[18F]FALLYPRIDE FOR INJECTION:STANDARD OPERATING PROCEDURES

PET Radiopharmaceutical Sciences Section,Date of review: 05/17/04

Molecular Imaging Branch,

National Institute of Mental Health,

National Institutes of Health,

Bldg. 10, Rm. B3 C338,

Bethesda, MD20892

List of SOPs for [18f]Fallypride for Injection

Name of SOP

/

SOP #

1

/

Preparation of Stock Solution of Kryptofix 2.2.2* and Potassium Carbonate

/

SOP#GP101

2 / Cleaning Procedure for Radiochemistry Glassware, Reactor Vials and Magnetic Bars /

SOP # GP102

3 / Fallypride HPLC Mobile Phase Preparation / SOP # GP103
4 / Micropore Filter Testing and Drug Product /Filter Compatibility (Bubble Point Test). / SOP # GP104
5 / Preparation of Stock Acetate Buffer /
SOP # GP105
6 / BOXCLEAN Program (GE Tracerlab FXFN) / SOP # MP201
7 / GE Tracerlab FXFNModule. Vacuum Pressure Check / SOP # MP202
8 / Production of [18F]Fallypridefor Injection; Part 1: Pre-Synthesis Procedures / SOP # MP203
9 / Production of [18F]Fallypride for Injection; Part 2: Synthesis and Purification / SOP # MP204
10 / Production of [18F]Fallypride for Injection; Part 3: Formulation / SOP # MP205
11 / Standard Curve of Reference Fallypride / SOP # QA301
12 / Analysis of Organic Residues by Gas Chromatography
in [18F]Fallypride forInjection / SOP # QA304
13 / Analytical HPLC QC-Method / SOP # QA305
14 / Sterility Test / SOP # QA306
15 / P Pyrogen Test (LAL Bacterial Endotoxin Test).
SiSimplified Procedure / SOP # QA307
16 / Analysis of The Purity of Fallypride and Tosyl-Fallypride by HP HPLC / SOP # QA308

SOP # GP101

Preparation of Stock Solution of Kryptofix 2.2.2* and Potassium Carbonate

Approved by: ______Initials______Date:______

Victor W Pike, Ph.D., Chief, PET Radiopharmaceutical Sciences,NIMH

1. Procedure:

  1. Using an analytical balance and a plastic screw-cap vial (20 mL size), weight out 25 mg of

potassium carbonate (Aldrich, anhydrous, 99.99%). Add deionized water (0.5 mL) and mix to dissolve.

  1. Using weighing paper, weigh 250 mg of Kryptofix 2.2.2 (anhydrous; 98%; Aldrich) and transfer to the same vial. Add dry acetonitrile (4.5 mL) and mix to dissolve.
  2. Place appropriate label showing preparation and expiry date (to be determined) and keep the solution in the refrigerator until use (do not store in glass vial).
  3. Record the data of Kryptofix 2.2.2-potassium carbonatecomplex in Table 1.

2.Record: Lot #______

Table 1. Reagents : [18F]fluoride-Kryptofix 2.2.2-potassium carbonate complex
Item / Reagent /

Specifications

/ Manufacturer / Lot # / Expiry date / Quantity
1 / Kryptofix 2.2.2
(250 mg) / Anhydrous
98% / Aldrich Chem. Co.
2 / Potassium carbonate
(25 mg) / Anhydrous
99.99% / Aldrich Chem. Co.
3 / Acetonitrile
(4.5 mL) / Anhydrous 99.8% / Aldrich Chem. Co.
4 / Water
(0.5 mL) / De-ionized / M; Millipore, In house

Note: 100 µLof solution contains 0.5 mg potassium carbonate and 5 mg Kryptofix 2.2.2

Chemist______Initials______Date______

SOP # GP102

Cleaning Procedure for Radiochemistry Glassware, Reactor Vials and Magnetic Bars

Approved by: ______Initials______Date:______

Victor W Pike, Ph.D., Chief, PET Radiopharmaceutical Sciences,NIMH,

Procedure:

  1. Rinse with acetone (HPLC grade) to remove organic residues.
  2. Rinse with hexane (HPLC grade) to remove residual silicon oil.
  3. Bathe the glassware in an aqueous 2% solution of Liqui-Nox (Valconox, made from dilution of a 10% stock solution).

a)Bring solution to vigorous boiling for 5–10 min.

b)Swab material being washed with wood, cotton-tipped applicators (6 in).

