Highly efficient solid phase supported radiosynthesis of [11C]PiB using tC18 cartridge as a “3-in-1” production entity

Article abstract of DOI:10.1002/jlcr.3569

Pittsburgh compound B ([¹¹C]PiB) is the gold standard positron emission tomography (PET) tracer for the in vivo imaging of amyloid plaques. Currently, it is synthesized by either solution chemistry or using a “dry loop” approach followed by HPLC purification within 30?minutes starting from [¹¹C]CO₂. Here, we report a novel, highly efficient solid phase supported carbon-11 radiolabeling procedure using commercially available disposable tC18 cartridge as a “3-in-1” entity: reactor, purifier, and solvent replacement system. [¹¹C]PiB is synthesized by passing gaseous [¹¹C]CH₃OTf through a tC18 cartridge preloaded with a solution of precursor. Successive elution with aqueous ethanol solutions allows for nearly quantitative separation of the reaction mixture to provide chemically and radiochemically pure PET tracer. [¹¹C]PiB suitable for human injection is produced within 10?minutes starting from [¹¹C]CH₃OTf (20?min from [¹¹C]CO₂) in 22% isolated yield not corrected for decay and molar activity of 190?GBq/μmol using 0.2?mg of precursor. This technique reduces the amount of precursor and other supplies, avoids use of preparative HPLC and toxic solvents, and decreases the time between consecutive production batches. Solid phase supported technique can facilitate [¹¹C]PiB production compliant with Good Manufacturing Practice (GMP) and improve synthesis reliability.

Full text of DOI:10.1002/jlcr.3569

Highly efficient solid phase-supported radiosynthesis
11
of [ C]PiB using tC18 cartridge as a “3-in-1”
production entity
11
Abbreviated title: Solid phase-supported [ C]PiB radiosynthesis
1
1
1
1
1
Mehdi Boudjemeline , Robert Hopewell , Pierre-Luc Rochon , Dean Jolly , Iness Hammami ,
2
,3
1,2,*
Sylvia Villeneuve , Alexey Kostikov
1
Montreal Neurological Institute, McGill University, 3801 University street, Montreal, QC,
Canada, H3A2B4
2
Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada.
3
Department of Psychiatry, McGill University, Montreal, Quebec, Canada.
*
Corresponding author. alexey.kostikov@mcgill.ca Tel. 1-514-398-1503 Fax 1-514-398-8948
Keywords: Labelling methods, carbon-11, Positron Emission Tomography, Amyloid imaging,
[11
C]PiB, Carbon-11 methylation, Solid phase supported synthesis, Solid phase extraction
cartridge, Automation
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/jlcr.3569
This article is protected by copyright. All rights reserved.

Abstract.
11
Pittsburgh compound B ([ C]PiB) is the gold standard PET tracer for the in vivo imaging of
amyloid plaques. Currently it is synthesized by either solution chemistry or using a “dry loop”
1
1
approach followed by HPLC purification within 30 minutes starting from [ C]CO . Here we
2
report a novel, highly efficient solid phase supported carbon-11 radiolabeling procedure using
commercially available disposable tC18 cartridge as a “3-in-1” entity: reactor, purifier and
11
11
solvent replacement system. [ C]PiB is synthesized by passing gaseous [ C]CH OTf through
3
a tC18 cartridge preloaded with a solution of precursor. Successive elution with aqueous
ethanol solutions allows for nearly quantitative separation of the reaction mixture to provide
1
1
chemically and radiochemically pure PET tracer. [ C]PiB suitable for human injection is
1
1
11
produced within 10 minutes starting from [ C]CH
3
OTf (20 minutes from [ C]CO ) in 22%
2
isolated yield not corrected for decay and molar activity of 190 GBq/µmol using 0.2 mg of
precursor. This technique reduces the amount of precursor and other supplies, avoids use of
preparative high performance liquid chromatography and toxic solvents and decreases the time
between consecutive production batches. Solid phase supported technique can facilitate
[11
reliability.
. Introduction.
