A preparation for preventing IL-2 derivative protein complex aggregation and non-covalently linked protein subunit shedding

Abstract

The present invention belongs to the field of biomedicine technology, and discloses a preparation for preventing IL-2 derivative protein complex aggregation and non-covalently linked protein subunit shedding; higher ionic strength will promote the dissociation of this non-covalently linked protein complex, and this problem is crucial during the patient infusion process, because the commonly used injection solution (such as 0.9% normal saline) with an ionic strength close to that of human body fluids has a high ionic strength, which is sufficient to dissociate the non-covalently linked protein complex of the interleukin-2 protein complex. The present invention systematically studies the effects of the solution pH, buffer salt type, surfactant and additives on the dissociation, colloidal stability and aggregation of the protein complex, reduces the dissociation of the interleukin-2 protein complex, and ensures that the protein does not form aggregates.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a preparation for preventing IL-2 derivative protein complex aggregation and non-covalently linked protein subunit shedding. Background Art

[0002] Due to the lack of covalent cross-linking, therapeutic non-covalently linked protein complexes are prone to non-covalently linked protein subunits falling off during storage and use. The biological activity of the protein complex after shedding will be adversely affected, so this phenomenon must be avoided as much as possible. Non-covalently linked protein complexes rely on a variety of non-covalent interactions to connect together, including but not limited to hydrophobic interactions, ionic bonds, and hydrogen bonds. Various interactions are affected differently by solutions and preparations. It is difficult to evaluate the size of non-covalent interactions of protein complexes through simple theoretical deduction and analysis. It is necessary to obtain the optimal preparation for a specific protein complex through systematic experimental research and analysis to balance the effects of various factors on the overall non-covalent interaction.

[0003] Most of the existing therapeutic protein preparations are for covalently linked protein complexes, such as antibodies. Such proteins do not have the problem of non-covalently linked protein subunits falling off, so the risks brought by this problem are not considered during the formulation development process. For example, surfactants reduce the hydrophobic interactions between protein subunits and are widely used in biological preparations to prevent protein aggregation. However, for protein complexes, surfactants may reduce the hydrophobic interactions between protein complex subunits, thereby causing the dissociation of protein complex subunits. For another example, in the development of covalently linked protein complex preparations, pH values ​​close to the isoelectric point of the protein will affect the colloidal stability of the protein, and being away from the isoelectric point usually increases the charge of the protein and reduces the aggregation tendency of the protein. However, in non-covalently linked protein complexes, being away from the isoelectric point usually increases the charge of the protein, which may increase the charge repulsion between subunits, causing the non-covalently linked protein subunits to fall off. In addition, since there are multiple non-covalent interactions in non-covalently linked protein complexes, including but not limited to hydrophobic interactions, ionic bond interactions, and hydrogen bond interactions, the contribution of various interactions in different protein complexes is different, and at the same time, various interactions are affected differently by solutions and preparations. Interleukin-2 derivatives are therapeutic non-covalent protein complexes (patent application publication number CN111018961A). Higher ionic strength promotes the dissociation of this non-covalently linked protein complex. This problem is crucial during the patient infusion process, because the commonly used injection solution (such as 0.9% saline) with a high ionic strength close to the ionic strength of human body fluids is high enough to dissociate the non-covalently linked protein complex of the IL-2 derivative protein complex. Existing preparations developed for covalently linked protein complexes do not have a solution to this problem. It is difficult to learn from commonly used preparations and preparations used for other different non-covalently linked protein complexes. These factors limit the preparation development and clinical application of therapeutic non-covalently linked protein complexes.

[0004] The present invention systematically studies the effects of solution pH, buffer salt type, ionic strength, surfactant and additives on the dissociation, colloidal stability and aggregation of protein complexes such as interleukin-2 (IL-2).

[0005] The present application found that the addition of surfactant is the key to preventing the non-covalently linked protein subunits of the IL-2 derivative protein complex from falling off, and can protect the protein complex from existing in the form of a complex at an ionic strength close to that of human body fluids, thus creating conditions for its clinical application. Summary of the invention

[0006] In order to overcome the deficiencies in the prior art, the present invention provides a preparation for preventing the aggregation of non-covalently linked protein complexes and the shedding of non-covalently linked protein subunits, particularly IL-2 derivative protein complexes.

[0007] To achieve the above-mentioned purpose, the present invention first discloses the composition of the preparation for preventing the aggregation of non-covalently linked protein complexes and the shedding of non-covalently linked protein subunits, which specifically includes 1-100 mM buffer, 2-12% (w / v) osmotic pressure regulator, 0.01-0.1% (w / v) surfactant, pH.5-7.5;

[0008] Preferably, the buffer is a phosphate buffer;

[0009] Preferably, the osmotic pressure regulator is sucrose;

[0010] Preferably, the surfactant is polysorbate 80;

[0011] Preferably, the non-covalently linked protein complex is an interleukin-2 derivative.

