# US 10,765,967 B2 · Process for purifying NADPH > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,765,967 B2 - Document ID: US10765967B2 - Status: Granted - Kind: B2 - Application number: 15/575,389 - Filed: 2016-12-14 - Published: 2019-11-07 - Granted: 2020-09-08 - Earliest claimed: 2016-12-14 - Assignees: Bontac Bio-Engineering (Shenzhen) Co., Ltd; Jiangxi Bontac Green-Biocatalysis Ecoindustrial Park Co.,Ltd - Applicants: Bontac Bio-Engineering(Shenzhen) Co.,Ltd; Jiangxi Bontac Green-Biocatalysis Ecoindustrial Park Co.,Ltd - Inventors: Qi Zhang; Dongmin Zhang; Rongzhao Fu - Web page: https://patentgrep.com/patent/US10765967B2 ## CPC - B01D 15/363: {Anion-exchange} - B01D 61/027: {Nanofiltration} - B01D 61/145: {Ultrafiltration} - B01D 61/147: {Microfiltration} - C07H 1/06: Separation; Purification - C07H 19/207: the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide - B01D 15/327: {with hydrophobic interaction} - B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28) - C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13) ## Abstract The present invention relates to a process for purifying crude NADPH product prepared by biocatalysis. The objective of the present invention is to solve the technical problems of low yield and low purity of the purified product in the existing ion exchange resin method. The present invention comprises sequentially the following steps: pretreatment, loading onto an ion column, elution of cations, pre-elution of impurities etc. The yield of the purification process disclosed in the present invention can be up to 85% or higher and the purified NADPH has a purity of up to 98% or higher. ## Claims ### Claim 1 A process for purifying NADPH, comprising the following steps: A. pretreatment: filtering a crude NADPH product prepared by biocatalysis through a microfiltration membrane to obtain a microfiltered crude NADPH product, wherein the microfiltration membrane is a hollow fiber membrane having a pore size of 0.1-0.5 μm; B. loading onto an ion column: loading the microfiltered crude NADPH product obtained in Step A onto an anion exchange resin column; C. elution of cations: after loading, washing the ion column with purified water until no cations are contained in the eluate; D. pre-elution of impurities: washing the ion column with a 0.07-0.5 mol/L sodium chloride solution until no substances having UV absorption at 260 nm are contained in the eluate; E. elution of product: washing the ion column with an aqueous solution containing 0.5 mol/L sodium chloride and 7% (V/V) ethanol, and collecting an eluate having UV absorption at 340 nm; and F. concentrating and drying the eluate obtained in Step E to obtain a purified NADPH product. ### Claim 2 (depends on 1) The process for purifying NADPH according to claim 1, wherein the anion exchange resin is a macroporous styrenic quaternary ammonium type I strong basic anion exchange resin. ### Claim 3 (depends on 2) The process for purifying NADPH according to claim 2, wherein the crude NADPH product is loaded in an amount of 9-15 g of NADPH per g of the anion exchange resin. ### Claim 4 (depends on 1) The process for purifying NADPH according to claim 1, wherein the process further comprises adjusting the crude NADPH product to pH 8.0-11.0 before Step A when the crude NADPH product has a pH less than 8.0-11.0. ### Claim 5 (depends on 4) The process for purifying NADPH according to claim 4, wherein the process further comprises adjusting the crude NADPH product to pH 9.0-10.0 before Step A when the crude NADPH product has a pH less than 9.0-10.0. ### Claim 6 (depends on 1) The process for purifying NADPH according to claim 1, wherein the process further comprises a step of pre-treating the anion exchange resin by soaking the anion exchange resin with a sodium hydroxide solution and washing with purified water. ### Claim 7 (depends on 1) The process for purifying NADPH according to claim 1, wherein the process further comprises a step of regenerating the anion exchange resin, wherein a regeneration solution used during regeneration is an aqueous solution comprising 1.0 mol/L sodium chloride and 0.1 mol/L hydrochloric acid, wherein a washing rate of the regeneration is 0.8-1.5 BV/H, and an amount of the regeneration solution used is 2.0 BV; wherein the resin is flushed with purified water after regeneration. ### Claim 8 (depends on 1) The process for purifying NADPH according to claim 1, wherein Steps C and D are under detection by a nucleic acid protein detector, wherein the eluate is collected when a reading of the nucleic acid protein detector starts to rise, and the collection is terminated when the reading starts to decline. ### Claim 9 (depends on 1) The process for purifying NADPH according to claim 1, wherein the concentrating of step F comprises microfiltration, ultrafiltration, and nanofiltration sequentially, wherein a hollow fiber membrane having a pore size of 0.1-0.5 μm is used for the microfiltration, an ultrafiltration membrane with a molecular weight cutoff of 10 K is used for the ultrafiltration, and a spiral wound membrane with a molecular weight cutoff of 100-400 is used for the nanofiltration. ### Claim 10 (depends on 1) The process for purifying NADPH according to claim 1, wherein the drying of step F is a vacuum freeze-drying. ### Claim 11 (depends on 2) The process for purifying NADPH according to claim 2, wherein the process further comprises adjusting the crude NADPH product to pH 8.0-11.0 before Step A when the crude NADPH product has a pH less than 8.0-11.0. ### Claim 12 (depends on 11) The process for purifying NADPH according to claim 11, wherein the process further comprises adjusting the crude NADPH product to pH 9.0-10.0 before Step A when the crude NADPH product has a pH less than 9.0-10.0. ### Claim 13 (depends on 2) The process for purifying NADPH according to claim 2, wherein the anion exchange resin is conducted with a pretreatment of soaking with a sodium hydroxide solution and washing with purified water. ### Claim 14 (depends on 2) The process for purifying NADPH according to claim 2, wherein the anion exchange resin is conducted with a regeneration, wherein a regeneration solution used during regeneration is an aqueous solution comprising 1.0 mol/L sodium chloride and 0.1 mol/L hydrochloric acid, wherein a washing rate of the regeneration is 0.8-1.5 BV/H, and an amount of the regeneration solution used is 2.0 BV; wherein the resin is flushed with purified water after regeneration. ### Claim 15 (depends on 2) The process for purifying NADPH according to claim 2, wherein Steps C and D are under detection by a nucleic acid protein detector, wherein the eluate is collected when a reading of the nucleic acid protein detector starts to rise, and the collection is terminated when the reading starts to decline. ### Claim 16 (depends on 2) The process for purifying NADPH according to claim 2, wherein the concentrating of step F comprises microfiltration, ultrafiltration, and nanofiltration sequentially, wherein a hollow fiber membrane having a pore size of 0.1-0.5 μm is used for the microfiltration, an ultrafiltration membrane with a molecular weight cutoff of 10 K is used for the ultrafiltration, and a spiral wound membrane with a molecular weight cutoff of 100-400 is used for the nanofiltration. ### Claim 17 (depends on 2) The process for purifying NADPH according to claim 2, wherein the drying of step F is a vacuum freeze-drying.