WO2017114384A1

WO2017114384A1 - Method for purifying and oxidizing polypeptide containing disulfide bond

Info

Publication number: WO2017114384A1
Application number: PCT/CN2016/112355
Authority: WO
Inventors: 尹传龙; 宓鹏程; 陶安进; 袁建成
Original assignee: 深圳翰宇药业股份有限公司
Priority date: 2015-12-31
Filing date: 2016-12-27
Publication date: 2017-07-06
Also published as: CN106928316B; CN106928316A

Abstract

The present invention provides a method for purifying and oxidizing polypeptide containing disulfide bond simultaneously. Specifically, the crude peptide on the chromatographic column is rinsed with mobile phase A and phase B, wherein the phase A contains an oxidizing agent for oxidizing disulfide bond, therefore, the disulfide bond is oxidized during the purification, and after the purification, the oxidized polypeptide is obtained.

Description

Method for purifying and oxidizing a disulfide-containing polypeptide

Technical field

The invention relates to a method for purifying a polypeptide drug.

Background technique

The disulfide bond formed between one or more pairs of cysteine residues plays an important role in maintaining the spatial conformation of the polypeptide as well as certain biological activities. Many existing drugs require the presence of disulfide bonds to be active, such as, for example, octreotide, nesiritide, eptifibatide, atosiban and terlipressin, etc., thus oxidation of disulfide bonds Has always been the focus of research in the peptide industry.

At present, the conventional method for oxidizing a disulfide bond of a polypeptide is first oxidized by using an oxidizing agent such as air, hydrogen peroxide or iodine, and then purified by reverse phase liquid chromatography to obtain a final product. Although the method can complete oxidation in 15min-30min, the oxidation rate is faster, but after the oxidation reaction is stopped, it needs to be purified by reverse phase liquid chromatography to obtain the desired product. Different oxidation methods are different in the process before the next reverse phase chromatography purification. For example, eptifibatide is oxidized by hydrogen peroxide according to a conventional method, and the solution volume is large, and multiple filtration is required before purification; nexilide is first oxidized with oxygen, and then subjected to rotary evaporation and filtration before purification. The steps of these methods are too cumbersome, and there are many parameters to be controlled, which is not conducive to the control of the quality of the polypeptide. The stability of the polypeptide itself is poor. Especially for some very unstable polypeptides, after the termination of the oxidation reaction, the strong oxidant may have destroyed the structure of the polypeptide itself and cause the polypeptide to denature before the subsequent step. Therefore, it is particularly important to reduce the oxidation operation steps and shorten the treatment time to increase the oxidation yield and control the product quality. In order to solve these problems, the present invention provides a method for completing oxidation of a polypeptide while purifying. The invention not only simplifies the operation process, but also shortens the processing time of the whole process, and further avoids structural instability caused by long-term contact between the polypeptide and the strong oxidant, and the yield is reduced.

There are several ways to oxidize the disulfide bonds of the polypeptide. One is to oxidize the peptide during solid phase or liquid phase synthesis. After cleavage, the oxidation product is obtained, and then purified, such as: CN 102827249 A; Solid phase synthesis of linear crude peptide, after cleavage, the linear crude peptide is dissolved and then subjected to liquid phase oxidation, and then purified, such as: CN 103304655 A, CN 104710509 A; or the linear crude peptide is purified to improve the purity and then disulfide oxidation. In order to improve the purity or reaction efficiency of the oxidation products, but each disulfide oxidation method has its advantages and disadvantages, solid phase oxidation is more suitable for some short peptides or some peptides that only need to form a pair of disulfide bonds; Oxidation is time consuming and laborious, but the purity after oxidation is relatively high, which facilitates subsequent purification. In order to improve the safety of the drug, the higher the purity of the existing peptide drugs, the better the purity is required to be greater than 99%, and the single impurity is required to be controlled. The single impurity is less than 0.15%, and the solid phase oxidation impurities are more, which is not conducive to quality. Control; if the liquid phase oxidation is used for purification, although the liquid phase oxidation is complete, the purity is high, but the oxidation treatment process More cumbersome, time-consuming and laborious.

