FASTlab Radiosynthesis of the 18F-labelled HER2-binding Affibody molecule [18F]GE-226

Article abstract of DOI:10.1002/jlcr.3789

An ¹⁸F-labelled human epidermal growth factor receptor (HER2) receptor binding radiotracer is a potential tool to non-invasively identify HER2 positive tumour lesions in subjects with recurrent metastatic breast cancer. Having explored the manu

Full text of DOI:10.1002/jlcr.3789

Iveson Peter (Orcid ID: 0000-0002-8573-8578)
FASTlab Radiosynthesis of the 18F-labelled HER2-binding Affibody Molecule [18F]GE-
226
Peter B. Iveson,1 Matthias Glaser,1,2 Bard Indrevoll,3 Jonathan Shales,1 Dimitrios Mantzilas,3
Lone Omtvedt,3 Sajinder K. Luthra,1 Duncan Hiscock,1, Julian Grigg1
1 GE Healthcare, Pharmaceutical Diagnostics, Pollards Wood, Nightingales Lane, Chalfont
St Giles, Buckinghamshire, HP8 4SP, UK.
2 current address: University College London, Institute of Nuclear Medicine –
Radiochemistry, Kathleen Lonsdale Building, 5 Gower Place, London WC1E 6BS, U.K.
3 GE Healthcare, Pharmaceutical Diagnostics, Nydalen, N-0401, Oslo, NORWAY
Abstract
An 18F-labelled HER2 receptor binding radiotracer is a potential tool to non-invasively
identify HER2 positive tumour lesions in subjects with recurrent metastatic breast cancer.
Having explored the manual radiochemistry to conjugate the Affibody molecule ZHER2:2891
with [18F]4-fluorobenzaldehyde, we have developed and optimized a full protocol for the
automated GE FASTlabTM synthesiser. Our chemometric model predicted the best
radiochemical purity for a short conjugation time (2.8 min), a low temperature (65oC), and a
medium Affibody molecule precursor amount (5.5 mg). Under these optimised conditions
[18F]GE-226 was produced after solid-phase extraction purification with activity yield of 30%
± 7 (n = 18) and a radiochemical purity of 94% ± 2 (n = 18). The synthesis and purification
was complete after 43 minutes and provided apparent molar activities of 12-30 GBq/μmol (n
= 12) at the end of synthesis.
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Introduction
Approximately 20-25 % of breast cancers express the human epidermal growth factor
receptor (HER2) which is associated with malignant disease.1 Currently, the standard practice
to determine HER2 status in tumours is by tissue biopsy. This technique is impractical to
assess multiple metastatic lesions and is generally restricted to a single lesion for assessment,
therefore non-invasive in vivo imaging of the HER2 biomarker could improve clinical
management of patients with recurrent metastatic disease. Our work is focussed around the
fluorine-18 radiolabelling of the ZHER2:2891 HER2-binding Affibody molecule.2 This versatile
class of high-affinity binder has already been subjected to 18F conjugation by other
laboratories. Successful studies have been carried out using the Affibody molecule labelled
with fluorine-18 using N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide (18F-FBEM) for the
maleimide-thiol based protocol,3-10 4-[18F]fluorobenzaldehyde ([18F]FBA) for
aminooxy/aldehyde coupling,11, 12 or the [18F]AlF/NOTA approach.13, 14 We have evaluated
various approaches to conjugate ZHER2:2891 with fluorine-18.15 The study concluded that
[18F]FBA conjugation to a C-terminal Cys-maleimide-aminooxy function was the preferred
method both with regard to in vivo data and prospective automation. We have consequently
translated the manual protocol to the GE FASTlabTM synthesiser. We report here the
development of an automated and robust synthesis of [18F]GE-226 for the commercial
FASTlab synthesiser with single-use disposable cassettes and on-cassette solid phase
extraction (SPE) purification.
