Moist Heat Sterilization

This course provides delegates with a comprehensive understanding of moist heat sterilisation processes from theoretical foundation through to the practical aspects of validation and biological indicators.

The course highlights the GMP requirements and current industry expectations for the routine operation, monitoring and control of sterilisation processes and gives practical examples of how these techniques are applied through appropriate engineering to ensure reliability in full compliance with all European, US and other Global regulatory requirements.

Importance moist heat sterilization topics also covered by this training course include:

Important definition and Understand SAL (Sterility Assurance Level)
Application of key terms including D-value, z-value, physical (F0), biological lethality (FBIO)
Spore Log Reduction (SLR), Probability of a Non-Sterile Unit (PNSU)
Steam quality and equilibration time
Considerations in the use of steam and super heated water for sterilization processes
Fundamentals in the selection and use of biological indicators and chemical indicators
Selection of a sterilization process type based on product attributes
Design considerations for key and critical parameters
Use the overkill approach and product specific approach
Use of master site and master solution approach
Use of bracketing approaches to optimize process qualification activities
Sterilizer equivalency
Impact assessment, Change Control, Investigation and CA-PA

Important definition and Understand Sterility Assurance Level

Bioburden: Population of viable microorganisms on or in raw materials, products, and labeling/packaging materials determined before sterilization.

Biological indicator (BI): A microbiological test system providing defined resistance to a specified sterilization process under defined conditions, which is used as an indicator of sterilization cycle efficacy.

Chemical indicator (CI): Test system that reveals fluctuation(s) in one or more predefined process variables based on a chemical or physical change resulting from exposure to a sterilization process.

D value: A value indicating the extinction rate of microorganisms killed under defined conditions. The D value is defined as the time or radiation dose required to inactivate 90% of a
population (one tenth of the starting value) of the test microorganism under stated exposure conditions.

Endotoxin: Lipopolysaccharide contained within the outer membrane of Gram-negative bacteria that may lead to pyrogenic reactions and other biological activities in humans.

F0 value: The number of equivalent minutes of steam sterilization at a temperature of 121.1°C delivered to a container or unit of product, calculated using a z value of 10.

Overkill sterilization: A sterilization process providing a sterility assurance level (SAL) of less than 10^-6, regardless of the bioburden count in the product being sterilized or the resistance of objective microorganisms to the sterilization. The process generally provides at least a 12-log reduction of indicator microorganism having a minimum D value of 1.0 minute

Sterile: Free from viable microorganisms

Sterility assurance level (SAL): Probability of viable microorganisms being present on a product unit after exposure to the proper sterilization process, normally expressed as 10^-n.

Sterility Assurance Level: The SAL for a given sterilisation process is expressed as the probability of micro-organisms surviving in a product item after exposure to the process. An
SAL of 10^-6, for example, denotes a probability of not more than 1 non-sterile item in 1 × 106 sterilised items of the final product.

Sterility: Sterility is the absence of viable microorganisms, as defined by a sterility assurance level equal to or less than 10^−6. The inactivation of microorganisms by physical or chemical means follows an exponential law; thus there is always a finite statistical probability that a micro-organism may survive the sterilising process. For a given process, the probability of survival is determined by the number, types and resistance of the microorganisms present and by the environment in which the organisms exist during treatment.

Sterilization: A process that destroys or eliminates all viable microbes to render a product free from viable microorganisms.

Sterilization cycle: A series of sterilization processes to be performed within a closed sterile chamber, consisting of dehumidification; conditioning; and the injection, product exposure, and removal of sterilizing agent.

Terminal sterilization: A process whereby a product is sterilized in its final container or packaging, which permits the measurement and evaluation of quantifiable microbial lethality. In
principle, the SAL should be less than 10^-6.

z value: Temperature change that results in a ten-fold change of the D value.

Biological indicators: Biological indicators are test systems containing viable microorganisms (usually spores of bacteria) that provide a defined challenge to verify the required effectiveness of a specified
sterilisation process.

CFU: A microbiological term that describes the formation of a single macroscopic colony after the introduction of one or more micro-organisms to microbiological growth media. One colony forming
unit is expressed as 1 CFU.

