STERILIZATION METHODS

We may define sterilization as any process that is carried out for eliminating or killing microorganisms, such as spore forms, viruses, bacteria, fungi, excluding prions from the food, equipment, surface, biological or medication culture medium. Sterilization is widely used in the surgical and medical fields. As surgical gloves have direct contact with sterile body tissue and blood stream, sterilization is necessary. Medical and implantable devices also employ sterilization. How the medication or surgical instruments are used in tissues, bone, blood, or skin decides the necessity of sterilization. Parenteral drugs demand high SAL (Sterility Assurance Level). The material being sterilized and sterilization’s purpose directs the method of sterilization that will be used. In order to prevent any mishap, different devices and materials are used, which in turn alters the method of sterilization too. pressured vapor sterilization and dry heat sterilization are the two major methods of sterilization.
Nevertheless, we have seen the advent of alternative surgical methods since 1950s as the technology in medical field has developed. Alternative sterilization methods, such as e-beam sterilization, gamma radiation sterilization, peracetic acid sterilization, gas plasma (H2O2) sterilization, formaldehyde sterilization, and ethylene oxide (EtO) sterilization developed after 1980s as the medical field advanced in the noninventional surgical methods. Methods like aceptic processing, sterilization by filtration, sterilization by ionizing radiation, gas sterilization, dry-heat sterilization, and steam sterilization are the components of pharmacopoeia (such as EP 5, BP, USP 30). Drugs’ sterilization also employs them. Nonetheless, numerous materials use various methods of sterilization. Table 1 depicts the merits and demerits associated with different sterilization methods.
Methods of sterilization are numerous and their efficacy depends on various factors e.g. type of organic protective material against sterilization and its amount in microorganisms, different types and number of the microorganisms, size and number of breaks in the instrument being used while the process is being carried out.
RADIOSTERILIZATION (RADIATION STERILIZATION)
Sterilization which is carried out with the help of gamma rays which is an ionizing radiation is called radiosterilization. These ionizing methods for sterilization were first used in 1895 and then its more frequent use started in the year 1921. The advantage of these radiations is that they can be used in final processing and containers of the drugs with minimal increase in the temperature. SAL of 10-6 is achieved by the reference dose of 25 kGy. Despite having its diverse uses, the radiolytic products formed at the end of the process alter the odor and color of the drug. This is because the process by which these radiations are produced is still unknown. From pharmaceutical perspective, for the drugs which are sensitive to heat, along with different methods of sterilization, radiosterilization is the method of choice. Techniques which are able to determine radio stability of any drug are only chromatographic.
The effects of radiations on the microorganisms and cells depend on dose rate, time of exposure and effects of the wavelength. Treatment of particles either with X-rays or gamma rays does not turn the materials into radioactive materials themselves. Type of particle, material on which the rays are being used and energy of the rays are the factors which may act on turning the material into radioactive. Particles with high energy and high penetration like neutrons can have these radioactive effects. Radiations can either act directly or indirectly on microorganisms. Ionizing the molecule via absorbing radioactive energy frankly is the direct effect. Primary target in this process is water molecule which is produced during the process. Free radicals H3O+ and OH- are the radiolytic products produced. OH- radicals have a known effect of damaging DNA having burly oxidant effects. O2 can also have some effects to the products.
When free radicals combine with the oxygen molecules they may start a chain of reactions and can also produce hydrogen peroxide. Numerous microorganisms die in the first place when they face DNA damage. Sometimes the damage is repaired because one of the filaments of DNA sustains its properties of ionization and exitation. Radiations can also cause formation of dimmers among pyrimidine base pairs. Covalent bonds are formed between thymine and cytosine bases or bacteria in both radiated and non radiated DNA. Presence of little quantity of water is the reason for the resistance of spores of bacteria. Therefore, spores are not damaged by OH- radicals. Formation of the dimmers among pyrimidine bases of a DNA chain is also another effect radiation on microorganisms. Covalent bonds were formed among thymine and cytosine bases in both non irradiated and irradiated chains of DNA. Protoplasm of bacteria have very scant amount of water. This is the main reason of resistance of spores of bacteria. Therefore, very little damage is done to the DNA of bacteria when they live in the form of spores. As compared to the bacteria, viruses aren't that sensitive to radiations. Among viruses some complex viruses are further less sensitive than single chain viruses of simple type. Various factors present throughput during the process of radiation influence the sensitivity of microorganisms. These factors include the pH, oxygen, temperature, ionic balance and water.
