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This paper examines the process of gamma irradiation of plastic materials used as part of single-use disposable systems in the pharmaceutical and biotechnology sectors, with a focus on validation requirements.
A relatively recent advance in biopharmaceutical processing has been the use of single-use disposable technologies. Single-use disposable technologies include tubing, capsule filters, single-use ion exchange membrane chromatography devices, single-use mixers, bioreactors, product holding sterile bags in place of stainless steel vessels (i.e., sterile fluid containment bags), connection devices, and sampling receptacles (1). The drivers for the implementation of such technology include improved sterility assurance, greater flexibility for biopharmaceutical manufacturing processes, reduced processing times, and cost and energy savings associated with the avoidance of washing, drying, and sterilizing stainless-steel equipment (2).
These advantages notwithstanding, the application of single-use sterile disposable technology in the biopharmaceutical industry remains in its infancy and there are several important validation steps to be considered. These steps relate to product compatibility issues (e.g., extractables and leachable) and to sterility assurance. This paper addresses an important aspect of sterility assurance: ensuring that single-use systems are sterile and that the process of rendering single-use systems sterile does not damage the device or cause adulteration of the product.
The primary method for the sterilization of single-use technology is by gamma irradiation. This is because plastics cannot be subjected to heat-based methods of sterilization without damaging the mould (i.e., styrene and other plastics are temperature sensitive) (3). Other alternative methods, such as gaseous sterilization by ethylene oxide, that although used on occasions, can leave unwanted and potentially toxic residuals (e.g., ethylene glycol and ethylene chlorhydrin) post-sterilization (4). Other alternative sterilization methods (e.g., liquid peracetic acid immersion system and plasma sterilization processes) are not sufficiently well established (5). Electronic beam irradiation is an alternative method of radiation to gamma. This is a concentrated, highly-charged stream of electrons generated by the accelera tion and conversion of electricity (6). Electronic beam radiation is not commonly used for single-use disposable systems due to its relatively limited ability to penetrate some types of plastics (7). Therefore, single-use systems are typically sterilized using gamma rays (i.e., electromagnetic wave radiation) (8). Despite the widespread application of the gamma irradiation, the process remains the only primary sterilization method not described in either the European Pharmacopoeia or United States Pharmacopeia (USP).
This paper outlines the process of gamma radiation and describes the important aspects of validation. The paper is designed to provide a guide to those wishing to understand more about the process and to offer advice for quality assurance personnel who are required to audit the sterility assurance of gamma radiation.
Single-use disposable technology
Single-use disposable technologies have been used in biopharmaceutical processing since the 1970s on a limited scale. It is only since the 21st century that such technologies are being implemented for large-scale manufacturing (e.g., upstream, downstream, and product filling applications). The forces driving the wider adoption include improving or eliminating cross contamination, sterility assurance, process efficiencies, operator protection, and cost savings.
Single-use disposable technologies are generally manufactured from plastic polymers involving processes of injection molding, extruding, and blow molding. The applications include devices for making aseptic connections, sampling devices, mixing devices, product-hold bags, and disposable manifold systems. Each of these systems is used to process or contain fluids including additives, buffers, bulk intermediates, and final formulations.
Before single-use technologies can be adopted, a number of tests must be performed in relation to design, microbial inhibition, chemical computability, and leachables and extractables (9).
Gamma radiation. The use of ionizing radiation dates back to the discovery of X-rays by Roentgen in 1895, and gamma radiation has been an established sterilization method since the early 1970s (10). It has only been since the 21st century, however, that the use of gamma irradiation has increased within the biopharmaceutical industry, replacing more established and costly methods like ethylene oxide. This arises from the use of gamma radiation to sterilize consumables and single-use technologies used for aseptic filling operations (11). It is the recent impetus for new cleanroom technologies that is leading to an increased use of gamma radiation as the optimal sterilization method.
The process of gamma radiation is a form of ionization (i.e., electron disruption). Gamma radiation is one of the three types of natural radioactivity, the other two being alpha and beta radiation (12). Gamma radiation is in the form of electromagnetic rays, like X-rays or ultraviolet light, of a short (less than one-tenth of a nanometer), and thus energetic, wavelength. It provides a physical means of sterilization or decontamination as the rays pass through the product being sterilized (i.e., irradiated) (13). In doing so, the radiation kills bacteria, where there is sufficient energy, at the molecular level by breaking down bacterial DNA and inhibiting bacterial division (14).
