CHAPTER 4
18
4.1 The PETtrace System
The production of the radionuclide requires the operation of the cyclotron. The machine installed at the Institute of Clinical Physiology is a PETtrace system (Fig. 13), manufactured by General Electric Positron Systems (Sweden,) and and specifically conceived for the production of positron emitting radionuclides for PET applications.
The operator interface to PETtrace control system is the Master Consolle. A private network connects the master to two slave units: the Accelerator Control System and the Chemistry Control Unit. The network ensure real-time control of all peripheral components and safety devices (signalling, intelocks, monitors). (Fig.14)
The Master reports on the operational condition of Cyclotron subsystems (see Fig.15):
Magnet System (1)
Radio-Frequency (RF) System (2) Ion Source System (3)
Beam Extraction System (4) Beam Diagnostic System (5) Vacuum System (6)
Target System (7)
Radiation Shield (option) (8)
Secondary Water Cooling System (9) Power Distribution System (10)
In addition, the Accelerator Control Unit (11) provides advanced control of the Cyclotron, including the following functions:
start-up and shutdown tuning, particle change
data logging, continuous monitoring of subsystems and interlocks.
Fig.16 reports a typical hardcopy of the cyclotron Master during start-up phase.
Fig.16: cyclotron Master during start-up phase.
Although tuning is automatically achieved by the system some check-points are under the control of the operator. These includes Cyclotron start-up/shut-down, target/radionuclide selection, target fill/empty/cleaning, beam intensity and irradiation time.
A safety chain prevent wrong commands are set; however continuous monitoring of system performance should be performed by the operator while beam and bombardment are active .
The master operates via dialogue windows that are related to specific environment (e.g. bombardment set up, fig.17).
Fig.17: Bombardment set-up
An important control that is made in-process during beam operation is via the Diagnostic System of the beam current. This system is able to monitor the current of the accelerated particles at different positions within the Cyclotron and in the Target System in order to control the beam intensity from the Ion Source to the target. The Diagnostic System includes an internal beam current probe (flip-in type) located at a small radius, and insulated extraction foils for monitoring the electron current during the extraction process. The collimators, placed on either side of the beam path (up/down) at the beam exits are used for beam centering and beam collimation.
The target bodies are insulated from ground to allow monitoring of the useful beam current entering into the target during isotope production. All signals from the beam monitoring components are connected to a multichannel Beam Current Analyzer (BCA).
.
Fig.18: vacuum system
Fig.19: Extraction Unit
The beam enters the target chamber through a double-foil assembly which is a part of the Target.
power dissipation (beam intensity x beam energy = watts). This heat is eliminated by a Helium Cooling System. This is a closed system, which facilitates cooling of the target foils (Two thin metal foil windows separate the target chamber from the Cyclotron vacuum chamber). The system includes the piping and valves for gas distribution, a heat exchanger, and a compressor for high-speed recirculation of the helium gas. Each target consists of a target chamber, front flange and rear flange.
Fig.20: Target system
All supplies to the target—target medium, cooling water and helium for foil cooling— are delivered at the rear flange of the target (Fig.21) by a single connection plate fixed to the target with a quick coupling. The 18O-enriched
target water is kept under a helium overpressure. (Fig.20) The back-pressure is also used to empty the target after irradiation. A syringe pump unit (LTF) is used for filling of the 18F-target. Supplies and their usage are
reported on the master and are available to the operator for review.
Lack or insufficient amount of supplies will prevent performing of irradiation
Fig.21: Target scheme
When the irradiation is ended a full report on supplies, irradiation conditions and eventual faults is elaborated by the system and both stored under a logfile and printed for batch record archiving. (Fig.22)
4.2 Tracerlab Fx FDG
The TRACERlab FX FDG is a computer controlled automated radiochemistry
system available in our laboratory, which produces 2-[18
F]-fluorodeoxyglucose (18FDG) from [18F]-fluoride. The system module
consists of the process module, an external vacuum pump, 24 V transformer, electronics unit and notebook and is designed as a closed system, so emission of radioactivity is minimized. The synthesis of the radiopharmaceutical is carried out from the target to the filling unit automatically. Only chemicals and disposable cartridges are needed for the synthesis . The process time is about 30 minutes. The radiochemical yield is >55 % depending on the purity of the starting materials. The 18FDG process is defined in a program sequence, which is
optimized for production with high yield and radiochemical purity as well as reliability. Chemical reactions of the radioactive compounds take place inside the reaction vessel of the closed system. The required reagents are filled into support vials.