One major concern in radiation sterilization of a material is microbiological investigation. The concerns are set in the following order:
a. Prior to sterilization, the bioburden i.e. the microbiological contamination is found out.
b. Probing the microorganism’s radio sensitivity.
c. Terminal products’ complete and pure hold.
d. Biological indicator’s formulation and utilization.
e. Looking at the environment’s hygienic conditions.
Soy bean-casein medium is utilized for sterility test purposes and facultative anaerobic as well as aerobic microorganisms. Moreover, incubation for 14 days is carried out at 30-32°C. For other detailed microorganisms, different types of appropriate medium can be utilized for suitable conditions of incubation. For the purpose of sterilization, chemicals, high pressure, heat, filtration applications or irradiation can be used. Since radiation sterilization has numerous advantages it is used more often as compared to these sterilization methods. Such systems include gamma rays, subatomic particles, electronic beams i.e. e-beam and Ultraviolet (UV) light irradiation.
Gamma radiation sterilization:
Self breaks of Cesium-137 (137Cs) or Cobalt-60 (60Co) make up the gamma rays. This type of sterilization method is very common and high penetrating and is used for disinfecting liquid, solid, gaseous materials homogenous and heterogeneous systems, disposable medical equipment which includes needles, density materials, syringes, cannulas, i.v. sets and cosmetics. Except with acetal, polytetrafluoroethylene (PTFE) and polyvinyl chloride, it can be utilized on various materials and is a batch or on going process. The material’s thickness determines the entire penetration. It is not dependant on heat or chemicals and provides energy saving. According to the rules of protecting radiation, the major source of radioactive should be shielded for purposes of operators’ safety. The continuous releasing gamma rays reveal the needed storage.
Since sterilization is not required, there could be an immediate release soon after the process is completed. One of other advantage is that at the end of sterilization process there would be no residue. In coming sections, detailed discussion would be done on Gamma sterilization procedure.
E-beam sterilization
The most common method for sterilizing medical devices is E-beam sterilization e.g. gamma radiation sterilization. This could be made by using e-beams, which are found with the help of accelerator method and also by isotope method. One of the advantages of these methods is that it requires very little exposition time that depends upon 10MeV of electron with high energy. Effectiveness of sterilization highly depends upon high energy. The time required by accelerator method is about 15 min. On the other hand, the time required for isotope method is at least 24 hours. In isotope method a source with co isotope is normally used. Design specific machines are used for the purpose of producing and increasing the accelerating of elector. The process is continuous and it can also be applied on any kind of materials dependending upon its penetration. Since sterilization test is not required when the process concludes, an immediate release could be done. As compared to X-ray or gamma, it has high dose rate and there is no residue when the process is concluded. These are few of the advantages of E-beam sterilization. Due to the use of high dose rate there would be shorter exposure time as well as there would be a less changes of degradation of polymers. One of the limitations of e-beams is that there is less penetration as compared to X-ray and gamma. E-beam sterilization will be discussed in detail in upcoming sections.
Discuses below are other kind of radiation sterilization techniques other then the two methods mentions above.
X-rays
Bremstrahlung is a type of ionized energy that is used in x-rays. Through this high ionized energy many medical devices and large packages could be sterilized. The x-rays have the potential to sterilize several packages that are of low density, effectively with consistent ratios. However, the process does not involve any use of radio-active or chemical material. Currently the process cannot be used officially for medical devices and drugs.
UV light irradiation
This light is can only be used as germicidal lamp which can be used in the purpose of sterilization of various surfaces and other transparent objects. However it can never be used for sterilizing contaminated areas. Plastics can therefore not be considered as an official method for medical devices and drugs.