Experimental
Materials and Methods
The aminoxy-modified Affibody molecule labelling precursor was prepared as described
previously.15 No-carrier-added aqueous [18F]fluoride was obtained from the 18O(p,n)18F
reaction (PETtrace cyclotron, GE Medical Systems) by the irradiation of an isotopically
enriched [18O]H2O target using a 16.4 MeV proton beam. The Sep-Pak AccellTM Plus QMA
carbonate Plus Light, Ion exchange Oasis® MCX Light and Sep-Pak® Plus Long tC2
cartridges were purchased from Waters. Analytical HPLC was carried out using a Gilson 322
pump with a UV/ViS 156 detector and a gamma detector (Bioscan Flow-count, PMT probe:
Thermo MS3083). Radioactivity measurements were performed using a Capintec CRC-15R
ion chamber. Radiochemical yields are reported as non-decay-corrected. The apparent molar
activities were estimated based on the starting amounts of peptide precursor.
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FASTlab synthesis of [18F]GE-226
All radiochemistry was performed on the FASTlab synthesiser with single-use cassettes. The
FASTlab sequence developed and optimised for the synthesis of [18F]GE-226 was
programmed in a step-by-step time-dependent sequence of events, e.g. syringe plunger
movement, nitrogen pressure, vacuum pressure and temperature regulation. A 4-
trimethylammonium benzaldehyde precursor (4-TMAB)16 was used to prepare [18F]FBA
which was then conjugated to the aminoxy functionalised ZHER2:2891 HER2-binding Affibody
molecule utilising aniline as a catalyst (Scheme 1). Optimisation of the conjugation reaction
between [18F]FBA and the aminoxy functionalised ZHER2:2891 HER2-binding Affibody
molecule was carried out by varying the amount of precursor (3.5 to 7.0 mg), reaction
temperature (60 to 80 °C), reaction time (2.8 to 20 min), amount of aniline catalyst (8.2 to 35
mg) and level of starting radioactivity (26 to 108 GBq) using a multivariate experimental
approach. Multivariate analysis was performed by Unscrambler (v. 9.8, Camo Inc). The
software was used to develop Design of Experiment (DoE) templates and the statistical
correlation between X- and Y-variables was determined by partial least squares regression
(PLS). Following the DoE approach allowed the number of experiments required to be
reduced as much as possible, whilst still having sufficient resolution to determine the design
space for the various responses and to make an assessment of how important the different
variables were in the values of the responses observed. In these initial experiments the
precursor was re-constituted manually to pH 2.7 in a citrate/phosphate buffer. Non-decay
corrected yields were determined by RP-18 reverse phase thin-layer chromatography (silica
gel 60 RP-18 on alumina sheets Merck KGaA) eluting with water/70% acetonitrile (labelled
Affibody molecule at origin).
Robustness studies
The Unscrambler software version 9.8 by Camo was used to establish suitable statistical
experimental designs and to build multiple linear regression models.
Starting activities of 42 to 71 GBq were used. Each batch of the [18F]GE-226 for robustness
testing was formulated in 25 mL phosphate buffered saline containing the radiostabiliser
sodium p-aminobenzoic acid (Na-pABA, 2.8 mg/mL). Radiochemical purity (RCP) and
chemical impurity values with UV detection at 280 nm were determined by HPLC
immediately after synthesis and approximately 3 hr post formulation according to the method
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shown in Table 1. Ethanol, acetonitrile and acetaldehyde amounts were determined using GC
while aniline was determined by both GC and HPLC.
Quality control
Details of the HPLC method developed for determination of radiochemical and chemical
purity of the radioactive samples are listed in Table 1. HPLC-UV/MS was used to identify
chemical impurities in the decayed samples, details of which are shown in Table 2. The
radiochemical purity was also determined by RP-18 reversed-phase thin-layer
chromatography as described above.
Results and Discussion
Conjugation optimisation
A detailed description of the FASTlab synthesis of [18F]GE-226 is shown in Figure 1. The
[18F]GE-226 FASTlab cassette is shown in Figure 2.
