Small nucleic acid drug purification process

Abstract

The invention discloses a small nucleic acid medicine purification process, and belongs to the technical field of nucleic acid purification. A small nucleic acid drug purification process comprises the following steps: S1, mixed mode chromatography: dissolving a small nucleic acid drug crude product in a buffer solution containing 20mM phosphate and 10% acetonitrile to obtain a solution A; loading the solution A to a mixed-mode chromatographic column with a mixed-mode chromatographic column medium comprising functionalized agarose gel, and carrying out gradient elution to remove impurities; eluting the target product with a buffer solution containing 2M NaCl and 10% acetonitrile; s2, size exclusion chromatography: diluting a small nucleic acid drug sample eluted by mixed mode chromatography with an equilibrium buffer solution, loading the sample into a size exclusion chromatography column, performing isocratic elution with the equilibrium buffer solution, monitoring an ultraviolet absorption signal at 260nm, collecting an elution peak, and freeze-drying to obtain a high-purity small nucleic acid drug; the functionalized agarose gel is agarose gel for simultaneously modifying a quaternary ammonium salt group and an alkyl group.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid purification, and in particular relates to a small nucleic acid drug purification process. Background Art

[0002] As a new generation of innovative drugs, small nucleic acid drugs have many advantages such as high specificity and high efficiency, and have shown great potential in the field of disease treatment. Small nucleic acid drugs mainly include antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), etc. Their mechanism of action is to directly target pathogenic target genes or RNA fragments through the principle of base complementary pairing, and regulate the transcription and translation process of pathogenic genes. At present, the preparation process of small nucleic acid drugs usually adopts solid-phase phosphoramidite chemistry, which synthesizes the target molecule through multiple cyclic steps such as deprotection, coupling, oxidation, and capping.

[0003] However, various impurities are inevitably generated during the synthesis process. Common types of impurities include: 1. Oligonucleotides with missing or added sequences, such as short nucleotides (Nx) and long nucleotides (N+x). Due to limitations of synthesis efficiency and raw material quality, not all oligonucleotides can synthesize complete oligonucleotide chains, resulting in such impurities. 2. Products with incomplete deprotection groups. During the synthesis process, some protecting groups may not be completely removed, resulting in the presence of such impurities in the product. 3. Oligonucleotides lacking purine bases and other degradation products. Factors such as reaction conditions during the synthesis process may lead to the loss of purine bases and may also produce other degradation products.

[0004] The existing technology has many defects and deficiencies in the purification of small nucleic acid drugs. Mainstream purification methods such as anion exchange chromatography, reverse phase chromatography and hydrophobic chromatography, although they can achieve the separation of small nucleic acid drug molecules and impurities to a certain extent, all have their own problems. For example, anion exchange chromatography is difficult to effectively separate some impurities with similar properties, resulting in insufficient purity of the purified product; reverse phase chromatography requires the use of a large amount of organic solvents, which is not only costly, but also faces environmental problems in the treatment of organic solvents; hydrophobic chromatography may not be effective for small nucleic acid drugs with certain specific structures, and also has the problem of high cost. In addition, the existing purification process often encounters problems such as decreased resolution and reduced yield during the scale-up production process, which makes it difficult to meet the needs of industrial large-scale production.

[0005] The patent application with publication number CN111944803A discloses a new microbial nucleic acid purification process, which uses silicon hydroxy magnetic beads as the core purification medium, combined with an optimized DNA binding solution (containing 1% sodium dodecyl sulfate), to significantly improve the binding efficiency of nucleic acids and magnetic beads. The patent claims that the purification yield can reach close to 98%, which is significantly higher than the traditional magnetic bead method (usually 80%-90%). The layered washing technology effectively removes impurities such as proteins and polysaccharides, and the final nucleic acid purity can reach 1.8-2.0, which meets the needs of downstream molecular detection. However, the component concentration of the magnetic bead binding solution (such as SDS content, buffer pH) needs to be strictly controlled, which can easily lead to nucleic acid degradation or impurity residues. In addition, although the layered washing step improves purity, it increases the operation steps and time, which is not conducive to large-scale production. Summary of the Invention

[0006] The purpose of the present invention is to provide a small nucleic acid drug purification process to improve the purity of the purified small nucleic acid drug.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] A small nucleic acid drug purification process comprises the following steps:

[0009] S1. Mixed-mode chromatography: Dissolve the crude small nucleic acid drug in a buffer containing 20 mM phosphate and 10% acetonitrile to obtain solution A; apply solution A to a mixed-mode chromatography column containing a functionalized agarose gel as the medium, and perform gradient elution to remove impurities; then elute the target product with a buffer containing 2 M NaCl and 10% acetonitrile;

[0010] S2. Size Exclusion Chromatography: The small nucleic acid drug sample eluted from the mixed-mode chromatography was diluted with equilibration buffer, loaded onto a size exclusion chromatography column, and isocratically eluted with equilibration buffer. The elution peak was collected by monitoring the UV absorption signal at 260 nm, and lyophilized to obtain a high-purity small nucleic acid drug;

[0011] The functionalized agarose gel is an agarose gel modified with a quaternary ammonium salt group and an alkyl group at the same time.

