Fully-automated production of [68Ga]Ga-PentixaFor on the module Modular Lab-PharmTracer

Background PentixaFor is a promising radiopharmaceutical for positron emission tomography in the detection of different tumor entities and other diseases. Until now, the synthesis of [68Ga]Ga-PentixaFor was reported for the automated synthesis module from Scintomics® only. Our aim was to evaluate the automated synthesis of this radiopharmaceutical on a different module in order to make it available for a broader community. Results The synthesis of [68Ga]Ga-PentixaFor with different amounts of PentixaFor (50 μg, 30 μg and 20 μg) on the Modular Lab PharmTracer (MLPT) from Eckert & Ziegler with the already established synthesis template for [68Ga]Ga-DOTATOC yielded best results with 50 μg PentixaFor for clinical multi-dose application. All different quality control parameters tested (e.g. sterility, stability and radiochemical purity) were in accordance with the European Pharmacopoeia. Conclusions [68Ga]Ga-PentixaFor was successfully synthesized fully-automated on the synthesis module Modular Lab PharmTracer and can be used for multi-dose application in clinical settings.


Background
In 2011, Demmer et al. first introduced chemokine receptor-4 (CXCR4)-binding peptidic probes for molecular imaging (Demmer et al., 2011). PentixaFor (Synonym for CPCR4.2, Fig. 1) is to date the most promising peptide with a high affinity to chemokine receptor-4. PentixaFor can be used as PET imaging agent when coupled to the positron emitting radionuclide gallium-68. Numerous clinical studies have already been performed in vivo and the interest is steadily increasing in new applications for different tumor entities and other diseases, e.g. inflammatory conditions (Bouter et al., 2018). Examples of clinical applications in oncology are the detection of neuroendocrine tumors, multiple myeloma (Pan et al., 2018), glioma, leukemia (Mayerhoefer et al., 2018) or lymphoma (Luo et al., 2019;Haug et al., 2019). Since the precursor PentixaFor was made available until summer 2018 only for use by the synthesis module from Scintomics®, only hospitals that operated a Scintomics® module were able to prepare [ 68 Ga]Ga-PentixaFor. Here, we would like to introduce the synthesis conditions of [ 68 Ga]Ga-PentixaFor on a different synthesis module, namely Modular Lab PharmTracer (MLPT) from Eckert & Ziegler Eurotope GmbHin order to introduce [ 68 Ga]Ga-PentixaFor to a broader community. For that purpose, different concentrations of PentixaFor were evaluated based on quality control parameters. Concerning the synthesis of 68 Ga-tracers in general, different modules are available on the market which have been reviewed by Boschi et al. In addition, generator post-processing steps like fractionation, anionic-exchange and cation-exchange are described (Boschi et al., 2013). The now proposed fully automated pre-purification of gallium-68 on a strong cation exchange (SCX) cartridge has the advantage that gallium-68 becomes concentrated and germanium-68 as well as non-radioactive impurities (e.g. Zn 2+ ) are trapped on the SCX cartridge.

Labeling results of different amounts of PentixaFor
The fully automated production of [ 68 Ga]Ga-PentixaFor was conducted on the commercial labeling synthesis module MLPT. Three different precursor amounts of PentixaFor were tested for radiolabeling with 1080-2500 MBq of gallium-68 (three generators from type GalliaPharm®) -50 μg, 30 μg and 20 μg. As shown in Table 1, the highest amount of PentixaFor (50 μg) resulted in 94.7% radiochemical yield (RCY, n.d.c.) in the product vial, 2.6% in the waste vial and 1.7% on the C 18 cartridge. By lowering the amount of Pentixa-For to 30 μg, the RCY of the product decreased to 80.5% (n.d.c.) and the amount on the C 18 cartridge increased to 13.3% (n.d.c.). By lowering the amount of starting material to 20 μg, the efficiency became worse. Only 20.0% (n.d.c.) of radiochemical yield was found  in the product vial and 69.4% on the C 18 cartridge. The distribution of the radioactivity in the cassette for the productions is presented in Fig. 2. The radioactivity found in the reactor and SCX cartridge did not change significantly by changing the amount of starting material.
In the waste fraction, with decreasing peptide amounts from 50 to 20 μg, the radioactivity increased from 2.6% to 9.3%. Interestingly, the activity measured in the waste vial consists of approximately 25% product and 75% free gallium-68 as seen in the radio-HPLC chromatogram as well as in the radio-iTLC (Supporting Information, Fig. 1).

