Seppak light c18 cartridge

Frozen renal cortical tissues were processed to extract membrane proteins. Membrane protein samples were then processed by Omics Technologies, Inc. using "Filter Assisted Sample Preparation" (FASP) method[117 (link)]. Briefly, protein samples in 9M UREA were reduced with 5 mM TCEP at 37 °C for 45 min and reduced cysteine were blocked using 50 mM iodoacetmide (IAA) at 25 °C for 15 min. Protein samples were then cleaned using 10 kDa Amicon Filter (UFC501096, Millipore) three times using 9M urea and two times using MyPro-Buffer 1 (Omics Technologies, Inc.). Samples were then proteolyzed with trypsin (V5111, Promega) for 12 hrs at 37 °C. The peptide solution then was acidified by adding 1% trifluoroacetic acid (TFA) and was incubated at room temperature for 15 min. A Sep-Pak light C18 cartridge (Waters Corporation) was activated by loading 5 mL 100% (vol/vol) acetonitrile and was washed by 3.5 mL 0.1% TFA solution two times. Acidified digested peptide solution was centrifuged at 1,800 × g for 5 min, and the supernatant was loaded into the cartridge. To desalt the peptides bound to the cartridge, 1 mL, 3 mL, and 4 mL of 0.1% TFA were used sequentially. To elute the peptides from the cartridge, 2 mL of 40% (vol/vol) acetonitrile with 0.1% TFA was used. The eluted peptides were lyophilized overnight and reconstituted in 37 μL MyPro-Buffer 3 (Omics Technologies, Inc.).

Synthesis of [¹⁸F]F13640 and quality controls pathways were previously described (Vidal et al., 2018a (link)). Briefly, after production of fluoride preparation ¹⁸O(p,n)¹⁸F cyclotron reaction, 5 mg of F13640 nitro precursor are introduced. After nucleophile substitution, [¹⁸F]F13640 is obtained by separation on a preparative HPLC column (SymmetryPrepC18, 7 μm, 7.80 mm × 300 mm, Waters). The radiotracer is formulated via solid phase extraction techniques using a Sep-Pak Light C18 cartridge (Waters). The final product is eluted with 1 mL of ethanol, diluted with saline and finally sterilized by filtration (sterile filter Millex-GS, 0.22 μm; Millipore).

Production of [¹⁸F]AmBF₃-TATE was conducted on a Trasis AllInOne module with 36 manifold actuators and an integrated HPLC system equipped with a Phenomenex Luna C-18 semi-preparative column (100 Å, 10 μm, 10 mm × 250 mm). The cassette setup is shown in Fig. 2 with tubing connections, syringes, and other disposables. The cassettes were pre-assembled in a cleanroom using disposable materials supplied by Trasis. Luer lock syringes (Becton Dickinson) were secured at Positions 3 (3 mL), 9, and 15 (10 mL). A Sep-Pak® Light Accell^TM Plus QMA-Cartridge (Waters) preconditioned with 10 mL saline followed by 20 mL DI water and a Sep-Pak® Light C-18 Cartridge (Waters) preconditioned with 10 mL ethanol followed by 20 mL DI water were installed on Positions 5 and 33, respectively.
Fig. 2

Setup of the synthesis cassette to produce [¹⁸F]AmBF₃-TATE with integrated HPLC purification on Trasis AllInOne module

Radiolabelling was conducted as previously described [17 (link)] (Additional file 1: Fig. S1). Briefly, the volume of the [^123/125I]Iodide (PerkinElmer Inc.) was reduced if necessary, by evaporation followed by redissolution in water (< 15 µL). 3 µL of a PARPi-01 precursor solution (Tributyl stannylated derivative of Olaparib, 15 mM in DCM) were evaporated to dryness, and re-dissolved in 20µL HOAc. 15 µL of the concentrated radio iodide solution were added to the acid precursor solution and labelling was started by adding 20 µL of a fresh chloramine-T solution (1 mM in MeCN). After 10 min the crude reaction mixture was purified using gradient RP HPLC with a classical C18 column (LiChrospher 100 RP18 EC 5µ, 250 × 4 mm, Merck, Germany). The product fraction was diluted with 15 mL water and was solid phase extracted by a preconditioned SepPak light C18 cartridge (WAT023501, Waters). The [^123/125I]PARPi-01 was eluted using 1 mL CH₃CN and evaporated to dryness. The residue was taken up in little EtOH and diluted by isotonic saline to keep the EtOH concentration below 8%. The resulting overall radiochemical yield was 78 ± 12% with purities of > 95%. Molar activity of obtained [¹²³I]PARPi-01 and [¹²⁵I]PARPi-01 were 279 ± 216 GBq/µmol (n = 3) and 30.5 ± 6.4 GBq/µmol (n = 4), respectively.

[⁶⁸Ga]Pentixafor was synthesized using a fully automated Scintomics GRP+ module. The eluate of a ⁶⁸Ge/⁶⁸Ga-generator (GalliaPharm, EZAG, Germany) was concentrated using a Chromafix PS-H⁺ and heated with a solution of 40 µg Pentixafor (unlabeled precursor; GMP quality) in 1.5 mL of HEPES buffer (N-(2-hydroxyethyl)piperazine-N´-(2-ethanesulfonic acid), 1.5 M at 125°C for six minutes. Unbound and colloidal gallium-68 was removed using a SepPak Light C18 cartridge (Waters). The purified product was formulated with PBS buffer. The chemical and radiochemical purity of [⁶⁸Ga]Pentixafor were analyzed by high performance liquid chromatography (HPLC) using an analytic Chromolith® Performance, RP-18e, 100-4.6 mm from Merck (Germany). Free ⁶⁸Ga³⁺-ions and ⁶⁸Ga-colloid were detected using radio-thin-layer chromatography (radio-TLC).
The glucose analogue [¹⁸F]FDG was synthesized using FASTlab FDG cassettes with a phosphate buffer formulation and a GE FASTlab platform (GE Healthcare).