[0012] Secondly, the present application also discloses a preparation for preventing aggregation of interleukin-2 derivative protein complexes and shedding of non-covalently linked protein subunits, the preparation comprising 20 mM phosphate buffer, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 6.5;

[0013] Preferably, the interleukin-2 derivative sequence is as described in application number 201911302355.5.

[0014] In addition, the present application also discloses the use of the above preparation in the preparation of anti-tumor and anti-inflammatory drugs.

[0015] Beneficial effects:

[0016] 1) The present invention has found a suitable surfactant so that even at extremely low (≤0.00014% w / v%) surfactant concentrations, the IL-2 derivative protein complex will not experience shedding of non-covalently linked protein subunits at higher ionic strengths and at lower concentrations at the ionic strength of human blood.

[0017] 2) The present invention creatively prevents the dissociation and aggregation of the IL-2 derivative protein complex by regulating the pH range.

[0018] 3) The present invention prevents the aggregation of IL-2 derivative protein complexes by adjusting the types and proportions of surfactants and additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Attached Figure 1 Size exclusion chromatography of IL-2 derivatives in 0.9% sodium chloride injection at 40°C accelerated experiments.

[0020] Attached Figure 2 The IL-2 derivatives were subjected to size exclusion chromatography in 0.9% sodium chloride injection solution containing 0.00014% w / v% Tween 80 at 25°C for 24 hours.

[0021] Attached Figure 3 The results of the dissociation of the LTC004 protein complex without Tween, with Tween 20 or Tween 80 at different pH values ​​are shown.

[0022] Attached Figure 4 is the size exclusion chromatogram of IL-2 derivatives.

[0023] Attached Figure 5 The size exclusion chromatograms of IL-2 derivatives at different pH values ​​show the percentage of monomers and aggregates.

[0024] Attached Figure 6 The percentage of monomers and aggregates in the size exclusion chromatograms of IL-2 derivatives under different surfactants. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with the accompanying drawings.

[0026] Example 1

[0027] Under higher ionic strength, IL-2 derivative protein complexes tend to dissociate.

[0028] Size exclusion chromatography: A SuperdexTM 200 Increase 10 / 300GL column (P / N: 28990944) was used; the liquid phase system was Agilent Technologies 1260 Infinity; the liquid phase workstation was Waters Empower3 network version; the dissociation products were analyzed by size exclusion chromatography; the flow rate was 0.5 mL / min, the column temperature was 25°C, the detection UV was 215 nm, the mobile phase was: 1xPBS, pH 7.4; the running time was 60 minutes.

[0029] Preparation method of 1xPBS mobile phase: Preparation of 10×PBS mother solution: Accurately weigh 2.7g KH2PO4, 35.8gNa2HPO4, 80g NaCl, 2g KCl, dissolve in purified water and make up to 1L, filter with 0.22um filter membrane; Preparation of 1×PBS mobile phase: transfer 100mL 10×PBS mother solution, add pure water to make up to 1000mL, mix well and degas.

[0030] Preparation method of 0.9% (w / v) sodium chloride solution: Accurately weigh 0.9g NaCl, add pure water to completely dissolve, and make up to 100mL, and then filter with a 0.22um filter membrane.

[0031] When therapeutic protein drugs are used, 0.9% sodium chloride injection is often used for dilution and intravenous administration. The ionic strength of 0.9% sodium chloride is relatively high. For IL-2 derivative protein complexes, higher ionic strength will weaken the strength of ionic interactions in non-covalent interactions. Therefore, it is necessary to investigate the dissociation of IL-2 derivative complexes in 0.9% sodium chloride injection. Figure 1 As shown, the IL-2 derivative complex was diluted into 0.9% sodium chloride injection to make the final concentration of the protein complex 0.02 mg / mL. Within 3 days of the accelerated experiment at 40°C, it was observed that the dissociation products of the IL-2 derivative gradually increased. Even in the freshly prepared data, partial dissociation could be observed, which caused stability problems for the injection of protein drugs and may cause changes in protein potency during the preparation and infusion of the drug.

[0032] Example 2

[0033] At higher ionic strength, the activity of IL-2 derivative protein complexes is significantly reduced.