[0012] The principle of mixed-mode chromatography (MMC) is to utilize two or more separation mechanisms (such as ion exchange and hydrophobic interaction) that mixed-mode chromatography media possess simultaneously, and to achieve separation based on the differences in charge properties, hydrophobicity, etc. between small nucleic acid drugs and impurities. Small nucleic acid drugs are negatively charged and can bind to the cation exchange groups on the medium. At the same time, their hydrophobic regions interact with the hydrophobic groups. Different impurities have different binding abilities due to their different charges and hydrophobicities, thus achieving preliminary separation. In terms of medium selection, agarose gel modified with quaternary ammonium salt cation exchange groups and hydrophobic alkyl groups (such as C4) is selected as a mixed-mode chromatography medium. This medium can provide ion exchange and hydrophobic interactions at the same time, enhancing the separation ability of small nucleic acid drugs and impurities.

[0013] Furthermore, the crude product concentration in the solution A is 1-2 mg / mL; the pH of the buffer solution containing 20 mM phosphate and 10% acetonitrile is 6.5-7.5.

[0014] Furthermore, the sample loading flow rate of the solution A is 0.8-1.2 mL / min.

[0015] Furthermore, the salt concentration increasing rate of the gradient elution is 0.05-0.2 M / min.

[0016] Furthermore, the elution flow rate of the gradient elution to remove impurities is 1-1.5 mL / min.

[0017] Furthermore, the eluent of the gradient elution is 4-5 times the volume of the washing column.

[0018] Furthermore, the elution flow rate of the buffer solution containing 2M NaCl and 10% acetonitrile is 1.5-2.0 mL / min.

[0019] The principle of size exclusion chromatography (SEC) is based on differences in molecular size. Small nucleic acid drugs and impurities have different diffusion and retention times in porous gel media. The target small nucleic acid drug molecules are of moderate size and cannot enter the interior of the gel particles. They flow between the particles and elute quickly. However, long nucleotides (N+x) or aggregates with larger molecular weights have a low probability of entering the gel pores and elute quickly. However, short nucleotides (Nx) and degradation products with smaller molecular weights can enter the gel and have a long retention time, thus achieving separation by molecular size. The medium selected is a dextran gel (such as Sephadex G-50) with a pore size range suitable for the molecular weight of small nucleic acid drugs as a size exclusion chromatography medium. This medium can effectively separate oligonucleotide fragments of different lengths.

[0020] Furthermore, the dilution ratio of the equilibration buffer is 5-10 times.

[0021] Furthermore, the equilibration buffer is a Tris-HCl buffer with a pH of 7.0.

[0022] Furthermore, the medium of the size exclusion chromatography column is one of Sephadex G-50 and Sephadex G-40.

[0023] Furthermore, the loading flow rate in the size exclusion chromatography is 0.5-1.0 mL / min; and the isocratic elution flow rate is 0.5-1.0 mL / min.

[0024] Furthermore, the temperature of the mixed mode chromatography is 20-25°C; the temperature of the size exclusion chromatography is 25-30°C.

[0025] Furthermore, the small nucleic acid drug is a small interfering RNA (siRNA) or an antisense oligonucleotide (ASO).

[0026] Furthermore, the functionalized agarose gel is prepared by the following steps:

[0027] (1) Take the agarose gel, wash it thoroughly with deionized water, suspend the gel in 0.5 M NaOH solution, and stir at room temperature for 15-30 minutes to obtain the pretreated agarose gel;

[0028] (2) mixing the pretreated agarose gel with the epoxy activation solution in a volume ratio of 1:1-5, shaking and reacting at 40-45°C for 4-6 hours. After the reaction is completed, washing with deionized water until neutral to obtain epoxy-activated agarose gel;

[0029] (3) mixing the epoxy-activated agarose gel with a quaternary ammonium salt solution in a volume ratio of 1:4-8, reacting at 45-50°C for 8-10 hours, and washing with 0.1M HCl and deionized water in sequence after the reaction to obtain a quaternary ammonium salt-modified intermediate;

[0030] (4) The quaternary ammonium salt-modified intermediate product is mixed with a hydrophobizing agent solution in a volume ratio of 1:2-6, the pH is adjusted to 9.0-10, and the mixture is reacted at 50-60°C for 6-8 hours. After the reaction is completed, the mixture is washed with ethanol and deionized water in sequence to obtain a functionalized agarose gel.

[0031] Furthermore, the epoxy activation solution is a 0.1 M NaOH solution containing 10% (v / v) epichlorohydrin and 20% (w / v) sodium sulfate.

[0032] Furthermore, the quaternary ammonium salt solution is a 0.1M carbonate buffer solution containing 2M trimethylamine hydrochloride (TMA-HCl) with a pH of 10-11.

[0033] Furthermore, the hydrophobizing agent solution is an isopropanol / water mixture containing 1M linear bromoalkane, and the volume ratio of the isopropanol to water is 1:1.

[0034] Furthermore, the linear bromoalkane is one or a combination of 1-bromobutane, 1-bromohexane and 1-bromooctane.

[0035] Beneficial effects of the present invention:

[0036] (1) The present invention provides a small nucleic acid drug purification process, which adopts a new combined chromatography strategy combining mixed mode chromatography (MMC) and size exclusion chromatography (SEC), and achieves high-purity and high-efficiency purification of small nucleic acid drugs by optimizing elution conditions and parameter control; mixed mode chromatography utilizes the dual effects of charge and hydrophobicity to effectively separate impurities with similar properties, such as incompletely deprotected products and partially degraded products; size exclusion chromatography separates oligonucleotides with sequence deletions or additions according to molecular size. The dual chromatography combination can more comprehensively remove various impurities and improve the purity of the purified small nucleic acid drugs.