The following are the main parts of the TRACERLab FxFDG (Fig.23).
The TRACERLab FxFDG is designed as a closed system. Synthesis of the radiopharmaceutical is automatically carried out from target to the product vial. The process time is about 30 minutes. The radiochemical yield is >55 % depending on the purity of the starting materials. The 18FDG
process is defined in a program sequence, which is optimized for production with high yield and radiochemical purity as well as reliability.
Fig.24: Reaction vessel
Chemical reactions of the radioactive compounds take place inside the reaction vessel of the closed system (Fig. 24). The required reagents are filled into support vials. The synthesis is based on separation of 18F from
[18O]H2O using an anion exchange column and productionof 18FDG by
nucleophilic substitution (Hamacher, Nebeling and Blessing, Appl. Radiat. Isot., 41, 49-54 (1990) (Hamacher, Coenen and Stˆcklin, J. Nucl. Med. 27, 235-238 (1986)). The synthesis process (Fig.25) is controlled by a computer and displayed graphically on the color screen along with all relevant conditions and values. Graphic visualization of the radiochemical synthesis on screen allows to follow each sequence of operation.
Fig.25: Recorded synthesis data
After synthesis, an automatic cleaning and purging procedure can be started. All components (valves, heaters, coolers etc.) can be controlled manually at any time even during automated synthesis process. Each synthesis is documented according to GLP/GMP guidelines. The important time--dependent signals of temperature, pressure and radioactivity detectors are recorded during synthesis and displayed graphically in a live display and stored permanently. The Process Module is a complete system housing the hardware for the FDG synthesis. The main parts are (Fig.26):
• Connectors for the daily assembly of purification column and separation cartridge as well as for the sterile filters.
• Several reagent vials, which are necessary to store the reagent fluids. • Target water vial with a GM-tube for indication of incoming radioactivity. • H218O vial to recover the used target water.
• Two reactors, each with a pneumatic cylinder for the reactor needle and a GM-tube for indication of radioactivity during the synthesis.
• Teflon tubes and solenoid valves for liquid handling.
Fig.26: TRACERlab Fx FDG
4.2.1 TRACERlab Fx FDG specifications
The reagent vials are tailor-made glass vessels. They are connected to tubing and valves through special connectors on top and bottom. To fill the vials with a reagent or liquid it is necessary to penetrate the septum on the front side with a sterile injection needle. The target water vial is a tapered 5 ml vial, sealed with a silicone septum and a cap. To transfer the target water out, the tube has to reach to the deepest point of the vial. Target water is sucked by vacuum through the separation cartridge into the H218O
Vial. Vacuum is generated by an external vacuum pump. The H218O vial is
a glass vessel to take up the rest of the H218O. The reactor is a special
vessel made from glassy carbon. It is closed with a tailormade PEEK reactor head. The head is sealed with a temperature resistant and inert washer. The reactor is heated by the use of a brass oven and cooled with forced convection by compressed air. The temperature is measured by a PT
needle is made of PEEK and can be moved to the deepest point by the use of a pneumatic cylinder. It empties the vessel in presence of inert gas pressure. On the reactor head teflon tubes are fastened. They connect reagent vials with the reaction vessel. The FDG collection vial is a tapered vial, sealed with a septum and cap. The liquid handling is obtained by the use of an inert gas supply. An internal pressure regulator as well as a needle valve control inert gas pressure and flow. Single distribution steps are controlled by the use of solenoid 2/2-way- or 3/2-way valves. Using GM-tubes the activity amounts of the incoming target water, the reactor vessel and the FDG collecting vials are indicated. The voltage for the GM-tube (550V) is generated by a high voltage module inside the synthesizer. The GM-tubes are collimated to reduce disturbance from external radiation sources. The amounts of radioactivity that has been recorded during the whole procedure is and is stored in an archive. It also appears in a graphic visualization screen. Tubing for compressed air inside the Synthesizer Module is made of polyamide. The outside diameters are 6 mm, 4 mm or 3 mm. The connectors are designed for a pressure up to 0,8 MPa (115 psi). Tubing for inert gas, vacuum and liquid handling is made of Teflon®. The outside diameters are either 1/8” or 1/16”. The connectors to the valves, the reagent vials and the reactor are flanged Tube End Fittings with male thread UNF” 28G. The connection to the Target water-, the H218O- and the FDG collection vials are tightened with septa, which have
optimized fitted borings for the Teflon®tubes. The connectors to the media supply tubes are stainless steel swagelok connectors, which are located on the backside of the module. The Control System for the TRACERLab FXFDG
consists of an Electronics Unit, a PC with control software and a cable set for connecting the Electronics Unit to the Computer and the Process Module. The Control System regulates the chemistry process within the parameters given by the program. It also reports the pressure-, the activity amount- and the temperature history of the synthesis. The purpose of the Electronics Unit is to regulate the FDG-synthesis process.
