Method for purifying oligodeoxynucleotides

Abstract

This invention relates to a method for manufacturing oligonucleotides (ODNs), which involves purifying a crude solution containing oligonucleotides, wherein the 5' end of the oligonucleotides does not have a protecting group, through a single reverse high-performance liquid chromatography column and under specific extraction conditions, by performing no more than two purification steps, thereby improving the purity of the oligonucleotides.

Description

Technical Field

[0001] The present invention relates to a method for producing an oligonucleotide, and in particular to a method for purifying an oligonucleotide having no protecting group at the 5' end and high purity. Prior Art

[0002] CpG oligodeoxynucleotides (CpG ODN) are oligonucleotides, short single-stranded DNA molecules, whose sequence contains a cytosine deoxynucleotide (C) followed by a guanine deoxynucleotide (G), and p is the phosphate bond connecting the two. CpG ODN has been developed as an immunomodulator or vaccine adjuvant.

[0003] Currently, the production process of CpG ODN is mainly carried out by two types of chromatography purification methods, namely reverse-phase high performance liquid chromatography (RP-HPLC) and anion exchange chromatography [ion (specifically, anion)-exchange liquid chromatography, IE-LC]. Among them, most processes use IE-LC to purify CpG ODN.

[0004] It is known that before purifying oligonucleotides by RP-HPLC, a protecting group, such as dimethoxytrityl (DMT), will be retained at the 5' end of the sequence of the main CpG ODN product. The main CpG ODN product having DMT at the 5' end of the sequence is also called a DMT-on main product, and the process of purifying CpG ODN using the DMT protecting group is also called a DMT-on purification process.

[0005] In the DMT-on purification process, the crude solution is first purified by the first purification step. The hydrophobicity of the 5'-DMT protecting group is used to retain the main product of DMT-on [the sequence is full-length, i.e., the length is n (-mer)] in the reversed-phase chromatography column. Some short-sequence impurities in the crude solution (the sequence is not full-length, for example, the length is n-1, n-2, n-3, nm, etc.) are not easily retained in the reversed-phase chromatography column and are washed out at the 5' end due to the lack of the DMT protecting group (also known as DMT-off).

[0006] After the DMT-off impurities are washed out of the reverse phase chromatography column, the DMT-on main product retained on the column is then washed with an acidic aqueous solution to remove the DMT protecting group at the 5' end. The aforementioned acidic aqueous solution generally contains a 1% to 3% dichloroacetic acid (DCA) aqueous solution, a 1% to 3% trichloroacetic acid (TCA) aqueous solution, a 1% to 3% trifluoroacetic acid (TFA) aqueous solution, or an acetic acid (acetic acid) aqueous solution greater than 50%, but does not contain an organic solvent, so the CpG ODN main product (also called the DMT-off main product) after the DMT protecting group at the 5' end is still retained in the column.

[0007] Afterwards, a second purification step was performed, which used a mobile phase containing an organic solvent (acetonitrile or methanol) with a gradient concentration to elute the reverse phase chromatography column to elute the main DMT-off product.

[0008] However, the conventional DMT-on purification process has the following risks. First, after the first purification, the removal of the 5'-DMT protecting group of the main product with an acidic aqueous solution is a chemical reaction, and it is difficult to determine whether the DMT is completely removed, and the step of removing the DMT also increases the process operation time. Second, the longer the main CpG ODN product is in an acidic environment, the higher the risk of depurination side reactions and the formation of depurination impurities. The depurination reaction mechanism is shown in the following formula (I).

[0009] In response to the above problems, the industry has proposed several process improvements. The first method is to use a tangential flow filtration system (TFF) to remove the DMT protecting group at the 5' end of the main CpG ODN product and perform a second purification. However, this purification method requires two different systems and is more cumbersome to operate. Secondly, the CpG ODN sequence product obtained by this purification method is a mixture of locked nucleic acid monomers (LNA monomers) and DNA monomers, and has a low purity (only 88%, less than 90%).

[0010] The second method is to purify the main CpG ODN product using anion-exchange liquid chromatography. The CpG ODN sequence product obtained by this purification method is mixed with 2'-modified RNA monomers (such as 2'-fluororibonucleosides and 2'-O-methylribonucleosides), but the column packing used is relatively expensive.

[0011] The third method is to use hydrophilic-interaction liquid chromatography (HILIC) to obtain a higher purity CpG ODN main product. However, this purification method does not specifically disclose which CpG ODN is suitable for purification, and the column packing used is relatively expensive.

[0012] The fourth method is to purify the main CpG ODN product by hydrophobic interaction chromatography (HIC). However, the CpG ODN sequence product obtained by this purification method is mixed with 2'-modified RNA monomers [2'-modified RNA monomers, such as 2'-fluororibonucleosides, 2'-O-methylribonucleosides] and DNA monomers, and the column packing used is relatively expensive.