[0037] (2) The functionalized agarose gel used in the present invention can enhance the selectivity for target molecules without sacrificing recovery rate by combining different types of interactions. The quaternary ammonium salt cation exchange group can specifically bind to negatively charged small nucleic acid drugs through electrostatic interactions, while the hydrophobic alkyl group can further regulate the strength and specificity of this binding through hydrophobic interactions. In addition, the mixed-mode chromatography medium and the size exclusion chromatography medium have good stability and can be reused multiple times, reducing material costs and being easy to scale up, meeting the needs of industrial large-scale production. DETAILED DESCRIPTION

[0038] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0039] Example 1

[0040] This embodiment provides a small nucleic acid drug purification process, comprising the following steps:

[0041] S1. Take the agarose gel, wash it thoroughly with deionized water, suspend the gel in 0.5 M NaOH solution, and stir at room temperature for 20 minutes to obtain the pretreated agarose gel;

[0042] The pretreated agarose gel was mixed with an epoxy activation solution at a volume ratio of 1:3. The epoxy activation solution was a 0.1 M NaOH solution containing 10% (v / v) epichlorohydrin and 20% (w / v) sodium sulfate. The mixture was shaken at 40°C for 4 h. After the reaction, the mixture was washed with deionized water until neutral to obtain epoxy-activated agarose gel.

[0043] The epoxy-activated agarose gel was mixed with a quaternary ammonium salt solution at a volume ratio of 1:5. The quaternary ammonium salt solution was a 0.1 M carbonate buffer solution containing 2 M trimethylamine hydrochloride (TMA-HCl) at a pH of 10. The mixture was reacted at 45°C for 8 h. After the reaction was completed, the mixture was washed with 0.1 M HCl and deionized water in sequence to obtain a quaternary ammonium salt-modified intermediate product.

[0044] The quaternary ammonium salt-modified intermediate product was mixed with a hydrophobicizing agent solution in a volume ratio of 1:4. The hydrophobicizing agent solution was an isopropanol / water mixture containing 1 M 1-bromobutane, and the volume ratio of isopropanol to water was 1:1. The pH was adjusted to 9.5, and the reaction was carried out at 50° C. for 6 h. After the reaction, the mixture was washed with ethanol and deionized water in sequence to obtain a functionalized agarose gel.

[0045] S2. Mixed-mode chromatography: The crude siRNA was dissolved in a buffer solution containing 20 mM phosphate and 10% acetonitrile to obtain solution A, wherein the crude product concentration in solution A was 1.5 mg / mL and the pH of the buffer solution was 7; solution A was loaded onto a mixed-mode chromatography column (column volume 10 mL) at a flow rate of 1 mL / min. The mixed-mode chromatography column medium included functionalized agarose gel, and impurities were removed by gradient elution. The salt concentration increasing rate of the gradient elution was 0.1 M / min, the elution flow rate was 1.2 mL / min, and the eluent volume was 5 times the wash column volume; the target product was then eluted with a buffer solution containing 2 M NaCl and 10% acetonitrile at a flow rate of 1.8 mL / min; the temperature for the mixed-mode chromatography was 24°C;

[0046] S3. Size exclusion chromatography: The small nucleic acid drug sample eluted from the mixed mode chromatography was diluted 5 times with the equilibration buffer, which was a Tris-HCl buffer with a pH of 7.0. The sample was loaded onto a size exclusion chromatography column (column volume 50 mL) at a flow rate of 0.8 mL / min. The medium of the size exclusion chromatography column was Sephadex G-50. Isocratic elution was performed with the equilibration buffer, the flow rate was maintained at 0.8 mL / min, and the temperature of the size exclusion chromatography was 28°C. The elution peak was collected by monitoring the ultraviolet absorption signal at 260 nm, and high-purity small nucleic acid drugs were obtained after lyophilization.

[0047] Example 2

[0048] The difference between this embodiment and embodiment 1 is that this embodiment is for the purification of ASO, mainly adjusting the chromatography column medium, and the specific implementation steps are as follows:

[0049] S1. Take the agarose gel, wash it thoroughly with deionized water, suspend the gel in 0.5 M NaOH solution, and stir at room temperature for 20 minutes to obtain the pretreated agarose gel;

[0050] The pretreated agarose gel was mixed with an epoxy activation solution at a volume ratio of 1:3. The epoxy activation solution was a 0.1 M NaOH solution containing 10% (v / v) epichlorohydrin and 20% (w / v) sodium sulfate. The mixture was shaken at 40°C for 4 h. After the reaction, the mixture was washed with deionized water until neutral to obtain epoxy-activated agarose gel.

[0051] The epoxy-activated agarose gel was mixed with a quaternary ammonium salt solution at a volume ratio of 1:5. The quaternary ammonium salt solution was a 0.1 M carbonate buffer solution containing 2 M trimethylamine hydrochloride (TMA-HCl) at a pH of 10. The mixture was reacted at 45°C for 8 h. After the reaction was completed, the mixture was washed with 0.1 M HCl and deionized water in sequence to obtain a quaternary ammonium salt-modified intermediate product.

[0052] The quaternary ammonium salt-modified intermediate product was mixed with a hydrophobicizing agent solution in a volume ratio of 1:4. The hydrophobicizing agent solution was an isopropanol / water mixture containing 1M 1-bromohexane, and the volume ratio of isopropanol to water was 1:1. The pH was adjusted to 9.5 and the reaction was carried out at 50°C for 6 hours. After the reaction, the gel was washed with ethanol and deionized water in sequence to obtain a functionalized agarose gel.

