O
N
NCS
89
Zr-Oxalate
H
N
S
N
H
NH
n
NH2
Pr
ot
ei
Pr
ot
ei
n
S
NH
Conjugate
n
+
HN
O
N O
OH HO N
O
O
N OH S
N N
H H
N
N O
ON
O
HN O
O
O
S
NO
N N
H
H
89 +4
Zr
tei
N O
OH HO N
HN O
O
O
N OH S
N N
H H
DFO-Bz-NCS
O
N
Pr
o
O
Figure 1 Schematic representation of DFO panitumumab (protein) conjugation reaction and
radiolabeling of DFO-panitumumab conjugate with 89Zr. At basic pH, isothiocynate group of
DFO reacts with lysine-NH2 of mAb to form a stable thiourea linkage. 89Zr-oxalate is used to
make stable 89Zr-DFO-panitumumab at room temperature.
MDA-MB-468
MDA-MB-231
BT-474
HER-1
β-Actin
Figure 2 A. Tumor uptake of 89Zr-panitumumab in various subcutaneous athymic nude female
xenograft models. 10.18 ± 1.24 MBq of 89Zr-panitumumab were administered intravenously via
tail-vein, and a 5 min CT scan followed by a 30 min static PET scan were performed at 96 h
post-injection; B. Comparative EGFR expression level of BT-474, MDA-MB-231, and MDAMB-468 cell lines by western blot analysis. Cartesian molecular imaging software v5.0.2.30 and
data is presented as EGFR/Actin ratio. The images are reprinted with the permission from
Elsevier Inc. Bhattacharyya et al., Nucl. Med. Biol. 2013, 40, 451-457.
mV
Detector A Ch1:280nm
350
300
250
200
(a)
150
100
50
0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
min
mV
Detector A Ch1:280nm
100
90
80
70
60
(b)
50
40
30
20
10
0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
mi n
Figure 3 HPLC chromatograms at 280 nm: (a) pure panitumumab; (b) DFO-panitumumab
conjugate.
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
y = 8.2761x + 0.0065
R² = 0.9999
0
0.05
0.1
0.15
0.2
0.25
(a)
mV
Detector A Ch1:280nm
3
2
1
0
0.0
5.0
10.0
15.0
20.0
25.0
min
(b)
Figure 4 (a) Standard calibration curve using DFO-panitumumab conjugate as an authentic
standard. X-axis represents the concentration in mg/mL (0.021 to 0.21mg/mL) and Y-axis
represents the absorbance (peak area). (b) HPLC chromatogram for a 20 µL injection of a 0.021
mg/mL DFO-panitumumab conjugate showing conjugate’s UV sensitivity at 280 nm.
Figure 5 Process flow of 89Zr-panitumumab synthesis for medical use. QC-1 and QC-2 are two
in-process QC.
Figure 6 HPLC chromatograms of 89Zr-panitumumab: (a) UV peak at 280 nm; (b)
corresponding radiation peak. Early small shoulder of radiation peak could be due the 89Zr
labeling to the trace amount of impurity (high molecular weight) present in the original protein.