| Blood is composed of blood cells and plasma.Plasma accounts for about 4%of human body weight,of which 10%is solute,and the remaining 90%is water.In plasma solutes,protein accounts for about 70%,,inorganic salt accounts for 9%,the others are sugars,lipids,inorganic salts and metabolites（uric acid,urea,creatinine,etc.）.The highest protein content in plasma is human serum albumin（HSA）,its content is about 35～55 g/L plasma,which accounts for more than half of the total protein.It is relatively easy to extract in large quantities and with high purity[1].Human serum albumin is widely used clinically and has important value.Due to its special function,high price and limited supply of raw materials,the shortage of human albumin in the market has become increasingly prominent[2].At present,domestic blood product manufacturers all use low-temperature ethanol process to produce human serum albumin,and the process is basically the same.The main problems of large-scale industrial production by low-temperature ethanol process need to control strictly low-temperature environment,high input cost,low purity and low yield,high polymer content,and many other shortcomings.When the polymer reaches a certain amount,it can promote the immune response of the body.In order to minimize the adverse reactions of human serum albumin products,it is necessary to study and improve the preparation process and methods in order to prepare products with higher purity and human albumin monomers.This paper mainly improves the process of preparing human serum albumin by low-temperature ethanol method,by combining the optimization research of ion exchange chromatography process steps,in order to further improve product quality,yield,improve the production environment and reduce the production cost.The details are as follows:（1）Research on the entry point of low-temperature ethanol process combined with ion exchange chromatography process1)First,confirm the HPLC method for measuring the molecular size distribution of human serum albumin,and the system suitability test results meet the requirements of the 2020 edition of the Pharmacopoeia.2)The HPLC method was used to detect the samples of the intermediate process of producing human serum albumin in the low-temperature ethanol process.The results showed that the human serum albumin FIV supernatant prepared by the low-temperature ethanol process was subjected to the ultrafiltration process.The purity of human serum albumin is greatly improved,which can meet the cost and efficiency requirements of the chromatography process.Confirm the ideal chromatographic process entry point through evaluation,and design the process flow.In the process of preparing human serum albumin by the low-temperature ethanol method,the entry point after the FIV precipitation process was selected as the entry point for the ion exchange chromatography process.（2）Selection of gel types for chromatography and optimization of conditions1)After ultrafiltration of the FIV supernatant separated in the low-temperature ethanol process,different types of ion gels are used to perform ion exchange chromatography on the samples,and the collected samples are tested by HPLC.According to the experimental results,the weak anion Capto DEAE was finally selected as the gel for low-temperature ethanol combined with ion exchange chromatography.2)Using the Box-Behnken design response surface method principle,according to the experience of low-temperature ethanol process application,select the three most important factors that can affect the chromatographic effect:the pH value,conductivity and loading volume of the sample（the amount of protein that passes through the chromatography gel per milliliter）,using these three factors as independent variables,and the purity of human albumin monomer as the response value（Y）to establish mathematics model.The experimental results show that the optimal conditions for chromatography are:pH 6.76,conductivity 5.43 mS/cm,and sample load 0.50 g protein/ml gel.3)Taking into account the convenience of process integration in the production process of human albumin,product quality and production cost,,the final determination of the chromatographic conditions is pH 6.90,conductivity 5.00 mS/cm,and sample load ≤ 1.20g protein/ml gel,and predict that the purity of human serum albumin monomer in the chromatographic flow-through fluid is 98.41%.Theoretically,it is 3.37%higher than the highest value of human serum albumin monomer purity in the low-temperature ethanol process（95.04%after ultrafiltration）,which can effectively improve the purity of human albumin monomer.（3）Comparative study before and after process improvement1)Select Capto DEAE gel as the chromatographic medium,and select the chromatographic conditions as follows:pH value 6.90,conductivity 5.00 mS/cm,and sample load less than 1.20g protein/ml gel.The experimental results show that the purity of human albumin monomer can reach 97.98%-99.20%by adding the intermediate product prepared by the chromatography process.After the pasteurization process,due to the polymerization of a small amount of protein,the purity of the finished product monomer is reduced（97.77%）,but still far higher than the monomer purity of the finished product prepared by the low-temperature ethanol process（93.82%）.2)The key indicators of human serum albumin products prepared by low-temperature ethanol combined with ion exchange chromatography process and low-temperature ethanol process:comparative analysis of purity,polymer,PKA,Al3+.The experimental results show that,compared with the finished human serum albumin prepared by the low-temperature ethanol process,the finished product of human serum albumin prepared by low-temperature ethanol combined with ion exchange chromatography has higher purity,the content of polymer,PKA,Al3+ and miscellaneous protein is lower,so it is safer for clinical use.At the same time,it has more advantages in terms of production cost control such as hardware investment,energy saving and consumption reduction.Therefore,the low-temperature ethanol combined with ion exchange chromatography described in this article can be applied in the production of human serum albumin. |
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