RU2658426C1 - Method for producing nicotinamide adenine dinucleotide (nad) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, namely formation of nicotinamide adenine dinucleotide (NAD) from the culture medium of the biosynthetic process. Culture liquid containing NAD is filtered on Buchner funnel mixed with kieselguhr. Purify the resulting filtrate on a weakly basic anionite in acetate form to give a slightly colored solution containing NAD. Concentrate the resulting slightly colored solution by transferring it to the solid phase at a temperature of -5±1 °C for 5 hours. Solid phase is then melted at a temperature of 2±2 °C to obtain a liquid fraction. Concentrate the obtained fraction by lyophilization and isolate NAD from the concentrate by introducing ethyl alcohol in two steps. Separate impurities are separated when the alcohol concentration reaches 55–60 %, then the precipitate of amorphous NAD is separated at an alcohol concentration of 85–90 %.

EFFECT: method provides 8-fold reduction in the volume of the NAD solution subjected to lyophilization, which ensures the availability of the method for industrial use.

1 cl, 1 ex

Description

The invention relates to the production of a drug substance - nicotinamide adenine dinucleotide (NAD), corresponding to the formula

Nicotinamide adenine dinucleotide (NAD) is one of the most important coenzymes responsible for redox processes in cells. The potential role of NAD in the treatment of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, has now been identified. Also, preparations containing NAD are used in medicine in the treatment of diseases of the heart, stomach and liver.

Known methods for producing NAD by isolating a product from a culture fluid generated during biosynthesis [CN 105481923, US 4515943, US 2016340378]. In the methods of the listed patents, the preparation of NAD from the culture fluid includes filtration and / or ultrafiltration of the culture fluid, purification of the resulting solution by ion exchange chromatography or on nonionic sorbents to obtain low concentration NAD solutions, which are then concentrated by lyophilization or reverse osmosis. From the obtained concentrated solutions, NAD is isolated by introducing into the aqueous solution a water-miscible organic solvent, for example ethyl alcohol, acetone.

In all known methods for the isolation of NAD, concentration of aqueous NAD solutions with a low content of the target product (not more than 3 g / l) is required. The process of concentrating such dilute solutions is complicated by the low thermal stability of NAD, and therefore it is necessary to conduct a low-throughput lyophilization process with a large solution volume or reverse osmosis, accompanied by significant product losses.

Closest to the claimed method is the method according to US 3140281, in accordance with which NAD is isolated from the culture fluid by filtration on a Buchner funnel mixed with kieselguhr, preliminary purification on weakly basic anion exchange resin in acetate form, chromatography on macroporous sulfocathionite to obtain a dilute aqueous solution of NAD. The resulting NAD aqueous solution was concentrated by distillation of water under vacuum. Isolation of NAD from the concentrate is carried out by introducing ethanol into the solution, first in two doses, with the separation of the inactive precipitate when the alcohol concentration reaches about 60%, then when adding alcohol to the filtrate until the ethanol concentration reaches 90%, the NAD precipitate is precipitated. The resulting amorphous precipitate is filtered off, washed with ethanol, then with diethyl ether and dried in vacuum. Obtain NAD with a purity of about 80-90%.

A significant disadvantage of the method according to US 3140281 is the low yield of the product at the stage of concentration of NAD by distillation of water under vacuum. The extremely low thermal stability of aqueous NAD solutions leads to the destruction of NAD during evaporation with the formation of decomposition products. Experimental reproduction of this stage revealed that even at a vacuum below 10 mm Hg. one stripped off solution contains not more than 50% of the product taken for concentration.

The inventive method is aimed at increasing the output allocated from the culture fluid NAD, reducing the cost of organic solvents and simplifying the process.

The problem is solved by obtaining nicotinamide adenine dinucleotide from the culture fluid of the biosynthesis process by filtering the culture fluid, pre-cleaning the resulting solution, concentrating it and isolating the target product from the concentrated solution.

Distinctive features of the proposed method are:

Concentration of NAD by transferring the solution to the solid phase at a temperature of (minus 5 ± 1) ° С, melting of the formed solid phase at a temperature of 2 ± 2 ° С and collection of concentrated fractions formed during melting of the solid phase.

