ChemComm
Communication
Fig. 3 Characterisation data for the one-pot photoradiochemical synthesis of [68Ga]GaNOTA-azepin-trastuzumab from pre-purified mAb. (A) Radio-
iTLC chromatograms, (B) analytical PD-10-SEC elution profiles, and (C) SEC-UHPLC chromatograms of the crude and purified product. Equivalent data
using compounds 3 and 4 are presented in the ESI.†
conjugation proceeds most efficiently under slightly basic Ongoing work has also found that photoradiochemistry is
conditions where the nucleophilicity of the lysine side-chain a viable tool for synthesising radiolabelled peptides, small-
is increased via deprotonation of the primary e-NH2 amine molecules and nanoparticles, and that entire process can be
(pKa
B
10.5). For this reason, the chelates were pre- automated (data to be reported elsewhere).
JPH thanks the Swiss National Science Foundation (SNSF
radiolabelled with [68Ga][Ga(H2O)6]3+ before adjusting the pH
in situ to 47.5 using NaHCO3 solution. Complex formation was Professorship PP00P2_163683), the Swiss Cancer League (Krebsliga
monitored by radio-iTLC and radio-size-exclusion chromatogra- Schweiz; KLS-4257-08-2017), and the University of Zurich (UZH)
phy (SEC) UHPLC. After complete complexation, an aliquot of for financial support. This project has received funding from
pre-purified trastuzumab was added with an initial chelate-to- the European Union’s Horizon 2020 research and innovation
mAb ratio of B10-to-1. Reaction mixtures were then irradiated programme/from the European Research Council under the
for 15 min at room temperature. Aliquots of the crude reaction Grant Agreement No. 676904, ERC-StG-2015, NanoSCAN. We
mixtures were analysed by radio-iTLC, manual size-exclusion thank all members of the Radiochemistry and Imaging Science
chromatography (PD-10-SEC) and radio-SEC-UHPLC. In addi- group at UZH for helpful discussions.
tion, a fraction was purified by preparative PD-10 and spin-
centrifugation methods to measure the absolute radiochemical
Conflicts of interest
yield (RCY), radiochemical purity (RCP) and molar activities
of the purified 68Ga-radiolabelled trastuzumab (Fig. 3 and
There are no conflicts to declare.
Fig. S24, S25, ESI†). Note, all experiments were performed in
triplicate with independent replicates.
Notes and references
1 E. Boros and J. P. Holland, J. Labelled Compd. Radiopharm., 2018, 61,
652–671.
2 P. Dennler, E. Fischer and R. Schibli, Antibodies, 2015, 4, 197–224.
3 B. M. Zeglis, K. K. Sevak, T. Reiner, P. Mohindra, S. D. Carlin,
P. Zanzonico, R. Weissleder and J. S. Lewis, J. Nucl. Med., 2013, 54,
1389–1396.
4 B. M. Zeglis, C. B. Davis, R. Aggeler, H. C. Kang, A. Chen, B. J. Agnew
and J. S. Lewis, Bioconjugate Chem., 2013, 24, 1057–1067.
5 J. P. Holland, Chem. – Eur. J., 2018, 24, 16472–16483.
Starting from either compound 1, 3 or 4, 68Ga-radiolabelled
trastuzumab was produced in crude radiochemical yields of
around 16–18%, as measured by analytical PD-10-SEC, and
11–16%, as measured by radioactive SEC-UHPLC (Fig. 3). Based
on the known initial concentrations of the reagents, the esti-
mated final chelate-to-mAb ratios were in the range 1.1 to 1.8.
For the radiochemical synthesis of [68Ga]GaNOTA-azepin-
trastuzumab, the purified sample was isolated in PBS with a
decay-corrected RCY of 10.1 Æ 0.7% (n = 3), a RCP 495%, and a
molar activity, Am of 0.46 Æ 0.09 MBq nmolÀ1 of protein (n = 3;
note, the protein concentration was remeasured after radio-
active decay to obtain an accurate value). Our previous work
found that photoradiochemical labelling does not compromise
the biological activity (immunoreactivity) of the antibody.18
In summary, experiments showed that photoactive deriva-
tives of widely used aza-macrocyclic chelates (NOTA, DOTA and
DOTAGA) are suitable for photochemical radiolabelling of
proteins. Introduction of these photoactive chelates opens the
possibility of using photoradiochemical methods with a broad
range of radionuclides taken from across the periodic table.
´
6 P. Klan and J. Wirz, Photochemistry of Organic Compounds: From
Concepts to Practice, 2009.
7 T. R. Sykes, T. K. Woo, R. P. Baum, P. Qi and A. Noujaim, J. Nucl.
Med., 1995, 36, 1913–1922.
8 T. R. Sykes, V. A. Somayaji, S. Bier, T. K. Woo, C. S. Kwok, V. Snieckus
and A. Noujaim, Appl. Radiat. Isot., 1997, 48, 899–906.
9 M. Nishikawa, T. Nakano, T. Okabe, N. Hamaguchi, Y. Yamasaki,
Y. Takakura, F. Yamashita and M. Hashida, Bioconjugate Chem.,
2003, 14, 955–961.
10 M. A. Stalteri and S. J. Mather, Eur. J. Nucl. Med., 1996, 23, 178–187.
11 K. Hashizume, N. Hashimoto and Y. Miyake, J. Org. Chem., 1995, 60,
6680–6681.
¨
12 H. J. Wester, K. Hamacher and G. Stocklin, Nucl. Med. Biol., 1996, 23,
365–372.
13 C. W. Lange, H. F. VanBrocklin and S. E. Taylor, J. Labelled Compd.
Radiopharm., 2002, 45, 257–268.
This journal is ©The Royal Society of Chemistry 2019
Chem. Commun.