Sterilization Methods and Pyrogen Testing
Qualitative vs. Quantitative Analysis
Qualitative Analysis: Results are expressed descriptively or in terms of presence/absence.
Quantitative Analysis: Results are expressed numerically.
The interpretation of an analysis can only be completely correct if the sample is representative. Both sampling and sample preparation are different for each type of analysis and each type of material to be analyzed. However, all methods must:
- Reflect the microbiological conditions of the lot.
- Be collected aseptically in sterile containers and protected from accidental contamination.
- Be kept refrigerated or frozen depending on the type of sample.
Cleaning, Disinfection, and Sterilization
Cleaning: A chemical and physical procedure that aims to remove any foreign material from the object being cleaned.
Disinfection: The elimination of pathogenic microorganisms.
Sterilization: Eliminates any form of life, including spores.
Sterilization Methods
Humid Heat
This method uses steam to denature and coagulate proteins. It is carried out using an autoclave, which sterilizes using pressurized steam. The most widely used method is the “Chamberland” method, which involves sterilizing the material with steam at 120°C and one atmosphere of pressure for 20-30 minutes.
Advantages:
- Fast heating and penetration.
- Destruction of bacteria and spores in a short time.
- Does not leave toxic residues.
- Economical.
Disadvantages:
- Cannot sterilize solutions that form emulsions with water.
- Corrosive to certain metal elements.
Dry Heat
This method dehydrates cells, causing their electrolytes to reach toxic concentrations. It also denatures lipids and proteins. However, a higher temperature is needed compared to moist heat: ovens are heated to between 140-170°C for 2-4 hours. It is used to sterilize glass material, such as pipettes, and other materials resistant to dry heat.
Advantages:
- Not corrosive to metals and instruments.
- Allows the sterilization of powder products, non-aqueous substances, and non-volatile viscous substances.
Disadvantages:
- Requires more time due to low penetration capacity.
Radiation
- Ultraviolet rays: Affect DNA molecules but have poor penetration. Mainly used for surface sterilization, such as operating rooms.
- Ionizers: Produce ions and free radicals that alter the structure of nucleic acids, proteins, and other essential components for the viability of microorganisms.
- Gamma rays: Highly penetrating and require specific conditions. Obtained from the disintegration of the cobalt-60 isotope.
Disadvantages of Radiation:
- Requires special structures.
- Radiation is cumulative, so only single-use materials are sterilized.
- Incompatible with other sterilization methods.
Filtration
This is the main method for sterilizing thermolabile solutions. Filtration through membranes retains microorganisms due to the small size of the filter pores and adsorption to the pore walls.
Disadvantages:
- While filtration achieves high levels of sterility, the sterility of the final product also depends on subsequent aseptic processing.
Ethylene Oxide
Ethylene oxide (C2H4O) is a cyclic ether with a boiling point of 10.7°C. It evaporates easily, has high penetration power, and is easily removed after treatment.
Aldehydes
Mechanism of action: Protein coagulation and alkylation, causing irreversible enzymatic structural changes that destroy microorganisms and spores.
Advantages:
- Low temperature.
- Short duration cycles.
- Certifiable.
Disadvantages:
- Incompatible with some materials.
- Not approved in the US.
- Toxic and carcinogenic.
Hydrogen Peroxide/Plasma Gas
This method exposes the material to hydrogen peroxide in the plasma phase for 55 minutes. It destroys microorganisms by oxidation without leaving toxic residues.
Advantages:
- Quick method (45-55 minutes).
- Automatic equipment.
- Non-toxic to personnel and the environment.
Disadvantages:
- Does not eliminate prions.
Sterilization Control
The effectiveness of sterilization methods is checked using chemical and biological indicators. Physical tests are conducted using thermometers, manometers, loading sensors, valves, and registration systems.
- Chemical indicators: Devices containing one or more chemical substances that change color or state. They are classified into six categories. External indicators, which respond to only one critical point of sterilization, are no longer used.
- Biological indicators: Devices prepared from non-pathogenic, highly resistant spores. They check for the destruction of specific microorganisms known to be the most resistant to the sterilization process being tested. A vial containing these bacteria is commonly used to perform a mandatory weekly test in steam autoclaves.
Sterilization Failure
Sterilization failure can be caused by:
- Equipment malfunction.
- Poor steam quality.
- Inadequate relative humidity in the processing area.
- Incorrect packaging type and method.
- Improper load configuration.
- Inappropriate cycle parameters for the load.
Sterility Testing
The sterility test applies to medicines considered sterile and must be performed under aseptic conditions. This test is not required for products subjected to terminal sterilization methods like steam, dry heat, or ionizing radiation. However, it is mandatory for products processed aseptically, sterilized by filtration, or sterilized by gas.
Sample analysis can be performed by filtration or direct inoculation onto a culture medium. Filtration is carried out under aseptic conditions. Once filtered, the membrane is transferred to the culture medium, or if the filtration instrument allows, the culture medium is added directly onto the membrane.
Pyrogen Testing
In Vivo Test
Variation in the rectal temperature of rabbits: This test involves the intravenous inoculation of the final product into three rabbits weighing over 1.5 kg. These rabbits must have been raised in special, highly controlled conditions to avoid false positives.
In Vitro Test
Limulus amebocyte lysate (LAL) assay for bacterial endotoxins: Horseshoe crabs (Limulus polyphemus) have amebocytes in their blood that can detect pyrogens, especially those from Gram-negative bacteria, which are the most significant source of bacterial endotoxins.
Products Requiring Pyrogen or Bacterial Endotoxin Testing
- Injectable medical specialties (solutions, powders, freeze-dried products, etc.).
- Water for injectables, eye drops, and products administered by inhalation.
- Raw materials of injectable quality (dextrose, mannitol, antibiotics, amino acids, etc.).
- Products such as plastic containers, elastomeric fences, and syringes associated with products for parenteral use.