RU2581019C1 - Method of purifying desmopressin (versions) - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to chemical-pharmaceutical industry and medicine. Invention discloses two methods of purifying desmopressin using low- and high-pressure liquid chromatography techniques.

EFFECT: methods provides removal of hard-to-remove impurities.

6 cl, 1 dwg, 1 tbl, 2 ex

Description

The invention relates to the field of chemical pharmaceutical industry and medicine.

Von Willebrand disease is a common hereditary bleeding disorder caused by quantitative or qualitative disorders of von Willebrand factor, a sticky glycoprotein commonly found in plasma, endothelial cells, subendothelial space, megakaryocytes, and platelets. The von Willebrand factor is involved in primary and secondary hemostasis, promotes platelet adhesion to the affected subendothelium, and is involved in platelet accumulation, especially in conditions of high breaking effects. It also serves as a protective courier for plasma factor VIII (FVIII). Therefore, factor defects in von Willebrand disease can cause bleeding and are expressed phenotypically by low plasma levels of coagulant VIII activity (FVIII: C) and prolonged skin bleeding time (BT), at least in the most severe and clinically obvious cases.

The main drugs used to treat spontaneous bleeding and to prevent bleeding during surgical procedures are desmopressin and plasma-derived coagulation factor concentrates.

Desmopressin (DDAVP) is a synthetic analogue of the antidiuretic hormone vasopressin. This is a well-tolerated and convenient hemostatic drug that can be used in many clinical conditions with hemorrhagic diathesis. The drug is available in concentrated forms for subcutaneous and intranasal use, which may be convenient for home treatment.

For use in medicine and for biomedical research, homogeneous highly purified preparations are required. Therefore, researchers in this field pay great attention to the development of new effective methods for the purification of hormonal drugs.

Patent SU 1747456 A1, July 15, 1992, relates to a method for purifying synthetic [8-arginine] vasopressin. The essence of the solution consists in column purification using a copolymer of 1-vinyl-2-pyrrolidone with N.N-dimethacroylhexamethylenediamine as a carrier.

US Pat. No. 5,500,413 describes a method for the production of desmopressin, which comprises the step of purifying by ion exchange chromatography on a cation exchange resin equilibrated with acid.

US20060148699 discloses a method for synthesizing peptides in the solid phase using a cationic amide resin followed by two purification steps to obtain pure desmopressin.

EP 0710243 (A1) discloses a process for the preparation and purification of cyclic peptides having a disulfide moiety in a single processing step, which simplifies synthesis and reduces production costs, but provides a high yield of pure peptide by direct ion exchange chromatography.

As the closest analogue, international application WO 2010119450 (A2) can be indicated, which relates to a method for producing 1-desamino-8-D-arginine of vasopressin (desmopressin) or its pharmaceutically acceptable salts, as well as to a method for purifying desmopressin or its pharmaceutically acceptable salts . The purification process of desmopressin is carried out by HPLC on a C-18 or C-8 type Novasep column with reverse phase. Purification of crude desmopressin is carried out by the gradient method, by elution with a gradient consisting of buffer A: water / acetic acid and buffer B: methanol / acetic acid. The purified pooled desmopressin fractions are then evaporated to remove the methanol solvent.

The concentrated pure pooled fractions thus obtained are lyophilized.

Upon receipt of desmopressin, a number of impurities are formed that affect the final effectiveness of the preparation, such as epimers and compounds with incompletely removed acetamidomethyl protection from one of the two thiol groups (impurities “hvost-l” and “hvost-2”). By the prototype method, epimers can be removed, however, impurities with protected groups are poorly separated by HPLC.

The present invention is to develop an improved new method for purification of desmopressin.

To solve the problem, two variants of a method for purifying desmopressin are proposed.

The first option involves dissolving the crude desmopressin in a 0.2-2% acetic acid solution, filtering, preliminary purification by low pressure liquid chromatography (LC) on a column with a sorbent representing a highly cross-linked styrene-divinylbenzene copolymer with a size of 75-150 microns, grade LPS-500, elution with an aqueous solution containing 5-15% isopropyl alcohol and 0.2-2% acetic acid, evaporation, purification by high performance liquid chromatography (HPLC (ACN) on a column with spherical silica gel type Vydac Denali C ₁₈ , elution e acetonitrile, evaporation and freeze drying.

