Purification method of phosphoryl diamine morpholino oligonucleotide

Abstract

The invention provides a method for purifying phosphoryl diamine morpholino oligonucleotide, and relates to the technical field of oligonucleotide purification. The purification method of the phosphoramide morpholino oligonucleotide comprises the following steps: carrying out ultrafiltration liquid change for the first time, carrying out ion pair reversed phase chromatography, carrying out ultrafiltration liquid change for the second time, and freeze-drying, so as to obtain the high-purity phosphoramide morpholino oligonucleotide. According to the invention, a purification process route of the phosphoric acid diamide morpholino oligonucleotide (PMO) is developed, the purity of the phosphoric acid diamide morpholino oligonucleotide (PMO) is effectively improved, the industrial requirements can be accurately met, and the development of the fields of scientific research and bioengineering is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biochemistry and bioengineering, and in particular to a method for purifying phosphorodiamidate morpholino oligonucleotides. BACKGROUND

[0002] Duchenne muscular dystrophy (DMD) is a rare X-linked recessive genetic disease caused by dystrophin protein expression defects, which can lead to progressive muscle weakness and atrophy. Phosphorodiamidate morpholino oligonucleotide (PMO) drugs bind to the pre-mRNA of dystrophin, change the splicing process of the pre-mRNA, skip the DMD exon carrying the genetic mutation, and generate a truncated protein with most of the dystrophin function to achieve the purpose of treatment.

[0003] Currently, four phosphorodiamidate morpholino oligonucleotide (PMO) drugs have been approved by regulatory agencies for different exon defects, including Eteplirsen, Golodirsen, Casimersen, and Viltolarsen. Although it can be known from existing literature and patents that such phosphorodiamidate morpholino oligonucleotide (PMO) drugs can be obtained through the process of'solid-phase synthesis >> aminolysis deprotection >> reverse phase / ion purification', the reported product purity is not high (<95%). Therefore, in order to obtain high-purity phosphorodiamidate morpholino oligonucleotide (PMO) drugs, it is urgent to develop an efficient purification method to meet the growing market demand.

[0004] The conventional phosphorodiamidate morpholino oligonucleotide (PMO) purification process includes precipitation, ion exchange chromatography, ordinary reverse phase chromatography, affinity chromatography, and gel electrophoresis. These methods have their own advantages and disadvantages, for example, precipitation is simple, fast, and low in cost, but has low resolution; ion exchange chromatography is widely used in oligonucleotide purification and has high efficiency, but in the purification of phosphorodiamidate morpholino oligonucleotide (PMO), the molecular electric neutrality of phosphorodiamidate morpholino oligonucleotide (PMO) makes the separation effect not obvious; phosphorodiamidate morpholino oligonucleotide (PMO) cannot be attached with a DMT group like oligonucleotides, and ordinary reverse phase chromatography cannot effectively separate the product by hydrophobicity difference; affinity chromatography uses DNA probes with complementary sequences to be fixed on the column to specifically capture target molecules, but the cost is high and not suitable for industrial production. Gel electrophoresis has high resolution, but the operation is complicated, the yield is low, and it is difficult to scale up, so it is only suitable for laboratory-scale identification. Therefore, it is necessary to develop a combination of two or three purification methods to avoid the limitations of a single method, meet the needs of industrial scaling, and reduce production costs.

[0005] The backbone of the phosphorodiamidate morpholino oligonucleotide (PMO) is electrically neutral and does not have a charged group, but the ion pair can interact with the weakly negatively charged or polar region in the phosphorodiamidate morpholino oligonucleotide (PMO) molecule in the mobile phase TEA+, effectively covering the surface of the phosphorodiamidate morpholino oligonucleotide (PMO) molecule to form a "ion pair complex" with stronger hydrophobicity, electrical neutrality or weak polarity. The hydrophobicity of this complex is much higher than that of the phosphorodiamidate morpholino oligonucleotide (PMO) itself, thereby enhancing the retention in reverse phase chromatography. This change in hydrophobicity makes the sample elute at a specific acetonitrile concentration during gradient elution, thereby improving the elution resolution and separating high-purity phosphorodiamidate morpholino oligonucleotide (PMO) molecules.

[0006] In addition, using triethylamine acetate (TEAA) as the ion pair reagent, there will be a certain amount of TEAA in the collected sample solution, and the presence of this salt will affect the freeze-drying crystal form, resulting in the inability to obtain a product with excellent properties. Therefore, an additional ultrafiltration and liquid exchange process is added to remove TEAA.

[0007] Therefore, the present application is proposed. SUMMARY

[0008] The purpose of the present application is to provide a phosphorodiamidate morpholino oligonucleotide purification method, which has high universality in the process of purifying phosphorodiamidate morpholino oligonucleotide (PMO) and can efficiently purify phosphorodiamidate morpholino oligonucleotide (PMO) molecules from complex mixtures.

[0009] In order to achieve the above-mentioned purpose of the present application, the following technical solutions are adopted: In a first aspect, the present application provides a phosphorodiamidate morpholino oligonucleotide purification method, which comprises: The phosphorodiamidate morpholino oligonucleotide solution is subjected to first ultrafiltration and liquid exchange, ion pair reverse phase chromatography, second ultrafiltration and liquid exchange, and freeze-drying to obtain high-purity phosphorodiamidate morpholino oligonucleotide.