[0013] The fifth method is to purify the main CpG ODN product by electrophoresis. However, this purification method does not reveal which CpG ODN is suitable for purification and is not suitable for mass production.

[0014] The above-mentioned conventional process cannot improve the mass production of oligonucleotides based on the existing reverse phase chromatography equipment, or the column filler used is relatively expensive. Therefore, it is urgent to propose an improved oligonucleotide purification method to solve the various shortcomings of the conventional process. Summary of the invention

[0015] Therefore, one aspect of the present invention is to provide a method for producing an oligonucleotide, wherein the oligonucleotide is a CpG ODN, the 5' end of the sequence of which does not have a protecting group, and a CpG ODN main product with a purity of not less than 90% can be obtained by a single reverse HPLC column and specific elution conditions for no more than two purification steps, while effectively overcoming the risks of incomplete DMT removal or the generation of depurinated impurities in the conventional purification process.

[0016] Secondly, another aspect of the present invention is to provide a method for producing an oligonucleotide, which utilizes an oligonucleotide with a CpG ODN purity of at least 80%, and whose 5' end does not have a DMT protecting group, and a single purification step is performed through a single reverse HPLC column and specific elution conditions to obtain a CpG ODN main product with a purity of not less than 90%.

[0017] Furthermore, another aspect of the present invention is to provide a method for producing an oligonucleotide, which utilizes an oligonucleotide with a CpG ODN purity of 30% to less than 80%, and the 5' end of the sequence does not have a DMT protecting group, and performs a secondary purification step through the same reverse HPLC column and specific elution conditions to obtain a CpG ODN main product with a purity of not less than 90%.

[0018] According to the above aspect of the present invention, a method for purifying oligonucleotides is provided. In one embodiment, the above method includes the following steps. First, a raw material containing CpG oligodeoxynucleotides (CpG ODN) is dissolved in a mobile phase A to form a crude solution, wherein the sequence of the CpG ODN can be, for example, as shown in the sequence identification number (SEQ ID NO): 1 or SEQ ID NO: 2, and the 5' end of the CpG ODN does not have a protecting group, and the mobile phase A contains a weak acidic reagent and a weak alkaline reagent, the weak acidic reagent contains 0.05M to 0.20M acetic acid or hexafluoroisopropanol (HFIP), the weak alkaline reagent contains triethylamine (TEA), N,N-diisopropylethylamine (DIPEA) or n-hexylamine (HA), and the pH value of the mobile phase A can be, for example, pH 7.0 to pH 8.4.

[0019] Next, the crude product solution is loaded into a reverse phase chromatography column to perform a first purification step, and a first solution is obtained by using mobile phase A and mobile phase B, wherein mobile phase B is acetonitrile or methanol, and the flow rate of mobile phase A and mobile phase B can be, for example, 0.2 to 0.3 mL / min / column volume (mL).

[0020] Then, when the purity of CpG ODN in the first solution is not less than 90%, the first solution is post-treated to obtain CpG ODN. In another way, when the purity of CpG ODN in the first solution is less than 90%, the first solution is loaded into the aforementioned reverse phase chromatography column, and a second purification step is performed using mobile phase C and mobile phase D to obtain a second solution, wherein mobile phase C contains a weak acidic reagent and a weak alkaline reagent but is different from mobile phase A, and mobile phase D is acetonitrile or methanol but is different from mobile phase B. When the purity of CpG ODN in the second solution is not less than 90%, the second solution is post-treated to obtain CpG ODN.

[0021] In the above embodiment, the purity of CpG ODN in one of the crude solutions may be, for example, at least 80%.

[0022] In the above embodiment, the protecting group comprises dimethoxytrityl (DMT).

[0023] In the above embodiment, when the aforementioned weakly acidic reagent is acetic acid, the concentration of the weakly alkaline reagent may be equal to the concentration of the weakly acidic reagent.

[0024] In the above embodiment, when the weak acidic reagent is hexafluoroisopropanol, the concentration of the weak alkaline reagent can be, for example, 1 / 5 to 1 / 15 of the concentration of the weak acidic reagent. In some examples, when the weak acidic reagent is hexafluoroisopropanol, the concentration of the weak alkaline reagent can be, for example, 1 / 10 to 1 / 12 of the concentration of the weak acidic reagent.

[0025] In the above embodiment, the purity of CpG ODN in the crude solution may be, for example, 30% to less than 80%. In this example, when the purity of CpG ODN in the first solution is less than 90%, the first solution is loaded into the reverse phase chromatography column, and the second purification step is performed by mobile phase C and mobile phase D to obtain the second solution. The mobile phase C and mobile phase A may be, for example, the same or different, and the mobile phase D and mobile phase B may be, for example, the same or different. When the purity of CpG ODN in the second solution is not less than 90%, the second solution is post-treated to obtain CpG ODN.

