A method for preparing a gradient pressurized concentration of an oligonucleotide high concentration API solution

Abstract

The application discloses a gradient pressurized concentration preparation method of an oligonucleotide high-concentration API solution and belongs to the technical field of medicines. The application first proposes a gradient pressurized-temperature control cooperative concentration process, introduces a pre-treatment step of osmotic pressure regulation, solves the problems of serious membrane pollution and low limit concentration in traditional constant pressure concentration, and can stably realize high-concentration concentration of 200-280 mg / mL. An amphoteric ion modified ultrafiltration membrane is adopted, online concentration monitoring and segmented temperature control transformation are carried out on traditional nanofiltration equipment, the anti-pollution ability of the membrane is obviously improved, the degree of automation of the concentration process is higher, and a reliable process scheme is provided for industrialized production of high-concentration oligonucleotide pharmaceutical preparations.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a gradient pressure concentration method for preparing a high-concentration API solution of oligonucleotides (single chain length 21-23nt). Background Technology

[0002] Currently, most oligonucleotide drugs are prepared via solid-phase synthesis. After the synthesis of two single chains, the mixture undergoes annealing, sterilization, and pyrogen removal, followed by lyophilization to obtain a solid API (active pharmaceutical ingredient). This API is then dissolved and reconstituted to prepare the formulation. The process flow is: solid-phase synthesis → cleavage & deprotection → purification → ultrafiltration → sterilization → lyophilization → solid API → reconstitution → formulation. If the solution API could be directly prepared into a formulation, eliminating the need for the lyophilization of the active pharmaceutical ingredient and the reconstitution of the formulation, production efficiency could be significantly improved and production costs reduced.

[0003] Currently, the clinical use concentration of siRNA small nucleic acid drugs for treating chronic diseases generally requires a concentration of 200 mg / mL or higher. For example, the concentration of Inclisiran injection is 200 mg / mL. However, the final limit concentration of the commonly used tangential flow ultrafiltration concentration process is usually below 100 mg / mL, which cannot achieve the industrial preparation of high-concentration API solutions above 200 mg / mL. At the same time, existing processes are prone to problems such as membrane fouling, uneven concentration, and increased degradation impurities during concentration, which seriously affect product quality and production stability.

[0004] Although existing studies have attempted to use nanofiltration equipment for concentration (constant pressure mode), key issues such as rapid membrane flux decay, poor product stability, and inability to achieve the required concentration at high concentrations have not yet been resolved. Therefore, exploring an industrial method that can efficiently concentrate oligonucleotide API solutions and ensure stable product quality is of great strategic significance for the development of high-concentration oligonucleotide formulations. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a gradient pressure concentration method for preparing high-concentration oligonucleotide API solutions. This invention is the first to propose a gradient pressure-temperature controlled synergistic concentration process, introducing an osmotic pressure regulation pretreatment step. This solves the problems of severe fouling and low limiting concentrations in traditional constant-pressure concentration membranes, achieving a stable high concentration of 200-280 mg / mL. The nanofiltration membrane concentration system uses a zwitterionic modified ultrafiltration membrane, and the traditional nanofiltration equipment has been modified with online concentration monitoring and segmented temperature control, significantly improving the membrane's antifouling ability and increasing the automation level of the concentration process. This provides a reliable process solution for the industrial production of high-concentration oligonucleotide drug formulations.

[0006] The technical solution of this invention is: a method for preparing a high-concentration oligonucleotide API solution by gradient pressure concentration, characterized in that, 1) Pretreatment: Osmotic pressure regulation pretreatment Add an osmotic pressure regulator to the sterilized and pyrogen-free oligonucleotide API solution (initial concentration is usually 20-50 mg / mL), stir well, and you will get the pretreated solution. 2) Concentration equipment modification The nanofiltration membrane concentration system uses zwitterionic modified ultrafiltration membranes with a molecular weight cutoff of 3 kDa-5 kDa; 3) Gradient pressurization-temperature control synergistic concentration The pretreated feed solution is transferred to the circulation tank of the nanofiltration membrane concentration system, and a stepped transmembrane pressure (TMP) control method is adopted, specifically as follows: ① First stage (concentration <100mg / mL): transmembrane pressure controlled at 1.0-1.5MPa, circulation pump frequency 15-20Hz, operating temperature (membrane module section) 15-20℃; ② Second stage (100mg / mL≤concentration<200mg / mL): Transmembrane pressure increased to 2.0-3.0MPa, circulation pump frequency 20-30Hz, operating temperature 20-25℃; ③ Third stage (concentration ≥200mg / mL): increase transmembrane pressure to 3.5-5.0MPa, circulation pump frequency 30-40Hz, operating temperature 25-30℃; stop concentration when the concentration reaches the target concentration of 200-280mg / mL.