The most common source of gamma rays for radiation processing comes from the radioactive isotope Cobalt 60, although other radionuclides can be used, such as Cesium 137. Each element decays at a specific rate and gives off energy in the form of gamma rays and other particles. Cobalt 60 is manufactured specifically for the gamma radiation process from non-radioactive Cobalt 59. The radioactive Cobalt 60 functions as the isotope source. High-energy photons are emitted from the Cobalt 60 to produce ionization (electron disruptions) throughout a product. The gamma process does not create residuals or impart radioactivity in processed products (15).
Gamma radiation is often referred to as a "cold process" for the temperature of the processed material that does not significantly increase (16). The sterilization process is not dependant on humidity, temperature, vacuum, or pressure, which means that the process is suitable for materials that cannot be subjected to high-temperature sterilization. The important variables for gamma radiation are the strength of the radiation dose (i.e., measurement of how much energy is absorbed when something is exposed to the radiation source) and the exposure time. The measurement of radiation is expressed in units called KiloGrays (KGy). One gray is the absorption of one joule of radiation energy by one kilogram of matter (17).
Standards. The regulatory requirements for gamma radiation are less defined than for sterilization by filtration, moist or dry heat, or by ethylene oxide. These methods of sterilization normally involve a direct biological challenge, as with biological indicators (preparations of a specific microorganism, with high resistance towards particular sterilization methods) for steam sterilization or a high population microbial challenge for filter validation. In the past Bacillus pumilus was used as a biological indicator to measure gamma irradiation. It was removed because the assessment of the product bioburden during validation was deemed to be a more accurate means of assessing potential resistance to the gamma radiation process. In contrast, with gamma radiation, the biological assessment is normally undertaken by an assessment of the product bioburden (i.e., the number of microorganisms on a certain amount of material prior to that material being sterilized) by assessing the total viable count (TVC) of the material, expressed as colony forming units (CFU).
For gamma radiation, the applicable standard is ISO 11137 "Sterilization of Health Care Products-Radiation" (18). The standard was developed in association with the Association for the Advancement of Medical Instrumentation (AAMI).
The standard is divided into three parts. The first part deals with validation and routine control methods. The second part deals with the establishment of radiation doses for items to be sterilized, and the third part relates to dosiometry. The standards function to determine how much radiation is permitted in order to achieve the desired level of sterilization when measured in terms of sterility assurance. The sterility assurance level is normally 106 (that is a theoretical concept where it is assumed that, in terms of probability, no more than one item sterilized out of one million would contain one or more microorganisms after the completion of the sterilization process) (19).
The official scope of the ISO 11137 standards is limited to medical devices. However, in the absence of any other applicable guidance, the standard is more often applied to other types of products and equipment.
Packaging and dose determination. Although gamma radiation is commonly used to sterilize plastics, not all types of plastics can be treated at a sufficient dose to achieve sterilization without degrading the plastic (20). The assessment of degradation is on-going and should be undertaken throughout the shelf life of the material (the application of ionizing radiation causes the excitation of polymer molecules, where, over time, the adsorbed dose can result in changes to the physical or chemical properties of the polymers). The plastic material to be irradiated is normally referred to as the "product." Most products are placed into an outer packaging in order to protect the irradiated product and to keep the product sterile once sterilized. The product remains sterile provided that the outer packaging remains intact. Occasionally, large or complex products cannot be tested in their entirety and a staged sterilization process is required. In such circumstances, thought should be given to the conditions under which final assembly will take place in order to avoid contamination from the environment or from personnel.
Given the range of different types of single-use sterilized disposable products being developed, and the range of different packaging configurations, the required gamma radiation dose to achieve sterilization or to protect the product from degradation will vary considerably. There is also considerable variation with types of plastic. For example, a relatively low dose of radiation is required to sterilize polypropylene when compared with polystyrene. Furthermore, the assessment of the dose is more straightforward for small items, such as a plastic container, and more complex for single-use systems. Single-use systems have variables including tubing length, different numbers and types of filters, and differences in the design of containers, bags, and valves, which make the determination of the irradiation dose more complicated. Considering these factors, a common radiation dose used for plastics is in the range 15–25 kGy (21).