[0026] In the above embodiment, the mobile phase C and the mobile phase A may be different, and the mobile phase D and the mobile phase B may be different.

[0027] In the above embodiment, the post-treatment may include a desalting step and a drying step. In this example, the drying step may selectively include a freeze-drying step.

[0028] According to another aspect of the present invention, a method for purifying oligonucleotides is provided. In one embodiment, the method comprises the following steps. First, a raw material containing CpG ODN is dissolved in a mobile phase A to form a crude solution, wherein the sequence of the CpG ODN is as shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the 5' end of the CpG ODN does not have a DMT protecting group, and the mobile phase A comprises a weak acidic reagent and a weak alkaline reagent, wherein the first weak acidic reagent comprises 0.10M to 0.20M acetic acid or hexafluoroisopropanol (HFIP), and the first weak alkaline reagent comprises triethylamine (TEA), N,N-diisopropylethylamine (DIPEA) or n-hexylamine (HA), and the pH value of the mobile phase A is pH 7.0 to pH 8.4, and the purity of the CpG ODN in the crude solution is at least 80%.

[0029] Next, the crude product solution is loaded into a reverse phase chromatography column, and a first purification step is performed by mobile phase A and mobile phase B to obtain a first solution, wherein mobile phase B is acetonitrile or methanol, and the flow rate of mobile phase A and mobile phase B is 0.2 to 0.3 mL / min / column volume (mL). When the purity of CpG ODN in the first solution is not less than 90%, the first solution is post-treated to obtain CpG ODN.

[0030] According to another aspect of the present invention, a method for purifying oligonucleotides is provided. In one embodiment, the aforementioned method includes the following steps. First, a raw material containing CpG oligodeoxynucleotide (CpG ODN) is dissolved in a mobile phase A to form a crude solution, wherein the sequence of the CpG ODN is as shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the 5' end of the CpG ODN does not have a DMT protecting group, and the mobile phase A comprises a weak acidic reagent and a weak alkaline reagent, the first weak acidic reagent comprises 0.05M to 0.20M acetic acid or hexafluoroisopropanol (HFIP), and the first weak alkaline reagent comprises triethylamine (TEA), N,N-diisopropylethylamine (DIPEA) or n-hexylamine (HA), and the pH value of the mobile phase A can be, for example, pH 7.0 to pH 8.4, and the purity of the CpG ODN in the crude solution is 30% to less than 80%.

[0031] Next, the crude product solution is loaded into a reverse phase chromatography column and a first purification step is performed using mobile phase A and mobile phase B to obtain a first solution, wherein mobile phase B is acetonitrile or methanol, and the flow rates of mobile phase A and mobile phase B can be, for example, 0.2 to 0.3 mL / min / column volume (mL).

[0032] When the purity of CpG ODN in the first solution is less than 90%, the first solution is loaded into the aforementioned reverse phase chromatography column, and a second purification step is performed using mobile phase C and mobile phase D to obtain a second solution, wherein the mobile phase C is the same as or different from the mobile phase A, and the mobile phase D is the same as or different from the mobile phase B, and the pH value of the mobile phase C is pH 7.0 to pH 8.4. When the purity of CpG ODN in the second solution is not less than 90%, the second solution is post-treated to obtain CpG ODN.

[0033] The oligonucleotide (ODN) manufacturing method of the present invention is applied to a crude solution containing oligonucleotides, wherein the 5' end of the oligonucleotides does not have a protecting group, and is subjected to no more than two purification steps through a single reverse HPLC column and specific elution conditions to obtain oligonucleotides with a purity of not less than 90%.

[0034] It is to be understood that the foregoing general description and the following detailed description are merely illustrative and are intended to provide further explanation of the invention as claimed. Simple diagram description

[0035] In order to make the above and other purposes, features, advantages and embodiments of the present invention more clearly understood, the detailed description of the attached drawings is as follows: [FIG. 1] to [FIG. 3] show the HPLC spectra according to Examples 8, 9 and 11 of the present invention. Implementation

[0036] Unless otherwise defined, the technical and scientific terms used below have the same meanings as those understood by those of ordinary skill in the art to which the present invention belongs. If the definition or usage of a term in a reference is inconsistent or contrary to the definition of the term herein, the definition of the term herein shall apply, and the definition of the term in the reference shall not apply. Secondly, unless otherwise defined in the context, a singular term may include the plural, and a plural term may include the singular.

[0037] As mentioned above, the present invention provides a method for producing an oligonucleotide, wherein the oligonucleotide is a CpG ODN, the 5' end of the sequence of which does not have a protecting group, and the purity of the oligonucleotide can be improved by performing no more than two purification steps through a single reverse HPLC column and specific elution conditions.

