METHOD OF PRODUCING THIOPHOSPHATE OLIGONUCLEOTIDES FOR THERAPEUTIC USE Russian patent published in 2025 - IPC C12N15/113 C12N15/10 

FIELD: medicine.

SUBSTANCE: invention refers to medicine and can be used in pharmaceutical industry, namely for producing a drug for treating spinal muscular atrophy. Invention discloses a method of producing highly purified antisense oligonucleotide nusinersen, which employs a combination of high-efficiency solid-phase synthesis and purification. Specifically, disclosed is a method of producing a modified thiophosphate oligonucleotide containing 2'-O-(2-methoxyethyl)ribonucleotides, structure UCACUUUCAUAAUGCUGG, where each nucleoside contains 2'-O-(2-methoxyethyl) modified sugar residue, wherein the oligonucleotide is synthesized on a solid-phase carrier using an amide phosphite method, wherein the activator used is 5-(ethylthio)-1H-tetrazole (ETT), wherein the method of production involves the following successive operations: to obtain thiophosphate groups, hydride of xanthan (5-amino-1,2,4-dithiazole-3-thione) is used in 20% pyridine solution in acetonitrile, ammonolysis is carried out in 23-35% ammonium hydroxide solution for 16 to 24 hours, bringing value of specific electrical conductivity to 145.0-170.0 mS/cm, then purification is carried out on a hydrophobic sorbent selected from a group comprising Butyl Sepharose FF, Phenyl Sepharose FF, Phenyl-650M, Capto Phenyl with a granule size of 45 to 165 mcm to remove the main amount of low-molecular weight components and related impurities, performing the final detritylation of the intermediate semi-product, then chromatographic purification is carried out in the presence of polar aprotic solvents on an anion-exchange sorbent selected from a group comprising Q Sepharose FF, 30 Q Source, GigaCap Q650M, BioPro IEX SmartSep Q30 (YMC), WorkBeads 40Q, wherein acetonitrile and NaCl are added to the mobile phase for effective removal of related impurities of the modified thiophosphate oligonucleotide, and then purification is carried out by gel filtration chromatography on a sorbent selected from a group comprising Sephadex G-25, Superdex 75, HW-50F for removal of residual organic solvents and elemental impurities to obtain a modified thiophosphate oligonucleotide, the purity of which is not less than 96%, as determined by HPLC-MS.

EFFECT: high efficiency of purifying thiophosphate oligonucleotides while maintaining high output of the end product.

6 cl, 8 dwg, 5 tbl, 2 ex