A few years ago, Genentech scientist Joseph Chen noticed—or rather didn’t notice—something in a biopharmaceutical compound that soon turned the field of endotoxin detection on its head. After adding low levels of purified Escherichia coli to certain undiluted biological formulations, Chen’s lab saw complete disappearance of the original inoculin.
As Houston would say, “We’ve got a problem.”
Researchers dubbed the phenomenon low endotoxin recovery (LER), and it has become such a significant issue that the organizers of this year’s PMF Bacterial Endotoxin Summit in Philadelphia devoted the entire meeting to the topic. Record attendance and a much larger slate of speakers from Big Pharma, the US Food and Drug Administration (FDA) and Charles River’s Endotoxin Microbial Detection division turned out for the May 15-16 meeting, which included lively group discussions as well as lectures from some of the leading lights in the field.
Endotoxins are highly toxic, heat-stable lipopolysaccharides (LPSs) that comprise the outer cell membrane of gram-negative bacteria, like the E. coli used in the Chen experiment. Other species include Neisseria, Salmonella, Shigella and Pseudomonas. Ubiquitous in nature, endotoxins can also trigger severe inflammatory fever reactions in patients, so regulatory authorities require companies to screen vaccines, drugs and medical devices like artificial kidneys that, for various reasons, remain particularly vulnerable to LPS contamination.
Endotoxin detection used to be done in rabbits, but in the mid-1970s the industry began using the Limulus Amebocyte Lysate (LAL) test, which contains the blood of the horseshoe crab (see Eureka blog.) Approximately 70 million LAL/TAL tests are performed every year.
Scientists attending the BED Summit largely agreed that the LER effect that Chen found is not a public health concern. There have been no reported endotoxin outbreaks due to a failure of the LAL test. But LER does present a problem in validating product hold-times. The chelating agents combined with surfactants that are common in biological formulations are particularly problematic. The combination of the surfactant Tween and sodium citrate, a buffering agent that resist changes in pH, appear to denature purified LPSs to the point where they are no longer detectable by LAL.
In light of the LER findings, the FDA now wants scientists to prove the endotoxin test works. How this gets resolved remains to be seen.