[0038] In other words, in one embodiment, the "CpG ODN" referred to herein is an artificially synthesized and modified oligonucleotide, such as a phosphorothioate oligonucleotide, and its sequence and length are not particularly limited. In some examples, the length of the oligonucleotide may be no more than 30 nucleotides (also known as 30-mer), or no more than 25-mer, or no more than 24-mer.

[0039] The "protecting group" referred to herein refers to the protecting group located at the 5' end of the oligonucleotide sequence, and its type is not particularly limited. In some specific examples, the crude oligonucleotide to be purified does not have a dimethoxytrityl (DMT) protecting group at the 5' end of the sequence, and the purified product can be referred to as a DMT-off main product.

[0040] In order to retain the main product of DMT-off in the column, in one embodiment, first, a raw material containing CpG oligodeoxynucleotides (CpG ODN) is dissolved in mobile phase A to form a crude solution, wherein the sequence of CpG ODN can be, for example, SEQ ID NO: 1 or SEQ ID NO: 2, and the 5' end of CpG ODN does not have a protecting group (i.e., DMT-off). In this embodiment, mobile phase A comprises a weak acidic reagent and a weak alkaline reagent, the weak acidic reagent comprises 0.05M to 0.20M acetic acid or hexafluoroisopropanol (HFIP) or 0.10M to 0.20M acetic acid or HFIP, and the weak alkaline reagent comprises triethylamine (TEA), N,N-diisopropylethylamine (DIPEA) or n-hexylamine (HA), and the pH value of mobile phase A can be, for example, pH 7.0 to pH 8.4. It is explained here that since the non-acidic mobile phase A is used in this case, no depurinated impurities are formed in the purification process. In some examples, when the aforementioned weak acidic reagent is acetic acid, the concentration of the weak alkaline reagent can be equal to the concentration of the weak acidic reagent. In other examples, when the weak acidic reagent is hexafluoroisopropanol, the concentration of the weak alkaline reagent can be, for example, 1 / 5 to 1 / 15 of the concentration of the weak acidic reagent, or the concentration of the weak alkaline reagent can be, for example, 1 / 10 to 1 / 12 of the concentration of the weak acidic reagent. Taking HFIP with a concentration of 0.05M to 0.20M as an example, the concentration of the weak acidic reagent can be, for example, 0.004M to 0.040M. If the mobile phase A does not use the aforementioned weak acidic reagent and weak alkaline reagent, the retention patterns of the DMT-off main product and the impurities in the reverse phase chromatography column will be relatively similar, which will be unfavorable for separating the two, resulting in the main product being difficult to achieve the desired purity (e.g., at least 90%).

[0041] Next, the crude product solution is loaded into a reverse phase chromatography column to perform a first purification step, and a first solution is obtained by mobile phase A and mobile phase B, wherein mobile phase B is acetonitrile or methanol. Generally speaking, the flow rates of mobile phase A and mobile phase B are not particularly limited, and depend on the volume of the selected reverse phase chromatography column. In some examples, the flow rates of mobile phase A and mobile phase B can be, for example, 0.2 to 0.3 mL / min / column volume (mL), or 0.24±10% mL / min / column volume (mL), so that the DMT-off main product is easily retained in the reverse phase chromatography column, and the DMT-off impurities are easily washed out of the reverse phase chromatography column. If the flow rates of mobile phase A and mobile phase B are less than 0.2 mL / min / column volume (mL), the purification process time will be too long, increasing the production time cost. If the flow rate of mobile phase A and mobile phase B is higher than 0.3 mL / min / column volume (mL), the yield will be lower.

[0042] Then, when the purity of CpG ODN in the first solution is not less than 90%, the first solution is post-treated to obtain CpG ODN. In another way, when the purity of CpG ODN in the first solution is less than 90%, the first solution is loaded into the same reverse phase chromatography column as above, and a second purification step is performed by mobile phase C and mobile phase D to obtain a second solution. In this embodiment, mobile phase C and mobile phase D are not particularly limited, mobile phase C and mobile phase A may be the same or different, and mobile phase D and mobile phase B may also be the same or different. In some examples, mobile phase C and mobile phase A may be, for example, different, and mobile phase D and mobile phase B may also be, for example, different. When the purity of CpG ODN in the second solution is not less than 90%, the second solution is post-treated to obtain CpG ODN.

[0043] In some embodiments, the purity of CpG ODN in the crude solution is not particularly limited. However, in some examples, the purity of CpG ODN in the crude solution may be, for example, at least 30%, or 30% to less than 80%, or at least 80%.

[0044] When the purity of the CpG ODN in the crude solution is at least 80%, a single purification step can be performed using the aforementioned oligonucleotide purification method to obtain an oligonucleotide with a purity of not less than 90%.

