Differential chemical behaviour of humanized monoclonal antibodies radiolabelled with 99mTc

In the radiopharmaceutical field the substitution of murine monoclonal antibodies (m-mAbs) by humanized monoclonal antibodies (h-mAbs) is one of the main works in our days. The use of h-mAbs involves many advantages, because the HAMA response is one of the principal drawbacks in the use of m-mAbs. However, the transference of the procedures from the m- MoAbs to the h-MoAbs is a cumbersome task, because these have a differential behaviour once reduced and radiolabeled with 99mTc. In Cuba the biotechnological industry has developed a great diversity of monoclonal antibodies, including new humanized antibodies. The use of these in therapy and diagnosis is very attractive. The procedures for radiolabeling the m-mAbs have been established and these are used in Cuba, but the radiolabeling of h-mAbs is a recent topic for our radiopharmacists. This work develops a simultaneous comparison of immunoglobulins from different origin. For this purpose the human-IgG, two h-MoAbs and a m-MoAb were employed. Our goal consists to demonstrate that there are differences in the behaviour of these immunoglobulins after their reduction with sodium metabisulfite (SMB) and radiolabeling with 99mTc. The four immunoglobulins were the human-IgG, the R3 and T1 humanized monoclonal antibodies (R3 and T1 h-mAbs), and the ior t3 murine monoclonal antibody (ior t3 m-mAb). The formation of fragments and aggregates, and the immunoreactivity of the monoclonal antibodies were studied once concluded the reduction process. Also, comparative studies of the radiochemical stability and challenges were carried out after radiolabeled. Furthermore four auxiliary ligands were compared in the radiolabelling of the h-IgG. The comparison of four ligands, showed a differential behaviour in the radiolabeling process. The tartrate and gluconate were the most suitable for the human-IgG. In the comparison, of the chemical behaviour of the h-IgG vs the humanized and murine monoclonal antibodies radiolabeled with 99mTc. The results showed differences in the collected data of these h-mAbs after the radiolabeling, the highest radiochemical purities were obtained with the human-IgG and m-mAb in comparison with the h-mAbs, also we observed a small difference between the h-mAbs which was confirmed statistically. However, for all immunoglobulins the radiochemical purity was higher to 90.0 % and the amount of 99mTc-radiocolloid was lower than 5.0%. The challenges showed the lowest dissociation for the 99mTc-h-IgG in comparison to the rest. The h-mAbs presented a differential behaviour, because the R3 h-mAb had lower dissociation in relation to the T1 h-mAb. These h-mAbs showed furthermore a higher dissociation compared to the m-mAb, this difference was confirmed statistically. The PAGE study showed a lower generation of fragments for the human IgG and the m-mAb. The humanized mAbs presented a higher generation and the T1 h-mAb showed more fragments in relation to the R3 h-mAb. There were not detected a molecular aggregates of the immunoglobulins by HPLC studies. However, the profile of the T1 h-mAb confirmed the results obtained in the fragmentation study, where appeared fragments of this MoAb. Ellman assay showed a significative difference among h-MoAbs and the rest of the inmunoglobulins. The immunoreactivity study showed the preservation of biological activity for all MoAbs. The results indicated differences in the chemical behaviour of the immunoglobulins after the reduction process. The percentage of fragmentation and generation of free sulphydryls groups for the evaluated immunoglobulins, demonstrated that the humanized MoAbs were more sensitive to the reduction process; this fact influenced without doubt their behaviour in the radiolabeling with 99mTc. After the radiolabeling, the humanized MoAbs showed a differential behaviour, which was consistent with the results of the pre-labeling evaluation, and demonstrated that the structural modification influenced in a determinant manner in their chemical and radiochemical behaviour