Development of ready to use kit formulation for trastuzumab radioimmunoconjugates and identification of radiochemical purity as the first step in quality control of the final product

Abstract

Aim The aim of this study is to present the part of our project dedicated to obtaining a stable, ready to use freeze dried kit formulation of antibody radioimmunoconjugates (trastuzumab immunoconjugates labelled with 90Y and 177Lu). As the first step in on-going in vitro stability of the final product and radiochemical purity determination, we used ITLC-SG method with different mobile phases. Methods Radioactive labelling of trastuzumab was performed with 90Y and 177Lu via DOTA, DTPA and 1B4M-DTPA in molar ratio 1:20. The specific activity of 1.425 mCi (90Y) and 8.150 mCi (177Lu) was achieved, using a solutions of 0.04 M HCl. Radiolabeling is performed by adding 8.5 µL of 90Y at pH 4.5-5 and 5 µL 177Lu at pH 6. Solutions with Tr-DTPA and Tr-1B4M-DTPA were incubated at room temperature for 30 min, while Tr-DOTA was incubated at 40 °C for 1 hour. Radiochemical purity of radioisotopes was tested with ITLC-SG using three mobile phases: 0.9% NaCl, 0.4 M methanol/sodium-acetate (1:1) and 0.1 M acetic buffer. The stability of radioimmunoconjugates was tested in 0.9% NaCl (177Lu) and 0.4 M methanol/sodium-acetate (1:1) (90Y), after incubation at room temperature for 1, 24, 48 and 72h. Results After choosing the most suitable mobile phase for determination of radiochemical purity by ITLC-SG of conjugates labeled with 90Y (99.87%) we used 0.4 M methanol/sodium acetate (1:1), and those with 177Lu (100%) with 0.9%. NaCl. Examination of radiochemical yield of radioimmunoconjugates showed the presence of radioactivity only at the start of the strip, due to the high Mw of Tr. The absence of radiolabeled fragments of the antibody, as well as radiolabeled chelators and free radioisotopes, proved that the stable radioimmunoconjugates were formulated. The highest yield of labeling with 90Y (>96%) is achieved in 0.4 M methanol/sodium-acetate (1:1), while with 177Lu (>99%) in 0.9% NaCl. Test stability after 24h showed the highest stability of 90Y-DOTA-Tr (>92.40%) and 177Lu-DOTA-Tr (>99.14%), with minimum released 90Y3+ (84.90%) and 177Lu-DOTA-Tr (>98.52%), with minimum released 90Y3+ (<15.10%) and 177Lu3+ (<1.48%). Conclusions After obtaining the final ready to use kit formulation, the results of the determination of radiochemical purity using ITLC-SG show a high radiolabeling efficiency (>95%), using both isotopes. However, radioactive yield with 177Lu (99%) was higher compared with 90Y (>96%). This method was used to monitor the stability of radiolabeled conjugates and after 72 hours of incubation, a small amount of free radioisotopes was released from radioimmunoconjugates (<5% of 177Lu and <25% of 90Y). The next planed step includes in vivo examinations in healthy mice and in a mouse model of HER2 positive breast tumor after the i.v. injection of radiolabeled trastuzumab radioimmunoconjugates in order to monitor and determine their pharmacokinetics and biodistribution in the whole body and critical organs/tumor