[0007] The zwitterionic modified ultrafiltration membrane used in this invention is described in Examples 1-2 of CN 120900449 A, "A Method for Preparing a Zwitterionic Copolymer Ultrafiltration Membrane." The preparation method of the zwitterionic copolymer is as follows: Step 1: N... methyl 4 Piperidone and 1,4 Butyryl lactone was dissolved in a solvent, heated to react, then cooled to room temperature, washed, and vacuum dried to obtain the zwitterionic piperidine monomer; Step 2: The zwitterionic piperidine monomer obtained in Step 1 was reacted with p-terphenyl and N... methyl 4 Piperidone is dispersed in a solvent, and trifluoroacetic acid and trifluoromethanesulfonic acid are added at low temperature to react, precipitate, wash, and vacuum dry to obtain zwitterionic copolymer; Step 3: Dissolve the zwitterionic copolymer obtained in step 2 in a solvent, degas, and scrape the membrane to obtain ultrafiltration membrane.

[0008] Furthermore, after concentration, the concentrate is transferred to a sterilized container, brought to the target concentration, and then directly filled into the container after sterilization filtration to prepare the formulation.

[0009] Furthermore, the nanofiltration membrane concentration system includes modules for monitoring the concentration of the feed solution in the circulation tank and modules for segmented temperature control of the circulation tank. This allows for real-time monitoring of the feed solution concentration in the circulation tank and control of the circulation tank temperature and membrane module temperature. Temperature gradient regulation promotes water molecule permeation while ensuring the stability of oligonucleotide molecules and reducing degradation.

[0010] Furthermore, the system alarm temperature in the gradient pressurization concentration is set to 10-40℃, and the transmembrane pressure alarm pressure is set to 0.5-5.5MPa.

[0011] Preferably, the osmotic pressure regulator is selected from one or more of mannitol, glucose, and sodium chloride, and the addition amount is 0.5-2.0% (w / v). This step can adjust the osmotic pressure of the solution, reduce the tendency of oligonucleotide molecules to aggregate at high concentrations, reduce adsorption fouling on the membrane surface, and improve the subsequent concentration efficiency by more than 30%.

[0012] This invention employs a gradient pressurization-temperature control synergistic concentration mechanism: After a low-concentration oligonucleotide solution enters the zwitterionic ultrafiltration membrane system, water molecules permeate through the membrane pores and flow out through the permeate end under the action of the gradient-increased transmembrane pressure, while larger oligonucleotide molecules are retained and returned to the circulation tank. Temperature gradient control creates a temperature difference on the membrane surface, promoting water molecule diffusion. Simultaneously, zwitterionic modification of the membrane surface reduces oligonucleotide adsorption, maintaining a stable membrane flux. After multiple cycles, water is continuously separated from the solution, ultimately achieving high-concentration concentration. The pre-concentration osmotic pressure adjustment step (osmotic pressure regulator) of this invention further reduces the aggregation effect of oligonucleotides at high concentrations, avoiding membrane pore blockage and ultimately achieving even higher concentrations.

[0013] Compared with the prior art, this invention has the following outstanding innovations and beneficial effects: 1. This invention proposes for the first time a gradient pressurization-temperature control synergistic concentration process, introducing an osmotic pressure regulation pretreatment step, which solves the problems of serious fouling and low limit concentration of traditional constant pressure concentration membranes, and can stably achieve high concentration concentration of 200-280mg / mL, breaking through the technical bottleneck of existing process concentration limit <100mg / mL. 2. This invention uses a zwitterionic copolymer ultrafiltration membrane, which modifies traditional nanofiltration equipment by implementing online concentration monitoring and segmented temperature control. The membrane's antifouling ability is significantly improved, the number of times it can be reused is increased by more than 2 times, the concentration process is more automated, and the concentration control is more precise. 3. Compared with traditional freeze-drying and reconstitution processes, this invention does not require freeze-drying and reconstitution, avoiding the problem of increased impurities generated during freeze-drying, resulting in higher product purity and more stable quality; at the same time, the production cycle is shortened by more than 40%, and the production cost is reduced by 35%, making it suitable for large-scale industrial production; it can be applied to the concentration preparation of various types of oligonucleotide drugs such as double-stranded siRNA, single-stranded oligonucleotides, and aptamers, providing key technical support for the development of high-concentration small nucleic acid preparations, and has extremely high industrial application value. Detailed Implementation

[0014] The effects are illustrated below with reference to specific examples.