Predefined parameters are stored within the application program, and these control the process components of the Process Module. Sensors in the system measure temperature, pressure and the radioactivity in the most important vessels.
4.2.2 The TRACERlab Fx FDG software
Only authorized personnel should install the TRACERlab FxFDG. The software starts automatically by double click the shortcut on the desktop of the PC (Fig.27). Then the graphic visualization screen appears . It is required to switch on the control unit before starting the program. In the Synthesis menu you can activate the following commands while synthesis is running:
• Stop the synthesis,
• Pause and Resume synthesis,
• Next Step: it is only possible in pause status. If at rest you can activate:
• Reset
• Manual Operation mode
• Clean: start the clean method
In manual operation mode you can use the mouse for controlling the module. If you move the mouse pointer on the visualization screen the mouse pointer’s shape changes from an arrow to a hand, if it is above device that can be operated with the mouse. Only if the mouse pointer has the hand shape you can perform operational actions.
The Listed devices can be controlled manually: • Valves
• Temperature set points • Needles
Fig.27: Graphic visualization of the FDGsoftware
After a run has finished the recorded data are saved automatically in the synthesis archive (Fig.28).
• A report can be shown and printed, • a log can be shown and
Fig.28: Recorded data
4.2.3 Synthesis process description.
Prior to synthesis, a PETtrace cyclotron produces 18F- externally. The 18F- within target water is then transferred to the target water reservoir on the TRACERLab FXFDG. The chemical process essentially comprises seven stages. In summary, 18O target water passes through an anion exchange column, where 18F- is adsorbed. The 18F- is eluted with K2CO3 solution. In the reactor the phase transfer catalyst TBA is added and transports the anion into the organic phase. Nucleophilic substitution Reaction takes place in acetonitrile with the precursor mannosetriflate. Hydrolysis is applied to remove all protection groups. After dilution with water and chromatographic purification the product is collected.
4.2.4 Steps of the process
Step 1: Separation of [18F]F- from [18O]H2O
The [18F]F- , produced via 18O [p, n] 18F nuclear reaction, is separated from remaining [18O]H2O using an anion exchange column. The 18F- ions are adsorbed on the ion exchange resin while the passing [18O]H2O water is collected in a vial.For elution of the adsorbed fluoride potassium carbonate is used. As phase transfer catalyst TBA is used to transport fluoride into organic phase where reaction takes place.
Step 2: Preparation of the nucleophilic substitution
The solution has to be evaporated and dried quantitatively to make sure no water is left. The OH- anion caused by dissociation of water competes with the F- anion during reaction and reduces the yield of [18F]FDG considerably. Drying is executedby azeotropic distillation of the water with acetonitrile. To eliminate all rests of water the distillation is followed by evaporation under vacuum.
Step 3: Nucleophilic substitution
In this step the FDG-precursor 1,3,4,6-tetra-O-acetyl-2-O trifluoro methane-sulphonyl-b-D-mannopyranose (dissolved in acetonitrile) is added to the reaction vessel. (Fig.29)
The strong triflate anion (triflouromethanesulphonate) in C2-position is substituted under the presence of TBA by F-. The protective groups (in C1-,
C3-, C4- and C6-/position) are acetyl groups.
Fig.30: Nucleophilic substitution
Reaction takes place under 85 °C for 5 min.
After complete substitution, the toxic acetonitrile has to be removed quantitatively. Again the solvent is removed by distillation flowed by evaporation under vacuum.
Step 4: Hydrolysis
HCl is applied to remove all protective groups from the reaction product 2-[18F]fluoro-1,3,4,6-tetra-O-acetyl-D-Glucose. The reaction below
is an example for removing Acetyl groups by using basic hydrolysis. Basic hydrolysis takes place at room temperature. The reaction product is 2-[18F]Fluoro-2-deoxy-D-glucose.
Step 5: Chromatographic Purification
of four layers of different resins. As FDG is retarded on the purification column the first 2 ml of the eluate are discarded to waste. With another 10 to 14 ml of sterile water, FDG is eluted. Finally, it is formulated as an isotonic solution of phosphate buffer pH 5.
