Gamma Sterilization of Pharmaceuticals—A Review of the Irradiation of Excipients, Active Pharmaceutical Ingredients, and Final Drug Product Formulations

Pharmaceuticals and Excipients

This review demonstrates a significant interest in the potential of gamma sterilization of pharmaceuticals as an alternative technology to the methods currently employed to ensure the sterility of the finished product. The data in Tables I and II was taken from 29 papers, reported from the late 1980s to 2012, that investigated 77 different drugs, APIs, or excipients. Table I is the collection of data that examined drugs in different conditions (i.e., solid, in water, different irradiation conditions, etc.) and used a variety of analytical techniques to determine whether or not gamma sterilization was suitable for the pharmaceutical.

As demonstrated in the table, a diverse range of analytical techniques were employed to appropriately characterize the sample, and the selected method largely depended on the overall objective of the research. For example, various chromatographic techniques, such as thin layer chromatography (TLC), gel permeation chromatography (GPC), and high-performance liquid chromatography (HPLC) were used to evaluate the degradation profile of the compound being irradiated by gamma irradiation. As well, various spectroscopic techniques including nuclear magnetic resonance (NMR), infrared (IR), ultraviolet (UV), and diode array (DAD) were reported. Other techniques employed include pH test and mass spectrometry (MS). The techniques selected for analysis were in large part driven by the chemical properties of the pharmaceutical/excipient as well as the sample matrix. Because the interaction between high-energy gamma irradiation and matter can lead to free radical formation, use of electron paramagnetic resonance (EPR), previously called electron spin resonance (ESR), to assess the formation and longevity of radicals is a critical analytical technique. The data shown in Table II are those drugs that were only evaluated for radical production after treatment with gamma irradiation.

The main categories included within Tables I and II are the pharmaceutical substance or excipient, indication, radiation conditions, investigation methods, and a brief summary of the effect of gamma sterilization. The list of pharmaceuticals has been organized in alphabetical order by drug name. In papers that evaluated more than one drug, the pharmaceuticals within that paper were organized alphabetically and the first drug listed dictated the location within the table. As shown, the research spans a wide variety of drug classes including medications for heart conditions, pregnancy management, asthma, and infections, as well as antibiotics, anti-inflammatory, proteins, and excipients used in pharmaceutical formulations.

The feasibility of gamma sterilization of pharmaceuticals depends on several factors including, but not limited to, the formulation and stability of the pharmaceutical, radiation dose necessary to attain sterility, product packaging, and irradiation conditions. As shown in Table I, minimal degradation (<1%) of the drug product was reported for several antibiotics (29, 30, 37, 43) when irradiated in the solid form. Radicals from sulfonamide antibacterial agents were identified at doses as low as 5 kGy under normal and accelerated stability conditions (43). No impact on the antimicrobial activity was reported against Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus. Although sterility was not investigated as part of this study, sterility assessment would have been an interesting addition to the discussion (43). In the case for solution irradiations, metocloprimide hydrochloride (39), an antiemetic, was irradiated as a 5 mg/mL solution at doses ranging between 5 and 25 kGy, and the outcome was several degradation products. However, Maquille et al. also demonstrated that this degradation could be mitigated with the addition of excipients. They reported greater than 90% recoveries from solutions containing excipients such as mannitol, nicotinamide, or pyridoxine (39). Conversely, research evaluating the effect of gamma sterilization on both cephradine (32) and ritodrine hydrochloride (42) showed significant degradation products when irradiated at 25 kGy. The conclusions were that these drugs were not suitable candidates for gamma sterilization. Interestingly, the antibiotics cephradine (32) and cefotaxime sodium salt (29, 30) have several similar moieties, suggesting that these molecules might behave similarly to radiation dose. However, less than 0.1% degradation was reported for cefotaxime sodium salt when irradiated to 25 kGy as a solid form (29, 30). This demonstrates that a generalized approach to irradiation of pharmaceuticals does not apply. The maximum acceptable dose is dependent on both the chemical moieties and the functionality of the moieties for a given application (52). Hence, irradiation of molecules can be acceptable if the effect of the radiation doesn't limit the functionality.