Preliminary cleaning is carried out by the sorbent LPS-500. This sorbent is actively used for sorption purification of flavonoids, peptides, proteins, medicinal substances and radioactive isotopes, has a surface area of 700-800 m / g, pore volume of 1.5-1.8 ml / g, pore size of 50-500 Å.

Final cleaning is carried out using a Grace Vydac Denali C ₁₈ column, which contains a new generation sorbent based on silicon 120 Å monomeric C ₁₈ . Previously, the best monomeric С ₁₈ sorbent had a carbon coating range from 2.8 to 3.6 μmol / m ² . Denali C18 has a coating in an amount of 4 μmol / m ² , which approaches the theoretical limit of the surface area.

It is possible to carry out additional filtration before cleaning at a flow ultrafiltration unit with 100 kDa membranes.

The second option includes dissolving the crude desmopressin in a 0.2-2% solution of acetic acid, filtering, preliminary purification by low pressure liquid chromatography (LC ND) on a column with an LPS-500 sorbent, eluting with an aqueous solution containing 5-15% isopropyl alcohol and 0.2-2% acetic acid, purification by low pressure liquid chromatography (LC) on an ATP-bio Sulfopropyl column (SPS-Bio), elute with an aqueous solution of ammonium acetate purification by high-performance liquid chromatography (HPLC (ACN)) on a column with spheres eskim silica type Vydac Denali _C18, eluting with acetonitrile, evaporation and freeze drying.

The polymer hydrophilic sorbents SPS-Bio Sulfopropyl are crosslinked polyacrylate with sulfopropyl groups with a capacity of ionic groups of 2.0-2.5 meq / g. They are used for chromatographic purification of proteins and are Russian analogues of popular foreign sorbents: Sepharose FF, Toyopearl 650, Fractogel EMD. They have higher microbiological and hydrolytic stability and throughput.

It is possible to carry out additional filtration before cleaning at a flow ultrafiltration unit with 100 kDa membranes.

The possibility of carrying out the invention is demonstrated by the following examples.

Example 1

Weighing, archival sampling and HPLC analysis of raw desmopressin

The container with raw desmopressin is weighed on a technical balance with an accuracy of 0.1 g.

Portions of raw, received in different banks, most often differ from each other (in the content of desmopressin and impurities in them, in color, in solubility). For this reason, an HPLC analysis of each incoming batch of raw material is performed.

To prepare the test solution, a 40-50 mg sample of crude desmopressin, weighed on an analytical balance with an accuracy of 1 mg, is placed in a plastic test tube with a 50 ml screw cap, and the value is entered in the Raw table. Add by weight a thousand-fold amount of “50 mM aqueous solution of KH ₂ PO ₄ , pH 3.0” (Appendix 4.2.2) with an accuracy of 0.1 g. For example, if the weighed portion of desmopressin was 45.8 mg, then 45.8 g was added "50 mm aqueous solution of KH ₂ PO ₄ , pH 3.0." In this case, a solution with a concentration of 1.0 mg / ml is obtained. After this, the tube is shaken several times and placed for 10-15 minutes in an ultrasonic bath at 35 ° C until a homogeneous clear solution is obtained.

Then about 1 ml of the solution is taken with a disposable syringe, filtered through a filter with a pore size of 0.22 μm into a 1.5 ml vial (for analytical chromatograph), closed with a lid provided with a gasket, and the vial is marked in accordance with the marking of the can, for example, “DES12- 01-AF. "

The prepared test solution was analyzed by HPLC (Plat_grad_DES.lcm method on a Platinum Rocket C18 100A 7 × 53 mm column) and by HPLC (Kinetex_grad_DES (MeOH) 1 cm method on a Kinetex C18 300A 2.6 μm 3 × 100 mm column), analysis results recorded in an electronic journal (table "Raw") and determine the content of desmopressin in the batch of raw (in percent and weight). See chromatogram 1 in FIG. 1. If in the analysis of raw desmopressin on a Kinetex C18, 100A, 2.6 μm, 3 × 100 mm column, the impurity is “hvost-1” <0.5% relative to the peak of desmopressin and the content is “hvost-2" <2%, then purification is preferred carried out by the method of embodiment 1. If “hvost-1”> 0.5% relative to the peak of desmopressin or the content of “hvost-2"> 2%, then purification is preferably carried out by the method of embodiment 2.

