[68Ga]Ga-FAPI-46 synthesis on a GAIA® module system: Thorough study of the automated radiolabeling reaction conditions.

The influence of several parameters involved in the 68Ga radiolabeling of FAPI-46 was studied at the scale of the automated reaction. Among the buffers tested, HEPES 0.3 M pH 4 allowed both high radiochemical purity (RCP) and radiochemical yield (RCY), without prepurification of 68Ga but after final purification of [68Ga]Ga-FAPI-46 on a C18 cartridge. A longer reaction time did not show significant benefit on the RCP, while higher loads of FAPI-46 and gentisic acid as anti-radiolysis compound allowed better RCY.

[68 Ga]Ga-PentixaFor: Development of a fully automated in hospital production on the Trasis miniAllinOne synthesizer.

[68 Ga]Ga-PentixaFor is a frequently used radiotracer to image the CXCR4/CXCL12 axis in various malignancies, infections, and cardiovascular diseases. To answer increasing clinical needs, an automatized synthesis process ensuring efficient and reproducible production and improving operator's radioprotection is needed. [68 Ga]Ga-PentixaFor synthesis has been described on other synthesizers but not on the miniAiO. In this work, we defined automated synthesis process and an analytical method for the quality control of [68 Ga]Ga-PentixaFor. Validation batches were performed under aseptic conditions in a class A hotcell. All the quality controls required by the European Pharmacopea (Eur. Ph) were performed. The analytical methods were validated according to the International Conference Harmonization (ICH) recommendations. Validation batches were performed with a radiochemical yield of 94.8 ± 2.6%. All the quality controls were in conformity with the Eur. Ph, and the validation of the analytical method complied with the ICH. The environmental monitoring performed during the synthesis process showed that the aseptic conditions were ensured. [68 Ga]Ga-PentixaFor was successfully synthesized with the miniAiO by a fully automated process. This robust production mode and the quality control have been validated in this study allowing to increase the access of patients to this new promising radiopharmaceutical.

A Reliable Production System of Large Quantities of [13N]Ammonia for Multiple Human Injections.

[13N]Ammonia is one of the most commonly used Positron Emission Tomography (PET) radiotracers in humans to assess myocardial perfusion and measure myocardial blood flow. Here, we report a reliable semi-automated process to manufacture large quantities of [13N]ammonia in high purity by proton-irradiation of a 10 mM aqueous ethanol solution using an in-target process under aseptic conditions. Our simplified production system is based on two syringe driver units and an in-line anion-exchange purification for up to three consecutive productions of ~30 GBq (~800 mCi) (radiochemical yield = 69 ± 3% n.d.c) per day. The total manufacturing time, including purification, sterile filtration, reformulation, and quality control (QC) analyses performed before batch release, is approximately 11 min from the End of Bombardment (EOB). The drug product complies with FDA/USP specifications and is supplied in a multidose vial allowing for two doses per patient, two patients per batch (4 doses/batch) on two separate PET scanners simultaneously. After four years of use, this production system has proved to be easy to operate and maintain at low costs. Over the last four years, more than 1000 patients have been imaged using this simplified procedure, demonstrating its reliability for the routine production of large quantities of current Good Manufacturing Practices (cGMP)-compliant [13N]ammonia for human use.

Automated production of [18F]MK-6240 on CFN-MPS200.

Automated production of [18F]MK-6240 was implemented for the first time in the CFN-MPS200 module. Three consecutive productions of [18F]MK-6240 complied with the product specifications. Formulated [18F]MK-6240 maintained stability, as measured by radio-high-performance liquid chromatography (HPLC), as well as clarity and pH, over a period of 8 h. Our established method can facilitate multi-center trials and widespread use of [18F]MK-6240.

Standardised Reconstructed Skin Models in Toxicology and Pharmacology: State of the Art and Future Development.

