Sterilization and Preservation by Radiation
John R. Logar
Elaine Daniell
The use of radiation for industrial application requires a multidisciplinary approach. There is a materials related component regarding the determination of the radiation effect (ie, cross-linking or degradation) of the product and/or packaging materials; a physicsrelated component regarding the determination of the absorbed-dose distribution throughout the product loading configuration used in the irradiation process; and, for sterile products, there is a microbiology-related component regarding the determination of the radiation resistance of the microbiological population on the product. The three main industrial applications of ionizing radiation are the sterilization of health care products, modification of materials, and the preservation of food. The industrial application of radiation for the sterilization of health care products is widespread, and its application is well established because it has been proven to be economical and reliable. Sterilization is used for the elimination of microorganisms on health care products to greatly reduce and/or eliminate the potential for patient infection from the sterilized product. Radiation sterilization uses energy from a radiation source to pass through the material to deactivate the microorganisms present. By contrast, the industrial application of radiation for both the preservation and enhanced microbial quality of food has experienced limited application due to consumer confidence in the process (benefit versus impact) even though the process is very similar to food pasteurization (the reduction and elimination of certain pathogenic microorganisms such as Listeria, Escherichia coli, and Salmonella). The third application of ionizing radiation is focused on the modification of materials by scission or cross-linking chemical bonds to deliver a predetermined effect and create an alternate form of the material (eg, cross-linked polyethylene, wire coating). For this chapter, we primarily focus on the use of ionizing radiation for the sterilization of health care products and the modification of materials used in health care products.
The history of processing with radiation in the health care industry began in the early 1950s. The first attempts were with electron beam irradiation, but due to the unreliability of the equipment available at the time, this method was abandoned. It was not until the advent of the use of cobalt 60 (60Co) isotope in the early 1960s that routine processing with radiation truly began. From the 1960s to today, there have been continual advances in the design and reliability of the irradiation equipment and thus the continued expansion in the use of both gamma and electron beam radiation applications. Additionally, in the late 1990s, with the development of high energy, high-current electron beam technology, the radiation processing industry renewed its interest in the use of electron beam and X-ray irradiation. That interest and expansion continues today. As a result, there are many types of irradiator designs and applications available to the health care industry that enables the selection of the most effective and efficient process for products and applications.
SOURCES OF RADIATION ENERGYIn general, radiation may be classified into two groups, electromagnetic and particle radiation. For the various types of radiation in the electromagnetic spectrum, they produce antimicrobial effects by transferring the energy of an electron or photon into characteristic ionizations in or near a biological target. In addition to creating pairs of positive and negative electrons, ions can also produce free radicals and activated molecules. These effects, which are produced without any appreciable rise (compared to other sterilization modalities) in temperature, have been termed cold sterilization because it relates to the destruction of microorganisms.
Electromagnetic Radiation
Of the types of electromagnetic radiation used for the destruction of microorganisms, microwave, ultraviolet, gamma, and X-rays, only the latter two are dealt within this chapter (for others, see chapters 9 and 10). The X-ray and gamma radiation, although nearly identical in nature, have different origins. The emission of an X-ray from an atom occurs when there is a transition of an electron from an outer shell to a vacancy in an inner shell; this is produced by bombarding a heavy metal target (eg, tantalum) with high-energy electrons from an electron beam accelerator. Gamma radiation is the result of a transition of an atomic nucleus from an excited state to a ground state, as seen in most radioactive materials, yielding electromagnetic radiation. The difference between gamma rays and X-rays are the wavelength frequency and energy spectrum of the emitted photons. X-rays and gamma rays have essentially the similar penetrating power because they carry no charge; however, the maximum energy of the electrons used to create the X-rays creates a subset of higher energy photons that will result in greater penetration.