A series of experiments were carried out to determine the optimum conditions for the
conjugation of [18F]FBA and the aminoxy functionalised ZHER2:2891 HER2-binding Affibody
molecule. The amounts of precursor and aniline and the conjugation temperature and time
were varied. The unpurified conjugation reaction mixtures were analysed by thin layer
chromatography.
Multivariate modelling of the results indicated that both time and temperature had a negative
effect on RCP. A short time (2.8 min) and a relatively low temperature (65°C) produced the
highest RCP values see (Figure 3(c)). The [18F]FBA recovery variable in Figure 3 was the
proportion of the starting activity that was returned, as purified [18F]FBA, to the reactor for
the conjugation reaction with the precursor.
An amount of 5.5 mg of the aminoxy precursor was selected as the optimum precursor amount
from the experiments as this gave acceptable RCP values and a [18F]GE-226 yield which would
allow the purification of [18F]GE-226 using tC2 SPE cartridges. When 3.5 mg precursor was
used, the RCP obtained (67%) was too low to allow adequate purification of the larger amount
of unreacted [18F]FBA and other radioactive impurities. Use of 7 mg precursor gave the highest
RCP values but this amount was not chosen to minimise the amount of precursor used.
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Purification
On-cassette SPE purification was desired as it is simpler, faster and potentially more
reproducible than purification by HPLC. A SPE method was therefore developed to purify
[18F]GE-226 using reverse phase tC2 SPE cartridges integrated on the cassette to remove
radioactive impurities such as unreacted [18F]FBA, [18F]Fluoride and some non-Affibody
molecule related chemical impurities. Separation of Affibody molecule related impurities was
not possible by SPE purification as these impurities were too structurally similar. However,
[18F]FBA was purified prior to the conjugation reaction to minimise the levels of these
Affibody molecule related impurities and their level was acceptable.
The two tC2 SPE cartridges used to purify [18F]GE-226 were preconditioned with ethanol,
acid/water and water during the conjugation reaction. The conjugation reaction solution was
then diluted with water and transferred through the tC2 cartridges to initiate the SPE
purification step. Washes using a 19% ethanol / H3PO4 (v/v) solution were then used to remove
unconjugated [18F]FBA and other uncharacterised, radioactive impurities from the tC2
cartridges.
[18F]GE-226 was eluted from the tC2 cartridges after purification using ca. 2 mL of 80:20
(v/v) ethanol/water solution followed by a 4 mL water rinse. Elution into 19 mL phosphate
buffered saline containing Na-pABA as radiostabiliser resulted in a final ethanol
concentration of ca. 5% (v/v).
Robustness testing
A robustness study was performed to challenge the [18F]GE-226 synthesis and SPE purification
on FASTlab with factors that could impact [18F]GE-226 yield and purity anticipated at
radiopharmaceutical manufacturing sites for potential clinical studies (e.g. FASTlab
synthesizer hardware, environmental factors and variations for selected materials used).
Reagent variables related to the [18F]FBA synthesis were excluded as 26 previous experiments
had shown the [18F]FBA production to be sufficiently robust (30-47% yield, non-decay
corrected, mean = 43% ± 4). The robustness study was therefore restricted to the conjugation
reaction and the SPE purification with assessment of chemical purity, RCP and radiochemical
yield (RCY).
Temperature was included as a variable to cover the possible range in manufacturing facilities
of 18 to 34°C. The highest temperature (34°C) was included as the temperature within a hot
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cell tends to be increased by the electrical equipment it contains. The lowest temperature
limitation of the process came from the freezing temperature of the DMSO in the 4-
trimethylammonium benzaldehyde (4-TMAB) precursor solution.
Previous testing of the amount of precursor had shown that the minimum acceptable amount
of precursor was 5.3 mg. Therefore, the robustness study included the amount of precursor as
a variable so that a 5.3 to 6.3 mg range was covered by the experiments.
As the aqueous-ethanolic solutions used during the SPE purification and elution steps were
mixed using automated movements of syringe 2 of the FASTlab cassette and, because the
ethanol % of these solutions was expected to be critical for reliable purification and elution,
deliberate variations in the syringe S2 plunger position in mm were investigated to assess
impact on purity and RCY.
