Fully automated synthesis and coupling of [18F]FBEM to glutathione using the iPHASE FlexLab module

Article abstract of DOI:10.1002/jlcr.3175

Site-specific radiolabelling of peptides or antibodies using [ ¹⁸F]FBEM is often preferred over non-site-specific radiolabelling with [¹⁸F]SFB because it does not affect the affinity of the antibody to its target. Unfortunately, the synthesis of [¹⁸F]FBEM and its conjugation to thiol containing macromolecules requires some manual intervention, which leads to radiation exposure of the radiochemist. In this publication, we report on the complete automation of [¹⁸F]FBEM production and its subsequent conjugation to glutathione using a slightly modified iPHASE FlexLab module. [¹⁸F]FBEM was produced in 1.185 ± 0.168 GBq (15-20%; n = 10; 0.75 ± 0.106 GBq non-decay corrected) with a specific activity of 57 ± 10 GBq/μmol. Radiochemical purity was 97 ± 1% and the synthesis time including HPLC purification and reformulation was 70 min. After evaporation to dryness, [¹⁸F]FBEM was conjugated to glutathione in PBS buffer pH 7.4 in quantitative yields. This fully automated method does not require any manual intervention and therefore reduces the radiation exposure to the operator. copy; 2014 John Wiley & Sons, Ltd.

Full text of DOI:10.1002/jlcr.3175

Note
Received 22 October 2013,
Revised 26 November 2013,
Accepted 27 November 2013
Published online 7 January 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3175
Fully automated synthesis and coupling of
18
[ F]FBEM to glutathione using the iPHASE
FlexLab module
d
b
Uwe Ackermann,a,b,c Lucie Plougastel, Christian Wichmann,
Yit Wooi Goh,a,b Shinn Dee Yeoh,a,b Stan S. Poniger,e
Henri J. Tochon-Danguy,a,b,c,e and Andrew M. Scotta,b,c
*
Site-specific radiolabelling of peptides or antibodies using [18F]FBEM is often preferred over non-site-specific radiolabelling
with [18F]SFB because it does not affect the affinity of the antibody to its target.
Unfortunately, the synthesis of [18F]FBEM and its conjugation to thiol containing macromolecules requires some manual
intervention, which leads to radiation exposure of the radiochemist. In this publication, we report on the complete
automation of [18F]FBEM production and its subsequent conjugation to glutathione using a slightly modified iPHASE
FlexLab module. [18F]FBEM was produced in 1.185 0.168 GBq (15–20%; n = 10; 0.75 0.106 GBq non-decay corrected) with
a specific activity of 57 10 GBq/μmol. Radiochemical purity was 97 1% and the synthesis time including HPLC purification
and reformulation was 70 min. After evaporation to dryness, [18F]FBEM was conjugated to glutathione in PBS buffer pH 7.4 in
quantitative yields. This fully automated method does not require any manual intervention and therefore reduces the
radiation exposure to the operator.
Keywords: peptide labelling; FBEM; radiolabelled synthons; PET
Furthermore, the synthesis and purification of [18F]FBEM
Introduction
followed by the subsequent sulphydryl coupling has never been
achieved in a single synthesis module and [18F]FBEM labelling of
The radiolabelling of peptides and antibodies using prosthetic
groups is becoming increasingly important in radiopha-
thiol containing macromolecules, therefore, still requires a
significant amount of time and resources.15 Also, this coupling
rmaceutical science. Due to their easy accessibility, NHS esters
such as succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) are widely
requires some degree of manual manipulations, thus leading to
used for this purpose.1–4 [18F]SFB reacts with free amino groups,
considerable radiation exposure to the operator. Our aim was
which are present as part of lysine residues in the chemical
to develop a fully automated procedure for the production and
subsequent antibody/peptide conjugation of [18F]FBEM using
structure of antibodies. However, the relatively short 109 min
half-life of fluorine-18 makes it only suitable for labelling of small
the commercially available iPHASE FlexLab module without the
need for any manual intervention.
Due to the shorter synthesis time and more robust production
antibody fragments such as single chain antibodies (scFvs).
Furthermore, [18F]SFB labelling also negatively affects the
binding affinity of the antibody to its target.5,6
because of less synthesis steps, we decided to implement the 4-
[18F]fluorobenzoic acid pathway on this synthesis module. Also, due
In order to achieve selective labelling at sites distant from the
active site, scFvs can be engineered to contain free thiol groups,
which can be reacted with thiol selective reagents such as N-(4-
[18F]fluorobenzyl)-2-bromoacetamide, N-[2-(4[18F]fluorobenzamido)
ethyl]maleimide ([18F]FBEM), N-[6-(4-[18F]fluorobenzylidene)aminoo-
xyhexyl]maleimide ([18F]FBAM), 1-[3-(2-[18F]fluoropyridin-3-yloxy)
propyl]pyrrole-2,5-dione ([18F]FPyME) or [18F]FPEGMA, which have
recently been developed (Figure 1).7–11
aDepartment of Nuclear Medicine and Centre for PET, Austin Health, Heidelberg,
Melbourne, Australia
bThe University of Melbourne, Parkville, Melbourne, Australia
cLudwig Institute for Cancer Research, Melbourne Branch, Melbourne, Australia
Of these, [18F]FBEM is of particular interest because it can be
produced from commercially available starting materials. [18F]
FBEM has been synthesised either from coupling of 4-[18F]
fluorobenzoic acid ([18F]FB) or [18F]SFB to N-(2-aminoethyl)
maleimide.9,12
dEcole National Supérior de Chimie de Rennes, Avenue du Général Leclerc -
CS50837, 35700 Rennes, France
eiPHASE Technologies, Melbourne, Australia
Despite recent advances in [18F]SFB production, this route still
is time consuming and requires numerous synthesis steps,
whereas the [18F]FB route is slightly shorter.13,14
*Correspondence to: Uwe Ackermann, Department of Nuclear Medicine and
Centre for PET, Austin Health, Heidelberg, Melbourne, Australia.
E-mail: ackerman@petnm.unimelb.edu.au
J. Label Compd. Radiopharm 2014, 57 115–120
Copyright © 2014 John Wiley & Sons, Ltd.

