A method for viral inactivation in the purification of recombinant human interleukin 10 and its application

Abstract

The application discloses a virus inactivation method in a recombinant human interleukin 10 (IL-10) purification process and application thereof. The pH of a sample containing the recombinant human IL-10 is adjusted to 7.0-9.0, mixed with a virus inactivator, and subjected to inactivation treatment, and the sample after the inactivation treatment is subjected to chromatography treatment by using a weak cation exchange column; the virus inactivator contains triphosylbutane and Tween-80. The application designs a new virus inactivation method, does not affect the physical and chemical stability and biological activity of IL-10, and has a virus inactivation index greater than or equal to 5 log values, can achieve inactivation effect in 2h, shortens the inactivation time of the sample, can effectively remove the organic reagents TNBP and Tween-80, and has a residual amount lower than the international standard requirement, and can also remove IL-10 monomer impurities generated by hydrolysis of IL-10 in the purification process, and improve the purity of the IL-10 sample.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for virus inactivation during the purification process of recombinant human interleukin-10 and its application. Background Technology

[0002] Because biotechnology products, cell matrices, and some raw materials used in production (pancreatic enzymes, serum) all carry the risk of viral contamination, and because the genomes of cells such as CHO and SP2 / 0 contain retroviral particles, the viral safety process validation for biopharmaceuticals must consider not only the cell matrix source and raw materials, but also the virus removal / inactivation capabilities of the purification process. Generally, treatment steps that reduce the viral infectivity titer by ≥4 log are considered effective virus removal / inactivation processes.

[0003] Natural human interleukin-10 (hIL-10) is a non-covalently bound oligodimer with a molecular weight of approximately 35-40 kDa. It contains two disulfide bonds within the monomer to maintain its structure and biological activity. hIL-10 is secreted by Th2 cells, activated B cells, monocytes, macrophages, and other cells. It can inhibit the secretion of IL2 and IFN-γ by Th1 cells, acting as an anti-inflammatory cytokine. It downregulates inflammatory responses and antagonizes inflammatory mediators, participating in the biological regulation of various cells, including immune cells, inflammatory cells, and tumor cells. It plays a crucial role in various diseases, including autoimmune diseases, severe infectious diseases, tumors, and transplant immunology.

[0004] The preparation process of biological products such as hIL-10 requires virus inactivation. Currently, commonly used virus inactivation methods include: (1) low pH incubation, which uses continuous low pH to cause certain viral components to deteriorate, thereby affecting viral replication and ultimately causing the virus to lose its infectivity. It is often used to inactivate lipid-enveloped viruses, but it is ineffective against non-lipid-enveloped viruses. However, since hIL-10 is unstable to acid, it is easy to form monomers under acidic conditions, which reduces its stability. Therefore, it is not suitable for low pH incubation to inactivate viruses in the production process; (2) organic reagent / detergent (S / D) treatment, that is, the mixture of organic solvent and detergent can destroy the lipid membrane of lipid-enveloped viruses, thereby causing the lipid to detach from the surface of the virus and causing the virus to lose its ability to adhere to and infect cells. However, organic reagents and detergents have an impact on the stability and activity of the sample. In addition, the removal of residual organic reagents and detergents also needs to be considered. This method requires examining the impact of organic reagents and detergents on the physicochemical stability and biological activity of IL-10, and secondly, solving the problem of residual removal of organic reagents and detergents.

[0005] In summary, there are few reported methods for effectively inactivating the virus during the preparation of hIL-10. Developing effective methods for inactivating the virus during the preparation of hIL-10 is of great significance for the production and application of hIL-10. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a method for virus inactivation during the purification process of recombinant human interleukin-10 and its application. This method achieves efficient virus inactivation and effective removal of inactivating agents while ensuring the high activity and purity of recombinant human interleukin-10.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for virus inactivation during the purification process of recombinant human interleukin-10, the virus inactivation method comprising:

[0009] The pH of the sample containing recombinant human interleukin-10 was adjusted to 7.0–9.0, mixed with a virus inactivating agent, and inactivated. The inactivated sample was then subjected to chromatography using a weak cation exchange column. The virus inactivating agent contained butyl triphosphate and Tween-80.

[0010] This invention designs a method for virus inactivation during the preparation of recombinant human interleukin-10 (hIL-10), including designing and controlling the pH of the inactivated sample to effectively maintain the purity and activity of hIL-10; designing a specific inactivating agent formulation to efficiently inactivate the virus while avoiding damage to the purity and activity of hIL-10; and coordinating the overall process to achieve efficient virus inactivation and removal of the inactivating agent while ensuring high activity and purity of recombinant human interleukin-10, and further removing IL-10 monomeric impurities.

[0011] Preferably, the mass percentage of butyl triphosphate in the virus inactivator is 0.1% to 5%, including but not limited to 0.2%, 0.3%, 0.5%, 1%, 2%, 3% or 4%, etc.