A secondary approach to evaluating gamma sterilization suitability was to assess the formation of radicals as a function of gamma dose by EPR analysis. Long-lived free radicals in gamma-sterilized/shelf-aged material were investigated due to a potential cause of long-term decomposition or degradation of the products. The free radical concentration indicates the rate of free radical transfer and stability of the final product. Table II is a summary of solid pharmaceuticals that were evaluated by EPR in an effort to characterize radicals produced as a consequence of gamma irradiation and to understand the stability of these species. As a general rule, a higher concentration of radicals generated at the same absorbed dose of radiation indicates a higher sensitivity of the drug towards gamma irradiation. However, the efficiency of microcrystalline matrices for trapping paramagnetic species also has to be considered.

There were three reports found in the literature utilizing EPR to assess the formation of free radicals from various pharmaceuticals following treatment with gamma irradiation (49⇓–51). In all cases, free radicals were present in samples treated with gamma irradiation as compared to the control (no irradiation), where either no or very weak EPR signals were observed. Köseoğle et al. showed that alkyl- and amine-type free radicals were very stable (>2 years) in some neurological and antihypertensive drugs (50). However, it should be noted that the free radicals generated in a solid matrix would not be preserved in solution, particularly those radicals that are carbon-, oxygen-, or sulfur-centered (49). These would rapidly convert into stable non-paramagnetic products.

Polymer Drug Delivery Systems

Gamma irradiation is a practical terminal sterilization method. The ability to sterilize the polymer drug system in its final container is very advantageous. Interaction of gamma radiation with the polymer can produce crosslinking, chain scission, and hydrogen evolution, which may influence the chemical and physical properties of the polymer material (53).

Controlled delivery of active drugs from biodegradable polymers is well established in the treatment of diseases. The process of incorporating the active drug into the polymer is done by various methods such as mixing or loading onto microparticles. Sterilization of biodegradable drug polymers by gamma irradiation can influence polymer stability due to the degradation process occurring in the polymer chains. Gamma irradiation enhances the formation of free radicals, which propagate chain scission over time. The major radiation effects on polymers results from excitation or ionization of atoms to cause crosslinking or chain scission. Crosslinking forms a higher molecular weight or more branched polymer with altered mechanical properties, while chain scission results in a low molecular weight polymer and change in crystallinity and density (53). In theory, both mechanisms occur at the same time but one dominates the other within the polymer chain. The ratio of resultant recombination, crosslinking, and chain scission is different from polymer to polymer based on the chemical composition and morphology of the polymer, absorbed dose, dose rate, oxygen, and storage conditions (temperature and oxygen level).

Similar to APIs, polymer stability after exposure to gamma irradiation has been extensively investigated by different methods such as NMR, differential scanning calorimetry (DSC), GPC, EPR, IR, and HPLC. These various characterization methods are useful to quantify any chemical and/or physical influence of gamma irradiation on the polymeric excipients.

This summary on the use of gamma sterilization on polymer drug delivery systems was taken from 45 papers published from 1989 to 2012 (Table III) that investigated the use of various biodegradable polymers such as poly(glycolide-co-glycolic) acid (PLGA), polyvinyl alcohol (PVA), hydrogels, glutaraldehyde, polyhydroxies, and polyanhydrides. These polymers are used as vehicles to deliver various drugs such as antibiotics, vaccines, contraceptives, chemotherapy drugs, and hormones.

The stability of the polymer drug delivery system depends not only on the polymer and drug chemical structure but also on the irradiation dose, irradiation conditions, and formulation process. Various sterilization methods/conditions have been proposed to reduce the degradation in the drug delivery systems, including the polymer material and the drug used. For example, papers in the literature reported that using conditions such as low temperature or an oxygen-deprived atmosphere may reduce the free radical formation, which will eventually enhance the product stability post–gamma irradiation. It was also reported that the polymer drug delivery system stability after gamma irradiation can be product-dependent due to the variation in chemical structures of the polymers and drugs, presence of excipients and/or additives, and the use of the final drug product. Various examples are shown in Table III.

Polyesters are the most commonly used biodegradable polymer in drug delivery systems. Irradiation sterilization is generally used to sterilize polyesters due to their instability to moisture and heat. The effect of gamma irradiation has been intensively investigated using various methods, which showed that the decrease in the mechanical properties was due to degradation through chain scission (100). Different polyesters may respond differently to gamma irradiation due to variation in their chemical structures and variations in the functional groups (100). Polyesters in general degrade by decreasing molecular weight, viscosity, and mechanical properties. Poly orthoesters (POE) can also be used as a carrier in controlled drug delivery. Merkli et al. reported that POE molecular weight and viscosity decreases resulted from chain scission at doses lower than 20 kGy (54). Evaluation of the structure and degradation mechanism was performed using ¹H NMR, ¹³C NMR, and IR analysis.

