Advanced pathway engineering for phototrophic putrescine production.

The polyamine putrescine (1,4-diaminobutane) contributes to cellular fitness in most organisms, where it is derived from the amino acids ornithine or arginine. In the chemical industry, putrescine serves as a versatile building block for polyamide synthesis. The green microalga Chlamydomonas reinhardtii accumulates relatively high putrescine amounts, which, together with recent advances in genetic engineering, enables the generation of a powerful green cell factory to promote sustainable biotechnology for base chemical production. Here, we report a systematic investigation of the native putrescine metabolism in C. reinhardtii, leading to the first CO2 -based bio-production of putrescine, by employing modern synthetic biology and metabolic engineering strategies. A CRISPR/Cas9-based knockout of key enzymes of the polyamine biosynthesis pathway identified ornithine decarboxylase 1 (ODC1) as a gatekeeper for putrescine accumulation and demonstrated that the arginine decarboxylase (ADC) route is likely inactive and that amine oxidase 2 (AMX2) is mainly responsible for putrescine degradation in C. reinhardtii. A 4.5-fold increase in cellular putrescine levels was achieved by engineered overexpression of potent candidate ornithine decarboxylases (ODCs). We identified unexpected substrate promiscuity in two bacterial ODCs, which exhibited co-production of cadaverine and 4-aminobutanol. Final pathway engineering included overexpression of recombinant arginases for improved substrate availability as well as functional knockout of putrescine degradation, which resulted in a 10-fold increase in cellular putrescine titres and yielded 200 mg/L in phototrophic high cell density cultivations after 10 days.

Engineering a powerful green cell factory for robust photoautotrophic diterpenoid production.

Diterpenoids display a large and structurally diverse class of natural compounds mainly found as specialized plant metabolites. Due to their diverse biological functions they represent an essential source for various industrially relevant applications as biopharmaceuticals, nutraceuticals, and fragrances. However, commercial production utilizing their native hosts is inhibited by low abundances, limited cultivability, and challenging extraction, while the precise stereochemistry displays a particular challenge for chemical synthesis. Due to a high carbon flux through their native 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway towards photosynthetically active pigments, green microalgae hold great potential as efficient and sustainable heterologous chassis for sustainable biosynthesis of plant-derived diterpenoids. In this study, innovative synthetic biology and efficient metabolic engineering strategies were systematically combined to re-direct the metabolic flux through the MEP pathway for efficient heterologous diterpenoid synthesis in C. reinhardtii. Engineering of the 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) as the main rate-limiting enzyme of the MEP pathway and overexpression of diterpene synthase fusion proteins increased the production of high-value diterpenoids. Applying fully photoautotrophic high cell density cultivations demonstrate potent and sustainable production of the high-value diterpenoid sclareol up to 656 mg L-1 with a maximal productivity of 78 mg L-1 day-1 in a 2.5 L scale photobioreactor, which is comparable to sclareol titers reached by highly engineered yeast. Consequently, this work represents a breakthrough in establishing a powerful phototrophic green cell factory for the competetive use in industrial biotechnology.

Psoralen as a Photosensitizers for Photodynamic Therapy by Means of In Vitro Cherenkov Light.

Possible enhancements of DNA damage with light of different wavelengths and ionizing radiation (Rhenium-188-a high energy beta emitter (Re-188)) on plasmid DNA and FaDu cells via psoralen were investigated. The biophysical experimental setup could also be used to investigate additional DNA damage due to photodynamic effects, resulting from Cherenkov light. Conformational changes of plasmid DNA due to DNA damage were detected and quantified by gel electrophoresis and fluorescent staining. The clonogene survival of the FaDu cells was analyzed with colony formation assays. Dimethyl sulfoxide was chosen as a chemical modulator, and Re-188 was used to evaluate the radiotoxicity and light (UVC: λ = 254 nm and UVA: λ = 366 nm) to determine the phototoxicity. Psoralen did not show chemotoxic effects on the plasmid DNA or FaDu cells. After additional treatment with light (only 366 nm-not seen with 254 nm), a concentration-dependent increase in single strand breaks (SSBs) was visible, resulting in a decrease in the survival fraction due to the photochemical activation of psoralen. Whilst UVC light was phototoxic, UVA light did not conclude in DNA strand breaks. Re-188 showed typical radiotoxic effects with SSBs, double strand breaks, and an overall reduced cell survival for both the plasmid DNA and FaDu cells. While psoralen and UVA light showed an increased toxicity on plasmid DNA and human cancer cells, Re-188, in combination with psoralen, did not provoke additional DNA damage via Cherenkov light.