[0012] Preferably, the mass percentage of Tween-80 in the virus inactivating agent is 0.1% to 5%, including but not limited to 0.2%, 0.3%, 0.5%, 1%, 2%, 3% or 4%, etc.

[0013] Preferably, the packing material for the chromatography process comprises a weak cation exchange packing material.

[0014] This invention designs a specific inactivator removal method, which uses weak cation exchange packing for chromatography. This method can not only efficiently remove the inactivator, but also effectively remove IL-10 monomer impurities generated by hydrolysis in the hIL-10 protein solution, thereby increasing the purity of the IL-10 sample to more than 95%.

[0015] Preferably, the inactivation treatment is performed at a temperature of 22–26°C for a time of not less than 2 hours.

[0016] In this invention, by controlling specific inactivation treatment conditions, viruses can be efficiently inactivated, reducing viral infectivity titers by ≥4 log while maintaining the purity and high activity of human interleukin-10.

[0017] Preferably, the ligand of the weak cation exchange packing includes carboxymethyl.

[0018] Preferably, the sample loading conditions for the chromatography process are: pH 6.5–6.8 (e.g., 6.6 or 6.7), and conductivity 3–5 mS / cm.

[0019] In this invention, specific loading conditions are designed to allow interleukin-10 to bind to the ion exchange column, while the inactivating agents, including butyl triphosphate and Tween-80, do not bind to the ion exchange column and flow through, which is beneficial for the removal of organic reagents.

[0020] Preferably, the equilibration buffer for the chromatography process includes any one of PB buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, disodium hydrogen phosphate-citrate buffer, or Bis-Tris buffer.

[0021] Preferably, the concentration of the PB buffer solution is 10-50 mM, including but not limited to 11 mM, 12 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 46 mM, 47 mM or 48 mM, etc., with 25 mM being the most preferred.

[0022] In this invention, a specific concentration of equilibrium buffer solution is used, which can not only meet the buffering capacity between pH 6.6 and 6.8, but also increase the adsorption capacity of the packing material for interleukin-10, increase the loading capacity of the packing material, and save economic costs.

[0023] Preferably, the pH of the PB buffer solution is 6.6 to 6.8.

[0024] Preferably, the elution buffer for the chromatography process includes any one of PB buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, disodium hydrogen phosphate-citrate buffer, or Bis-Tris buffer.

[0025] Preferably, the concentration of the PB buffer solution is 10-50 mM, including but not limited to 11 mM, 12 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 46 mM, 47 mM or 48 mM, etc.

[0026] Preferably, the PB buffer solution further contains NaCl, and the solution conductivity is 85-92 mS / cm, including but not limited to 86 mS / cm, 87 mS / cm, 88 mS / cm, 89 mS / cm, 90 mS / cm or 91 mS / cm.

[0027] Preferably, the pH of the PB buffer solution is 6.6 to 6.8.

[0028] Preferably, the elution conditions for the chromatography process involve eluting with 3-5% elution buffer at a volume percentage of ≥15 CV, more preferably 3% CV.

[0029] Preferably, the elution conditions for the chromatography process involve eluting with 15-25% elution buffer (volume percentage) for ≥5 CV, more preferably 20% vol, and adjusting the pH of the eluted sample to 7.2-7.4.

[0030] In this invention, specific washing and elution conditions are designed to efficiently remove inactivating agents and impurities from the sample, ensuring high purity and high recovery rate of the sample.

[0031] As a preferred technical solution, the method for virus inactivation during the purification process of recombinant human interleukin-10 includes:

[0032] The pH of the sample containing recombinant human interleukin-10 was adjusted to 7.0–9.0, and it was mixed with a virus inactivating agent containing butyl triphosphate and Tween-80. The inactivation was carried out at 22–26°C for no less than 4 hours. The inactivated sample was then subjected to chromatography using a weak cation exchange column.

[0033] The chromatography process includes pre-equilibration with elution buffer, equilibration with equilibration buffer, adjusting the sample pH to 6.5–6.8 and conductivity to 3–5 mS / cm, loading the sample, rinsing with equilibration buffer, eluting ≥15 CV with 3–5% elution buffer, washing away impurities, eluting ≥5 CV with 15–25% elution buffer, and adjusting the pH of the eluted sample to 7.2–7.4.

[0034] In a second aspect, the present invention provides the application of the virus inactivation method described in the first aspect during the purification process of recombinant human interleukin-10 in the preparation of recombinant human interleukin-10.