For the process of sterilization, the wrapped product is normally packed into a special container, typically manufactured from aluminum, called a tote. A tote has fixed internal dimensions and is designed to transport product through the radiation process. The weight and dimensions of the tote must be accounted for when establishing the radiation dose.
The dose determination is the key validation step when using gamma radiation. The dose is the amount of gamma radiation absorbed by an item undergoing sterilization. This is normally set as a range, where a minimum and maximum dose is established. The minimum dose is established as the point where sterilization occurs, and the maximum dose is the point beyond which the product is no longer compatible with the sterilization process. In general, the higher the dose rate, the lower the adverse effects upon polymer products. This is mainly due to the diffusion of oxygen during the irradiation process.
To validate a load, there are three aspects to consider: establishing the dose range, measuring the effectiveness of the sterilization, and dose mapping.
Irradiation validation. Irradiation validation is designed to set the dose range. The primary focus is to determine if the irradiation process damages the packaging material or the product to be sterilized. Damage is assessed by calculating the maximum dose. This assessment is examined through stability trials, whereby samples are held at under defined storage conditions (i.e., temperature and relative humidity) and examined at periodic intervals for discoloration, brittleness, and other damage.
Ionizing radiation generates free radicals in plastic polymers leading to degradation from chain scission (i.e., changes in molecular weight) or alterations to cross-linking. Potential radiation effects on some materials include embrittlement (i.e., change to material hardness), discoloration (i.e., often yellowing caused by surface oxidation), unpleasant odor (i.e., from volatile material formed by reactions from within the polymers), or lack of functionality due to a compromised physical trait, such as tensile strength (22).
Sterilization validation. The aim of this sterilization validation is to determine the dose required to achieve a sterility assurance of 106. The probability of sterilization is commonly assessed by bioburden determination (determination of the bacterial and fungal load), as recommended by ISO 11137. This involves the following four steps.
Determination of the bioburden of the product. This is normally done by taking 10 units per batch from three different batches of product, post-irradiation. In doing so, it is important to be sure that the product that was subjected to gamma radiation was representative of the product normally manufactured. The manufacturer of the product normally carries out the bioburden determination. It is important to ensure that the bioburden recovery method is accurate because insufficient recovery of microorganisms during bioburden tests would result in an underestimation of the true bioburden of the product and lead to an inadequate sterilization dose applied to the product.
The bioburden of the product is dependent upon several factors. These include the nature and source of the raw material, the components used in manufacturing, the product design and size, the manufacturing process, the manufacturing equipment, and the manufacturing environment (e.g., the type of cleanroom used). For products manufactured using approved suppliers and assembled within cleanrooms certified as ISO 14644 class 7 under localized unidirectional airflow protection, the expected bioburden would be relatively low (e.g., not more than 10 CFU per device). Additional data relating to the risk from the manufacturing environment can be provided through microbiological environmental monitoring.
There are different methods for bioburden determination. One of the most common methods is the repetitive (exhaustive) recovery method. This method involves washing the sample product repeatedly until it is estimated that no further microorganisms will be recovered. The washing process can include the addition of sterile glass beads or ultrasonication to facilitate microbial recovery. The eluent from the washing should be tested using an appropriate TVC test method (where membrane filtration is the method of choice, followed by the pour plate technique).
The microbial counts from all washes are compared in order to assess the total bioburden. Such extraction methods require validating. Method validation involves deliberately inoculating a sterile disposable item with a known number of microorganisms and then assessing the number recovered from the washing steps to the theoretical inoculum challenge. A valid method should recover the microbial challenge as per the guidance in USP <1227> for microbial method validation. Where the challenge cannot be recovered, one factor may be the variation with the process of drying the microbial challenge organism onto the plastic item prior to washing (23).
Calculation of appropriate dose based on the resistance of an identified microbial population. This is based on the total number of bacteria and fungi isolated and an assessment of the types of species recovered, as characterized using microbiological identification techniques. This is an important distinction as it is not simply the total numbers recovered as some microorganisms have a theoretically greater resistance to gamma radiation processes than others, most notably Streptococcus faecium and Micrococcus radiodurans (24).
Radiation dose assignment. To select the appropriate radiation dose in relation to the theoretical resistance of the microorganism, ISO 1137 provides a guidance table.