[0010] Further, the first ultrafiltration and liquid exchange further comprises the following pretreatment steps: Water is added to the phosphorodiamidate morpholino oligonucleotide ammonolysis solution to terminate the ammonolysis reaction, and a terminated reaction solution is obtained. An organic solvent is added to the terminated reaction solution to obtain a mixed solution. The mixed solution is filtered to obtain a solution to be subjected to ultrafiltration.

[0011] Further, the phosphorodiamidate morpholino oligonucleotide ammonolysis solution comprises, by mass percentage: 5-40% phosphorodiamidate morpholino oligonucleotide, 5-30% ammonia, 1-10% hydrophobic small molecule, and the balance being water.

[0012] Further, the volume ratio of the water and phosphorodiamidate morpholino oligonucleotide ammonolysis solution is (4-10):1.

[0013] Further, the volume of the organic solvent accounts for 40-60% of the total volume of the mixed solution.

[0014] Further, the organic solvent is selected from any one or a combination of at least two of acetonitrile, methanol, and dimethyl sulfoxide, and is further preferably acetonitrile.

[0015] Further, the regenerated cellulose membrane is used for the first and second ultrafiltration liquid changes.

[0016] Further, the regenerated cellulose membrane used for the first and second ultrafiltration liquid changes has a molecular weight cut-off of ≤2 kD.

[0017] Further, the first ultrafiltration liquid change includes one liquid change using a mixed solution of an organic solvent and water, and a second liquid change using water.

[0018] Further, the volume of the organic solvent accounts for 40-60% of the total volume of the mixed solution of the organic solvent and water.

[0019] Further, the organic solvent is selected from any one or a combination of at least two of acetonitrile, methanol, and dimethyl sulfoxide, and is preferably acetonitrile.

[0020] Further, the mixed solution of the organic solvent and water is continuously supplemented until the supplemented volume is 6-10 times the initial volume of the sample.

[0021] Further, the water is continuously supplemented until the supplemented volume is 6-8 times the initial volume of the sample.

[0022] Further, the process parameters of the first ultrafiltration liquid change include a temperature of 20-30°C, a transmembrane pressure (TMP) of 1.0-1.5 Bar, and an inlet flow rate of 100-300 LMH.

[0023] Further, the chromatographic column used for the ion pair reverse phase chromatography is an octadecylsilane-bonded silica gel column.

[0024] Further, the loading capacity of the chromatographic column used for the ion pair reverse phase chromatography is 1-3% (ratio of sample mass to packing mass).

[0025] Further, in the ion pair reverse phase chromatography, the mobile phase A is a triethylamine acetate aqueous solution, and the mobile phase B is an acetonitrile aqueous solution.

[0026] Further, in the mobile phase A, the concentration of the aqueous triethylamine acetate solution is 20-100 mM, and the pH of the aqueous triethylamine acetate solution is 6.0-8.0.

[0027] Further, in the mobile phase B, the volume of the acetonitrile accounts for 40-60% of the total volume of the acetonitrile aqueous solution.

[0028] Further, the ion pair reverse phase chromatography adopts the following gradient elution procedure: The first stage: 10-40% B, for 20-40 min; The second stage: 40-80% B, for 150-170 min.

[0029] Further, in the ion pair reverse phase chromatography, the eluate with a main peak absorption value of 1000 mAu or more is collected.

[0030] Further, the second ultrafiltration liquid exchange includes: using water for liquid exchange; Further, the liquid exchange is continuously supplemented with water until the supplemented volume is 6-8 times the initial volume of the sample.

[0031] Further, the process parameters of the second ultrafiltration liquid exchange include: temperature 20-30℃, transmembrane pressure TMP 1.0-1.5 Bar, and inlet flow rate of liquid exchange 100-300 LMH.

[0032] Further, the temperature of the freeze-drying is -80-35℃, and the time of the freeze-drying is 36-48 h.

[0033] Compared with the prior art, the present application has the following beneficial effects: (1) The present application provides a high-efficiency and stable high-purity phosphorodiamidate morpholino oligonucleotide (PMO) purification process, which significantly improves the purity of the phosphorodiamidate morpholino oligonucleotide product to more than 98%, breaking through the bottleneck that the PMO purity in the prior art is generally less than 95%; (2) The purification method uses a 2 kD ultrafiltration membrane combined with ultrafiltration liquid exchange to effectively remove ammonia, hydrophobic small molecule impurities and synthesis by-products, effectively reduce the subsequent chromatography burden, and prevent column pressure rise and blockage; then ion pair reverse phase chromatography is used to realize high-resolution separation under TEAA-acetonitrile gradient elution, and the adjacent impurities of the main peak are accurately removed; then the salt used in the reverse phase chromatography is removed by ultrafiltration liquid exchange, and finally a solid powder with excellent properties is obtained by using freeze-drying technology; (3) The purification method can accurately meet the industrial demand, is suitable for the large-scale production of various PMO drugs, has the advantages of controllable operation and good repeatability, and promotes the development of scientific research and biological engineering fields. Attached Figure Description

[0034] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 This is a process flow diagram of the purification method for phosphorylated diamine morpholino oligonucleotides described in this invention.