[0053] S2. Mixed-mode chromatography: The crude ASO product was dissolved in a buffer solution containing 20 mM phosphate and 10% acetonitrile to obtain solution A, wherein the crude product concentration in solution A was 2 mg / mL and the pH of the buffer solution was 7; solution A was loaded onto a mixed-mode chromatography column (column volume 15 mL) at a flow rate of 1.2 mL / min. The mixed-mode chromatography column medium included functionalized agarose gel, and impurities were removed by gradient elution. The salt concentration increasing rate of the gradient elution was 0.1 M / min, the elution flow rate was 1.5 mL / min, and the eluent volume was 4 times the wash column volume; the target product was then eluted with a buffer solution containing 2 M NaCl and 10% acetonitrile at a flow rate of 2 mL / min; the temperature of the mixed-mode chromatography was 24°C;

[0054] S3. Size exclusion chromatography: The small nucleic acid drug sample eluted from the mixed mode chromatography was diluted 8 times with the equilibration buffer, which was a Tris-HCl buffer with a pH of 7.0. The sample was loaded onto a size exclusion chromatography column (column volume 50 mL) at a flow rate of 0.6 mL / min. The medium of the size exclusion chromatography column was Sephadex G-40. Isocratic elution was performed with the equilibration buffer, the flow rate was maintained at 0.6 mL / min, and the temperature of the size exclusion chromatography was 28°C. The elution peak was collected by monitoring the ultraviolet absorption signal at 260 nm, and high-purity small nucleic acid drugs were obtained after lyophilization.

[0055] The remaining raw materials and preparation process remain the same as in Example 1.

[0056] Example 3

[0057] Compared with Example 1, this example is different in that 1-bromobutane in S1 is replaced by 1-bromooctane, and the experimental parameters are adaptively adjusted. The specific implementation steps are as follows:

[0058] S1. Take the agarose gel, wash it thoroughly with deionized water, suspend the gel in 0.5 M NaOH solution, and stir at room temperature for 20 minutes to obtain the pretreated agarose gel;

[0059] The pretreated agarose gel was mixed with an epoxy activation solution at a volume ratio of 1:3. The epoxy activation solution was a 0.1 M NaOH solution containing 10% (v / v) epichlorohydrin and 20% (w / v) sodium sulfate. The mixture was shaken at 40°C for 4 h. After the reaction, the mixture was washed with deionized water until neutral to obtain epoxy-activated agarose gel.

[0060] The epoxy-activated agarose gel was mixed with a quaternary ammonium salt solution at a volume ratio of 1:5. The quaternary ammonium salt solution was a 0.1 M carbonate buffer solution containing 2 M trimethylamine hydrochloride (TMA-HCl) at a pH of 10. The mixture was reacted at 45°C for 8 h. After the reaction was completed, the mixture was washed with 0.1 M HCl and deionized water in sequence to obtain a quaternary ammonium salt-modified intermediate product.

[0061] The quaternary ammonium salt-modified intermediate product was mixed with a hydrophobicizing agent solution in a volume ratio of 1:4. The hydrophobicizing agent solution was an isopropanol / water mixture containing 1M 1-bromooctane, and the volume ratio of isopropanol to water was 1:1. The pH was adjusted to 10 and the reaction was carried out at 60°C for 7 hours. After the reaction, the gel was washed with ethanol and deionized water in sequence to obtain a functionalized agarose gel.

[0062] S2. Mixed-mode chromatography: The crude siRNA was dissolved in a buffer solution containing 20 mM phosphate and 10% acetonitrile to obtain solution A, wherein the crude product concentration in solution A was 1.5 mg / mL and the pH of the buffer solution was 7; solution A was loaded onto a mixed-mode chromatography column (column volume 10 mL) at a flow rate of 1 mL / min. The mixed-mode chromatography column medium included functionalized agarose gel, and impurities were removed by gradient elution. The salt concentration increasing rate of the gradient elution was 0.1 M / min, the elution flow rate was 1.2 mL / min, and the eluent volume was 5 times the wash column volume; the target product was then eluted with a buffer solution containing 2 M NaCl and 10% acetonitrile at a flow rate of 1.8 mL / min; the temperature for the mixed-mode chromatography was 24°C;

[0063] S3. Size exclusion chromatography: The small nucleic acid drug sample eluted from the mixed mode chromatography was diluted 5 times with the equilibration buffer, which was a Tris-HCl buffer with a pH of 7.0. The sample was loaded onto a size exclusion chromatography column (column volume 50 mL) at a flow rate of 0.8 mL / min. The medium of the size exclusion chromatography column was Sephadex G-50. Isocratic elution was performed with the equilibration buffer, the flow rate was maintained at 0.8 mL / min, and the temperature of the size exclusion chromatography was 28°C. The elution peak was collected by monitoring the ultraviolet absorption signal at 260 nm, and high-purity small nucleic acid drugs were obtained after lyophilization.

[0064] The remaining raw materials and preparation process remain the same as in Example 1.

[0065] Example 4

[0066] Compared with Example 1, this embodiment differs in that the volume ratio of agarose gel to solution is increased. The specific implementation steps are as follows:

[0067] S1. Take the agarose gel, wash it thoroughly with deionized water, suspend the gel in 0.5 M NaOH solution, and stir at room temperature for 20 minutes to obtain the pretreated agarose gel;

[0068] The pretreated agarose gel was mixed with an epoxy activation solution at a volume ratio of 1:1. The epoxy activation solution was a 0.1 M NaOH solution containing 10% (v / v) epichlorohydrin and 20% (w / v) sodium sulfate. The mixture was shaken at 40°C for 4 h. After the reaction, the mixture was washed with deionized water until neutral to obtain epoxy-activated agarose gel.