Amyloid beta protein (Aβ) accumulates in the brains of patients with Alzheimer’s
C]PiB production compliant with Good Manufacturing Practice and improve synthesis
1
disease (AD) [1] and the resulting Aβ plaques are currently one of the main targets for AD
diagnostic and preventive therapies [2,3,4]. The radiochemical synthesis of carbon-11 labeled
thioflavin-T derivatives [5] resulted in development of PET radiotracer Pittsburgh compound
1
1
B ([ C]PiB) for Aβ imaging in 2003 [6]. The results of the first trials in humans were published
shortly after and revolutionized the field of AD diagnostics [7]. For the first time, the Aβ
plaques were visualized in the brains of living AD patients, and the correlation between
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amyloid deposition and radioactivity accumulation patterns was later validated by the autopsy
1
1
in post-mortem studies of typical AD brains [8,9]. By highlighting Aβ deposition, [ C]PiB
provides earlier and more specific diagnosis in different stages of AD as well as distinguishes
AD from other types of non-Aβ forms of dementia such as the frontotemporal lobar
degeneration (FTLD) [10]. Amyloid PET for clinical diagnostics has recently been approved
by FDA in the US to rule out AD based on the absence of Aβ in the brain, and other countries
are expected to follow shortly. Despite recent emergence of various fluorine-18 labeled
amyloid tracers, such as florbetapir [11], florbetaben [12], flutemetamol [13], NAV4694 [14]
1
1
and others, [ C]PiB remains the most studied tracer for Aβ PET imaging due to high affinity
for Aβ plaques (K = 1.4 nM) [6], fast uptake and low non-specific binding.
d
11
11
The original radiosynthetic procedure of [ C]PiB ([ C]6-OH-BTA-1) involved N-
methylation of the O-methoxymethyl (MOM) protected compound 6-OMOM-BTA-0 using
1
1
11
C-methyl iodide ([ C]CH
3
I) followed by the deprotection of the MOM group with
hydrochloric acid (Figure 1, A) [6]. Subsequently, a one-step synthesis from the unprotected
11
compound 6-OH-BTA-0 (0.4 mg) was developed by Wilson [15] using C-methyl triflate
1
1
(
[ C]CH
3
OTf) either in methylethylketone solution or by elegant captive solvent “loop”
method [16,17]. Consequent purification by means of high performance liquid chromatography
11
(
HPLC), reformulation and sterile filtration affords [ C]PiB for clinical imaging within 30
minutes after end of bombardment. Currently, most PET centres use between 0.8-1 mg of
precursor methods utilizing “loop”, solution or on-column [18] approach followed by semi-
preparative HPLC purification and reformulation.
A recent trend in PET radiochemistry is the development of cassette-based kits that
allow for production of various fluorine-18 labeled tracers with reduced technical effort from
the laboratory personnel and minimal maintenance of the equipment between syntheses. The
first kit-based synthesis was introduced for the radiolabeling of the most widely used PET
This article is protected by copyright. All rights reserved.

1
8
tracer [ F]FDG; lately, similar kits have been developed for other tracers including FMISO,
FMC, and others. These kits are commercially available from ABX advanced biochemical
compounds [19], Huayi Isotopes [20] and other vendors for most of the existing automated
modules. Outside of the physical half-life limitations (T1/2 = 20.3 min), one of the major reasons
carbon-11 remains a less popular isotope for PET radiochemistry than fluorine-18 is a lack of
similar GMP-grade production kits that would allow for fast and reliable radiolabeling.
1
8
Subsequently, while many F-tracers have found application in clinical trials and have been
11
successfully commercialized, C-tracers are mostly used in highly specialized research PET
1
1
centres worldwide. Development of similar kits for C-labeled tracers would significantly
simplify their production and eliminate the need for cleaning and drying of the module between
consecutive batches. Finally, fully disposable kits would prolong the intervals between
preventive maintenance of the production modules, improve synthesis reliability and allow
avoiding sterilization of the reusable equipment.
