• Pharma

  Removing Pyrogens – An Important Step in Pharma Packaging

  Posted by Frank Davis on October 24, 2022 at 4:55 pm

  What is Depyrogenation in Pharma?

  Depyrogenation refers to the removal of pyrogens from solution, from injectable pharmaceuticals. A pyrogen is defined as any substance that can cause a fever. Bacterial pyrogens include endotoxins and exotoxins, although many pyrogens are endogenous to the host. Endotoxins include lipopolysaccharide (LPS) molecules found as part of the cell wall of Gram-negative bacteria, and are released upon bacterial cell lysis.

  Endotoxins may become pyrogenic when released into the bloodstream or other tissue where they are not usually found. Although the colon contains Gram-negative bacteria in abundance, they do not cause a pyrogenic effect as the bacteria are not undergoing gross lysis, and the immune system is not exposed to free endotoxin while the colonic wall is intact.

  When LPS is released upon bacterial cell lysis, the lipid A component is first bound by serum LPS-Binding Protein (LBP) and then transferred to CD14 (either free CD14 in the serum or bound to the cell surface of macrophages or monocytes). This monomerises the aggregated LPS, as the LPS receptor Toll-like Receptor 4 (TLR4) cannot recognise LPS while aggregated. Monomeric LPS is then transferred to MD-2 pre-complexed with TLR4 on macrophages and monocytes. This leads to release of pro-inflammatory cytokines and nitric oxide, which may lead ultimately to septic shock depending on the strength of response. Vascular endothelial cells also express TLR4 and MD-2 and so respond to LPS directly, as well as via cytokines and nitric oxide. Bronchial epithelial cells and colonic epithelial cells also express TLR4, but as they do not express MD-2 they rely on LPS pre complexed with serum MD-2 in order to signal to LPS.

  

  Does autoclaving remove endotoxin?

  Autoclaving can sterilize the surface of glassware, however, endotoxins that cling to sides tend to be too heat-resistant to be removed. It is believed that autoclaving and boiling doesn’t destroy all the endotoxin present. That is to say depyrogenation is more difficult than sterilization.

  Packaging components used in pharmaceutical drugs and medical devices are scrupulously cleaned before use to ensure patient safety. Regulators expect drug manufacturers to demonstrate compliance with federal requirements intended to assure clean, sterile and safe drug products enter the medical marketplace.

  Depyrogenation, the reduction of bacterial endotoxin, is critical in preparing packaging components for use in injectable drug products.

  Pharmaceutical packaging components such as ampoules, vials and stoppers require specific depyrogenation procedures appropriate for the component’s material. The main depyrogenation processes that will be referenced are dry heat and intense washing.

  The FDA defines a sterile pharmaceutical product as the probability of 1 in a million of finished products being contaminated (-6). This is referred to as the Sterility Assurance Level (SAL). There are seven basic characteristics of sterile pharmaceutical packaging components, described in Table 1. All of these characteristics must be achieved for safe pharmaceutical packaging. One particularly challenging characteristic is that materials must be free from pyrogenic endotoxin contamination.

  Depyrogenation can be defined as the reduction of pyrogenic substances, including bacterial endotoxin, and is generally achieved by removal or inactivation. There are several techniques in which depyrogenation can be achieved. The more common techniques are shown in table 3. The two most utilized techniques for pharmaceutical packaging components are dry heat exposure via a depyrogenation oven and rinsing by USP and WFI water.

  Endotoxins are strongly regulated and strictly tested for within the pharmaceutical industry. A common test to detect endotoxins from gram-negative bacteria is known as Bacterial Endotoxins Test (BET) which has three methods.

  • Method A – (gel-clot technique) is based on gel formation.
  • Method B – (turbidimetric technique) is based on the development of turbidity after cleavage of an endogenous substrate.
  • Method C – (chromogenic technique) is based on the development of color after cleavage of a synthetic peptide-chromogen complex.

   

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