[0069] The epoxy-activated agarose gel was mixed with a quaternary ammonium salt solution at a volume ratio of 1:4. The quaternary ammonium salt solution was a 0.1 M carbonate buffer solution containing 2 M trimethylamine hydrochloride (TMA-HCl) at a pH of 10. The mixture was reacted at 45°C for 8 h. After the reaction was completed, the mixture was washed with 0.1 M HCl and deionized water in sequence to obtain a quaternary ammonium salt-modified intermediate product.

[0070] The quaternary ammonium salt-modified intermediate product was mixed with a hydrophobicizing agent solution in a volume ratio of 1:2. The hydrophobicizing agent solution was an isopropanol / water mixture containing 1 M 1-bromobutane, and the volume ratio of isopropanol to water was 1:1. The pH was adjusted to 9, and the reaction was carried out at 50° C. for 6 h. After the reaction, the mixture was washed with ethanol and deionized water in sequence to obtain a functionalized agarose gel.

[0071] S2. Mixed-mode chromatography: The crude siRNA was dissolved in a buffer solution containing 20 mM phosphate and 10% acetonitrile to obtain solution A, wherein the crude product concentration in solution A was 1.5 mg / mL and the pH of the buffer solution was 7; solution A was loaded onto a mixed-mode chromatography column (column volume 10 mL) at a flow rate of 1 mL / min. The mixed-mode chromatography column medium included functionalized agarose gel, and impurities were removed by gradient elution. The salt concentration increasing rate of the gradient elution was 0.1 M / min, the elution flow rate was 1.2 mL / min, and the eluent volume was 5 times the wash column volume; the target product was then eluted with a buffer solution containing 2 M NaCl and 10% acetonitrile at a flow rate of 1.8 mL / min; the temperature for the mixed-mode chromatography was 24°C;

[0072] S3. Size exclusion chromatography: The small nucleic acid drug sample eluted from the mixed mode chromatography was diluted 5 times with the equilibration buffer, which was a Tris-HCl buffer with a pH of 7.0. The sample was loaded onto a size exclusion chromatography column (column volume 50 mL) at a flow rate of 0.8 mL / min. The medium of the size exclusion chromatography column was Sephadex G-50. Isocratic elution was performed with the equilibration buffer, the flow rate was maintained at 0.8 mL / min, and the temperature of the size exclusion chromatography was 28°C. The elution peak was collected by monitoring the ultraviolet absorption signal at 260 nm, and high-purity small nucleic acid drugs were obtained after lyophilization.

[0073] The remaining raw materials and preparation process remain the same as in Example 1.

[0074] Example 5

[0075] Compared with Example 1, this example is different in that the volume ratio of agarose gel to solution is reduced. The specific implementation steps are as follows:

[0076] S1. Take the agarose gel, wash it thoroughly with deionized water, suspend the gel in 0.5 M NaOH solution, and stir at room temperature for 20 minutes to obtain the pretreated agarose gel;

[0077] The pretreated agarose gel was mixed with an epoxy activation solution at a volume ratio of 1:5. The epoxy activation solution was a 0.1 M NaOH solution containing 10% (v / v) epichlorohydrin and 20% (w / v) sodium sulfate. The mixture was shaken at 40°C for 6 h. After the reaction, the mixture was washed with deionized water until neutral to obtain epoxy-activated agarose gel.

[0078] The epoxy-activated agarose gel was mixed with a quaternary ammonium salt solution at a volume ratio of 1:8. The quaternary ammonium salt solution was a 0.1 M carbonate buffer solution containing 2 M trimethylamine hydrochloride (TMA-HCl) at a pH of 10. The mixture was reacted at 45°C for 10 h. After the reaction was completed, the mixture was washed with 0.1 M HCl and deionized water in sequence to obtain a quaternary ammonium salt-modified intermediate product.

[0079] The quaternary ammonium salt-modified intermediate product was mixed with a hydrophobicizing agent solution at a volume ratio of 1:6. The hydrophobicizing agent solution was an isopropanol / water mixture containing 1 M 1-bromobutane, and the volume ratio of isopropanol to water was 1:1. The pH was adjusted to 10, and the reaction was carried out at 50° C. for 8 h. After the reaction, the gel was washed with ethanol and deionized water in sequence to obtain a functionalized agarose gel.

[0080] S2. Mixed-mode chromatography: The crude siRNA was dissolved in a buffer solution containing 20 mM phosphate and 10% acetonitrile to obtain solution A, wherein the crude product concentration in solution A was 1.5 mg / mL and the pH of the buffer solution was 7; solution A was loaded onto a mixed-mode chromatography column (column volume 10 mL) at a flow rate of 1 mL / min. The mixed-mode chromatography column medium included functionalized agarose gel, and impurities were removed by gradient elution. The salt concentration increasing rate of the gradient elution was 0.1 M / min, the elution flow rate was 1.2 mL / min, and the eluent volume was 5 times the wash column volume; the target product was then eluted with a buffer solution containing 2 M NaCl and 10% acetonitrile at a flow rate of 1.8 mL / min; the temperature for the mixed-mode chromatography was 24°C;

[0081] S3. Size exclusion chromatography: The small nucleic acid drug sample eluted from the mixed mode chromatography was diluted 5 times with the equilibration buffer, which was a Tris-HCl buffer with a pH of 7.0. The sample was loaded onto a size exclusion chromatography column (column volume 50 mL) at a flow rate of 0.8 mL / min. The medium of the size exclusion chromatography column was Sephadex G-50. Isocratic elution was performed with the equilibration buffer, the flow rate was maintained at 0.8 mL / min, and the temperature of the size exclusion chromatography was 28°C. The elution peak was collected by monitoring the ultraviolet absorption signal at 260 nm, and high-purity small nucleic acid drugs were obtained after lyophilization.