Of special interest for the kit development are the reactions that can proceed at room
18
temperature on a disposable cartridge, for example [ F]fluoromethylation of
1
8
dimethylaminoethanol in production of [ F]fluoromethylcholine, or hydrolysis of
[18
F]fluorodeoxyglucose tetraacetate in the [ F]FDG synthesis. Although sorbent-supported
18
1
1
radiolabeling of several C-tracers have been reported, including pioneering work by the
University of Michigan PET center [21,22], most of the radiosyntheses rely on time and labor
consuming HPLC purification and reformulation of the tracers. As part of our latest endeavours
in simplifying the labeling with carbon-11 [23] and fluorine-18 PET isotopes [24], we wish to
1
1
report a new highly efficient solid phase-supported radiosynthesis of [ C]PiB by
[11
C]methylation of the 6-OH-BTA-0 on a disposable cartridge used as “3-in-1” entity for
1
1
reaction, purification and formulation. Briefly, gaseous [ C]CH OTf is passed through a
3
disposable cartridge preloaded with 6-OH-BTA-0 precursor and nearly quantitative separation
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11
of synthesized [ C]PiB from starting material and radioactive impurities is achieved using a
biocompatible aqueous ethanol as an eluent. Using this very reliable technique, we developed
11
a method for [ C]PiB production using automated module and disposable cassette kits. The
resulting tracer meets all specifications to be compliant with requirements of multicenter
Dominantly Inherited Alzheimer Network Trials Unit (DIAN-TU) [25] clinical trials:
radiochemical and radionuclidic identity; radiochemical purity > 95%; chemical purity –
amount of residual precursor or other UV impurities < 1.3 μg; pH 4-8; ethanol content < 10%,
sterile and endotoxin free.
2
. Experimental.
2
.1. Materials and general procedures
All radiochemistry procedures were performed at the PET radiochemistry facility of the
Montreal Neurological Institute (MNI) as described before [26]. Briefly, the isotope was
11
14
11
obtained in the form of [ C]CO
2
via N(p,α) C nuclear reaction by irradiation of N
2
/O
2
(99.5:0.5) with 18 MeV protons in the gas target of the cyclotron (Cyclone® 18/9 IBA,
1
1
11
Louvain-La-Neuve, Belgium). The resulting [ C]CO
2
was converted to [ C]CH
3
OTf using a
1
1
commercially available Synthra module. The [ C]CH
3
OTf output line was directly attached to
1
1
the manifold secured on the Scintomics GRP automated module for C-methylation.
Compounds 6-OH-BTA-0 (precursor) and 6-OH-BTA-1 (cold standard) were
purchased from ABX advanced biochemical compounds (Cat No 5101 and 5140, respectively).
Disposable cartridges Oasis HLB (Cat No. 186000132), Sep-Pak tC18 Plus (Cat No.
WAT036810) and Sep-Pak C18 Plus (Cat No. WAT020515) were provided by Waters®.
Disposable polycarbonate and solvent-resistant manifolds were purchased from Scintomics
(Cat. No. ACC-101 and ACC-201, respectively). HPLC solvents were purchased from Fisher
and inorganic salts for buffer preparation from Sigma-Aldrich. Buffer solutions were prepared
according to Sigma-Aldrich buffer reference center [27] and their pH verified with pH strips.
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Quality control procedures were performed according to the validated SOPs used at the PET
radiochemistry facility of the MNI for routine production of radiotracers intended for use in
humans. Specifically, Agilent 1200 HPLC instrument equipped with a reversed phase column
(MZ Analytical PerfectSil 120 C8 5μm, 100 x 4.0 mm; 40/60 acetonitrile/water at 0.7 mL/min),
UV (350 nm) and radioactivity detector (Raytest Gaby) was used to confirm radiochemical
identity and purity as well as chemical purity and molar activity. Perkin Elmer Clarus 480 gas
chromatograph (GC) equipped with Restek MTX-Wax column (30 m, 0.53 mm) was used for
detection and quantification of the residual solvents.
2
.2. Separation of 6-OH-BTA-0 and 6-OH-BTA-1.
Our first goal was to find the optimal conditions with regards to solid phase extraction
SPE) sorbent, pH of the aqueous solution and ethanol content for efficient separation of non-
(
radioactive 6-OH-BTA-0 and 6-OH-BTA-1 on a commercially available disposable cartridge.
In a typical experiment, premixed aqueous ethanol (ca. 10%) solution containing both
compounds was passed through the preconditioned cartridge followed by sequential elution
with aqueous solutions of increasing ethanol concentration and all eluate fractions were
analyzed by HPLC.