U. Ackermann et al.
Figure 1. Structures of the most important [18F]F labelled prosthetic groups for thiollabelling.
to the reported instability of N-(2-aminoethyl)maleimide in the detectors to initiate injection of the product onto the column. Both
presence of diisopropyl ethylamine (DIEA) and diethyl reactor vials have five reagent vials (vials 2–6 for R1 and vials 15–19
cyanophosphonate, we decided to use N,N′-dicyclohexylcarbodiimide for R2) for the addition of reagents or liquids. Vial 1 is used for the
(DCC) without DIEA to promote the coupling reaction between 4-[18F] elution of F-18 fluoride from the quaternary methylammonium
fluorobenzoic acid and N-(2-aminoethyl)maleimide.12
(QMA) cartridge and the remaining vials 7–14 are used for the
The coupling of [18F]FBEM to glutathione was then purification and reformulation of products or intermediates. The
investigated as a model for future coupling reactions to suitably temperature in both reactor vials can be independently heated
modify single chain antibodies (Figure 2).
to 255 °C and cooled to ~4 °C using vortex tube cooling with
magnetic stirrer bars for mixing. Programming is performed within
the Movicon interface using a Microsoft Excel recipe template. This
can be done offline as well as in ‘live’ mode.
Materials and methods
For the purpose of this study, the HPLC loop vial 1 was
converted into an intermediate reaction vial. This was achieved
by wrapping a nichrome (NiCr)-resistive heating wire around
the intermediate vial. The wire was insulated with heatshrink
wrap and supplied with 6 V DC power. This method performed
well and as a result, iPHASE is currently developing a drop-in
aluminium heater insert for the FlexLab module to perform such
intermediate heating on these vials.
General
Figure 3 shows a schematic of the iPHASE FlexLab module. The
module is a dual reactor system that allows HPLC purification of
intermediates as well as HPLC purification of the final product.
For this purpose, the module is equipped with two HPLC columns,
a column selector and a 5 mL injection loop for each HPLC column.
HPLC injections are performed via dedicated loop vials using fluid
Figure 2. Synthesis of [18F]FBEM and conjugation to glutathione.
www.jlcr.org
Copyright © 2014 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 115–120