[0036] Figure 2 The diagram shows the structure of Eteplirsen provided in Example 1.

[0037] Figure 3 The liquid phase diagram shows the phosphorylated diamine morpholino oligonucleotide purified by the method provided in Example 1.

[0038] Figure 4 This is a structural diagram of the Golodirsen provided in Example 2.

[0039] Figure 5 The liquid phase diagram shows the phosphorylated diamine morpholino oligonucleotide purified by the method provided in Example 2.

[0040] Figure 6 Liquid phase diagram of the phosphorylated diamine morpholino oligonucleotide obtained by the method provided in Comparative Example 1. Detailed Implementation

[0041] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0042] Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present application are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0043] The technical solutions of the present application will be described clearly and completely below in connection with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0044] In a first aspect, the present application provides a method for purifying a phosphorodiamidate morpholino oligonucleotide, as shown in the formula: Figure 1 The method for purifying the phosphorodiamidate morpholino oligonucleotide comprises: The phosphorodiamidate morpholino oligonucleotide solution is subjected to first ultrafiltration, ion pair reverse phase chromatography, second ultrafiltration, and freeze-drying to obtain high-purity phosphorodiamidate morpholino oligonucleotide.

[0045] In the present application, the process route of the method for purifying the phosphorodiamidate morpholino oligonucleotide comprises continuous ultrafiltration, ion pair reverse phase chromatography, ultrafiltration desalination, and freeze-drying performed in sequence. This process for purifying phosphorodiamidate morpholino oligonucleotide (PMO) in series has high universality, can efficiently purify phosphorodiamidate morpholino oligonucleotide (PMO) molecules from complex mixtures, and significantly improves the purity of phosphorodiamidate morpholino oligonucleotide products to more than 98%, breaking through the bottleneck that the purity of PMO in the prior art is generally less than 95%.

[0046] It should be noted that if the first ultrafiltration is not performed and the ion pair reverse phase chromatography is directly performed, the alkalinity of the first ammonia solution is too strong, which can damage the stationary phase for a long time, increase the cost, and is not a good scale-up process. In addition, too many impurities can affect the resolution of the reverse phase chromatography, resulting in an unqualified purity of the final product.

[0047] As an optional embodiment, the first ultrafiltration further comprises the following pretreatment steps before the first ultrafiltration: adding water to the phosphorodiamidate morpholino oligonucleotide ammonolysis solution to terminate the ammonolysis reaction to obtain a terminated reaction solution; adding an organic solvent to the terminated reaction solution to obtain a mixed solution; filtering the mixed solution to obtain a solution to be ultrafiltrated.

[0048] It should be noted that after the ammonolysis solution of most phosphorodiamidate morpholino oligonucleotide (PMO) molecules is added with purified water to stop the ammonolysis reaction for a period of time, turbidity phenomenon occurs, and the main precipitated substance is a small molecule impurity with strong hydrophobicity; therefore, the sample needs to be filtered with a filter before ultrafiltration to remove visible foreign matter, reduce the risk of ultrafiltration membrane blockage, and improve the efficiency of liquid exchange.

[0049] As an optional implementation, the phosphorodiamidate morpholino oligonucleotide ammonolysis solution includes, by mass percentage: phosphorodiamidate morpholino oligonucleotide 5-40% (for example, it can be 5%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, etc.), ammonia 5-30% (for example, it can be 5%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, etc.), a small hydrophobic molecule 1-10% (for example, it can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc.), and the balance is water.

[0050] As an optional implementation, the phosphorodiamidate morpholino oligonucleotide ammonolysis solution includes, by mass percentage: phosphorodiamidate morpholino oligonucleotide 10-20%, ammonia 10-25%, a small hydrophobic molecule 2-5%, and the balance is water.

[0051] It should be noted that after the ammonolysis solution of most phosphorodiamidate morpholino oligonucleotide (PMO) molecules is added with purified water to stop the ammonolysis reaction for a period of time, turbidity phenomenon occurs, and the main precipitated substance is a small molecule impurity with strong hydrophobicity; therefore, the sample needs to be filtered with a filter before ultrafiltration to remove visible foreign matter, reduce the risk of ultrafiltration membrane blockage, and improve the efficiency of liquid exchange.

[0052] As an optional implementation, the volume ratio of the water to the phosphorodiamidate morpholino oligonucleotide ammonolysis solution is (4-10):1, for example, it can be 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc.

[0053] As an optional implementation, the volume of the organic solvent accounts for 40-60% of the total volume of the mixed solution, for example, it can be 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, etc.

[0054] As an optional implementation, the organic solvent is selected from any one or a combination of at least two of acetonitrile, methanol, and dimethyl sulfoxide.

[0055] As a preferred implementation, the organic solvent is acetonitrile.

[0056] As an optional implementation, the first ultrafiltration liquid exchange uses regenerated cellulose membrane.

[0057] As an optional implementation, the regenerated cellulose membrane used in the first ultrafiltration liquid exchange has a molecular weight cut-off of ≤2 kD.

[0058] As an optional implementation, the first ultrafiltration liquid exchange includes: first liquid exchange using a mixture of an organic solvent and water, and second liquid exchange using water.

