Radiopharmaceutical
Production
FDG Synthesizer
Operation
STOP
Operation of synthesizers
All the commercial syntheizers are
based on the Hamacher synthesis [1].
Some use base hydrolysis while others
use acid hydrolysis. Each has some
advantages and disadvantages. The
final sterilization may be done either
with a 0.22 micron membrane filter or by
steam autoclaving.
Content
1.
2.
3.
Key Features of the Synthesis
Extraction of F-18 from target water
Drying of Fluoride with TBA or
Cryptand
4.
Labeling of glucose with F-18
5.
Removal of protecting groups
6.
Purification of final product
7.
Sterilization
8.
QC sample removal
9.
Dispensing
10. QC Control During Synthesis
1. HAMACHER, K., COENEN, H.H., STOCKLIN, G. Efficient stereospecific synthesis
of no-carrier-added 2-[18F]-fluoro-2-deoxy-D-glucose using aminopolyether supported
nucleophilic substitution. J Nucl Med 1986;27(2):235-8.
STOP
SYNTHESIS OF
[18F]FDG
PRODUCT
Preparation
18F-
FDG
“NAKED”
FLUORIDE
Cleaning
O
O H
O
H
Synthesis Module
18F
PRECURSOR
Nucleophilic
substitution
O
H
Deprotection
INTERMEDIATE
STOP
18F
O
H
[18F]FDG
Radiopharmaceutical
Production
Overall FDG Synthesis
Scheme
Starting with the MannoseTriflate
FDG Synthesizer
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
The protecting groups keep the fluoride
from reacting at positions other than the 2
position on the ring
Both FDG and glucose
are formed in the
synthesis
See the following slides for
some key features of the
synthesis.
Key Features of the Synthesis
Radiopharmaceutical
Production
FDG Synthesizer
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
•
•
Why the precursor molecule needs acetyl protection?
This is to make substitution reaction possible. Fluoride anion
tends to form very strong hydrogen bonds with any type of polar
groups such as hydroxyl groups present in sugars. This strong
hydrogen bond formation prevents the fluoride from reacting in
the desired nucleophilic substitution. Therefore, all hydroxyl
groups of precursor are protected by acetyl ester protection.
Key Features of the Synthesis
Radiopharmaceutical
Production
FDG Synthesizer
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
•
•
Why Kryptofix 2.2.2 is used?
Kryptofix 2.2.2 (4,7,13,16,21,24-hexaoxa-1,10diazabicyclo[8,8,8]hexacosane) is a phase transfer catalyst. On
one hand, it helps to solubilize the [18F]fluoride anion in an
anhydrous reaction solvent (acetonitrile). On the other hand, it
helps to separate the strong ion pair formed between
[18F]fluoride and potassium by ‘caging’ the potassium cation.
This helps to make fluoride free from interaction with potassium
and make it more available for nucleophilic displacement
reaction.
Key Features of the Synthesis
Radiopharmaceutical
Production
FDG Synthesizer
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
•
•
What is TBA?
TBA stands for tetrabutylammonium carbonate, which is an
alternative phase transfer catalyst, and may be used instead of
Kryptofix 2.2.2.. Some synthesizers could use either TBA or
Kryptofix. When TBA is used in a synthesis, a suitable quality
control procedure for estimation of TBA present in the FDG
product is needed instead of Kryptofix 2.2.2. A minor
disadvantage of TBA use is that it needs to be converted from
commercially available hydroxide into carbonate form. In this
book we make assumption that Kryptofix is used as the phase
transfer catalyst.
Key Features of the Synthesis
Radiopharmaceutical
Production
FDG Synthesizer
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
•
•
How is D-glucose (DG) (4) formed in the reaction?
D-glucose is always formed as a by-product of FDG synthesis
from the unreacted precursor (1) by hydrolysis of
tetraacetylmannose triflate into D-glucose. D-Glucose is
mentioned in International Pharmacopoeia as a principal peak
in the quality control test for chemical purity. There are no limits
for its presence in the final formulation, because it is a natural
compound present in human blood in large concentrations.