Quality control of [ 68 Ga]Ga-PentixaFor
The radiochemical purity was evaluated with a standard protocol by radio-HPLC and radio-iTLC.
With radio-HPLC, free gallium-68 would be detected at t R = 2.5 min, whereas gallium-68 bound to PentixaFor was detected at t R = 11 min. Radioactive impurities could not be detected with this method (Fig. 3a).
With radio-iTLC, no 68 Ga-colloide could be detected at R f = 0.2 but the product at R f = 0.8 (Fig. 4).
The cold standard [ nat Ga]Ga-PentixaFor shows a similar retention time of t R = 11 min as seen in Fig. 3b.
In addition, the product solution was tested for endotoxins. For this purpose, the solution was diluted with endotoxin-free-water in a ratio 1:10. For all samples, an endotoxin concentration below 0.5 IE/mL was detected. This is in accordance with the European Pharmacopoeia (9.0/0125).
With regards to sterility, all products were sterile. The stability of [ 68 Ga]Ga-PentixaFor in aqueous solution at room temperature was tested up to 4 h via radio-HPLC. As seen in Fig. 5, [ 68 Ga]Ga-PentixaFor is stable at those conditions. No radioactive by-products or free gallium-68 could be detected during this time period. In Table 2, product specifications of the most promising approach with 50 μg Pentix-aFor are summarized. Acceptance criteria are based on the European Pharmacopoeia (9.0/0125).

Discussion
For establishing the automated synthesis of [ 68 Ga]Ga-PentixaFor on the Modular Lab PharmTracer from Eckert & Ziegler Eurotope GmbH for the first time, three different amounts of PentixaFor were evaluated for the production of this tracer with the same standard synthesis template as used for [ 68 Ga]Ga-DOTATOC on the synthesis module MLPT. The results summarized in Table 1 and Fig. 2 demonstrate that the amount of 50 μg of PentixaFor yields the best radioactivity distribution on the cassette. The low standard deviations for 50 μg confirm the high reproducibility of the production.
For 30 μg and 20 μg, an increasing amount of radioactivity on the C 18 cartridge and a decreasing yield of activity in the product vial was observed. Theoretically, two scenarios are to our knowledge possible to explain the increased radiochemical yield on the C 18 cartridge. Either the product is trapped on the C 18 cartridge or uncomplexed 68 Gacolloide. Product could be eluted from the C 18 cartridge with pure ethanol. After the automated synthesis is completed, we tried to elute the activity. This resulted in no change of activity measured on the C 18 cartridge. As a consequence, no product is stuck on the C 18 cartridge. The most likely explanation is that 20 μg and 30 μg are too low to complex 68 Ga 3+ ions completely resulting in a trapping of unbound gallium-68 in the form of 68 Ga 3+ -colloide on the C 18 cartridge. Based on the analyzation of the waste fraction by radio-HPLC and radio-iTLC, 25% of the activity found in the waste fraction was product. With respect to the total radioactivity used in 50 μg experiments, this means a negligibly product activity loss of under 1%.
The minimum amount needed to fully complex PentixaFor under the described conditions was 50 μg. In former studies in which [ 68 Ga]Ga-PentixaFor was synthesized on a synthesis module from Scintomics®, 40 μg and 20 μg of PentixaFor were used in a final product volume of 15 mL and 14 mL, respectively. The amount of PentixaFor that is safe to be used in vivo is therefore < 20 μg per patient based on those results (Lapa et al., 2016;Wester et al., 2015). In the frame of the here described synthesis protocol, it is important to make sure via standard operating procedures (SOPs), that the maximal injected amount of tracer is below 20 μg/patient. Thus, the maximum injected volume needs to be below 3.6 mL.
With regard to the quality control parameters, we achieved an endotoxine-free, sterile and stable solution of [ 68 Ga]Ga-PentixaFor with a radiochemical purity > 95% over a period of 4 h. Moreover, all tested quality control parameters were in accordance with the European Pharmacopoeia (see Table 2). Calculating the ethanol content based on the parameters chosen in the synthesis template of the MPLT software, results in an ethanol content that cannot exceed 6.2%. Nevertheless, we recommend to either measure the ethanol content of every batch by gas chromatography before releasing it for clinical application, or validate the ethanol content by an external laboratory if a gas chromatography is not available onsite.