Nuclear Targeting with an Auger Electron Emitter Potentiates the Action of a Widely Used Antineoplastic Drug.

We present the combination of the clinically well-proven chemotherapeutic agent, Doxorubicin, and (99m)Tc, an Auger and internal conversion electron emitter, into a dual-action agent for therapy. Chemical conjugation of Doxorubicin to (99m)Tc afforded a construct which autonomously ferries a radioactive payload into the cell nucleus. At this site, damage is exerted by dose deposition from Auger radiation. The (99m)Tc-conjugate exhibited a dose-dependent inhibition of survival in a selected panel of cancer cells and an in vivo study in healthy mice evidenced a biodistribution which is comparable to that of the parent drug. The homologous Rhenium conjugate was found to effectively bind to DNA, inhibited human Topoisomerase II, and exhibited cytotoxicity in vitro. The collective in vitro and in vivo data demonstrate that the presented metallo-conjugates closely mimic native Doxorubicin.

Gamma camera imaging characteristics of 166Ho and 99mTc used in Selective Internal Radiation Therapy.

BACKGROUND: The administration of a 166Ho scout dose is available as an alternative to 99mTc particles for pre-treatment imaging in Selective Internal Radiation Therapy (SIRT). It has been reported that the 166Ho scout dose may be more accurate for the prediction of microsphere distribution and the associated therapy planning. The aim of the current study is to compare the scintigraphic imaging characteristics of both isotopes, considering the objectives of the pre-treatment imaging using clinically geared phantoms. METHODS: Planar and SPECT/CT images were obtained using a NEMA image quality phantom in different phantom setups and another body-shaped phantom with several inserts. The influence of collimator type, count statistics, dead time effects, isotope properties and patient obesity on spatial resolution, contrast recovery and the detectability of small activity accumulations was investigated. Furthermore, the effects of the imaging characteristics on personalized dosimetry are discussed. RESULTS: The images with 99mTc showed up to 3 mm better spatial resolution, up to two times higher contrast recovery and significantly lower image noise than those with 166Ho. The contrast-to-noise ratio was up to five times higher for 99mTc than for 166Ho. Only when using 99mTc all activity-filled spheres could be distinguished from the activity-filled background. The measurements mimicking an obese patient resulted in a degraded image quality for both isotopes. CONCLUSIONS: Our measurements demonstrate better scintigraphic imaging properties for 99mTc compared to 166Ho in terms of spatial resolution, contrast recovery, image noise, and lesion detectability. While the 166Ho scout dose promises better prediction of the microsphere distribution, it is important to consider the inferior imaging characteristics of 166Ho, which may affect individualized treatment planning in SIRT.

Investigation of Photodynamic Therapy Promoted by Cherenkov Light Activated Photosensitizers-New Aspects and Revelations.

This work investigates the proposed enhanced efficacy of photodynamic therapy (PDT) by activating photosensitizers (PSs) with Cherenkov light (CL). The approaches of Yoon et al. to test the effect of CL with external radiation were taken up and refined. The results were used to transfer the applied scheme from external radiation therapy to radionuclide therapy in nuclear medicine. Here, the CL for the activation of the PSs (psoralen and trioxsalen) is generated by the ionizing radiation from rhenium-188 (a high-energy beta-emitter, Re-188). In vitro cell survival studies were performed on FaDu, B16 and 4T1 cells. A characterization of the PSs (absorbance measurement and gel electrophoresis) and the CL produced by Re-188 (luminescence measurement) was performed as well as a comparison of clonogenic assays with and without PSs. The methods of Yoon et al. were reproduced with a beam line at our facility to validate their results. In our studies with different concentrations of PS and considering the negative controls without PS, the statements of Yoon et al. regarding the positive effect of CL could not be confirmed. There are slight differences in survival fractions, but they are not significant when considering the differences in the controls. Gel electrophoresis showed a dominance of trioxsalen over psoralen in conclusion of single and double strand breaks in plasmid DNA, suggesting a superiority of trioxsalen as a PS (when irradiated with UVA). In addition, absorption measurements showed that these PSs do not need to be shielded from ambient light during the experiment. An observational test setup for a PDT nuclear medicine approach was found. The CL spectrum of Re-188 was measured. Fluctuating inconclusive results from clonogenic assays were found.