The invention provides a new liquid phase disulfide bond oxidation method, which is different from the traditional oxidation idea, can make up for the solid phase oxidation yield and low purity, and the traditional liquid phase oxidation method has the disadvantages of time-consuming and laborious operation, and the operation is simple and the oxidation purity is greatly improved. And yield, and easy to scale up production.

Summary of the invention

The invention relates to a novel liquid phase oxidation method, which realizes disulfide oxidation of a polypeptide drug linear crude peptide directly on a preparative chromatography column. The main method is to use the oxidant-containing solution as the A1 phase and the organic solvent as the B phase. The peptide drug is oxidized while the peptide drug is gradient-eluted on the column, and the collected sample is the oxidized peptide drug substance. .

The present invention relates to a method for purifying a oxidized disulfide-containing polypeptide, wherein the crude peptide to be purified and oxidized is separated on a chromatographic column while being oxidized; the crude peptide is eluted while being eluted with a mobile phase containing an oxidizing agent;

Preferably, the following steps are included:

The crude peptide is washed with mobile phase A and phase B,

Wherein the phase A is an aqueous solution of an inorganic salt, and the phase A further comprises an oxidizing agent that oxidizes a disulfide bond;

Further, the oxidizing agent in the phase A is selected from one or more of hydrogen peroxide, DMSO, iodine, and a metal ion oxidizing agent (Fe ³⁺ , Cu ²⁺ , Ag ⁺ ); preferably, the metal ion oxidizing agent is selected from the group consisting of Fe ₂ O ₃ .

Further, the oxidizing agent in the phase A is hydrogen peroxide or DMSO, and the pH of the phase A is 7.5-9.0.

Further, the oxidizing agent in the phase A is iodine and has a pH of 2.5 to 9.0, preferably 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0.

Further, the number of moles of the oxidizing agent is 2 to 10 times, preferably 3, 4, 5, 6, 7, 8, 9 times the number of moles of the crude peptide.

Further, the phase A contains water and an inorganic salt, and the phase B contains an organic solvent. Preferably, the phase B contains one or more of acetonitrile, methanol, isopropanol, ethanol, and tetrahydrofuran, and the inorganic salt is selected from the group consisting of phosphoric acid. One or more of potassium dihydrogenate, sodium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, and sodium chloride.

Further, the column stationary phase is selected from the group consisting of a silica gel matrix, a polymer polymer, and zirconia.

Further, the disulfide-containing polypeptide is selected from the group consisting of octreotide, eptinol, nesiceptide, atosiban, terlipressin, ziconotide, linaclotide, desmopressin, and the like.

The elution procedure and eluent used in the present invention may employ any procedure and eluent capable of separating the polypeptide.

Further, the elution procedure is that the inorganic salt solution is used as the mobile phase A phase, the oxidant is added to the phase A, and the mixture is uniformly mixed; and the chromatographically pure methanol is used as the phase B. The wave was detected at a flow rate of 60-80 ml/min at 230 nm. Phase B elution gradient: B%: 15% - 35%, 50-70 min.

Further, the elution procedure is such that the inorganic salt solution is used as the mobile phase A, and is added to the oxidizing agent to be uniformly mixed; the chromatographically pure methanol is used as the phase B. Flow rate: 200 ml/min. Detection wavelength: 230 nm. Phase B elution Gradient: B%: 8% - 20%, 50-70 min.

Further, the elution procedure is such that the inorganic salt solution is used as the mobile phase A, and is added to the oxidizing agent to be uniformly mixed; acetonitrile is used as the phase B. Flow rate: 200 ml/min. Detection wavelength: 230 nm. B phase acetonitrile elution gradient: B%: 18% - 28%, 50-70 min.