[0059] In the present application, first, the molecular weight difference between phosphor diamide morpholino oligonucleotide (PMO) and small molecular impurities in the synthetic crude product is utilized, the concentration of the organic solvent (preferably acetonitrile) of the replacement solution is adjusted, and ammonia and hydrophobic impurities are removed; this process not only effectively removes part of the impurities, but also ingeniously solves the problem of column blockage and pressure increase during reverse phase chromatography; in addition, pure water is used for the second liquid exchange, and the purpose is to remove the organic solvent (such as acetonitrile) used in the first liquid exchange.

[0060] As an optional implementation, in the first ultrafiltration liquid exchange, the volume of the organic solvent accounts for 40-60% of the total volume of the mixture of the organic solvent and water, for example, it can be 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, etc.

[0061] As an optional implementation, in the first ultrafiltration liquid exchange, the organic solvent is selected from any one or a combination of at least two of acetonitrile, methanol, and dimethyl sulfoxide.

[0062] As a preferred implementation, in the first ultrafiltration liquid exchange, the organic solvent is acetonitrile.

[0063] As an optional implementation, the first liquid exchange continues to supplement the mixture of the organic solvent and water until the supplemented volume is 6-10 times the initial volume of the sample, for example, it can be 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, etc.

[0064] As an optional implementation, the second liquid exchange continues to supplement water until the supplemented volume is 6-8 times the initial volume of the sample, for example, it can be 6 times, 6.5 times, 7 times, 7.5 times, 8 times, etc.

[0065] It should be noted that when ultrafiltration is used to remove small molecules, the displacement multiple can be determined by analyzing the liquid phase, detecting the residual small molecules, and judging the final displacement multiple.

[0066] As an optional implementation, the process parameters of the first ultrafiltration displacement include: temperature 20-30℃ (for example, it can be 20℃, 22℃, 24℃, 25℃, 26℃, 28℃, 30℃, etc.), transmembrane pressure (TMP) 1.0-1.5 Bar (for example, it can be 1.0 Bar, 1.1 Bar, 1.2 Bar, 1.3 Bar, 1.4 Bar, 1.5 Bar, etc.), inlet flow rate of displacement 100-300 LMH (for example, it can be 100 LMH, 120 LMH, 140 LMH, 150 LMH, 160 LMH, 180 LMH, 200 LMH, 220 LMH, 240 LMH, 250 LMH, 260 LMH, 280 LMH, 300 LMH, etc.).

[0067] As an optional implementation, the ion pair reverse phase chromatography uses an octadecylsilane bonded silica column.

[0068] As an optional implementation, the ion pair reverse phase chromatography uses an octadecylsilane bonded silica column.

[0069] As an optional implementation, in the ion pair reverse phase chromatography, the mobile phase A is a triethylamine acetate aqueous solution, and the mobile phase B is an acetonitrile aqueous solution.

[0070] In the present application, the triethylamine acetate (TEAA) aqueous solution is selected as the mobile phase A, and the acetonitrile aqueous solution is selected as the mobile phase B, so that a stable ion pair complex can be formed, the retention capacity of PMO on the C18 column is enhanced, the polarity change is accurately controlled through gradient elution, the separation resolution is significantly improved, the adjacent impurities before and after the main peak are effectively removed, and the accurate collection of the target product with high purity (>98%) is realized.

[0071] As an optional implementation, in the mobile phase A, the concentration of the triethylamine acetate aqueous solution is 20-100 mM, for example, it can be 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, etc., and the pH of the triethylamine acetate aqueous solution is 6.0-8.0, for example, it can be 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, etc.

[0072] As an optional implementation, in the mobile phase B, the volume of the acetonitrile accounts for 40-60% of the total volume of the acetonitrile aqueous solution, for example, it can be 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, etc.

[0073] As an optional implementation, the ion-pair reversed-phase chromatography employs the following gradient elution procedure: Phase 1: 10-40% B, for 20-40 minutes (e.g., 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min, 40 min, etc., preferably 30 minutes); Second stage: 40~80%B, lasting 150~170 min (e.g., 150 min, 152 min, 154 min, 156 min, 158 min, 160 min, 162 min, 164 min, 166 min, 168 min, 170 min, etc., preferably 160 min).

[0074] In this invention, impurities near the main peak are removed by ion-pair reversed-phase chromatography using a specific gradient elution procedure to obtain high-purity phosphoryldiamine morpholino oligonucleotides (PMO).

[0075] As an optional implementation, during the ion-pair reversed-phase chromatography process, the chromatographic eluent with a main peak absorbance value of 1000 mAu or higher is collected.

[0076] As an optional implementation, the second ultrafiltration fluid exchange uses a regenerated cellulose membrane.

[0077] As an optional implementation, the regenerated cellulose membrane used for the second ultrafiltration fluid exchange has a molecular weight cutoff of ≤2kD.

[0078] As an optional implementation, the second ultrafiltration fluid replacement includes: using water for fluid replacement; As an optional implementation, the liquid replacement is continuously replenished with water until the replenished volume is 6 to 8 times the initial volume of the sample.