[0034] MO7E and NK92 cell activity experiment: MO7E and NK92 cell lines were cultured in complete medium at 37°C and 5% CO2. Cells in the logarithmic growth phase were harvested, washed twice with PBS to remove cytokines, and then resuspended in medium without cytokines. Cell counts were performed using a platelet counter, and cell viability was detected by trypan blue exclusion to ensure that cell viability was above 90%. Cell density was adjusted using medium without cytokines, and then inoculated in a 96-well cell culture plate, 90 μL per well, for a total of 5,000 cells. The cells in the 96-well plate were cultured at 37°C and 5% CO2 for standby use. A 10-fold drug concentration was prepared so that the final working concentration was: 1) The highest concentration of MO7E cells was 200 nM, a 3.16-fold dilution ratio, 9 dilutions, and three replicates for each drug concentration; 2) The highest concentration of NK92 cells was 20 nM, a 5-fold dilution ratio, 9 dilutions, and three replicates for each drug concentration. Then transfer 10 μL of each serial dilution sample to the corresponding experimental wells of the 96-well cell plate. Incubate the cells in the 96-well plate with the drug at 37°C and 5% CO2 for 72 hours before performing CTG analysis. Thaw the CTG reagent and equilibrate the cell plate to room temperature for 30 minutes. Add an equal volume of CTG solution to each well. Shake on an orbital shaker for 5 minutes to lyse the cells. Place the cell plate at room temperature for 20 minutes to stabilize the luminescence signal. Read the luminescence value and collect data.

[0035] The data were analyzed using GraphPad Prism 7.0 software, and the dose-effect curve was fitted using nonlinear S-curve regression to obtain the EC50 value.

[0036] Cell survival rate (%) = (Lum test drug-Lum culture medium control) / (Lum solvent control-Lum culture medium control) × 100%.

[0037] As can be seen from Example 1, the IL-2 derivative protein complex will dissociate in 0.9% sodium chloride injection, and the effect of this dissociation process on drug potency directly affects the formulation development and drug use of the drug. The IL-2 derivative protein complex will bind to the β and γ receptors of interleukin 2, promote the growth of MO7E and NK92 cells, and thus exert immunosuppression on tumor cells (patent application publication number CN111018961A). The change in potency of the IL-2 derivative protein complex in 0.9% sodium chloride can be studied by the activity experiment of MO7E and NK92 cells. The EC50 of MO7E and NK92 cells can reflect the biological activity of the IL-2 derivative protein complex. The higher the value, the lower the biological activity. In the accelerated experiment of IL-2 derivatives in 0.9% sodium chloride, when stored at 40°C for 5 days, it can be found that the EC50 of the protein complex increased significantly, indicating that the activity was significantly reduced and the drug potency was significantly reduced. Combined with Example 1, the IL-2 derivative complex will dissociate in 0.9% sodium chloride, and the biological activity will be significantly reduced. Therefore, the dissociation of the IL-2 derivative complex should be avoided as much as possible during the formulation development process.

[0038] Table 1 Results of cell activity acceleration test of IL-2 derivatives in 0.9% sodium chloride

[0039]

[0040] Example 3

[0041] Very low concentrations of Tween 80 can still protect the IL-2 derivative protein complex from dissociation

[0042] Ultra-high pressure size exclusion chromatography: AdvanceBio SEC 200A 1.9um 4.6x150mm chromatographic column (P / N: PL1580-3201) was used, and the liquid phase system was Waters Acquity UPLC H-CLASS PLUS. The liquid phase workstation was Waters' network version of Empower3, and the main peak and dissociation products were analyzed by ultra-high pressure size exclusion chromatography. The flow rate was 0.35mL / min, the column temperature was 25℃, the detection UV was 280nm, and the mobile phase was: 2xPBS, pH 7.0; the running time was 10 minutes.

[0043] Mobile phase 2xPBS, pH7.0 solution preparation method: 10×PBS stock solution preparation: accurately weigh 2.7g KH2PO4, 35.8g Na2HPO4, 80g NaCl, 2g KCl, dissolve in purified water and dilute to 1L, filter with 0.22um filter membrane. 2×PBS, pH7.0 mobile phase preparation: pipette 200mL 10×PBS stock solution, add purified water to 900mL, adjust pH to 7.0, dilute to 1L with water, mix and degas.

[0044] Preparation method of 0.9% (w / v) sodium chloride solution: Accurately weigh 0.9g NaCl, add pure water to completely dissolve, and make up to 100mL, and then filter with a 0.22um filter membrane.

