Application of choline and / or choline ionic liquid in reducing viscosity of high-concentration macromolecular preparation

Abstract

The invention relates to an application of choline and / or choline ionic liquid in reducing the viscosity of a high-concentration macromolecular preparation. The working concentrations of the choline and the choline ionic liquid are respectively and independently 0-300mM. The invention provides a novel viscosity reducer for a protein liquid preparation with relatively high viscosity, which can effectively reduce the viscosity of a solution without influencing the protein stability, ensure the stability of a protein medicine under the conditions of high-temperature acceleration, normal-temperature shaking and repeated freezing and thawing, reduce the risk possibly caused by high viscosity in the production process of the medicine, and improve the safety of the medicine. The preparation method has a wide application prospect in the field of preparation of subcutaneous injection preparations.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology pharmaceutical preparations, and in particular to the application of choline and / or choline-based ionic liquids in reducing the viscosity of high-concentration macromolecular preparations. Background Art

[0002] High-concentration preparations have developed rapidly in recent years. They can reduce the volume of administration, reduce production and transportation costs, and meet individualized medical needs. Such drugs are administered by subcutaneous injection, intravenous injection, etc., which helps to improve patient compliance and reduce the number of visits. However, high protein concentrations (≥50 mg / mL) pose significant challenges to formulation development. In addition to problems with drug solubility and stability, higher protein concentrations lead to higher solution viscosity, which causes a series of problems in formulation formulation and manufacturing process. For example, high-viscosity preparations may cause insufficient fluidity during the filling process, increase the pressure of the filling equipment, and affect production efficiency and consistency of the finished product. In addition, excessive viscosity may lead to incomplete filling or bubble formation, thereby affecting product quality. Therefore, in the development process of high-concentration formulation prescriptions, special attention should be paid to how to reduce the viscosity of the preparation as much as possible.

[0003] The fundamental reason for the increase in viscosity is the strong interaction between proteins, including hydrogen bonds, ionic interactions, etc., which is particularly significant in high-concentration formulations. Under high concentration conditions, the interaction between protein molecules is enhanced, which significantly increases the viscosity of the solution. These interactions cause protein molecules to entangle with each other in the solution, increasing the flow resistance. In addition, at high concentrations, proteins may undergo conformational changes to form more compact stacking and cross-linked structures, which also lead to an increase in viscosity. In addition, changes in the ionic strength and pH of the solution can directly affect the charge and solubility of the protein, further exacerbating the interaction between protein molecules. For example, at low ionic strength, the electrostatic repulsion of proteins is weakened, aggregation is prone to occur, and viscosity increases. Therefore, when developing high-concentration formulations, understanding and controlling these interactions is crucial to ensuring the fluidity and stability of the drug. Effective formulation optimization, such as the use of viscosity-reducing excipients, can effectively reduce the interaction between protein molecules, thereby helping to overcome the challenges caused by increased viscosity and ensure the quality and safety of the final product.

[0004] Currently, commonly used viscosity-reducing excipients include sodium chloride and some amino acids, such as arginine, lysine, histidine and proline, which are salts or organic compounds containing hydrophilic and lipophilic parts. The viscosity-reducing mechanisms of viscosity-reducing excipients can be divided into two categories: the first is to reduce viscosity by increasing the ionic strength of the solution. Salt produces an electrostatic shielding effect on the surface of protein molecules, which weakens the interaction between protein molecules by affecting the interaction between ions and proteins. This shielding effect destroys the mutual attraction between protein molecules and reduces the viscosity of the solution. Through this mechanism, salt can effectively improve the fluidity of protein solutions and enhance their stability. The second is to reduce viscosity through the hydrophobic effect. The hydrophobic part of some amino acids can interact with the hydrophobic region of protein molecules to reduce the aggregation tendency of proteins. For example, arginine can interact with aromatic residues in a cation-π interaction. This interaction reduces the hydrophobic interaction between protein molecules, thereby reducing the viscosity of the solution. Through these two mechanisms, viscosity-reducing excipients can effectively improve the fluidity and stability of biologics. For example, CN102258464A discloses a high-concentration antibody and protein preparation, which relates to a highly concentrated antibody and protein preparation with reduced viscosity, stability, relative isotonicity and low turbidity, which is particularly suitable for subcutaneous administration, and the preparation contains arginine-HCl, histidine and polysorbate.