F0 value: The F0 value of a saturated steam sterilisation process is the lethality expressed in terms of the equivalent time in minutes at a temperature of 121 °C delivered by the process to the load in its container with reference to micro-organisms possessing a theoretical Z-value of 10. A process with a lethality of F0BIO > 12 minutes. For example a process that provides at least a 12 log reduction of biological indicator microorganisms having a minimum D value of 1 minute.

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Application of key terms including D-value, z-value, physical (F0)

D-value, z-value, and F0 (pronounced F-zero) are key terms commonly used in the field of food microbiology and food processing. They are important parameters for understanding and optimizing the thermal processing of food to ensure safety and quality. Here’s a brief explanation of each term and its application:

1. D-value (Decimal Reduction Time):

– Definition: The D-value represents the time required at a specific temperature to achieve a 10-fold reduction (1 log cycle reduction) in the population of a specific microorganism.

– Application: D-values are crucial for determining the lethality of a heat treatment process. They help in designing thermal processes to reduce the microbial load in food products. The higher the D-value, the more heat-resistant the microorganism.

2. Z-value:

-Definition: The Z-value is the change in temperature required to cause a tenfold (1 log cycle) change in the D-value.

– Application: Z-values are used to predict how a microorganism’s heat resistance changes with temperature. In other words, the Z-value helps in estimating the impact of temperature fluctuations on the microbial destruction rate. A higher Z-value indicates that the microorganism is more sensitive to temperature changes.

3. F0 (F-zero):

– Definition: F0 is a term used in the context of thermal processing and sterilization. It represents the equivalent time at a reference temperature (usually 121.1°C) that is equivalent to the lethal effects of a specific heat treatment at a different temperature.

– Application: F0 is used in the calculation of thermal processes, especially in autoclaves and retort systems, to ensure the safety of canned and processed foods. It allows for the standardization of thermal processes at different temperatures, taking into account the cumulative lethal effects of heat over time.

In summary, these terms are critical in the design and validation of thermal processes in the food industry, helping to ensure the safety and quality of processed foods by understanding and controlling the microbial population through heat treatments.

Biological lethality (FBIO), Spore Log Reduction (SLR), Probability of a Non-Sterile Unit (PNSU)

Biological lethality (FBIO), Spore Log Reduction (SLR), and Probability of a Non-Sterile Unit (PNSU) are terms commonly used in the context of microbial reduction and sterilization processes. These terms are often associated with the validation of sterilization processes, particularly in pharmaceutical and healthcare industries. Let’s break down each term:

1. Biological lethality (FBIO):

– Definition: Biological lethality (FBIO) refers to the cumulative lethal effects of a sterilization process on the microbial population. It is a measure of the overall effectiveness of a sterilization method in reducing or eliminating microorganisms.

– Application: FBIO is used to assess the success of a sterilization process, especially in situations where a specified level of microbial reduction is required to ensure product safety. It takes into account the time and temperature parameters of the sterilization process.

2. Spore Log Reduction (SLR):

– Definition: Spore Log Reduction (SLR) represents the logarithmic reduction in the number of viable spores (typically bacterial spores) achieved by a sterilization process. It is a measure of the effectiveness of a sterilization method in killing or inactivating spores.

– Application: SLR is commonly used in the validation of sterilization processes, particularly in the pharmaceutical industry. For example, if a process achieves a 3-log reduction in spore count, it means that the process has reduced the spore population by a factor of 1,000 (10^3).

3. Probability of a Non-Sterile Unit (PNSU):

– Definition: Probability of a Non-Sterile Unit (PNSU) is a statistical measure that quantifies the likelihood of a unit or item not being sterile after undergoing a sterilization process. It is expressed as a probability or percentage.

– Application: PNSU is used to assess the reliability and assurance level of a sterilization process. A lower PNSU indicates a higher level of confidence that the sterilization process has effectively rendered the units sterile. The calculation of PNSU takes into account factors such as the initial bioburden, the microbial reduction achieved by the sterilization process, and the statistical variability associated with the process.