4.2.5 Preparation for the synthesis process.
The Process Module is prepared by adding the columns, cartridges and filters as well as filling the reagent vials with chemicals according to the operator guide. TRACERlab allows you to run two consecutive synthesis after both sides of the synthesis module are prepared; this is due to a load once, run twice design. The steps to follow are:
1. Pour 0.5 ml of TBA into vials 1 and 7. 2. Pour 1ml of Acetonitrile into vials 2 and 8.
3. Pour 1ml of Acetonitrile plus 20mg of Mannose Triflate into reagent vials 3 and 9.
4. Pour 1.0 ml of HCl 1N into vials 4 and 10.
5. Pour 2 ml of sterile, injectable water into vials 5 and 11. 6. Pour 14 ml of sterile, injectable water into vials 6 and 12. 7. Pour 3 ml of Phosphate Buffer pH 5 into the collection vial.
The 18F separation cartridge , an ANION EXCHANGE CARTRIDGE
(PS-HCO3-), must have been previously activated with 1 ml ethanol, then
rinsed with 1 ml deionized water, finally drained with helium or vacuum. The FDG purification column, a CHROMABOND SET IV is a single cartridge consisting of 4 different resins:
• PS-H: CATION EXCHANGE CARTRIDGE • PS-HCO3: ANION EXCHANGE CARTRIDGE • ALOX N: ALUMINUM OXIDE CARTRIDGE • HR-P: REVERSED PHASE CARTRIDGE
The cartridge must be activated slowly (i.e. single drops) with 20 ml Ethanol, then washed with 100 ml sterile water for injection. After power-up, the system needs some affirmations in the preparation screen. The preparation screen will open automatically by using the start button in the main screen. By using the Start Synthesis button the synthesis process will start.
Transfer of Target Water and Start of Synthesis
In the main screen the value of the incoming radioactivity is displayed. After all target water has reached the vial the F4 button must be pressed to start the synthesis.
Separation of [18F]F- from [18O]H2O
[18F]F- is separated from remaining [18O]H2O using an anion exchange
column. The 18O-enriched water is collected in a separate vial. 18F- is
eluted with K2CO3 solution and transferred to the reaction vessel.
Preparation of Nucleophilic Substitution
The reaction vessel is heated and the mixture is evaporated under helium flow. When a defined temperature is reached the drying procedure is continued under vacuum for approximately one minute. The reaction vessel is cooled down and flushed with helium. After a few seconds the phase transfer catalyst dissolved in solvent is transferred into the reaction vessel. The reaction vessel is closed and heated for 5 min. Under these conditions the nucleophilic substitution takes place. Afterwards, the vessel is cooled down. Under helium flow the solvent is evaporated. After a few seconds, the temperature is increased to 105 °C. Vacuum is applied to remove the solvent completely. After approx 80 sec. the reaction vessel is dry.
Hydrolysis
After removal of the solvent the reaction vessel is flushed with helium at 90 °C and 3 ml H2O are transferred from support vial 5. The solution is cooled down to 33°C. After reaching the temperature 1 ml NaOH is added from
vial 4 to the reaction vessel. The hydrolysis takes place under these conditions for approximately 90 seconds.
Chromatographic Purification
After hydrolysis V18 is opened and with helium pressure the solution is transferred to the FDG purification column. The water is discarded to waste. Afterwards the sterile water from support vial 6 is transferred in the same way through the reaction vessel and the FDG purification column to the collect vial.
End of Synthesis
The activity of 2[18F]FDG solution is determined and the lamp at the collect
vial is switched on. The synthesis has finished and a protocol is automatically printed.
4.3 Dispensing Unit
After the FDG production has been successfully completed, you can dispense the FDG solution into a sterile vial. The dispenser base unit is installed into a customized lead shielded hot cell (Fig.31).