Dissolution and filtration

Prepare a solution of "0.6% acetic acid (UK)" at the rate of 1.2 l per 100 g of raw. “0.6% of the UK” is poured into a 5-liter container at the rate of 1.0 liter per 100 g of raw desmopressin, but not more than 4 liters. When stirring on a magnetic stirrer, add raw desmopressin in portions of 20-40 g (tablespoon), then alter at room temperature for 1 hour. During dissolution, the pH of the crude solution should be periodically measured with indicator paper. If the pH value is above 5, you should lower it by adding acetic acid (in portions of 10 ml per glass, followed by checking the pH).

If a clear solution is formed (yellow or brown), then it is filtered through a glass pore filter. 3, laying the bottom of the glass filter with filter paper in a Bunsen flask. When filtering, a solution of raw desmopressin foams. It is necessary to filter with caution, preventing the transfer of foam into vacuum hoses.

If a cloudy or opalescent solution forms as a result, then the solution is clarified on a Pellicon flow ultrafiltration unit with 100 kDa membranes.

The filtered solution is circulated using a peristaltic pump under a pressure of 1.3-1.8 atm above the filters and the filtered filtrate is collected in a container. After passing through the filter elements more than 0.8 of the initial volume of the solution, it is diluted with purified water to 0.5 of the initial volume and the filtration is continued. The dilution is repeated three times. The last time filtering is carried out to the end (the pressure in the system spontaneously rises to 2.0 atm). The obtained filtrate (2.5-3 times larger in volume than the initial solution) does not contain visible mechanical impurities.

The filter elements are thoroughly washed with purified water and a 0.1 M solution of sodium hydroxide in purified water.

Isopropyl alcohol (IPA) was added to the filtered solution to a concentration of 2% by volume and mixed thoroughly.

Preliminary purification of crude desmopressin using low pressure liquid chromatography on a LPS-500 column

The choice of column depends on the amount of raw material. In this case, the load on the sorbent (the number of grams of raw material per liter of sorbent) is taken into account. The optimal value is 50-60 g of raw per liter of sorbent. At lower values, the column remains underloaded, while the separation results in an excessively dilute solution. At large values, the column is overloaded, separation is worsened.

200-400 g of crude are separated on column No. 1 (150 × 320 mm, 5.6 L, 75-150 μm).

400-700 g of crude are separated on a column No. 3 (150 × 480 mm, 8.5 l, 75-150 microns).

700-1300 g of raw material is separated on column No. 5 (200 × 530 mm, 16.6 L, 60 μm).

Column balancing

Prepare 20 l of "2.0% IPA and 0.6% of the Criminal Code." If necessary, additional quantities are prepared as they are consumed.

Connect column No. 3 to the K-Prime II and open the upper (input) and lower (output) cranes.

Balance the column with LPS-500 with a solution of “2.0% IPA and 0.6% CC". A crude desmopressin solution is applied to the LPS-500 column.

Desmopressin was eluted from the LPS-500 column. To do this, prepare 60 l of "8% IPA and 0.6% of the Criminal Code." Connect the capacitance with "8% IPA and 0.6% CC" to the input channel. The supply line valve is opened, which is omitted in “8% IPA and 0.6% of the Criminal Code”, while the line for applying the desmopressin-filtrate solution is closed with another valve. Start the execution of the method.

At the first separation, when the detector 1 AU reaches the reading, fractions are collected, and only 1 and 2 channels of the fraction collector are used:

- 1-5 fraction of 1.5 l,

- 6 - subsequent fractions - 3 l.

After the relative content of "hvost-2" exceeds 50% or the value of mAU drops below 0.1 AU, the collection of fractions is stopped, the separation method is stopped (the "Stop" button).

Fractions in which the purity of desmopressin by chromatogram <50% or the concentration of desmopressin <0.5 g / L are discarded. The combined fractions were evaporated on a Buchi Rotavapor R220 rotary evaporator to a concentration of about 100 g / L and sent to high pressure HPLC.

A column with LPS-500 is washed with “80% IPA and 2% CC” (15-25 L, the moment of washing is determined by the detector and visually by the disappearance of the color of the sorbent).

Purification of desmopressin by HPLC on a Grace 101mm column with Vydac Denali.