[0045] As described in Example 1, at higher ionic strengths, the IL-2 derivative protein complex will dissociate. As described in Example 2, the dissociation of the IL-2 derivative protein complex will significantly reduce the potency, so the dissociation of the protein complex should be avoided. By diluting the IL-2 derivative protein complex containing 0.04% Tween 80 with 0.9% sodium chloride injection to a concentration of 10 μg / mL, the final concentration of Tween 80 is reduced to 0.00014% w / v%. This experimental condition simulates the drug use conditions of dissolving a drug containing Tween 80 in 0.9% sodium chloride injection for intravenous drip. Figure 2 , no obvious dissociation products were observed within 24 hours at room temperature of 25°C, indicating that even extremely low concentrations of Tween 80 can still protect the IL-2 derivative protein complex from dissociation. Although in theory, low concentrations of protein complexes are more likely to dissociate, and surfactants may reduce hydrophobic interactions in non-covalent protein interactions. However, the unexpected discovery of the present application proves that Tween 80 actually protects the non-covalent protein complex of IL-2 derivatives. The overall result is that the protein complex is prevented from dissociation, thereby creating the necessary conditions for the clinical application of the protein complex in high ionic strength and ionic strength close to that of human body fluids.

[0046] Example 4

[0047] Protective effects of Tween 20 and Tween 80 on the dissociation of IL-2 derivative protein complexes at different pH values.

[0048] The sample size exclusion chromatography was performed at different pH values. In this method, a SuperdexTM 200 Increase 10 / 300 GL column (P / N: 28990944) was used, and the liquid phase system was Agilent Technologies 1260 Infinity. The liquid phase workstation was the network version of Waters Empower3, and the dissociation products were analyzed by size exclusion chromatography. The flow rate was 0.5 mL / min, the column temperature was room temperature 25°C, the detection UV was 280 nm, and the mobile phase was: 1xPBS, pH 7.4; the running time was 60 minutes.

[0049] Preparation method of 1xPBS mobile phase: Preparation of 10×PBS stock solution: accurately weigh 2.7g KH2PO4, 35.8g Na2HPO4, 80g NaCl, 2g KCl, dissolve in purified water and dilute to 1L, filter with 0.22um filter membrane. Preparation of 1×PBS mobile phase: pipette 100mL 10×PBS stock solution, add pure water to dilute to 1000mL, mix and degas. Preparation method of 0.9% (w / v) sodium chloride solution: accurately weigh 0.9g NaCl, add pure water to completely dissolve, dilute to 100mL, and then filter with 0.22um filter membrane.

[0050] Preparation method of pH4.5, 0.1M citric acid solution: weigh 1.92g citric acid, add pure water to completely dissolve, and make up to 90mL, adjust the pH to 4.5, add pure water to make up to 100mL, and then filter with a 0.22um filter membrane.

[0051] Preparation method of pH5.5, 1M sodium acetate solution: weigh 4.1g sodium acetate, add pure water to completely dissolve, and make up to 40mL, adjust the pH to 5.5, add pure water to make up to 50mL, and then filter with a 0.22um filter membrane.

[0052] pH 6.5, 0.2M PB solution preparation method: Prepare 0.2M NaH2PO4 solution: weigh 12g NaH2PO4, add pure water to completely dissolve, and dilute to 500mL, filter with 0.22um filter membrane

[0053] Prepare 0.2M Na2HPO4 solution: weigh 14.2g Na2HPO4, add pure water to completely dissolve, and dilute to 500mL, filter with 0.22um filter membrane, measure 34.25mL 0.2M NaH2PO4 solution, and 15.75mL 0.2MNa2HPO4 solution respectively; after mixing, it is 50mL pH6.5, 0.2M PB solution.

[0054] Preparation method of pH7.5, 0.1M Tris solution: weigh 0.61g Tris, add pure water to completely dissolve, and make up to 40mL, adjust the pH to 7.5, add pure water to make up to 50mL, and then filter with a 0.22um filter membrane.

[0055] Preparation of pH 4.5, 0.9% (w / v) sodium chloride solution: measure 28.5mL 0.9% NaCl solution and 1.5mL pH 4.5, 0.1M citric acid solution respectively; after mixing, 30mL pH 4.5, 0.9% NaCl solution is obtained.

[0056] Preparation of pH 5.5, 0.9% (w / v) sodium chloride solution: measure 29.85mL 0.9% NaCl solution and 0.15mL pH 5.5, 1M sodium acetate solution respectively; after mixing, 30mL pH 5.5, 0.9% NaCl solution is obtained.

[0057] Preparation of pH 6.5, 0.9% (w / v) sodium chloride solution: measure 29.25mL 0.9% NaCl solution and 0.75mL pH 6.5, 0.2M PB solution respectively; after mixing, 30mL pH 6.5, 0.9% NaCl solution is obtained.

[0058] Preparation of pH 7.5, 0.9% (w / v) sodium chloride solution: measure 28.5mL 0.9% NaCl solution and 1.5mL pH 7.5, 0.1M Tris solution respectively; after mixing, it is 30mL pH 7.5, 0.9% NaCl solution.