  1. Rinse glassware 3 times with Millipore water.

a)Carefully spot-check each item.

b)Give final rinses with de-ionized water bottle (3 times).

c)Leave the clean material to drip, over paper towel.

  1. Place small V-vials, magnetic bars and reaction vials in beakers (Pyrex) and cover with aluminum foil. Place material in the oven at 170−180 ºC and allow to dry overnight.
  2. Keep all the material in the oven until use.

Notes:

a) Except for the aqueous Valconox that can be disposed in the sink, all other washing solvents must be placed in their respective waste container.

b) In the Oven Log enter date and temperature IN and date and temperature OUT.

Chemist______Initials______Date______
SOP # GP103

Fallypride HPLC Mobile Phase Preparation

Approved by: ______Initials______Date:______

Victor W Pike, Ph.D., Chief, PET Radiopharmaceutical Sciences,NIMH,

Procedure:

  1. Preparation of HPLC solvent for the preparative column

a) Fill up a flask (1 L size) for HPLC solvent with acetonitrile (HPLC grade) containing 0.6% TEA (6 mL of TEA /L).

b) In a similar flask, prepare 1 L of HPLC water only.

  1. Preparation of HPLC solvent for analytical column.

a) To prepare stock ammonium formate solution (100 mM), weigh out ammonium formate (6.013 g) on weighing paper and then dissolve in HPLC water (1 L), mix well and store at room temperature.

b) Put stock ammonium formate solution (50 mL; 100 mM) into a volumetric cylinder (1 L size), then dilute with HPLC water (450 mL; HPLC Grade)

c) Put acetonitrile (HPLC grade; 500 mL) into another volumetric cylinder. Mix

solutions from (a) and (b).

Chemist______Initials______Date______

SOP # GP104

Micropore Filter Testing and Drug Product /Filter Compatibility

(Bubble Point Test).

Approved by: ______Initials______Date:______

Victor W Pike, Ph.D., Chief, PET Radiopharmaceutical Sciences,NIMH

  • Filter-Integrity test. The integrity of the Millex-MP sterile filter that was used for sterile filtration of [18F]Fallypride for Injection is tested by the following method. The knob on the pressure regulator that is hooked to the house compressed air gas supply is turned counter clockwise so as to minimize outlet pressure. The filter assembly is removed from the sterile dose vial and placed on the male luer-lock fitting of the pressure-regulated 1/8” Teflon line. A disposable needle (1.5 inch 22 gauge) is placed on the male luer fitting of the Millex-MP filter. A portion of this needle is submerged in a test tube (12 x 50 mm) containing about 3 mL of HPLC grade water. The knob of the pressure regulator is slowly turned clockwise and the pressure gauge is monitored visually. The pressure is brought to 45 p.s.i. If there is an absence of a steady stream of bubbles in the test tube when the pressure gauge reads 45 p.s.i., then the filter passes the test. The result of the filter test is recorded on the batch record and in the summary section of the quality control test form.

Chemist______Initials______Date______

SOP # GP105

Preparation of Stock Acetate Buffer

Approved by: ______Initials______Date:______

Victor W Pike, Ph.D., Chief, PET Radiopharmaceutical Sciences.NIMH

A. Reagents:

Name of reagent / Manufacturer / Quantity
Sodium acetate / Aldrich Chem. Co.
Cat. # 241245, ACS reagent
Acetic acid / Aldrich Chem. Co.
Cat. # A6283, 500 mL
Water / EMD Chemicals Inc.
Cat. # WX0004-1

B. Procedure:

1.On weighing paper, weigh out sodium acetate (14.45 g) and then dissolve this in HPLC water (500 mL) in a bottle (500 mL size).

2.Take acetic acid (2.15 mL) and put it into above solution.

3.Mix well.

4.Measured the pH of the buffer solution; the pH of this buffer should be 5.3−5.7.