Subatomic particles
The particles can either be generated through a radioisotope or a device. It depends what type of particle it is the ability to penetrate mat always vary. It is again not a kind of method that could be officially used for medical devices and drugs.
GAMMA RADIATION STERILIZATION
Gamma radiations can sterilize not only the ingredients of pharmaceutical but the forms of final dosage as well the method of sterilization through gamma radiations was known as the industrial method of sterilization, as declared by USP 30, BP and EP 5. The benefits and the advantages that are gained through the process of gamma radiation could be defined.
1. Penetration

The raw materials or the final products can be easily sterilized when they are in their final packaging. These pharmaceutical ingredients would need terminal sterilization
2. Formulation of the product/package
Like all other packaged products, vials, delivery system of new drugs such as liposome, microspheres or other anti bodies could be easily sterilized successfully and effectively through irradiation. These materials also include syringes and sets of infusion etc. Since, the risk of diffusing the gas into the product or out of it is not involved here as it was there with sterilization along EtO and in multilayer materials, it can be utilized.
3. Easy Validation Process
It is quite simple to validate the process of radiation sterilization as it involves just one variable which is time. Time will vary only when a constant speed is maintained with 60Co source is decomposite. In each situation, in order to control the time span of conveyor as the source turns around time meters are utilized, after determining the required dose and placement of source. Significantly, validation is an easy process as sterilization is compared with vapor or gas and control of many issues has to be maintained.
4. Guarantee After Process
The conformation of the results has been indicated by dosimetry systems which are used throughout the process and after the completion of it. Since, the product’s absorbed dose has been demonstrated by this system, so sterility test is not required. Once sterilization is done, no further process is required before delivering the product to the customer.
5. Decreasing the Endotoxin Level
Through sterilization of gamma radiation this can be attained and it can be applied to toxic hood gases, drogs, drugs, animal feeds. In spite of having various uses, for numerous medical instruments sterilization, gamma radiation sterilization can be very useful. Group (24) can be denoted for these medical instruments. Air filters, test tubes, urine analysing tubes, petri plaques, vaccine vehicles, brushes, masks, rubbers etc are the material which have several medical utilities. Drains, speculums, hermodialyses, air tubes, clips, syringes, adhesive tapes, surgical sets, gloves, sutures, pets etc. are the surgical instruments or that are exposed directly to the patients.
Implants and machine used for short time or for long time for example artherio-venous shunts, periton dialysis sets, aortic valves, peripheralvascular prothesis, dental implants, artificial eyelids, joint prothesis.
E-BEAM RADIATION
E-beam irradiation techniques are getting more popular for sterilization of medical instruments ,they have many benefits like they are safe, without any emission with highly pace dealing out .Although low intensity medical ornaments can be cleaned by e-beam sterilization normally, highly intensity medical devices like vessel bases can be cleaned with high competency constant e-beam .
The capacity to manage the energy levels through and within beams is the logics for adopting of the procedures. Though the first time electronic beam was used in 1950s in sterilization, its common use as sterilization was properly started in 1970s. In 1960s e-beam was initiated to be used in medical packaging as a secure method. From that time these procedures were initially adopted in medical field depending on being companionable with quantity of other material. It can be used for increasing some kind of material. In this system, electrons are concerted and increased as much as higher as of light speed which makes quick reaction on molecules or microorganism on the product or sample that will be cleaned and sterilized. Product which went through e-beam at a particular pace, which helps messenger or card system to get the required electron dosage for sterilization process. Through this way, a connected association can be acquired for product. Thickness and magnitude of the product is dependent on the energy of the electron and their intensity.
The difference between E-beam irradiation and sterilization of gamma radiation is just of less piercing and more dosage ratio in spite of these both are similar in terms of energy ionization. One more distinction among them is the usage of e-beams in production of electricity as it can produce chargeable electrons.