[0005] However, the two viscosity-reducing excipients mentioned above each have their own disadvantages. The prior art shows that the function of viscosity-reducing excipients is affected by the properties of protein molecules. In a study of bovine serum albumin, when 150 mM sodium chloride was added to the protein solution, the viscosity curve shifted significantly upward and became flat. There are several possible reasons for this result: (1) the addition of sodium chloride increases the viscosity over the entire pH range through steric repulsion, and (2) the electrostatic shielding effect of sodium chloride reduces the viscosity of the solution at alkaline and acidic pH, but not at neutral pH. At the same time, excessive salt concentration may cause protein denaturation or precipitation, thereby affecting its biological activity. Amino acids are relatively expensive and cannot play a viscosity-reducing role in all types of protein solutions. Therefore, when selecting viscosity-reducing excipients in the study of formulations, molecules that can interact with proteins through π-π or cation-π interactions should be given priority. These interactions help to form a more ordered structure in the solution, reduce direct contact between proteins, and thus reduce aggregation and entanglement between protein molecules, thereby reducing the overall viscosity while ensuring the molecular stability of the protein.

[0006] Due to their unique properties, ionic liquids are widely used in many fields, such as solvents, catalysts, separation processes, material synthesis, and battery electrolytes. In biological preparations, choosing ionic liquids as viscosity reducers has many advantages. First, ionic liquids have low volatility at room temperature, which allows them to maintain good stability in preparations, reduce component loss, and extend the shelf life of drugs. Secondly, ionic liquids can dissolve a variety of biomolecules, including proteins and polysaccharides, effectively reduce the viscosity of biological preparations, and improve their fluidity and application performance. In addition, the properties of ionic liquids can be adjusted by selecting different anions and cations. For example, specific ionic liquids can produce cation-π interactions and π-π interactions with protein molecules to meet specific application requirements and give preparations better functionality. This adjustability makes ionic liquids flexible and diverse in biological preparations and can be optimized according to actual needs. It is worth mentioning that some ionic liquids have good biocompatibility and can be used safely in biological environments without negatively affecting biological activity. In some studies on small molecule drug preparations, the solubility of drugs after adding ionic liquids is always higher than the solubility of drugs in water. At the same time, ionic liquids do not affect the free radical activity or cell activity of small molecule drugs. These properties make ionic liquids an ideal choice for developing new biologics. Therefore, ionic liquids can be used as viscosity reducers in biologics, combining multiple advantages such as superior performance, flexibility and biocompatibility, providing important support for modern biotechnology and formulation development.

[0007] In summary, how to provide a viscosity reducer suitable for high-concentration macromolecular preparations has become one of the problems to be solved urgently in this field. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides the use of choline and / or choline-based ionic liquids in reducing the viscosity of high-concentration macromolecular preparations, which can effectively reduce the viscosity of the solution without affecting the stability of the protein, ensure the stability of protein drugs under high-temperature acceleration, room-temperature shaking and repeated freeze-thaw conditions, and reduce the risks that may be caused by high viscosity during the drug production process.

[0009] To achieve this object, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides the use of choline and / or choline-based ionic liquids in reducing the viscosity of high-concentration macromolecular preparations.

[0011] The choline and ionic liquid thereof provided by the present invention can be used as green functional excipients, significantly improving the solubility and drug loading of drugs at non-toxic concentrations without affecting the activity of protein drugs and the stability of cell drug products, and can effectively reduce the viscosity of the solution without affecting the stability of the protein.

[0012] Preferably, the working concentration of choline is 0-300mM (for example, it can be 0mM, 0.05mM, 0.1mM, 0.2mM, 0.5mM, 1mM, 5mM, 10mM, 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM or 300mM, etc.), preferably 75-200mM (for example, it can be 75mM, 100mM, 125mM, 150mM, 175mM or 200mM, etc.).