[0079] As an optional embodiment, the process parameters of the second ultrafiltration and liquid exchange include: temperature 20-30℃ (for example, it can be 20℃, 22℃, 24℃, 25℃, 26℃, 28℃, 30℃, etc.), transmembrane pressure (TMP) 1.0-1.5 Bar (for example, it can be 1.0 Bar, 1.1 Bar, 1.2 Bar, 1.3 Bar, 1.4 Bar, 1.5 Bar, etc.), inlet flow rate of liquid exchange 100-300 LMH (for example, it can be 100 LMH, 120 LMH, 140 LMH, 150 LMH, 160 LMH, 180 LMH, 200 LMH, 220 LMH, 240 LMH, 250 LMH, 260 LMH, 280 LMH, 300 LMH, etc.).

[0080] As an optional embodiment, the temperature of the freeze-drying is -80-35℃, for example, it can be -80℃, -75℃, -70℃, -65℃, -60℃, -55℃, -50℃, -45℃, -40℃, -35℃, -30℃, -25℃, -20℃, -15℃, -10℃, -5℃, 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, etc., and the time of the freeze-drying is 36-48h, for example, it can be 36h, 38h, 40h, 42h, 44h, 46h, 48h, etc.

[0081] As an optional embodiment, the temperature of the freeze-drying is -40-25℃.

[0082] As an optional embodiment, the method for purifying the phosphorodiamidate morpholino oligonucleotide specifically includes the following steps: (1) terminating the ammonolysis reaction: water is added to the phosphorodiamidate morpholino oligonucleotide ammonolysis solution to terminate the ammonolysis reaction, and a terminated reaction solution is obtained; wherein the volume ratio of the water to the phosphorodiamidate morpholino oligonucleotide ammonolysis solution is (4-10):1; (2) adjusting the concentration of the organic solvent in the sample: an organic solvent is added to the terminated reaction solution to obtain a mixed solution; wherein the volume of the organic solvent accounts for 40-60% of the total volume of the mixed solution; the organic solvent is selected from any one or a combination of at least two of acetonitrile, methanol and dimethyl sulfoxide; (to prevent hydrophobic impurities from precipitating, causing filtration difficulty) (3) filtration: the mixed solution is filtered to obtain a solution to be ultrafiltrated; (to remove visible impurities, reduce the risk of ultrafiltration membrane blockage, and improve the efficiency of liquid exchange) (4) first ultrafiltration and liquid exchange (continuous ultrafiltration and liquid exchange): The ultrafiltration is performed using regenerated cellulose membrane with a molecular weight cut-off of ≥2 kD; wherein, a mixture of machine solvent and water is used as the displacement solution, and the volume of the displacement solution is controlled to be 6-10 times; (the purpose is to remove ammonia and small molecular impurities in the crude product); then, the displacement solution is changed to purified water, and the displacement is continued for 6-10 times, so as to reduce the proportion of acetonitrile in the sample solution, and the sample after ultrafiltration is obtained (the purpose is to better combine with the stationary phase when the sample is loaded on the reversed-phase chromatography); (5) Ion pair reversed-phase chromatography loading: The sample after ultrafiltration is pumped into a C18 reversed-phase column, and the loading capacity is controlled to be in the range of 1-3%; (6) Ion pair reversed-phase chromatography elution: The mobile phase A uses 20-100 mM TEAA, pH 7.0±1.0, and the mobile phase B uses 40-90% acetonitrile aqueous solution, and the elution gradient is divided into two sections: the first section is 10-40% B, 30 min; and the second section is 40-80% B, 160 min; the main peak absorption value of more than 1000 mAu is collected, and one tube is collected every 1 min, so as to obtain the eluate; (7) Second ultrafiltration displacement (ultrafiltration desalination): The ultrafiltration is performed using regenerated cellulose membrane with a molecular weight cut-off of ≥2 kD; if the sample volume is too large, the collected sample can be concentrated by a proper multiple, so as to reduce the displacement time, and then the sample is continuously displaced by purified water for 6-10 times (the purpose is to remove TEAA in the sample); (8) Freeze drying: 80% ethanol aqueous solution is prepared as a pre-freezing solution, and a proper amount of dry ice is added to the pre-freezing solution for cooling. Then, the sample after ultrafiltration desalination is transferred to a freeze-drying bottle, and is pre-frozen to a solid state. Finally, the sample is transferred to a hanging bottle freeze dryer, and is freeze-dried for 36-48 h.

[0083] The application is further described below through examples. Unless otherwise specified, the materials in the examples are prepared according to the existing method, or are directly purchased from the market.