Three-dimensional (3D) reconstructed human skin (RhS) models featuring fully-differentiated characteristics of in vivo human epidermis have been known for almost 40 years. In this chapter, the topic of commercial in vitro tissue models is described, taking RhS models as an example. The need for highly standardised models is evident for regulatory testing purposes, e.g. the classification and labelling of chemicals and formulations, as well as for pharmacology-oriented research and drug development. Following the standardisation of RhS model production by commercial developers, international validation studies and regulatory acceptance, 3D RhS models are now used globally in both industrial and academic research laboratories. Industrial production of standardised 3D RhS models involves GMP-compliant processes together with ISO 9001 documentation in order to control and ensure reproducibility and quality. Key biological, functional, and performance features that are addressed in industrial production include barrier properties, histological and immunohistochemical characterisation, lipid profile characterisation, and tissue viability before and after transport. An up-to-date survey of commercial RhS tissue producers and the regulatory acceptance status of major safety, hazard, and efficacy assays currently available to chemical and pharmaceutical industries is presented in this chapter. Safety and ethical concerns related to the use of human tissue in the industrial production of RhS models are discussed. Finally, innovative approaches to the production of standardised 3D RhS models including automated production, development of more representative 3D RhS models using advanced additive manufacturing tools, microfluidics technologies, and bioprinting are presented. The future outlook for 3D RhS models includes a prevalence of high-quality models which will be fabricated by end-users rather than commercial producers. These will overcome problems with shipments and customs clearance that many users still face when buying RhS from overseas commercial suppliers. Open-source technologies and commercial components for "do-it-yourself" RhS will significantly change the skin model market as well as regulatory acceptance of open-source models during the next decade. All of these developments and improvements will together allow more widespread use of in vitro RhS models for broader application as animal replacements in areas ranging from industrial and regulatory toxicology and pharmacology, to drug development and personalised medicine.

Fully-automated production of [68Ga]Ga-FAPI-46 for clinical application.

BACKGROUND: [68Ga]Ga-FAPI-46 is a promising radiopharmaceutical for in vivo detection of the fibroblast activation protein by positron emission tomography. Until now, the synthesis of [68Ga]Ga-FAPI-46 has been only performed manually. Our aim was to evaluate the automated synthesis of this radiopharmaceutical on two different commercially available synthesis modules in order to make the tracer readily available for clinical application. RESULTS: The synthesis of [68Ga]Ga-FAPI-46 with different amounts of precursor (10-50 µg) on the Modular Lab PharmTracer (MLPT) and Modular Lab eazy (ML eazy) from Eckert & Ziegler with a customized synthesis template and a customized single-use cassette yielded best results with 50 µg FAPI-46 for clinical multi-dose application. All relevant quality control parameters tested (e.g. sterility, stability and radiochemical purity) were in accordance with the European Pharmacopoeia. CONCLUSIONS: [68Ga]Ga-FAPI-46 was successfully synthesized fully-automated on the synthesis modules Modular Lab PharmTracer and ML eazy and is, thus, available for multi-dose application in clinical settings.

Three-step two-pot automated production of NCA [18F]FDOPA with FlexLab module.

Automated three-step two-pot production of no-carrier-added (NCA) [18F]FDOPA was first implemented in the iPHASE FlexLab module. Decay-corrected radiochemical yield (RCY) of [18F]FDOPA synthesized by this method was 10~14% (n = 7) with a synthesis time of ~110 min [18F]FDOPA was obtained in > 95% of radiochemical purity with a molar activity of ~431 GBq/µmol. With the method successfully implementing on the commercial FlexLab module and its built-in step-by-step activity monitoring, further processes optimization would be achieved.

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.

GMP-Compliant Manufacturing of NKG2D CAR Memory T Cells Using CliniMACS Prodigy.