[0013] Preferably, the working concentration of the choline ionic liquid is 0 to 300 mM (for example, 0 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM or 300 mM, etc.).

[0014] Preferably, the choline-based ionic liquid comprises a choline amino acid ionic liquid.

[0015] Preferably, the choline amino acid ionic liquid includes any one of a choline-phenylalanine ionic liquid, a choline-arginine ionic liquid or a choline-glycine ionic liquid, or a combination of at least two thereof.

[0016] Preferably, the working concentration of the choline-phenylalanine ionic liquid is 0-300mM (for example, it can be 0mM, 0.05mM, 0.1mM, 0.2mM, 0.5mM, 1mM, 5mM, 10mM, 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM or 300mM, etc.), preferably 75-200mM (for example, it can be 75mM, 100mM, 125mM, 150mM, 175mM or 200mM, etc.).

[0017] Preferably, the working concentration of the choline-arginine ionic liquid is 0-150 mM (for example, 0 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM or 150 mM, etc.), preferably 75-100 mM (for example, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM or 100 mM, etc.).

[0018] Preferably, the preparation method of the choline amino acid ionic liquid comprises the following steps:

[0019] (1) rotary evaporating the methanol solution of choline chloride at 35-45° C. (e.g., 35° C., 37° C., 39° C., 40° C., 41° C., 43° C., or 45° C.) to remove the solvent methanol;

[0020] (2) dissolving an amino acid in an amount equimolar to that of choline in water, mixing with choline chloride, cooling in an ice-water bath, stirring for 10 to 14 hours (e.g., 10 hours, 11 hours, 12 hours, 13 hours, or 14 hours), and removing the solvent water by rotary evaporation at 45 to 55° C. (e.g., 45° C., 47° C., 49° C., 50° C., 51° C., 53° C., or 55° C.);

[0021] (3) adding a mixed solvent of acetonitrile and methanol in a volume ratio of (8-10):1 to precipitate the unreacted amino acid;

[0022] (4) filtering, evaporating under reduced pressure to remove the solvent, and vacuum drying at 55-65° C. (for example, 55° C., 57° C., 59° C., 60° C., 61° C., 63° C. or 65° C.) to obtain the choline amino acid ionic liquid.

[0023] The specific point values ​​in the above (8-10) can be 8, 8.5, 8.8, 9, 9.2, 9.5 or 10, etc.

[0024] Preferably, the high-concentration macromolecule preparation comprises any one of a high-concentration protein preparation, a high-concentration nucleic acid preparation or a high-concentration vaccine preparation, or a combination of at least two thereof.

[0025] Preferably, the high-concentration protein preparation comprises any one of a high-concentration monoclonal antibody preparation, a high-concentration biclonal antibody preparation or a high-concentration fusion protein preparation, or a combination of at least two thereof.

[0026] Preferably, the protein concentration of the high-concentration protein preparation is ≥50 mg / mL (for example, it may be 50 mg / mL, 75 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL or 300 mg / mL, etc.).

[0027] In a second aspect, the present invention provides a high-concentration monoclonal antibody solution, wherein the high-concentration monoclonal antibody solution contains a monoclonal antibody and a viscosity-reducing auxiliary material, wherein the viscosity-reducing auxiliary material includes choline and / or a choline-based ionic liquid.

[0028] Preferably, the high concentration monoclonal antibody solution contains 100-300 mg / mL (for example, 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, 200 mg / mL, 225 mg / mL, 250 mg / mL, 275 mg / mL or 300 mg / mL, etc.) of monoclonal antibody and 0-300 mM (for example, 0 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM or 300 mM, etc.) of viscosity reducing excipient.

[0029] Preferably, the high concentration monoclonal antibody solution contains 0-300mM (for example, 0mM, 0.05mM, 0.1mM, 0.2mM, 0.5mM, 1mM, 5mM, 10mM, 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM or 300mM, etc.) choline, preferably 75-200mM (for example, 75mM, 100mM, 125mM, 150mM, 175mM or 200mM, etc.) choline.

[0030] Preferably, the high concentration monoclonal antibody solution contains 0 to 300 mM (for example, 0 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM or 300 mM, etc.) of choline ionic liquid.