Further, the elution procedure is such that the inorganic salt solution is used as the mobile phase A, and is added to the oxidizing agent to be uniformly mixed; ethanol is used as the phase B. Flow rate: 200 ml/min. Detection wavelength: 220 nm. Phase B ethanol elution gradient: B%: 25% - 45%, 50-70 min. The invention combines oxidation and purification into one, one step, saves time and effort, realizes effective separation of isomer impurities and other difficult separation impurities in the crude peptide while oxidizing, and then converts to acetic acid by reverse phase HPLC method. Salt or other salt forming forms ultimately increase the yield and purity of the product. At the same time, the solid phase oxidation yield, low purity, and liquid phase oxidation are disadvantageous. The method is simple and convenient to operate, and is advantageous for realizing large-scale preparation, and provides a new method for liquid phase oxidation.

The column described in the present invention is a silica gel matrix, a polymer polymer, a zirconia type reversed-phase chromatography column, and includes C1, C4, C8, C18 and other types of columns. Both can be used for the stationary phase of the liquid phase disulfide oxidation process.

All solutions which can dissolve the disulfide oxidizing agent can be used as the eluted flowing phase A of the present invention, including water, various inorganic salts and the like. Phase B can be various organic phases such as acetonitrile, methanol, isopropanol and the like.

The pH limitation range of the present invention varies according to the disulfide bond oxidation mode, and the oxidation mode of the hydrogen peroxide, air, and DMSO in an alkaline environment is limited to a pH range of 7.5-9.0; when the iodine is used, the pH is limited. It is 2.5-9.0; the metal ion is used as the disulfide oxidant for all pH, preferably the pH range is 1.5-11.5.

The disulfide oxidizing agent according to the present invention is many, and is not limited to one or more of DMSO, hydrogen peroxide, iodine, and metal ion oxidizing agent, and any oxidizing agent dissolved in the eluting mobile phase can be used as the oxidizing agent of the present invention.

The concentration of the oxidizing agent of the present invention is mainly 2 to 10 times the number of moles of the substance to be purified.

DRAWINGS

Figure 1 is a linear crude peptide mass spectrum.

Figure 2 shows the sperm peptide mass spectrum.

3 is an HPLC chromatogram after separation and purification of Example 1.

4 is an HPLC chromatogram after separation and purification of Example 2.

Figure 5 is a HPLC chromatogram after separation and purification of Example 3.

Figure 6 is a HPLC chromatogram after separation and purification of Example 4.

Figure 7 is a HPLC chromatogram after separation and purification of Example 5.

detailed description

The different column sizes that can be used include: 5 cm x 25 cm (column diameter x length), 10 cm x 25 cm, 15 cm x 25 cm.

Example 1: Purification of crude octreotide peptide

2.0 g of octreotide linear crude peptide was dissolved and filtered, and the filtrate was collected for use.

Column: octadecyl bonded silica gel column, the column size is 5cm × 25cm.

making process:

100 mmol/L potassium dihydrogen phosphate solution was adjusted to pH 8.5 with aqueous ammonia as mobile phase A phase, 10 g of hydrogen peroxide was added to phase A, and the mixture was uniformly mixed; and chromatographically pure methanol was used as phase B. The wave was detected at a flow rate of 60-80 ml/min at 230 nm. Phase B elution gradient: Gradient elution of the sample with a gradient of B%: 15% - 35%, 50-70 min. The purification is completed while the oxidation is completed.

The oxidized purified polypeptide obtained in the above step is formed into a stable salt, and after lyophilization, a standard octreotide having a purity greater than 99.0% can be obtained.

After lyophilization, 0.75 g of a white powdery solid sperm peptide was obtained. The purity is 99.28%, and the individual impurities are less than 0.15%. The purification yield was 68% (calculated as the octreotide content in the crude product), the total yield was 37.5%, and the purified batch required a total of 1.6 h.

Example 2: Purification of octreotide crude peptide

15 g of octreotide crude peptide was dissolved and filtered, and the filtrate was collected for use.

1 column: octadecyl bonded silica gel column, the column size is 10cm × 25cm.

A 100 mmol/L sodium dihydrogen phosphate solution was adjusted to pH 8.0 with aqueous ammonia as mobile phase A, and 150 g of hydrogen peroxide was added; and chromatographically pure methanol was used as phase B. Flow rate: 200 ml/min. Detection wavelength: 230 nm. Phase B elution gradient: Gradient elution of the sample with a gradient of B%: 8%-20%, 50-70 min. The purification is completed while the oxidation is completed.