A plastic insert is fitted into the ion chamber and covers the flange on the hot cell floor. This insert acts as an electrical insulator to protect from the high voltage of the ionization chamber. The ion chamber itself is located under the hot cell floor inside a lead shielded safe. The ion chamber is sealed air tight against the hot cell floor by pushing it upwards into a soft seal with a scissors jack. On the left side of the vial tray a black cup is mounted. This cup is used as a base to park the collection vessel. (Fig.32)
Fig.32: Dispensing unit and its components
This collection vessel is connected to the FDG input lines and is housed in a black cage with aknob on it. The knob is used to transport the collection vessel with the robot. On the left wall of the hot cell a frame is mounted,
and ethanol) and an optional third button with saline solution for dilution of the product. A separate small lead box below the ionization chamber stores 2 waste bottles. The larger of them stores waste water from the sterilizer while the smaller one stores the waste from the cleaning cycles. The dispenser is electrically connected to the control unit and both are further connect to the ComServer. The Com Server communicates with the control computer via Ethernet and with the robot and control unit via RS232. Electrical connections between the dispenser and outside electronics can be found underneath the top cover inside the dispenser. The connectors are located in the back half of the dispenser. On the back left side next to the electrical connectors inside the dispenser lie the gas connectors and pressure gauges. Compressed air and helium are passed into the hot cell through separate fittings on the top of the hot cell. Cables are running through a circular cable passage. FDG capillary lines are passed through a shielded flange. Each line connects to one FDG synthesizer. The input lines are connected to an electrical valve which allows the selection of FDG input through the control computer. The sterilizer inside the dispenser is supplied with deionized water from a bottle on top of the dispenser. Waste water and steam from the sterilizer is running through a Teflon line through the hot cell floor on the left side into a waste water tank in a shielded compartment underneath the ion chamber. Both waste water tank and fresh water bottle of the dispenser require a minimum water level which is clearly marked on each container. The sterilizer is supplied with mains by a power point inside the hot cell. It can be switched on and off from outside. In front of the sterilizer drawer is the air lock, that transfers the lead containers with the filled vials out of the hot cell. The air lock is either a pneumatically driven lift or a simple hand driven drawer. The base unit consists out of four major components: diluter (syringe pump), clean room, sterilizer and robot.
4.3.1 Diluter
The diluter is responsible for all liquid handling. It is based on a 5 ml CAVRO XL 3000 high resolution syringe pump with a 4/2 port valve and is fitted onto the left front panel. This unit is controlled by the dispenser software and can be operated fully automatic or manually. The syringe requires conditioning before first use to prevent from air bubbles in the system. Seven additional valves are located behind the left front panel. Valve V1 and V2 select the desired input line and connect via valve V3 to the collection vessel. The collection vessel is vented and this exhaust is sealed with a sterile filter to maintain a closed system. Valve V3 is connected via the main line to the syringe pump branching off three further vessels. (Fig.33)
Fig.33: Dispensing software
V4 connects a waste bottle, V5 connects the cleaning liquids and V6 connects the saline input for dilution. Valve V7 is used to purge the system with helium. Helium pressure is usually set to 100 kPa. From the syringe
area. It ends in a Luer connector which connects a sterile filter and needle. Only the provided filter and needle types shall be used with the system. Height and position of the filter needle combination can be adjusted to ensure, that the needle punctuates the septum in the middle. The collection vessel is built into a black cage to be handled by the robot. To remove the vessel rotate the upper plastic spring ring until its opening allows the tubes to pass. The collection vessel is fitted with a stirrer inside the base. The stirrer is plugged into a socket on the front panel. Make sure a stirrer magnet is inside the vessel.
4.3.2 Clean room
For the purpose of aseptic dispensing and to keep the needle sterile the dispenser is fitted with a small horizontal laminar flow unit to provide clean room conditions. In this clean room environment the septum is pierced. No moving parts are mounted in this area to prevent particle contamination. The sterile and evacuated vials are brought into this area by a rotating arm from outside. Air velocity can be set and regulated by the computer and is controlled by a sensor inside the clean room area. Surfaces are kept uniform and free from holes and screws for easier sanitation and disinfection. The particle filter (H/EU 14) provides a clean room class 100 (US-Federal Standard (Fig.34)
). Laminar flow is achieved by a nylon mesh which is protected by a Stainless Steel grid. The laminar flow unit circulates the air inside the hot cell. Thus a faster recovery of the clean room environment around the dispenser can be obtained.
4.3.3 Sterilizer
For terminal sterilization of the product inside the vial a small sterilizer is included with the dispenser (Fig. 35 and 36).
Fig.35: The sterilizer
The sterilizer has a capacity of max. 17 vials (12 vials, former versions). It is equipped with an independent temperature sensor inside a reference vial and pressure sensor to monitor sterilization conditions. Thus sterilization conditions are continuously monitored and the F0-value is calculated to proof sterility of the product according to the overkill method. The sterilization cycle heats 132°C for 3.5 minutes. The sterilizer may only be operated while the hot cell is closed.