[0015] The main instruments used in this invention are: high performance liquid chromatograph: Agilent 1260, Agilent Technologies; endotoxin detection system: Endosafe PTS, Charles River.

[0016] For zwitterionic copolymer ultrafiltration membranes, see Example 1 and Example 2 of CN 120900449 A, which describes a method for preparing a zwitterionic copolymer ultrafiltration membrane.

[0017] Modified nanofiltration membrane concentration system: BPNF-1812-40BAR, manufactured by Shanghai Bopu Environmental Protection Technology Co., Ltd. Based on this system, the following components are added: ① Online UV concentration monitoring module: Real-time monitoring of the feed concentration in the circulation tank; the online UV concentration monitoring module can directly use commercially available industrial online UV detectors (such as online UV detection units from Agilent or Thermo Fisher Scientific, or online concentration monitoring devices from domestic brands), installed on the outlet pipeline of the ultrafiltration circulation tank. Utilizing the characteristic UV absorption of oligonucleotides at 260nm, the absorbance of the feed solution can be detected in real time and converted into concentration. This technology is a publicly available and mature technology, requiring no special development. ② Segmented temperature control module: Controls the temperature of the circulation tank and the membrane module segment temperature, promoting water molecule permeation through temperature gradient regulation while ensuring the stability of oligonucleotide molecules and reducing degradation. The segmented temperature control module is a conventional industrial temperature control solution. It only requires configuring independent heat exchange structures (jacket or heat exchange coil) in the circulation tank and membrane module sections respectively, and connecting them to two independent constant temperature water bath circulation devices to achieve segmented temperature control. All components (constant temperature water bath, temperature sensor, PID temperature controller) are standard industrial products that can be directly purchased.

[0018] The siRNA used in the embodiments of this invention has the following positive strand: 5'-AAG GAG AAG AAG AGC CCG CAA-3'; and the negative strand: 5'-UUG CGG GCT CTT CTT CTC CTT-3'. See: Elbashir, SM, Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., & Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature ,411(6836), 494-498. Example 1: Preparation of API solution of 200 mg / mL siRNA 1. Sample pretreatment Take the API solution of siRNA that has been purified, sterilized and depyrogenated after solid-phase synthesis, with an initial concentration of 32.42 mg / mL and a total volume of 10 L, add 0.8% (w / v) mannitol, stir to dissolve, and set aside; one portion (5 L) is used for concentration in the process of this invention, and the other portion (5 L) is used for conventional freeze-drying process as a comparison.

[0019] 2. System Preparation and Concentration The modified nanofiltration unit-wound membrane system was disinfected and cleaned, and a 3KD zwitterionic copolymer ultrafiltration membrane was installed. The pretreated feed solution was added to the ultrafiltration circulation tank, online concentration monitoring was activated, and concentration was performed according to the following gradient parameters: ① First stage: TMP=1.2MPa, pump frequency 18Hz, temperature 16℃, concentrate to a concentration of 85mg / mL; ② Second stage: Increase TMP to 2.5MPa, pump frequency 25Hz, temperature 22℃, concentrate to a concentration of 190mg / mL; ③ Third stage: Increase TMP to 4.0MPa, pump frequency 35Hz, temperature 28℃, concentrate to a concentration of 212mg / mL and then stop.

[0020] Compared to traditional constant-pressure concentration, gradient pressurization can significantly alleviate membrane concentration polarization at high concentrations, increase the ultimate concentration, and reduce the deposition of oligonucleotide molecules on the membrane surface, thus extending the membrane's lifespan.

[0021] 3. Post-processing and testing After concentration, the liquid was transferred to a sterile container, and its concentration, purity, sodium content, pH, and endotoxin were tested. The results are shown in Table 1.

[0022] Example 2: Preparation of 250 mg / mL siRNA solution API Following the same procedure as in Example 1, the endpoint concentration was adjusted to 250 mg / mL, and the gradient parameters were adjusted as follows: TMP = 4.5 MPa in the third stage, ultimately yielding a siRNA solution API with a concentration of 252.3 mg / mL. The concentrated solution was transferred to a sterile container, and its concentration, purity, sodium content, pH, and endotoxin levels were measured. The results are shown in Table 1.

