Study On The Protein Content Of Human Serum Albumin

Human serum albumin(HSA)is a biological product prepared by isolating healthy human plasma from low temperature ethanol and being inactivated by virus.It is used clinically for hemorrhagic,traumatic body,burn,cirrhosis With ascites and edema,malignancy,edema of kidney disease.At present,domestic enterprises use low temperature ethanol method to separate plasma protein,the process has high protein yield,low production cost,good economic benefits,and thus in the industrial production of large-scale application.However,the specificity of low temperature ethanol is not high,in the production process can not completely remove the protein composition,the enterprise process parameters are not the same,so there will be a certain amount of finished protein in the final protein.According to the literature,these o-proteins present in the preparation are one of the major causes of clinical adverse reactions [1].The types of adverse reactions are systemic hypersensitivity and multiple organ damage,including anaphylactic shock,heat-like reactions,kidney damage Wait.Therefore,the exploration of unknown proteins in blood products can provide a reference for clinical adverse reactions.The purpose of this study is to establish the method of isolating and isolating proteins and to determine the composition of heterozygous proteins in human serum albumin preparations.Data support,while laying the foundation for product consistency evaluation.In order to obtain a preliminary understanding of the differences in the content and types of heterozygous proteins in the enterprises,a total of 323 batches of albumin preparations were obtained for statistical analysis of the two batches of batches of data published in the past two years.The results showed that the purity of albumin preparations produced in the five enterprises ranged from 96.2% to 98.3%,and the differences among the enterprises were large.Which A production of the preparation of the purity of the preparation is more uniform,suggesting that the production process is more stable;B production of the preparation of the purity of the four companies than the other low,showing that there is a certain amount of unknown protein products.In order to further analyze the protein composition of each enterprise,the albumin preparations were analyzed by polyacrylamide gel electrophoresis(SDS-PAGE),and the similarities and differences of the components were explored.The results showed that the albumin preparations produced between enterprises were different,There are 8 to 9 bands of the protein of each enterprise preparation.In view of the limitations of cellulose acetate membrane electrophoresis and SDS-PAGE,we can not specifically identify the protein composition.In this study,we try to establish an unknown protein spectrum identification method,and then determine the unknown protein composition of human serum albumin.In the establishment of mass spectrometry method,the first test of the concentration of the test,and then optimize the mass spectrometry parameters.The results showed that albumin concentration exceeded 5mg / ml,more than the instrument identification threshold,resulting in albumin main components failed to be successfully identified,suggesting that protein concentration can not be too high.Protein concentration at 1mg/ml-2mg/ml,the main ingredient albumin peptide map is complete,but the unknown protein detection efficiency is low;the albumin peptide in the b/y ion continuity analysis found that protein identification score More than 150 points,the result has a high degree of credibility.The above results suggest that the main component protein concentration is too high to cover the unknown protein signal,the identification of unknown proteins need to further separation of unknown proteins.The separation and purification of albumin in different enterprises were carried out in combination with the preliminary results.The method of separation and purification was established and the method of separation and purification was established.The types,quantity,molecular weight,isoelectric point distribution and process related Sex.Alcohol was separated by anion exchange column and gel filtration column.The results showed that only one principal peak of principal component was obtained by using anion exchange chromatography,and three peaks were obtained by gel filtration chromatography.Therefore,gel filtration Chromatography separates the positional components of albumin.The results showed that the results of pepsin digestion were poor and the protein score was low,while the use of trypsin reproducibility was good and the protein identification score was higher.The effect of trypsin digestion was further confirmed.The results showed that there were 11 proteins detected in three parallel experiments.Among them,8 proteins were detected every time,indicating that the method was stable.Finally,trypsin was used to identify the unknown protein components in human serum albumin.The results showed that the products of the five production enterprises were divided into apo protein A-II Apolipoprotein A-II,Haptoglobin,Hempoexin,Alpha-1B-glycoprotein,α-2-HSglycoprotein(Alpha-2HS-Glycoprotein,and slightly different.The analysis of the isoelectric point of the unknown protein revealed that the isomeric point of the unknown protein was close to that of albumin and could be precipitated with albumin in the co-precipitation of ethanol.It was suggested that the unknown protein was a process-related protein.The molecular weight of the unknown protein was analyzed,Unknown proteins are present in albumin in the form of multimers.The above results show that in clinical applications,should pay more attention to the unknown protein hypersensitivity effect,in industrial production,should optimize the production process,as far as possible to remove the unrelated protein,improve product quality.The method of albumin preparation of hybrid protein mass spectrometry provides a data support for the study of clinical adverse reactions,which provides a new method for the evaluation of the consistency of albumin preparations,laying a foundation for the quality control of blood products,providing a new idea.