Natural killer group 2D (NKG2D) is a natural killer (NK) cell-activating receptor that recognizes different stress-induced ligands that are overexpressed in a variety of childhood and adult tumors. NKG2D chimeric antigen receptor (CAR) T cells have shown potent anticancer effects against different cancer types. A second-generation NKG2D CAR was generated by fusing full-length human NKG2D to 4-1BB costimulatory molecule and CD3ζ signaling domain. Patient-derived CAR T cells show limitations including inability to manufacture CAR T cells from the patients' own T cells, disease progression, and death prior to return of engineered cells. The use of allogeneic T cells for CAR therapy could be an attractive alternative, although undesirable graft vs. host reactions may occur. To avoid such adverse effects, we used CD45RA- memory T cells, a T-cell subset with less alloreactivity, as effector cells to express NKG2D CAR. In this study, we developed a protocol to obtain large-scale NKG2D CAR memory T cells for clinical use by using CliniMACS Prodigy, an automated closed system compliant with Good Manufacturing Practice (GMP) guidelines. CD45RA+ fraction was depleted from healthy donors' non-mobilized apheresis using CliniMACS CD45RA Reagent and CliniMACS Plus device. A total of 108 CD45RA- cells were cultured in TexMACS media supplemented with 100 IU/mL IL-2 and activated at day 0 with T Cell TransAct. Then, we used NKG2D-CD8TM-4-1BB-CD3ζ lentiviral vector for cell transduction (MOI = 2). NKG2D CAR T cells expanded between 10 and 13 days. Final cell products were analyzed to comply with the specifications derived from the quality and complementary controls carried out in accordance with the instructions of the Spanish Regulatory Agency of Medicines and Medical Devices (AEMPS) for the manufacture of investigational advanced therapy medicinal products (ATMPs). We performed four validations. The manufacturing protocol here described achieved large numbers of viable NKG2D CAR memory T cells with elevated levels of NKG2D CAR expression and highly cytotoxic against Jurkat and 531MII tumor target cells. CAR T cell final products met release criteria, except for one showing myc overexpression and another with viral copy number higher than five. Manufacturing of clinical-grade NKG2D CAR memory T cells using CliniMACS Prodigy is feasible and reproducible, widening clinical application of CAR T cell therapies.

Automated and Continuous Production of Polymeric Nanoparticles.

Polymeric nanoparticles (NPs) are increasingly used as therapeutics, diagnostics, and building blocks in (bio)materials science. Current barriers to translation are limited control over NP physicochemical properties and robust scale-up of their production. Flow-based devices have emerged for controlled production of polymeric NPs, both for rapid formulation screening (~µg min-1) and on-scale production (~mg min-1). While flow-based devices have improved NP production compared to traditional batch processes, automated processes are desired for robust NP production at scale. Therefore, we engineered an automated coaxial jet mixer (CJM), which controlled the mixing of an organic stream containing block copolymer and an aqueous stream, for the continuous nanoprecipitation of polymeric NPs. The CJM was operated stably under computer control for up to 24 h and automated control over the flow conditions tuned poly(ethylene glycol)-block-polylactide (PEG5K -b-PLA20K ) NP size between ≈56 nm and ≈79 nm. In addition, the automated CJM enabled production of NPs of similar size (D h ≈ 50 nm) from chemically diverse block copolymers, PEG5K -b-PLA20K , PEG-block-poly(lactide-co-glycolide) (PEG5K -b-PLGA20K ), and PEG-block-polycaprolactone (PEG5K -b-PCL20K ), by tuning the flow conditions for each block copolymer. Further, the automated CJM was used to produce model nanotherapeutics in a reproducible manner without user intervention. Finally, NPs produced with the automated CJM were used to scale the formation of injectable polymer-nanoparticle (PNP) hydrogels, without modifying the mechanical properties of the PNP gel. In conclusion, the automated CJM enabled stable, tunable, and continuous production of polymeric NPs, which are needed for the scale-up and translation of this important class of biomaterials.

Simplified and robust one-step radiosynthesis of [18 F]DCFPyL via direct radiofluorination and cartridge-based purification.