Validation of calculated dose. As part of the validation, the calculated dose is verified by selecting an appropriate sample size (normally 100 units of the product) to determine if the dose is efficacious. The test is undertaken by placing individual units of product into sterile bottles containing microbiological culture media (e.g., soybean casein digest medium) and incubated for 14 d. This is the "sterility test," although it bears some similarities to the direct inoculation sterility test described in the US or European pharmacopoeias, it is not equivalent (25). Any bottle of media that exhibits microbial growth (turbidity) is indicative of the product not being sterile and that the sterilization cycle is inappropriate for the product.
As with the bioburden determination method, the test for sterility requires validation. The object of the validation is to show that the product material does not inhibit the growth of microorganisms (a method suitability test). Inhibition of microorganisms would lead to the risk of a false negative result occurring. The method suitability determination involves using the same type and volume of culture media used for the sterility test. The product is inoculated with known numbers of a bacteria culture and a fungal culture. The inoculated product is then incubated. Any product that shows no growth or slowed growth is considered bacteriostatic or fungistatic and the method declared unsuitable (26). Within the medical device industry, an acceptance criterion of a sterility assurance level of 102 has been used (i.e., two units could fail the sterility test and results could be deemed as acceptable). This level of assurance is unacceptable for single-use technologies used in conjunction with aseptically-manufactured products. In such cases, further modification of the method is required, such as increasing the volume of culture media or using culture media with an added neutralizer.
There are complications with the testing of large products for the bioburden and sterility tests. To carry out tests as commonly practiced, disassembly (i.e., "sectioning") would be required so that the product can be immersed into microbiological culture media (i.e., broth). This is an activity that could result in adventitious contamination due to the need for personnel to manipulate the product. With such devices, an alternative approach is more often adopted that involves sterility of a product's fluid path (i.e., passing a sterile buffer through the product and examining it for microbial growth). The test is assessed by incubating the media in the presence of the product (or product rinse) for 14 days and examining the broth, post-incubation, for microbial growth.
When assessing the data to determine the final sterilization dose for routine batches of single-use products, it is normal to use the highest dose (Dmax) established in the validation in order to set a level of overkill. This provides additional assurance should the product bioburden shift upwards or should more resistant strains appear.
A further important consideration is that the bioburden test and sterility test are often carried out by the product manufacturer and gamma irradiation plant, respectively. In order that the data are comparable, it is important that similar test methods, microbiological culture media, and test incubation parameters are employed. If this is not done, then the reporting of a sterility test as zero microorganisms may be incorrect if the sterility test method is not capable of detecting all of the microorganisms detected in the bioburden test. This can arise if different microbiological culture media are employed or if the medium is incubated at a different temperature for a shorter time period. The sterility test result may then be due to the inability of the organism to grow under the test conditions rather than an indication that the organism has been destroyed by the sterilization process.
Variations to the bioburden method are permitted within the ISO 11137 standard, such as the VDmax25, which permits fewer units of product to be tested and for similar types of product items to be grouped together for the validation (for 25 kGy doses). This variation can only be used where it has been established that the bioburden level is relatively low (at less than 1000 CFU per device). When considering whether different products can be grouped together, account must be made of the plastic materials, construction processes, surface area and handling.
Dose mapping. The third aspect of the validation is dose mapping. For this, the product, in its final packaging configuration, is profiled in order to identify the high and low zones of absorbed dose in the product load. Dose mapping determines the loading configuration that will be used during routine sterilization.
Understanding Gamma Sterilization, by Jerold Martin, Pall Life Sciences
The object of the validation is to set processing parameters and the product release specification. The validation parameters are established through a performance qualification (dose mappings), which is typically run three times using the maximum packaging size. The main steps for undertaking a performance qualification, for each product, are as follows:
The level of radiation is assessed using devices called dosimeters (i.e., a device, instrument, or system that measures the exposure to radiation). It is important to assess the number of dosimeters required to assess the radiation dose. With a standard tote, it is typical to use 15–20 dosimeters. This number is necessary in order to achieve an accurate assessment because the radiation dose applied to the products packaged at the outer edge of the tote is often higher than the dose received by the material in the inner center of the tote, as illustrated in Figure 1.
