Development of the purification process of gallium-68 eluated from Ge-68/Ga-68
generator
Tossaporn Siriprapa1,*, Nopamon Sritongkul1,#, Malulee Tuntawiroon1,#, Nipavan
Poramatikul2,#, Jatupol Sangsuriyan 2,#
1
Radiation Science Program, Division of Nuclear Medicine, Faculty of Medicine Siriraj
Hospital, Mahidol University, Bangkok, Thailand
2
Thailand Institute of Nuclear Technology, Bangkok, Thailand
*e-mail: Tossaporn.sir@mahidol.ac.th #e-mail: sinsu@mahidol.ac.th, simtt@mahidol.ac.th,
pomnipa@yahoo.com, j_sangsuriyan@hotmail.com
Abstract
Introduction: Gallium-68 have a short half-lived of 68 min, 89.14% positron decay,
produced by generator system and have suitable chemical property for labelling
radioligand, make it a promising tracer for PET imaging.
Methods: In this work, development of purification method for Ga-68 eluted from
68Ge/68Ga generator prior to perform radio labeling had been investigated. Cation and
anion exchange chromatography techniques were used in combination to separate trace
amount of competing metal ions and Ge-68 parent nuclide. The eluant Ga-68 in 5 ml 0.6N
HCl was loaded onto AG50W-X8 cation exchange column, wash with
80%Acetone/0.15NHCl, eluted the Ga-68 by 98%Acetone/0.2NHCl mixture. The Ga-68
was then pass through AG1-X8 anion exchange column and purified Ga-68 was recovered
by eluted with small volume of ultra pure water. Metal content, after purification process,
analysed by ICP-AES, were reduced by 61%, 38% and 44% for Fe(II), Zn(II) and Al(III)
respectively ,68Ge breakthrough was in the range of ∼10−3% by MCA spectrometer with
HPGe detector. Labeling efficiency with NOTA-RGD,a tracer for angiogenesis imaging,
gave average yields Ga-68 NOTA-RGD around 50% (not corrected for decay) and 9899% radiochemical purity(by HPLC).
Conclusions: Cation exchange incombination with anion exchange chromatography had
been proven to be an efficient method for purification of Ga-68 eluant from Ge-68/Ga-68
generator prior to radiolabeling of Ga-68 PET radiotracer.
Keywords: 68GE/68GA GENERATOR, PURIFICATION, RADIOLAELING, ION
EXCHANGE PURIFICATION, PET
Introduction
Positron emission tomography
(PET)
is an excellent diagnostic
imaging technique and has become the
dominant imaging method in the field of
nuclear medicine with high resolution,
high sensitivity and offering a precise
quantitative analysis.
Gallium-68 is a decay product of its
parent Ge-68(half-life [t1/2] = 270.95
days) and can be available by 68Ge/68Ga
generator system. Gallium-68 have
short half-life 68 min decay by positron
emission(89.14%) and have been
developed
as
potential
PET
radiopharmaceuticals without on site
cyclotron eg.Ga-68DOTA-TOC/DOTA
-TATE
for
neuroendocrine,Ga-68
NOTA-RGD for angiogenesis or Ga-68
Citrate for infection imaging. Currently,
several 68Ge/68Ga generator systems for
68
Ga are based on metal oxide, organic
resin
or
inorganic
support
such as TiO2, SnO2, Al2O3, pyrogallolformaldehyde resins and silica based as
sorbents. The principle drawback of
these system is that the Ga-68 eluant
usually contaminate with long life Ge68 and trace metal impurities which will
potentially compete Ga-68 ion when
radiolabeling with nanomole level of
conjugated peptides or other carrier
ligands. In addition, the eluant from Ge68/Ga-68 generator are relatively large
volume and high HCl concentration
from 0.1-1 N
which make
problem on labeling process. Therefore,
dedicated procedures to purify and
concentrate Ga-68 before radiolabeling
are need to be explored.
Recently, Zhernosekov et al. (2007)
[1] reported a third method to purify
68
Ga from generator eluates on a micro
cation-exchange column eluted with
HCl/acetone mixtures for preparation of
68
Ga-labelled
radiopharmaceuticals.
Ocak et al. (2009) [2] summarized the
three purification and concentration
techniques of the characteristics of the
68
eluate
of
TiO2-based
Ge/68Ga
generators by fractionated elution,
cation exchange chromatography and
anion exchange chromatography. An
important aspect for wide use of 68Garadiolabelling in clinical PET is the
large volume after elution from the
generator, high [H+], Moreover, there is
concern about 68Ge breakthrough and
contamination of metal ion impurities.