[0023] Comparative Example: Traditional Freeze-Dried Reconstitution Process An equal initial volume of siRNA solution was used in accordance with the traditional process: the sterilized and pyrogen-free oligonucleotide API solution was directly transferred to a lyophilizer for lyophilization to obtain solid API. This solid API was then reconstituted with purified water and brought to a final volume of 200 mg / mL to prepare the formulation, which served as a control. The solution was transferred to a sterile container, and its concentration, purity, sodium content, pH, and endotoxin levels were measured. The results are shown in Table 1.

[0024] Table 1 Comparison of Product Quality by Different Processes

[0025] As can be seen from the results in Table 1, the API solution obtained by the process of this invention has higher purity than that obtained by the traditional freeze-drying process, and the total impurities and characteristic impurities are lower than those obtained by the traditional freeze-drying process. The pH is closer to that of the initial solution, and the product quality is better.

[0026] Table 2 shows a comparison of production efficiencies for different processes. The production process of this invention significantly reduces the production cycle, lowers production costs, and improves the service life of the membrane.

[0027] Table 2 Comparison of Production Efficiency of Different Processes

Claims

1. A method for preparing a high-concentration oligonucleotide API solution using gradient pressure concentration, characterized in that, 1) Pretreatment: Osmotic pressure regulation pretreatment The sterilized and pyrogen-free oligonucleotide API solution is mixed with an osmotic pressure regulator to obtain a pretreated solution; the initial concentration of the oligonucleotide API solution is 20-50 mg / mL. 2) Concentration equipment modification The nanofiltration membrane concentration system uses zwitterionic modified ultrafiltration membranes with a molecular weight cutoff of 3 kDa-5 kDa; 3) Gradient pressurization-temperature control synergistic concentration The pretreated feed solution is transferred to the circulation tank of the nanofiltration membrane concentration system, and a stepped transmembrane pressure control method is adopted, specifically: ① First stage, concentration <100mg / mL: transmembrane pressure controlled at 1.0-1.5MPa, operating temperature 15-20℃; ② Second stage, concentration ≤ 100mg / mL < 200mg / mL: transmembrane pressure increased to 2.0-3.0MPa, operating temperature 20-25℃; ③ Third stage, concentration ≥200mg / mL: increase transmembrane pressure to 3.5-5.0MPa, operating temperature 25-30℃; stop concentration when the concentration reaches the target concentration of 200-280mg / mL.

2. The preparation method according to claim 1, characterized in that, After concentration, the concentrate is transferred to a sterilized container, brought to the target concentration, and then directly filled into the container after sterilization filtration to prepare the formulation.

3. The preparation method according to claim 1, characterized in that, The nanofiltration membrane concentration system includes a module for monitoring the concentration of the feed liquid in the circulation tank and a module for segmented temperature control of the circulation tank, thereby monitoring the concentration of the feed liquid in the circulation tank in real time and controlling the temperature of the circulation tank and the membrane module section.

4. The preparation method according to claim 1, characterized in that, In the first stage, the circulation pump frequency is 15-20Hz; in the second stage, the circulation pump frequency is 20-30Hz; and in the third stage, the circulation pump frequency is 30-40Hz.

5. The preparation method according to claim 1, characterized in that, In gradient pressurization concentration, the system alarm temperature is set to 10-40℃, and the transmembrane pressure alarm pressure is set to 0.5-5.5MPa.

6. The preparation method according to claim 1, characterized in that, The osmotic pressure regulator is selected from one or more of mannitol, glucose, and sodium chloride.

7. The preparation method according to claim 6, characterized in that, The amount of the osmotic pressure regulator added is 0.5-2.0% (w / v).

8. The preparation method according to any one of claims 1-7, characterized in that, The preparation method of the zwitterionic copolymer is as follows: Step 1: N-methyl-4-piperidinone and 1,4-butyryl lactone are mixed and dissolved in a solvent, heated to react, then cooled to room temperature, washed, and vacuum dried to obtain zwitterionic piperidine monomer; Step 2: The zwitterionic piperidine monomer obtained in Step 1 is mixed and dispersed with p-terphenyl and N-methyl-4-piperidinone in a solvent, and trifluoroacetic acid and trifluoromethanesulfonic acid are added at low temperature to react, precipitate, wash, and vacuum dry to obtain zwitterionic copolymer; Step 3: The zwitterionic copolymer obtained in Step 2 is dissolved in a solvent, degassed, and then coated to obtain an ultrafiltration membrane.

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