Method for preparing VcMMAE using supercritical fluid chromatography

Abstract

The invention relates to a method for preparing VcMMAE by using supercritical fluid chromatography separation, comprising the following steps: mixing a dissolving reagent with a crude VcMMAE product to prepare a test solution; performing supercritical fluid chromatography detection on the test solution; the conditions for supercritical fluid chromatography separation include: a mobile phase including a mixture of supercritical carbon dioxide and an alcohol solvent; a stationary phase including one of a cyano group bonded to the surface of silica gel, silica gel, a 2-(1-pyrene)ethyl group bonded to the surface of silica gel, and an imidazole group bonded to the surface of silica gel; the method utilizes the unique mobile phase properties after supercritical CO2 is mixed with an alcohol solvent, and after selecting a suitable stationary phase, the separation and preparation of low-purity VcMMAE can be achieved, the separation time is short, the separation effect is good, the reagent cost can be effectively reduced, and a higher production capacity can be achieved, and the method has broad application prospects in the field of drug research and development of antibody drug conjugates.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a method for preparing VcMMAE by using supercritical fluid chromatography. Background Art

[0002] VcMMAE (mc-vc-PAB-MMAE), a MMAE derivative with a valine-citrulline (Vc) linker, is a drug-linker conjugate with anti-tumor activity, used in the synthesis of antibody-drug conjugates (ADCs). The structural formula of VcMMAE is shown in the following formula (I). VcMMAE is formed by the connection of MMAE and Vc, wherein MMAE is a synthetic anti-tumor drug that can be effectively released from VcMMAE in vitro and exert cytotoxic activity; MMAE's Chinese name is monomethyl auristatin E, which belongs to the statin class of drugs. Its main use is to reduce the cholesterol level in the blood, thereby preventing and treating high cholesterol and hyperlipidemia. It reduces the synthesis of cholesterol, increases the expression of low-density lipoprotein (LDL) receptors, and promotes the clearance of LDL, thereby reducing the cholesterol content in the blood.

[0003] (I)

[0004] Currently, the purification of VcMMAE on the market mostly relies on high-performance liquid chromatography to increase its purity from 70%-80% to more than 99%. However, high-performance liquid chromatography requires a huge amount of organic reagents, and some impurities are difficult to remove. The sample post-processing process is cumbersome and costly. Summary of the invention

[0005] Based on this, it is necessary to provide a method for preparing VcMMAE using supercritical fluid chromatography, which can separate low-purity VcMMAE crude products and prepare high-purity VcMMAE, while effectively reducing the amount of organic reagents used and achieving higher production capacity.

[0006] The technical solution is as follows:

[0007] The present invention provides a method for preparing VcMMAE using supercritical fluid chromatography, comprising the following steps:

[0008] Mix the dissolving reagent with the crude VcMMAE to prepare the test solution;

[0009] The test solution is subjected to supercritical fluid chromatography separation to prepare VcMMAE;

[0010] The conditions for supercritical fluid chromatography separation include: the mobile phase includes a mixture of supercritical carbon dioxide and an alcohol solvent; the stationary phase includes one of silica gel surface bonded cyano, silica gel, silica gel surface bonded 2-(1-pyrene)ethyl and silica gel surface bonded imidazole;

[0011] The VcMMAE has the following structure:

[0012] .

[0013] In one embodiment, in the mobile phase, the volume ratio of supercritical carbon dioxide to alcohol solvent is (95-60): (5-40).

[0014] In one embodiment, the alcohol solvent is one or more of methanol, ethanol and isopropanol.

[0015] In one embodiment, the supercritical fluid chromatography separation uses isocratic elution.

[0016] In one embodiment, the supercritical fluid chromatography separation conditions include: column temperature is 30°C-35°C.

[0017] In one embodiment, the conditions for supercritical fluid chromatography separation include: an injection volume of 10 μL-500 μL.

[0018] In one embodiment, the conditions of supercritical fluid chromatography separation include: a detection wavelength of 210nm-230nm; and / or a flow rate of 10mL / min-15mL / min.

[0019] In one embodiment, the supercritical fluid chromatography separation conditions include: the back pressure of the supercritical fluid chromatography system is set to 10MPa-15MPa.

[0020] In one embodiment, the dissolving agent includes an alcohol solvent;

[0021] Optionally, the alcohol solvent is methanol and / or isopropanol.