[0059] In the experiment, 0.5 mg / mL of IL-2 derivative dissolved in 20 mM PB, pH 6.5 solution was taken and divided into three portions, each 500 μL, and water, Tween 20, and Tween 80 were added to obtain three samples without Tween, with 0.4 w / v% Tween 20 and 0.4 w / v% Tween 80. 100 μL of each sample was diluted in 900 μL of the above 0.9% NaCl buffer solution with different pH values, so that the final concentration of Tween was 0.04 w / v%, and stored at 25°C and 40°C for 24 hours, and then the degree of dissociation was analyzed using size exclusion chromatography.

[0060] At pH values ​​of 4.5, 5.5, 6.5, and 7.5, in 0.9% sodium chloride injection, the IL-2 derivative protein complex was observed to dissociate to varying degrees at room temperature (25°C) and under accelerated test conditions (40°C). In general, the dissociation was more obvious under accelerated test conditions (40°C). When Tween 20 or Tween 80 was added to the sample before dilution and then diluted to 0.04% w / v% Tween, SEC was used to observe the protection of Tween on the dissociation of the IL-2 derivative protein complex under different pH conditions. Under pH 4.5 ( Figure 3 A). At room temperature (25°C), in the presence of Tween 20, Tween 80, or without Tween, the dissociation of the IL-2 derivative protein complex was observed to be obvious. In the accelerated experiment (40°C), the dissociation of the IL-2 derivative protein complex was more serious when Tween 20 and Tween 80 were present than when Tween was absent, indicating that Tween accelerated the dissociation of the IL-2 derivative protein complex at pH 4.5. At pH 5.5, 6.5, and 7.5 ( Figure 3 BD), at room temperature (25°C), the IL-2 derivative protein complex dissociated when there was no Tween, while no obvious dissociation was observed when there were Tween 20 and Tween 80; under accelerated test (40°C), the IL-2 derivative protein complex dissociated more obviously when there was no Tween, while no obvious dissociation was observed when there were Tween 20 and Tween 80. This result fully demonstrates that within the pH range of 5.5 to 7.5, Tween can protect the IL-2 derivative protein complex from dissociation.

[0061] Example 5

[0062] Systematic screening of the effects of pH and buffer solution on the stability and aggregation of IL-2 derivative protein complexes.

[0063] Appearance: Wipe the sample bottle clean, place it under a clarity tester in a dark room, adjust the illumination to 2000-3750 Lux, place the sample at the edge of the light shield (25 cm), hold the neck of the test bottle, and visually inspect the color, clarity, and visible foreign matter against a black and white background.

[0064] Protein concentration: After the sample is mixed evenly, 2.5 μL of the sample is taken with a pipette and the concentration of the sample at a wavelength of 280 nm is detected by NanoDrop 2000. The extinction coefficient is 1.130 AU*mL*mg-1*cm-1. Repeat the measurement twice and take the average as the final result.

[0065] Size exclusion chromatography: if the sample concentration is higher than 5.0 mg / mL, dilute it with ultrapure water to 5.0 mg / mL. If the sample concentration is lower than or equal to 5.0 mg / mL, no dilution is required. SEC-UPLC detection uses ACQUITY UPLC@Protein BEH SEC Column, 200A, 1.7μm, 4.6mm×150mm / Waters chromatographic column, using ultra-high performance liquid chromatography UPLC (WatersH-Class Bio Core System), set the column temperature to 25±3℃, the mobile phase is 50mM PB, 300mM NaCl, pH 6.8±0.1, the injection volume is 5μg, at a flow rate of 0.4mL / min, isocratic elution for 8min, detection at a wavelength of 214nm, and the peak area normalization method is used to calculate the monomer content.

[0066] Differential scanning calorimetry: When the sample concentration is greater than 2 mg / mL, dilute the sample to a concentration of 1 mg / mL with the corresponding buffer. When the sample concentration is between 0.5 and 2 mg / mL, no dilution is required. Add 400 μL of the corresponding two pairs of buffers to a 96-well plate, add 400 μL of buffer to the left well of the next pair of wells, and add 400 μL of sample to the right well. Experimental parameter settings for differential scanning calorimeter (MalvernSYS12907): scan start temperature is 10°C, scan end temperature is 95°C, and heating rate is 200°C / h. Use software for data analysis.

[0067] Dynamic light scattering: The sample was centrifuged at 12000rpm for 5min, then 20μL of the sample was added to a 384-well sample plate, and 10μL of paraffin oil was added to the sample, and the 384-well sample plate was centrifuged at 2000rpm for 5min to remove bubbles. The 384-well sample plate was placed on a dynamic light scattering instrument (WYATT Dynaproplate Reader Ⅱ), and the temperature program was set, the scanning time was 3 seconds, and the scan was 3 times. The program was run, and the data were analyzed using the instrument software DYNAMICS.