Fig.37: Sterilisation graphic
To avoid decomposition, the FDG must be adjusted to pH = 5.5. It is important, that it does not reach pH = 6, because at this pH a considerable degradation starts. To achieve a proper pH value a buffer solution is added into the collect vessel of the synthesis module. The dispensing unit uses steam sterilization with a sterilization temperature well below 140°C. The possible decomposition products are well known and can be detected in the chromatographic quality control.4 So, any decomposition that may take place in the case of malfunction of the sterilization unit or too high pH value would be detected in the quality control. In order to check the proper sterilization, the F0 value is determined for each sterilization cycle. The F0 value has the dimension of a time and is directly proportional to the reduction of microorganisms. It is defined by:
F0=D121 (logA-logB )
whereD121 is equal to the time required at 121°C to reduce the population of the most heat resistant organism in the product by 90% (D value). A is the microbial count per vial (bio burden), B is the maximum acceptable probability of survival of microorganisms. This means, that a sterilization
with an arbitrary temperature causes the same reduction of microbes than a sterilization at 121°C and a duration that is given by the F0 value.
To be sure that the sterilisation has been successful there is a second temperature and pressure system control which allows us to assume the final product is steril (Fig.37 and 38).
Fig.38: Sterilisation graphic
4.3.4 Robot
All transport tasks and movements are carried out by a 3 axis robot with the following directions:
X-axis> right-left
Y-axis>forward-backward Z-axis>up-down
The robot has its home position in the left back corner where it is initialized before operation. Attached to the Z-axis is an extension and a small pneumatic gripper. This gripper is designed to hold standard 20 mm crimping caps (Fig. 39).
Fig.39: Robot
Robot positions are stored within the dispenser software and need to be reviewed regularly. Speed and acceleration for each axis can be controlled separately. For calibration operation, which has to be performed with open hot cell, you may only start robot movements if no person is able to access the hot cell during movement. Visual inspection of the robot positions may only be performed after the movement is finished as indicated by the computer. Moving the robot arm by hand is only permitted, if the software is either not active or in “at rest state” (neither manual nor automatic operation is started). In addition, the reset button of the control unit must be pressed before.
4.3.5 Activimeter
The dispenser is equipped with an activimeter (dose calibrator, ionization chamber) for measuring the activity of the batch the activities of the filled vials. The activimeter has 7 amplification ranges which are switched automatically. It is calibrated with the help of a Cs-137 radiator. Range 2 is absolutely calibrated with the Cs-radiator while the other ranges are calibrated relative to each other using test currents that are provided by an internal current source.
4.3.6 Preparation for dispensing
Before operation please you have to be sure the dispenser is in best conditions and all equipment is functioning to the specification. A quick system check may prevent from fatal errors or robot crashes, that is:
• notebook - on
• control unit, comserver - on • compressed air - open
• helium - open
• power outlet for sterilizer - on
• water supply for sterilizer - sufficient • waste water tank capacity - enough space • waste - enough space
Before dispensing FDG the dispenser has to be fitted with fresh consumables.
• sterile filter for dispensing (Braun Sterifix Paed)
• sterile needle - check position with a vial in manual mode • sterile filter for ventilation
• sterile spike (Braun MiniSpike) • water for injection for cleaning • ethanol (70%) for cleaning
• if dilution is required: isotonic NaCl solution
Finally make sure all consumables and vials are in place and the robot is in home position. Carefully check the filter and needle position with a spare vial. Adjust the needle by opening the locking nut on the dispensing pin.
4.3.7 Dispensing operations
The operator enters the nominal activity value and the name of the customer for each vial. Before the hot cell is closed, lead container and vial labels are printed and the latter ones stuck onto the vials. They contain the
calibration time. For the activity the nominal value is used, that is matched by the filling process within 10%. After receiving the active FDG solution from the FDG synthesis module the dispensing process is started either automatically or by an operator command. Total activity of the FDG stock solution is determined in a calibrated ionization chamber. The volume of this stock solution is determined by measuring the activity before and after removing a defined volume in three steps. The measurement results are fitted into a linear function (activity against remaining volume). This function is used to calculate the total volume of the stock. One after another vial is filled with the nominal activity. The robot picks up the vials from the storage tray and hands it over to the rotating arm. It is then brought into the clean room by rotating it inside and lifting the vial up to pierce the septum with the sterile needle. The syringe now picks up the calculated volume and dispenses the FDG solution into the vial. After dispensing the vial is transferred to the integrated miniature sterilizer. If all vials are filled they are sterilized. After that the activity of each vial is checked and it is transferred to the air lock into the transport container. After all vials have been locked out, a dispensing report (Fig.40) and optional labels for the lead containers are printed, which contain the same information as the pre-printed labels, but the measured activity values. Alternatively each container label may be printed for each vial immediately after it is locked out with an optional single label printer.