After lyophilization, 6.1 g of a white powdery solid peptide was obtained. The purity is 99.30%, and the individual impurities are less than 0.15%. The purification yield was 73.9% (calculated as the octreotide content in the crude product), the total yield was 40.6%, and the purified batch required a total of 1.6 h.

Example 3: Purification of octreotide crude peptide

25 g of octreotide crude peptide was dissolved and filtered, and the filtrate was collected for use.

Column: Octadecyl bonded silica gel column with a column size of 15 cm x 25 cm.

A 100 mmol/L sodium dihydrogen phosphate solution was adjusted to pH 7.5 with aqueous ammonia, and 150 g of hydrogen peroxide was added as the phase A; and chromatographically pure methanol was used as the phase B. Flow rate: 200 ml/min. Detection wavelength: 230 nm. Phase B elution gradient: Gradient elution of the sample with a gradient of B%: 8%-20%, 50-70 min. The purification is completed while the oxidation is completed.

After lyophilization, 8.9 g of a white powdery solid peptide was obtained. The purity is 99.30%, and the individual impurities are less than 0.15%. Purification yield 64% (calculated as octreotide in crude), total yield 35.6%, total purification required 1.6h.

Example 4: Purification of morphine peptides

250 g of the eptipeptide crude peptide was dissolved, and then the solution was passed through a 0.45 μm filter for about 1 hour, and the filtrate was collected for use.

Column: Octadecyl bonded silica gel column with a column size of 15 cm x 25 cm.

A 50 mmol/L sodium dihydrogen phosphate solution was adjusted to pH 8.0 with aqueous ammonia, and 140 g of hydrogen peroxide was added as phase A; chromatographically pure acetonitrile was used as phase B. Flow rate: 200 ml/min. Detection wavelength: 230 nm. Phase B acetonitrile elution gradient: Gradient elution of the sample with a gradient of B%: 18% - 28%, 50-70 min. Purification is completed while oxidation is complete

The oxidized purified polypeptide obtained in the above step is formed into a stable salt, and after lyophilization, a standard morphine having a purity greater than 99.0% can be obtained.

After lyophilization, 85 g of a white powdery solid sperm peptide was obtained. The purity is 99.40%, and the individual impurities are less than 0.15%. The purification yield was 58% (calculated as the morphine content in the crude product) with a total yield of 34%.

The time is as follows:

过滤时间 Filter time	1小时1 hour
纯化时间Purification time	30小时30 hours
总时间total time	31小时31 hours

Example 5: Ziconotide purification

15 g of the ziconotide linear crude peptide was dissolved and filtered, and the filtrate was collected for use.

Column: Octadecyl bonded silica gel column with a column size of 10 cm x 25 cm.

61.5 g of sodium hydroxide was dissolved in 10 L of water, pH 7.0 was adjusted with hydrochloric acid, and 8 g of ferric oxide was added as mobile phase A; chromatographically pure ethanol was used as phase B. Flow rate: 200 ml/min. Detection wavelength: 220 nm. Phase B ethanol elution gradient: B%: 25% - 45%, gradient change of 50-70 min to gradient elution of the sample. The purification is completed while the oxidation is completed.

The oxidized purified polypeptide obtained in the above step is formed into a stable salt, and after lyophilization, a standard acetate-based kanonotide having a purity greater than 99.0% can be obtained.

After lyophilization, 3.2 g of a white powdery solid sperm peptide was obtained. The purity is 99.32%, and the individual impurities are less than 0.15%. The purification yield was 61% (calculated as the linear peptide content of the zocorin in the crude product), and the total yield was 32%.

Comparative Example 1: Purification of crude peptides by eptinin amplification

The crude morphine peptide 250g was dissolved and filtered, and subjected to liquid phase oxidation to dissolve the concentration of 1 mg/mL. After 1 hour, the oxidation was completed, and then filtered. The impurities were extremely difficult to be filtered due to the change of impurities after oxidation, and it was subjected to two-step filtration and one-step coarse filtration. After one step of fine filtration, coarse filtration to remove most of the insoluble matter, and then filtering the 0.45 μm filter, which takes about 10 hours, and then subjected to reverse phase purification.

