Yttrium-90 and Rhenium-188 Radiopharmaceuticals for Radionuclide Therapy

6.2.1.2. Determination of the number of DOTA molecules bound to rituximab
The number of chelator (p-NCS-Bz-DOTA) molecules bound to rituximab
was determined by two methods: radioassay using cold 89 YCl 3·6H 2 O spiked
with a trace of 90 Y acetate and spectroscopic assay using the Cu(II) arsenazo(III)
complex [6.8].
(a)
Radioassay
Aliquots approximating to 100 µg of rituximab of each of the reaction
mixtures corresponding to different antibody:DOTA molar ratios (1:10, 1:25,
1:50 and 1:100) were taken. Approximately 200 µCi of 90 Y acetate was added,
together with cold 89 YCl 3 in the ratio of 1:1 (yttrium:DOTA). The reaction
was carried out at 40°C for 2 h, and the reaction mixture was purified using a
PD-10 column. Fractions of 1 mL were collected using 0.05M phosphate buffer
(pH7.4) and the radioactivity associated with each fraction was measured.
The elution patterns are as shown in Fig. 6.5. The percentage of radioactivity
associated with rituximab in comparison with the total radioactivity gave an
indication of the numbers of DOTA molecules bound to the antibody, which were
estimated to be ~5, 12, 20 and 36 for rituximab:DOTA conjugated at 1:10, 1:25,
1:50 and 1:100 ratios, respectively.
The reaction mixture of a 1:10 ratio of DOTA rituximab was spiked with
cold 89 YCl 3 corresponding to 0.5:1, 1:1 and 2:1 yttrium:DOTA ratios to confirm
the number of DOTA bound to rituximab. Radiolabelling was carried out as
described in Section 6.2.1.4 and purification was carried out using a PD-10
column. The elution patterns (see Fig. 6.6) show ~67.9%, 49% and 56.1% for
0.5:1, 1:1 and 2:1 yttrium:DOTA ratios, respectively. These patterns indicated
that approximately five molecules of DOTA were bound to the antibody.
(b)
Spectroscopic assay using Cu(II) arsenazo(III) assay
The number of DOTA molecules bound to rituximab was also determined
using the Cu(II) arsenazo(III) assay, as reported by Brady et al. [6.8]. A stock
solution referred to as copper reagent composed of 25 µM of Cu(II) and 100 µM
of arsenazo(III) in 0.15M NH 4 OAc (OAc = acetate anion), pH7.0, was prepared.
Serial dilutions of this reagent were made to ensure that this reagent obeyed Beer
Lambert’s law by determining the absorbance at 652 nm. A spectrum of the copper
reagent was taken and absorbance at 652 nm was recorded. The experiment
indicated that for 1 mL of the copper arsenazo (III) reagent, absorbance (A) was
0.079. Using Beer’s law, the molar extinction coefficient ε was calculated to be
3160 L·cm –1 ⋅mol –1 .
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