Among three tested cartridges commercially available by Waters®, trifunctional tC18
sorbent provided the best results for separation of 6-OH-BTA-0 and 6-OH-BTA-1 in cold
simulation experiment and were later translated to radiolabeling procedure. Other cartridges
(Oasis HLB and Sep-Pak C18) performed poorly and were not tested in radiolabeling
experiments (data not shown). Excellent separation of the compounds in both “cold”
11
simulations and later in radiosynthesis of [ C]PiB was achieved using 0.2M acetate buffer at
pH 3.7, prepared by mixing 0.2M sodium acetate solution (50 mL) with 0.2M acetic acid (450
mL). We rationalized that these aromatic amines have sharper elution profiles and therefore
better separation in moderately acidic conditions where they exist predominantly in protonated
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forms. Thus, a 20% aqueous ethanol solution in 0.2M acetic buffer at pH 3.7 (80 mL) allowed
for almost quantitative washout of the 6-OH-BTA-0 from tC18 cartridge, while the 6-OH-
BTA-1 remained trapped. The latter was eluted by increasing the concentration of ethanol to
4
0% in 20 mL total volume. These conditions were later slightly modified for production of
1
1
radiochemically and chemically pure [ C]PiB as described below.
2
.3. General radiolabelling procedure
In a typical radiolabeling experiment, preconditioned (10 mL water followed by 5 mL
acetone) and dried by the stream of nitrogen (50 mL/min, 1 min) tC18 cartridge was slowly
loaded with a 2 mg/mL solution of 6-OH-BTA-0 in acetone (50, 100 or 150 μL).
[11
C]methylation was performed by passing gaseous [ C]CH
11
OTf directly through the loaded
3
1
1
cartridge secured on a manifold (Fig 2A). Once all the [ C]CH OTf was trapped on the
3
cartridge as monitored by the radioactivity detector mounted behind the cartridge holder, it was
allowed to react with the precursor for 2-3 min at room temperature and then successively
eluted with aqueous (pH 3.7) ethanol solutions of increasing concentrations: first, 12.5% EtOH
(92 mL), then 15% EtOH (55 mL) into the waste bottle (Fig 2B, 2C); and finally with 50%
EtOH (2.5 mL) followed by sterile phosphate buffer (10 mL) into the final vial (Fig 2D) .
3
. Results and discussion.
The solid-state radiochemical 11C-alkylations in general require lower precursor
amount than the comparable reactions in solution as previously reported by Wilson [15-17] and
more recently Scott [28]. Our first goal was to investigate if even lower amounts of precursor
(0.1, 0.2 and 0.3 mg) would provide high radiochemical yield of the product using on-cartridge
technique. The results of the radiochemical yield optimization experiments are summarized in
Table 1.
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To our delight, as little as 0.2 mg of precursor per synthesis provides excellent
11
radiochemical yields of [ C]PiB in SP-supported procedure (Table 1, entry 2). Notably,
reducing the amount even further to 0.1 mg (entry 1) still provides a reasonable quantity of the
1
1
tracer, although the final amount of [ C]PiB is lower and the yield is less reliable. Increasing
the amount to 0.3 mg (entry 3) further improves the yield, while still providing a reasonable
chemical purity of the final product despite a higher amount of the precursor in the final
product. However, we observed that the acetone used to solubilize the precursor can affect the
stability of the polycarbonate manifolds used in the synthesis, which are particularly vulnerable
to this solvent. Thus, in two experiments using 150 μL of the precursor solution (0.3 mg), the
manifold was destroyed, prompting us to manually intervene in the synthesis to recover the
labeled product. This problem can be alleviated by using higher quality solvent-resistant
manifolds (See Supporting Information). An optimal amount of precursor will depend on
particular needs of the PET center in terms of yield of radiotracer and other considerations. Our
attempt to repeat the tC18-suported synthesis using ethanol (Table 1, entry 4) only yielded 3%
[11
C]PiB (from [ C]CH
11
3
OTf) with less than 70% RCP, deeming this solvent unsuitable for the
proposed alkylation technique. Further investigation of ethanol compatibility with solid phase-
1
1
supported C-methylation might be required.
1
1
Table 1. Optimization of precursor amount, C-methylating agent and solvent.