Radiosynthesis of Stable 198Au-Nanoparticles by Neutron Activation of αvß3-Specific AuNPs for Therapy of Tumor Angiogenesis.

This paper reports on the development of stable tumor-specific gold nanoparticles (AuNPs) activated by neutron irradiation as a therapeutic option for the treatment of cancer with high tumor angiogenesis. The AuNPs were designed with different mono- or dithiol-ligands and decorated with different amounts of Arg-Gly-Asp (RGD) peptides as a tumor-targeting vector for αvß3 integrin, which is overexpressed in tissues with high tumor angiogenesis. The AuNPs were evaluated for avidity in vitro and showed favorable properties with respect to tumor cell accumulation. Furthermore, the therapeutic properties of the [198Au]AuNPs were evaluated in vitro on U87MG cells in terms of cell survival, suggesting that these [198Au]AuNPs are a useful basis for future therapeutic concepts.

[Evaluation of radionuclide impurities in several radiopharmaceuticals]. / Überprüfung der Radionuklidreinheit in ausgewählten Radiotherapeutika.

PURPOSE: Revisions to German radiation protection laws have resulted in updated limit values, which could affect the unrestricted release of waste produced by nuclear medicine therapy. In addition, signs of long-lived concomitant nuclides in 153Sm and 223Ra radiopharmaceuticals have been seen in the past. Therefore, the goal of this article was to analyze the radionuclidic purity of selected radiopharmaceuticals. METHOD: 48 samples from 12 different radiopharmaceuticals were examined. A high purity germanium semiconductor detector (HPGe detector) was used for the qualitative and quantitative evaluation of concomitant nuclides. RESULTS: Various europium isotopes were identified in 90Y-citrate, 153Sm-Quadramet, 166Ho-QuiremSpheres, and 169Er-erbium citrate, with the greatest amount being found in 153Sm (7.0 ppm (152Eu), 8.4 ppm (154Eu), and 2.1 ppm (155Eu)). 169Yb was the most significant impurity in 169Er (513 ppm). In the case of 177Lu radiopharmaceuticals, there was a significant difference in the 177mLu content (0.8 ppm vs. 0.0024 ppm) between two different manufacturers. No concomitant nuclides could be found within the detection limits in the case of 90Y spheres, 223Ra, and 225Ac. CONCLUSION: The limit values for unrestricted release are exceeded manyfold in the case of the identified concomitant nuclides. As a result, alternative release procedures (extension of the decay time, specific release, release in the individual case) or transfer to collection facilities must be considered. Technical methods for reducing or preventing impurities could also be a possible solution. Consequences for patient radiation exposure were able to be ruled out.

The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing.

Biotechnological application of the green microalga Chlamydomonas reinhardtii hinges on the availability of selectable markers for effective expression of multiple transgenes. However, biological safety concerns limit the establishment of new antibiotic resistance genes and until today, only a few auxotrophic markers exist for C. reinhardtii. The recent improvements in gene editing via CRISPR/Cas allow directed exploration of new endogenous selectable markers. Since editing frequencies remain comparably low, a Cas9-sgRNA ribonucleoprotein (RNP) delivery protocol was strategically optimized by applying nitrogen starvation to the pre-culture, which improved successful gene edits from 10% to 66% after pre-selection. Probing the essential polyamine biosynthesis pathway, the spermidine synthase gene (SPD1) is shown to be a potent selectable marker with versatile biotechnological applicability. Very low levels of spermidine (0.75 mg/L) were required to maintain normal mixotrophic and phototrophic growth in newly designed spermidine auxotrophic strains. Complementation of these strains with a synthetic SPD1 gene was achieved when the mature protein was expressed in the cytosol or targeted to the chloroplast. This work highlights the potential of new selectable markers for biotechnology as well as basic research and proposes an effective pipeline for the identification of new auxotrophies in C. reinhardtii.

Epigenetic-Like Stimulation of Receptor Expression in SSTR2 Transfected HEK293 Cells as a New Therapeutic Strategy.