[0045] When the purity of CpG ODN in the crude solution is 30% to less than 80%, the aforementioned oligonucleotide purification method can also be applied, and the purification steps are performed no more than twice in the same reverse phase chromatography column and specific mobile phase, and oligonucleotides with a purity of not less than 90% can also be obtained. In this embodiment, the raw material containing CpG ODN is dissolved in mobile phase A to form a crude solution with a CpG ODN purity of 30% to less than 80%, wherein the 5' end of the CpG ODN does not have a protecting group.

[0046] Next, the crude product solution is loaded into a reverse phase chromatography column, and a first purification step is performed using mobile phase A and mobile phase B to obtain a first solution, wherein mobile phase B is acetonitrile or methanol, and the flow rates of mobile phase A and mobile phase B can be, for example, 0.2 to 0.3 mL / min / column volume (mL), or 0.24±10% mL / min / column volume (mL).

[0047] When the purity of CpG ODN in the first solution is less than 90%, the first solution is loaded into the same reverse phase chromatography column as above, and a second purification step is performed by mobile phase C and mobile phase D to obtain a second solution. In this embodiment, mobile phase C and mobile phase D are not particularly limited, and mobile phase C and mobile phase A may be the same or different, and mobile phase D and mobile phase B may also be the same or different. In some examples, mobile phase C and mobile phase A may be different, and mobile phase D and mobile phase B may also be different. When the purity of CpG ODN in the second solution is not less than 90%, the second solution is post-treated to obtain CpG ODN.

[0048] In other examples, the CpG ODN main product with a purity of not less than 90% obtained from the first solution and the second solution can be selectively combined and post-processed, wherein the post-processing can include a desalting step and a drying step. In this example, the aforementioned drying step can selectively include a freeze-drying step.

[0049] The above oligonucleotide manufacturing method effectively overcomes the risk of incomplete DMT excision or generation of depurinated impurities in the conventional purification process, and the obtained CpG ODN main product with a purity of not less than 90% can be used in pharmaceutical compositions, for example, as a vaccine adjuvant, or in combination with existing drugs.

[0050] It is understood that the following specific configurations, viewpoints, illustrations and embodiments are for illustration only and are not intended to be limiting conditions of the present invention. The main features of the present invention may be applied to various embodiments without departing from the spirit and scope of the present invention. Therefore, a person with ordinary knowledge in the technical field to which the present invention belongs can easily determine the necessary technical features of the present case without departing from the spirit and scope of the present invention, and make various changes and modifications to the present invention to adapt it to different uses and conditions.

[0051] Example 1

[0052] 1.1 Sample pretreatment

[0053] 22 mg of artificially synthesized 22-mer CpG ODN (sequence: 5'-T*G*A*C*T*G*T*G*A*A*C*G*T*T*C*G*A*G*A*T*G*A-3', where * represents phosphorothioate bond, A, G, C, T represent bases, and its sequence is shown in SEQ ID NO:1; purity 83%) was dissolved in mobile phase A [triethylammonium acetate (TEAA) formed by 0.1 M acetic acid and 0.1 M triethylamine, pH 7] to prepare a crude solution.

[0054] 1.2 Purification

[0055] The crude product solution was filtered through a microporous filter membrane with an average pore size of 0.45 μm and then loaded into a preparative high performance liquid chromatography (Prep-HPLC) device for purification. The filler of the reverse phase chromatography column was C18 silica gel particles with an average particle size of 10 μm. The column diameter and length were 4.6 mm × 250 mm and the column volume was 4.2 mL, respectively, as shown in Table 1. Purification was carried out under the following conditions: (1) mobile phase A: 0.1 M TEAA, pH 7; (2) mobile phase B: acetonitrile; (3) flow rate: 1 mL / min; (4) elution gradient (as shown in Table 4).

[0056] Please refer to Table 1, which shows the purification conditions and results of Example 1. In the component solution obtained in the main interval, the purity of CpG ODN is 93%. After the above-mentioned component solution is washed with water to remove excess salt through the same branch column, the main product is washed out of the column with a mixture of water and acetonitrile, and a solid product is obtained after freeze-drying. Its yield is shown in Table 1.

[0057] Examples 2 to 7

[0058] The sample pretreatment and purification of Examples 2 to 7 are the same as those of Example 1, except for the crude purity of CpG ODN, loading capacity, column volume, mobile phase, flow rate, elution gradient, etc., as shown in Tables 1 and 4.

[0059] Please refer to Table 1, which shows the purification conditions and results of Examples 2 to 7. In the component solutions obtained in the main interval, the purity of CpG ODN is greater than or equal to 91%. After the above-mentioned component solutions are washed with water to remove excess salts through the same branch column, the main product is washed out of the column with a mixture of water and acetonitrile, and a solid product is obtained after freeze-drying. The yield is shown in Table 1.

