Radiopharmaceuticals are injections or infusions for diagnostic or therapeutic use which effect is caused by the incorporated radionuclide. Radioactivity has, depending on its character, a very high energy that can cause single- or even double-strand breaks of DNA that eventually lead to apoptosis of the respective cells. In nuclear medicine, this feature is used to detect or even treat various diseases. As radiopharmaceuticals are applied parenterally, they must be sterilized before use. In the European Pharmacopoeia, six different methods are described to achieve sterility with a sterility assurance level (SAL) of 10− 6 – steam sterilization, dry heat, radiation, gas, membrane filtration and working under aseptic conditions (Ph. Eur. Vol 9.0, 5.1.1). For radiopharmaceuticals, aseptic preparation in combination with membrane filtration is the most chosen sterilizing method. Other methods are usually not applicable due to the short half-lives of the radionuclides and/or incompatibility of the radiopharmaceutical with elevated temperatures as they may contain heat-sensitive biomolecules. In general, the production of radiopharmaceuticals needs to fulfil the requirements of good manufacturing practice (GMP) and sterile (starting) materials are used wherever possible. The (automated) production is performed under aseptic conditions in laminar-air flow hoods class A or in hot cells/ isolators using synthesis modules in GMP clean rooms. The final step of the production is mostly the filtration of the radiopharmaceutical through a 0.22 µm membrane filter for end-sterilization. In order to check for sterility conformity, the radiopharmaceutical is incubated (retrospectively) for two weeks on growth media as described in the European Pharmacopoeia (Ph. Eur. Vol 9.0, 2.6.1).
Our hypothesis is, that radiopharmaceuticals might be self-sterilizing due to the fact that they contain a high radioactivity concentration and high-energy radionuclides. To the best of our knowledge, this hypothesis was thus far only tested for [99mTc]-radiopharmaceuticals[2, 3] and [18F]-radiopharmaceuticals. Brown et al. state, that the time lag between preparation and the sterility test of [99mTc]-radiopharmaceuticals should be as short as possible. Their reasoning is, that longer time lags have a greater chance to obtain negative sterility results although the preparation might have been already contaminated at the time of dispensing. Jörg et al. investigated the “autosterilization” effect of [18F]-radiopharmaceuticals and concluded, that intrinsic [18F]-radiation is not sufficient for achieving sterility of the radiopharmaceutical. In our opinion, the main drawbacks, which we will avoid in our experiment set-up of these studies, were the long incubation times of up to 11 hours with the radiation source and the direct inoculation of the radiopharmaceutical of interest with the microorganisms. These long incubation times are not realistic in the daily routine because in-house synthesized radiopharmaceuticals in nuclear medicine departments are usually applied within one hour after preparation to the patients. Furthermore, samples taken directly out of the inoculated microorganisms-radiopharmaceutical solution contain remaining radioactivity. Thus, the seeded sample on growth media may be effected by the remaining radioactivity.
In the scope of this work, we will focus on the positron emitter gallium-68 and on the therapeutically used beta- and gamma-emitter lutetium-177, as they are routinely used for in-house synthesis of radiopharmaceuticals in nuclear medicine departments. Lutetium-177 is primarily a beta-emitter (490 keV) that decays after 6.7 days to the stable hafnium-177 but also emits gamma rays with an energy of 113 keV (3%) and 210 keV (11%). Gallium-68 is a positron emitter (1899 keV (88%) and 822 keV (1%)) with gamma energies of 511 keV (178%) and 1077 keV (3%) with a half-life of 68 minutes. Both radionuclides can be linked to peptides such as DOTATOC (edotreotide) and PSMA.
The growth behavior of two different germs have been evaluated at two different steps during the preparation – either pre-dispensing or post-dispensing. Bacillus pumilus is a known radiation resistant species and can be used to validate ionizing radiation sterilization as used in the European Pharmacopoeia as sterility marker. Additionally, this germ is resistant to environmental stresses. Staphylococcus succinus was chosen as a member of the wide-spread genus staphylococcus. Both germs are Gram-positive and categorized in the lowest biological safety class and can be handled in normal laboratories.
As gallium-68 and lutetium-177 labeled radiopharmaceuticals are mostly prepared in-house, we assumed that the time of injection will be within 30 minutes post-dispensing. Consequently, we chose 30 minutes as first incubation time point. We also added a 5 hours incubation point post-dispensing, as this might be relevant for lutetium-177 radiopharmaceuticals when they are produced ahead of injection.
Additionally, pre-dispensing measures were chosen. During the synthesis of e.g. [68Ga]Ga-DOTATOC, the starting materials are heated to 95 °C for approximately 15 min. Here, we tested the effect of an elevated temperature on the bacteria after 15 minutes. Additionally, gallium-68 radiopharmaceuticals are often post-processed with a maximum of 10% (V/V) ethanol. To take this also into account, we tested the bacterial growth after 30 min incubation with 10% (V/V) ethanol.