The most challenging part of the described technique was to find conditions for
quantitative separation of the reaction mixture on a relatively short SPE cartridge. After
thorough optimization of the ethanol content and volumes of washing solutions, we found the
optimal conditions: first 12.5% EtOH (92 mL) is used to elute the radioactive impurities and
majority of the 6-OH-BTA-0, then 15% EtOH (55 mL) washes out the residual precursor and
11
a negligible amount of the product (~5%). Finally, the elution of desired [ C]PiB, pure both
radiochemically (> 95% RCP) and chemically (<1.3 μg of 6-OH-BTA-0) is achieved at the
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5
0% ethanol concentration. Results of purification optimization can be found in supplementary
information. Typical chromatograms and radioactivity distribution between fractions are
represented on Figure 3
11
After the procedure optimisations described above, [ C]PiB is synthesised and
1
1
prepared for direct injection from a starting activity of [ C]CO
2
ranging from 13 to 37.5 GBq
11
as follows. [ C]CH
3
OTf is passed through the preconditioned, dried and loaded with 0.2 mg
of 6-OH-BTA-0 tC18 Plus cartridge, allowed to react with precursor for 2-3 min and the
reaction mixture is efficiently separated using successive elution with aqueous ethanol
solutions of increasing concentrations. From the end of bombardment to the end of the
synthesis, the whole procedure is completed within 20 minutes. The final tracer synthesized
under optimized conditions meets all the criteria specified in the certificate of analysis (CofA)
for use in humans under DIAN-TU clinical trial protocol: radiochemical purity is consistently
above 95%, the 6-OH-BTA-0 content is below 1.3 μg per 10 mL dose and the ethanol content
11
is below 10%. [ C]PiB is stable within 1 hour in the final solution, sterile and free from
pyrogens and bacterial endotoxins. The results of full batch successful validation runs from
[11
C]CH
OTf generated by dry chemistry method are summarized in Table 2.
3
11
Table 2. Summary of the [ C]PiB production validation batches under optimized conditions.
11
In an attempt to develop a fully disposable all-in-one kit for production of [ C]PiB
11
from [ C]CO released from the cyclotron target, we combined this technique with our
2
11
11
previously reported “[ C]kits” for cassette-based conversion of [ C]CO
2
into
[11
C]CH
I/[ C]CH
11
OTf using wet method. Despite a higher radiochemical yield, relatively
3
3
low molar activity leads to formation of the unidentified radiochemical impurity co-eluting
11
with [ C]PiB, which we attributed to a double methylation product. Although selective elution
11
of radiochemically pure [ C]PiB was achieved (27 mL of 30% aqueous ethanol), low molar
activity and high ethanol content would make tracer batches produced by all-in-one kits
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unsuitable for clinical applications. We are currently working on improving the molar activity
11
11
of [ C]CH
. Conclusion
We have developed a solid phase-supported technique to produce a clinically relevant [ C]PiB
3
OTf produced with “[ C]kits” by wet method.
4
1
1
1
1
tracer from [ C]CH OTf in an automated mode and avoiding HPLC purification. Our method
3
takes advantage of the disposable tC18 cartridges as “3-in-1” entity for production, purification
and formulation of this tracer. In our work, we optimized the RCY with regards to precursor
amount and reaction solvent; and purification with regards to cartridge type, pH of the buffer,
1
1
ethanol content and volume of the eluent. This synthesis from [ C]CH OTf is completed
3
11
within 10 minutes and affords [ C]PiB in high yield and molar activity as well as excellent
radiochemical and chemical purity as an injectable solution for clinical or preclinical imaging.
The described methodology allows for reduction of chemicals (including precursor and HPLC
solvents) used in the synthesis, shortens the synthesis time and reduces the technical
involvement of the personnel. Most importantly, high reliability is achieved by avoiding
HPLC-related failures, while use of biocompatible ethanol instead of most commonly used
acetonitrile for tracer purification simplifies GMP-compliant quality control procedures.
1
1
Furthermore, it opens new avenues to develop a fully automated disposable kits for [ C]PiB
production. Development of similar highly efficient solid phase-supported syntheses of other
1
1
clinically relevant tracers produced by C-methylation is currently underway in our lab.