U. Ackermann et al.
Figure 3. Schematic of the modified iPHASE FlexLab module with HPLC loop vial 1 converted into an intermediate reactor vial. This figure is available in colour online at
wileyonlinelibrary.com/journal/jlcr
Solvents were purchased from MERCK and used without further UV-vis detector and two LC-20 AD solvent pumps for high
purification. [19F]FBEM standard was purchased from ABX, Radeberg, pressure mixing of mobile phase was used. The stationary phase
Germany. All other reagents were purchased from Sigma-Aldrich was a Phenomenex Gemini C-18, 5 μ RP column, 150 × 4.6 mm.
and used without further purification. (4-ethoxycarbonylphenyl) Acetonitrile (A) and water (B) with 0.1% formic acid were used
trimethylammonium triflate was synthesised according to a as the mobile phase and a gradient elution technique was used
literature procedure.16
for analysis: 0–18 min: 5–90% A, 18–30 min: isocratic 90% A at a
Chromafix PS-HCO3 cartridges were purchased from ABX, flow rate of 0.5 mL/min. The Bioscan FC-4000 dual BGO
Radeberg. NBu4HCO3 used for the elution was generated by coincidence detector was used for the detection of radioactive
diluting 50 μL of NBu4OH (1 M in methanol) in 450 μL of water compounds. Table 1 shows the preparation of the modified
followed by purging with CO2 for 3 h.
FlexLab module for the production of [18F]FBEM and coupling
Strata-X cartridges (33 μm, 60mg/1mL tube, part no: 8B-S100- to glutathione.
UAK) were purchased from Phenomenex, preconditioned with
2 mL of ethanol and washed with 2 mL of water before use. No-
Radiochemistry
carrier-added [18F]fluoride was produced by the 18O(p,n)18
F
After loading of the reagents, [18F]fluoride from the target was
trapped on the ion exchange cartridge and eluted using eluent
from vial 1. The solution was dried with 1 mL of dry acetonitrile
at 110 °C for 6 min under vacuum with argon flow. After drying,
the precursor was added from vial 2, and labelling was achieved
by heating to 110 °C for 15 min. Base solution was then added
from vial 3, and the solution heated to 90 °C for 15 min under
vacuum with argon flow. This achieved hydrolysis of ethyl
4-[18F]fluorobenzoate to 4-[18F]fluorobenzoate (4-[18F]FB) during
the evaporation step as well as removal of the methanol, which
was added as part of the 1 M tetrabutylammonium hydroxide
stock solution. 4-[18F]FB was then diluted with 10 mL of acetic
acid solution from vial 6 and 4-[18F]FB trapped on the Strata-X
SPE cartridge in position A. The cartridge was washed with water
from vial 5 and 4-[18F]FB was then eluted with 0.5 mL acetonitrile
containing 40 mg of DCC from vial 7 into intermediate reaction
(loop) vial 1, which was then heated to 56 °C for 10min. After this
time, 4.5mL of water from vial 4 was added to the intermediate
reaction vial through R1 and the mixture injected into HPLC system
1. A Phenomenex Gemini 10 μm C18 250 × 10mm (part no
00G-4436-N0) column was used as a stationary phase and
acetonitrile (A) and water (B) with 0.1% ammonium formate were
used as the mobile phase. A gradient elution technique was used
to purify [18F]FBEM: 0–20 min: 10–90% A at a flow rate of
4 mL/min. The peak at 12.5 min was collected in HPLC vial 1, trapped
on Strata-X SPE cartridge B and transferred to reactor vial 2 where the
[18F]FBEM solution was evaporated at 40 °C under vacuum with
argon flow. Glutathione (1 mg in 1 mL of PBS buffer, pH 7.4) was then
added from vial 15 and the mixture heated to 40 °C for 10 min. The
crude reaction mixture was then transferred to a collection vial and
analysed by radio-LCMS to confirm product identity.
nuclear reaction with a 10 MeV proton beam generated by the
IBA Cyclone 10/5 cyclotron in a titanium target using [18O]H2O at
Austin Health, Centre for PET. Typical irradiation parameters were
20 μA for 30 min, which resulted in 5.4–8.1GBq (146–219 mCi) of
[18F]fluoride being transferred into the synthesis module. These
yields represent around 25% of the theoretical yield and are
explained by the use of recycled [18O]H2O of unknown isotopic
enrichment. Isolation of the [18F]fluoride ion from [18O]H2O was
achieved by trapping on a Chromafix PS-HCO3 ion exchange
column. The column was eluted with a solution containing
NBu4HCO3 (50 μmol in a mixture of 450 μL water and 50μL of
methanol) and 300 μL of acetonitrile. Evaporation to dryness with
1 mL of acetonitrile gave the anhydrous [18F]fluoride used in the
labelling experiments.
The quality control and specific activity measurement of [18F]
FBEM was performed using a Shimadzu HPLC system equipped
with a 20 μL injection loop, a SPD-20A UV-vis detector and two
LC-20 AD solvent pumps for high pressure mixing of mobile
phase was used. The stationary phase was a Phenomenex
Gemini NX C-18, 5 μ RP column, 150 × 4.6 mm. Acetonitrile (A)
and water (B) with 0.1% formic acid were used as the mobile
phase and a gradient elution technique was used for analysis:
0–18 min: 5–90% A, 18–30 min: isocratic 90% A at a flow rate of
0.5 mL/min. The Bioscan FC-4000 dual BGO coincidence detector
was used for the detection of radioactive compounds. Specific
activity was measured using this HPLC with a cold mass standard
curve of known concentrations of [19F]FBEM at 230 nm.
The quality control of the coupling of [18F]FBEM to glutathione
was performed using radio-liquid chromatography–mass
spectrometry (LCMS) measurements with a Shimadzu 2010
LCMS system equipped with a 5 μL injection loop, a SPD-20A
J. Label Compd. Radiopharm 2014, 57 115–120
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org