The key validation parameters are: product weight and volume, dimensions of packaging components and density, and the configuration of the packaging components. With the dimensions, it is important that the product is evenly distributed because the radiation dose is applied at the same level from both sides. With the issue of load configuration, this point is sometimes overlooked. It is nonetheless important that the way that the components are packaged during validation be replicated for all successive radiation runs. This is important because if the orientation alters, then this can cause changes to the density mix and thus the effectiveness of the irradiation. Once the validation parameters are established, the parameters are used for routine processing and no parameter should be permitted to vary significantly from the established parameter.
Once established, it is necessary that any future changes in product, its package, or the presentation of product for sterilization are re-assessed for the effect on the appropriateness of the sterilization process. It is prudent to re-assess the validation parameters at least on an annual basis and to assess the bioburden of the product quarterly, in order to determine that the gamma radiation process remains effective (27). This assessment may include fractional studies. Here the product is irradiated and tested at sublethal doses (i.e., at levels of gamma radiation below the minimum established in the validation study) to check for continued dose efficacy. This is sometimes referred to as dose auditing.
Gamma radiation process
On completion of the validation, the standard manufactured batches of the product can be subjected to routine sterilization. The sterilization process is based on the establishment of validation parameters.
To ensure good levels of quality control, it is recommended that the following steps be put in place:
There are two methods of gamma radiation: continuous or batch. For both it is important that the distribution of gamma radiation applied to the product is even. With the continuous method, an automated conveyance system functions to move the product past a gamma source and back out on a continuous basis until the end of the cycle is achieved. With the batch methods, a set number of totes are used at set positions within the irradiation chamber. Special chemical indicator labels should be fixed to each item to indicate if irradiation has been successful. The radioisotope is then moved into an exposure position, and the product is irradiated for a specified period of time. The method selected is dependent upon the method used during the validation.
Auditing gamma irradiation plants
The wide application of single-use sterile disposable technology means that the biotechnology company purchasing the materials should undertake an audit of the sterilization process in order to be satisfied that the process is robust and that the possibility of non-sterility is minimal (28). Furthermore, the diversity of radiation sterilization systems available today places renewed emphasis on the need for thorough quality assurance audits of these facilities. As with any sterilization process, gamma radiation should be subject to quality auditing to ISO 9001 standards and to good manufacturing practice as applicable (29, 30). When conducting an audit of gamma irradiation processes, the following areas should be considered when developing the audit checklist:
In addition to the above, the plant should be checked to determine if it is compliant with appropriate regulations for nuclear facilities and that appropriate segregation and labeling of components is in place.
These points should be assessed, against the radiation plant's standard operating procedures, in order to establish confidence that the plant undertaking the gamma radiation process does so in a consistent and effective way.
Single-use sterile disposable technology is rapidly becoming the norm for biotechnology and biopharmaceutical organizations. The application of the technology requires that the products are sterile and remain robust in terms of their construction. The material of construction, plastic, means that the primary method for sterilization is gamma radiation. Gamma radiation, as a sterilization method, confers the advantage of being relatively low cost, effective (in having a deep material penetration), and in avoiding leaving toxic residues. However, the radiation process may degrade some polymers rendering it unsuitable from some materials. In addition, the process parameters must be correctly defined in order for the sterilization process to be effective. There are several variables that could, if not carefully addressed, lead to non-sterility or to material degradation. To counter this possibility, an effective validation strategy must be developed and be supported by quality audits of the irradiation process. The end-user of the sterile disposable products, not the manufacturer or the sterilization plant, should adopt the lead role when undertaking such assessments.
This paper set out to provide a framework for establishing sterilization cycles and auditing such processes. In doing so, the paper has examined some of the key parameters required for sterilization by gamma radiation. The sterilization technique remains one of the less defined by regulatory agencies and yet it is seemingly the fastest growing technique used within the pharmaceutical industry as the trend towards sterile, plastic disposable consumables increases.
Tim Sandle, PhD*, is the head of the microbiology department at Bio Products Laboratory Limited, email@example.com. Madhu Raju Saghee is a quality assurance professional at Gland Pharma Limited, India.
*To whom all correspondence should be addressed.
1. T. Sandle and M. Saghee, Jrnl of Comm. Biotechnol. (2011).
2. G. Rao, A. Moreira, and K. Brorso, Biotechnology and Bioengineering 102 (2) 348–356 (2008).
3. Radiation Sterilization Working Group (AAMI Sterilization Standards Committee). Radiation Sterilization—Material Qualification. Technical Information Report No. 17–1997. Association for the Advancement of Medical Instrumentation (Arlington, VA, 1998).