The stability of polymers after the irradiation process was also affected by the presence or absence of oxygen. Copolymers such as β-caprolactone and ethylene oxide (CL₆E₉₀CL₆), used as implantable polymeric systems, were stable when sterilized with gamma irradiation. Martini et al. reported that this copolymer showed no negative effect when gamma-sterilized at 72 kGy in the solid state and in aqueous solutions, as well as in the presence and absence of oxygen as indicated by the thermal analysis of the polymer using DSC (55). More degradation was observed in the aqueous state compared to the solid state but this copolymer was considered stable up to 54 kGy as indicated by reduction in the molar mass (55). Indication of the influence of gamma irradiation on the mobility of the polymer was observed by pulsed NMR analysis. The effect on molecular mass as determined by GPC was only measurable with doses less than 40 kGy due to the insolubility of the highly crosslinked copolymers formed (55).

A common form of drug delivery systems is polymeric microspheres loaded with a drug. One of the most common microspheres is PLG microspheres because of their biodegradability and ease of use. Montanari et al. reported that PLG microspheres were hard to evaluate by EPR due to the unstable free radicals resulting from chain scission after the irradiation, which led to a decrease in molecular weight (56). GPC and DSC analysis indicated a decrease in the molecular weight as a consequence of chain scission. Another study, by Bittner et al. in 1999, investigated the stability of PLG microspheres after gamma irradiation and reported that when samples were purged with nitrogen before sealing and irradiated at low temperature using dry ice (−80 °C), the negative impact of gamma irradiation on the polymer was reduced (57). Other reports in the literature indicate that an increase in the stability of various polymers was observed when the thermal effects during the irradiation process were controlled (58⇓⇓⇓–62).

Natural biopolymers such as chitosan can be used for drug delivery due to their biodegradability, low toxicity, and good biocompatibility. Desai et al. investigated the compatibility of chitosan microparticles to gamma irradiation and reported no drug degradation as observed by UV spectroscopy and no polymer crosslinking as observed by FT-IR analysis (63). Drug release behavior, swelling, and surface morphology were affected slightly by gamma sterilization (63).

Hydrogels, which are hydrophilic polymers, are currently being used in drug delivery systems. Sterilization of hydrogels using gamma irradiation did not alter the shape or the dimension of the hydrogel sponges (64). Mechanically, the irradiated sponges were slightly stiffer than non-irradiated samples with a small decrease in water absorbance (64).

For contraception use, levonogestrel (LNG) was loaded on microparticles made of the biodegradable polymer glutaraldehyde and tested for compatibility with gamma irradiation. A study by Puthli et al. in 2008 indicated that irradiation did not affect the flow of the product and did not cause a tendency of clumping and aggregation (65). The color of the microspheres (solid matrix particle) did not change while the size of the microparticles (coated microspheres containing active agent) changed slightly, but this change was not significant (65). Another study by Puthli et al., in 2009, investigated the use of LNG microspheres loaded on PLGA polymer (66). This study also reported that there was no change in the color or size of microspheres and that the system was free-flowing with no clumping or aggregation behavior (66).

Regulatory Validation Guidelines and Decision Tree for Pharmaceutical Terminal Irradiation

Based on the results shown in many of the papers reviewed, lower doses of radiation are often preferable to higher doses when a pharmaceutical product is shown to be radiation-sensitive. Similarly, several mitigation strategies were explored that lessened the radiation-induced effects in the end product. Using this information, along with published standards, a decision tree can be created to aid pharmaceutical manufacturers in establishing an irradiation process that may be successful for their product.

Terminal sterilization is preferred where possible to ensure patient safety. As previously mentioned, an SAL of 10⁻⁶ where achievable is prescribed for any devices or substances that will come into contact with compromised human tissue unless a risk assessment can be performed to justify a higher SAL (101, 102). A sterilization dose of 25 kGy has traditionally been regarded as a adequate to address products with high pre-sterilization bioburden (up to 1000 CFU/product unit) (103, 104). This may lead to overprocessing of the products, however, as most pharmaceuticals are manufactured in clean environment and have low bioburden.

