Purification Of High Purity Bovine Serum Albumin And Quantity Method Of Two Serum Proteins

Bovine serum albumin(BSA)is the most abundant protein component in bovine plasma.It has the functions of scavenging free radicals,anticoagulation,maintaining plasma osmotic pressure and body fluid balance.Preparation of high purity BSA is of great significance for quality control of clinical pharmaceutical products.In this paper,the process of preparing high purity albumin by chromatography was established based on the crude commercially available bovine serum albumin.The process amplification research was carried out to achieve the scale of gram production.The product was freeze-dried and the powder with high purity was obtained.The albumin and transferrin were characterized and analyzed by mass balance method.One-step anion exchange chromatography purification process was established,and BSA with high purity was prepared from crude material.The factors such as pH gradient elution method,feeding pH,protein concentration and buffer composition are investigated.The optimal operating conditions were determined as follows: protein concentration 10 mg/mL,pH A;chromatographic medium DEAE Sepharose FF;buffer system with acetic acid-sodium acetate,concentration 20 mM,pH 5.2,elution buffer concentration 20 mM and pH was 4.6.Under the optimal separation conditions,the purity of the albumin product reached 99.5% and the yield was 62%.Then the process was enlarged to verify the feasibility and repeatability of the process.30 grams BSA was prepared.The purity of product was more than 99% by electrophoresis,which provided a basis for promoting the large-scale preparation of the purification process.In order to solve the problem of stability of liquid albumin preparations,freeze-drying experiments were carried out.The conditions were optimized and sodium octanate was added as a protective agent to ensure the stability of the molecular structure of albumin after freeze-drying.BSA and human transferrin(HTF)were characterized.The secondary structure and molecular weight of the two proteins were identified by a circular dichroism spectrometer and mass spectrometer respectively.The activity of BSA was determined by pyrogallol autooxidation.Transferrin can enhance the inhibitory effect of artesunate(Artesunate)on the proliferation of cancer cells.It was found that the cell survival rate of artesunate combined with transferrin was significantly lower than that of artesunate alone,indicating that the structure of transferrin was intact and its activity was maintained after purification.The water,main component and ash of the product were determined and the purity values of two proteins were determined by mass balance method.The purity of BSA was 96.71%,in which the principal component content was 99.70%,water content was 2.99% and ash content was 0.07%.The purity of transferrin was 91.21%,in which the principal component content was 95.86%,water content was 4.85% and ash content was 0.06%.