A positive result in the claimed method is achieved due to the following identified in the development process features of the allocation of NAD from the culture fluid:

• Translation of the diluted NAD solution into the solid phase at a temperature of (minus 5 ± 1) ° С, which is not accompanied by NAD destruction;

• Melting of the solid phase at a temperature of 2 ± 2 ° C occurs with selective accumulation of NAD in the liquid phase without the destruction of the target product;

Example

A 6.0 L culture fluid containing 15 g of NAD is centrifuged in a centrifuge. The filtrate is cooled and mixed with 1 kg of kieselguhr.

The mixture is filtered through a large diameter Buchner funnel, and the precipitate is washed with water. A combined filtrate of about 8 L volume is obtained, which is a yellow clear solution.

The resulting solution was passed through a column containing weakly basic anion exchange resin in acetate form. During the passage of the solution, most of the impurities consisting of nucleotide substances and pigments are adsorbed on the anion exchange resin. NAD is practically not adsorbed on anion exchange resin and passes into the filtrate. The first portions of the filtrate not containing NAD are separated from the main filtrate. The column is washed with a small amount of water to displace NAD from the anion exchange resin.

As a result of preliminary purification of the NAD solution on anion exchange resin, 8 L of a slightly colored solution containing 13.5 g of NAD (1.7 g / L) is obtained.

The solution is transferred to a polyethylene container and kept at a temperature of (minus 5 ± 1) ° C for 5 hours. The resulting solid phase (ice mass) is crushed and transferred to a vertical column with a diameter of 12 cm and a height of 100 cm, equipped with a jacket for supplying brine to maintain the temperature in the reaction mass at a level of 2 ± 2 ° C.

The liquid phase formed as a result of melting is collected in fractions of 500 ml. The first 2 fractions with a volume of 1.0 l with a concentration of 8.5 g / l are used for further concentration of NAD by lyophilization. The next 3 fractions with a volume of 1.5 l containing 4.2 g of NAD (concentration of 2.8 g / l) are returned to the cycle for subsequent concentration by freezing. The product yield at this stage is 94%.

A 1.0 L fraction with a concentration of 8.5 g / L is transferred to further concentration by lyophilization to obtain a NAD solution with a concentration of 25-30%. The resulting concentrate is adjusted with nitric acid to a pH of 2-3 and ethanol is introduced into the solution to achieve an ethanol concentration of 55-60%. Precipitated impurities consisting mainly of peptide compounds are separated by filtration. Ethanol is additionally added to the resulting solution until an ethanol concentration of 85-90% is reached. The resulting amorphous NAD precipitate was separated by centrifugation, washed with ethanol, then with diethyl ether and dried in vacuum. Obtain 7.7 g of an amorphous powder NAD with a purity of 85%.

Thus, the inventive method provides an 8-fold reduction in the volume of the NAD solution subjected to lyophilization, which ensures the availability of the method for industrial use.

The obtained amorphous NAD can be further purified by the method of DE 3141030 to obtain crystalline NAD with a purity of at least 95%.

Claims (1)

A method for producing nicotinamide adenine dinucleotide (NAD) from a biosynthesis culture fluid, including filtering a culture fluid containing NAD on a Buechner funnel mixed with kieselguhr, purifying the resulting filtrate on a weakly basic anion exchange resin in acetate form to obtain a slightly colored solution containing NAD, followed by concentration and the selection of the target product from a concentrated solution, characterized in that the concentration is carried out by transferring a slightly colored solution containing NAD, into the solid phase at a temperature of -5 ± 1 ° C for 5 hours and subsequent melting of the solid phase at a temperature of 2 ± 2 ° C to obtain a liquid fraction, then the obtained fraction is concentrated by lyophilization and NAD is isolated from the concentrate by adding ethyl alcohol in two doses with the separation of precipitated impurities when the alcohol concentration reaches 55-60%, then at an alcohol concentration of 85-90% the precipitate of amorphous NAD is separated.