The final purification of the raw desmopressin is performed on an AZURA high-pressure chromatographic apparatus (Knauer, Germany).

Preparing for the HPLC process on a Grace 101mm. Prepare an aqueous solution of "12% acetonitrile (ACN) and 0.25% acetic acid UK" at the rate of: 15 l of eluent for one separation. Connect to pump flow switch A, input 1.

Prepare an aqueous solution of “50% ACN and 0.25% of the UK” at the rate of: 1.3 l of eluent per division. Connect to pump flow switch B, input 2.

Balance the column "12% ACN and 0.25% of the Criminal Code."

Switch the injector valve to the “Inject” position, flush the ASM2.1 pump with a balancing solution of “12% ACN and 0.25% UK” at a flow rate of 20 ml / min. At the same time, the leaving eluent is collected in a 50 ml graduated cylinder. Rinse until flow rate stabilizes.

Transfer the inlet capillary of the ASM2.1 pump into a container with a solution of desmopressin.

Using a pump ASM2.1, a solution volume containing 20 g of desmopressin is introduced into the column.

HPLC purification process. First division

Apply desmopressin. About 20 min (± 4 min), when the detector reaches 200 mAU, they begin to collect fractions of 0.25 L each, after passing the maximum intensity in the region of 23 min - 0.5 L to 40 min, then the last gradient fraction is collected.

The collected fractions were analyzed by HPLC. When sampling for analysis from fractions collected with a detector reading of 50 to 200, the solution is diluted 2 times, 200 to 1000 5 times, 1000 to 1500 10 times, more than 1500 20 times. Fractions with a desmopressin purity of less than 75% or a concentration of less than 1.8 mg / ml are discarded.

For the remaining fractions, the possibility of combining them into “Target HPLC fractions” is calculated.

- For this, the total purity (P) is determined by the formula when sequentially adding to fractions with desmopressin content not less than 98.5% of less pure fractions (where mi is the mass of desmopressin in the i fraction, pi is the percentage of desmopressin in the i fraction):

- For those impurities whose content in at least one of the combined fractions exceeds 0.5%, before combining, the total content of these impurities (A) is calculated after a possible combination of fractions according to the formula (where Si is the area of the peak of the impurity in the i fraction, Vi is the volume fractions, di - sample dilution, ai - percentage of impurity in i fraction):

The fractions for which in the "Target fraction of HPLC" P> 98.5% and the content of single peaks <0.5% are combined.

Subsequent HPLC Separation

The moment of the beginning of the collection of fractions in subsequent separations is determined based on the results of the first separation so that the beginning of the collection of 3 fractions in subsequent separations corresponds to the time of the start of collection of the first fraction that fell into the “Target” during the first separation. The collection of 1 fraction begins 2 minutes before the start of the collection of the third fraction. In this way:

- 1 fraction (discharge) - 1 minute (0.25 L);

- 2 fraction (according to the result of the analysis, it is determined either in “recyclable waste - prepeak” or in “target”) - 1 minute (0.25 L);

- 3 fraction (target) - up to the detector 100 mAU

- 4 fraction ("utilized waste - tail") - the moment of a sharp increase in signal intensity until the detector reads 20 mAU (about 3 min)

Fractions of the following separations are combined (respectively, “Recycled waste - prepeak” - 3 l plastic canister, “HPLC target fractions” - 10 l plastic canister, “Recycled waste - tail” - 5 l plastic canister) and stored in the refrigerator until evaporation. As 9-10 L of “target HPLC fractions” accumulate, they are analyzed by HPLC (Yupi_grad_DES.lcm Method). If the content of "hvost-l" <0.5%, evaporate to a concentration of 10-15% by weight and subjected to repeated HPLC-purification on a GRACE column of 101 mm

Using pump A, a solution volume containing 20 g of desmopressin is introduced into the column.

Desmopressin Concentration

After purification by HPLC and evaporation to an approximate concentration of 20 g / L, concentration is possible to remove excess acetic acid from the mixture. To do this, prepare a solution for separation on HPLC.

Preparing for the concentration process using HPLC.

Prepare an aqueous solution of "3.0% ACN and 0.25% of the UK" at the rate of: 10 l of eluent for one concentration. Connect to pump flow switch A, input 1.

Prepare "25% ACN and 0.25% UK" at the rate of 7 l per division. Connect to pump flow switch B, input 1.