U. Ackermann et al.
Table 1. Preparation of the modified FlexLab module for the production of [18F]FBEM and coupling to glutathione
Entry
Position
Reagents or materials
Quantities
1
2
Vial 1
Vial 2
NBu4HCO3
4-(ethoxycarbonylphenyl)
500 μL (50 μmol) plus 300 μL of acetonitrile
5 mg (20 μmol) in 300 μL of dry DMSO
trimethylammonium triflate
1 M tetrabutylammonium hydroxide
3
Vial 3
40 μL (40 μmol) in methanol plus 4 mL of
dry acetonitrile
4
5
6
7
8
9
10
11
12
Vial 4
Vial 5
Vial 6
Vial 7
Vial 8
Vial 9
Vial 10
Water
Water
1.8% acetic acid
DCC
Empty
Acetonitrile
Water
Empty
4.5 mL
3 mL
10 mL
40 mg (193 μmol) in 0.5 mL of acetonitrile
1 mL
10 mL
Vials 11–14
Vial 15
Glutathione
1 mg (3.25 μmol) in 1 mL of PBS buffer
pH 7.4
13
14
Vials 16–19
Intermediate reaction vial
(loop vial 1)
Empty
N-(2-aminoethyl)maleimide
trifluoroacetate
7 mg (27.5 μmol) as dry solid
15
16
17
18
19
Reactor 1
HPLC vial 1
Cartridge A
Cartridge B
Acetonitrile (dry)
Water
Strata-X SPE cartridge
Strata-X SPE cartridge
Chromafix PS-HCO3
1 mL
60 mL
1
1
1
F-18 trapping cartridge
to instal a heating mantle around this vial, which was capable of
heating the vial contents to 56 °C.
Results and discussion
The aim of this project was to develop a fully automated process
Purification of [18F]FBEM was achieved using semi-preparative
from the production of [18F]FBEM to the conjugation of free thiol reversed phase HPLC with a gradient elution technique. This
containing peptides or antibodies using a single synthesis module. achieved good separation of [18F]FBEM from other radioactive
In order to achieve this aim, we used the dual reactor iPHASE compounds.
FlexLab module and performed the coupling of [18F]FBEM to
In the semi-preparative HPLC chromatogram of the crude
glutathione in reactor vial 2. The synthesis of [18F]FBEM was reaction mixture (Figure 4, top panel), [18F]FBEM was observed at
achieved by producing 4-[18F]FB in reactor 1 followed by the 12.5min. A second radioactive peak was also observed at
coupling to N-(2-aminoethyl) maleimide in the intermediate vial 1. 22.5min. When isolated and analysed via LCMS, the characteristic
In order to achieve optimum yields of [18F]FBEM, it was necessary ions associated with this peak were consistent with the ‘activated’
Figure 4. Top: Semi-preparative HPLC chromatogram of [18F]FBEM purification: 254 nm UV trace (left), radioactivity (right); bottom: quality control of [18F]FBEM after
purification: 230 nm UV trace (left), radioactivity (right). This figure is available in colour online at wileyonlinelibrary.com/journal/jlcr
www.jlcr.org
Copyright © 2014 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 115–120