The robustness study also included the tC2 SPE cartridge fill mass as a variable as the mass of
fill material potentially affected the performance of the SPE cartridges during the SPE
purification step. Multivariate modelling focused on the three responses RCY, RCP and total
chemical impurities.
The main variation in RCY was due to the total tC2 SPE cartridge fill mass and syringe S2
position variables, both of which were important during the SPE purification stage. Higher
RCY was associated with higher total tC2 SPE cartridge fill mass and lower offset syringe S2
value. Variables of starting activity, precursor mass and hot cell temperature did not give
significant variation over the design space tested.
RCP values varied from ca. 90 to 95 % at approximately 3 hr post formulation. A time of 3 hr
post formulation was chosen as this would allow sufficient time for QC testing and transport
before clinical use. The main effects on RCP were found to be syringe S2 position and
temperature with activity as a negative correlation. Variables of precursor mass and tC2
cartridge mass did not give a significant variation in RCP over the design space tested. All RCP
values were acceptable over the design space (≥ 90%) except one of the experiments which
gave an RCP value of 89%, 3 hr post formulation. This experiment was carried out at a low hot
cell temperature (18.9°C).
It can be concluded from the modelling results that, with the SPE purification conditions used,
the synthesis should be carried out at hot cell temperatures between 20 to 34°C as RCP values
> 90% are predicted to be obtained post formulation between t = 0 and 3 hr in this temperature
range (see Figure 4).
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The main variation in chemical impurities was due to the purification stage and the main
variables were tC2 SPE fill mass and syringe S2 position. There was a positive correlation
between the level of chemical impurities with precursor mass. (Figure 5) Variables of starting
activity and hot cell temperature did not give a significant variation in the level of chemical
impurities over the design space tested.
The results from the robustness study were used to calculate the amounts of chemical impurities
which would be present in a 370 MBq injection at 3 hr post formulation. This was achieved by
determining the volume of formulated product required to provide a 370 MBq injection at 3 hr
and then calculating the amount of chemical impurities in this volume. The amounts of
chemical impurities, estimated by HPLC, varied from 87 to 226 µg in 25 mL of which 60 to
86% were Affibody molecule related. HPLC-UV/MS analysis of the decayed formulation
samples showed that the most abundant Affibody molecule related impurities were the
aminoxy precursor, GE-226, oxidised GE-226 and the Affibody molecule acetate, acetaldehyde
and formaldehyde conjugates.
A recent study in metastatic breast cancer patients with 68Ga-ABY025 (ZHER2:2891 Affibody
molecule) indicated that higher contrast images were obtained with 427 µg ZHER2:2891 Affibody
molecule at 2 hr post injection.17 Higher uptake in metastases in all locations and lower uptake
in normal hepatic tissue were observed with the higher Affibody molecule dose.17 It may
therefore be beneficial to add in additional ZHER2:2891 Affibody molecule to the [18F]GE-226
formulation to optimize imaging for clinical applications.
Measurement of pH values (5.9 to 6.1) showed that all formulations were suitable for injection
with respect to pH. The % ethanol values (4.6 to 5.9 % (v/v)) were acceptable for intravenous
use. All other values for chemical impurities and residual solvents (acetonitrile, acetaldehyde,
aniline and K222) were significantly below those which would have an impact on efficacy or
safety of the [18F]GE-226 injection (see Table 3).
Manufacturing Process
The non-decay corrected radiochemical yield for a number of productions is shown in Figure
6. Starting activities were 49-88 GBq. The average non-corrected radiochemical yield was
30% ± 7 (n = 18).
The variation in end of synthesis RCP values for several productions at different radioactive
concentrations (RAC) is shown in Figure 7. RCP values above the minimum required value
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of 90% were obtained in each production. Increasing the RAC from 400 to 1100 MBq/mL
had little or no effect on the RCP. These results show that starting activities up to ca. 88 GBq
could be routinely used to produce up to 30 GBq of [18F]GE-226 in a 25 mL formulation.