Balance the column "3% ACN and 0.25% of the Criminal Code."

The inlet capillary equipped with a filter is immersed in a container with a solution of desmopressin to the bottom. Connect to pump flow switch A, input 2. Set the application time of 100 g of desmopressin at F = 200 ml / min. The calculated volume of desmopressin solution is applied to the column.

HPLC Concentration Process

The collection of 1 fraction begins with the beginning of the elution method. Collection of the second fraction - from the moment when the detector readings reach 500 mAU, until the detector readings are reduced to 200 mAU.

All collected fractions were analyzed by HPLC. When sampling analytics from fractions collected at a detector reading of up to 300, the solution is not diluted, from 300 to 1000 - diluted 5 times, from 1000 to 2000 10 times, over 2000 20 times.

Fractions containing less than 75% desmopressin are discarded. For the remaining fractions, the possibility of combining them into “Target HPLC fractions” is calculated.

To do this, the total purity (P) is determined by the formula when sequentially adding to fractions with a desmopressin content of at least 98.5% less than pure fractions (where mi is the mass of desmopressin in the i fraction, pi is the percentage of desmopressin in the i fraction):

- For those impurities whose content in at least one of the combined fractions exceeds 0.5%, before combining, the total content of these impurities (A) is calculated after a possible combination of fractions according to the formula (where Si is the area of the impurity peak in the i fraction, Vi - fraction volume, di - sample dilution, ai - percentage of impurity in i fraction):

The fractions for which in the "Target fraction of HPLC" P> 98.5% and the content of any single peaks <0.5% are combined.

Fractions of the following separations are combined (“concentrated HPLC fractions” - 10 L plastic container) and stored in a refrigerator until evaporated.

As 9-10 L of “concentrated HPLC fractions” accumulated, they were analyzed by HPLC and evaporated to an approximate concentration of 15 g / L. At the end of the series, the “50% IPA” column is washed.

Evaporation and lyophilization

The desired fractions are evaporated on a Buchi Rotavapor R220 vacuum evaporator at a bath temperature of not higher than 40 ° C to a concentration of about 100-200 g / l.

Pour into 2-3 kg pre-weighed pear-shaped flasks, mark and lyophilize.

The lyophilized substance desmopressin is stored in sealed flasks in a freezer (-19 ° C).

Example 2

Weighing, archival sampling and HPLC analysis of raw desmopressin

and Dissolving and filtering

As in example 1.

Preliminary purification of desmopressin raw using low pressure liquid chromatography on a column with LPS-500 (for example, column No. 2).

Column balancing

Prepare 20 l of "2.0% IPA and 0.6% of the Criminal Code." If necessary, additional quantities are prepared as they are consumed.

Connect column No. 2 to the K-Prime II and open the upper (input) and lower (output) cranes.

Balance the column with LPS-500 with a solution of “2.0% IPA and 0.6% CC". A crude desmopressin solution is applied to the LPS-500 column.

Desmopressin was eluted from the LPS-500 column. To do this, prepare 60 l of "8% IPA and 0.6% of the Criminal Code." Connect the capacitance with "8% IPA and 0.6% CC" to the input channel. The supply line valve is opened, which is omitted in “8% IPA and 0.6% of the Criminal Code”, while the line for applying the desmopressin-filtrate solution is closed with another valve. Start the execution of the method.

At the first separation, when the detector 1 AU reaches the reading, fractions are collected, and only 1 and 2 channels of the fraction collector are used:

- 1-5 fraction of 1.5 l,

- 6 - subsequent fractions - 3 l.

After the relative content of "hvost-2" exceeds 50% or the value of mAU drops below 0.1 AU, the collection of fractions is stopped, the separation method is stopped (the "Stop" button).

Fractions in which the purity of desmopressin by chromatogram <50% or the concentration of desmopressin <0.5 g / L are discarded. The combined fractions are evaporated on a Buchi Rotavapor R220 rotary evaporator to a concentration of about 100 g / l and sent to IOX on ATP bio (SPS bio).

A column with LPS-500 is washed with “80% IPA and 2% CC” (15-25 L, the moment of washing is determined by the detector and visually by the disappearance of the color of the sorbent).