U. Ackermann et al.
mV(x100)
AD2
5.0
4.0
3.0
2.0
1.0
0.0
0.0
mV(x10)
Detector A:254nm
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
min
2.50
2.25
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
min
(x1,000,000)
570.0000 (100.00)
4.00
3.75
3.50
3.25
3.00
2.75
2.50
2.25
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
Figure 5. Radio-LCMS trace of [18F]FBEM-glutathione: radioactivity (left), UV (centre) and m/z = 570 (right).
intermediate [18F]4 where 4-[18F]FB is coupled to DCC. Although
After semi-preparative HPLC purification, [18F]FBEM was collected
the chromatogram suggests that the conversion of 4-[18F]FB to in HPLC vial 1, trapped on a Strata-X cartridge, washed with water
[18F]FBEM is around 50%, analysis of the intermediate reaction vial and eluted into reactor 2 with 1 mL of acetonitrile for subsequent
content revealed that only 30% of [18F]4 is injected into the HPLC drying and coupling to glutathione. [18F]FBEM was obtained in
column, whereas no [18F]FBEM could be detected in the decay-corrected radiochemical yields of 1.185 0.168 GBq (15–20%;
intermediate reaction vial.
n= 10; 0.75 0.106 GBq non-decay corrected) with a specific activity
The presence of [18F]4 also suggests that higher yields of [18F] of 57 10 GBq/μmol. Radiochemical purity was 97 1%, and the
FBEM could be achieved if this compound was fully reacted with synthesis time including HPLC purification and reformulation was
the N-(2-aminoethyl) maleimide precursor. We hypothesised that 70 min. Figure 4 (bottom panel) shows the quality control HPLC
this could be accomplished by adding an organic base to the chromatogram of [18F]FBEM.
reaction mixture. However, when even small amounts of bases
Coupling of [18F]FBEM to glutathione
such as N,N-dimethylamino pyridine or DIEA were used, we
obtained complex reaction mixtures where [18F]FBEM yields
were low and [18F]FBEM could not be separated from other The coupling of [18F]FBEM to glutathione was achieved by
radioactive compounds.
evaporating the acetonitrile from the [18F]FBEM at 40 °C under
J. Label Compd. Radiopharm 2014, 57 115–120
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org

U. Ackermann et al.
vacuum with argon flow. The coupling to glutathione was our laboratory, this method is now routinely used to label
performed in PBS buffer pH 7.4 at 40 °C for 10 min. After transfer suitably modified single chain antibodies.
to the product vial, the crude reaction mixture was analysed
using radio-LCMS to confirm product identity and measure
Acknowledgement
labelling yields. The yield for the coupling of [18F]FBEM to
glutathione was found to be 99 0.5% (n = 3). In this process,
evaporation of [18F]FBEM to dryness is a key step to obtaining
high yields. Otherwise, the coupling yield between [18F]FBEM
and glutathione is only 50%. Figure 5 shows the LCMS profile
of the conjugation product [18F]5. Although [18F]5 represents a
mixture of diastereomers, there was no resolution of those
compounds using the HPLC conditions employed.
This work was supported by NHMRC project grants 1011418 and
1029249.
Conflict of Interest
The authors reported the following conflict of interest: Henri
Tochon-Danguy and Stan Poniger are directors of iPhase
technologies..
These experiments have shown that a fully automated
production of [18F]FBEM and subsequent labelling of thiol
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up-scaled, and we are typically producing 3.7 GBq of [18F]FBEM
(non-decay corrected yields of radioactivity) from 33.3 GBq of
[18F]fluoride.
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The fully automated synthesis of [18F]FBEM and its coupling to
thiols was achieved in a single hot cell using the modified iPHASE
FlexLab module. [18F]FBEM was synthesised in 1.185 0.168 GBq
(15–20%; n = 10; 0.75 0.106 GBq non-decay corrected)
radiochemical yield with a specific activity of 57 10 GBq/μmol.
Radiochemical purity was 97 1%, and the synthesis time
including HPLC purification and reformulation was 70 min.
Quantitative coupling to glutathione was achieved using dried
[18F]FBEM after 10 min reaction time in PBS buffer pH 7.4. The
most significant advantage of this protocol is that radiation
exposure to the radiochemist is minimised because no manual
handling of [18F]FBEM is required. Up-scaling of this process to
produce 3.7 GBq of [18F]FBEM (non-decay corrected yields of
radioactivity) from 33.3 GBq of [18F]fluoride is possible and, in
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J. Label Compd. Radiopharm 2014, 57 115–120