[0082] The remaining raw materials and preparation process remain the same as in Example 1.

[0083] Comparative Example 1

[0084] This comparative example differs from Example 2 in that only mixed-mode chromatography was used to purify ASO without the SEC purification step. The specific implementation steps are as follows:

[0085] S1. Take the agarose gel, wash it thoroughly with deionized water, suspend the gel in 0.5 M NaOH solution, and stir at room temperature for 20 minutes to obtain the pretreated agarose gel;

[0086] The pretreated agarose gel was mixed with an epoxy activation solution at a volume ratio of 1:3. The epoxy activation solution was a 0.1 M NaOH solution containing 10% (v / v) epichlorohydrin and 20% (w / v) sodium sulfate. The mixture was shaken at 40°C for 4 h. After the reaction, the mixture was washed with deionized water until neutral to obtain epoxy-activated agarose gel.

[0087] The epoxy-activated agarose gel was mixed with a quaternary ammonium salt solution at a volume ratio of 1:5. The quaternary ammonium salt solution was a 0.1 M carbonate buffer solution containing 2 M trimethylamine hydrochloride (TMA-HCl) at a pH of 10. The mixture was reacted at 45°C for 8 h. After the reaction was completed, the mixture was washed with 0.1 M HCl and deionized water in sequence to obtain a quaternary ammonium salt-modified intermediate product.

[0088] The quaternary ammonium salt-modified intermediate product was mixed with a hydrophobicizing agent solution in a volume ratio of 1:4. The hydrophobicizing agent solution was an isopropanol / water mixture containing 1M 1-bromohexane, and the volume ratio of isopropanol to water was 1:1. The pH was adjusted to 9.5 and the reaction was carried out at 50°C for 6 hours. After the reaction, the gel was washed with ethanol and deionized water in sequence to obtain a functionalized agarose gel.

[0089] S2. Mixed-mode chromatography: The crude ASO product was dissolved in a buffer solution containing 20 mM phosphate and 10% acetonitrile to obtain solution A, wherein the crude product concentration in solution A was 2 mg / mL and the pH of the buffer solution was 7; solution A was loaded onto a mixed-mode chromatography column (column volume 15 mL) at a flow rate of 1.2 mL / min. The mixed-mode chromatography column medium included functionalized agarose gel, and impurities were removed by gradient elution. The salt concentration increasing rate of the gradient elution was 0.1 M / min, the elution flow rate was 1.5 mL / min, and the eluent volume was 4 times the volume of the washing column; the target product was then eluted with a buffer solution containing 2 M NaCl and 10% acetonitrile at an elution flow rate of 2 mL / min; the temperature of the mixed-mode chromatography was 24°C; the elution peak was collected and lyophilized to obtain a high-purity small nucleic acid drug.

[0090] The remaining raw materials and preparation process remain the same as in Example 2.

[0091] Comparative Example 2

[0092] This comparative example is different from Example 1 in that the agarose gel is not alkyl-modified. The specific implementation steps are as follows:

[0093] S1. Take the agarose gel, wash it thoroughly with deionized water, suspend the gel in 0.5 M NaOH solution, and stir at room temperature for 20 minutes to obtain the pretreated agarose gel;

[0094] The pretreated agarose gel was mixed with an epoxy activation solution at a volume ratio of 1:3. The epoxy activation solution was a 0.1 M NaOH solution containing 10% (v / v) epichlorohydrin and 20% (w / v) sodium sulfate. The mixture was shaken at 40°C for 4 h. After the reaction, the mixture was washed with deionized water until neutral to obtain epoxy-activated agarose gel.

[0095] The epoxy-activated agarose gel was mixed with a quaternary ammonium salt solution in a volume ratio of 1:5. The quaternary ammonium salt solution was a 0.1 M carbonate buffer solution containing 2 M trimethylamine hydrochloride (TMA-HCl) with a pH of 10. The mixture was reacted at 45°C for 8 h. After the reaction was completed, the mixture was washed with 0.1 M HCl and deionized water in sequence to obtain the functionalized agarose gel.

[0096] S2. Mixed-mode chromatography: The crude siRNA was dissolved in a buffer solution containing 20 mM phosphate and 10% acetonitrile to obtain solution A, wherein the crude product concentration in solution A was 1.5 mg / mL and the pH of the buffer solution was 7; solution A was loaded onto a mixed-mode chromatography column (column volume 10 mL) at a flow rate of 1 mL / min. The mixed-mode chromatography column medium included functionalized agarose gel, and impurities were removed by gradient elution. The salt concentration increasing rate of the gradient elution was 0.1 M / min, the elution flow rate was 1.2 mL / min, and the eluent volume was 5 times the wash column volume; the target product was then eluted with a buffer solution containing 2 M NaCl and 10% acetonitrile at a flow rate of 1.8 mL / min; the temperature for the mixed-mode chromatography was 24°C;

[0097] S3. Size exclusion chromatography: The small nucleic acid drug sample eluted from the mixed mode chromatography was diluted 5 times with the equilibration buffer, which was a Tris-HCl buffer with a pH of 7.0. The sample was loaded onto a size exclusion chromatography column (column volume 50 mL) at a flow rate of 0.8 mL / min. The medium of the size exclusion chromatography column was Sephadex G-50. Isocratic elution was performed with the equilibration buffer, the flow rate was maintained at 0.8 mL / min, and the temperature of the size exclusion chromatography was 28°C. The elution peak was collected by monitoring the ultraviolet absorption signal at 260 nm, and high-purity small nucleic acid drugs were obtained after lyophilization.

[0098] The remaining raw materials and preparation process remain the same as in Example 1.