AAMI has published several methods for sterilization dose substantiation in ANSI/AAMI/ISO 11137-2 (4) for all irradiation modalities, including e-beam and x-ray radiation. With current validation methods, lower and lower doses can be substantiated for a similar initial bioburden. For example, at low initial bioburden levels, the minimum dose substantiated using Method VD_max is 15 kGy, using Method 1 the minimum dose is 11.0 kGy, and using Method 2B the minimum dose is 8.2 kGy. For a product that exhibits radiation sensitivity, there is an advantage to looking at alternate validation methods to substantiate a lower dose.

Product which has very low or no measurable bioburden can be challenging to validate with gamma irradiation. Attention is called to the need to employ a bioburden correction factor for determination of the average bioburden. For average bioburden values <0.1, a verification dose of 0 kGy is specified (VD_max methods). This can create difficulties because bioburden may not be uniformly distributed across the product, resulting in random bioburden spikes that may lead to failure of verification dose testing. Kowalski et al. have proposed alternate approaches for estimating the average bioburden values of products with low bioburden (105).

In conjunction with the initial validation studies, routine dose audits are necessary to maintain the established sterilization dose for the product. These are frequently performed on a quarterly basis or at a lesser frequency once a successful dose audit history and control of the process have been established.

Figure 2 presents a decision tree that can guide a user through the process of validating a terminal dose. The decision tree has been arranged in order of increasing cost and complexity of the validation and process or product changes.

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Figure 2

Radiation sterilization validation decision tree.

The first step in establishing a sterilization process is to determine the initial bioburden. This includes the determination of a bioburden correction factor and screening for the release of substances that may affect bioburden determinations as outlined in ISO 11737-1:2006 (52). Likewise, the test of sterility performed as part of dose verification testing must also be shown to be free of any microbiostatic or microbiocidal substances (106). Then, using published dose setting methods, the achievable minimum dose and the validation effort required can be estimated. If the bioburden on the product results in a sterilization dose that adversely affects the integrity of the product, process changes such as improvements to clean room processes, and the use of clean or previously sterilized components and equipment, can be used to further reduce the microbial load on the product.

The publications reviewed in this paper described several methods to evaluate the suitability of the pharmaceutical product after irradiation, including investigation of the impurities and free radicals present as well as degradation and stability over time. The method of investigation and criteria for pass/fail will depend on both the drug indication and formulation.

If the pharmaceutical product does not pass the evaluation at the dose established for the minimum achievable bioburden, then changes to the irradiation conditions that do not alter the product may be considered. Some of the irradiation conditions used in the publications reviewed include reduced- or controlled-temperature irradiations, the presence or absence of oxygen, and the dose rate (refer to Tables I and III). Any changes to irradiation conditions that affect the degradation of the product may also affect the microbiological kill efficiency of the resulting process; therefore dose establishment validation must be assessed.

If the process is already aseptic and the incoming bioburden cannot be further reduced, then the concept of adjunct processing may be applied (2, 107). This is a method that applies an SAL to an aseptic process, which then allows a reduced radiation dose for the final SAL to meet 10⁻⁶—for example, if an SAL of 10⁻² may be applied to the aseptic process, then a radiation dose which gives and SAL of 10⁻⁴ may be used to achieve 10⁻⁶. There are currently no published methods or guidelines for adjunct processing, so there must be a strong rational in place to justify its use. This rationale may require prior review and approval by the appropriate regulatory authorities.

Alternately, ANSI/AAMI ST 67 gives guidance on choosing an SAL greater than 10⁻⁶ for products that cannot be terminally sterilized at 10⁻⁶ by any modality (i.e., methods other than gamma sterilization). Risk assessment that weighs the benefit of the particular product against the possibility of harm due to a non-sterile unit can be used to justify an SAL other than 10⁻⁶. If a higher SAL can be justified, then the same AAMI/ISO 11137-2 dose setting methods can be used to substantiate the dose required, and in all cases the resultant doses will be significantly lower than those required for an SAL of 10⁻⁶. For both adjunct processing and alternate SALs, the resultant terminal dose can be well beneath 10 kGy. If at these low dose levels the pharmaceutical product is still unusable, then an alternate sterilization methodology should be applied.

Another approach described in some of the papers is modification of the product to increase radiation resistance, including the use of additives that also act as radioprotectants. Certain excipients have been shown to have radioprotective properties, as reported in Tables I through III. Similarly, drug products irradiated in dry or solid form performed better under irradiation then certain liquid forms. Changes to the drug product require a complete revalidation of the product bioburden and functionality before and after irradiation.

Conflict of Interest Declaration

The authors declare that they have no competing interests.