Low Pressure Ion Exchange Chromatography on an ATP-Bio Packed Column (SPS-bio)

The combined fractions after preliminary purification on the LPS-500 are subjected to purification on a K-Prime-II chromatographic apparatus using ion exchange chromatography on a column packed with ATP-bio. Balance the column of 20 l of "0.6% of the Criminal Code." Connect column No. 2 to K-Prime II, open the upper (inlet) and lower (outlet) taps, substitute a drain capacity of 10 l under the outlet tubes of the fraction collector. Balance the column with SPS-bio “0.6% CC". A solution of desmopressin is applied to the SPS-bio column (70 μm, 4.2L, 150 × 240 mm).

After completion of the application process, the container from the solution is washed with 5 l of purified water, this solution is applied to the column. In the manual pump control mode, purified water is pumped through the column until the conductometer reading at the outlet of the column drops below 1 mS.

Desmopressin was eluted from the ATP-bio column. To do this, prepare 5 l of concentrate "4.0 M ammonium acetate." Prepare 30 l of “0.2 M NH40Ac”. Upon reaching the detector AT-5 (254 nm) 1 AU, the collection of fractions of 3 l is started. As the fractions are collected, they are analyzed by HPLC (we use the Kinetex_grad_DES (MeOH) .l cm method (Shimadzu Nexera2).) When taking samples for analysis from fractions collected when the detector was shown below 2, the solution is not diluted, from 2 to 3 the solution is diluted in 2 times, from 3 to 4 times 5 times, more than 4 times 10 times. After the detector readings drop below 0.5 AU, the collection of fractions is stopped, the separation method is stopped.

Fractions in which either the purity of desmopressin by chromatogram <50% or the concentration of desmopressin <0.1 g / l are discarded. The column with ATP-bio is washed, pumping 20 L of purified water (flow direction from top to bottom, a given flow rate of the mobile phase 300 ml / min, input line No. 1).

The column is washed with ATP-bio, pumping "0.1 M hydrochloric acid" until the pH at the outlet of the column is less than 2 (need about 30 l), then the column is washed with purified water until the conductometer reading at the outlet of the column drops below 1 mCm (need approximately 15 L). Balance the column with ATP bio by pumping 20 L of “0.6% CC” (Method DES_SPS_eq.opn; flow direction from top to bottom; target flow rate of the mobile phase 300 ml / min). Purification of desmopressin by HPLC on a Grace 101mm column with Vydac Denali.

The final purification of the raw desmopressin is performed on an AZURA high-pressure chromatographic apparatus (Knauer, Germany).

Preparation of HPLC purification

Prepare an aqueous solution of "3% ACN and 0.25% of the Criminal Code" at the rate of: 5 l of eluent for one separation. Connect to pump flow switch A, input 3.

Prepare an aqueous solution of "11% ACN and 0.25%" UK "at the rate of: 11 l of eluent for one separation. Connect to pump flow switch A, input 1.

Prepare an aqueous solution of “50% ACN and 0.25% of the UK” at the rate of: 1.3 l of eluent per division. Connect to pump flow switch B, input 2.

Balance the column “3% ACN and 0.25% of the AC” using the method “DES_eq_3% .met". In the container with desmopressin is immersed to the bottom equipped with a filter inlet capillary of pump A, input 2.

Using pump A, a solution volume containing 20 g of desmopressin is introduced into the column.

HPLC purification process. First division

Apply a solution of desmopressin.

After 25 minutes, when the detector reaches 200 mAU, they begin to collect fractions of 0.25 L, first, after passing the maximum intensity in the region of 30 minutes, 0.5 L to 45 minutes, then the gradient fraction is collected.

The collected fractions were analyzed by HPLC. When sampling analytics from fractions collected at a detector reading of 50 to 100, the solution is diluted 2 times, from 100 to 500 5 times, from 500 to 1,500 10 times, over 1,500 20 times. Fractions with a desmopressin purity of less than 75% or a concentration of less than 1.8 mg / ml are discarded.

For the remaining fractions, the possibility of combining them into “Target HPLC fractions” is calculated.

To do this, the total purity (P) is determined by the formula when sequentially adding to fractions with a desmopressin content of at least 98.5% less than pure fractions (where mi is the mass of desmopressin in the i fraction, pi is the percentage of desmopressin in the i fraction):

- For those impurities whose content in at least one of the combined fractions exceeds 0.5%, before combining, the total content of these impurities (A) is calculated after a possible combination of fractions according to the formula (where Si is the area of the impurity peak in the i fraction, Vi - fraction volume, di - sample dilution, ai - percentage of impurity in i fraction):

The fractions for which in the "Target fraction of HPLC" P> 98.5% and the content of single peaks <0.5% are combined.