The aim of the study was to increase the uptake of the SSTR2-targeted radioligand Lu-177-DOTATATE using the DNA methyltransferase inhibitor (DNMTi) 5-aza-2'-deoxycytidine (5-aza-dC) and the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The HEKsst2 and PC3 cells were incubated with variable concentrations of 5-aza-dC and VPA to investigate the uptake of Lu-177-DOTATATE. Cell survival, subsequent to external X-rays (0.6 or 1.2 Gy) and a 24 h incubation with 57.5 or 136 kBq/mL Lu-177-DOTATATE, was investigated via colony formation assay to examine the effect of the epidrugs. In the case of stimulated HEKsst2 cells, the uptake of Lu-177-DOTATATE increased by a factor of 28 in comparison to the unstimulated cells. Further, stimulated HEKsst2 cells demonstrated lower survival fractions (factor 4). The survival fractions of the PC3 cells remained almost unchanged. VPA and 5-aza-dC did not induce changes to the intrinsic radiosensitivity of the cells after X-ray irradiation. Clear stimulatory effects on HEKsst2 cells were demonstrated by increased cell uptake of the radioligand and enhanced SST2 receptor quantity. In conclusion, the investigated approach is suitable to stimulate the somatostatin receptor expression and thus the uptake of Lu-177-DOTATATE, enabling a more efficient treatment for patients with poor response to peptide radionuclide therapy (PRRT).

Comparison of image quality and spatial resolution between 18F, 68Ga, and 64Cu phantom measurements using a digital Biograph Vision PET/CT.

BACKGROUND: PET nuclides can have a considerable influence on the spatial resolution and image quality of PET/CT scans, which can influence diagnostics in oncology, for example. The individual impact of the positron energy of 18F, 68Ga, and 64Cu on spatial resolution and image quality was compared for PET/CT scans acquired using a clinical, digital scanner. METHODS: A Jaszczak phantom and a NEMA PET body phantom were filled with 18F-FDG, 68Ga-HCl, or 64Cu-HCl, and PET/CT scans were performed on a Siemens Biograph Vision. Acquired images were analyzed regarding spatial resolution and image quality (recovery coefficients (RC), coefficient of variation within the background, contrast recovery coefficient (CRC), contrast-noise ratio (CNR), and relative count error in the lung insert). Data were compared between scans with different nuclides. RESULTS: We found that image quality was comparable between 18F-FDG and 64Cu-HCl PET/CT measurements featuring similar maximal endpoint energies of the positrons. In comparison, RC, CRC, and CNR were degraded in 68Ga-HCl data despite similar count rates. In particular, the two smallest spheres of 10 mm and 13 mm diameter revealed lower RC, CRC, and CNR values. The spatial resolution was similar between 18F-FDG and 64Cu-HCl but up to 18% and 23% worse compared with PET/CT images of the NEMA PET body phantom filled with 68Ga-HCl. CONCLUSIONS: The positron energy of the PET nuclide influences the spatial resolution and image quality of a digital PET/CT scan. The image quality and spatial resolution of 68Ga-HCl PET/CT images were worse than those of 18F-FDG or 64Cu-HCl despite similar count rates.

Transarterial Radioembolization Planning and Treatment with Microspheres Containing Holmium-166: Determination of Renal and Intestinal Radionuclide Elimination, Effective Half-Life, and Regulatory Aspects.

After transarterial radioembolization (TARE) with microspheres loaded with holmium-166, radioactivity is excreted from the body. The aim of this study was to evaluate radioactive renal and intestinal excretions after TARE planning and treatment procedures with holmium-166-loaded microspheres and to correlate the findings with the intratherapeutic effective half-life. Urinary and intestinal excretions of patients who underwent TARE procedures were collected during postinterventional intervals of 24 h (TARE planning) and 48 h (TARE treatment). Whole-body effective half-life measurements were performed. Calibrations of the 166Ho measuring system showed evidence of long-living nuclides. For excretion determination, 22 TARE planning procedures and 29 TARE treatment procedures were evaluated. Mean/maximum total excretion proportions of the injected 166Ho were 0.0038%/0.0096% for TARE planning procedures and 0.0061%/0.0184% for TARE treatment procedures. The mean renal fractions of all measured excretions were 97.1% and 98.1%, respectively. Weak correlations were apparent between the injected and excreted activities (R2 planning/treatment: 0.11/0.32). Mean effective 166Ho half-lives of 24.03 h (planning) and 25.62 h (treatment) confirmed low excretions. Radioactive waste disposal regulations of selected jurisdictions can be met but must be reviewed before implementing this method into clinical practice. Inherent long-living nuclide impurities should be considered.