Fig.40: Distribution report
4.3.8 Cleaning the dispenser
Cleaning of the dispenser is performed in three steps by automatic procedures. At first the remaining activity is purged (Rinse), then the apparatus is sterilized with ethanol (Clean 1). Finally the ethanol is removed by rinsing with water again (Clean 2). Every day, the connection line between synthesizer and dispenser should be rinsed with ethanol. After that the line should be dried with helium and rinsed with water for injection in order to remove the remaining ethanol. For this procedure you must prepare the synthesizer to receive the ethanol and water.
4.4 Matherials 4.4.1Chemicals
18F-FDG Synthesis:
Tetrabutylammonium Hydrogen Carbonate (0,075M)-ABX; Mannose triflate plus, ultra pure-ABX;
Acetonitril (Merk);
Hydrochloric solution (Sigma); Injectable Water (Fresenius Kabi) ; Aceton (Riedel de Haen);
Ethanol solution (Fluka).
18F-SFB Synthesis:
Methanol for analysis (Merk); Acetonitril for HPLC (Carlo Erba);
O-(N-succinimidyl)-N,N,N’,N’-tetrametiluronium tetrafluoroborate (TSTU) ≥ 98% (Fluka);
Di-tert-butil dicarbonate (Boc)2O ≥99.5% (Fluka); Pure Trifluoroacetyc acid for spectroscopy (Merk);
Na+ ricombinant insulin salt expressed in E.Coli (Sigma); Acetonitril for DNA synthesis max 10 ppm H2O (Merk); Potassium carbonate plus 99.995% (Aldrich);
Kryptofix 98% (Aldrich);
Dimetilsulfoxide 99.5% (Carlo Erba); Triethylammine 99.5% (Aldrich); Methyliodure 99% (Fluka);
Etil 4-fluorobenzoate 99% (Aldrich); 4-fluorobenzoic acid 98% (Aldrich);
Ethyl 4-dimetilammine benzoate >98% (Fluka); Silver triflate 99.95%+ (Aldrich);
Metil triflate 99% (Aldrich);
Potassium fluoride 99% (Aldrich); Demineralized water ultra-milli Q;
Sodium hydroxide 1M (Riedel de Haën); Hydrochloric acid1M (Titolchimica); Acetic acid 100% (Merk);
Di ethylic eter (Carlo Erba);
Tetrabutylammonium idroxyde 40% (Fluka); N-idrossi succinimide 98% (Aldrich);
Ethanolammine (Fluka)
Buffer pH 8.0 (Riedel de Haën); Molecular Sieves 4Å (Scantec);
Strata cartridge SCX 55µm 70Å 500 mg/3 ml;
Strata cartridge X 33µm Polymeric Sorbent 200 mg/3 ml; Sephadex G25 superfine (Pharmacia).
4.4.2 Quality Control System
18F-FDG Quality Control:
Binary HPLC pump (Waters 1525) combined with a Dual λ Absorbance Detector (Waters 2487);
Isocratic HPLC pump (Waters 1515) combined with an Electrochemical Detector (Waters 2465) and a radioactivity detector GABI ( Raytest );
AR 2000 TLC scanner (Bioscan); GC Clarus 500 (Perkin Elmer); Gabistar (Raytest).
18F-SFB Quality Control:
Double pump Waters Chromatograph (mod.501) coupled with an Automated Gradient Controller (Waters) (mod.484).
Phmeter : Microcomputer pHmeter (HI 8424 Hanna Instruments). Applied Biosystem Sciex API 4000 Mass Spectrometer.
High resolution Mass Spectrometer Bruker Microthof. NMR Gemini 200 (200 MHz) Spectrometer.
4.5 18F-SFB-insulin
N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) was shown to be a suitable
acylation agent for radiolabelling of peptides, proteins, and antibodies (G. Vaidyanathan et al, 1992), (H.J. Wester et al, 1996), ( S. Zijlstra et al, 2003). Such bioactive compounds when labelled with a [18F]fluorobenzoyl group can be useful radiotracers for in vivo studies of physiological processes by positron emission tomography (PET). Imaging with 18F-SFB
insulin is important to study insulin metabolism and to carry on in vivo receptor binding experiments. The manual synthesis and purification of SFB insulin was developed in our laboratory. The procedure itself is quite complex and the main steps are the labelling of the intermediate succinimidyl ester and the reaction with the protected insulin. The knowledge of insulin behaviour in vivo, during different fasting situations or under variable lipidemia/glycemia levels would be functional to a better understanding of diabete desease and/or dismetabolic syndromes.