[18 F]DCFPyL is a clinical-stage PET radiotracer used to image prostate cancer. This report details the efficient production of [18 F]DCFPyL using single-step direct radiofluorination, without the use of carboxylic acid-protecting groups. Radiolabeling reaction optimization studies revealed an inverse correlation between the amount of precursor used and the radiochemical yield. This simplified approach enabled automated preparation of [18 F]DCFPyL within 28 minutes using HPLC purification (26% ± 6%, at EOS, n = 4), which was then scaled up for large-batch production to generate 1.46 ± 0.23 Ci of [18 F]DCFPyL at EOS (n = 7) in high molar activity (37 933 ± 4158 mCi/µmol, 1403 ± 153 GBq/µmol, at EOS, n = 7). Further, this work enabled the development of [18 F]DCFPyL production in 21 minutes using an easy cartridge-based purification (25% ± 9% radiochemical yield, at EOS, n = 3).

Cationic eluate pretreatment for automated synthesis of [68Ga]CPCR4.2.

Fostered by the clinical success of sst-ligands, the development and evaluation of (68)Ga-labeled peptides have become a very active field in radiopharmaceutical chemistry. Consequently, various new peptide tracers have been developed, e.g. [(68)Ga]CPCR4.2 for in vivo imaging of solid and haematological tumors or [(68)Ga]TRAP(RGD)3 for imaging of α(v)ß3 integrin expression. As a consequence of different matrices (TiO2, SnO2, polymers) exploited in commercial (68)Ge/(68)Ga-generators, HCl of different concentrations (0.05...1.0 M) is used to obtain (68)Ga as starting material for automated syntheses. We have developed a purification method which reduces the eluate volume and adjusts the HCl concentration. The method may potentially allow standardization of the eluate composition of different commercial generators prior to labeling. Recently, a cationic purification process has been reported which allows the pre-fixation of (68)Ga on a Varian SCX cation exchange cartridge and subsequent elution of (68)Ga with acidified NaCl solutions. As part of the development of ready-to-use cassettes for the automated production of (68)Ga-CPCR4.2 using a SCINTOMICS GRP module and an iThemba Labs generator that is eluted with 0.6...1.0 M HCl, we tested and compared the (68)Ga-trapping efficiency of various commercial available cation exchange cartridges, the efficiency of subsequent (68)Ga-elution from these cartridges by means of various protocols and the influence of these variations on the labeling efficiency of [(68)Ga]CPCR4.2, [(68)Ga]TRAP(RGD)3 and [(68)Ga]DOTATATE/[(68)Ga]DOTANOC. Finally, we transferred the optimized method to the automated, cassette based synthesis of [(68)Ga]CPCR4.2 and the aforementioned peptides. From seven tested cation exchange cartridges, Chromafix PS-H(+) gave the best extraction results (>95%). Moreover, we observed that acidified solutions of 5 M NaCl or 2.5 M CaCl2 can be used for efficient cartridge elution. Using a disposable cGMP-compliant cassette system, we obtained [(68)Ga]CPCR4.2 in 80% decay-corrected yield and >99% purity. These data were confirmed by the production of [(68)Ga]DOTATATE, [(68)Ga]DOTANOC and [(68)Ga]TRAP(RGD)3 on the otherwise identical cassette system.

The OrbiSac system: results and organizational impact.

BACKGROUND AND OBJECTIVES: Growing health care demands, the uneven distribution of requirements for highly specialised services and the limits to the resources available to ensure an adequate response to the health care demands all make clear the managerial and macroeconomic need to adapt the organizational set-up of the health care facilities, redistributing the productive activities and care services in the territory, to meet the demands. We also found that it had become necessary to identify a different organizational model from the one in use in order to match the availability of platelet concentrates to the dynamics of the increased requests for this blood component in the setting of an Interhospital Immunotransfusion Department that involves the hospitals of Rimini, Forlì and Cesena. METHODS: Observational and statistical methods were used to determine the production, productivity, efficiency, efficacy and cost-benefit ratio of the production of platelet concentrates and the appropriateness, with respect to the dynamics and territorial distribution of the requests, before and after a profound procedural and methodological reorganisation which included centralisation of the production and the adoption of an automatic instrument for processing platelets from buffy-coats (OrbiSac System). RESULTS: The reorganization enabled the Department to reach almost complete self-sufficiency, to produce concentrates with a platelet content conforming with the indications of ministerial decrees, to uniform the quality of this blood component within the Department and to improve the use of human resources.