Chemist______Initials______Date______

SOP # MP201

BOXCLEAN program (GE TRACERlab FXFN)

Approved by: ______Initials______Date: ______

Victor W Pike, Ph.D., Chief, NIMH, PET Radiopharmaceutical Sciences

1. Procedure:

  1. If present, remove used Sep-Pak and re-connect line to its holder-connector.
  2. Disconnect the following three lines placing the output into the waste reservoir:

a)The one coming from the Sep-Pak holder-connector into waste.

b)The one coming from the Sep-Pak holder-connector, going into the collection V-vial (10 mL size)

c)The one going from the HPLC loop waste into a V-vial (5 mL size) connected to an external empty syringe (20 mL size), to pull diluted reaction mixture into injector loop.

d)A fourth line, from the injector to a Blue Max (50 mL size) or to a vial (20 mL size) (waste) remains the same.

  1. Check that nitrogen and air compressed air valves are open. Check nitrogen cylinder is open. Be sure there is a reactor vessel connected.
  2. Place dry-ice in the vacuum trap.
  3. Start the TracerLab automated software program.

Select Synthesis: "Boxclean", ENTER.

Follow instructions.

Add acetone as follows:

a)1−2 mL acetone to target vial

b)1–2 mL acetone to support vials V1 to V9 (fill small ones and half fill large ones)

c)25 mL acetone to collection bulb vessel

d)0.5 mL acetone to V-vial (10 mL size) (leave the spinal needle way up)

  1. In Preparation CHECK LIST, check each item in the list, going down with repeated ENTER. Finally, enter operator initials.
  2. The screen will show the list of timed events for BOXCLEAN and the process will start with the vacuum pup ON and the temperature increasing to 100oC.

Check that the temperature reaches 100oC and the pressure in reactor goes down.

  1. At the end of the Program, the system will be flushed with nitrogen, go under vacuum to remove most of the acetone vapors and then stop.
  2. Remove the acetone that may be trapped in the vacuum trap. Dispose of it in the appropriate container.

Copy of the BOXCLEAN Program see Attached file: Document 9: Validation Runs, attached BOX Clean Program on the batch: FAY-032404.

Chemist______Initials______Date______

SOP # MP202

GE TRACERlab FXFNModule. Vacuum Pressure check

Approved by: ______Initials______Date: ______

Victor W Pike, Ph.D., Chief, NIMH, PET Radiopharmaceutical Sciences

Procedure:

  1. Open nitrogen and compressed air valves located outside hot-cell
  2. Start Dell Computer-Windows 98. Enter Name and Password. After Virus Scan

Computer will open. Click twice on TRACERLab icon. Enter "Admin" and "Tracerlab"

  1. Once on the Instrument SCHEME, look at the square area in the right side of the screen that shows pressure of compressed air and nitrogen.
  2. Turn on the vacuum on by click “ON” of the "Power”, which represents the vacuum on the Instrument SCHEME. (Note: this can be turned OFF in the same way).
  3. Compressed air pressure should be around _____kPa and nitrogen around______kPa.
  4. Click the mouse in V24 and V25 should open with space bar (1 = open, 0 = close), this will set full vacuum on reactor vessel, reaching around ≤ 3 kPa. Record the lowest pressure. ______kPa
  5. Close V24 (space bar), pressure should hold (change should be ≤ 1 kPa/min).
  6. If pressure is not holding find and correct the source of leak; e.g. tighten stoppers in support vials, check valves 2 to 6, 13, 14, 20, 24; tight reactor vial, check O-ring.
  7. With mouse, go to V20 (Nitrogen), open V20 with space bar (can also close with space bar). Pressure should go up. Close with space bar and open V24. Check the pressure again. It should go below 2 kPa again.
  8. If pressure holds, go ahead with the next step.

Note. When both vacuum and pressure are open during the procedure, pressure should hold between 21.5 to 22 kPa (V20 and V24 open).

