Arev, Marija and Dzodic, Predrag and Makreski, Petre and Ruskovska, Tatjana and Janevik-Ivanovska, Emilija (2018) Physico-chemical assessment of labeled freeze dried kits of trastuzumab-immunoconjugates significant for breast cancer therapy. In: 19th European Symposium on Radiopharmacy and Radiopharmaceuticals, 05-08 Apr 2018, Groningen, Netherlands.
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Abstract
Introduction: Monoclonal antibodies appears as an important therapeutic agents for cancer treatment and have shown high complexity in the manner of action and their biological properties. Due to the significant potency in various malignancies and easy detection of radioactivity with outside scintigraphy, radioimmunoconjugates have become a part of many clinical trials. The antibody manipulation and exposure to stress conditions during the processes of conjugation, lyophilization and labeling, can cause disruption of the native structure of the protein. The most appropriate technique for protein integrity and purity examinations is reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Attenuated total reflectance-infrared (ATR-IR) and Raman spectroscopy as powerful, non-destructive and easy-to-use techniques provide valuable molecular structure information and are convenient for verification of changes in the secondary structure. The successful experience in formulation of ready-to-use kits of rituximab-immunoconjugates, inspire us to use another attractive monoclonal antibody for therapy of HER2 positive breast cancer. The same protocols, with minor adjustments, were implemented for structural characterization of labeled and non-labeled lyophilized trastuzumab-immunoconjugates formulations.
Material and Methods: The purified trastuzumab from Herceptin® was conjugated with bifunctional chelators (BFCAs), p-SCN-Bn-DTPA, p-SCN-Bn-DOTA, p-SCN-Bn-1B4M-DTPA in ratio of 1:20 and lyophilized to solid state. The freeze dried conjugates and cold labeled samples with LuCl3 and YCl3 were used for further examinations and physico-chemical characterization by applying of SDS-PAGE, ATR-IR and Raman spectroscopy.
Results: Under reducing conditions migration of the trastuzumab provided separation of two bands of fragments with molecular weight of 25 kDa for light chain and 50 kDa for heavy chain, proven with the Wide Range, Molecular Weight marker. The same intensity of the fragments of lyophilized and labeled conjugates with the fragments of pure trastuzumab was indicated that there is no degradation of the antibody. ATR-IR and Raman spectra also have indicated that all samples have retained native structure expressed in terms of assignment of the amide bands (amide I, II and III bands), characteristic for IgG1 structures. Characteristic amide I band at ~1670 cm-1 and amide III band (1230-1300 cm-1) were detected in Raman spectra. IR spectra also contain the amide I (1700-1600 cm-1), amide II (1480-1575 cm-1) and amide III bands (1255-1244 cm-1) specific for secondary structure of the proteins.
Conclusion: The promising results from electrophoresis and vibrational spectroscopy are good basis for further radiolabeling of immunoconjugates with 177Lu and 90Y for treatment and imaging of HER2 positive lesions.
| Item Type: | Conference or Workshop Item (Poster) |
|---|---|
| Subjects: | Medical and Health Sciences > Other medical sciences |
| Divisions: | Faculty of Medical Science |
| Depositing User: | Marija Arev |
| Date Deposited: | 03 Dec 2018 08:54 |
| Last Modified: | 25 May 2023 10:56 |
| URI: | https://eprints.ugd.edu.mk/id/eprint/20911 |
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