Column: Octadecyl bonded silica gel column with a column size of 15 cm x 25 cm.

A 50 mmol/L sodium dihydrogen phosphate solution was adjusted to pH 8.0 with aqueous ammonia to obtain phase A pure acetonitrile as phase B. Flow rate: 200 ml/min. Detection wavelength: 230 nm. Take B%: 18% - 28%, 50-70min The gradient changes the sample to a gradient elution.

The oxidized purified polypeptide obtained in the above step is formed into a stable salt, and after lyophilization, a standard-compliant morphin peptide having a purity greater than 99.0% can be obtained.

After lyophilization, 58 g of a white powdery solid sperm peptide was obtained. The purity is 99.31%, and the individual impurities are less than 0.15%. The purification yield was 40% (calculated as the eptipeptide content in the crude product) with a total yield of 23%. The time is as follows:

过滤时间Filter time	10小时10 hours
纯化时间Purification time	30小时30 hours

氧化时间Oxidation time	1小时1 hour
总共耗时Total time consuming	41小时41 hours

Purification of a batch of 250g eptipeptide crude peptide took 41h, and the crude peptide after oxidation was allowed to be placed for 20h. In this process, product stability is difficult to control.

The oxidative purification method of the present invention can increase the yield from the previous 23% to 34%, increase the 50% while increasing the productivity (41-31) / 31 = 32%, and increase the yield by 50%. Advantage. In the prior art method, since the oxidation time is too long, the oxidation product changes, and it is difficult to remove, thereby affecting the purification effect. In the case of unstable products, the quality is more difficult to ensure, and the method of the present invention creatively oxidizes the step. It is carried out in a column, and the method of adding an oxidizing agent ensures that the quality of the crude peptide after oxidation is the same, and the method of the present invention can increase the productivity by 30% and the yield by 50%.

Claims

A method for purifying a oxidized disulfide-containing polypeptide, wherein the crude peptide to be purified and oxidized is separated on a chromatography column while being oxidized; and the crude peptide is eluted while being eluted with a mobile phase containing an oxidizing agent;

Preferably, the crude peptide is eluted with mobile phase A and phase B,

Among them, the phase A is an aqueous solution of an inorganic salt, and the phase A further contains an oxidizing agent which oxidizes a disulfide bond.
The method according to claim 1, wherein the oxidizing agent in the phase A is selected from the group consisting of hydrogen peroxide, DMSO, iodine, and a metal ion oxidizing agent (Fe 3+ , Cu 2+ , Ag + ). Or several; preferably, the metal ion oxidizing agent is selected from the group consisting of Fe 2 O 3 .
The method for purifying a oxidized disulfide-containing polypeptide according to any one of claims 1 to 2, wherein the oxidizing agent in the phase A is hydrogen peroxide or DMSO, and the pH of the phase A is 7.5 to 9.0.
The method for purifying a oxidized disulfide-containing polypeptide according to any one of claims 1 to 2, wherein the oxidizing agent in the phase A is iodine and has a pH of from 2.5 to 9.0.
The method for purifying a oxidized disulfide-containing polypeptide according to any one of claims 1 to 4, wherein the oxidizing agent is 2 to 10 times the number of moles of the crude peptide.
The method for purifying a oxidized disulfide-containing polypeptide according to any one of claims 1 to 5, wherein the phase B contains an organic solvent, and preferably, the phase B contains acetonitrile, methanol, isopropanol, ethanol, tetrahydrofuran. One or more.
The method for purifying a oxidized disulfide-containing polypeptide according to any one of claims 1 to 6, wherein the column stationary phase is selected from the group consisting of a silica gel matrix, a polymer polymer, and zirconia.
The method for purifying a oxidized disulfide-containing polypeptide according to any one of claims 1 to 7, wherein the disulfide-containing polypeptide is selected from the group consisting of octreotide, eptinide, nesiritide, atosiban, and traniga Calfin, ziconotide, linaclotide, desmopressin.