[0084] Example 1 The present example provides a purification process for obtaining high-purity phosphorodiamidate morpholino oligonucleotide (tipirsen, the structural formula is as shown in Figure 2 The present example provides a purification process for obtaining high-purity phosphorodiamidate morpholino oligonucleotide (tipirsen, the structural formula is as shown in (1) Termination of ammonolysis reaction: A 6-fold volume of purified water is slowly added to the high-concentration phosphorodiamidate morpholino oligonucleotide (PMO) ammonolysis solution, so as to terminate the ammonolysis reaction of the sample, and a terminated reaction solution is obtained; The phosphorodiamidate morpholino oligonucleotide ammonolysis solution includes, in terms of mass percentage: 14% phosphorodiamidate morpholino oligonucleotide, 25% ammonia, 5% hydrophobic small molecules, and the balance is water; (2) Adjust the sample acetonitrile concentration: After adding purified water, then add pure acetonitrile, stirring while adding, finally control the acetonitrile volume concentration at 50%, prevent hydrophobic impurities from precipitating, get the mixed solution; (3) Filtration: The mixed solution sample is filtered with a 0.45 μm filter before ultrafiltration, to remove visible impurities, reduce the risk of ultrafiltration membrane blockage, and improve the efficiency of liquid exchange; (4) First ultrafiltration liquid exchange (continuous ultrafiltration liquid exchange): Ultrafiltration uses a 2 kD Sartorius regenerated cellulose membrane package for ultrafiltration liquid exchange, first uses 50 vol% acetonitrile solution as the displacement solution, the liquid exchange volume is 6 times the sample volume, to separate the large amount of ammonia water and hydrophobic small molecules in the crude product; then, change the displacement solution to purified water and continue ultrafiltration liquid exchange, the displacement volume is 7 times the sample volume, the purpose is to enable the sample to better combine with the stationary phase during reverse phase loading, and the acetonitrile in the sample needs to be removed; The process parameters of the first ultrafiltration liquid exchange include: temperature 25℃, transmembrane pressure (TMP) 1.5 Bar, inlet flow rate of liquid exchange 200 LMH.

[0085] (5) Ion pair reverse phase chromatography loading: Pump the ultrafiltrated sample into an Ultimate XB-C18, 10 μm, 300 Å C18 reverse phase chromatography column, and control the loading capacity at 2%; (6) Ion pair reverse phase chromatography elution: Mobile phase A uses 50 mM TEAA aqueous solution, pH 7.0, mobile phase B uses 50% acetonitrile aqueous solution; the elution gradient is divided into two sections, the first section is 10~40%B for 30 min, and the second section is 40~80%B for 160 min; collect the main peak absorption value above 1000 mAu part, 1 tube per 1 min; (7) Second ultrafiltration liquid exchange (ultrafiltration desalination): Ultrafiltration uses a 2 kD Sartorius regenerated cellulose membrane package for ultrafiltration liquid exchange, uses purified water as the displacement solution, the liquid exchange volume is 7 times the sample volume, the purpose is to enable the freeze-dried product to present excellent properties, and the collected sample needs to be desalted; The process parameters of the second ultrafiltration liquid exchange include: temperature 25℃, transmembrane pressure (TMP) 1.5 Bar, inlet flow rate of liquid exchange 200 LMH.

[0086] (8) Freeze-drying: Prepare 80 vol% ethanol aqueous solution as pre-freezing solution, add appropriate amount of dry ice to the pre-freezing solution to cool to -45±5℃; then, transfer the ultrafiltration desalted sample to a freeze-drying bottle, pre-freeze to solid state. Finally, transfer to a hanging bottle freeze dryer, freeze-dry for 36 h to obtain high-purity phosphorodiamidate morpholino oligonucleotide (PMO) product.

[0087] After freeze-drying, analyze the purity of the phosphorodiamidate morpholino oligonucleotide (PMO) product by liquid chromatography, and the purity of the main peak can reach 98.67%, and the single impurity is controlled within 1.5% (such as Figure 3

[0088] Example 2 This example provides a purification process for obtaining high-purity phosphorodiamidate morpholino oligonucleotide (Golodirsen, structural formula as shown in Figure 4 (1) Terminate the ammonolysis reaction: Slowly add 6 times the volume of purified water to the high-concentration phosphorodiamidate morpholino oligonucleotide (PMO) ammonolysis solution to terminate the sample ammonolysis reaction to obtain a terminated reaction solution; The phosphorodiamidate morpholino oligonucleotide ammonolysis solution includes, by mass percentage: phosphorodiamidate morpholino oligonucleotide 16%, ammonia 21%, hydrophobic small molecule 3%, and the balance is water; (2) Adjust the acetonitrile concentration of the sample: After adding purified water, then add pure acetonitrile, stir while adding, and finally control the volume concentration of acetonitrile at 50% to prevent hydrophobic impurities from precipitating to obtain a mixed solution; (3) Filtration: Filter the sample with a 0.45 μm filter before ultrafiltration to remove visible impurities, reduce the risk of ultrafiltration membrane blockage, and improve the efficiency of liquid exchange; (4) First ultrafiltration liquid exchange (continuous ultrafiltration liquid exchange): Ultrafiltration uses a 2 kD Sartorius regenerated cellulose membrane to perform ultrafiltration liquid exchange. First, use 50 vol% acetonitrile aqueous solution as the displacement solution, and the displacement volume is 6 times the sample volume to separate the large amount of ammonia water and hydrophobic small molecules in the crude product. Then, change the displacement solution to purified water to continue ultrafiltration liquid exchange, and the displacement volume is 7 times the sample volume. The purpose is to enable the sample to better combine with the stationary phase during reverse phase loading, and to remove acetonitrile in the sample. The process parameters of the first ultrafiltration liquid exchange include: temperature 25℃, transmembrane pressure (TMP) 1.5 Bar, and inlet flow rate of liquid exchange 200 LMH.