The radioisotopes that are typically used for gamma processing are 60Co and cesium 137 (137Cs). The 60Co is produced as an intentional by-product of nuclear reactors used to produce electricity. It is formed when naturally occurring 59Co absorbs an additional neutron, creating the radioisotope 60Co. This radioisotope has a half-life of 5.27 years and decays with the emission of two highenergy gamma rays (1.17 and 1.33 MeV) and a lower energy (0.318 MeV) β particle to become the nonradioactive element nickel 60 (60Ni). Because of an isotope’s decay, it is necessary to periodically replenish the isotope to maintain a desired throughput capacity. The 60Co is the radioisotope most commonly used for industrial radiation sterilization due to its availability and effectiveness in delivering the intended effect.
The 137Cs is also produced as a nuclear reactor byproduct. It is formed during the separation of chemicals from other nuclear wastes and is then encapsulated. The 137Cs exists as cesium chloride, which is readily soluble in water. This radioisotope has a half-life of 30 years and decays with the emission of two β particles (0.51 and 1.17 MeV) and one gamma ray (0.66 MeV) to become barium 137 (137Ba). The lower energy gamma ray produced as 137Cs decays results in a reduction in penetrating power compared with 60Co. Typically, the capsules for storage of 137Cs are intended for dry storage, due to the repeated thermo-quenching required with wet storage and the increased risk of encapsulation failures. Therefore, this isotope is not currently used for the industrial radiation processing applications. Other radioisotopes have been investigated for industrial application but have not been developed further because of high production costs, limited commercial availability, low penetrating power, or short half-life.
TABLE 29.1 Machine classification, design, and application | |||||||||||||||||||||||||
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Particle Radiation
Particles usually considered of importance in radiation biology are the α, β, neutron, meson, positron, and neutrino. The only particle currently applicable to sterilization is the β particle or electron. The α particles, although capable of causing dense ionizations, have limited penetrating ability; neutrons, which are uncharged, have great penetrating power but are unacceptable because they can induce radioactivity. Mesons and protons are produced only by expensive, high-energy machines and are therefore not used for industrial application. The β radiation, arising from the decay of an isotope source, consists of electrons with a single negative charge, a low mass and low energy; because of their low energy, these electrons cannot penetrate materials deeply.
Electrons produced by machines can be emitted or accelerated to a predetermined energy. The higher the energy electron produced, the greater the penetration into materials, products, and packages. Machines that are used in the acceleration of electrons vary in both their design and output with regard to energy and power. This flexibility enables the user to customize the machine to the application. The range of machine energy and power, and examples of their applications, are provided in Table 29.1. Electron beam emitters produce low-energy electrons in the range of 80 to 300 kV and machine-accelerated electrons fall into low-, medium-, and high-energy categories; 100 keV to 25 MeV. For machine-accelerated electrons, the accelerating field
is generated using direct current, radiofrequency or microwave energy. Once the electrons are accelerated to the desired energy, the beam can then be managed by magnetic fields to achieve such results as increasing its size, changing its shape, or scanning over a predetermined area.
is generated using direct current, radiofrequency or microwave energy. Once the electrons are accelerated to the desired energy, the beam can then be managed by magnetic fields to achieve such results as increasing its size, changing its shape, or scanning over a predetermined area.
TABLE 29.2 Units and conversion factors in radiation | ||||||||||||
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Radiation Units
The fundamental measurement parameter in radiation processing is the amount of energy deposited per unit mass, which is referred to as absorbed dose. The unit of absorbed dose is the gray (Gy), where 1 Gy is equivalent to absorption of 1 J/kg (Table 29.2). Dose measurements are typically related to the amount of absorbed dose in water. This relationship is used because most of the products (health care products) that undergo radiation processing are focused on delivering a dose to microbiological flora and thus the targeted density is similar to water. The dose equivalent is used to express the quantity of dose used in radiation protection; it expresses all radiation on a common scale for calculating the effective absorbed dose. The units of dose equivalent are the sievert and rem. The radiation dose used for sterilization is typically expressed in the kGy unit of measure.