Radiopharmaceutical
Production
FDG Synthesizer
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
Extraction of F-18 from target
water
•
After irradiation of enriched water, the mixture 18F-/18O[H2O] is
transferred from the target onto a pre-conditioned anion
exchange separation cartridge such as QMA (quaternary
ammonium anion exchange) SepPakTM column. The 18F- ions
are retained on the cartridge while unused [18O]H2O and the
cationic and other impurities from the target are removed and
collected in a waste vial. The trapped 18F- is subsequently
eluted from the cartridge with a solution containing K2CO3 and
the phase transfer catalyst (Kryptofix 2.2.2), in acetonitrile and
pharmaceutical grade water. 18F-KF/Krytofix complex eluted is
collected in the reaction vial. Quantities of K2CO3 and Kryptofix
2.2.2 vary depending upon the synthesis module.
Radiopharmaceutical
Production
Drying of Fluoride with TBA or
Cryptand
•
Effective dryness of fluoride (virtual freedom from water) is a
critical factor for the nucleophilic reaction to occur efficiently and
with eventual high yield of FDG. The required dryness is
achieved through repeated azeotropic evaporation (typically 3
times) with anhydrous acetonitrile at ~85C under inert gas or
vacuum. It is essential that during each solvent removal step,
the mixture to taken to complete dryness, and also that the
vessel temperature does not exceed 100ºC in order to prevent
decomposition of Kryptofix 2.2.2.
•
Nucleophilic displacement reactions with fluoride are known to
be quite difficult and unpredictable because of the fluoride ion
being a weak nucleophile in an aqueous media, leading to very
poor yields. In a polar aprotic media such as acetonitrile,
fluoride undergoes nucleophilic reactions rather quickly.
However, for fluoride to become an effective nucleophile, it must
be available as reactive fluoride. The 18F-/K2CO3/Kryptofix
2.2.2 complex is effectively an organic cation/inorganic anion
salt soluble in acetonitrile making the [18F]fluoride available in a
highly reactive form.
FDG Synthesizer
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
Labeling of glucose with F-18
Radiopharmaceutical
Production
FDG Synthesizer
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
•
In the synthesis of 18F-FDG based upon the method of
Hamacher et. al. [1], the 1,3,4,6-tetraO-acetyl-2-O-trifluoromethanesulfonyl-beta-D-mannopyranose
(mannose triflate) precursor reacts with [18F]Fluoride through
nucleophilic displacement. Although various precursor
substrates have been described in the literature, the use of
triflate as the leaving group in the nucleophilic reaction with
18F-fluoride is found to be the most efficient, rapid and clean.
The reaction between the dry mixture of 18F-/K2CO3/Kryptofix
prepared in the previous step and mannose triflate in anhydrous
acetonitrile at ~85C provides a high yield of the 18F labeled
intermediate in less than 5 minutes. An added advantage of
using the acetylated mannose triflate is that after the
nucleophilic displacement of the triflate group by 18F, the acetyl
groups can easily be removed by hydrolysis (acid or base) to
give rise to FDG.
Removal of protecting groups
Radiopharmaceutical
Production
•
The final chemical step in the synthesis of FDG is the removal
of the four acetyl protecting groups, which is easily
accomplished through hydrolysis with either mild acid or base.
Both methods are equally effective.
•
The alkaline hydrolysis is the most commonly used method in
commercial synthesis modules since it requires less time and
lower temperature. In this case, the 18F-labelled intermediate
with intact acetyl groups is treated with mild base (1-2 M NaOH)
at room temperature to remove the acetyl groups. The alkaline
hydrolysis may be performed directly on a C-18 cartridge at
room temperature instead of adding base to the reaction vessel.
•
One possible drawback is that alkaline hydrolysis has a
potential for epimerization of FDG to [18F]Fluorodeoxymannose
(FDM) which is a radiochemical impurity that should be
controlled in the FDG manufacturing process. It has been
shown, however, that formation of FDM is a possible only if the
hydrolysis is performed at higher temperature [7]; epimerization
at room temperature is negligible.
FDG Synthesizer
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
Purification of final product
Radiopharmaceutical
Production
•
FDG Synthesizer
– Cation exchange for removal of Kryptofix2.2.2 or TBA
– C-18 or similar lipophilic column for removal of unhydrolyzed and
partially hydrolyzed intermediates
– Alumina for removal of unreacted fluoride
– Ion retardation for neutralization and pH adjustment
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
FDG is purified by passing through a series of columns (e.g.,
SepPakTM cartridges) for removal of impurities. These columns
include (not necessarily in this order):
•
In some synthesis modules, the unhydrolyzed intermediates are
trapped on a C-18 column and impurities are washed out prior
to hydrolysis. Final formulation includes adjustment of
isotonicity, pH and volume adjustment. Some synthesis units
utilize cation exchange and ion retardation resins to neutralize
the solution, while others use buffers and addition of calculated
quantity of hypertonic NaCl or Na2CO3 to achieve the required
pH, isotonicity and required volume of the final product.