[0022] In one embodiment, the concentration of the test solution is ≤50 mg / mL.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present application provides a method for preparing VcMMAE using a supercritical fluid chromatography system. The method uses supercritical carbon dioxide and alcohol solvents as mobile phases, utilizes the unique mobile phase properties of supercritical CO2 mixed with alcohol solvents, and selects a suitable stationary phase to achieve the separation and preparation of low-purity VcMMAE. The separation time is short and the separation effect is good, and the purity can be increased from 70%-80% to more than 99%. In addition, fewer organic reagents are used in the preparation process, which can effectively reduce reagent costs and achieve higher production capacity, simplify post-processing procedures, and achieve cost reduction and efficiency improvement. It has broad application prospects in the field of drug research and development of antibody-drug conjugates. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 This is the separation spectrum of the VcMMAE sample in Example 1 in the supercritical fluid chromatography system.

[0027] Figure 2 This is the separation spectrum of the VcMMAE sample in Example 2 in the supercritical fluid chromatography system.

[0028] Figure 3 This is the separation spectrum of the VcMMAE sample in Example 3 in the supercritical fluid chromatography system.

[0029] Figure 4 This is the detection and analysis spectrum of the samples collected in Example 3 in a supercritical fluid chromatography system.

[0030] Figure 5 This is the separation spectrum of the VcMMAE sample in Example 4 in the supercritical fluid chromatography system.

[0031] Figure 6 This is the separation spectrum of the VcMMAE sample in Example 5 in the supercritical fluid chromatography system.

[0032] Figure 7 This is the separation spectrum of the VcMMAE sample in Example 6 in the supercritical fluid chromatography system. DETAILED DESCRIPTION

[0033] The present application is further described in detail below in conjunction with specific embodiments. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0035] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0036] As used herein, "one or more" refers to any one, any two, or any two or more of the listed items.

[0037] Herein, the optional scope of "and / or", "or / and", and "and / or" includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, wherein said any and all combinations include any two relevant listed items, any more relevant listed items, or a combination of all relevant listed items.

[0038] Herein, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0039] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0040] The percentage content involved in the present invention, unless otherwise specified, refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0041] The percentage concentrations involved in the present invention, unless otherwise specified, refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0042] The temperature parameters in the present invention, if not specifically limited, allow both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±2°C, ±1°C, ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, ±0.1°C are allowed. Normal temperature or room temperature in the present invention refers to no temperature control operation, generally refers to 4°C~35°C, preferably refers to 20±5°C.

[0043] The existing high performance liquid chromatography technology for separating and purifying VcMMAE has the problem of large consumption of organic reagents and difficult removal of individual impurities. The use of supercritical fluid chromatography can effectively make up for the defects of the existing methods. Supercritical fluid chromatography (SFC) utilizes the characteristics of supercritical fluids, that is, under the state above the critical pressure and critical temperature, the substance is neither a gas nor a liquid, but a state between gas and liquid. Under the supercritical state, different components in the sample can be separated according to their different solubility in the fluid. By changing the pressure and temperature, the solubility of the supercritical fluid can be controlled, thereby achieving sample separation and detection. Supercritical fluid chromatography can be used to analyze substances with different polarity, boiling points, and molecular weights, and has a wide range of applications. However, there is currently no method for separating and preparing VcMMAE using SFC.

[0044] During the research process, the technical personnel of the present application unexpectedly discovered that by using supercritical carbon dioxide and alcohol solvents as mobile phases, utilizing the unique mobile phase properties of the mixture of supercritical CO2 and alcohol solvents, and selecting a suitable stationary phase for separation and purification of crude VcMMAE, the optimal separation of VcMMAE can be achieved, and fewer organic reagents are used in the preparation process, which can effectively reduce reagent costs, simplify post-processing procedures, and achieve cost reduction and efficiency improvement. Therefore, a more green, environmentally friendly, cost-reducing and efficiency-enhancing method can be provided for the preparation of high-purity VcMMAE, which can replace and upgrade the current high-performance liquid chromatography method.

[0045] The present invention provides a method for preparing VcMMAE using supercritical fluid chromatography, comprising the following steps:

[0046] Mix the dissolving reagent with the crude VcMMAE to prepare the test solution;

[0047] The test solution is subjected to supercritical fluid chromatography separation to prepare VcMMAE;

[0048] The conditions for supercritical fluid chromatography separation include: the mobile phase includes a mixture of supercritical carbon dioxide and an alcohol solvent; the stationary phase includes one of silica gel surface bonded cyano, silica gel, silica gel surface bonded 2-(1-pyrene)ethyl and silica gel surface bonded imidazole;

[0049] The VcMMAE has the following structure:

[0050] .