The electrons formed by the accelerator of e-beams are some time uninterrupted but few times they are quiver. The cause behind the desolation of DNA chain is the preoccupation of electron by the product that is a part of e-beam sterilization and also they are behind the changes occurred in chemical and molecular bonds. Product sterilization needs such electrons that have more energy so that they can easily penetrate in product. Packaging material also depend on compactness and size. Second thing that is more considerable is irradiation as it can harm the packaging material due to its high efficiency level. This collapse gives birth to radicals that are made by polymers and name as “chain scissioning”. This takes only seconds to be happened.
The characteristics of e-beams can be gathered in a sequence;
The process of e-beam sterilization is endorsed from FDA and also identified and approved by international standards organizations,
It has an ability to pass from different material of product packaging like foils,
There is no harm on packaging that have sterile seals,
It becomes easy to manage temperature during process of irradiation.
Restrained dose assortment can be attained,
The institution needed for e-beam sterilization are expensive to be built instead of this its process is productive in relating to cost,
This process is high speeded as it take very short time that influence the effectiveness of routine methodology and also can harm shippable and sterilized goods,
-With a quick pace it provide the dosage for the protection of the properties of the product,
-It merely affects the atmosphere. Though it does affect the structuring of slight amount of ozone,
-In order to be on the safer side personnel should wear protective suites to save them from harmful effets of e-beam,
- For the purpose of implementation and procedure validation guidance documents can be brought to use for the process of sterilization (25, 26, 28):
a. Absorbed dose
From the chain cleavage of DNA Microorganisms are departed based upon the impacts between accelerated electrons and breed radicals. The thing that has utmost importance is basically the dose that has been absorbed and the quantity of interaction product and e-beam which will be untainted. It is basically the absorbed energy in joules divided by mass per unit ([J.kg-1] = [Gy]). The small portion of survival of microorganisms is inversely proportional to the dose absorbed. One of the most vital concerns in the e-beam sterilization is the D value that is needed for the reduction of the survival fraction to 1 by 10 and the value of D is basically the value for single microorganism. The amount of dose needed is based upon the targeted reduction level.
b. Acceleration energy
The basic connection between the dose that has been absorbed and the depth mostly depends upon the energy accelerated energy. For serving this purpose, it is very essential to carry out appropriate setting for serving the properties of the product.
c. Necessity of optimum system
The decline in the sterilization efficacy, variation in the strength and color is entirely based upon the additional dose. Another setback of increasing the energy and dose is the considerable rise in cost. It should also be noted that higher energy is generally falling under the parameter of 10 MeV. For the purpose of generating the optimum irradiation system attaining the uniform dose to articles with appropriate e-beam energy is crucial.
As it is delimitated by the Committee for Proprietary Medicinal Products and European Pharmacopoeia, the shape of pharmaceutical dosage that is sterilized in its terminal stage is known as a sterile product. The factors that influence the selection of a sterilization technique includes; the nature of the product, the sterilization dose and microorganisms’ sensitivity to that sterilization technique, and the sensitivity of the product to the radiation and the SAL value that is required (28).
The use of e-beam sterilization in pharmaceutical industry
Those products which have a difficult formulation and wrapping procedures, this process is suitable for them, for the reason that, it seems tough to sustain sterile environment in each phase, and as the conditions are sterile, so the substantiation can not be done easily of these difficult aseptic products. In order to assure and sustain the sterility of pharmaceuticals and medical devices, terminal sterilization can be more beneficial if selected, but there is a disadvantage beside its advantages, that is, to build up huge irradiation institutions, high cost would incurred. In order to sustain protection and usefulness to the FDA’s fulfillment, a time overwhelming and expensive technique is used by the number of pharmaceutical companies, that is, terminal sterilization. In order to reduce the drug debasement, the mechanism that is used to manage the whole bioburden in the product, is the main aspect in the e-beam sterilization of pharmaceuticals. With the e-beam’s elasticity, the little batches are used to benefit the e-beam sterilization, so that the debasement effect on drugs can be reduced. As the formation of chemical changes diminishes, that’s why the right adjustment of dose is required. A lesser sterilization dose is desirable by cleaner raw materials and manufacturing operations. The consumption of e-beam can be extended through the usage of some molecules. In order to decrease the effect of free radicals, antioxidants like ascorbate, or compound which have sulphydral or SH bonds are used, so that their interaction with an active molecular structure of a rug can also be minimized.
