BET
Creation of an In-house Naturally Ocurring Endotoxin Preparation for Use in Endotoxin Spiking Studies and LAL Sample Hold Time Analysis
This article was first published in Volume 14 Issue 6 of American Pharmaceutical Review. Abstract
Kim Bowers and Lynn Johnson Pfizer
Detection of endotoxin that may be present in biopharmaceutical products is critical to patient safety. Although the endotoxin molecule itself is highly stable, various factors such as matrix composition, storage temperature and container makeup can affect its stability in manufacturing samples that are collected and subsequently assayed for endotoxin content. Pfizer in Andover, MA developed a procedure for creating an in-house naturally occurring endotoxin (NOE) preparation to assess the stability of endotoxins across various matrices, temperatures and containers. The use of NOE provides benefits over using commercially available Control Standard Endotoxin (CSE), such as increased laboratory flexibility and control, and has been used by Pfizer to assess endotoxin stability over time in various matrices and temperatures.
Introduction
Endotoxin is a lipopolysaccharide structure located in the cell wall of Gram-negative bacteria. Since endotoxins belong to a group of fever-causing substances called pyrogens, parenteral drug products that may contain endotoxin can elicit a pyrogenic reaction in patients. The FDA has established a pyrogenic threshold of 5-endotoxin units/kilogram (EU/kg) of body weight. Endotoxin exposure beyond this level may induce fever, shock, and death. It is critical that endotoxin levels are monitored and controlled in biomanufacturing processes and products for reasons of safety and compliance. The Limulus Amebocyte Lysate (LAL) assay is often used to measure the level of endotoxins in biological products. Testing occurs at a variety of points throughout the purification process, as well as in bulk drug substance and finished drug product. Effective endotoxin testing is critical to the safety of a product, and as such it is crucial that any endotoxins that may be present throughout the manufacture and release of a product be accurately detected.
Kim Bowers is Manager, Quality Science and
Technology at Pfizer Global Supply. Ms. Bowers holds a Bachelor of Arts in Biology from Mount Holyoke College and has been employed by Wyeth/Pfizer for over 10 years.
Lynn Johnson is a Scientist III at Pfizer. Her education background includes a Bachelor’s
Degree in Microbiology from the University of New Hampshireand a Master’s Degree in Public Health from Boston University.
Although it is generally accepted that the endotoxin molecule itself is highly stable, various matrices, temperatures and/or storage containers may affect the ability of the Limulus Amebocyte Lysate (LAL) assay to detect the presence of endotoxin. At Pfizer Specialty Care in Andover, MA, a variety of studies were initiated to study these effects. In order to measure the effect of time and/or temperature on the recovery of endotoxins, it was necessary to add endotoxins into the starting biopharmaceutical samples. Since there were multiple matrices and temperatures being assessed, a large volume of endotoxin was required to complete these studies.
Control Standard Endotoxin (CSE) is a type of endotoxin that is commercially available. CSE is used to prepare standard curves and positive product controls (PPCs) for the LAL assay. CSE is prepared by licensed LAL vendors as a reagent from a purified E. coli strain and contains fillers in the formula. As such, CSE is not representative of endotoxin that may be present during an actual contamination event. Also, CSE is generally not available in high EU/mL formulations. Naturally occurring endotoxin (NOE) is produced by Gram-negative bacteria and is filtered but not further processed, making it representative of a contamination event. Additionally, since it can be grown to very high EU/mL concentrations, it is suitable for use in spiking studies.
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