[0060] Example 8

[0061] 8.1 Sample preparation

[0062] 1168 mg of artificially synthesized 22-mer CpG ODN (sequence: 5'-T*G*A*C*T*G*T*G*A*A*C*G*T*T*C*G*A*G*A*T*G*A-3', where * represents phosphorothioate bond, A, G, C, T represent bases, and its sequence is shown in SEQ ID NO:1; purity 81%) was dissolved in mobile phase A [containing 0.1 M triethylammonium acetate (TEAA), pH 7] to prepare a crude solution.

[0063] 8.2 Purification

[0064] The crude product solution (volume 508 mL) was filtered through a microporous filter membrane with an average pore size of 0.45 μm and then loaded into a preparative high performance liquid chromatography (Prep-HPLC) device for purification, wherein the filler of the reverse phase chromatography column was C18 silica gel particles with an average particle size of 10 μm, the column diameter and length were 2.0 cm × 25.0 cm, and the column volume was 78 mL, as shown in Table 1. Purification was carried out under the following conditions: (1) mobile phase A: 0.1 M TEAA, pH 7; (2) mobile phase B: acetonitrile; (3) flow rate: 19 mL / min; (4) elution gradient (as shown in Table 4).

[0065] Please refer to Table 1, which shows the purification conditions and results of Example 8. In the component solution obtained in the main interval, the purity of CpG ODN was 93%. After the component solution was washed with water to remove excess salts through the same branch column, the main product was washed out of the column with a mixture of water and acetonitrile, and 0.67 g of solid product was obtained after freeze drying. Its HPLC spectrum is shown in Figure 1, and its peak results are shown in Table 5.

[0066] Example 9

[0067] 9.1 Sample preparation

[0068] 7890 mg of the artificially synthesized 24-mer CpG ODN (sequence: 5'-T*C*G*T*C*G*T*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T-3, where * represents phosphorothioate bond, G, C, and T represent bases, as shown in SEQ ID NO:2; purity 80%) was dissolved in mobile phase A [containing 0.1 M HFIP and diisopropylethylamine (DIPEA), pH 8.3] to prepare a crude solution.

[0069] 9.2 Purification

[0070] The crude product solution (volume 263 mL) was filtered through a microporous filter membrane with an average pore size of 0.45 μm and then loaded into a preparative high performance liquid chromatography (Prep-HPLC) device for purification, wherein the filler of the reverse phase chromatography column was C18, silica gel particles with an average particle size of 10 μm, the column diameter and length were 5.0 cm × 16.5 cm, and the column volume was 324 mL. Purification was carried out under the following conditions: (1) mobile phase A: 0.1 M HFIP and DIPEA, pH 8.3; (2) mobile phase B: acetonitrile; (3) flow rate: 78 mL / min; (4) elution gradient (as shown in Table 4), as shown in Table 1 and Table 4.

[0071] In the component solution obtained in the main interval, the purity of CpG ODN was 92%. After the above-mentioned component solution was washed with water to remove excess salt through the same branch column, the main product was washed out of the column with a mixture of water and acetonitrile, and 6.6 g of solid product was obtained after freeze drying. Its HPLC spectrum is shown in Figure 2, and its peak results are shown in Table 5.

[0072] Example 10

[0073] 10.1 Sample preparation

[0074] The sample pretreatment of Example 10 is the same as that of Example 8, except that the crude purity (52%) and loading (3639 mg) of CpG ODN are as shown in Table 1.

[0075] 10.2 First purification

[0076] The first purification of Example 10 was the same as that of Example 1, except for the CpG ODN loading capacity (3639 mg, of which the volume was 9098 mL), the column diameter and length (5.0 cm × 16.5 cm, respectively), the column volume was 324 mL, the flow rate (78 mL / min), the elution gradient, etc., as shown in Tables 1 and 4.

[0077] Example 10 The purity of CpG ODN in the component solution obtained in the main interval was 82%, which was less than 90%, so a second purification was performed.

[0078] 10.3 Second purification

[0079] Load the main interval component solution collected in the first purification in Section 10.2 into the same column of the same preparative HPLC equipment and purify under the following conditions: (1) Mobile phase C: 0.1 M hexafluoroisopropanol (HFIP) and triethylamine (TEA, 0.0086 M), pH 8.3; (2) Mobile phase D: methanol; (3) Flow rate: 78 mL / min; (4) Elution gradient (as shown in Table 4).

[0080] The purity of CpG ODN in the component solution obtained in the main section of Example 10 was 91%. After the component solution was washed with water to remove excess salt through the same branch column, the main product was washed out of the column with a mixture of water and acetonitrile, and 1.3 g of solid product was obtained after freeze drying.

[0081] Embodiment 11

[0082] 11.1 Sample preparation

[0083] 5795 mg of artificially synthesized 22-mer CpG ODN (sequence is the same as in Example 1, as shown in SEQ ID NO: 1; purity 32%) was dissolved in mobile phase A (containing 0.1 M TEAA, pH 7) to prepare a crude solution.