In summary, these terms are essential in the validation and verification of sterilization processes, ensuring that they meet the required microbial reduction levels and provide a high degree of confidence in the sterility of the treated products or units.

Steam quality and equilibration time

Steam Quality:

Steam quality refers to the degree of dryness or the fraction of steam in a mixture that is in the vapor phase. It is expressed as a percentage and indicates the amount of vapor (dry steam) present in the total steam mixture. Steam can exist in different qualities, ranging from wet steam (containing significant liquid water) to dry steam (comprised mostly of vapor). The quality of steam is important in various industrial processes, especially those that involve heat transfer, as it affects the efficiency of heat exchange equipment.

In the context of steam quality, there are two key terms:

1. Dryness Fraction (x):

– The dryness fraction (x) is a measure of the steam quality and represents the ratio of the mass of vapor to the total mass of the steam (vapor + liquid). It is expressed as a fraction or percentage.

– For dry steam, the dryness fraction is 1 (or 100%), indicating that the steam is entirely in the vapor phase. For wet steam, the dryness fraction is less than 1.

2. Superheated Steam:

– Superheated steam is steam that has been heated to a temperature higher than its saturation temperature at a given pressure. It contains only vapor and no liquid phase.

Accurate control of steam quality is crucial in applications such as power generation, heating, and various industrial processes. Wet steam can lead to inefficiencies and potential damage to equipment due to water droplets impacting surfaces.

Equilibration Time:

Equilibration time refers to the time required for a system or substance to reach a state of equilibrium, where certain properties or conditions become constant. In the context of steam or heat exchange systems, equilibration time is relevant to ensure that the system stabilizes and operates consistently.

For example, when introducing steam into a heat exchanger or a system, it takes time for the temperature, pressure, and steam quality to reach a steady state. Equilibration time depends on factors such as the size of the system, the rate of heat transfer, and the control mechanisms in place.

Proper consideration of equilibration time is important in designing and operating steam-based systems to achieve optimal performance and efficiency. It ensures that the system reaches a stable condition where the desired steam quality is maintained, and heat transfer processes operate effectively.

Considerations in the use of steam and super heated water for sterilization processes

Principle of Steam Sterilization:

Steam sterilization, also known as autoclaving, is a widely used method for the sterilization of equipment, instruments, and other objects in various industries, including healthcare, pharmaceuticals, and laboratories. The principle behind steam sterilization is the use of high-pressure saturated steam to kill or inactivate microorganisms, including bacteria, viruses, and spores. The key steps in steam sterilization include:

1. Exposure to High-Temperature Steam: The items to be sterilized are exposed to steam at a high temperature, typically around 121°C (250°F) or higher. The high temperature is maintained for a specific duration to ensure the destruction of microorganisms.

2. Moist Heat Penetration: The presence of moisture (steam) enhances the efficiency of the sterilization process. Moist heat is generally more effective than dry heat in penetrating and killing microorganisms.

3. Denaturation of Proteins: The high temperature of steam causes the denaturation of proteins in microorganisms, leading to their destruction. The process is lethal to a wide range of microorganisms, making steam sterilization highly effective.

Benefits of Steam Sterilization:

1. Broad Spectrum of Microbial Destruction: Steam sterilization is effective against a wide range of microorganisms, including bacteria, viruses, and spores. It is one of the most reliable methods for achieving sterility.

2. Penetration and Uniformity: Steam can penetrate porous materials effectively, ensuring that all surfaces of the items being sterilized are exposed to the sterilizing conditions. This is crucial for ensuring the complete destruction of microorganisms.

3. Moist Heat Advantage: The presence of moisture in steam enhances the microbial-killing effect compared to dry heat. Moist heat is particularly effective in disrupting the structure of bacterial spores.

4. Compatibility: Steam sterilization is suitable for a variety of materials, including glassware, metal instruments, rubber, and most heat-stable plastics. It is widely used in healthcare settings and laboratory environments.