[0089] (5) Ion pair reverse phase chromatography loading: ​​The ultrafiltrated sample was pumped into a chromatographic column of Ultimate XB-C18, 10 μm, 300 Å, and the loading capacity was controlled at 2%; (6) Ion pair reverse phase chromatography elution: The mobile phase A was 50 mM TEAA aqueous solution, pH 7.0, and the mobile phase B was 50% acetonitrile aqueous solution; the elution gradient was divided into two sections, the first section was 10~40% B for 30 min, and the second section was 40~80% B for 160 min; the main peak absorption value was collected at 1000 mAu or more, and 1 tube was collected every 1 min; (7) Second ultrafiltration and replacement (ultrafiltration desalination): The ultrafiltration and replacement were performed using a 2 kD Sartorius regenerated cellulose membrane, and purified water was used as the replacement solution, and the replacement volume was 7 times the sample volume, and the purpose was to present excellent properties of the freeze-dried product, and the desalination treatment was required for the elution collected sample; The process parameters of the second ultrafiltration and replacement include: temperature 25℃, transmembrane pressure (TMP) 1.5 Bar, and inlet flow rate 200 LMH.

[0090] (8) Freeze-drying: An 80 vol% ethanol aqueous solution was prepared as a pre-freezing solution, and the pre-freezing solution was cooled to-50℃ by adding a proper amount of dry ice; then, the sample after ultrafiltration and desalination was transferred to a freeze-drying bottle and pre-frozen to a solid state. Finally, it was transferred to a hanging bottle freeze-drying machine and freeze-dried for 36 h to obtain high-purity phosphorodiamidate morpholino oligonucleotide (PMO) product.

[0091] After freeze-drying, the purity of the phosphorodiamidate morpholino oligonucleotide (PMO) product was detected by analytical liquid chromatography, and the main peak purity could reach 99.79% (as shown in Figure 5 ).

[0092] Comparative Example 1 This comparative example provides a purification process for obtaining phosphorodiamidate morpholino oligonucleotide (Tegoprazan, structural formula as shown in Figure 2 ). (1) Termination of ammonolysis reaction: A 6-fold volume of purified water was slowly added to the high-concentration phosphorodiamidate morpholino oligonucleotide (PMO) ammonolysis solution to terminate the ammonolysis reaction of the sample, and a terminated reaction solution was obtained; The phosphorodiamidate morpholino oligonucleotide ammonolysis solution includes, by mass percentage: phosphorodiamidate morpholino oligonucleotide 14%, ammonia 25%, and hydrophobic small molecule 5%, and the balance is water; (2) Adjusting the acetonitrile concentration of the sample: After adding purified water, then add pure acetonitrile, stirring while adding, finally control the volume concentration of acetonitrile at 50%, to prevent the precipitation of hydrophobic impurities, to obtain a mixed solution; (3) Filtration: The mixed solution sample is filtered with a 0.45 μm filter before ultrafiltration to remove visible impurities, reduce the risk of ultrafiltration membrane blockage, and improve the efficiency of liquid exchange; (4) First ultrafiltration liquid exchange (continuous ultrafiltration liquid exchange): The ultrafiltration uses a 2 kD Sartorius regenerated cellulose membrane package for ultrafiltration liquid exchange, and directly uses a pure water solution as the displacement solution, and the liquid exchange volume is 10 times the sample volume, to separate a large amount of ammonia water and hydrophobic small molecules in the crude product; The process parameters of the first ultrafiltration liquid exchange include: temperature 25℃, transmembrane pressure (TMP) 1.5 Bar, and inlet flow rate of liquid exchange 200 LMH.

[0093] (5) Normal reverse phase chromatography loading: The ultrafiltrated sample is pumped into an Ultimate XB-C18, 10 μm, 300 Å chromatography column, and the loading capacity is controlled at 2%; (6) Normal reverse phase chromatography elution: The mobile phase A uses 0.1% formic acid aqueous solution, pH 7.0, and the mobile phase B uses pure acetonitrile; the elution gradient is 12~30vol%B, 144 min, and the main peak absorption value is collected above 1500 mAu, and 1 tube is collected every 1 min; (7) Second ultrafiltration liquid exchange (ultrafiltration desalination): The ultrafiltration uses a 2 kD Sartorius regenerated cellulose membrane package for ultrafiltration liquid exchange, and uses purified water as the displacement solution, and the liquid exchange volume is 7 times the sample volume, to control the pH of the product at neutral.

[0094] The process parameters of the second ultrafiltration liquid exchange include: temperature 25℃, transmembrane pressure (TMP) 1.5 Bar, and inlet flow rate of liquid exchange 200 LMH.

[0095] (8) Freeze-drying: An 80 vol% ethanol aqueous solution is prepared as a pre-freezing solution, and an appropriate amount of dry ice is added to the pre-freezing solution to cool it to -50℃; then, the sample after ultrafiltration desalination is transferred to a freeze-drying bottle and pre-frozen to a solid state. Finally, it is transferred to a hanging bottle freeze-drying machine, the cold trap temperature is set to -80℃, and freeze-drying is performed for 36 h to obtain a higher purity phosphorodiamidate morpholino oligonucleotide (PMO) product.

[0096] After freeze-drying, the purity of the phosphorodiamidate morpholino oligonucleotide (PMO) product is detected by analytical liquid chromatography, and the main peak purity is only 95.33% (as shown inFigure 6 As shown in the figure).