[0031] Preferably, the choline-based ionic liquid includes a choline-phenylalanine ionic liquid and / or a choline-arginine ionic liquid.

[0032] Preferably, the high concentration monoclonal antibody solution contains 0-300mM (for example, 0mM, 0.05mM, 0.1mM, 0.2mM, 0.5mM, 1mM, 5mM, 10mM, 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM or 300mM, etc.) of choline-phenylalanine ionic liquid, preferably 75-200mM (for example, 75mM, 100mM, 125mM, 150mM, 175mM or 200mM, etc.) of choline-phenylalanine ionic liquid.

[0033] Preferably, the high concentration monoclonal antibody solution contains 0-150mM (for example, 0mM, 0.05mM, 0.1mM, 0.2mM, 0.5mM, 1mM, 5mM, 10mM, 25mM, 50mM, 75mM, 100mM, 125mM or 150mM, etc.) choline-arginine ionic liquid, preferably 75-100mM (for example, 75mM, 80mM, 85mM, 90mM, 95mM or 100mM, etc.) choline-arginine ionic liquid.

[0034] Preferably, the high concentration monoclonal antibody solution also contains a buffer solution.

[0035] Preferably, the buffer solution comprises a 15-25 mM (eg, 15 mM, 17 mM, 19 mM, 20 mM, 21 mM, 23 mM or 25 mM, etc.) histidine hydrochloride solution with a pH of 5-7 (eg, 5, 5.5, 6, 6.5 or 7, etc.).

[0036] Preferably, the high concentration monoclonal antibody solution further contains a surfactant.

[0037] Preferably, the surfactant comprises 0.03% to 0.05% (eg, 0.03%, 0.035%, 0.04%, 0.045% or 0.05%) w / v Tween 80.

[0038] Preferably, the high concentration monoclonal antibody solution further contains a stabilizer.

[0039] Preferably, the stabilizer comprises 2% to 10% (eg, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, etc.) w / v sucrose.

[0040] In a third aspect, the present invention provides use of the high-concentration monoclonal antibody solution as described in the second aspect in the preparation of a subcutaneous injection medicament.

[0041] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention provides a green functional excipient based on choline, which significantly improves the solubility and drug loading of drugs at non-toxic concentrations without affecting the activity of protein drugs and the stability of cell drug products. It is a new viscosity reducer for protein liquid preparations with high viscosity, which can effectively reduce the viscosity of the solution and ensure the stability of protein drugs under high-temperature acceleration, room-temperature shaking and repeated freezing and thawing conditions, thereby reducing the risks that may be caused by high viscosity during the production process of drugs. DETAILED DESCRIPTION

[0044] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0045] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0046] In the present invention, the macromolecular preparation refers to a composition containing or consisting of a biological product. For example, the macromolecular preparation can be a macromolecular drug product stock solution, a simulated solution containing a macromolecular drug product, or the macromolecular drug product itself.

[0047] General implementation method:

[0048] Appearance: Inspect the appearance of the samples, including color, clarity, and visible foreign matter, in front of the black and white background of the YB-2 clarity tester. The light intensity of the clarity tester is set to 2000-3750 lux.

[0049] Concentration: Use Thermo Fisher's UV spectrophotometer to test the protein concentration, accurately input the extinction coefficient of the molecule, load 2.5 μL of sample each time, repeat the test twice and report the average value. Pure water should be used as a blank control before each sample test.

[0050] pH: pH was detected using a multi-parameter tester with a glass electrode. Before use, pH calibration solution (pH 4.01, 7.00, 9.21) was used for calibration, and the calibration slope was in the range of 95.0%-105%. Each sample was tested twice and the average value was calculated.

[0051] Osmotic pressure: Use Advanced 2020 Multi-Sample osmometer for osmotic pressure testing. During the test, add the standard Clinitrol 290mOsm / kg before and after the sample. The sample volume for each sample is 20μL, and each sample is tested once.