Sterilization
Radiopharmaceutical
Production
FDG Synthesizer
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
•
Sterilizing filtration is performed by passing the purified FDG
solution through a vented 0.22µm filter. Sterilization with steam
(autoclave) may also be applied, but is not required. The filtered
product is essentially the final product which is held in
quarantine until the time it is qualified for patient use.
QC sample removal
Radiopharmaceutical
Production
FDG Synthesizer
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
•
A representative test sample is removed from the well-mixed
bulk solution. If the production protocol entails dilution, sample
must be drawn after dilution. Moreover, sampling must be done
aseptically, ensuring not to microbiologically contaminate the
final product. The sample size is usually about 0.5 mL for QC,
and about 1.0 mL for retention in case of repeat tests and
product complaints.
Dispensing
Radiopharmaceutical
Production
•
The FDG may be dispensed either into unit doses or in
multidose vials using either manual or automated systems.
This process must be done in an aseptic manner. If the product
is dispensed into open vials, it must be done in a class A
environment. The applicable GMP regulation should be
followed. Practically, the typical injection volume is 2-5 mL.
•
The product vial (or the syringe) and the outer lead shielding
should be affixed with label containing product information,
including: product name; total activity at reference time;
expiration time; etc.
FDG Synthesizer
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
Radiopharmaceutical
Production
QC Parameter Control
During Synthesis
Character
FDG Synthesizer
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
Aim
How to control during synthesis
Appearance Clear,
colourless, and
free of
particulate
matter
Carefully prepare the purification
cartridges for efficient purification of
the product. .
Carefully assemble the reagents
and ‘kit’ on the synthesis module
during pre-synthesis set-up by
practicing aseptic handling of
materials to render the solution free
of particulate matter and
microorganisms.
Radionuclidi ≥ 99%
c Purity
[18O]water of >95% enrichment is
recommended. In case of recycling
the irradiated water, the water must
undergo effective purification. The
use of recycled water must be
validated prior to use in the
production of a patient dose. .
Radiopharmaceutical
Production
QC Parameter Control
During Synthesis
Character
FDG Synthesizer
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
Aim
Radiochemi ≥ 95%
cal
Purity
Kryptofix
 50µg/mL
How to control during synthesis
Raw materials and supplies must
be controlled and care must be
taken to ensure efficient
purification at the end of
synthesis through proper
preparation and conditioning of
the purification cartridges and
leak-free assembly.
Using only the amount of Kryptofix
2.2.2 that has been validated
during set-up, and efficient
purification at the end of
synthesis.
Radiopharmaceutical
Production
FDG Synthesizer
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
QC Parameter Control During
Synthesis
Character
Aim
How to control during synthesis
Residual
solvents
Acetonitrile:
Controlled during synthesis (efficient
 0.04%;
evaporation is the key parameter,
Ethanol:  0.5% which is equipment related and not
necessarily controllable by the
operator);
The operator must ensure complete
removal of solvents used for
conditioning the cartridges.
Impurities
[18F]FDM 
10%;
Cl-DG  0.5 mg
per maximum
injectable
volume
[18F]FDM can be controlled by
optimizing the hydrolysis
temperature, time and concentration
of sodium hydroxide;
Cl-DG is likely to result from the
presence of chloride ion in reaction
mixture arising from improperly
treated anion exchange resin used
for collection of [18F]fluoride. Proper
rinsing of the cartridge will remove
the unwanted chloride ions.
Radiopharmaceutical
Production
QC Parameter Control
During Synthesis
Character
FDG Synthesizer
Content
Key Features of the
Synthesis
Extraction of F-18 from
target water
Drying of Fluoride with
TBA or Cryptand
Labeling of glucose with
F-18
Removal of protecting
groups
Purification of final
product
Sterilization
QC sample removal
Dispensing
QC Control During
Synthesis
STOP
Endotoxin
(pyrogen)
and Sterility
Aim
 175 EU per
maximum
injectable
volume
How to control during synthesis
Microbial contamination of the
product should be controlled
through use of low bioburden raw
materials and supplies used in
synthesis; and ensuring aseptic
handling of materials and
components during set-up.
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