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Immunization has been considered a successful global health program that saves many persons' lives each year. The vaccines reduce the risk of getting the disease by building immunity in the body. Therefore, the constant availability of essential vaccines is an important factor in community health. One of the most important vaccines is the diphtheria vaccine, which is usually used asiMultivalent diphtheria-tetanus-pertussis/i(DTP) combination vaccines. The production of this vaccine takes about 45 days, from the initial bacterial culture to the end of toxin production. However, the production of this vaccine can be optimized in case the production stages are carried out under normal conditions. In this study, a significant amount of impurities was removed after washing with phosphate buffer saline, and the toxin was then purified by Sephadex G-50. In this method, the toxin was concentrated to be stored in a smaller space (this removes the concerns for the provision of a suitable space). Another problem with the diphtheria vaccine is that it is reversible after detoxification of the toxin using formaldehyde. For this reason, it is suggested to use MPEG for detoxification, which will produce more stable covalent bonds between PEG and the first type of amine groups in the toxin chain. Tests were performed to evaluate factors, such as in vivo cytotoxicity, lack of edemas formation, the neutralizing activity of serum from guinea pigs immunized with the diphtheria toxoid inactivated with MPEG, and the immunogenic activity of the purified and modified toxin. Comparison of this PEG detoxification toxoid with the standard toxoid produced in Razi Vaccine and Serum Institution, Karaj, Iran, showed that washing with PBS and purification with Sephadex G-50 was an efficient method. The stability and reversibility of the toxoid approved by MPEG were acceptable. Therefore, the results of animal tests showed that the obtained product was stable and caused no wound or necrosis in the tested animals.