[0097] In summary, the present application utilizes the characteristics of ultrafiltration and reverse phase chromatography respectively to remove different impurities in two-step purification in a cross-removing manner, and the purity of the freeze-dried product can reach more than 98%. The phosphorodiamidite morpholino oligonucleotide (PMO) purification process has the advantages of high efficiency, low cost, mature technology and easy scale-up production, and is suitable for separating and purifying phosphorodiamidite morpholino oligonucleotide (PMO) ammonolysis solution to obtain high-quality phosphorodiamidite morpholino oligonucleotide (PMO) product.

[0098] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for purifying phosphorylated diamine morpholino oligonucleotides, characterized in that, The purification method for the phosphorylated diamine morpholino oligonucleotide includes: The solution of phosphorylated diamine morpholino oligonucleotide was subjected to a first ultrafiltration, ion-pair reversed-phase chromatography, a second ultrafiltration, and freeze-drying to obtain high-purity phosphorylated diamine morpholino oligonucleotide.

2. The purification method for phosphorylated diamine morpholino oligonucleotides according to claim 1, characterized in that, The following pretreatment steps are also included before the first ultrafiltration fluid change: Water is added to the ammonolysis solution of the phosphorylated diamine morpholine oligonucleotide to terminate the ammonolysis reaction and obtain a terminated reaction solution; an organic solvent is added to the terminated reaction solution to obtain a mixture; the mixture is filtered to obtain a solution to be ultrafiltered.

3. The purification method for phosphorylated diamine morpholino oligonucleotides according to claim 1 or 2, characterized in that, The phosphoryldiamine morpholine oligonucleotide ammonolysate comprises, by mass percentage: 5-40% phosphoryldiamine morpholine oligonucleotide, 5-30% ammonia, 1-10% hydrophobic small molecules, and the remainder being water. Preferably, the volume ratio of the water to the phosphoryl diamine morpholino oligonucleotide ammonolysate is (4~10):1; Preferably, the volume of the organic solvent accounts for 40-60% of the total volume of the mixture; Preferably, the organic solvent is selected from any one or a combination of at least two of acetonitrile, methanol, and dimethyl sulfoxide, with acetonitrile being the most preferred.

4. The purification method for phosphorylated diamine morpholino oligonucleotides according to claim 1, characterized in that, Both the first and second ultrafiltration fluid changes used regenerated cellulose membranes. Preferably, the regenerated cellulose membrane used for the first and second ultrafiltration fluid replacements has a molecular weight cutoff of ≤2 kD.

5. The purification method for phosphorylated diamine morpholino oligonucleotides according to claim 1, characterized in that, The first ultrafiltration fluid exchange includes: first, a mixture of organic solvent and water is used for the first fluid exchange, and then water is used for the second fluid exchange; Preferably, the volume of the organic solvent accounts for 40-60% of the total volume of the mixture of the organic solvent and water; Preferably, the organic solvent is selected from any one or a combination of at least two of acetonitrile, methanol, and dimethyl sulfoxide, with acetonitrile being the most preferred. Preferably, the solution is continuously replenished with a mixture of organic solvent and water during each solution change until the replenished volume is 6 to 10 times the initial volume of the sample. Preferably, the secondary solution replacement involves continuously replenishing water until the replenished volume is 6 to 8 times the initial volume of the sample; Preferably, the process parameters for the first ultrafiltration fluid exchange include: temperature 20~30℃, transmembrane pressure TMP 1.0~1.5 Bar, and inlet flow rate of 100~300 LMH.

6. The purification method for phosphorylated diamine morpholino oligonucleotides according to claim 1, characterized in that, The ion-pair reversed-phase chromatography used an octadecylsilane-bonded silica column; Preferably, the loading capacity of the chromatographic column used for the ion-pair reversed-phase chromatography is 1-3%.

7. The purification method for phosphorylated diamine morpholino oligonucleotides according to claim 1, characterized in that, In the ion-pair reversed-phase chromatography, mobile phase A is an aqueous solution of triethylamine acetate, and mobile phase B is an aqueous solution of acetonitrile. Preferably, in the mobile phase A, the concentration of the triethylamine acetate aqueous solution is 20~100 mM, and the pH of the triethylamine acetate aqueous solution is 6.0~8.0; Preferably, in the mobile phase B, the volume of the acetonitrile accounts for 40-60% of the total volume of the acetonitrile aqueous solution.

8. The purification method for phosphorylated diamine morpholino oligonucleotides according to claim 1, characterized in that, The ion-pair reversed-phase chromatography employed the following gradient elution procedure: Phase 1: 10-40% B, lasting 20-40 minutes; Phase 2: 40-80% B, lasting 150-170 minutes; Preferably, during the ion-pair reversed-phase chromatography process, the eluent with a main peak absorbance of 1000 mAu or higher is collected.

9. The purification method for phosphorylated diamine morpholino oligonucleotides according to claim 1, characterized in that, The second ultrafiltration fluid replacement includes: using water for fluid replacement; Preferably, the solution is continuously replenished with water until the replenished volume is 6 to 8 times the initial volume of the sample; Preferably, the process parameters for the second ultrafiltration fluid exchange include: temperature 20~30℃, transmembrane pressure TMP 1.0~1.5 Bar, and inlet flow rate of 100~300 LMH.

10. The purification method for phosphorylated diamine morpholino oligonucleotide according to claim 1, characterized in that, The freeze-drying temperature is -80~35℃, and the freeze-drying time is 36~48 h.

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