EFFECTS OF IONIZING RADIATIONThe primary effect of the absorption of high-energy radiation is ionization in matter. Ionization is the product of either direct interaction of charged particles with matter that dislodges both ions and individual atomic particles or an indirect interaction of a photon with atoms that causes electrons to be ejected. Ionization is the transformation of individual atoms or molecules from an uncharged or stable state to a charged or excited state. It is the formation of these excited state particles and their interactions that alter materials exposed to radiation.
At any given time, all portions of a material are not equally subjected to the energy of ionizing radiation. Ionizing radiation is by nature discrete, and in passage through a material, the photons or electrons produce a number of localized events along their passage or track. Certain portions of the material exposed to radiation may experience a slight alteration, whereas an adjacent area is subjected to intense energy. Along a track, photons of energy ionize the material and can produce free radicals and excited atoms. Secondary electrons, if they possess sufficient energy, ionize or excite additional adjacent atoms, forming a spur of delta rays (Figure 29.1). The sequence of events along a track is therefore localized and intense, and the alterations in affected molecules can be severe. Examples of ionization include the radiolysis of water or the formation of oxygen radicals. This breaking of chemical bonds and formation of free radicals results in changes to the structures of both materials and microorganisms.
Effects on Material
Polymers are typically described as chain-like molecules that contain either a carbon or silicone backbone structure. In general, the longer the chain, the greater the strength of the polymer. Exposure to radiation causes breakage of this backbone structure, and subsequently one of three reactions can occur. The first type of reaction that might occur is simply a recombination of the structure that does not result in a physical change. The second type of reaction is cross-linking, which is the combination of multiple chains to produce even longer chains with greater interaction between chains. This type of reaction can result in a physical change toward greater strength and consequently a decrease in the elongation properties of the polymer. The third type of reaction is chain scission, where the broken chain is terminated by something other than the original backbone molecule. The free radicals that are produced by the irradiation process often terminate this structure, and this results in a physical change characterized by a decrease in strength and elongation. Other types of radiation-induced changes to polymers can include color changes and odor inducement. In the manufacture of health care products that are composed of polymers, either a radiation-resistant polymer can be used or antioxidants can be added to the polymer base to minimize the effects of radiation processing.1 The effects of radiation on metals are typically very small, especially at the doses necessary for sterilization.
The interaction of radiation with food also produces changes. At the doses required for food irradiation
(0.4-8 kGy), the changes are usually limited to nutritional quality, odor, taste, and color; however, the degree of change(s) is mostly related to the overall dose delivered (either no change up to significant change). The interaction with biological liquids, tissues, or drug components can also produce changes. At the doses used in the sterilization process, changes may occur that cause a degradation of the drug content or functional aspects of the biological liquids or tissue such as protein-based substances (eg, collagen, human or recombinant albumin, tissue). Radiation may modify these by breaking of the protein chains and disrupting the ability of the amino acid to combine with other polymers, which can change the function of the material. Fluids may undergo changes when exposed to radiation, the type and degree of which are based on the chemical makeup of the fluid, the dose, and the dose rate of the radiation process. As with all other materials, free radicals are produced that combine to result in a change in the fluid. The most observed and easiest to quantitate change is that of pH. Reactions in fluids are completed in less time than those in solid materials, thus resulting in less additional change over time.
(0.4-8 kGy), the changes are usually limited to nutritional quality, odor, taste, and color; however, the degree of change(s) is mostly related to the overall dose delivered (either no change up to significant change). The interaction with biological liquids, tissues, or drug components can also produce changes. At the doses used in the sterilization process, changes may occur that cause a degradation of the drug content or functional aspects of the biological liquids or tissue such as protein-based substances (eg, collagen, human or recombinant albumin, tissue). Radiation may modify these by breaking of the protein chains and disrupting the ability of the amino acid to combine with other polymers, which can change the function of the material. Fluids may undergo changes when exposed to radiation, the type and degree of which are based on the chemical makeup of the fluid, the dose, and the dose rate of the radiation process. As with all other materials, free radicals are produced that combine to result in a change in the fluid. The most observed and easiest to quantitate change is that of pH. Reactions in fluids are completed in less time than those in solid materials, thus resulting in less additional change over time.
