Advancements in Microfluidic Cassette-Based iMiDEV™ Technology for Production of L-[11C]Methionine and [11C]Choline.

Microfluidic technology is a highly efficient technique used in positron emission tomography (PET) radiochemical synthesis. This approach enables the precise control of reactant flows and reaction conditions, leading to improved yields and reduced synthesis time. The synthesis of two radiotracers, L-[11C]methionine and [11C]choline, was performed, using a microfluidic cassette and an iMiDEVTM module by employing a dose-on-demand approach for the synthesis process. We focused on optimizing the precursor amounts and radiosynthesis on the microfluidic cassette. L-[11C]methionine and [11C]choline were synthesized using a microreactor filled with a suitable resin for the radiochemical reaction. Trapping of the [11C]methyl iodide, its reaction, and solid-phase extraction purification were performed on a microreactor, achieving radiochemical yields of >80% for L-[11C]methionine and >60% for [11C]choline (n = 3). The total synthesis time for both the radiotracers was approximately 20 min. All quality control tests complied with the European Pharmacopeia standards. The dose-on-demand model allows for real-time adaptation to patient schedules, making it suitable for preclinical and clinical settings. Precursor optimization enhanced the cost efficiency without compromising the yield. The importance of dose-on-demand synthesis and optimized precursor utilization to produce L-[11C]methionine and [11C]choline was emphasized in this study. The results demonstrated the feasibility of dose-on-demand adaptations for clinical applications with reduced precursor quantities and high radiochemical yields.

Clinically Applicable Cyclotron-Produced Gallium-68 Gives High-Yield Radiolabeling of DOTA-Based Tracers.

By using solid targets in medical cyclotrons, it is possible to produce large amounts of 68GaCl3. Purification of Ga3+ from metal ion impurities is a critical step, as these metals compete with Ga3+ in the complexation with different chelators, which negatively affects the radiolabeling yields. In this work, we significantly lowered the level of iron (Fe) impurities by adding ascorbate in the purification, and the resulting 68GaCl3could be utilized for high-yield radiolabeling of clinically relevant DOTA-based tracers. 68GaCl3 was cyclotron-produced and purified with ascorbate added in the wash solutions through the UTEVA resins. The 68Ga eluate was analyzed for radionuclidic purity (RNP) by gamma spectroscopy, metal content by ICP-MS, and by titrations with the chelators DOTA, NOTA, and HBED. The 68GaCl3eluate was utilized for GMP-radiolabeling of the DOTA-based tracers DOTATOC and FAPI-46 using an automated synthesis module. DOTA chelator titrations gave an apparent molar activity (AMA) of 491 ± 204 GBq/µmol. GMP-compliant syntheses yielded up to 7 GBq/batch [68Ga]Ga-DOTATOC and [68Ga]Ga-FAPI-46 (radiochemical yield, RCY ~ 60%, corresponding to ten times higher compared to generator-based productions). Full quality control (QC) of 68Ga-labelled tracers showed radiochemically pure and stable products at least four hours from end-of-synthesis.

Fully automated production of the fibroblast activation protein radiotracer [18 F]FAPI-74.

We report herein an efficient and fully automated protocol for the radiosynthesis of [18 F]FAPI-74, a new positron emission tomography (PET) radiopharmaceutical for in vivo detection of the fibroblast activation protein. [18 F]FAPI-74 was synthesized via a rapid [18 F]aluminum fluoride coordination reaction, which was first developed on the flexible GE TRACERLab FX2N (FXN) platform and later translated to the cassette-based module Trasis AllInOne (AIO). The results obtained with both modules were comparable in terms of yield and reproducibility. Automation of [18 F]FAPI-74 radiosynthesis on the FXN was carried out in 35 min with a radiochemical yield (RCY) of 18.5 ± 2.5% (n = 5, relative to starting [18 F]fluoride). Method transfer to the AIO platform following minor optimizations allowed for the production of [18 F]FAPI-74 in an isolated RCY of 20 ± 2.5% [n = 3] with an overall synthesis time of 40 min. The radiochemical purity was greater than 95% for [18 F]FAPI-74, obtained from both modules. Overall, the protocol reliably provides a sterile and pyrogen-free good manufacturing practice (GMP) compliant product of [18 F]FAPI-74 suitable for clinical PET imaging.

Automation of cell line development.

An automated platform for development of high producing cell lines for biopharmaceutical production has been established in order to increase throughput and reduce development costs. The concept is based on the Cello robotic system (The Automation Partnership) and covers screening for colonies and expansion of static cultures. In this study, the glutamine synthetase expression system (Lonza Biologics) for production of therapeutic monoclonal antibodies in Chinese hamster ovary cells was used for evaluation of the automation approach. It is shown that the automated procedure is capable of producing cell lines of equal quality to the traditionally generated cell lines in terms of colony detection following transfection and distribution of IgG titer in the screening steps. In a generic fed-batch evaluation in stirred tank bioreactors, IgG titers of 4.7 and 5.0 g/L were obtained for best expressing cell lines. We have estimated that the number of completed cell line development projects can be increased up to three times using the automated process without increasing manual workload, compared to the manual process. Correlation between IgG titers obtained in early screens and titers achieved in fed-batch cultures in shake flasks was found to be poor. This further implies the benefits of utilizing a high throughput system capable of screening and expanding a high number of transfectants. Two concentrations, 56 and 75 muM, of selection agent, methionine sulphoximine (MSX), were applied to evaluate the impact on the number of colonies obtained post transfection. When applying selection medium containing 75 muM MSX, fewer low producing transfectants were obtained, compared to cell lines selected with 56 muM MSX, but an equal number of high producing cell lines were found. By using the higher MSX concentration, the number of cell line development projects run in parallel could be increased and thereby increasing the overall capacity of the automated platform process.

Transcription regulation of the multiple endocrine neoplasia type 1 gene in human and mouse.

Multiple endocrine neoplasia type I (MEN1) is an autosomal dominant tumor syndrome, with the presence of tumors in parathyroid, pancreatic, and anterior pituitary. The tumor suppressor gene MEN1, located on chromosome 11q13, encodes a 610 amino acid, 68-kDa protein, menin. Menin is conserved among species but has no similarity with any known protein. To investigate how the expression is regulated in both man and mouse, we assayed a greater than 1-kb region upstream of the second exon for promoter activity in luciferase reporter vectors. The basic promoter was located closely upstream the most commonly expressed first exon. The region further upstream modified the activity. Repetitive elements of the short interspersed/Alu type covered the entire human upstream regulatory region and were the only apparent motif in common with its murine ortholog. Previous studies have indicated a compensatory induction of the second allele because of inactivation of the first allele. We found that overexpression of menin in an inducible cell culture system down-regulated the proximal promoter. In response to down-regulation of MEN1 expression by RNA interference, the regulatory region activated the promoter in a compensatory manner. Our data confirm that the expression of the MEN1 gene is regulated by a feedback from its product menin.