Preliminary Study On Separation And Purification Of Recombinant Human Serum Albumin From The Fermentation Broth Of Pichia Pastoris

Recombinant human serum albumin is an alternative to blood proteins,having the same physiological function with the human serum albumin. Due to have the advantage of not carry infectious virus, the recombinant human serum albumin have achieved widely attention. Recombinant human serum albumin mainly relies on the Pichia Pastoris gene recombinant to produce presently. The fermentation process would produce many impurities,so the separation and purification of target products is an important part of the industrialization of recombinant human serum albumin.This paper takes Pichia Pastoris recombinant human serum albumin fermented broth as the research object, using the technology such as membrane separation and chromatography to separation of recombinant human serum albumin. The path of separation and purification is fermented broth centrifugal process:centrifuging the fermentation broth?inactivated the protease of supernatant fluid through heating?the primary ultrafiltration separation?the secondary ultrafiltration separation?the adsorption decolorization?cation exchange chromatography?anion exchange chromatography. The main results are as follows:(1) The studied of recombinant human serum albumin solution through ultrafiltration separation technology.The molecular weight of recombinant human serum albumin is about 67 k D. Firstly,using the membrane with the molecular weight of 80 k D to wipe out the impurities with the molecular weight greater than 67 k D; Secondly, using the membrane with the molecular weight of 30 k D to wipe out the impurities with the molecular weight less than67 k D. During the process of ultrafiltration membrane separation, the best operating pressure is 0.15 MPa and the best operating p H value of 7.0.The concentration of the recombinant human serum albumin increasing from 2 % to 12 %, the purity of target achieved preliminary separation.(2) The studied of adsorption decolourization process of recombinant human serum albumin solution of ultrafiltrate.Through the static adsorption experiment, we have known that the adsorption capacity of LS-305 and LS-610 B is 68.0 ?AV·g-1 and 78.0 ?AV·g-1. The rate of protein loss is 3.32 % and 3.39 % respectively. Through the experimental data fitting, we have known that the correlation coefficient of Langmuir model is 0.9602 and 0.9761, the correlation coefficient of Second-order dynamic model is 0.9877 and 0.9975 respectively.Through the dynamic adsorption experiment, the pigment removal rate can reach 81.5 %,and the loss rate of recombinant human serum albumin was 3 %,when the initial conditions were as follows: the material liquid p H value is 6.0, flow of 40 m L·h-1, feed volume 200 ml(live macroporous resin filling amount to 20 m L).(3) The studied of the decoloring liquid chromatography purification technology of recombinant human serum albumin.The purity of recombinant human serum albumin can achieved 71 %,when the initial conditions were as follows: the balance of the buffer p H value of 3.7,and the elution of the buffer p H value of 5.9,the Na Cl concentration of 0.1 mol·L-1,during cation exchange chromatography experiment. The purity of recombinant human serum albumin purification is 95 %, when the initial conditions were as follows: the elution of the buffer p H value of 7.6, the Na Cl concentration of 0.5 mol·L-1. during the anion exchange chromatography experiment.The results showed that the process route of purification of recombinant human serum albumin from Pichia Pastoris fermentation broth is feasible. This study has certain reference significance to further study and enlarges industrial scale and optimization.