[3-4].
The purpose of this study is to
study a method for purification of
Gallium-68 with high radiochemical
purity and short purification times using
the system of ion-exchange columns
combining cation and anion exchange
processes with a high labelling
efficiency of 68Ga-NOTA-RGD with
small volume and low acidity useful for
direct radiolabeling reactions.
68Ga
labelled peptides for clinical use
Gallium-68 labeled peptide has been
explored for different receptors and
68
Ga-NOTA-RGD(fig.1) has also been
applied for imaging of tumors and
angiogenesis.
The integrin binds to extracellular
matrix proteins such as vitronetic,
fibrinogen and laminin, the amino acid
sequence. Moreover, arginine-glycineaspartic acid (RGD) peptide sequence
(Fig. 1) has the most efficient for
targeting the integrin αvβ3 receptor.
The highly expressed of the
integrin αvβ3 on osteoclasts and active
endothelial cells of the neovasculature
of various tumors. It has been
demonstrated that it is expressed in
tumor cells in human such as late-stage
glioblastomas, breast tumor, prostate
tumor, malignant melanomas, and
ovarian carcinomas [5-8]. Thus, the αvβ3
integrin-positive patients have proved to
be a valid target and can be quantify
integrin αvβ3 expression level for
angiogenesis imaging.
O
OH
N
Ga+3
N N
K : Lysine
O
HN
O HO
HO
S
y : D-Tyrosine
OH
NH
O
O HN
O
HO
N
H
O
HN
O
D : Aspartic acid
R : Arginine
H
N
NH2
NH
NH
NH
O G : Glycine
Figure 1 Chemical structure of
NOTA–RGD
68
Ga-
Materials and methods
Two types of 68Ge/68Ga generators
were used in this study. The 1st
68
Ge/68Ga generator was the product of
iThemba Labs (Somerset West, SouthAfrica) and had been checked for
metallic impurity. The elution was done
by using steriled 0.6N HCl prepared
from ultra pured Hydrochloric Acid and
tri-distilled water.
The 2nd 68Ge/68Ga generator was
the product Eckert&Ziegler (Berlin,
Germany). The elution was done by
using steriled 0.1N HCl prepared from
ultra pured Hydrochloric Acid and tridistilled water, was
used for
radiolabeling.
Ga-68 purification and ICP-AES
analysis for metal ions
Using iThemba Lab’s 68Ge/68Ga
generator, initially, the generator was
eluted with 5 mL 0.6N HCl, flow rate
<1 mL/min, the eluate was loaded on to
cation exchange column (AG 50W-X8,
100–200 Mesh, Bio-Rad). Secondly, 0.5
mL of 80%Acetone/0.15N HCL was
used to wash the cation column and
eluted with 0.5 mL mixture of
98%Acetone and various concentration
of HCL (0.05, 0.1, 0.15 and 0.2N) on
four column of micro-chromatography
column. Thirdly, each eluant from
cation exchange column was loaded
onto anion exchange column (AG1-X8,
100–200 Mesh Bio-Rad) separately,
discard first eluant and then 68Ga was
recovered in 1 mL tri-distilled water.
The 68Ga products after purification
process were analyzed for metal ion
content by ICP-AES (Inductively
Coupled Plasma atomic emission
spectrometer)
By
ICP-AES,
concentrations of the following metal
ions were determined: Fe, Zn, Ge and
Al. The standard solutions were used to
prepare
one
standard
solution
containing 1000 ppb of each of these
metals. The 1000 ppb standard solution
was diluted to prepare a 50, 100, 200
and 400 ppb standard solutions.
Labeling efficiency of purify 68Ga
Using
Eckert&Ziegler
68
68
Ge/ Ga generator which has higher
radioactivity, Initially, the generator
was eluted with 5 mL HCl 0.1N flow
rate <1 mL/min, the eluate was loaded
on the cation exchange column (AG
50W-X8, 100–200 Mesh, BioRad).
Secondly,
0.5
mL
of
80%Acetone/0.15N HCL was used to
wash the cation column and eluted with
0.5 mL 98%Acetone/0.2N HCL.