[Treatment Planning and Dose Verification for Combined Internal and External Radiotherapy (CIERT)]. / Bestrahlungsplanung und Dosisverifikation für die kombinierte interne und externe Strahlentherapie (CIERT).

AIM: The combined internal and external radiotherapy (CIERT) take advantage of the benefits from radionuclide therapy and external beam irradiation. These include steep dose gradients and a low toxicity to normal tissue due to the use of unsealed radioisotopes as well as homogeneous dose distribution within the tumor due to external beam irradiation. For a combined irradiation planning, an infrastructure has to be developed that takes into account the dose contributions from both modalities. A physical verification of the absorbed dose distribution should follow by measurements using OSL detectors. METHOD: Internal irradiation was performed using Re-188 in a cylindrical phantom with three inserts. SPECT images were acquired to calculate the internal dose using the software STRATOS. The dose distribution was exported as DICOM-RT data and imported in the software Pinnacle. Based on the internal dose distribution the external irradiation using 6 MV photons was planned. The dose contributions of both modalities separately as well as for combined irradiation was measured using OSL detectors made out of Beryllium oxide. RESULTS: The planed doses of combined irradiation (1 Gy, 2 Gy, 4 Gy) could be verified within the uncertainty of the detectors. The mean energy response to Re-188 was (88.6 ± 2.4) % with respect to the calibration with 200 kV X-ray irradiation. The energy response to 6 MV photons was (146.0 ± 4.9) %. CONCLUSION: A workflow for the treatment planning of combined internal and external radiotherapy has been developed and tested. Measurements verified the calculated doses. Therefore, the physical and technical basis for the dosimetry of combined irradiation were worked out.

Comparison of conventional and integrated fixed-film activated sludge systems: attached- and suspended-growth functions and quantitative polymerase chain reaction measurements.

Pilot-scale integrated fixed-film activated sludge (IFAS) and non-IFAS control systems were compared, with respect to overall performance and functional behaviors and microbial population composition in the attached and suspended phases. The suspended phases of the control and IFAS systems exhibited similar rates of ammonia consumption; the attached phase in the second aerobic IFAS reactor had significantly higher rates of ammonia consumption and nitrate production than any other biomass source, and the attached biomass from the first aerobic reactor had the lowest ammonia consumption rates. Quantitative polymerase chain reaction (qPCR) indicated the presence of the ammonia-oxidizing bacteria Nitrosomonas oligotropha and the nitrite-oxidizing bacteria Nitrospira spp. and Nitrobacter spp. Mathematical modeling and qPCR both indicated greater concentrations of nitrifiers in the attached phases of a downstream aerobic reactor relative to the upstream reactor, possibly because of increased competition from heterotrophs for space in the attached phase of the upstream aerobic reactor.

High cell density cultivation enables efficient and sustainable recombinant polyamine production in the microalga Chlamydomonas reinhardtii.

Modern chemical industry calls for new resource-efficient and sustainable value chains for production of key base chemicals such as polyamines. The green microalga Chlamydomonas reinhardtii offers great potential as an innovative green-cell factory by combining fast and inexpensive, phototrophic growth with mature genetic engineering. Here, overexpression of recombinant lysine decarboxylases in C. reinhardtii enabled the robust accumulation of the non-native polyamine cadaverine, which serves as building block for bio-polyamides. The issue of low cell densities, limiting most microalgal cultivation processes was resolved by systematically optimizing cultivation parameters. A new, easy-to-apply and fully phototrophic medium enables high cell density cultivations of C. reinhardtii with a 6-fold increase in biomass and cell count (20 g/L biomass dry weight, ~2·108 cells/mL). Application of high cell density cultivations in established photobioreactors resulted in a 10-fold increase of cadaverine yields, with up to 0.24 g/L after 9 days and maximal productivity of 0.1 g/L/d.

Rational Promoter Engineering Enables Robust Terpene Production in Microalgae.