Subsequent HPLC Separation

The moment of the start of collection of fractions in subsequent separations is determined on the basis of the results of the first separation so that the start of collection of 3 fractions in subsequent separations corresponds to the time of the start of collection of the first fraction that fell into the “Target” during the first separation. The collection of 1 fraction begins 2 minutes before the start of the collection of the third fraction. In this way:

- 1 fraction (discharge) - 1 minute (0.25 L);

- 2 fraction (according to the result of the analysis, it is determined either in “recyclable waste - prepeak” or in “target”) - 1 minute (0.25 L);

- 3 fraction (target) - until the detector reads 100 mAU;

- 4 fraction ("utilized waste - tail") - the moment of a sharp increase in signal intensity until the detector reads 20 mAU (about 3 min). As 9-10 L of “target HPLC fractions” accumulate, they are analyzed by HPLC. If the content of "hvost-1" <0.5%, evaporate to a concentration of 10-15% by weight and subjected to repeated HPLC-purification on a GRACE column of 101 mm

At the end of the series, the column “50% IPA and 1% CC” is washed.

Desmopressin Concentration and Evaporation and Lyophilization

As in example 1.

The method allows to obtain synthetic desmopressin with a purity of not less than 98.5% and a single impurity of less than 0.5%.

Effect: better cleaning of the target product.

As can be seen from the table, the separate use of the cleaning stages does not lead to high-quality cleaning of all impurities. When using the claimed options for the complete separation of gradient and epimeric impurities, there may be insignificant amounts of Hvost-1 and Hvost-1- up to 0.5%.

Claims (6)

1. The method of purification of desmopressin, including dissolving the crude desmopressin, filtering, purification by low pressure liquid chromatography, evaporation and freeze drying, characterized in that the purification by low pressure liquid chromatography is carried out on a sorbent column representing a highly cross-linked styrene-divinylbenzene copolymer with size 75-150 microns of the LPS-500 brand, using an aqueous solution containing 5-15% isopropyl alcohol and 0.2-2% acetic acid as an eluent, evaporate the obtained fractions, carry out complete purification by high performance liquid chromatography on a column with a spherical silica gel of the type Vydac Denali C ₁₈ , using an aqueous solution of acetonitrile and acetic acid as an eluent, followed by evaporation and freeze drying of the target product. 2. The method of purification of desmopressin according to claim 1, characterized in that they carry out additional filtration before cleaning at the flow ultrafiltration unit with 100 kDa membranes. 3. The method of purification of desmopressin according to claim 1 or 2, characterized in that after purification and evaporation to an approximate concentration of 20 g / l, the solution is concentrated until an excess of acetic acid is removed. 4. A method for purifying desmopressin, including dissolving the crude desmopressin, filtering, purification by low pressure liquid chromatography, evaporation and freeze drying, characterized in that the purification by low pressure liquid chromatography is carried out on a column with a sorbent representing a highly cross-linked styrene-divinylbenzene copolymer with size 75-150 microns of the LPS-500 brand, using an aqueous solution containing 8% isopropyl alcohol and 0.6% acetic acid as an eluent, the obtained fractions are evaporated, additional underbody purification by low pressure liquid chromatography on a column with a sorbent, which is a cross-linked polyacrylate with sulfopropyl groups with a capacity of ion groups 2.0-2.5 meq / g of the SPS-bio Sulfopropyl brand, using an aqueous solution of ammonium acetate as an eluent, followed by purification using a highly efficient liquid chromatography on a column with a spherical silica gel of the type Vydac Denali C ₁₈ using an aqueous solution of acetonitrile and acetic acid as eluent, and then evaporated and lyophilized to obtain I am the target product. 5. The method of purification of desmopressin according to claim 4, characterized in that they carry out additional filtration before cleaning at the flow ultrafiltration unit with 100 kDa membranes. 6. The method of purification of desmopressin according to claim 4 or 5, characterized in that after purification and evaporation to an approximate concentration of 20 g / l, the solution is concentrated until an excess of acetic acid is removed.

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