[0084] 11.2 First purification

[0085] The crude product solution (volume 1190 mL) was filtered through a microporous filter membrane with an average pore size of 0.45 μm and then loaded into a preparative high performance liquid chromatography (Prep-HPLC) device for purification. The filler of the reverse phase chromatography column was C18 silica gel particles with an average particle size of 10 μm. The column diameter and length were 5.0 cm × 16.5 cm, respectively, and the column volume was 324 mL. Purification was carried out under the following conditions: (1) mobile phase A: 0.1 M TEAA, pH 7; (2) mobile phase B: acetonitrile; (3) flow rate: 78 mL / min; (4) elution gradient (as shown in Table 4).

[0086] The purity of CpG ODN in the component solution obtained from the main interval was 84%, which was less than 90%, so a second purification was performed.

[0087] 11.3 Second purification

[0088] Load the main interval component solution collected in the first purification in Section 11.2 into the same column of the same set of preparative HPLC equipment and purify it under the following conditions: (1) Mobile phase C: 0.1 M HFIP and TEA (0.0086 M), pH 8.3; (2) Mobile phase D: methanol; (3) Flow rate: 78 mL / min; (4) Elution gradient (as shown in Table 4).

[0089] In the component solution obtained in the main section of Example 11, the purity of CpG ODN was 92%. After the component solution was washed with water to remove excess salts through the same branch column, the main product was washed out of the column with a mixture of water and acetonitrile, and 1.7 g of solid product was obtained after freeze drying. Its HPLC spectrum is shown in Figure 3, and its peak results are shown in Table 5.

[0090] Table 1

[0091] Table 2

[0092] Table 3

[0093] Table 4

[0094] Table 5

[0095] It should be noted that those with ordinary knowledge in the technical field to which the present invention belongs should understand that Examples 1 to 7 are small-scale tests, while Examples 8 to 11 are examples of actual mass production. In summary, the above-mentioned oligonucleotides of specific sequence and length, specific reagents, specific solvents, specific processing steps, specific analysis modes or specific evaluation methods are only used to illustrate the method for producing oligonucleotides. However, those with ordinary knowledge in the technical field to which the present invention belongs should understand that, without departing from the spirit and scope of the present invention, oligonucleotides of other sequences and lengths, other reagents, other solvents, other processing steps, other analysis modes or other evaluation methods can also be used in the method for producing oligonucleotides, and are not limited to the above. For example, the above-mentioned method for producing oligonucleotides can be used to purify oligonucleotides of longer or shorter lengths, and can also improve the purity of oligonucleotides.

[0096] As can be seen from the above examples, the method for producing oligonucleotides (ODN) of the present invention has the advantage that the crude DMT-off oligonucleotides can be purified by no more than two purification steps through a single reverse HPLC column under specific elution conditions to obtain oligonucleotides with a purity of not less than 90%. The obtained high-purity CpG ODN main product can be used in pharmaceutical compositions, such as as a vaccine adjuvant, or used in combination with existing drugs.

[0097] Although the present invention has been disclosed above with respect to several specific embodiments, other embodiments are also possible. Therefore, the spirit and scope of the present invention as claimed herewith should not be limited to the embodiments contained herein.

[0098] none

[0099] Domestic storage information (please note the order of storage institution, date and number) none Overseas deposit information (please note the order of deposit country, institution, date and number) none

[0100] TW202521152A_112145197_SEQL.xml

Claims

1. A method for purifying oligonucleotides, comprising: dissolving a starting material containing CpG oligodeoxynucleotides (CpG ODN) in a mobile phase A to form a crude solution, wherein the sequence of the CpG ODN is as shown in sequence identification number (SEQ ID NO): 1 or SEQ ID NO: 2, and the CpG The 5' end of the ODN does not have a protecting group. The mobile phase A contains a weak acid reagent and a weak base reagent. The weak acid reagent contains 0.05 M to 0.20 M of acetic acid or hexafluoroisopropanol (HFIP). The weak base reagent contains at least 0.0065 M of triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), or n-hexylamine (HA). The pH of the mobile phase A is from pH 7.0 to pH 8.

4. When the weak acid reagent is acetic acid, the concentration of one of the weak base reagents is equal to the concentration of one of the weak acid reagents. When the weak acid reagent is hexafluoroisopropanol, the concentration of one of the weak base reagents is 1 / 5 to 1 / 15 of the concentration of one of the weak acid reagents. The crude solution is loaded into a reversed-phase chromatography column, and a first purification step is performed using mobile phase A and mobile phase B to obtain a first solution, wherein mobile phase B is acetonitrile or methanol, and the flow rate of one of mobile phase A and mobile phase B is 0.2 to 0.3 mL / min / column volume (mL); and when the purity of CpG ODN in the first solution is not less than 90%, the first solution is post-processed to obtain the CpG ODN.

