Radiosensitivity Study of Freeze-dried Antibodies to Gamma Irradiation
Radiosensitivity Study of Freeze-dried Antibodies to Gamma Irradiation
July/August 2004
I. Caballero(1), S. Altan?s(3), A. Castillo(1), V. Deridder(2), T. Gomez(1), Y. Miralles(1), V. Naydenov(2), E. Prieto(3) and B. Tilquin(2)
(1)Center of Molecular Immunology, 216 esq. 15. Siboney. Playa, C. Habana. Cuba
(2)Universit? Catholique de Louvain, Facult? de M?decine, 7230 Avenue Mounier, 72 1200 Bruxelles, Belgique
(3)Center of Applied Studies to Development of Nuclear Energy. 5ta y 30. Miramar, Playa, Cuba
Analysis of degradation freeze-dried antibodies by 60Co gamma irradiation was performed using the UV Scanning Spectroscopy, Gas Chromatography, High Performance Liquid Chromatography and Electron Paramagnetic Resonance techniques. The byproducts formed after irradiation are in very low abundance. They were not detected even when using high sensitivity GC and UV Scanning Spectroscopy. We also anticipate that the formed byproducts will be similar to the non-damaged product. The EPR spectra of all monoclonal antibodies are asymmetric features centered around 332 T. The signal intensity depends on the irradiation dose linearly up to 15 kGy, while at higher doses the linearity disappears apparently by radical recombination despite the low diffusion properties of the freeze-dried compounds. The most radiosensitive antibodies were humanized monoclonal antibodies and the glucose played a radioprotector role. The HPLC results indicate that it is an adequate technique to detect the byproducts. The formed radical are very stable and they can be detected up to nine months.
Key words: gamma radiation, antibodies, radical stability
The regulations for radiosterilization are different among countries, but with the publication of ANSI/AAMI/ISO 11137 by American National Standard at least there now exists a recognized guidance for implementing this technology. The publication describes specific requirements for validation, process control and routine monitoring in the radiation sterilization of health care products. This guidance may be applied to continuous and batch type gamma irradiators, using the radionuclides 60Co and 137Cs, and also to irradiators using a beam from an electron or x-ray generator.
Gamma radiation is generated by the spontaneous decay of radioisotopes. Exposure to gamma rays sterilizes the product by disrupting the DNA structure of microorganisms located on or within the product, thereby eliminating its ability to reproduce life-sustaining cells. The deep penetration of gamma rays makes it an ideal solution for products having various densities and types of product packaging. The gamma ray sterilization process is reproducible, easy to use, validate and has a proven track record. Using dosimetric release procedures allows for the shipment of products immediately after the sterilization processing [1].
On the other hand, during the irradiation of the biopharmaceutical products there occurs the appearance of free radicals, with a high chemical reactivity that can induce changes in the pharmacological, organoleptical and toxicological drug properties [2-4]. These new byproducts are usually at a very low concentration in drugs, approximately 10-7 M [5].
A suitable technique for the detection of free radicals should be specific for the radiation treatment. The sensitivity of the technique should be high enough to allow signal recording over the self life of the product. The most common techniques we found useful for providing qualitative and quantitative data, whether or not sample has been irradiated, were UV-Spectroscopy, Gas Chromatography High Performance Liquid Chromatography and Electron Paramagnetic Resonance techniques [6].
The objectives of this study are to establish the techniques to discriminate between irradiated and nonirradiated freeze-dried antibodies and to evaluate the stability of the formed radiolitycal byproducts.
Materials and Methods
For our study, we have used formulations containing two murine monoclonal antibodies obtained from mouse liquid ascitic (ior/egf r3 and ior cea1), one chimeric (t3Q) and one humanized monoclonal antibodies (hr3) produced from bioreactor supernatants in the Center of Molecular Immunology in Cuba. The antibodies were freeze-dried and sealed under vacuum.
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