Silica gel impregnated glass microfiber chromatography paper

Gallium-68 was obtained by elution of a ⁶⁸Ga/⁶⁸Ge-generator (Cyclotron Co. Obninsk, Russia) with 0.1 M HCl. Eluate was collected in fractions of 400 µl at a speed of 800 µl/min. The third fraction containing in average 452 ± 147 MBq was used for radiolabeling.
All conjugates were radiolabeled according to the following protocol. Solution of 25 µg of affibody molecule in 300 µL ascorbic acid buffer (1 M, pH 3.6) was incubated with 200 µl of gallium-68 eluate (150-200 MBq) for 15 min at 85 °C. Labeling yields were determined with ITLC (Instant Thin Layered Chromatography). Samples were applied to silica gel-impregnated glass microfiber chromatography paper (Agilent Technologies, Santa Clara, CA, USA) and eluted with 0.2 M citric acid. If necessary, the radiolabeled conjugates were purified with NAP-5 size-exclusion columns (GE Healthcare), pre-washed with 1% BSA/PBS.
To test the stability, 2 µg of labeled conjugates were incubated for 1 h in PBS at room temperature or in human serum at 37 °C. The samples were analyzed with ITLC.

For labeling, 10 µg (HE)₃-Z_HER3-X was dissolved in 55 µL sodium acetate (0.2 M, pH 5.5) and incubated with 7.5–14 MBq [⁵⁷Co]CoCl₂ for 45 min at 60 °C. Instant thin-layered liquid chromatography (ITLC) was used to determine the labeling yields. For analysis, a sample of the radiolabeling mixture was applied to silica gel-impregnated glass micro-fiber chromatography paper (Agilent Technologies, Santa Clara, CA, USA). Samples were eluted with citric acid (0.2 M, pH 2) and scanned with the Cyclone Storage Phosphor System, and OptiQuant image analysis software (PerkinElmer, Waltham, MA, USA) was used to determine the labeling yield.
To test the in vitro stability of [⁵⁷Co]Co-(HE)₃-Z_HER3-X, 1 µg of radiolabeled compound was incubated in PBS or 500-fold molar excess of EDTA for 24 h at room temperature. Release of the radiolabel was analyzed with ITLC.
(HE)₃-Z_HER3-NODAGA was labeled with gallium-68 according to the method previously described [31 (link)]. In brief, 25 µg of (HE)₃-Z_HER3-NODAGA was incubated with 300 µL ascorbic acid (1 M, pH 3.6) and 200 µL of gallium-68 eluate (150 MBq) for 15 min at 85 °C and thereafter purified with NAP-5 size-exclusion columns (GE Healthcare, Uppsala, Sweden).

Conjugates (25 µg in 50 µl ammonium acetate buffer 0.2 M, pH 5.5) were incubated with 25 µl of ¹¹¹In-chloride (20 MBq) for 40 minutes at 85 °C. For Z₀₈₆₉₈-DOTA incubation was prolonged to 60 minutes and performed in presence of acetonitrile. Radiolabeled conjugates were purified with disposable NAP5-columns (GE Healthcare) pre-equilibrated with 1% BSA/PBS and eluted with PBS. Labeling yield and radiochemical purity was analyzed by instant thin layered chromatography (ITLC). For analysis, 1 µl of radiolabeled solution was applied to silica gel-impregnated glass microfiber chromatography paper (Agilent Technologies, CA, USA), and samples were developed in citric acid (0.2 M). Radioactivity distribution was then measured with the Cyclone Storage Phosphor System and analyzed with OptiQuant image analysis software (Perkin Elmer, Waltham, MA, USA).
Stability of the radiolabeled conjugates was evaluated by incubating 2.5 µg of ¹¹¹In-Z₀₈₆₉₈-X (here and further “radiolabeled affibody molecules”) at 37 °C with 100 µl human serum for 24 h. The samples were thereafter analyzed with ITLC.