample. A significant decrease of endotoxin recovery (<50%) has been observed in some cases already after a few minutes, e.g. 30 min. On the other hand, there have been cases where masking was only found after several days, e.g. 5 days.
  The incubation temperature shall be chosen to match the sample's ambient temperature which commonly occurs in the course of its handling.
  
• Which value is used as start in a masking kinetic study?
  The endotoxin recovery in LRW (LAL reagent water) can be used as start value (100%).
  
• Which concentration shall the endotoxin spike have?
  The optimum spike concentration depends on the sample to be analyzed and shall be adjusted to the endotoxin limit to be detected and the MVD (maximum valid dilution). Low spike concentrations, e.g. <10 EU/ml, may even happen to behave unfavorably in pure LRW.
  
• How do you ensure that LRW and/or the sample tube do not mask endotoxin?
  To exclude endotoxin masking in LRW or by the sample tube, the endotoxin spike (in a single sample tube) shall be monitored together with the masking sample over time.
  
• Which test systems can be used for detection?
  All common detection systems can be used for conducting endotoxin masking experiments, e.g. LAL (turbidometric, chromogenic etc.), recombinant detection systems or monocyte activation tests (MAT).
  
• Which measures shall be taken after observing masking?
  After observing masking, the optimization of the used test system, e.g. with respect to sample preparation, or the replacement with an alternative method are advised.
  
• What is the principle of demasking?
  It is assumed that the detectability of endotoxin depends on its supramolecular structure. This structure may exhibit various aggregation forms and thus different activities depending on the ambient conditions. In the course of masking, the endotoxin structure is converted into an undetectable state, e.g. monomers. To return masked endotoxin into an active state, the ambient conditions have to be chosen in a way that shifts the balance back toward "self organization" into active aggregation forms."