• airborne endotoxins
• injectable drugs
• LAL test
• pharmaceutical quality control

Endotoxins are fragments of the Gram negative bacteria membrane. Endotoxins contain lipopolysaccharide (LPS), an amphiphilic macromolecule which can exert toxic effects on human, such as fever, inflammation, disseminated intravascular coagulation (DIC), systemic inflammatory response (SIRS) and even including shock and death.

For this reason Pharmacopoeias establish endotoxin limits for injectable pharmaceutical products and provide compendial methods for the determination of such pyrogens (29a, 7a and 28a).

The Bacterial Endotoxins Test (BET), commonly known as the LAL test (Limulus Amebocyte Lysate Test), is based on the activation of an enzymatic cascade produced by endotoxins and leads to the coagulation of an amebocyte lysate (i.e. LAL reagent) obtained from the “horseshoe crab” i.e. Limulus polyphemus.

Not only do endotoxins exist in solutions, they are also present in airborne form during all the "open" processes that affect solid or liquid matrices, which could be contaminated by Gram-negative bacteria known as ubiquitous microorganisms.

The aim of this research is to develop a method for detecting and quantifying airborne endotoxins in classified production environments in order to evaluate the quality of the process and detect the presence of an objectionable past or current Gram negative bacteria contamination.

To quantify airborne endotoxins (airborne endotoxin test) we chose to follow the standard method described by EN 14031:2005 ("Workplace atmospheres - Determination of airborne endotoxins") involving air sampling on glass fiber filters, extracting endotoxins from the matrix and quantifying them with the LAL kinetic test method.

To quantify airborne Gram-negative bacteria we used settling plates, containing a selective medium for the growth of such bacteria ( VRBGA : Violet Red Bile Glucose Agar ), while for surface

sampling we used contact plates, containing the same medium added with inactivating agents to neutralize disinfectants.

To ensure the reliability of the airborne endotoxins test, it was necessary to conduct early testing to assess glass fiber filter compatibility, endotoxin extraction parameters (e.g. solution type, volume of the solution and agitation speed and time) and sample storage conditions.

To ensure the reliability of the microbial test, it was necessary to conduct early growth promotion with the medium challenged using disinfectants.

Finally, environmental monitoring was conducted on three production cleanrooms, identified as most at risk for the presence of water.

The early results on the glass fiber filter with the kinetic colorimetric method were not compliant with acceptance criteria, presumably because the reagent volume is not sufficient to neutralize the matrix interference. Therefore we chose to use the kinetic turbidimetric method, which did not show interference and allowed endotoxins to be recovered not only in the solution but also if absorbed on the matrix.

The best sample storage conditions were at a frozen temperature of -80 °C for not more than 4 days.

The results of the microbial test demonstrated that the medium composition was capable of efficiently neutralizing our disinfectants.

The tests conducted in the selected environments revealed the presence of detectable amounts of airborne endotoxins but lower than the quantification limit for the assay (i.e. <0.005 IU /ml corresponding to < 1 IU/m3).

The low concentrations of airborne endotoxins detected in the cleanrooms did not represent a risk for the product processed herein and for the workers’ health, since they are far inferior to the Health -based Occupational Exposure Limit (HBROEL = 11,25 UI/m3h) recommended by the Dutch Expert Committee on Occupational Standards (DECOS).

Finally, we found viable Gram negative bacteria on the room surfaces samples but not on the settling plates so the detection of airborne endotoxins could be an index of their presence (past or current) in the environment giving an early warning; however further tests should be conducted to verify this relationship.