METHOD FOR PREPARING HUMAN INSULIN COMBINED PREPARATION Russian patent published in 2005 - IPC

FIELD: chemical-pharmaceutical industry, pharmacy.

SUBSTANCE: method involves preparing human insulin ester by transpeptidation reaction of porcine insulin in the excess of threonine di-tert.-butyl ester in an aqueous-organic medium in the presence of trypsin, inhibition of reaction by acidification, purification of prepared insulin ester by chromatography method, removal of protecting groups with trifluoroacetic acid and purification of prepared human crude insulin. Preparing part of the combined preparation of short effect by dissolving the prepared insulin with concentrations 15%, 25% or 50% in diluted acid and mixing with preserving agent solutions, isotonic agent and substances with buffer capacity. Preparing part of the combined preparation of prolonged effect by dissolving remaining insulin in diluted acid also followed by addition of zinc ions acid solutions and protamine sulfate, mixing with buffered solutions of m-cresol, phenol and glycerol, keeping the solution up to formation of crystals and combination of prepared parts of the combined insulin preparation. The transpeptidation reaction is carried out in the weight ratio trypsin to porcine insulin = 1:(300-1000) and before acidification of reaction medium additional inhibition of reaction is carried out by dilution of the reaction mixture with water by 2-3 times. Insulin ester is purified by HPLC method followed by precipitation of fractions with ester derivative in the presence of zinc ions, removal of protecting groups and preparing crystals of crude insulin that are purified by repeated HPLC method. Both purifying processes are carried out by using sorbent DIASOGEL ODS (C18) as immobile phase with particles size from 15 mcm and pores size 120 , and at the first stage 0.06 M glycine-HCl buffer with 0.015 M of ammonium sulfate and propanol-2 with concentration from 20% to 35% at pH 2.5 is used as mobile phase, and at the second stage 0.05 M acetate buffer with the content of propanol-2 from 10% to 20% at pH 2.5 is used. In preparing both parts of the combined preparation crystals of insulin are dissolved in diluted acid by stage wherein firstly finely dispersed suspension of insulin crystals in water is prepared followed by addition of diluted acid to it. In preparing the combined preparation of prolonged effect a mixture obtained after mixing with buffered solutions of m-cresol, phenol and glycerol is kept at temperature 18-21°C for 20-22 h. Invention provides the development of effective, economy method for preparing the combined preparation of human insulin with low immunological properties that allows enhancing yield of insulin in the process of its manufacturing.

EFFECT: improved preparing method.

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