Thirdly,
the eluant from cation
exchange column was loaded onto
anion exchange column (AG1-X8, 200–
400 Mesh Bio-Rad), discard first eluant
and then 68Ga was recovered in 1 mL
tri-distilled water. Labeling was
performed as the method previously
described [9] briefly 20 µg NOTA-RGD
peptide, 20 µL Ga-68(20 µCi) were
added to 200 µL 0.1M NaOAc (Sodium
Acetate), pH was adjusted to 5.0-5.5
and the mixture was heated at 90-95 0C
for 10 min. The labeled product of Ga68NOTA-RGD was purified by SepPak C-18 mini column. The labelling
efficiencies and radiochemical purities
of purified 68Ga-NOTA-RGD, were
determined by High-performance liquid
chromatography (HPLC), (Agilent
tech., Serie 1200) with Phenomenex
Jupiter column C18, 5 µm, 4.6x250
mm. The solvents were 0.1% (m/v)
trifluoroacetic acid in de-ionised water
(A) and 100% acetonitrile (B), flow rate
1 mL/min,detector UV-visible (200, 280
nm) and gamma-ray detector (Raytest
Gabi Star). Determination of 68Ge
breakthrough was done after completed
decay of Ga-68(>48 hr), using γ-ray
spectrometer (MCA) equipped with a
coaxial HPGe detector.
Breakthrough and Radionuclidic
purity (RNP)
Breakthrough is use to express the
ratio of activities in the eluate of the
parent radionuclide (68Ge) versus
daughter radionuclide (68Ga). Another
radionuclidic purity (RNP) is defined as
the ratio of activity of a radionuclide
versus the activity of another
radionuclide. We define radionuclidic
purity (RNP) here as the ratio of
activities of 68Ge versus 68Ga in the
eluate, expressed in % by this equation.
Breakthrough (%) =
68Ge activity product
x 100%
68Ga activity
Results and discussion
Concentration of cationic metallic
impurities
The Inductively Coupled Plasma
atomic emission spectrometer (ICPAES) analysis of the eluates verified the
presence of metal impurities that could
interfere in Ga (III) complexation
chemistry. In the solutions, Metal ions
(Fe (III), Zn (II), Ge (IV) and Al (III))
in the eluate, Germanium (IV) was
always ˂1 ppb (Fig. 1).
Figure 1 metal ion in several eluates.
And for all other metals ˂1 ppm.
(Fe(II), Zn(II) and Al(III) were reduced
from the solution 61, 38 and 44%
respectively, after purification process)
compared to the non-purified eluate.
68Ge
Breakthrough
68
For
quantification,
Ge
breakthrough was also measured after
complete 68Ga decay (>48 h) in
spectrometer equipped with a coaxial
HPGe detector for determination of
68
68
Ge
breakthrough.
The
Ge
breakthrough to the generator was
found to be ∼10−3% of the eluted 68Ga
activity after eluate purification.
Radiolabelling of NOTA-RGD
The labeling yields of 68Ga-NOTARGD were 96.0-99.3% from the loaded
activity on the first column. When
combined the two purification methods
(cation exchange and anion exchange
chromatography respectively). The
elution yields decreased to 50% after
the first elutions. The radiochemical
purity (RCP) of 68Ga-NOTA-RGD was
higher than 99%. The time for this
experiment was completed within 30
minutes. The preparation process,
including HPLC quality control.
Conclusion
The 68Ge/68Ga generator was eluted
every day for radiotracer production for
reduce the amount of metal impurities
at the time of 68Ga-NOTA-RGD
synthesis. After eluate purification, the
68
Ga (III) preparation could be obtained
in a minimized volume, the mean
elution yield was 50% (not corrected
for decay). Most importantly, The 68Ge
breakthrough to the generator was
found to be ∼10−3% of the eluted 68Ga
activity after eluate purification. And
the radiochemical purity (RCP) of
68
Ga-NOTA-RGD used for patient
doses were greater than 90% (up to
99%), overall processing time was
accomplished within 30 minutes. This
resulted in a high labelling efficiency
of 68Ga-NOTA-RGD with small
volume and low acidity useful for
direct radiolabeling reactions.
Acknowledgements:
The dissertation was financially
supported under Research Fellowship,
provided
by
Siriraj
Graduate
Scholarship.
We are grateful for Radioisotope
Center, Thailand Institute of Nuclear
Technology to include copyrighted
photographs as part of my thesis. I
would like to thank Mr. Jatupol
Sangsuriyan
and
Miss
Nipavan
Poramatikul for his valuable and
constructive suggestions during the
planning and development of this
research work.
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