Further details are presented in the Supplemental Materials. Schematics and sample code for operating the microcontrollers are available on request.
Data and code availability statement
An electronic computer-aided design file containing the full assembly and all components of the radiosynthesizer required for 3D-printing, as well as the Arduino program (sketch) is available from the corresponding author.
Formulated HerceptinTM (Roche/Genentech, South San Francisco, CA) and human serum albumin (HSA; Merck, Darmstadt, Germany) were reconstituted in water (>18.2MΩ·cm at 25oC). Protein concentrations were determined by using a NanodropTM OneC Microvolume UV-Vis Spectrophotometer.
The ALISI radiosynthesizer was constructed by using the computer assisted design (CAD) software Solidworks2020 (Dassault Systèmes, Vélizy-Villacoublay, France). Components were prepared through additive manufacturing, laser cutting or purchased directly from commercial vendors. Additive manufacturing components were produced by selective laser sintering and made from PA2200, a fine powder based on polyamide-12. The synthesizer case was manufactured from laser cut, 5mm plates of high tensile strength, black polyoxymethylene (POM) and an aluminum profile modular assembly system (Kanya AG, Rüti, Switzerland).
Control and electronics
Electronic components are based around the single-chip ATmega328 microcontroller. Microcontrollers, breakout boards, additional circuit boards, and electronic components were either custom-manufactured or purchased from Arduino.cc (Ivrea, Italy), Adafruit Industries (New York City, NY, USA), RS Components (Frankfurt-am-Main, Deutschland), or Distrelec AG (Nänikon, Switzerland). The photoreactor consists of an electropolished stainless steel tube containing an array of three, high-powered light-emitting diodes (LEDs; Nichia, Anan, Japan) with a light output of 1.03W per LED at 365nm. Valve modulation is performed with standard digital servos (Savöx, Salt Lake City, UT, USA). All liquid transfer steps are driven pneumatically with a syringe pump controlled by a NEMA-17 bipolar stepper motor (Distrelec AG, Nänikon, Switzerland).
Liquid handling and cassettes
All components for the disposable cassette system were either custom-manufactured or purchased from commercial vendors (B. Braun Melsungen AG, Meslungen, Germany, or BD, Heidelberg, Germany). The cassettes use flexible, high chemical resistance Tygon tubing. For purification, custom-made SK-10 separation columns (Econo-Pac chromatography columns, Bio-Rad Laboratories, Hercules, CA, USA) filled with 8.3mL of pre-soaked Sephadex® G-100 (Merck, Darmstadt, Germany) were constructed. The stationary phase of the SK-10 columns was capped with filter-frits and columns were eluted with sterile PBS (pH7.4).
Synthesis and radiochemistry
The photoactivatable chelate DFO-PEG3-ArN3 and the fluorescent photoactivatable fluorophore (PhotoTag) RhodB-PEG3-ArN3 were synthesized and characterized as described previously.(24) The stock solution of 89Zr-oxalate ([89Zr(C2O4)4]4-(aq.) in ~1M oxalic acid) was obtained from PerkinElmer (Waltham, MA, USA; manufactured by the BV Cyclotron VU, Amsterdam, The Netherlands) and was used without further purification. Full experimental details on the model chemistry using RhodB-PEG3-ArN3, 68GaDFO-PEG3-ArN3 and 89Zr-oxalate in combination with DFO-PEG3-ArN3 are given in the Supplemental Information. All reported RCC and RCY values are decay corrected.
Setup for automated radiolabeling
Reactions with ALISI were performed on new liquid handling equipment, freshly assembled from sterile packaging. Briefly, individual reservoirs were assigned to stock solutions of Na2CO3(aq.), DFO-PEG3-ArN3, 89Zr-oxalate, protein, reaction buffer, and sterile PBS for purification by size-exclusion chromatography (SEC) (see Supplemental Tables 1–4). A minimum volume of ~50mL was necessary to achieve adequate liquid transfer. For test reactions where reagent volumes were below this threshold, the total volume was adjusted with an appropriate volume of water.
Description of the automated 89Zr-photoradiolabeling procedure
A schematic of the plumbing diagram and a photograph of the ALISI system set-up for the synthesis of 89Zr-mAbs is shown in Figure 2. The radiosynthesizer unit is initialized by pushing the power button. After initialization, the system provides a digital prompt on the built-in LCD-display to indicate that the device is ready to start. Automated radiosynthesis and purification is initiated by pressing the start button. Thereafter, the radiosynthesizer transfers all reagents and components to the reaction vial, located inside the photoreactor, in the following sequence: i) 89Zr-oxalate in ~1M oxalic acid is transferred. ii) An equal volume of 1M Na2CO3(aq.) is transferred. iii) DFO-PEG3-ArN3 in a solution of ~10% DMSO and aqueous sodium borate buffer (0.25M, pH8.0) is removed from the reservoir and first used to wash the 89Zr-stock solution reservoir before being delivered to the reaction vial. iv) The protein solution is transferred. v) Additional sodium borate buffer (0.25M, pH8.0) is used to wash the protein reservoir and then transferred.
After the reagent transfer sequence, the reaction mixture is irradiated with 365nm light for 90s. Tests indicated that the temperature of the reaction mixture does not change during this time, but the high-powered LEDs require cooling with an aluminum heat-sink attached to a fan. After irradiation, the crude mixture is transferred to the SK-10 size-exclusion column (SEC) for purification. After automatic separation, the product fraction containing the high molecular weight protein is filtered through a standard 0.22 mm sterile filter and collected in a sterile vial. The product is formulated in sterile PBS (pH7.4), and after quality control, is ready for use in radiochemical, cellular, or in vivo assays.
Data were plotted by using the GraphPad Prism 9.0 software (GraphPad Software Inc., San Diego, California USA).