Chemist______Initials______Date______

SOP # MP203

Production of [18F]Fallypride for Injection; Part 1: Pre-synthesis Procedures

Approved by: ______Initials______Date: ______

Victor W Pike, Ph.D., Chief, NIMH, PET Radiopharmaceutical Sciences

Procedures:

  1. Check that nitrogen and compressed air valves are open.
  2. Check nitrogen cylinder is open.
  3. Turn on the computer with TRACERlab FXFNsoftware and with Beckman HPLC software.
  4. Open the TRACERlab FXFNsoftware and check the pressure of compressed air and nitrogen pressure. The pressure of compressed air should be 400 p.s.i and nitrogen pressure > 100 p.s.i.
5.Clean GE TRACERlab FXFN Module and hot-cell 5.
4.1 Follow the program "CLEAN BOX" (SOP # MP201). Clean and dry the GE TRACERlab FXFN module on the day of preparation or the day before the preparation. All the reagent vessels and transfer lines of the module to be used in the preparation should be washed with acetone and dried under nitrogen flush before using.

4.2 Inspect hot-cell compounding/dispensing area for cleanliness. Remove extraneous materials and labels. Spray ethanol to clean this area if necessary.

5.3 Inspect and set if necessary charcoal trap in the module

6.Prepare [18F]fluoride ion-Kryptofix 2.2.2-potassium carbonatecomplex solution

5.1Add stock Kryptofix 2.2.2-potassium carbonate (70−100 µL) into V-Vial (1 mL size).

5.2Take V-Vial 1 to Cyclotron Facility (CC, NIH) to collect aqueous [18F]fluoride ion; the activity of [18F]fluoride ionshould be between 150−250 mCi water ( ≤ 500 µL).

6.Verify integrity of glassy reaction vessel assembly (containing small magnetic bar stirrer) in TRACERlab FXFN Module. Follow SOP # MP202and record the data in the sheet of Summary Records of [18F]Fallypride for Injection run:

6.1Vacuum pressure check (gauge, kPa).

6.2The compress air pressure (kPa)

6.3Nitrogen pressure (kPa)

7. Prepare the preparative HPLC system

7.1Start the HPLC computer software, enter the necessary information and select the right method “ Fally_Production”

7.2 Verify that mobile phase bottles in the right position on the Beckman System Gold Deliverymodule.

7.2.1The solvent bottle of 0.6% TEA connects to B1 line (markedred)

7.2.2The solvent bottle of HPLC water connects to A1 line (markedred)

Above connection should match the setup in the method “Fally_Production”.

7.2.3Set Acetonitrile w 0.6%TEA /HPLC water (30: 70 v/v) as initial conditions. Equilibrate the Luna column (Phenomenex) with fallypride preparative mobile phase (~ 200 mL)

7.3Note the pump pressure while increasing the flow rate to 2 mL/min.

7.4 Pump the HPLC mobile phase through the preparative column at a low flow (1−2 mL/min) and maintain this flow until purification time.

8.Prepare in the clean bench a sterile filtration/collection Unit, using aseptic technique:

8.1.Prepare the final sterile product vial. The final sterile empty vial, product needle, vent needle and twofilters (Millex 0.22 µm) are assembled in a certified laminar flow sterile cabinet. Attach a prepared label for [18F]fallypride with the current batch number and date to the product vial.

8.2.Move a prepared product vial unit from the laminar flow sterile cabinet and attach it to the product line of TracerLab FN Module.

9.Open the Tracerlab Fx Computer Controlling System ("Admin" and "Tracerlab") and Select Synthesis method: "Fallypride". Click “Synthesis-Start” to start preparation of the synthesis.

10.Enter all the required information (including date, radiotracer, batch #, human preparation #) into the corresponding method of the HPLC computer software program.

11. Check that you have the correct UV absorbance (254 nm) and the correct settings for the gamma 12. Place the solvents and precursor solution in the corresponding module support vials:

______Support vial 1:acetonitrile (0.5 mL).

______Support vial 2 [Used as a port (V-vial; 1 mL size)]: [18F]fluoride-K 2.2.2-potassium carbonate solution; 100 µL).

______Support vial 4:dry acetonitrile (1 mL) plus tosyl fallypride (2.0±0.5 mg).

______Support vial 5:dry acetonitrile (0.5 mL)

______Support vial 6:dry acetonitrile (1.5 mL)

______Support vial 7:sterile saline solution (9–14 mL)

______Support vial 8:Ethyl alcohol for Injection (1 mL)

______Support vial 9sterile water (8 mL)

______Large (10 mL size) V-vial: 0.5 mL sterile water with 0.6%TEA.

______Large bulb vessel: HPLC water (90 mL) and sodium acetate (250 mM; pH 5.5; 10 mL; see SOP # GP104).

12.Check that ‘complex solution’ delivery line goes through valve V2 and into reactor vessel (glassy carbon).