[0068] pH measurement: The pH meter (METTLER TOLEDO S400) was calibrated with three standard solutions (pH 4.01, 7.00, and 9.21, respectively). The calibration slope was required to be between 95.0% and 105.0%. 100 μL of the sample was dispensed to determine the pH value.

[0069] Under heated and pressurized conditions, the effects of different pH and buffer salts on the stability, aggregation and dissociation of IL-2 derivative protein complexes were studied by using appearance observation, protein concentration determination, size exclusion chromatography (SEC), differential scanning calorimetry (DSC), dynamic light scattering (DLS), etc.

[0070] The pH and buffer solution compositions tested in the experiment are shown in the following table

[0071] Table 2 pH and buffer solution composition tested

[0072]

[0073]

[0074] Table 3 DSC determination of protein melting temperature at different pH values

[0075]

[0076] The melting onset temperature and melting temperature show the stability of the protein structure. The higher the temperature, the more stable the protein structure. As shown in Table 3, the IL-2 derivative protein complex has two melting temperatures. In different pH and buffer salt systems, the melting onset temperature, melting temperature 1 and melting temperature 2 have the same change trend, showing a synchronous rise or a synchronous fall. Taking the melting onset temperature as an example, in the range of pH 4.5-6.5, the melting onset temperature increases with the increase of pH, and in the range of pH 6.5-8.5, the melting onset temperature decreases with the increase of pH. It can be seen from the data that the structural stability of the protein is affected by the pH value, and the stability near pH 6.5 is better.

[0077] Table 4 DLS determination of protein aggregation temperature at different pH values

[0078]

[0079] The protein aggregation temperature shows the colloidal stability of the protein. The higher the temperature, the higher the colloidal stability of the protein. As shown in Table 4, in the pH range of 4.5-6.5, in the same type of buffer salt, the protein aggregation temperature of the IL-2 derivative protein complex increases with the increase of pH. In the 20mM phosphate and 20mM citrate / arginine buffer systems, the protein aggregation temperature is higher than the temperature range of the experimental determination, showing good colloidal stability. As can be seen from the data, the colloidal stability of the protein is affected by the pH value, and the stability is better at pH 6.5 and above.

[0080] Table 5 Changes in appearance and protein concentration during accelerated stability experiments

[0081]

[0082]

[0083] Table 5

[0084]

[0085]

[0086] Table 5

[0087]

[0088]

[0089] Among them, C (colorless), CL (transparent), SO (slightly milky white), PF (no visible particles), PO (visible particles, ++ more than 5 visible particles observed). The appearance and concentration of the protein show the aggregation and adsorption of the protein. The slight milky white color and the observation of visible particles prove that the protein has obviously aggregated, and the significant decrease in protein concentration reflects that the protein has obviously aggregated or has obviously adsorbed. The accelerated experiment was placed at 40°C for 4 weeks, and samples were taken for testing at 0, 1, 2, and 4 weeks, which can simulate the performance of the protein at a higher temperature and has important reference significance for the long-term stability of the protein. The protein complex was observed to generate opalescence in the 4-week stability experiment from pH 4.5 to pH 6.5, and even visible particles were observed under some conditions. All show that the protein complex is prone to aggregation at pH 6.0 and below. At pH 6.5 and above, no opalescence and visible particles were observed, indicating that the long-term stability of the protein is good at pH 6.5 and above.

[0090] Size exclusion chromatography (SEC) can reflect the aggregation and dissociation of IL-2 derivative protein complexes. At different pH values ​​and buffer salts, placed at 40°C for 4 weeks, the generation of aggregates and dissociation products of protein complexes can be observed ( Figure 4 ). Through quantitative analysis of the proportion of monomers and aggregates of protein complexes in SEC ( Figure 5 ), we can observe the trend of the influence of pH value on protein stability. From the data, it can be seen that when the pH is 5.0 and below, the protein complex is prone to form aggregates when placed at 40°C for 4 weeks. At pH 5.5 and above, the proportion of monomers is above 90% in most cases, and the proportion of aggregates is low. This shows that the protein meets the poor colloidal stability at pH 5.0 and below and is prone to form aggregates. At pH 5.5 and above, the formation of aggregates is not obvious.

[0091] Example 6

[0092] Systematic screening of the effects of surfactants and additives on the stability and aggregation of IL-2 derivative protein complexes.