Microalgal biotechnology promises sustainable light-driven production of valuable bioproducts and addresses urgent demands to attain a sustainable economy. However, to unfold its full potential as a platform for biotechnology, new and powerful tools for nuclear engineering need to be established. Chlamydomonas reinhardtii, the model for microalgal synthetic biology and genetic engineering has already been used to produce various bioproducts. Nevertheless, low transgene titers, the lack of potent expression elements, and sparse comparative evaluation prevents further development of C. reinhardtii as a biotechnological host. By systematically evaluating existing expression elements combined with rational promoter engineering, we established novel synthetic expression elements, improved the standardized application of synthetic biology tools, and unveiled an existing synergism between the PSAD 5' UTR and its corresponding chloroplast targeting peptide. Promoter engineering strategies, implemented in a newly designed synthetic algal promoter, increased the production of the sesquiterpene (E)-α-bisabolene by 18-fold compared to its native version and 4-fold to commonly used expression elements. Our results improve the application of synthetic biology in microalgae and display a significant step toward establishing C. reinhardtii as a sustainable green cell-factory.

A novel, robust and mating-competent Chlamydomonas reinhardtii strain with an enhanced transgene expression capacity for algal biotechnology.

In the future, algae biotechnology could play an important role in sustainable development, especially with regard to the production of valuable chemicals. Among the established laboratory strains with efficient transgene expression, there are none that have demonstrated the required robustness for industrial applications, which generally require growth at larger scale. Here, we created a robust and mating-competent cell line of the green microalga Chlamydomonas reinhardtii, which also possesses a high transgene expression capacity. This strain shows a comparably high resistance to shear stress by accumulating increased amounts of biomass under these conditions. As a proof-of-concept, a high phototrophic productivity of cadaverine from CO2 and nitrate was demonstrated by efficiently expressing a bacterial l-lysine decarboxylase. In contrast to other established strains, this novel chassis strain for phototrophic production schemes is equipped with the traits required for industrial applications, by combining mating-competence, cell wall-mediated robustness and high level transgene expression.

αvß3-Specific Gold Nanoparticles for Fluorescence Imaging of Tumor Angiogenesis.

This paper reports on the development of tumor-specific gold nanoparticles (AuNPs) as theranostic tools intended for target accumulation and the detection of tumor angiogenesis via optical imaging (OI) before therapy is performed, being initiated via an external X-ray irradiation source. The AuNPs were decorated with a near-infrared dye, and RGD peptides as the tumor targeting vector for αvß3-integrin, which is overexpressed in tissue with high tumor angiogenesis. The AuNPs were evaluated in an optical imaging setting in vitro and in vivo exhibiting favorable diagnostic properties with regards to tumor cell accumulation, biodistribution, and clearance. Furthermore, the therapeutic properties of the AuNPs were evaluated in vitro on pUC19 DNA and on A431 cells concerning acute and long-term toxicity, indicating that these AuNPs could be useful as radiosensitizers in therapeutic concepts in the future.

Ac-EAZY! Towards GMP-Compliant Module Syntheses of 225Ac-Labeled Peptides for Clinical Application.

The application of 225Ac (half-life T1/2 = 9.92 d) dramatically reduces the activity used for peptide receptor radionuclide therapy by a factor of 1000 in comparison to 90Y, 177Lu or 188Re while maintaining the therapeutic outcome. Additionally, the range of alpha particles of 225Ac and its daughter nuclides in tissue is much lower (47-85 µm for alpha energies Eα = 5.8-8.4 MeV), which results in a very precise dose deposition within the tumor. DOTA-conjugated commercially available peptides used for endoradiotherapy, which can readily be labeled with 177Lu or 90Y, can also accommodate 225Ac. The benefits are lower doses in normal tissue for the patient, dose reduction of the employees and environment and less shielding material. The low availability of 225Ac activity is preventing its application in clinical practice. Overcoming this barrier would open a broad field of 225Ac therapy. Independent which production pathway of 225Ac proves the most feasible, the use of automated synthesis and feasible and reproducible patient doses are needed. The Modular-Lab EAZY is one example of a GMP-compliant system, and the cassettes used for synthesis are small. Therefore, also the waste after the synthesis can be minimized. In this work, two different automated setups with different purification systems are presented. In its final configuration, three masterbatches were performed on the ML EAZY for DOTA-TATE and PSMA-I&T, respectively, fulfilling all quality criteria with final radiochemical yields of 80-90% for the 225Ac-labeled peptides.