13.Place acetonitrile (0.5 mL) in a syringe (3 mL) connected to an external line going into the "complex" vial for recovery of [18F]fluoride-K 2.2.2-potassium carbonate solution left in transportation vial and its addition to the reaction vessel.

14.Activate one C-18 plus Sep-Pak with ethanol (10 mL) sterile water (10 mL) and place in its corresponding connection site in the module.

  1. Place the small double-neck round bottom flask to receive the ethanolic eluate of the Sep-Pak in its corresponding location in the Box.

16. In a lead pot, lace a sterile empty vial (30 mL size) fitted with a vent needle/filter and a second needle and Millev GV filter for sterilization of the final product.

17. Place enough dry-ice/acetone mixture in the vacuum trap.

  1. Check that the nitrogen and compressed air valves close to the hot cell are open (also check the nitrogen cylinder).
  2. Assure that syringes and needles have been changed. Use depyrogenated reaction vessel (with magnetic stirrer), HPLC collection bulb (10 mL size) V-vial, two neck round-bottom flask, sterile vial etc.
  3. Keep the HPLC column flowat 2 mL/min.

Synthesis comments:

______

The approved procedure has been followed without any deviation

Chemist______Initials______Date______

SOP # MP204

Production of [18F]Fallypride for Injection; Part 2: synthesis and purification

Approved by: ______Initials______Date: ______

Victor W Pike, Ph.D., Chief, NIMH, PET Radiopharmaceutical Sciences

Procedures: [18F]Fallypride synthesis and purification

  1. Preparation of [18F]fluoride ion/Kryptofix 2.2.2-potassium carbonate complex ([18O]water-acetonitrile-Kryptofix 2.2.2-[18F]fluoride ion-potassium carbonate).
  2. Using the dose calibrator (Biodex AtomLab 300) set at F-18, determine the amount of radioactivity that will be transferred to the clean glassy reaction vessel (20 mL size). Record the data: EOB, starting activity of [18F]fluoride-[18O]water and Volume of [18F]fluoride-[18O]water.
  3. Connect one vent needle, one needle and line from external syringe containing acetonitrile (0.5 mL) and one spinal needle and line going from the radioactive solution through V2 to reactor vial. Make sure that spinal needle is all the way down to the bottom of the radioactive solution.
  4. Starting the preparation and transferring the F-18 complex.

2.1 Start the TRACERLab automated software program. Select Synthesis: "Fallypride" in method box, click “Synthesis-Start” in main menu bar, follows process instructions. Organize "Instrument screen", "Program" and "Graphics" in the computer screen.

2.2The program will have already started setting the vacuum pump ON and transferring the F-18 complex from the V-vial (1 mL size) into the reaction vessel, then MESSAGE BOX comes out. At this moment add acetonitrile (0.5 mL) from the outside syringe into the transport F-18 vial (pushing with air until the line is empty and the vial is full). Press “Enter” or click “OK” when done.

  1. [18F]Fallypride synthesis step 1: remove water from the F-18 complex.

The reactor temperature will be increased first to 65 ºC and later to 88 ºC (T1 + 3 min) and then back to 60 ºC. At this point, the contents of V5 are added and the temperature cycle starts again: heat at 65ºC, then at 88ºC and finally at 60 ºC (drying process).

4.[18F]Fallypride synthesis step 2: 18F-Fallypride reaction. Check V4 and reaction temperature.

4.1Once drying is complete, V4 will open and the tosylate precursor in acetonitrile (1 mL) will be added to the reaction vessel and the mixture heated at 100 ºC for 30 min. At the end of the reaction, the reaction vessel will be cooled at 35 ºC and the first part of the Program will finish.