Kanamycin A, B Component Separation Purification Of New Technology Research

The domestic current Kanamycin A separating technology is static adsorption and dynamic eluation processing based on 732 strong acid cation-exchange resin. A new extraction and purification technology of Kanamycin A and B was developed in this work.Kanamycin A and B are soluble weak polybases. According to the determined data in our potentiometric titration experiments ,the polybasic ionization constants of Kanamycin A and B were estimated by means of two algorithm, Stepwise Linear Regression and Simultaneous Equations Algorithm and Rosenbrock Algorithm. The calculated polybasic ionization constants of Kanamycin A were similar to the reported data. The stepwise ionization constants of Kanamycin B pKa,=5.61, pKa2=6.56, pKa3=7.43, pKa4=8.22, pKaj =9.11, were first reported. The distribution of different Kanamycin A and B charged-ion in different pH conditions was simulated.After comparison test of eight cation-exchange resins , D-186 cation resin was sorted out to separate Kanamycin B from the fermented broth, which was characterized of higher adsorption rate of Kanamycin B compared with Kanamycin A and lower impurity adsorption rate. The orthogonal design and analysis of experiments was applied to optimize the operation condition during adsorption ( pHV.O, flow rate 1.5V/V/h. ) and the condition during impurity elution ((NH4)2SO concentration 1.0%(VAO, pH7.5, flow rate 1.25V/V/h ). A new technology of dynamic absorption and dynamic eluation involving one D-186 resin and three 732 resins column was developed, which was able to concentrate Kanamycin B effectively and guarantee the quality of final product of Kanamycin A in abnormal fermentation.D-186 resin was also selected to separate Kanamycin A and B from crystallization residue. Then the optimum operation condition of extracting Kanamycin and purging away impurity is also determined. The concentration of diluted crystallization residue was 12600 u/ml, pH 7.0, flow rate 1.5V/V/h; and the concentration of (NH4)2SO4 was 1.5%(V/V), pH 7.5, flow rate 1.75V/V/h. The dynamic absorption and dynamic eluation technology incuding three columns of D-186 resin was established. The recovery rate ofKanamycin A and B was respectively 80% and 93%. The elimination rate of impurity was about 99%.The collected fractions Kanamycin B from fermented broth and crystallization residue solution weremixed and then concentrated to 30-35 X 105u/ml, contained about 35% Kanamycin B. Based on the principle of separation chromatography, Kanamycin B was separated and purified by H401 anion-exchange resin. The optimum technological conditions were determined: the diameter of resin 0.20mm, the linked-degree of resin 2.5 , and the adder quantities 1.05ug/ml (resin), the eluant concentration of NH4OH 0.6%(V/V), the elution rate 0.5 ml/min for a column of 1> 1.8cm X 300cm. The purity of Kanamycin A\B was 99.5% and 90%.