[0052] Viscosity: by Rheosense Initium one plus viscometer test, Initiumone Plus uses a syringe pump and a syringe needle to drive the fluid through the sample at a certain flow rate. Chip. When the sample passes through When the channel of the chip is opened, the pressure sensor in the chip measures the drop in fluid pressure. Using the Hagen-Poiseuille equation, the software can automatically calculate the viscosity of the fluid at a certain shear rate. The test is performed on an A05 chip with the chip temperature set to 25±0.1℃. The sample viscosity is tested at 5 different shear rates, and the measurement is repeated 3 times at each shear rate. For Newtonian fluids, the average of the valid data is reported as the final viscosity result.

[0053] Size Exclusion High Performance Liquid Chromatography (SEC-HPLC): SEC-HPLC is a purity analysis method that separates molecules based on their size in a solution. It is run on an Agilent HPLC system using a size exclusion chromatography column. Particulate impurities and other substances in the mobile phase must be filtered before use (using a 0.22μm or finer filter membrane). The sample temperature was set to 5°C and the column temperature was set to 25°C. The mobile phase composition was 50mM phosphate buffer, 300mM sodium chloride, pH 6.8, and the flow rate was set to 1.0mL / min. The sample was diluted to a concentration of 10mg / mL using the mobile phase, 100μg of the sample was injected into the system and run for 20min, and the detection wavelength was set to 280nm. Data analysis was performed using Agilent CDS software.

[0054] Imaging Capillary Isoelectric Focusing (iCIEF): is a purity analysis instrument used to monitor the distribution of protein charge variants. The isoelectric point (pI) is an intrinsic property of a specific protein and is the pH value at which the protein molecule carries no net charge. Under an external electric field, the charge variants migrate along a continuous pH gradient formed by carrier ampholytes and stop at a pH equal to their pI. 20 μg of sample was mixed with 80 μL of a premix consisting of carrier ampholytes, pI markers, methylcellulose, and urea. Detection was performed on a ProteinSimple iCE3 analyzer equipped with a fluorocarbon-coated full-column detection capillary at a wavelength of 280 nm to evaluate the distribution of charge variants in different pI ranges. The pI values ​​and relative abundance of the separated peaks were quantified using the software ChromPerfect Analysis.

[0055] Non-reduced Caliper-SDS (SDS, sodium dodecyl sulfate) (Caliper-SDS-NR): Performed on a PerkinElmer Caliper automated electrophoresis instrument, proteins can be separated by size. In the Caliper-SDS-NR analysis, the sample was diluted to 1 mg / mL with pure water. 2 μL of the diluted sample was thoroughly mixed with 7 μL of sample denaturation solution (a mixture of sample buffer, 10% SDS solution, and 100 mM N-ethylmaleimide, with a volume ratio of 100:10:4) and incubated at 70°C for 10 min. 35 μL of water was added to each sample, vortexed for 10 seconds, and centrifuged at 14000 rpm for 1 min. 42 μL of the prepared sample was transferred to a 96-well plate, centrifuged at 4000 rpm for 15 min, and placed on the plate holder of the Labchip GXII. Separation and detection were performed on the filled decolorized gel, dye gel, and chip (HT-protein Express LabChip). The results were analyzed using LabChip GX Reviewer software.

[0056] Reduced Caliper-SDS (Caliper-SDS-R): Performed on a PerkinElmer Caliper automated electrophoresis instrument, proteins can be separated by molecular weight after the sample is reduced. In the Caliper-SDS-R analysis, the sample is diluted to 1 mg / mL with pure water. 2 μL of the diluted sample is thoroughly mixed with 7 μL of sample denaturing solution (a mixture of sample buffer, 10% SDS solution, and 1M dithiothreitol (relative to the reducing agent) in a volume ratio of 100:10:4) and incubated at 70°C for 10 min. 35 μL of water is added to each sample, vortexed for 10 seconds, and centrifuged at 14000 rpm for 1 min. 42 μL of the prepared sample is transferred to a 96-well plate, centrifuged at 4000 rpm for 15 min, and placed on the plate holder of the Labchip GXII. Separation and detection are performed on a decolorizing gel, gel dye, and chip (HT-protein Express LabChip). The results are analyzed using LabChip GX Reviewer software.