[0099] Comparative Example 3

[0100] This comparative example is different from Example 1 in that the agarose gel is not modified with quaternary ammonium salt. The specific implementation steps are as follows:

[0101] S1. Take the agarose gel, wash it thoroughly with deionized water, suspend the gel in 0.5 M NaOH solution, and stir at room temperature for 20 minutes to obtain the pretreated agarose gel;

[0102] The pretreated agarose gel was mixed with an epoxy activation solution at a volume ratio of 1:3. The epoxy activation solution was a 0.1 M NaOH solution containing 10% (v / v) epichlorohydrin and 20% (w / v) sodium sulfate. The mixture was shaken at 40°C for 4 h. After the reaction, the mixture was washed with deionized water until neutral to obtain epoxy-activated agarose gel.

[0103] The epoxy-activated agarose gel was mixed with a hydrophobicizing agent solution in a volume ratio of 1:4. The hydrophobicizing agent solution was an isopropanol / water mixture containing 1M 1-bromobutane, and the volume ratio of isopropanol to water was 1:1. The pH was adjusted to 9.5 and the reaction was carried out at 50°C for 6 hours. After the reaction, the gel was washed with ethanol and deionized water in sequence to obtain a functionalized agarose gel.

[0104] S2. Mixed-mode chromatography: The crude siRNA was dissolved in a buffer solution containing 20 mM phosphate and 10% acetonitrile to obtain solution A, wherein the crude product concentration in solution A was 1.5 mg / mL and the pH of the buffer solution was 7; solution A was loaded onto a mixed-mode chromatography column (column volume 10 mL) at a flow rate of 1 mL / min. The mixed-mode chromatography column medium included functionalized agarose gel, and impurities were removed by gradient elution. The salt concentration increasing rate of the gradient elution was 0.1 M / min, the elution flow rate was 1.2 mL / min, and the eluent volume was 5 times the wash column volume; the target product was then eluted with a buffer solution containing 2 M NaCl and 10% acetonitrile at a flow rate of 1.8 mL / min; the temperature for the mixed-mode chromatography was 24°C;

[0105] S3. Size exclusion chromatography: The small nucleic acid drug sample eluted from the mixed mode chromatography was diluted 5 times with the equilibration buffer, which was a Tris-HCl buffer with a pH of 7.0. The sample was loaded onto a size exclusion chromatography column (column volume 50 mL) at a flow rate of 0.8 mL / min. The medium of the size exclusion chromatography column was Sephadex G-50. Isocratic elution was performed with the equilibration buffer, the flow rate was maintained at 0.8 mL / min, and the temperature of the size exclusion chromatography was 28°C. The elution peak was collected by monitoring the ultraviolet absorption signal at 260 nm, and high-purity small nucleic acid drugs were obtained after lyophilization.

[0106] The remaining raw materials and preparation process remain the same as in Example 1.

[0107] Comparative Example 4

[0108] This comparative example is different from Example 1 in that alkyl and quaternary ammonium salt modifications are not performed at the same time. The specific implementation steps are as follows:

[0109] S1. Mixed-mode chromatography: The crude siRNA was dissolved in a buffer solution containing 20 mM phosphate and 10% acetonitrile to obtain solution A, wherein the crude product concentration in solution A was 1.5 mg / mL and the pH of the buffer solution was 7; solution A was loaded onto a mixed-mode chromatography column (column volume 10 mL) at a flow rate of 1 mL / min. The mixed-mode chromatography column medium included agarose gel, and impurities were removed by gradient elution. The salt concentration increasing rate of the gradient elution was 0.1 M / min, the elution flow rate was 1.2 mL / min, and the eluent volume was 5 times the washing column volume; the target product was then eluted with a buffer solution containing 2 M NaCl and 10% acetonitrile at a flow rate of 1.8 mL / min; the temperature of the mixed-mode chromatography was 24°C;

[0110] S2. Size exclusion chromatography: The small nucleic acid drug sample eluted from the mixed mode chromatography was diluted 5 times with the equilibration buffer, which was a Tris-HCl buffer with a pH of 7.0. The sample was loaded onto a size exclusion chromatography column (column volume 50 mL) at a flow rate of 0.8 mL / min. The medium of the size exclusion chromatography column was Sephadex G-50. Isocratic elution was performed with the equilibration buffer, the flow rate was maintained at 0.8 mL / min, and the temperature of the size exclusion chromatography was 28°C. The elution peak was collected by monitoring the ultraviolet absorption signal at 260 nm, and high-purity small nucleic acid drugs were obtained after lyophilization.

[0111] The remaining raw materials and preparation process remain the same as in Example 1.

[0112] Performance Testing

[0113] The high-purity small nucleic acid drugs obtained by the purification processes of Examples 1 to 5 and Comparative Examples 1 to 4 were tested for purity. The results are shown in Table 1:

[0114] Table 1

[0115]

[0116]

[0117] As can be seen from Table 1, Example 1 is for the purification of siRNA, and the results show that the final yield is 84.8% and the purity is 96.8%; Example 2 is for the purification of ASO, and the results show that the final yield is 85.1% and the purity is 97.5%; Example 3 is different from Example 1 in that the carbon chain of the hydrophobic group is extended, and the results show a downward trend compared with Example 1. This is because the carbon chain extension leads to excessive hydrophobicity, requiring high concentration organic solvent elution, enhancing the adsorption of hydrophilic impurities and reducing selectivity, thereby resulting in a decrease in the purification effect; Examples 4-5 are different from Example 1 in that the volume ratio of the functionalized reagent and agarose gel affects the purification effect. The yields of all of them are greater than 84% and the purities are greater than 95%.