4. W. Wozniak-Parnowska and A. Najer, Acta Microbiol. Pol. 27 161–168 (1978).
5. W.A. Rutala, M.F. Gergen, and D.J. Weber, American Jour. of Infection Control 26 (4) 393-398 (1998).
6. A.G. Chmielewskia, M. Haji-Saeida, and S. Ahmedb, "Progress In Radiation Processing Of Polymer," Nuclear Instruments and Methods in Physics Research 236 (1-4) 44-54 (2005).
7. S. Baloda, BioProcess Inter. Supplement 10-22 (May 2008).
8. K.J.R. Clark and J. Furey, BioProcess Inter. 4 (6) S16-S20 (2006).
9. H. Pora, and B. Rawlings, BioProcess Int. 7 (14): 9-16 (2009).
10. M.A. Sebold, and J.A. Williams, "Radiation Sterilization" in M.R. Saghee, T. Sandle, and E.C. Tidswell (Eds.), Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices, Business Horizons, 841-872 (New Delhi, 2011).
11. E. Zandbergen and M. Monge, BioProcess Inter. Supplement 48-51 (June 2006).
12. R. Tykva and D. Berg, Man-Made and Natural Radioactivity in Environmental Pollution and Radiochronology, Kluwer Academic Publishers (Amsterdam, 2004).
13. E.R.L. Gaughran, and R.F. Morrissey, Sterilization of Medical Products, Volume 2, Multiscience 35-39, (Montréal, 1981).
14. A. Booth, Sterilization of Medical Devices, Buffalo Grove: Interpharm Press, 1998.
15. P. P. Dendy and B. Heaton, Physics for Diagnostic Radiology, CRC Press. p. 12. (1999).
16. P. Whaid, An Introduction to Isotopes and Radiations, Allied Publishers Limited, (Mumbai, 2001).
17. A. Vértes, S. Nagy, and Z. Klencsár, Handbook of Nuclear Chemistry, Volume 3. Kluwer Academic Publishers (Amsterdam, 2003).
18. ANSI/AAMI/ISO 11137-1: 2006. Sterilization of health care products-Radiation–Part 1: Requirements for development, validation, and routine control of a sterilization process for medical devices.
19. N.A. Halls, Achieving Sterility in Medical and Pharmaceutical Products, Marcel Dekker (New York, 1994).
20. N. Martakis, M. Niaounakis, and D. Pissimissis, Journal of Applied Polymer Science 51 (2) 313–328 (2003).
21. A. Tallentire, Radiat. Phys. Chem. 15 83–89 (1980).
22. Majewski, S., Bowen, M., Zorn, C., et al., "Radiation Damage Studies In Plastic Scintillators With a 2.5-MeV Electron Beam," Nuclear Instruments and Methods in Physics Research 281 (3) 500-507 (1989).
23. ISO, Sterilization of Medical Devices–Microbiological Methods–Part 1: Estimation of Population of Microorganisms on Products, ISO 11737-1: 1995, ISO (Geneva, 1995).
24. A. Anellis, D. Berkowitz, and D. Kemper, Appl Microbiol. 25(4) 517–523, (1973).
25. T. Sandle, "Practical Approaches to Sterility Testing" in M.R. Saghee, T. Sandle, and E.C. Tidswell, (Eds.), Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices, Business Horizons, 173192 (New Delhi, 2011).
26. ISO, Sterilization of Medical Devices–Microbiological Methods–Part 2: Tests of Sterility Performed in the Validation of a Sterilization Process, ISO 11737-2: 1998, ISO (Geneva, 1998).
27. J-M Cappia and N.B. T. Holman, BioProcess Inter. 2 (9) 56-63 (2004).
28. J.A. Beck, International Journal of Radiation Applications and Instrumentation, Part C. Radiation Physics and Chemistry 35 Issues 4-6, 811-815 (1990).
29. ISO, Quality Management Systems–Requirements, ISO 9001: 2000, ISO, (Geneva, 2005).
30. Genova, Hollis, Crowell and Schady, Journal of Parenteral Science and Technology 41 (1) 33-36 (January 1987).
31. D. Jenke, Journal of Pharmaceutical Science and Technology 64 (6) 527-535 (2010).