Ultra-high performance liquid chromatography method for determining molecular size distribution of human serum albumin

Abstract

The invention discloses an ultra-high performance liquid chromatography method for determining the molecular size distribution of human serum albumin. The method comprises the step of determining themolecular size distribution of the human serum albumin by means of ultra performance liquid chromatography. In the ultra high performance liquid chromatography analysis, ACQUITY UPLC PROTEIN BEH SEC Column and a chromatographic column with 200 angstrom, the particle size of 1.7 micron, the column length of 150 mm and the column diameter phi of 4.6 mm are adopted, the column temperature is room temperature, a PBS buffer solution with the mobile phase of 0.2 mol L<-1> and the flow rate of 0.6 ml min<-1> is adopted, a Waters UPLC TUV ultraviolet detector is used for collecting data at 280 nm, thesample injection volume is 10 microliters, the volume of a strong probe washing solution is 200 microliters, the volume of a weak probe washing solution is 600 microliters, and a quantitative loop option is partial loop with needle overfill. According to the UPLC method for determining the molecular size distribution of the human serum albumin in the research, compared with original HPLC methods,the detection speed is increased by more than 10 times, and the chromatographic response is increased by more than 5 times. The UPLC method for determine the polymer content of a human serum albuminproduct is high in detection speed, simple and good in repeatability. The UPLC method helps to achieve high-throughput determination of human serum albumin multimers, and the efficiency of testing thehuman serum albumin is substantially improved.

Description

technical field [0001] The invention relates to the technical field of measuring human serum albumin, in particular to an ultra-high performance liquid chromatography measuring method for the molecular size distribution of human serum albumin. Background technique [0002] Polymers in human serum albumin mainly appear after the Bartholin virus inactivation process. With the completion of the virus inactivation step by heating at 60°C for 10 hours, a small part of albumin forms polymers by itself after heating, and A small part polymerizes with the existing heteroproteins to form multimers, that is, in addition to the factors affecting Barthera virus inactivation, the purity of albumin will also affect the multimer content. Human albumin is mainly used to treat hypoalbuminemia caused by various reasons, shock caused by blood loss trauma or burns, etc. The State Drug Administration issued a new batch release management method in 2018, which stipulates that both domestic and i...

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Problems solved by technology

The current statutory testing methods such as the 2015 edition of "Chinese Pharmacopoeia" and foreign pharmacopoeias including "European Pharmacopoeia", "British Pharmacopoeia" and other human serum albumin polymer determination methods are traditional HPLC methods, and the analysis process takes at least 60 minutes. Incremental release of human serum albumin from the factory and the time limit requirements for batch release inspection have conflicted, and the disadvantages of high inspection time and cost have begun to emerge. Therefore, it is particularly necessary to establish a new inspection method that is fast, accurate, and repeatable.

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