A reversed-phase high-performance liquid chromatography analysis method for recombinant human lysozyme

Abstract

The invention discloses a reversed-phase high-performance liquid-phase chromatographic analysis method of recombinant human lysozyme. The method adopts octadecyl silane bonded silica gel with an aperture of 20 to 50 nm and a particle size of 2 to 10 micrometers as a chromatographic column filler, so that not only can the macromolecular proteins such as the recombinant human lysozyme have a certain retention on the column, but also the excessively strong combination between the proteins and the filler can be prevented from leading to the incapability of elution; and the sodium chloride is added into a flow phase, so that the solubility of the recombinant human lysozyme in the reversed-phase high-performance liquid-phase chromatographic analysis process is ensured, the recombinant human lysozyme can be completely blended with the flow phase, a step number, symmetry, repetition and accuracy in quantitative analysis of the chromatographic peak are ensured, and the method is used for analyzing and quantitatively detecting the recombinant human lysozyme.

Description

technical field [0001] The invention relates to a reverse-phase high performance liquid chromatography analysis method of recombinant human lysozyme. Background technique [0002] High-performance liquid chromatography is a chromatographic method that uses a high-pressure infusion pump to pump the specified mobile phase into a chromatographic column filled with fillers to separate and measure the test product. [0003] The injected test substance is carried into the column by the mobile phase, and when the mixture carried in the mobile phase flows through the stationary phase, it interacts with the stationary phase. Due to the differences in the nature and structure of each component in the mixture, and the magnitude and strength of the force generated between the stationary phases, as the mobile phase moves, the mixture undergoes repeated distribution and equilibrium between the two phases, so that Each component is retained by the stationary phase for a different time, so...

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Problems solved by technology

This protein of recombinant human lysozyme has its special physical and chemical properties, and the inventor finds that there are following problems when analyzing and detecting by reversed-phase high-performance liquid chromatography: (1) recombinant human lysozyme has a molecular weight of 14000 Da, and its molecular weight is relatively large. The reversed-phase C18 column analysis often shows that the protein is too strongly bound to the filler to cause elution, which cannot be analyzed normally and the chromatographic column is damaged; (2) Recombinant human lysozyme is a salt-soluble protein, which can be dissolved in an aqueous solution of sodium chloride above 100mM Recombinant human lysozyme solution in aqueous sodium chloride solution lower than 100mM will appear in a suspended state; the mobile phase of commonly used reversed-phase high-performance liquid chromatography does not use high concentration of sodium chloride, which will lead to Recombinant human lysozyme is in an incompletely dissolved state, even if there are chromatographic peaks, it cannot accurately reflect its dose-effect relationship, and the incompletely dissolved protein will also damage the chromatographic system

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