[0057] Example 1

[0058] This embodiment prepares a choline amino acid ionic liquid, comprising the following steps: 15.6 mL of a methanol solution of choline chloride (57.76 mmol of choline chloride) is subjected to rotary evaporation at 40°C to remove the solvent methanol. An equimolar amount of amino acid is dissolved in 50 mL of water and added to choline to form a mixture. The mixture is cooled in an ice-water bath and stirred for 12 hours. Rotary evaporation is performed at 50°C to remove the solvent water. A mixed solvent of acetonitrile and methanol in a ratio of 9:1 is then added to the system to precipitate unreacted amino acids. The mixture is stirred and filtered, and the solvent is removed by evaporation under reduced pressure. The purified ionic liquid is dried overnight at 60°C in a vacuum oven and stored in a moisture-proof state for later use.

[0059] Example 2

[0060] This example compares the viscosity of solutions with different protein concentrations under the same prescription. A certain concentration of monoclonal antibody is replaced by ultrafiltration into a buffer salt system of 20mM histidine hydrochloride and pH 6.0, and sucrose and Tween 80 are added to make the sucrose content in the final solution 8% w / v and the Tween 80 content 0.04% w / v. The protein concentrations are adjusted to about 200 mg / mL, 150 mg / mL, 100 mg / mL and 50 mg / mL respectively. Under 25°C, the viscosity of the protein preparation is measured. The results are shown in Table 1. The solution viscosity increases significantly with the increase of protein concentration.

[0061] Table 1

[0062] Protein concentration (mg / mL) Preparation viscosity (25℃, mPa·s) 214.6 609.8 156.4 59.4 113.0 12.2 50 2.5

[0063] Example 3

[0064] This example explores the effect of different viscosity reducing agent formulations on the viscosity of the preparation. The monoclonal antibody is replaced by ultrafiltration into a buffer salt system of 20 mM histidine hydrochloride, pH 6.0, and different concentrations and types of viscosity reducing agent excipients are added to adjust the protein concentration to close to 140 mg / mL. The viscosity of the protein preparation is measured at 25°C, and the excipient information and measurement results are shown in Table 2. Among them, the choline and arginine mixture in the prescriptions F18-F21 is an equimolar mixture of choline and arginine.

[0065] Table 2

[0066]

[0067]

[0068] As can be seen from Table 2, choline or choline-phenylalanine ionic liquid added to the monoclonal antibody solution has the effect of reducing the viscosity of the liquid, and the effect of choline-phenylalanine ionic liquid in reducing viscosity is more obvious than that of choline alone, and the viscosity of the formulation decreases with the increase of the content of choline or choline-phenylalanine ionic liquid added. Choline-arginine ionic liquid also has a certain viscosity reduction effect on the monoclonal antibody solution within a specific concentration range, and arginine hydrochloride and a mixture of choline and arginine have no viscosity reduction effect.

[0069] Example 4

[0070] This example explores the stability of different excipients. A certain concentration of monoclonal antibody is replaced by ultrafiltration to 20mM histidine hydrochloride, pH 6.0 buffer salt system, and different concentrations of excipients are added according to the scheme in Table 3. The protein concentration is adjusted to 150mg / mL, sterile filtration, rubber plugs and aluminum caps are added for standby use. The prepared sample to be tested is placed at 40°C, shaken and frozen and thawed for storage and sampled and tested according to the scheme in Table 4. The results of pH, osmotic pressure and viscosity are shown in Table 5, the results of appearance observation are shown in Table 6, the results of protein concentration are shown in Table 7, the results of SEC-HPLC are shown in Tables 8 and 9, the results of iCIEF are shown in Tables 10 and 11, and the results of Caliper-SDS-NR and Caliper-SDS-R are shown in Table 12.

[0071] Table 3

[0072]

[0073]

[0074] Table 4

[0075]

[0076] Among them, X, Y, Z, and V represent the test items, X = appearance, protein concentration, SEC-UPLC, Y = iCIEF, Caliper-SDS NR&R, Z = pH value, and V = osmotic pressure and viscosity.

