The Purification And Purity Determination Of Albumin Reference Material

Albumin,accountting for more than 50% of the total plasma protein,is one of the most widely used protein in clinical.The reference material of human albumin is of great significance to the quality control of products and the evaluation of related analytical methods.In this paper,the purification method of human albumin from serum and pure urinary albumin was established.The process was further multigram-scaled up successfully.Then the structure and activity of the obtained product was characterized,and a quantitative method based on isotopic dilution mass spectrometry(ID-MS)was established.The human serum albumin(HSA)purification process of cold ethanol precipitation combined with two chromatography step from plasma was firstly studied.Most of the impurity proteins such as immunoglobulin and fibrinogen were removed with cold ethanol precipitation.On the basis of molecular structure and isoelectric point of HSA,the purification scheme of double ion exchange chromatography was selected.The chromatographic condition of pH6.0 and CM Sepharose FF was finally determined.Furtherly investigated the effect of different buffer on the cation exchange chromatography,the CEC had a acetate buffer system,loading sample at pH5.2 with a gradient elution of pH4.6.The purity of the purified product was more than 99.0% and yield reached 79%.In addition,study on the purification of human urine products was developed.One step process of gel filtration chromatography effectively separate impurities and the main components was established,and the purity of the final product reached 99% as well.Herein,we describe an accurate method for protein quantification based on conventional acid hydrolysis with an isotope dilution-high performance liquid chromatography–tandem mass spectrometry method.The analyte protein,human albumin,was characterized by HPSEC and circular dichroism(CD).The HSA secondary structure as well as function was proved no significantly varied.And the molecular weight was 66546 Da detected by MALDI-TOF Mass Spectrum.This experiment was based on key comprision CCQM-K78,the value assignment of four amino acids including Phenylalanine,leucine,isoleucine and proline in the hydrolysate was measured to calculate the protein content.In addition,the chromatographic and hydrolysis conditions were optimized.Methodological evaluation consisting of sensitivity,repeatability and accuracy proved the reliable method.The quantitative results can be traced to the SI unit.