A drug being freeze during and after the process of irradiaton increase the chances of recombination, because of freezing of free radicals, and decreases the chances of degradation. Their ability of migration and interaction with other radicals also reduces. By moving oxygen react with nitrogen and argon gas maintains greater stability in product while reducing the oxidative reactions.
Comparison of sterilization methods and their applications
In the comparison for some of the sterilization techniques, in the gamma radiation sterilization the doses for biomedical applications for bulk materials are ranging in between 10-30 kGy. E-beam radiation is used successfully in a variety of materials as a bactericide but the sterilization of polymers only has a disadvantage that irradiations can exert reactions in molecular bonds like chain breaks, photo-oxidation reactions or cross-linkings. Besides the technique of radiation sterilization, EtO sterilization has the most important drawback that in the presence of a residue it causes some cytotoxic reactions blocking the use of EtO of the polymers for the sterilization process and causing degredations in some molecules like hydrolysis in the polymers.
E-beam generators is usually having its single energy ranging in between 3-12 MeV for its operations but in these days better choice for usage of e-beam equipments is in different energies for operations. There are two most important major points in e-beam sterilization, first is that a strict control of the current scan energy and e-beam is required and the other is that the transporting of the product by means of the beam in the conveyor. The process of sterilization can be accommodated by the speed of the conveyor which depends on the beam current, which can be managed by the feedback circuitry which ensures that till the last part of sterilization process keeping the dose constant.
It is very applicative for various types of materials which are used for medical purposes or for packaging. If we consider how different this is from gamma radiation sterilization, the advantage of e-beam sterilization is highlighted with its less degradation effect which relies on how short the exposure time has been in order to link the dose rate. Another advantage of this e-beam sterilization is the dissymmetric release which is also known as the immediate release. The dosage of the radiation makes this achievable. This method was adopted by the FDA and the American National Standard, ANSI/AAMI/ISO 11137-1994 also linked to this issue. It is not an obligation to maintain the sterility but it is however important to follow the instructions properly so that as soon as the product finishes, it can be sent out.
The gamma sterilization and e-beam sterilization are significant processes that are quite useful in the sterilization of pharmaceuticals. The gamma radiation possesses such characteristics as to transform a process of a few minutes to hours. This is dependent on the volume and mass of the product. It is capable of providing the same dose to products that have a relatively small mass. The pharmaceutical formations differ in sterilization methods based on their mechanisms and their functioning processes. The amount of dose given firstly depends on the bioburden, SAL and the level of radio sensitivity that an organism possesses. The normal amount that SAL is set at is 10−6 m.o/ml or g for the pharmaceuticals that can be injected, ophtalmic ointment and ophtalmic drops and is 10-3 for certain products such as the gloves used in the aseptic conditions. Normally for an effectivity (F-value) of n = 8 is used for sterilization of Bacillus pumilus for the standard dose of 25 kGy which is equal to almost eight times that of its D10 (2.2-3 kGy). Due to this factor, the optimum sterilization dose is 25 kGy at the above level of bioburden.
The relative impact of sterilizing oxorubicin-loaded poly (butyl cyanoacrylate) nanoparticles was investigated on e-beam and gamma irradiation. There were made by a method of anionic polymerization. The doses of irradiation were between 10-35 kGy and to see whether the sterilization is successful or unsuccessful, Bacillus pumilus was utilized. Studies in the field of microbiology showed that irradiation dose of 15 kGy was enough for sterilizing techniques of e-beam and gamma radiation for the purpose of sterilizing 100 CFU.g-1 PBCA nanoparticles of bioburden. It was found out that the two chosen techniques of sterilizing resulted well in the dose that was investigated. The firmness of the preparation as well as active ingredient were unaffected by an irradiation dose of 35 kGy. The physiochemical properties of drug loaded as well as empty nanoparticles such as molecular weight, particle size, aggregation stability and polydispersity index were not impacted by the process.
