Separation Of Allergic Impurities In Penicillin G Sodium

Penicillin is one of the most common causes of allergy. Most allergic drug reactions are induced by macromolecular impurities but not Penicillin itself, so remove of these macromolecular impurities is significant to reduce the incidence of allergy. Gel chromatography method as an efficient separation mode is easy to operate, and suitable for separating macromolecular impurities. Therefore, the theory and the process of the separation of macromolecular impurities with gel chromatography were studied in this thesis.The instability of penicillin G sodium (PGS) affects the separation of impurities. On this account, the effects of media, buffer concentration, pH and temperature on the stability of PGS were investigated. The stability of PGS in various media was in the order of: citrate buffer > acetate buffer > phosphate buffer > sodium bicarbonate > 0.9 % NaCl and 5 % glucose. In the case of the citrate buffer, to maintain stability for a long time, the molar concentration ratio of citrate buffer to PGS should be higher than 0.75. Low temperature and neutral circumstance were favorable for the stability of PGS.The separation of macomolecular impurities from PGS was studied on the gels of Sephadex G-25, G-10. The effects of media, buffer concentration and temperature on the separation of macromolecular impurities and the chromatographic behavior of PGS were investigated. Interactions between PGS and gels were discussed. The chromatographic behavior of PGS on the gels was not only dominated by size exclusion mechanism, but also affected by the secondary effects. The separation of macromolecular impurities was better on G-10 than that on G-25, but the broad peak of PGS on G-10 was not favorable for removing impurities. In buffers with high PGS loading, the secondary effects were turn to be small, and most of PGS eluted as size exclusion mechanism.The separation of macromolecular impurities on Toyopearl HW-40F gel was studied. Compared to G-10 column, the column efficiency of HW-40F column was higher and the separation of macromolecular impurities was more effective on it. At the same chromatographic condition, the interactions between PGS and HW-40F gel were larger. The adsorption effect between PGS and HW-40F was induced by hydrophobic interactions. High ion strength and low pH values of mobile phase were helpful to enhance the hydrophobic interactions. Even with a high loading, penicilloylated macromolecular impurities and penicilloic acid could be effectively removed when low concentration neutral citrate buffer was used as mobile phase.The adsorption equilibrium of PGS on HW-40F was studied by frontal analysis method. The effects of buffer species, concentration and temperature were investigated. The amount of PGS adsorbed on HW-40F decreased in the order of: citrate buffer, phosphate buffer, acetate buffer. The adsorption equilibrium data of PGS increased with increasing buffer concentration, and decreased with increasing temperature. In 0.005 mol/L citrate buffer, the adsorption equilibrium data could be modeled by Freundlich equation with average relative error of 1.881 %; in 0.02, 0.03, 0.05 mol/L citrate buffer, the adsorption equilibrium data could be modeled by Redlich-Peterson equation with average relative error of 3.573, 2.646, 2.118 %, respectively.The allergenicity of fractions separated by HW-40F column from degraded PGS solution and fresh PGS solution was examined by passive cutaneous anaphylaxis. The results showed that commercial PGS contained allergic impurities. Macromolecular impurities which could induce serious allergy might be constituted by penicilloylated derivative.