[0077] Table 5

[0078] prescription pH Osmotic pressure (mOsm / kg) Viscosity(cP) F1 6.1 97 2.4 F2 6.1 91 2.2 F3 6.1 236 2.6 F4 6.0 220 2.4 F5 6.0 203 11.9 F6 6.0 200 28.6

[0079] It can be seen from Table 5 that choline-phenylalanine ionic liquid, choline-arginine ionic liquid and choline all have the effect of reducing the viscosity of high-concentration monoclonal antibody preparations. Since the types of monoclonal antibodies used in this example and Example 3 are different, the viscosity reducing effects of the viscosity reducers are different.

[0080] Table 6

[0081]

[0082] As can be seen from Table 6, the appearance results show that all results are light yellow, slightly opalescent liquids. Except for F1, which has rare visible particles (diameter greater than 50 μm, number less than or equal to 3) after shaking for three days, the rest show no visible particles, indicating that the stabilizers selected in the prescription will not cause protein molecules to aggregate.

[0083] Table 7

[0084] prescription T0 40℃-2 weeks 40℃-4 weeks Shaking - 3 days Freeze-thaw - 3 cycles F1 148.8 156.1 152.8 147.4 151.7 F2 148.1 153.9 152.1 144.3 150.4 F3 143.7 155.7 154.0 148.5 151.5 F4 147.1 155.0 152.1 148.4 152.1 F5 151.4 154.4 151.3 149.2 152.9 F6 149.5 154.6 151.6 150.6 150.6

[0085] It can be seen from Table 7 that after high temperature incubation, room temperature shaking and repeated freezing and thawing, the protein concentrations of the F1-F6 formulations did not change significantly.

[0086] Table 8

[0087]

[0088] Table 9

[0089]

[0090]

[0091] Among them, Main peak is the main peak of protein molecule, HMW is the high molecular weight peak, and LMW is the low molecular weight peak.

[0092] As can be seen from Tables 8 and 9, after the protein preparation was shaken at room temperature and repeatedly frozen and thawed, the main peak percentage did not change significantly. After two weeks and four weeks of high temperature incubation, the main peak showed a clear trend of continuous decline, but the degree of decline was within an acceptable range. This shows that the choline-phenylalanine ionic liquid did not cause the aggregation of protein molecules in the protein preparation.

[0093] Table 10

[0094]

[0095] Table 11

[0096]

[0097]

[0098] Among them, Main peak is the main peak of protein molecule, Acidic Peak is the acidic peak, and Basic Peak is the basic peak.

[0099] It can be seen from Tables 10 and 11 that as the high temperature incubation time increases, the acidic peak in the protein preparation increases and the main peak decreases. Among them, the acidic peak of F5 increases the least, which may be because the added sucrose and arginine have a certain effect of stabilizing the protein. However, this stabilization effect is not obvious in the performance of shaking at room temperature and repeated freezing and thawing.

[0100] Table 12

[0101]

[0102]

[0103] Among them, NR stands for Caliper-SDS-NR and R stands for Caliper-SDS-R.

[0104] It can be seen from Table 12 that the purity of protein preparations after high temperature incubation, room temperature shaking and repeated freezing and thawing has decreased to a certain extent. Among them, with the increase of high temperature incubation time, the decrease rate of protein purity is higher than that of shaking and freezing and thawing, but the decrease rate is relatively small.

[0105] The above results show that the liquid preparation prepared by using the viscosity reducer, buffer system and therapeutic protein of the present invention, especially high-concentration protein, has a lower viscosity. At the same time, the protein preparation added with the viscosity reducer can remain stable under conditions such as high-temperature incubation, room-temperature shaking and repeated freezing and thawing. It shows that choline-phenylalanine ionic liquid can effectively reduce viscosity in macromolecular prescriptions while ensuring good stability of protein drugs.

[0106] In summary, the present invention provides the use of choline and / or choline-based ionic liquids in reducing the viscosity of high-concentration macromolecular preparations, which can effectively reduce the viscosity of the solution without affecting the stability of the protein, and significantly improve the drug solubility and drug loading at non-toxic concentrations without affecting the activity of protein drugs or the stability of cell drug products.