[0118] In addition, compared with Example 2, Comparative Example 1 only uses mixed-mode chromatography to purify ASO without the SEC purification step, resulting in a decrease in the final purity. This is because the mixed-mode chromatography has limited separation ability for short sequences with minimal molecular weight differences. In comparison with Comparative Examples 2-3 and Example 1, whether alkyl modification or quaternary ammonium salt modification is performed, the purity and yield will decrease. Compared with Example 1, Comparative Example 4 uses agarose gel as the medium of the mixed-mode chromatography column. There is no quaternary ammonium salt cation exchange group that specifically binds to the negatively charged small nucleic acid drug through electrostatic interaction, and the hydrophobic alkyl group further regulates the strength and specificity of this binding through hydrophobic interaction, thereby resulting in a further decrease in purity and yield. In addition, the functionalized agarose gel prepared has good stability, can be reused multiple times, and is easy to scale up production.

[0119] The above disclosures are only a few specific embodiments of the present invention. However, the embodiments of the present invention are not limited thereto. Any changes that can be conceived by those skilled in the art should fall within the scope of protection of the present invention.

Claims

1. A small nucleic acid drug purification process, characterized in that: The following steps are involved: S1. Mixed-mode chromatography: Dissolve the crude small nucleic acid drug in a buffer containing 20 mM phosphate and 10% acetonitrile to obtain solution A; Solution A is loaded onto a mixed-mode chromatography column whose medium includes functionalized agarose gel, and impurities are removed by gradient elution; The target product was then eluted with a buffer containing 2 M NaCl and 10% acetonitrile; S2. Size Exclusion Chromatography: The small nucleic acid drug sample eluted from the mixed-mode chromatography was diluted with equilibration buffer, loaded onto a size exclusion chromatography column, and isocratically eluted with equilibration buffer. The elution peak was collected by monitoring the UV absorption signal at 260 nm, and lyophilized to obtain a high-purity small nucleic acid drug; The functionalized agarose gel is an agarose gel modified with a quaternary ammonium salt group and an alkyl group at the same time.

2. A small nucleic acid drug purification process according to claim 1, characterized in that: The crude product concentration in the solution A is 1-2 mg / mL; the pH of the buffer solution containing 20 mM phosphate and 10% acetonitrile is 6.5-7.

5.

3. A small nucleic acid drug purification process according to claim 1, characterized in that: The loading flow rate of solution A is 0.8-1.2 mL / min; the salt concentration increasing rate of the gradient elution is 0.05-0.2 M / min; the elution flow rate of the gradient elution to remove impurities is 1-1.5 mL / min; the eluent of the gradient elution is 4-5 times the volume of the washing column; the elution flow rate of the buffer elution containing 2 M NaCl and 10% acetonitrile is 1.5-2.0 mL / min.

4. A small nucleic acid drug purification process according to claim 1, characterized in that: The dilution ratio of the equilibration buffer is 5-10 times; the equilibration buffer is a Tris-HCl buffer with a pH of 7.

0.

5. A small nucleic acid drug purification process according to claim 1, characterized in that: The medium of the size exclusion chromatography column is one of Sephadex G-50 and Sephadex G-40.

6. A small nucleic acid drug purification process according to claim 1, characterized in that: The sample loading flow rate in the size exclusion chromatography is 0.5-1.0 mL / min; the isocratic elution flow rate is 0.5-1.0 mL / min.

7. A small nucleic acid drug purification process according to claim 1, characterized in that: The temperature of the mixed mode chromatography is 20-25° C.; the temperature of the size exclusion chromatography is 25-30° C.; and the small nucleic acid drug is siRNA or ASO.

8. A small nucleic acid drug purification process according to claim 1, characterized in that: The functionalized agarose gel is prepared by the following steps: (1) Take the agarose gel, wash it thoroughly with deionized water, suspend the gel in 0.5 M NaOH solution, and stir at room temperature for 15-30 minutes to obtain the pretreated agarose gel; (2) mixing the pretreated agarose gel with the epoxy activation solution in a volume ratio of 1:1-5, shaking and reacting at 40-45°C for 4-6 hours. After the reaction is completed, washing with deionized water until neutral to obtain epoxy-activated agarose gel; (3) mixing the epoxy-activated agarose gel with a quaternary ammonium salt solution in a volume ratio of 1:4-8, reacting at 45-50°C for 8-10 hours, and washing with 0.1M HCl and deionized water in sequence after the reaction to obtain a quaternary ammonium salt-modified intermediate; (4) The quaternary ammonium salt-modified intermediate product is mixed with a hydrophobizing agent solution in a volume ratio of 1:2-6, the pH is adjusted to 9.0-10, and the mixture is reacted at 50-60°C for 6-8 hours. After the reaction is completed, the mixture is washed with ethanol and deionized water in sequence to obtain a functionalized agarose gel.

9. A small nucleic acid drug purification process according to claim 8, characterized in that: The epoxy activation solution is a 0.1M NaOH solution containing 10% (v / v) epichlorohydrin and 20% (w / v) sodium sulfate; the quaternary ammonium salt solution is a 0.1M carbonate buffer solution containing 2M trimethylamine hydrochloride (TMA-HCl) with a pH of 10-11; the hydrophobizing agent solution is an isopropanol / water mixture containing 1M linear bromoalkane, and the volume ratio of the isopropanol to water is 1:

1.

10. A small nucleic acid drug purification process according to claim 9, characterized in that: The linear bromoalkane is one or a combination of 1-bromobutane, 1-bromohexane and 1-bromooctane.

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