[0051] In some of the examples, the composition of the VcMMAE crude product includes VcMMAE and other related impurities. When the purity of the crude product is 78% or above, it is possible to prepare qualified samples after fine-tuning using the method proposed in this application.

[0052] In some of the examples, the volume ratio of supercritical carbon dioxide to alcohol solvent in the mobile phase is (95-60): (5-40). Further, the volume ratio of supercritical carbon dioxide to alcohol solvent in the mobile phase is (80-70): (20-30). Furthermore, the volume ratio of supercritical carbon dioxide to alcohol solvent in the mobile phase is 75:25.

[0053] In some examples, the alcohol solvent is one or more of methanol, ethanol and isopropanol. The present application utilizes the unique mobile phase properties of supercritical CO2 mixed with alcohol solvents to select a corresponding stationary phase to separate and purify the VcMMAE crude product.

[0054] In some examples, the supercritical fluid chromatography separation uses isocratic elution.

[0055] In some examples, the conditions for supercritical fluid chromatography separation include: a column temperature of 30° C.-35° C. It can be understood that the column temperature includes but is not limited to 30° C., 31° C., 32° C., 33° C., 34° C., and 35° C.

[0056] In some examples, the conditions for supercritical fluid chromatography separation include: an injection volume of 10 μL-500 μL. It is understandable that the injection volume includes but is not limited to 10 μL, 30 μL, 50 μL, 70 μL, 90 μL, 110 μL, 130 μL, 150 μL, 170 μL, 200 μL, 250 μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL.

[0057] In some examples, the supercritical fluid chromatography separation conditions include: a detection wavelength of 210nm-230nm. It can be understood that the detection wavelength includes but is not limited to 210nm, 215nm, 220nm, 225nm, and 230nm.

[0058] In some examples, the conditions for supercritical fluid chromatography separation include: a flow rate of 10 mL / min-15 mL / min. It is understood that the flow rate includes but is not limited to 10 mL / min, 11 mL / min, 12 mL / min, 13 mL / min, 14 mL / min, and 15 mL / min.

[0059] In some examples, the conditions for supercritical fluid chromatography separation include: the back pressure set by the supercritical fluid chromatography system is 10MPa-15MPa. It can be understood that the back pressure set by the supercritical fluid chromatography system includes but is not limited to 10MPa, 11MPa, 12MPa, 13MPa, 14MPa, 15MPa.

[0060] In some examples, the dissolving agent includes an alcohol solvent;

[0061] Optionally, the alcohol solvent is methanol and / or isopropanol.

[0062] In some examples, the concentration of the test solution is ≤50 mg / mL. Further, the concentration of the test solution is 30 mg / mL-50 mg / mL. It is understood that the concentration of the test solution includes but is not limited to 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, and 50 mg / mL.

[0063] In some examples, the method for preparing VcMMAE by supercritical fluid chromatography separation further comprises the following steps:

[0064] The eluate was collected according to the retention time corresponding to VcMMAE in the spectrum obtained after supercritical fluid chromatography detection of the test solution to prepare VcMMAE.

[0065] Without limitation, the eluent may be subjected to high performance liquid chromatography to verify the purity of VcMMAE. The conditions of the high performance liquid chromatography detection include: mobile phase A is ammonium acetate buffer, mobile phase B is acetonitrile, and gradient elution is used.

[0066] In some of these examples, the HPLC detection also satisfies at least one of the following conditions:

[0067] (1) Flow rate is 1 mL / min-2 mL / min;

[0068] (2) The detection wavelength is 210nm-220nm;

[0069] (3) Column temperature is 25°C-35°C;

[0070] (4) The stationary phase comprises octadecylsilane bonded silica gel;

[0071] (5) The volume ratio of mobile phase A to mobile phase B is (70~20): (30~80).

[0072] In some examples, the gradient elution procedure of the HPLC detection includes:

[0073] From 0 to 25 min, the volume percentage of the mobile phase A decreased from 70% to 35%;

[0074] 25min~30min, the volume percentage of the mobile phase A decreases from 35% to 20%;

[0075] 30min-35min, the volume percentage of the mobile phase A is maintained at 20%;

[0076] From 35min to 35.1min, the volume percentage of the mobile phase A increased from 20% to 70%;

[0077] From 35.1 min to 40 min, the volume percentage of the mobile phase A was maintained at 70%.