 FIGURE 29.1 Interaction of gamma rays and electrons with matter. |
LETHAL EFFECTS OF RADIATION ON MICROORGANISMSIntracellular Effects
Radiation can cause a wide variety of physical and biochemical effects in microorganisms. It is most likely that the primary cellular target governing loss of viability is the DNA molecule.2 The radiation sensitivity of 79 organisms, ranging from viruses to higher plants and animals, was correlated with their nucleic acid volume.3 The larger the nucleic acid volume, the more sensitive the microorganism was to radiation. It also appears that appreciable differences in radiation sensitivity may be the result of an ability to repair DNA damage or cellular protective properties such as the spore coats rather than an inherent radiation resistance of the DNA target.4,5,6,7 Such protective and repair mechanisms are known to be important in microorganisms with higher resistance to radiation inactivation.8 The damage to the DNA molecule by the radiation energy renders the microorganism unable to replicate, thereby eliminating the ability of the cell to remain viable.
For the purpose of this discussion, microbial survival is defined as the ability of a microorganism either to reproduce significantly in nutrient broth or to produce a colony-forming units (CFUs) on a recovery medium after exposure to radiation.
Survival Curves
The destruction or inactivation of microorganisms by radiation occurs in geometric progression. In other words, the same fraction of microorganisms is inactivated with application of successive increments of radiation; therefore, the radiation effect is cumulative. Graphing the logarithm of the surviving organisms against the amount of radiation dose can best plot this relationship. Some representative types of curves are shown in Figure 29.2. Curve 1 represents the exponential relationship previously described.9 Curve 2 is representative of organisms
that exhibit a shoulder effect before exponential inactivation, which may be due to repair mechanisms. Examples of these organisms are Deinococcus (originally Micrococcus) radiodurans10 and radiation-resistant mutants of Salmonella ser Typhimurium.4 These curves are indicative of typical homogeneous populations.
that exhibit a shoulder effect before exponential inactivation, which may be due to repair mechanisms. Examples of these organisms are Deinococcus (originally Micrococcus) radiodurans10 and radiation-resistant mutants of Salmonella ser Typhimurium.4 These curves are indicative of typical homogeneous populations.
 FIGURE 29.2 Schematic diagram of survival curves. The initial number, N0, of microorganisms was 1000 colony-forming units. Curve 1 represents an exponential relationship, and curve 2 shows a shoulder effect before the exponential relationship is achieved. |
The number and types of microorganisms found on products before sterilization is typically referred to as the bioburden. Bioburden typically consists of a heterogeneous population of microorganisms, with a mixture of minimally to highly resistant microorganisms based on microorganism type and product environment (see the following text and chapter 64). The number of each type of microorganism varies based on raw materials, control of the manufacturing environment, type of manufacturing process (manual or automated), and geographic location of the manufacturing site. The resistance of a heterogeneous population is typically evaluated by eliminating the minimally resistant population of microorganisms using a low radiation dose and then characterizing the radiation resistance of the remaining population of highly resistant microorganisms. For populations of microorganisms that have a radiation resistance similar to curve 1 in Figure 29.2, the D10 value can be obtained from any portion of the straight line. The D10 value in this case is defined as the radiation dose required to kill 90% of a homogeneous microbial population, where it is assumed that the death of microorganisms follows first-order kinetics. For populations of microorganisms that have a radiation resistance similar to curve 2, the D10 value can be best obtained from the straight-line portion. To determine the extent of radiation treatment, the shoulder effect may need to be considered.
