METHOD TO PRODUCE RECOMBINANT INSULIN GLARGINE Russian patent published in 2013 - IPC C12P21/02 C07K14/62 

FIELD: biotechnologies.

SUBSTANCE: method includes a stage of yeast cultivation, transformed by a vector containing a DNA sequence, determined by the formula X-B-Y-A, coding the precursor of insulin glargine, where X is a sequence of leader peptide, containing at least one amino acid. B is a B1-B30 sequence of amino acids of B-chain of the insulin glargine molecule. Y is a linker peptide containing at least two amino acids. A is an A1-A21 sequence of amino acids of an A-chain of a molecule insulin glargine, a stage of extraction of an expressing precursor of insulin glargine, a stage of crystallisation of the extracted precursor of insulin glargine, a stage of completion of fermentative conversion of insulin glargine precursor crystals at pH from 8 to 10 in presence of tripsin or tripsin-like ferment and water soluble organic dissolvents at the ratio from 40% to 60% of the reaction mix with formation of insulin glargine, containing at least one related admixture. Then the stage of insulin glargine treatment by reverse phase highly efficient liquid chromatography is carried out on a chromatographic matrix, using a polar organic buffer dissolvent in a water phase, containing a buffer based on organic acid, in which the matrix is first balanced with 10% acetonitrile in 250 mM of acetic acid with further elution of insulin glargine in the specified acetonitrile. Then the matrix is again balanced with 10% acetonitrile in the buffer on the basis of organic acid in concentration from 20 mM to 200 mM at pH from 3 to 8.5 with subsequent elution of insulin glargine in the specified acetonitrile, and further repeatedly the matrix is balanced with 6% ethanol in the buffer on the basis of organic acid in concentration from 10 mM to 50 mM with subsequent elution of the specified insulin glargine in the specified ethanol. Further the treated insulin glargine is deposited by means of addition of the buffer on the basis of citric acid and zinc chloride at pH from 6 to 8.

EFFECT: invention makes it possible to produce insulin glargine with high purity and low content of glycolised admixtures.

12 cl, 9 dwg, 8 tbl, 9 ex