[0078] The following is further described in conjunction with specific embodiments. The raw materials involved in the following specific embodiments, unless otherwise specified, can all be commercially available; the instruments used, unless otherwise specified, can all be commercially available; the processes involved, unless otherwise specified, are all routinely selected by those skilled in the art.

[0079] The following are specific embodiments.

[0080] Example 1

[0081] The instrument used in this embodiment is a supercritical fluid chromatograph of Jiangsu Hanbang Technology Co., Ltd. The chromatographic column specification is 10*250mm, the mobile phase flow rate is 10mL / min, the back pressure is set to 10Mpa, the VcMMAE concentration is 50mg / mL (purity 78%), the injection volume is 10μL, and the detection wavelength is 220nm.

[0082] Table 1 shows other relevant conditions selected in the method for separating and preparing high-purity VcMMAE using a supercritical fluid chromatography system in Example 1.

[0083] Table 1 SFC separation conditions in Example 1

[0084]

[0085] The specific steps corresponding to Example 1 are as follows: a VcMMAE sample with a purity of 78% is dissolved in methanol, ultrasonically assisted to prepare a 50 mg / mL solution, and NuciferaCN (5um, 10*250mm) is selected as a chromatographic column on a supercritical fluid chromatograph of Jiangsu Hanbang Technology Co., Ltd., and the volume ratio, total flow rate, back pressure and chromatographic column temperature of the supercritical carbon dioxide and alcohol solvent in the mobile phase are controlled according to Table 1, and the temperature of the carbon dioxide entering the pump is ensured to be 8°C. VcMMAE is separated, and the separation effect diagram is shown in FIG. Figure 1 As shown. Figure 1 It can be seen that the peak corresponding to VcMMAE appears at a retention time of about 14 minutes, and is clearly separated from the impurities. The distribution of impurity points is conducive to stacked needle injection, proving that the above conditions can be used on SFC to achieve effective separation of VcMMAE.

[0086] Example 2

[0087] The instrument used in this embodiment is a supercritical fluid chromatograph produced by Jiangsu Hanbang Technology Co., Ltd. The chromatographic column specification is 10*250mm, the mobile phase flow rate is 10mL / min, the back pressure is set to 10Mpa, the VcMMAE concentration is 50mg / mL (purity 78%), the injection volume is 200μL, and the detection wavelength is 220m.

[0088] Table 2 shows other relevant conditions selected in the method for separating and preparing high-purity VcMMAE using a supercritical fluid chromatography system in Example 2.

[0089] Table 2 SFC separation conditions in Example 2

[0090]

[0091] The specific steps corresponding to Example 2 are as follows: a VcMMAE sample with a purity of 30% is dissolved in ethyl acetate, ultrasonically assisted dissolution, and a 50 mg / mL solution is prepared. On a supercritical fluid chromatograph of Jiangsu Hanbang Technology Co., Ltd., Nucifera CN (5um, 10*250mm) is selected as a chromatographic column, and the volume ratio of supercritical carbon dioxide and alcohol solvent in the mobile phase, the total flow rate, the back pressure and the column temperature of the chromatographic column are controlled according to Table 2, and the temperature of carbon dioxide entering the pump is ensured to be 8°C. VcMMAE is separated, and the separation effect diagram is shown in FIG. Figure 2 As shown. Figure 2 It can be seen that when the sample amount is increased to 10 mg, there is still a certain degree of separation between the impurities and the target peak, indicating that the preparation of VcMMAE can be achieved on SFC.

[0092] Example 3

[0093] The instrument used in this embodiment is a supercritical fluid chromatograph produced by Jiangsu Hanbang Technology Co., Ltd. The chromatographic column specification is 10*250mm, the mobile phase flow rate is 10mL / min, the back pressure is set to 10Mpa, the VcMMAE concentration is 50mg / mL (purity 78%), the injection volume is 200μL, the sample is injected every 9min, a total of 3 times, and the detection wavelength is 220nm.

[0094] Table 3 shows other relevant conditions selected in the method for separating and preparing high-purity VcMMAE using a supercritical fluid chromatography system in Example 3.

