Horseshoe Crab
  • Conduct hold-time studies with NOE

 

Low Endotoxin Recovery (LER) is a controversial topic that has been circulating throughout the endotoxin detection community for the last couple of years.  LER is defined as the inability to recover a known amount of spiked endotoxin and is claimed to be distinctly different than the interference commonly seen with the LAL assay that is normally overcome by dilution.  

Two common drug excipients, polysorbate and citrate, have been identified as probable causes of the masking effect more commonly referred to as LER.  These substances are estimated to be used in more than 70% of protein formulations (Maggio, 2012).  There is also some evidence that phosphate-containing formulations may also be affected by LER.  However, the LER effect has only been observed in combination formulations of the aforementioned excipients, and not in individual raw materials.

The U.S. FDA included a stability screen requirement in their 2012 Guidance for Industry Pyrogen and Endotoxins Testing: Questions and Answers.  This guidance recommends that the industry perform hold-time studies to verify the amount of time a sample can be reasonably stored prior to final release testing.

Endotoxin normally exists in an aggregated state and not as monomers.  According to Mueller et al.* only the aggregated forms of endotoxin are biologically active as it appears that monomeric endotoxin does not bind well to Factor C and this inhibits the activation of the LAL cascade.  There are claims that the disaggregation problem is primarily seen with purified forms of endotoxin such as RSE or CSE and that a solution to the problem is to conduct hold studies using an NOE source.   

There have been several published articles demonstrating that purified endotoxin performs differently as compared to NOE in recovery studies.  In some cases the inability to recover purified endotoxin happens almost immediately where recovery of NOE is stable over an extended period.  Lonza has done some initial testing using a polysorbate/citrate-based buffer and compared recovery of RSE and NOE over a 10-day period.  In this study, RSE recovery dropped significantly the following day and was undetectable by day two.  NOE, however, was recoverable over a 10-day period. 

If you are looking for an NOE source that can be used as a tool in your hold-time studies, Lonza would like to invite you to participate in our alpha testing program.  By participating in our program you will receive NOE preparations free of charge, and in turn we would like to receive your feedback on the performance of NOE as it pertains to LER.

 

For more information about the NOE alpha testing program, please contact Lonza Scientific Support .

 

*Mueller et al., Aggregates are the biologically active units of endotoxin, JBC , March 2004