[0093] Appearance: Wipe the sample bottle clean, place it under a clarity tester in a dark room, adjust the illumination to 2000-3750 Lux, place the sample at the edge of the light shield (25 cm), hold the neck of the test bottle, and visually inspect the color, clarity, and visible foreign matter against a black and white background.

[0094] Protein concentration: After the sample is mixed evenly, 2.5 μL of the sample is taken with a pipette and the concentration of the sample at a wavelength of 280 nm is detected by NanoDrop 2000. The extinction coefficient is 1.130 AU*mL*mg-1*cm-1. Repeat the measurement twice and take the average as the final result.

[0095] Size exclusion chromatography: if the sample concentration is higher than 5.0 mg / mL, dilute it with ultrapure water to 5.0 mg / mL. If the sample concentration is lower than or equal to 5.0 mg / mL, no dilution is required. SEC-UPLC detection uses ACQUITY UPLC@Protein BEH SEC Column, 200A, 1.7μm, 4.6mm×150mm / Waters chromatographic column, using ultra-high performance liquid chromatography UPLC (WatersH-Class Bio Core System), set the column temperature to 25±3℃, the mobile phase is 50mM PB, 300mM NaCl, pH 6.8±0.1, the injection volume is 5μg, at a flow rate of 0.4mL / min, isocratic elution for 8min, detection at a wavelength of 214nm, and the peak area normalization method is used to calculate the monomer content.

[0096] Differential scanning calorimetry: When the sample concentration is greater than 2 mg / mL, dilute the sample to a concentration of 1 mg / mL with the corresponding buffer. When the sample concentration is between 0.5 and 2 mg / mL, no dilution is required. Add 400 μL of the corresponding two pairs of buffers to a 96-well plate, add 400 μL of buffer to the left well of the next pair of wells, and add 400 μL of sample to the right well. Experimental parameter settings for differential scanning calorimeter (MalvernSYS12907): scan start temperature is 10°C, scan end temperature is 95°C, and heating rate is 200°C / h. Use software for data analysis.

[0097] Dynamic light scattering: Centrifuge the sample at 12000rpm for 5min, then take 20μL of the sample and add it to the 384-well sample plate, add 10μL of paraffin oil to the sample, and then centrifuge the 384-well sample plate at 2000rpm for 5min to remove bubbles. Place the 384-well sample plate on the dynamic light scattering instrument (WYATT Dynaproplate Reader Ⅱ), set the temperature program, scan time for 3 seconds, scan 3 times, and run the program. Data are analyzed using the instrument software DYNAMICS.

[0098] pH value measurement: The pH meter (METTLER TOLEDO S400) was calibrated with three standard solutions (pH 4.01, 7.00, 9.21, respectively), and the calibration slope was required to be between 95.0% and 105.0%. 100 μL of sample was dispensed and the pH value was determined.

[0099] Two pH and buffer solution systems with relatively stable proteins were selected from Example 5, and different surfactants and additives were further added for further formulation research. The effects of different surfactants and additives on the stability, aggregation and dissociation of IL-2 derivative protein complexes were studied by using appearance observation, protein concentration determination, size exclusion chromatography (SEC), differential scanning calorimetry (DSC), dynamic light scattering (DLS), etc. The composition of the surfactants and additives tested in the experiment is shown in Table 6.

[0100] Table 6 DLS determination of protein aggregation temperature in different surfactants

[0101]

[0102] The protein aggregation temperature shows the colloidal stability of the protein. As shown in Table 6, various surfactants of different concentrations were screened, and the colloidal stability of the protein was determined by the protein aggregation temperature. As can be seen from the data, the colloidal stability of the protein was good in Tween 20, Tween 80, and Poloxamer 188.

[0103] Table 7 DSC determination of protein aggregation temperature in different surfactants

[0104]

[0105] The melting onset temperature and melting temperature show the stability of the protein structure. As shown in Table 7, in Poloxamer 188, as the concentration of surfactant increases, the melting onset temperature gradually decreases, indicating that Poloxamer 188 will destroy the structural stability of the IL-2 derivative protein complex. No obvious changes in the melting onset temperature and melting temperature 1 were observed in Tween 20 and Tween 80 at various concentrations, and they were close to the control sample without adding surfactant, indicating that Tween 20 and Tween 80 would not destroy the structural stability of the protein complex.