The impact of EtO gas sterilization and e-beam irradiation was studied on the mechanical properties and structure of multiblock copolymer i.e. a biomedical material. A number of doses on the material had been employed to find out the best e-beam irradiation. IR spectroscopy, tensile testing, DSC, gel permeation chromatography and dynamic mechanical thermal analysis were carried out to find out the probable changes that can occur in the mechanical and structural properties of multiblock copolymer. The most suitable sterilization dose is described as 25 kGy after classification had been carried out. It was also discovered that treatment of Eto gas, like e-beam sterilization, did not impact the physiochemical characteristics of polymer and believed it as a substitutive technique of sterilization.
After using the various doses of high energy electrons and gamma radiation, the structure of metoclopramide hydrochloride solid samples were studied. The products’ degredation was classified with several methods such as atmospheric pressure chemical ionization/liquid chromatography/tandem mass spectrometry. No noteworthy difference was found between the metoclopramide hydrochloride’s e-beam and gamma irradiations.
Following the sterilization, the prepared degredation producrs were not significantly different from metoclopramide and in the solid-state was found chemically stable. Macromolecules can be ionized by radiation ionization and depending on the chemical bonds’ decomposition it can affect radiolysis. H3O and OH are the radiolysis products of protein solution and depend upon the existence of water. The indirect impact of irradiation depends on the impact of such radicals macromolecules that make up a huge part of the damage. Despite the radiation of diffusion being very limited, till now the mean frozen form issue is water. A study was carried out for examining the impact of high energy electrons and gamma rays on protein macromolecules. It was found that a large part of the damage to proteins is linked to the primary ionizations to these molecules directly and that in the frozen state proteins are more sensitive to radiation as compared to the liquid state. The measurement of mass active structures can help in identifying survival frozen proteins that from the destroyed ones.
Several studies have also been conducted on the impacts of radiating ionization on animal diets. The impacts of gamma and e-beam rays have been compared on animal diets’ laboratory. A linear accelerator created 10MeV electrons for the laboratory animals’ power diets as well as solid sterilization for e-beam. A resource of 60Co gamma rays was required for sterilizing gamma rays min 20 kGy. Solid diets that had various thicknesses required various sterilizing procedures.
There were two way in which irradiation was given. To diets which have 30 – 45 mm thickness were given one-sided irradiation while dual-sided irradiation was given to diets having thickness ranging form 75 to 90 mm. Through different observation they found that, there was no considerable difference between diet’s nutrition quality when sterilizing was done with the help of gamma radiation or e-beam. Through this it could be said that e-beam sterilization could be an alternative of gamma rays.
There were many studies, but one of them research about the effects on samples of gum Arabic samples if gamma radiation and e-beam was used. At initial stage gum arabic samples were contaminated due to different bacteria like Bacillus cereus, Clostridum perfringens and Enteroccus faecalis. With the help of 10kGy of e-beam or gamma ray a complete process of decontamination was performed. It was observed that material degradation was directly proportional to dose of gum arabic sample. There could be a change in properties due to high doses link decrease in viscosity and darkening of color. By considering SEM results, in sample gamma rays produces more colors and also the crystal size changes. For both, medial and food industry, it was found through sampling that the optimum level for the purpose of sterilization is 5 kGy. Through all the arguments this could be concluded that for the purpose of terminal sterilization e-beam could be used and can also be an alternative of gamma rays.
E-beam irradiation is also used for non-tissue materials such as forming hydrogles for blood vessels and artificial kidneys as well as for tissue materials like bones, aortas and aortic valves. It was reported by a researcher that in tissue materials, a dose of e-beam irradiation is normally in range of 2 Mrad. Without any negative reactions, during different clinical procedures irradiated bones can be used. If we look at from the perspective of sterility and host acceptance, we will find that the usage of e-beam irradiation of tissue material (such as aortic or aorta valves) resulted in optimum conditions. One of the study has also examined the surface characteristics of poly(L-lactide-cocaprolactone) (PLCL) biopolymers, which are basically used in tissue engineering.