The primary method for determining a D10 value is that of Stumbo et al. (1950).11 This method adapted the most probable number (MPN) technique for obtaining the D10 value and derived an equation capable of describing either inoculated or indigenous microorganisms. From the MPN equation,12
where [x with bar above] is the MPN of surviving organisms per unit tested, n is the number of units irradiated, and q is the number of units rendered sterile. The D10 value is derived by using the equation
This model assumes exponential inactivation.
Radiation Resistance
The radiation resistance of microorganisms can be altered, either increased or decreased, by altering the environment that exists during irradiation. Representative factors that alter the radiation resistance are presented in Table 29.3. For example, if the radiation conditions are such that the supply of oxygen is not limited, the resistance of the organism can be decreased. In other words, if the supply of oxygen is limited for an aerobic organism or an anaerobic environment exists, the resistance of the aerobic organism may be increased.
Although many external factors may influence the radiation resistance of various microorganisms, in general, microorganisms can be grouped based on their relative resistance (Table 29.4). Bacterial spores, with few exceptions, are generally the most resistant to radiation, gram-negative rods are the least resistant, and yeast and fungi have an intermediate resistance. There are specific organisms that have been found to show resistance to radiation such as Roseomonas mucosa and D radiodurans.8 In some cases, the manufacturing environment may lead to increased resistance such as a harsh dry environment or freezing of product may increase resistance of spore-forming organisms and a bacterial biofilm in water sources or liquid vessels
may increase organism resistance due to repeated contact with chemical disinfectants. This increased resistance may require a radiation pretreatment of raw materials or components to mitigate resistance developed by the microbial flora. Alternatively, another sterilization method might be selected for pretreatment of raw materials or components, depending on the microorganism observed and the root cause of the increased radiation resistance.
may increase organism resistance due to repeated contact with chemical disinfectants. This increased resistance may require a radiation pretreatment of raw materials or components to mitigate resistance developed by the microbial flora. Alternatively, another sterilization method might be selected for pretreatment of raw materials or components, depending on the microorganism observed and the root cause of the increased radiation resistance.
TABLE 29.3 Factors that modify radiation resistance | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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MICROBIAL VALIDATION AND ESTABLISHMENT OF DOSETypically, the product radiation sterilization dose is validated using the published methods by International Organization for Standardization (ISO)28,29 along with other technical publications.30,31 The method chosen for establishing the sterilization dose may be based on the natural
bioburden population count or bioburden resistance. Method 1 and VDmax are both based on the bioburden count that fall within the microorganism standard distribution of resistance (SDR). The SDR specifies resistances of microorganisms in terms of D10 values and the probability of occurrence of values in the total population.29 For families of products where the product microbial resistance is less than or equal to the SDR, method 1 may be used to validate a lower sterilization dose; however, if groups of products need to run together at a standardized dose or
products are of high value (eg, implants), then the VDmax method that requires less samples may be more favorable. If the product microbial resistance is not less than or equal to the SDR, then the method must be based on the determination of microbial resistance, which would require the use of method 2. The method of choice may also be governed by the functional dose limitations of the product due to material compatibility that would require a dose establishment method capable of qualifying a lower sterilization dose such as method 1 or method 2.
bioburden population count or bioburden resistance. Method 1 and VDmax are both based on the bioburden count that fall within the microorganism standard distribution of resistance (SDR). The SDR specifies resistances of microorganisms in terms of D10 values and the probability of occurrence of values in the total population.29 For families of products where the product microbial resistance is less than or equal to the SDR, method 1 may be used to validate a lower sterilization dose; however, if groups of products need to run together at a standardized dose or
products are of high value (eg, implants), then the VDmax method that requires less samples may be more favorable. If the product microbial resistance is not less than or equal to the SDR, then the method must be based on the determination of microbial resistance, which would require the use of method 2. The method of choice may also be governed by the functional dose limitations of the product due to material compatibility that would require a dose establishment method capable of qualifying a lower sterilization dose such as method 1 or method 2.
TABLE 29.4 Radiation resistance of microorganisms and other biological units | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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