2. The method for purifying oligonucleotides as described in claim 1, wherein one of the crude solutions has a CpG ODN purity of at least 80%.

3. The method for purifying oligonucleotides as described in claim 1, wherein the protecting group comprises dimethoxytriphenylmethyl (DMT).

4. The method for purifying oligonucleotides as described in claim 1, wherein when the weak acidic reagent is hexafluoroisopropanol, the concentration of one of the weak basic reagents is 1 / 10 of the concentration of one of the weak acidic reagents.

5. The method for purifying oligonucleotides as described in claim 1, wherein one of the crude solutions has a CpG ODN purity of 30% to less than 80%.

6. The method for purifying oligonucleotides as described in claim 5, wherein when the purity of one of the CpG ODNs in the first solution is less than 90%, the first solution is loaded into the reversed-phase chromatography column, and a second purification step is performed using a mobile phase C and a mobile phase D to obtain a second solution, wherein the mobile phase C is the same as or different from the mobile phase A, and the mobile phase D is the same as or different from the mobile phase B; when the purity of one of the CpG ODNs in the second solution is not less than 90%, the second solution is subjected to the post-processing to obtain the CpG ODN.

7. The method for purifying oligonucleotides as described in claim 6, wherein mobile phase A comprises acetic acid and triethylamine, and mobile phase B is acetonitrile, mobile phase C comprises hexafluoroisopropanol and triethylamine, and mobile phase D is methanol.

8. The method for purifying oligonucleotides as described in claim 1, wherein the post-processing comprises a desalting step and a drying step.

9. The method for purifying oligonucleotides as described in claim 6, wherein the drying step comprises a freeze-drying step.

10. A method for purifying an oligonucleotide, comprising: dissolving a starting material containing CpG oligodeoxynucleotide (CpG ODN) in a mobile phase A to form a crude solution, wherein the sequence of the CpG ODN is as shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the CpG The 5' end of the ODN does not have a protecting group. The mobile phase A contains a weak acid reagent and a weak base reagent. The weak acid reagent contains 0.05 M to 0.20 M acetic acid or hexafluoroisopropanol (HFIP). The weak base reagent contains triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), or n-hexylamine (HA). The pH of the mobile phase A is from pH 7.0 to pH 8.

4. When the weak acid reagent is acetic acid, the concentration of one of the weak base reagents is equal to the concentration of the weak acid reagent. When the weak acid reagent is hexafluoroisopropanol, the concentration of one of the weak base reagents is 1 / 10 of the concentration of the weak acid reagent. The purity of the CpG ODN in the crude product solution is at least 80%. The crude solution is loaded into a reversed-phase chromatography column, and a first purification step is performed using mobile phase A and mobile phase B to obtain a first solution, wherein mobile phase B is acetonitrile or methanol, and the flow rate of one of mobile phase A and mobile phase B is 0.2 to 0.3 mL / min / column volume (mL); and when the purity of CpG ODN in the first solution is not less than 90%, the first solution is post-processed to obtain the CpG ODN.

11. A method for purifying an oligonucleotide, comprising: dissolving a starting material containing CpG oligodeoxynucleotide (CpG ODN) in a mobile phase A to form a crude solution, wherein the sequence of the CpG ODN is as shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the CpG The 5' end of the ODN does not have a protecting group. The mobile phase A contains a weak acid reagent and a weak base reagent. The weak acid reagent contains 0.10 M to 0.20 M acetic acid or hexafluoroisopropanol (HFIP). The weak base reagent contains triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), or n-hexylamine (HA). The pH of the mobile phase A is from pH 7.0 to pH 8.

4. When the weak acid reagent is acetic acid, the concentration of one of the weak base reagents is equal to the concentration of the weak acid reagent. When the weak acid reagent is hexafluoroisopropanol, the concentration of one of the weak base reagents is 1 / 10 of the concentration of the weak acid reagent. The purity of the CpG ODN in the crude product solution is 30% to less than 80%. The crude solution is loaded into a reversed-phase chromatography column and purified using mobile phase A and mobile phase B to obtain a first solution, wherein mobile phase B is acetonitrile or methanol, and the flow rate of either mobile phase A or mobile phase B is 0.2 to 0.3 mL / min / column volume (mL). When the purity of CpG ODN in the first solution is less than 90%, the first solution is loaded into the reversed-phase chromatography column and purified using mobile phase C and mobile phase D to obtain a second solution, wherein mobile phase A contains acetic acid and triethylamine, mobile phase B is acetonitrile, mobile phase C contains hexafluoroisopropanol and triethylamine, and mobile phase D is methanol, and the pH of mobile phase C is between pH 7.0 and pH 8.

4. When the purity of CpG ODN in the second solution is not less than 90%, the second solution is post-treated to obtain the CpG ODN.

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