[0095] Table 3.1 SFC separation conditions in Example 3

[0096]

[0097] The specific steps corresponding to Example 3 are as follows: a VcMMAE sample with a purity of 78% is dissolved in methanol, ultrasonically assisted dissolution, and a 50 mg / mL solution is prepared. On a supercritical fluid chromatograph of Jiangsu Hanbang Technology Co., Ltd., NuciferaCN (5um, 10*250mm) is selected as a chromatographic column, and the volume ratio, total flow rate, back pressure and chromatographic column temperature of the supercritical carbon dioxide and alcohol solvent in the mobile phase are controlled according to Table 3.1, and the temperature of the carbon dioxide entering the pump is ensured to be 8°C. VcMMAE is separated, and the separation effect diagram is shown in FIG. Figure 3 As shown. Figure 3 It can be seen that under the stacked needle injection method, the crude VcMMAE product can still be stably separated under supercritical fluid chromatography and the separation efficiency is greatly improved.

[0098] Then follow Figure 3 The samples were collected at the red box position and tested by HPLC gradient elution according to the method in Table 3.2. The test results are as follows: Figure 4 As shown, the purity of the collected and tested VcMMAE is 99.96%, which fully meets the standard of high-purity VcMMAE required on the market.

[0099] Table 3.2 Detection conditions for collecting VcMMAE samples in Example 3

[0100] Example 4

[0101] The instrument used in this embodiment is a supercritical fluid chromatograph produced by Jiangsu Hanbang Technology Co., Ltd. The chromatographic column specification is 10*250mm, the mobile phase flow rate is 10mL / min, the back pressure is set to 10Mpa, the VcMMAE concentration is 50mg / mL (purity is 78%), the injection volume is 500μL, the sample is injected every 15min, a total of 3 injections, and the detection wavelength is 220nm.

[0102] Table 4 shows other relevant conditions selected in the method for separating and preparing high-purity VcMMAE using a supercritical fluid chromatography system in Example 4.

[0103] Table 4 SFC separation conditions in Example 4

[0104]

[0105] The specific steps corresponding to Example 4 are as follows: a VcMMAE sample with a purity of 78% is dissolved in methanol, ultrasonically assisted to prepare a 200 mg / mL solution, and Nucifera SI (10um, 10*250mm) is selected as a chromatographic column on a supercritical fluid chromatograph of Jiangsu Hanbang Technology Co., Ltd. The volume ratio of supercritical carbon dioxide and alcohol solvent in the mobile phase, the total flow rate, the back pressure and the column temperature of the chromatographic column are controlled according to Table 4, and the temperature of carbon dioxide entering the pump is ensured to be 8°C. VcMMAE is separated, and the separation effect diagram is shown in FIG. Figure 5 As shown. Figure 5 It can be seen that when using SI chromatographic column for separation and preparation, its separation degree for the front impurities is better than CN, but the separation peak shape of the back impurities is slightly worse. Using the above conditions on SFC can still achieve effective separation of VcMMAE.

[0106] Example 5

[0107] The chromatograph used in this embodiment is a supercritical fluid chromatograph of Jiangsu Hanbang Technology Co., Ltd., with a chromatographic column specification of 10*250mm, a mobile phase flow rate of 10mL / min, a VcMMAE concentration of 50mg / mL (purity of 78%), an injection volume of 50μL, and a detection wavelength of 220nm.

[0108] Table 5 shows other relevant conditions selected in the method for separating and preparing high-purity VcMMAE using a supercritical fluid chromatography system in Example 5.

[0109] Table 5 SFC separation conditions in Example 5

[0110]

[0111] The specific steps corresponding to Example 5 are as follows: a VcMMAE sample with a purity of 78% is dissolved in methanol, ultrasonically assisted to prepare a 50 mg / mL solution, and Nucifera PYE (10 um, 10*250 mm) is selected as the chromatographic column on the supercritical fluid chromatograph of Jiangsu Hanbang Technology Co., Ltd., and the volume ratio of A and B in the mobile phase, the total flow rate, the back pressure and the column temperature of the chromatographic column are controlled according to Table 5, and the temperature of the carbon dioxide entering the pump is ensured to be 8°C, and VcMMAE is separated, and the separation effect diagram is shown in the figure. Figure 6 As shown. Figure 6 It can be seen that when using the PYE chromatographic column for analysis and preparation, it can be seen that the impurities are distributed farther away. Using the above conditions on SFC can still achieve effective separation of VcMMAE, but from the preparation perspective, the overall stacking time is long and the production efficiency is low.