[0106] Table 8 pH value, buffer salt, surfactant and additive composition tested

[0107]

[0108]

[0109] Table 9 DSC determination of protein melting temperature under different additive compositions

[0110]

[0111]

[0112] Melting onset temperature and melting temperature show the stability of protein structure. As shown in Table 9, in different pH, buffer salt, surfactant and additive systems (Table 8), the changing trends of melting onset temperature, melting temperature 1 and melting temperature 2 are consistent, showing synchronous rise or synchronous decline. Taking melting onset temperature as an example, comparing F1 with F6, F2 with F7, F3 with F8, F4 with F9, F5 with F10, 20mM phosphate pH 6.5 shows a higher melting onset temperature relative to 20mM histidine salt pH 5.5, i.e., higher protein structure stability. Taking melting onset temperature as an example, comparing F1 with F2, F6 with F7, in the presence of 0.02% w / v% Tween 80, 8% w / v% sucrose and 8.8% w / v% dihydrate trehalose have little effect on protein melting onset temperature, and the stability of protein structure is similar. Taking the melting start temperature as an example, comparing F1 with F3, F6 with F8, in the presence of 0.02% w / v% Tween 80, 8% w / v% sucrose is higher than 140mM arginine hydrochloride, that is, the stability of the protein structure is higher. Taking the melting start temperature as an example, comparing F1 with F4, F6 with F9, 0.02% w / v% Tween 80 and 0.04% w / v% Tween 80 have little effect on the protein melting start temperature, and the stability of the protein structure is similar. Taking the melting start temperature as an example, comparing F4 with F5, F9 with F10, 0.04% w / v% Tween 80 and 0.04% w / v% Tween 20 have little effect on the protein melting start temperature, and the stability of the protein structure is similar.

[0113] Table 10 Appearance changes in accelerated and room temperature stability tests

[0114] Serial number Fresh configuration 40℃-2 weeks(w) 25℃-2 weeks(w) F1 C,CL,PF C,SO,PO++ C,CL,PF F2 C,CL,PF C,SO,PO++ C,CL,PF F3 C,CL,PF C, SO, PF C,CL,PF F4 C,CL,PF C,SO,PO++ C,CL,PF F5 C,CL,PF C,SO,PO++ C,CL,PF F6 C,CL,PF C,CL,PF C,CL,PF F7 C,CL,PF C,CL,PF C,CL,PF F8 C,CL,PF C,CL,PF C,CL,PF F9 C,CL,PF C,CL,PF C,CL,PF F10 C,CL,PF C,CL,PF C,CL,PF

[0115] Among them, C (colorless), CL (transparent), SO (slightly milky white), PF (no visible particles), PO (visible particles, ++ more than 5 visible particles were observed).

[0116] Table 11 Changes in concentration in accelerated and room temperature stability experiments

[0117] Serial number Fresh configuration 40℃-2 weeks(w) 25℃-2 weeks(w) F1 1.0 1.1 1.0 F2 1.0 1.1 1.0 F3 1.0 1.0 1.0 F4 1.0 1.1 1.0 F5 1.0 1.1 1.0 F6 1.0 1.0 1.0 F7 1.0 1.0 1.0 F8 1.0 1.0 1.0 F9 1.0 1.0 1.0 F10 1.0 1.0 1.0

[0118] The protein appearance (Table 10) and concentration (Table 11) show the aggregation and adsorption of the protein. The accelerated experiment was placed at 40°C for 2 weeks, and the room temperature stability experiment was placed at 25°C for 2 weeks. At pH 5.5, even with the use of surfactants and additives, the generation of opalescence can still be observed in the accelerated stability experiment, and under some conditions, the generation of visible particles is even observed. All show that the protein complex is prone to aggregation at pH 5.5. The addition of surfactants and additives failed to effectively avoid it. At pH 6.5, various combinations of surfactants and additives were used, and no opalescence and visible particles were observed, indicating that the long-term stability of the protein is good at pH 6.5.

[0119] Size exclusion chromatography (SEC) can reflect the aggregation and dissociation of IL-2 derivative protein complexes. Figure 6 In different combinations of surfactants and additives, after being placed at 40°C for 2 weeks and at 25°C for 2 weeks, the aggregates produced in the pH 5.5 buffer system were significantly more than those in the pH 6.5 buffer system. Especially in the F3 combination, 140mM arginine hydrochloride made the protein aggregation trend more obvious. On the contrary, in the F8 combination, in the pH 6.5 buffer system, 140mM arginine hydrochloride did not have a significant effect on the protein aggregate production and monomer ratio. This shows that the colloidal stability of the protein complex at pH 5.5 is poor and it is easy to form aggregates. Even with the use of surfactants and additives, it is still worse than under pH 6.5 conditions.

[0120] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A preparation for preventing aggregation of interleukin-2 derivative protein complexes and shedding of non-covalently linked protein subunits, characterized in that: The preparation consists of an interleukin-2 derivative at a concentration of 0.02 mg / mL, 20 mM phosphate buffer, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 6.5, and the sequence of the interleukin-2 derivative is as described in application number 201911302355.5.

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