Acknowledgments
We thank McGill University, Montreal Neurological Institute and McConnell Brain Imaging
Centre for support of this work. We also thank Dr. Gassan Massarweh for access to
radioisotopes and radiochemistry facility, Mrs. Monica Lacatus-Samoila for help with quality
control and Mr. Vadim Bernard-Gauthier for proof-reading the manuscript and valuable
suggestions.
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1
1
Table 1. Optimization of precursor amount, C-methylating agent and solvent.
1
1
Entry
Precursor amount
0.1 mg
[ C]CH
3
X
n
3
Solvent
acetone
acetone
acetone
ethanol
RCY*
18.1±3.8%
22.0±3.1%
32.1±3.7%
<3%
1
1
1
2
3
4
[ C]CH
3
3
3
3
OTf
OTf
OTf
OTf
1
1
0.2 mg
[ C]CH
11
3
1
1
0.3 mg
[ C]CH
1
1
0.2 mg
[ C]CH
1
OTf
1
1
*
Not corrected for decay yield starting from [ C]CH
3
11
Table 2. Summary of the [ C]PiB production validation batches under optimized conditions.
Batch
PiB161025
9.21
PiB161027
11.25
2.37
PiB161110
7.84
PiB161118
6.44
[11
C]MeOTf, GBq
Amount, GBq
RCY, %*
2.26
2.11
1.41
24.5
21.1
26.9
21.8
RCP, %
98.0
97.2
97.8
99.2
Molar activity,
GBq/µmol
1
54.6
322.6
121.1
162.1
Residual precursor, μg
pH
0.32
5
0.55
0.58
0.87
5
8.8
5
7.7
5
8.1
EtOH content, %
Acetone content, ppm
BET test
9.4
33
38
46
33
N/A
N/A
<10 EU/mL
No Growth
<10 EU/mL
No Growth
<10 EU/mL
No Growth
Sterility test
11
*
From [ C]MeOTf, not corrected for decay
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Figures
1
1
11
Figure 1. (A) The original radiosynthetic procedure of [ C]PiB ([ C]6-OH-BTA-1)
1
1
involved N-methylation of the 6-OMOM-BTA-0 using [ C]CH
3
I and (B) the one-step
11
11
radiosynthesis of [ C]PiB from the unprotected 6-OH-BTA-0 using [ C]CH
3
OTf.
1
1
Figure 2. Step-by-step synthesis and purification of [ C]PiIB on tC18 cartridge: A) gaseous
11
[
C]CH
3
OTf is passed through the cartridge loaded with 6-OH-BTA-0; B) aqueous ethanol
(
18.5 mL) is withdrawn into the 20 mL dispenser syringe (100 mL/min); C) the impurities are
11
washed out into a waste bottle (50 mL/min); D) [ C]PiB is eluted into the final vial through a
sterile filter.
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1
1
[
C]PiB
Radioactive
impurities
PiB
6
-OH-BTA-0
Elution
Wash 2
Wash 1
Elution
Wash 2
Wash 1
UV detection
Radioactivity detection
[11
C]CH
X*
[11C]PiB
6-OH-BTA-0
3
Solutio
n
V,
mL
MBq**
MBq
**
%**
%**
%****
μg
*
*
~180*
***
<
0.0
1
Wash 1 92.0 62.3 4070
<4
~90
*
Wash 2 55.0
Elution 12.5
0.2
0.3
15
18
2.4
163
~10
0.3
~20*****
0.58
34.8
2276
1
1
11
11
*
[ C]CH
3
I, [ C]CH
3
OTf and [ C]CH OH; **Quantity expressed as a percentage of
3
the total activity corrected for decay; ***Decay-corrected values; ****Percentage of the total
mass of 6-OH-BTA-0 eluted from the cartridge; *****Absolute values outside of the
calibration curve.
Figure 3. Elution profile with aqueous (pH 3.7) ethanol solution of increasing
concentrations. Wash 1 (92 mL of 12.5% EtOH), Wash 2 (55 mL of 15% EtOH) and Elution
(
2.5 mL of 50% EtOH) were sequentially passed through the cartridge to elute the radioactive
11
impurities, the residual 6-OH-BTA-0 and finally the [ C]PiB, respectively.
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