Not too long ago, my wife and I took a vacation to Kiawah Island in South Carolina to celebrate her birthday. Early one morning we took a walk along the beach, and after a few hundred yards I came across numerous horseshoe crab exoskeletons lying in the sand. I immediately recognized the arthropod for its importance in pharmaceutical quality assurance; more specifically for the detection of endotoxins.

 

Bacterial endotoxins, often called pyrogens, are poisonous substances produced within living cells or organisms. These fever-producing materials most often originate from gram-negative bacterial cell walls, but can also originate as leachates from some chemicals and materials. In pharmaceutical production, especially sterile compounding, it is necessary to reduce traces of endotoxins in drug products as even small amounts will cause illness in humans.

According to USP 797 guidelines, “All high-risk level compounded sterile products (CSPs) … before they are sterilized shall be tested to ensure that they do not contain excessive bacterial endotoxins.”

Here at Hartley Medical, we allocate a substantial annual budget solely to quality assurance testing. We perform both sterility and endotoxin testing per USP requirements.

A very sensitive procedure for detecting the presence of endotoxins in drugs is the Limulus Amebocyte Lysate (LAL) test. The LAL assay utilizes the amoebocytes (blood cells) of a horseshoe crab. The lysate found in the blood binds with the dangerous endotoxins present creating a clot via a cascade of reactions; thus, forming the basis of their detection and quantification.

When it comes to LAL testing for bacterial endotoxins, there are three techniques: 1) the gel-clot technique, which is based on gel formation; 2) the turbidimetric technique, based on the development of turbidity after cleavage (the division or splitting of form) of an endogenous substrate; and 3) chromogenic technique, based on the development of color after cleavage of a synthetic peptide-chromogen complex.[1]

Hartley Medical began endotoxin testing in 1999 utilizing the gel-clot test. In 2003, we advanced our technology to test using the turbidimetric technique; a method we still use today. This is a photometric test to measure the increase in turbidity (the cloudiness or haziness of a fluid caused by individual particles that are generally invisible to the naked eye). More specifically, we utilize kinetic-turbidimetric testing, which measures both the time (onset time) needed for the reaction mixture to reach a predetermined absorbance, and the rate of turbidity development. In the presence of endotoxins, the lysate begins to gel – causing the solution to become turbid. Higher concentrations of endotoxins cause the increase in solution turbidity to occur faster than lower concentrations. A standard curve is always run with each test. The concentration of the unknown is extrapolated from the standard curve. The time required for the appearance of turbidity is inversely proportional to the amount of endotoxins present.[2]

It is fascinating to think of how intertwined nature and science are. Bacteria are everywhere. And, for the most part, bacteria are beneficial as they reduce organic waste and recycle nutrients back into the food chain. Sometimes, however, bacteria cause disease if they enter parts of our body that are usually bacteria-free, such as the bloodstream or the intra-spinal space– causing severe illness (such as sepsis) and/or death. Therefore, pharmaceutical companies, especially sterile compounding pharmacies, must take great care in producing drugs that are free of bacteria and non-pyrogenic. Hartley Medical recognizes the horseshoe crab’s contribution to the world of pain management; and we are happy to change and save lives.


[1] “Bacterial Endotoxins.” European Pharmacopoeia 5.0. <http://iccvam.niehs.nih.gov/docs/pyrogen/regulatory/20614e.pdf>. 27 Dec. 2011

[2] “Endotoxin Detection: Products and Services.” Lonza. 2010 < http://www.biocenter.hu/teszt/pdf/ lalkat10.pdf>. 27 Dec. 2011.