A Novel Approach For Purification Of The Recombinant Human Serum Albumin From Transgenic Pig Plasma

At present,human serum albumin(HSA)is extracted by human blood.Due to the limited source to meet the market demand and the extremely complex source of human blood,there may be potential threats such as blood pollution,a safe and reliable HSA substitute with relatively low cost is urgently needed.Based on the rapidly developing gene recombinant technology,this technology can be used to produce recombinant human serum albumin(r HSA).As this technology can overcome the deficiency of blood supply and blood pollution in traditional methods,it has become the focus of r HSA research and production.The isolation and purification of r HSA from transgenic pig plasma must meet the requirements of clinical medication and biochemical research,therefore the approach for purification of r HSA is the key.In this study,a r HSA separation and purification method with high purity and yield from pig blood was established,laying a foundation for large-scale preparation of r HSA.This paper consists of four chapters as follows:In the preface of the first chapter,HSA and r HSA were summarized,several common albumin purification methods were briefly introduced,and the research objectives and significance of this topic were proposed.In the second chapter,r HSA was extracted from pig plasma by the improved method of low-temperature ethanol precipitation – thermal ethanol precipitation.We investigated and optimized the influence of p H,heating temperature,constant temperature,reaction time and dilution ratio of the reaction system on the extraction effect of r HSA.By reversed-phase high performance liquid chromatography(RP-HPLC)purity characterization results shows that the p H6.5,65? and 40 min and 2 times dilution multiple optimal reaction conditions,the purity of r HSA from pig plasma of 9.3% to 69.5% of hot ethanol precipitation.In chapter 3,we investigated the effect of cationic,anion,cationic and anion-cation exchange chromatography on r HSA after rough purification by thermal ethanol precipitation,and optimized the chromatographic conditions.The results showed that the r HSA crude purified by thermal ethanol precipitation was purified by anion exchange chromatography,and the best purification effect could be achieved.The purity of the product obtained by RP-HPLC was 85.0%.In chapter 4,in order to further improve the purity,two routes(reversed phase chromatography(route 1)and size exclusion chromatography(route 2))were used for the secondary fine purification of r HSA purified by anion exchange chromatography.RP-HPLC characterization results showed that the product purity of RP-HPLC and size exclusion chromatography reached 100.0%.By HPLC-MS /MS characterization,the purity of the two routes was about 97.33% and 93.77%,respectively,and the total recovery was about 41.1% and 38.6%,respectively.Although the thermal ethanol precipitation—anion exchange chromatography—reverse-phase chromatography(route 1)the purity and recovery rate relative to the thermal ethanol precipitation—anion exchange chromatography—size exclusion chromatography(route 2)are higher,but the chromatography of mobile phase organic solvent may lead to a target protein degeneration occurs,the structure of the protein may undermine limiting the application of the purified route;However,size exclusion chromatography uses low-concentration salt buffer with more mild conditions,which is beneficial to maintain the stability of protein in the purification process and is more suitable for r HSA purification.Therefore,route 2 is finally adopted as the r HSA purification scheme in transgenic pig blood.The established method is expected to extract high purity r HSA from transgenic pig plasma for its wider application.