Chromatographic purification method for acylated insulin

Abstract

The invention discloses a chromatographic purification method for acylated insulin and belongs to the field of preparation of recombinant insulin or insulin analogs. According to the chromatographic purification method for acylated insulin, high-grade silica gel carriers (like C4, C8 and C18 alkanes taken as reversed phase filler of ligands) are taken as chromatographic filler, a high-pressure chromatographic system is used for performing fine separation and purification on acylated insulin, the purity of a coarse product can be improved from 50%-60% to 99.0% or higher in the alkaline eluent environment with pH (potential of hydrogen) being 6.5-8.0, and the yield of the purified sample can be higher than 90%. Besides, according to the method, the loading quantity of coarse samples is larger, and the purification cost is greatly saved; the linear flow speed is high in the chromatographic purification process, and the insulin analogs can be rapidly purified. The chromatographic purification method is simple, stable and reliable to operate, has higher amplification and is suitable for industrial preparation of the insulin analogs.

Description

technical field [0001] The invention relates to a reverse-phase chromatographic purification method for acylated insulin, which belongs to the field of preparation of recombinant insulin or insulin analogues. Background technique [0002] Diabetes is a common metabolic endocrine disease that seriously endangers human health. Acylated insulins are neutral, soluble, long-acting insulin analogs, such as insulin detemir, lysine B29N(ε-tetradecanoyl) to (B30) human insulin, the first to use chemical The modified method is produced by acylation of amino acids on the peptide chain with fatty acids that can reversibly bind to albumin in blood. Acylated fatty acids can stabilize the self-aggregation of insulin molecules and reversibly bind to albumin, making acylated insulin absorb slowly from the subcutaneous injection site and prolong the duration of action, which can reduce the risk of hypoglycemia while controlling blood sugar. [0003] In the history of insulin development, th...

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Problems solved by technology

In comparison, although the equipment maintenance cost of using organic polymer fillers for purification is low, it has many obvious disadvantages: first, the particle size of organic polymer fillers (generally above 30 microns, up to hundreds of microns) is smaller than that of The particle size of ordinary high-grade silica gel carrier filler (generally between 5-20 microns) is much larger. Due to the poor pressure resistance of the medium itself, the pressure bearing capacity of the column packing process is limited, and the medium inside the packed column will not be dense and uniform. The number of theoretical plates generally does not exceed 20,000, and the efficiency of the newly packed chromatographic column is not high, resulting in a low degree of separation of the sample. It is difficult to achieve a purity of more than 98% after purification and the yield is low, which will significantly increase the product Second, the particle size of the organic polymer filler is generally between 5-300 μm, the particle uniformity is poor, and the pressure resistance is low. The filler will be broken during the packing of the chromatography column and the purification process, resulting in Collapse inside the chromatography column leads to a decrease in column efficiency, a decrease in resolution, and a further decrease in yield

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