4.5.1 18F-SFB insulin manual synthesis
[18F]Fluoride was produced with the PETtrace cyclotron. After irradiation,
the [18O]H2O containing [18F]F− was transferred in the radiopharmaceutical
laboratory. The synthesis started by adding [18F]fluoride to a solution of
Kryptofix 222 (11,6mg) and potassium carbonate (4,26mg) in MeCN (250µl). The radioactive eluate was dried by means of a nitrogen stream at 90 °C. The reaction mixture was carefully dried by addition and evaporation of anhydrous MeCN (250µl x 5). The triflate salt of ethyl 4-N,N,N-trimethylammoniumbenzoate (10mg) and 250 µl DMSO were added
and heated at 90°C for 10 minutes without helium flow. This led to the formation of ethyl 4-[18F]fluorobenzoate. Then 0,1ml NaOH 1N were added
and the mixture was heated at 90°C for 8 minutes. After, 0,65ml HCl 1N were added and the mixture was diluted till 3 ml total volume with water. The purification of the intermediate 4-[18F]fluorobenzoic acid was carried
out by a strata X cartridge (33µm Polymeric Sorbent 200 mg/3 ml) previously activated with 1ml H2O and 1 ml MeOH. The cartridge has been then washed with 1ml water and dried under N2 flow. The resultant mixture containing 4-[18F]fluorobenzoate and 4-[18F]fluorobenzoic acid was
eluted with 3 ml di MeOH. Then a SCX cartridge (55µm 70Å 500 mg/3 ml), previously activated with 5 ml di HCl 0.1M, H2O till neutrality and 2 ml MeOH was used to separated the two components. The 4-[18F]fluorobenzoic
acid solution was heated at 90°C and 28 µl TBA idroxyde 40% were added; then the solution was dried by adding MeCN (250µl x 5). Then a solution of TSTU (13 mg) in anhydrous MeCN (250 µl) was added, and the mixture was heated at 90 °C for 2 min. The mixture was cooled down and diluted with 5% aqueous acetic acid (8 ml) before being passed through a strata X (33µm Polymeric Sorbent 200 mg/3 ml). The cartridge was then washed with a mixture of water:MeCN=80:20 (10 ml) and eluted with MeCN (1,5 ml) to obtain the final solution of purified 18F-SFB. The final solution has been
dried under N2 flow and 1.5 mg di-Boc-Insulin with 200 µl borate buffer (pH = 8.5) were added. After 15 minutes at ambient temperature the reaction led to the formation of the 18F-SFB-insulin. To determine the
extent of the reaction conversion, and the radiochemical and chemical purity of the intermediate and final products, an HPLC system was used. (Fig.41)
COOEt (Me)2N COOEt (Me)3N+ TfO -MeTfO Et2O COOEt (Me)3N+ TfO -COOEt 18F (K222)+/18F DMSO COOEt 18F COOH 18F NaOH HCl COOH 18F (But)4N+OH -TSTU 18F COO N O O Fig.41: 18F-SFB synthesis
4.5.2 18F-SFB insulin automated synthesis
Here is represented the project of automated synthesis of 18F-SFB we
developed, based on the principle of unit operations, in which a complex synthetic procedure is reduced to a series of simple operations. The apparatus makes possible the sequence of processes of the synthesis of
18F-SFB. (Fig.42) Vacuum 18O H2O rec TMAPB DMSO H2O H2O 40°C 90°C 18F CO 2H 18F CO 2 N O O NaOH MeCN K222 HCl MeCN 18F-/K 222 MeOH 18F -water K2CO3 (aq) TBA MeCN TSTU/AcCN AcOH MeCN/H2O Di-Boc-insulin pH 8,5 90°C N2liq N2liq Helium
Fig.42: 18F-SFB automated model
The three-step manual radiosynthesis of N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) was adapted to three reaction vessels
synthesis module, where the main steps of the reaction take place. In the first reaction vessel the 4-[18F]fluorobenzoic acid is synthesized, which is
F-SFB is synthesized and transferred in the last reaction vessel where the coniugation with the protected insulin take place.
The reaction vessels are connected with heaters, a vacuum system and a helium flow system, in order to reach the temperatures and the conditions requested for the synthesis steps.
Conclusions
The experience with the automatic synthesizer of 18F-FDG has been
fundamental to the acquisition of the practical experience to
analyze the methodology of synthesis of 18F-SFB-ins in the perspective of
process automation; define the basic layout;
identify the process checkpoint.
The final result is represented by the project of a prototype that could be built using commercially available components. The system would have a convenient cost and could be developed into a commercial automatic synthesizer.
