Are radiopharmaceuticals self-sterilizing? Radiation effect of gallium-68 and lutetium-177 on bacillus pumilus and staphylococcus succinus

Simon Poetzsch Charite Universitatsmedizin Berlin Campus Virchow-Klinikum Winfried Brenner (  winfried.brenner@charite.de ) https://orcid.org/0000-0003-2478-6004 Sarah Spreckelmeyer Charite Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität and Berlin Institute of Health, Department of Nuclear Medicine, Augustenburger Platz 1, 13353 Berlin https://orcid.org/0000-0003-1348-0309


Results
Incubation with gallium-68 and lutetium-177 for both 30 minutes and 5 hours post-dispensing did not cause any signi cant effect on bacteria growth. As the theoretical dose is only 0.1-0.6% of the Ph. Eur. recommended dose of 25 kGy, we conclude that the beta and positron energy of lutetium-177 and gallium-68 as used for standard radiopharmaceutical in-house production is not su cient to decrease the number of colony forming units compared to the control values.

Conclusions
Based on these ndings, gallium-68 and lutetium-177 labeled radiopharmaceuticals are not selfsterilizing under the tested conditions with respect to bacillus pumilus and staphylococcus succinus.
Consequently, strict aseptic preparation conditions in addition to end-sterilization of the radiopharmaceutical e.g. through membrane ltration are strongly advised for in-house productions.

Background:
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. [1] 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 ltration and working under aseptic conditions (Ph. Eur. Vol 9.0, 5.1.1). For radiopharmaceuticals, aseptic preparation in combination with membrane ltration 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 ful l 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 ow hoods class A or in hot cells/ isolators using synthesis modules in GMP clean rooms. The nal step of the production is mostly the ltration of the radiopharmaceutical through a 0.22 µm membrane lter 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 [ 99m Tc]-radiopharmaceuticals [2,3]  F]-radiation is not su cient 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 microorganismsradiopharmaceutical 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.
[5] Both radionuclides can be linked to peptides such as DOTATOC (edotreotide) and PSMA. [6] 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. [7] Additionally, this germ is resistant to environmental stresses. Staphylococcus succinus was chosen as a member of the wide-spread genus staphylococcus. [8] 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 rst 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. [ 68 Ga]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.

Staphylococcus succinus
The

Bacillus Pumilus
The results of the 30 minutes and 5 hours incubation experiment post-dispensing are shown in Fig. 3. After 30 minutes, the amount of cfu in the control fraction does not change compared to the start value.

Theoretical Dose
The theoretical dose was calculated using standard radiochemical formulas (Supporting Information). The theoretically achievable dose in the radiopharmaceuticals produced in-house ranges from 45 Gy to 161 Gy for gallium-68 and 12 Gy to 119 Gy for lutetium-177. Compared to the radiation dose to achieve an SAL of 10^-6, we achieve only 0.1-0.6% of the necessary dose of 25 kGy (Table 1). Our results match those results described in the literature. Although these results may be promising in regards to achieving sterility with elevated temperatures during the preparation step, the radiopharmaceutical could still get contaminated during the subsequent dispensing step.

Conclusions:
Based on these ndings, gallium-68 and lutetium-177 labeled radiopharmaceuticals are not selfsterilizing under the tested in-house production conditions with respect to bacillus pumilus and staphylococcus succinus. Consequently, strict aseptic preparation conditions in addition to endsterilization of the radiopharmaceutical e.g. through membrane ltration are strongly advised for in-house productions.

Material And Methods
All materials used were sterile single use materials e.g. syringes.

Post-dispensing Experiments
The overnight culture was homogenized. The bacteria vials for the control and gallium-68/lutetium-177 treatment were prepared as follows: One mL (approximately 1 × 10^9 cfu) of the overnight culture was added to 9 mL 0.9% NaCl solution and homogenized. A sample from this solution was taken to estimate the cfu at the beginning of the experiment (start). As control values, samples were taken from the untreated bacteria-solution after 30 minutes and 5 hours (control).
100 µl of the bacteria vial was used for further dilutions and seeding on counting agar plates. After 24 h, the cfu were counted and the total cfu calculated as described below.
9. Thomas P. Long-term survival of Bacillus spores in alcohol and identi cation of 90% ethanol as relatively more spori/bactericidal. Curr Microbiol. 2012;64(2):130-9. Supplementary Files This is a list of supplementary les associated with this preprint. Click to download. PoetzschBrennerSpreckelmeyerSupportingInformation.docx