Quality control
Representative HPLC chromatograms used to determine the radiochemical and chemical
purity of the formulated [18F]GE-226 are shown in Figure 8.
Conclusion
We have successfully automated the manual radiolabelling production of [18F]GE-226 by
transferring to the FASTlab synthesiser. This automated process has been optimised and
[18F]GE-226 was produced after solid-phase extraction purification with a non-corrected
radiochemical yield of 30% ± 7 (n = 18) and a radiochemical purity of 94% ± 2 (n = 18). The
synthesis and purification was complete after 43 minutes and provided apparent molar
activities of 12-30 GBq/μmol (n = 12). Our results demonstrate that this reliable automated
manufacture for [18F]GE-226 will allow the production of doses for clinical studies. The
automated FASTlab process has already been successfully used to supply [18F]GE-226 for a
pre-clinical study to detect HER2 expression and pharmacodynamic response to HER2
modulating therapies.18
Acknowledgements
We thank Robert Forrest for assistance with the FASTlab preparations and Ole Henrik
Hansteen and Inger Oddbjorg Fremstad for assistance with the analyses.
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Scheme 1. Radiosynthesis of [18F]FBA and Affibody molecule conjugation reaction forming
[18F]GE 226.
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Figure 1. Detailed description of the optimised FASTlab synthesis of [18F]GE-226.
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Figure 2. FASTlab cassette layout for radiosynthesis of [18F]GE-226 including SPE
purification.
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a)
b)
c)
d)
Figure 3. Chemometrics modelling data: a) regression coefficients for time, temperature,
precursor amount, aniline amount, [18F]FBA recovery and starting activity (+ interaction and
squared terms); b) co-relation of measured RCP and predicted values; c) RCP response
surface for time and temperature; d) RCP response surface for time and precursor amount.
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Figure 4. The dependence of RCP % (3 hr post formulation) on temperature and the syringe
S2 position (mm) at start of synthesis activity of 70 GBq.
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Figure 5. The dependence of total chemical impurity mass (µg) on the precursor mass (mg)
and the total tC2 SPE fill mass (g).
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36
34
32
30
28
26
24
22
20
RCY (%)
0
5
10
15
20
No. of productions
Figure 6. Radiochemical yield (% RCY) (non-decay corrected) of [18F]GE-226 for a number
of FASTlab productions.
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99
97
95
93
91
89
87
85
RCP (%)
400
600
800
1000
1200
RAC MBq/mL
Figure 7. Radiochemical purity (% RCP) versus radioactive concentration (RAC, MBq/mL)
of [18F]GE-226 for a number of FASTlab productions. The specification requirement is
RCP not less than 90% at EOS.
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Figure 8. Representative HPLC radiochromatogram of formulated [18F]GE-226 (tR = 18.5
min).
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Table 1: HPLC conditions for determination of radiochemical purity and
chemical impurities by UV detection
Parameter
Description
10 – 30 % B in 25 min
30mM ammonium formate pH 4
MeCN
Phenomenex Kinetex C18, 2.6µm, 50 x 4.6 mm
UV 280 nm and radiometric detector
Phenomenex KrudKatcher
1.0 mL / min
Mobile phase
Mobile phase A
Mobile phase B
Column
Detector
Guard Column
Flow rate
31 min
Run time
Table 2: HPLC-UV/MS conditions for identification of chemical impurities in
decayed samples
Parameter
Description
0 – 1 min 10% B, 1 -15min 10 – 40% B, 15 – 20 min 40 – 70% B
0.1% (v/v) formic acid, 20 mM ammonium acetate, 10%
acetonitrile (v/v) in H2O
Mobile phase
Mobile phase A
0.1% (v/v) formic acid, 20 mM ammonium acetate in 85.9% (v/v)
acetonitrile
Mobile phase B
Phenomenex Kinetex C18, 2.6µm, 100 x 4.6 mm
UV 215 nm, mass spectrometry, mass range m/z 100 – 2000
Phenomenex KrudKatcher
1.0 mL / min
30 min
Column
Detector
Guard Column
Flow rate
Run time
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