[0112] Example 6

[0113] The chromatograph used in this embodiment is a supercritical fluid chromatograph of Jiangsu Hanbang Technology Co., Ltd., with a chromatographic column specification of 10*250mm, a mobile phase flow rate of 10mL / min, a VcMMAE concentration of 50mg / mL (purity of 78%), an injection volume of 50μL, and a detection wavelength of 220nm.

[0114] Table 6 shows other relevant conditions selected in the method for separating and preparing high-purity VcMMAE using a supercritical fluid chromatography system in Example 6.

[0115] Table 6 SFC separation conditions in Example 6

[0116]

[0117] The specific steps corresponding to Example 6 are as follows: a VcMMAE sample with a purity of 78% is dissolved in methanol, ultrasonically assisted to prepare a 50 mg / mL solution, and Nucifera IMU (5um, 10*250mm) is selected as the chromatographic column on the supercritical fluid chromatograph of Jiangsu Hanbang Technology Co., Ltd., and the volume ratio of A and B in the mobile phase, the total flow rate, the back pressure and the column temperature of the chromatographic column are controlled according to Table 6, while ensuring that the temperature of carbon dioxide entering the pump is 8°C, and VcMMAE is separated. The separation effect diagram is shown in the figure. Figure 7 As shown. Figure 7 It can be seen that when using IMU for preparation, it is similar to the separation of chromatographic column PYE, the location of the impurities is far away, the stacked injection time is long, and the production efficiency is low. However, using the above conditions on SFC can still achieve effective separation of VcMMAE.

[0118] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0119] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for preparing VcMMAE using supercritical fluid chromatography, characterized in that: The following steps are involved: Mixing a dissolving agent with the crude VcMMAE product to prepare a test solution; the dissolving agent is selected from methanol or ethyl acetate; The test solution is subjected to supercritical fluid chromatography separation to prepare VcMMAE; The conditions for the supercritical fluid chromatography separation include: the mobile phase includes a mixture of supercritical carbon dioxide and methanol; the stationary phase is a cyano group bonded to the surface of silica gel; the column temperature is 30°C-35°C; the injection volume is 10μL-200μL; the flow rate is 10mL / min-15mL / min; the back pressure set by the supercritical fluid chromatography system is 10MPa-15MPa; the temperature of carbon dioxide when entering the pump is 8°C; the volume ratio of supercritical carbon dioxide and methanol in the mobile phase is controlled according to the following table or ； The VcMMAE has the following structure: 。 2. The method for preparing VcMMAE using supercritical fluid chromatography according to claim 1, characterized in that: The conditions for supercritical fluid chromatography separation include: the detection wavelength is 210nm-230nm.

3. The method for preparing VcMMAE using supercritical fluid chromatography according to claim 1, characterized in that: The concentration of the test solution is ≤50 mg / mL.

4. The method for preparing VcMMAE using supercritical fluid chromatography according to claim 1, characterized in that: The method for preparing VcMMAE by supercritical fluid chromatography separation also includes the following steps: The eluate was collected according to the retention time corresponding to VcMMAE in the spectrum obtained after supercritical fluid chromatography detection of the test solution to prepare VcMMAE.

5. The method for preparing VcMMAE using supercritical fluid chromatography according to claim 4, characterized in that: The method also includes performing high performance liquid chromatography on the eluent to verify the purity of VcMMAE; the conditions of the high performance liquid chromatography detection include: mobile phase A is ammonium acetate buffer, mobile phase B is acetonitrile, and gradient elution is used; The high performance liquid chromatography detection also meets the following conditions: (1) Flow rate is 1 mL / min-2 mL / min; (2) The detection wavelength is 210nm-220nm; (3) Column temperature is 25°C-35°C; (4) The stationary phase comprises octadecylsilane bonded silica gel; and (5) The volume ratio of mobile phase A to mobile phase B is (70~20): (30~80).

6. The method for preparing VcMMAE using supercritical fluid chromatography according to claim 5, characterized in that: The gradient elution procedure of the high performance liquid chromatography detection comprises: From 0 to 25 min, the volume percentage of the mobile phase A decreased from 70% to 35%; 25min~30min, the volume percentage of the mobile phase A decreases from 35% to 20%; 30min-35min, the volume percentage of the mobile phase A is maintained at 20%; From 35min to 35.1min, the volume percentage of the mobile phase A increased from 20% to 70%; From 35.1 min to 40 min, the volume percentage of the mobile phase A was maintained at 70%.

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