Method of production of human insulin methyl ester
Inventors
Buddha, Madhavan • Hazra, Partha • Goudar, Dinesh Channabasappa • Sathyanarayan, Srikanth G.
Assignees
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Publication Number
US-10611813-B2
Publication Date
2020-04-07
Expiration Date
Abstract
The present invention relates to production of Human Insulin methyl ester by enzymatic reaction. The present invention further relates to production and enhancement of purity of Human Insulin Methyl ester.
Core Innovation
The invention relates to enzymatic production of human insulin methyl ester (HIME). The disclosed method contacts insulin desB30 with L-threonine methyl ester in the presence of trypsin in a solvent system comprising water, dimethyl sulfoxide and dimethylformamide in specified ratios, and dioxane is present at about 1:0.2 (v/v) or less relative to water.
The dioxane/trypsin environment is directed to improve purity by reducing impurities described as desB(23-29) and the cyclized form of desB-30 during hydrolysis and subsequent coupling of L-threonine methyl ester to desB30.
The document also describes downstream crystallization of produced HIME by using citrate buffer and zinc chloride while maintaining the solution at about 12 °C or less to yield HIME microcrystals with improved purity.
Claims Coverage
The provided content includes one independent claim directed to a method for obtaining human insulin methyl ester and several dependent claim refinements, for a total of multiple inventive feature limitations covering solvent composition, dioxane level, trypsin and pH constraints, collection conditions, and crystallization conditions.
Enzymatic conversion using desB30 and LTME with trypsin in a specified multi-solvent system
Contacting, in the presence of trypsin, insulin desB30 with L-threonine methyl ester in a solvent comprising water, dimethyl sulfoxide and dimethylformamide in defined water/dimethyl sulfoxide and water/dimethylformamide ratios.
Including dioxane at or below a defined level in the trypsin solvent
The solvent further comprises dioxane, wherein dioxane is present in the solvent in a ratio of water/dioxane of about 1:0.2 (v/v) or less.
Defined trypsin amount during contacting
The method specifies contacting desB30 with L-threonine methyl ester in the presence of trypsin at selected trypsin amounts per mg of desB30, including about 20 U or more or about 50 U or more.
Defined contacting pH range
The method specifies contacting desB30 with L-threonine methyl ester in the presence of trypsin at a pH between about 6.0 and about 7.5.
Collecting the produced HIME after defined time and temperature
The method includes collecting the produced human insulin methyl ester after at least 7 hours at a temperature between about 18 °C and about 30 °C.
Crystallization using citrate buffer and zinc chloride at low temperature
The method further crystallizes produced human insulin methyl ester by adding citrate buffer and zinc chloride, while maintaining the solution at about 12 °C or less.
The claim coverage centers on enzymatic contacting of insulin desB30 with L-threonine methyl ester using trypsin in a defined water/dimethyl sulfoxide and water/dimethylformamide solvent system that includes limited dioxane, followed by collection under defined time and temperature conditions and crystallization using citrate buffer and zinc chloride at low temperature.
Stated Advantages
Improved purity by reducing impurities including desB(23-29) and the cyclized form of desB-30 during hydrolysis and coupling.
Reported purity enhancement versus DMF-based processes.
Improved purity of HIME microcrystals obtained by crystallization at about 12 °C or less with citrate buffer and zinc chloride.
Documented Applications
Production of human insulin methyl ester (HIME) using enzymatic processing of insulin desB30 with L-threonine methyl ester in the presence of trypsin and a solvent system including water, dimethyl sulfoxide, dimethylformamide, and limited dioxane.
Purification of produced HIME by downstream crystallization with citrate buffer and zinc chloride at about 12 °C or less to yield HIME microcrystals with improved purity.
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