Conclusions
[ 68 Ga]Ga-PentixaFor was successfully synthesized fully-automated on the synthesis module MLPT for the first time. All the tested quality parameters for the radiochemical purity, pH, endotoxins and sterility are in accordance with the European Pharmacopoeia. In addition, the product solution is stable for at least 4 h after production, as shown by radio-HPLC. Thus, [ 68 Ga]Ga-PentixaFor can be easily and reliably produced on the module MLPT for clinical application.

Materials
PentixaFor was obtained from PentixaPharm. An aqueous stock solution of 1 mg/ mL was prepared and kept at − 15°C. All chemicals were of pure chemical grade and solvents for high-pressure-liquid-chromatography (HPLC) were obtained as HPLC grade. TraceSelect water (Sigma-Aldrich) was used in all experiments. The pharmaceutical grade 68 Ge/ 68 Ga generator (GalliaPharm®, Eckert & Ziegler Radiopharma GmbH, Germany), Modular Lab PharmTracer (Eckert & Ziegler Eurotope GmbH, Germany) and reagent set EZ-102 (Eckert & Ziegler Eurotope GmbH, Germany) were used. The amount of detected metal impurities/ 68 Ge breakthrough as provided by the manufacturer was less than the defined limit in the European Pharmacopeia monograph. Activity counting was determined using a borehole counter (Nuklear-Medizintechnik Dresden GmbH, Germany). HPLC was performed using the HPLC system Knauer Azura (UVD: 2.1 L; P6.1L) coupled with UV and  (Schultz et al., 2013). The reaction vial containing the reaction mixture was preheated to 50°C, and after the 68 Ga-activity was transferred to the reaction vial, the temperature was increased to 95°C. After 5 min at 95°C, the reaction mixture was cooled down by adding 3 mL of 0.9% NaCl. The crude reaction solution was subsequently transferred to the C 18 cartridge for purification. The final product was eluted from the C 18 cartridge using a EtOH/H 2 O (50/50) solution and passed through a 0.22 μm sterile filter into the sterile product vial. The resulting product was diluted to 9 mL with 0.9% NaCl. A sample for quality control was taken.

Quality control
After synthesis, the product vial is removed from the MLPT module and further evaluated for quality control determining the following parameters: total product activity, 68 Ga 3+ -identity via half-life time, chemical purity (pH, sterility and endotoxins) and radiochemical purity (high-pressure-liquid chromatography). The stability of the product at room temperature was monitored by radio-HPLC for 4 h. Radio-HPLC was performed with a standardized method. A: water + 0.1% TFA; B: acetonitrile + 0.1% TFA, gradient: 0-25 min A: 0-100%; 25-28 min A: 100-0%. The limit of detection of the radio-HPLC is 10 kBq/ 20 μL injected volume and the recovery is approximately 80%. For iTLC, ammonium acetate/methanol (1/1) was used as mobile phase and iTLC-SG strips as stationary phase. The sterility tests were performed as described in the European Pharmacopoeia at the Institut für Hygiene, Charité.