Principle of Superheated Water Sterilization:

Superheated water sterilization is an alternative sterilization method that utilizes water at temperatures above its boiling point. The principle involves exposing items to superheated water, typically at temperatures ranging from 150°C to 200°C, under high pressure. The key steps in superheated water sterilization include:

1. Pressurized Superheated Water: Water is pressurized to prevent boiling at normal temperatures. The temperature of the water is then increased above its boiling point at atmospheric pressure, creating superheated water.

2. Exposure to Superheated Water: Items to be sterilized are exposed to the superheated water for a specified duration. The high temperature and pressure contribute to the destruction of microorganisms.

Benefits of Superheated Water Sterilization:

1. Reduced Processing Time: Superheated water sterilization can achieve sterilization more rapidly than traditional autoclaving methods, leading to shorter processing times.

2. Material Compatibility: Superheated water is less corrosive than steam, which may be beneficial for certain materials and instruments.

3. Energy Efficiency: Superheated water sterilization can be more energy-efficient compared to steam sterilization in some cases, as it may require less energy to heat water to superheated temperatures.

It’s important to note that the choice between steam and superheated water sterilization depends on factors such as the nature of the items to be sterilized, material compatibility, processing time requirements, and specific application needs. Each method has its advantages and considerations based on the unique characteristics of the sterilization process.

Fundamentals in the selection and use of biological indicators and chemical indicators

Biological indicators (BIs) and chemical indicators (CIs) are essential tools in sterilization and disinfection processes to ensure the efficacy of the procedures. Here are the fundamentals in the selection and use of these indicators:

Biological Indicators (BIs):

1. Purpose:

– Definition: Biological indicators are test systems containing viable microorganisms, usually spores, that are used to assess the sterilization or disinfection process’s effectiveness.

– Purpose: BIs provide a direct measure of the lethality of the process on living organisms, making them highly reliable indicators of microbial destruction.

2. Selection Criteria:

– Resistant Microorganisms: Choose BIs with microorganisms that are resistant to the specific sterilization method being used (e.g., bacterial spores for steam sterilization).

– Relevance: Select BIs that are relevant to the application (e.g., Geobacillus stearothermophilus spores for steam sterilization).

3. Placement:

– Challenging Areas: Place BIs in the most challenging areas within the load, where microbial kill may be the most difficult to achieve.

4. Incubation and Reading:

– Incubation Conditions: Follow recommended incubation conditions and temperature to allow the surviving microorganisms to grow.

– Reading Time: Adhere to recommended reading times for accurate interpretation of results.

5. Interpretation:

– Positive Result: Growth of microorganisms indicates a failure in the sterilization process.

– Negative Result: No growth suggests successful microbial destruction.

Chemical Indicators (CIs):

1. Purpose:

– Definition: Chemical indicators are substances that undergo a chemical or physical change, usually a color change, when exposed to specific conditions.

– Purpose: CIs provide a visual indication that the items have been exposed to the sterilization process, but they do not provide information about microbial destruction.

2. Types of CIs:

– Class 1 – Process Indicators: Monitor exposure to the sterilization process but do not confirm microbial kill. Examples include indicator tapes and strips.

– Class 2 – Bowie-Dick Test: Specific for steam sterilization and used to detect air removal issues in pre-vacuum cycles.

– Class 3 – Integrators: Monitor both time and temperature during the sterilization process, providing a more comprehensive assessment.

3. Placement:

– Externally Visible: Place CIs where they are externally visible on the packaging to provide a quick visual indication.

4. Interpretation:*

– Color Change: A color change in the CI indicates exposure to the sterilization process. The specific color change varies based on the type of CI used.

5. Complementary Use:

– Combine with BIs: Chemical indicators are often used in conjunction with biological indicators to provide both a visual and biological confirmation of the sterilization process.

6. Frequency of Use:

– Every Load: Process indicators are typically used with every load to verify that items have been exposed to the sterilization process.

In summary, the selection and use of biological indicators and chemical indicators are critical for ensuring the effectiveness of sterilization and disinfection processes. Biological indicators provide direct evidence of microbial destruction, while chemical indicators offer a visual confirmation of exposure to the sterilization process. The choice between them often involves a combination to provide a comprehensive assessment of the sterilization process.