[0107] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. Application of choline and / or choline-based ionic liquids in reducing the viscosity of high-concentration macromolecular preparations.

2. The use according to claim 1, characterized in that: The working concentration of choline is 0 to 300 mM, preferably 75 to 200 mM; Preferably, the working concentration of the choline ionic liquid is 0-300 mM.

3. The use according to claim 1 or 2, characterized in that: The choline ionic liquid includes a choline amino acid ionic liquid; Preferably, the choline amino acid ionic liquid comprises any one of a choline-phenylalanine ionic liquid, a choline-arginine ionic liquid or a choline-glycine ionic liquid, or a combination of at least two thereof; Preferably, the working concentration of the choline-phenylalanine ionic liquid is 0 to 300 mM, preferably 75 to 200 mM; Preferably, the working concentration of the choline-arginine ionic liquid is 0 to 150 mM, preferably 75 to 100 mM.

4. The use according to claim 3, characterized in that: The preparation method of the choline amino acid ionic liquid comprises the following steps: (1) Rotary evaporation of a methanol solution of choline chloride at 35-45° C. to remove the solvent methanol; (2) dissolving an amino acid in an amount equimolar to that of choline in water, mixing with choline chloride, cooling in an ice-water bath, stirring for 10 to 14 hours, and removing the solvent water by rotary evaporation at 45 to 55°C; (3) adding a mixed solvent of acetonitrile and methanol in a volume ratio of (8-10):1 to precipitate the unreacted amino acid; (4) filtering, evaporating under reduced pressure to remove the solvent, and vacuum drying at 55-65° C. to obtain the choline amino acid ionic liquid.

5. The use according to any one of claims 1 to 4, characterized in that: The high-concentration macromolecular preparation includes any one of a high-concentration protein preparation, a high-concentration nucleic acid preparation or a high-concentration vaccine preparation, or a combination of at least two thereof; Preferably, the high-concentration protein preparation comprises any one of a high-concentration monoclonal antibody preparation, a high-concentration biclonal antibody preparation or a high-concentration fusion protein preparation, or a combination of at least two thereof; Preferably, the protein concentration of the high-concentration protein preparation is ≥50 mg / mL.

6. A high concentration monoclonal antibody solution, characterized in that: The high-concentration monoclonal antibody solution contains monoclonal antibodies and viscosity-reducing auxiliary materials, and the viscosity-reducing auxiliary materials include choline and / or choline-based ionic liquids.

7. The high concentration monoclonal antibody solution according to claim 6, characterized in that: The high-concentration monoclonal antibody solution contains 100-300 mg / mL monoclonal antibody and 0-300 mM viscosity-reducing auxiliary material; Preferably, the high concentration monoclonal antibody solution contains 0 to 300 mM choline, preferably 75 to 200 mM choline; Preferably, the high concentration monoclonal antibody solution contains 0-300 mM choline ionic liquid.

8. The high concentration monoclonal antibody solution according to claim 6 or 7, characterized in that: Choline ionic liquids include choline-phenylalanine ionic liquids and / or choline-arginine ionic liquids; Preferably, the high concentration monoclonal antibody solution contains 0 to 300 mM choline-phenylalanine ionic liquid, preferably 75 to 200 mM choline-phenylalanine ionic liquid; Preferably, the high concentration monoclonal antibody solution contains 0 to 150 mM choline-arginine ionic liquid, preferably 75 to 100 mM choline-arginine ionic liquid.

9. The high concentration monoclonal antibody solution according to any one of claims 6 to 8, characterized in that: The high concentration monoclonal antibody solution also contains a buffer solution; Preferably, the buffer solution comprises a 15-25 mM histidine hydrochloride solution at a pH of 5-7; Preferably, the high concentration monoclonal antibody solution further contains a surfactant; Preferably, the surfactant comprises 0.03% to 0.05% w / v Tween 80; Preferably, the high concentration monoclonal antibody solution further contains a stabilizer; Preferably, the stabilizer comprises 2% to 10% w / v sucrose.

10. Use of the high concentration monoclonal antibody solution according to any one of claims 6 to 9 in the preparation of a subcutaneous injection medicament.

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