In pharmaceutical manufacturing, the presence of endotoxin poses a risk to final product quality and patient safety. A few micrograms of endotoxin in a vial of injectable drug could trigger an innate immune response that can lead to severe reactions in sensitive patients. This article will discuss what endotoxins are, how they can be detected, and how you can prevent them contaminating your sterile manufacturing facilities and cleanrooms.
What Are Endotoxins?
A lipopolysaccharide, endotoxin molecule from E.Coli
Also known as pyrogens, endotoxins are a major component of the cell walls of gram-negative bacteria. They are released as lipopolysaccharides during bacterial cell death. The presence of even a small amount of endotoxin can cause a dangerous inflammatory response (‘pyro’ is derived from the Greek word for fire). These molecules have been implicated in numerous adverse events, including hypersensitivity and anaphylaxis, after administration of parenteral products contaminated with gram-negative bacteria and their cell debris. Endotoxins given intravenously at concentrations as low as 1 mg/mL have been demonstrated to be fatal. When endotoxins enter the bloodstream, they cause endotoxemia, which results in respiratory failure, a drop in blood pressure, and reduced oxygen levels. This eventually causes sepsis and death.
Preventing Contamination by Endotoxins During Sterile Manufacturing
Endotoxin contamination in parenteral pharmaceutical manufacturing can be particularly dangerous
Numerous controls can be implemented in pharmaceutical manufacturing to minimize gram-negative bacterial growth and eliminate the possibility of endotoxin contamination. It is always preferrable to control endotoxin levels upstream in the manufacturing process rather than having to remove contamination in the final steps.
Indirect controls include taking measures to limit the growth and spread of gram-negative bacteria during production. The greatest danger of exposure and proliferation of gram-negative microbes is posed to materials derived from natural sources or with high water input. However, water is often a key component in pharmaceutical manufacturing, especially in the production of parenteral products. Biofilm formation can be avoided with proper validation and control of water quality, water generation, and water distribution.
Standing water in the cleanroom can be a source of contamination
Auditing suppliers to ensure they are exercising consistent and documented control over their manufacturing processes is one approach to regulating endotoxin levels in raw materials and packaging. A vendor audit should specifically include bioburden and endotoxin control since microbial populations and microbe types directly impact endotoxin levels in materials.
Endotoxin levels can be managed with aseptic technique and good manufacturing practices. First, cleanroom operators must be educated on how to lessen their microbial footprint and equipped with appropriate sterile protective apparel (coveralls, masks, gloves, etc.). Secondly, the spread of bacteria and other microorganisms is prevented, at least in part, by employing cleaning and disinfection procedures in critical areas.
As highlighted in the new addition of GMP Annex 1, cleaning and disinfecting must be performed separately. Cleaning by mopping and/or wiping cleanroom surfaces with specialized products will physically remove debris and residue so disinfectants subsequently applied will be at their most effective.
Suite Ultra-Fiber mop is scientifically proven to remove >99.8% of endotoxins
The choice of cleaning products is critical for endotoxin removal, as not all materials are able to pick up and retain sub-micron cell debris. The Suite® family of cleanroom products contain Ultra-Fiber technology which has been scientifically proven to remove >99.8% of endotoxin units[i]. Less than 0.02% can be transferred from the Ultra-Fiber, eliminating any cross-contamination concerns.
Endotoxin Detection and Testing
Quality control and risk reduction at critical points in the manufacturing process includes routinely evaluating endotoxin limits. Endotoxin levels should also be checked after any process changes have been made (such as the introduction of new raw material or equipment).
There are 3 methodologies to detect or quantify endotoxins in compliance with EP, USP and ANSI/AAMI ST72 to meet FDA and MHRA requirements: Gel Clot, Turbidimetric and Chromogenic. All three methods utilize the same reagent, often referred to as Limulus Amoebocyte Lysate or LAL. The Gel Clot method is qualitative only, while Turbidimetric and Chromogenic are both quantitative and known for their low limits of detection.
Depyrogenation (Endotoxin Removal) Techniques
Common depyrogenation methods include using dry heat to destroy endotoxins through high temperature exposure, and filtration to remove endotoxins from solutions through adsorption and size exclusion. Heat-stable materials (glass for instance) can be subjected to dry heat because depyrogenation procedures are carried out at temperatures between 250°C and about 400°C. Dry heat depyrogenation has the added benefit of sterilizing the materials as it removes endotoxins. Depyrogenation by filtration can be used only on liquid solutions, suspensions, emulsions, and products made of proteins or peptides. The destruction of the finished product makes using dry heat or filtration in many situations an impractical choice.
Endotoxins are a major component of the cell walls of gram-negative bacteria. They are toxic substances that stimulate the immune system and are a hazard to patient safety if present in medical products. In order to prevent contamination by endotoxins in sterile manufacturing, it is important to maintain upstream control of endotoxin levels in raw materials, (water in particular), and packaging. Maintaining strict aseptic procedures during the manufacturing process, as well as using cleaning products designed to pick up endotoxin-level debris, are added safeguards. Finally, depyrongenation techniques can be employed to directly remove endotoxins, however this may result in destruction of the final product itself. As in the healthcare industry that pharmaceutical manufacturers serve, prevention is far better than cure.
[i] Data on File, Benchmark Products
- FDA Bacterial Endotoxins/Pyrogens Inspection Guide 2014 (current): https://www.fda.gov/inspections-compliance-enforcement-and-criminal-investigations/inspection-technical-guides/bacterial-endotoxinspyrogens
- Endotoxin Detection Methods for Pharmaceuticals and Medical Devices: https://www.rapidmicrobiology.com/test-method/endotoxin-detection-methods-for-pharmaceuticals-and-medical-devices