[0035] Thirdly, the present invention provides a method for preparing recombinant human interleukin-10, wherein the preparation method employs the virus inactivation method described in the first aspect during the purification process of recombinant human interleukin-10 to inactivate the virus in the sample containing recombinant human interleukin-10, thereby obtaining the product.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] This invention addresses the need for virus inactivation in the preparation of recombinant human interleukin-10 products by designing a novel virus inactivation method. This method controls the sample pH range to 7.5–9.0 and ensures that specific inactivating agents do not affect the physicochemical stability and biological activity of IL-10. The virus inactivation index is greater than or equal to 5log, and inactivation can be achieved in 2 hours, shortening the sample inactivation time. The use of a weak cation exchange column effectively removes organic reagents TNBP and Tween-80, with residual amounts below international standards. It also removes IL-10 monomer impurities generated during the purification process, improving the purity of the IL-10 sample. Attached Figure Description

[0038] Figure 1 Images showing the results of SDS-PAGE with and without reduction;

[0039] Figure 2 This is the SEC-HPLC chromatogram of sample 1 in Example 2;

[0040] Figure 3 This is the SEC-HPLC chromatogram of sample 2 in Example 2;

[0041] Figure 4 This is the SEC-HPLC chromatogram of sample 3 in Example 2;

[0042] Figure 5 The biomechanical curve of PRV virus inactivation;

[0043] Figure 6 The biomechanical curve for the inactivation of Sindbis virus;

[0044] Figure 7 The image shows the results of reduced SDS-PAGE chromatography using 25 mM PB equilibration buffer.

[0045] Figure 8 The image shows the reduced SDS-PAGE results of weak cation exchange chromatography using 50 mM PB equilibration buffer.

[0046] Figure 9 The image shows the reduced SDS-PAGE results of weak cation exchange chromatography using washing / elution conditions (3% elution buffer / 20% elution buffer; 5% elution buffer / 20% elution buffer);

[0047] Figure 10 The image shows the reduced SDS-PAGE results of weak cation exchange chromatography using washing / elution conditions (12% elution buffer / 20% elution buffer; 10% elution buffer / 20% elution buffer).

[0048] Figure 11The image shows the results of reduced SDS-PAGE using weak cation exchange chromatography with washing / elution conditions (3% elution buffer / 50% elution buffer). Detailed Implementation

[0049] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0050] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0051] Definitions of abbreviations and key terms in this invention.

[0052] 1. S / D Virus Inactivation Technology: (solvent / detergent, organic reagent / cleaning agent) This technology uses a mixture of organic solvents and cleaning agents to destroy the lipid envelope of viruses, thus inactivating enveloped viruses, but it has no effect on non-enveloped viruses. It can also be used for plasma, blood products, and biological products.

[0053] 2. Indicator virus: refers to an infectious live virus used in virus removal / inactivation process validation studies to demonstrate the effectiveness of the process treatment.

[0054] 3. Virus inactivation: A process designed to "kill" viruses in order to enhance their safety.

[0055] 4. TNBP: Butyl triphosphate, an organic reagent used for virus inactivation.

[0056] 5. Tween-80: Tween-80 is a cleaning agent used for virus inactivation.

[0057] 6. SDS-PAGE: Polyacrylamide gel electrophoresis, which uses the difference in molecular weight of proteins to separate samples.

[0058] 7. SEC-HPLC: Size exclusion high performance liquid chromatography.

[0059] 8. ED50: Half-maximal effective concentration, used to characterize the biological activity of IL-10. The higher this value, the lower the sample activity; the lower this value, the higher the sample biological activity.

[0060] Example 1

[0061] This embodiment designs a stable pH for the hIL-10 protein solution.

[0062] 1. Sample preparation: Take about 135 mL of solution containing hIL-10 protein, divide the sample into two parts, add hydrochloric acid dropwise to one part and sodium hydroxide dropwise to the other part, adjust the pH of the sample as shown in Table 1 below, and then place it in a shaker at 24℃ for 4 h, take a sample to test its purity and biological activity, and then let it stand for another 6 h before taking a sample to test its purity.

[0063] 2. Sample purity was determined using the SEC-HPLC method: The HPLC system used was a Waters 2695 separation unit, a 2487 UV detector, and Empower2 software; the chromatographic column was a TOSOH TSKgel G3000 SWXL (5µm, 7.8mm ID*30cm); the mobile phase was 250mM phosphate buffer, pH 7.0, room temperature, flow rate 0.8mL / min, injection volume 20µL, and detection wavelength 214nm. Empower2 software was used to analyze the chromatograms and calculate relevant data. The SEC-HPLC purity results are shown in Table 1.

[0064] 3. Bioactivity assay method: Dilute the sample concentration to 5 μg / mL, then sequentially dilute 1, 2, 4, 8, 16, 32, 64, and 128 times, for a total of 8 dilution gradients; MC / 9 cells were diluted with fresh complete culture medium to a concentration of 2.0 × 10⁻⁶. 4 Cells were plated at 1000 mL / mL, and after 72 h of culture, IL-4 and diluted sample were added to each well. Cell count was measured after 24 ± 4 h of culture. Data were then processed using GraphPad Prism 6 software, with cell count as the ordinate and sample dilution concentration as the abscissa to obtain an activity curve. The ED50 value of the fitted curve is the activity curve of IL-10.

[0065] Table 1. Sample pH and purity results after 4 hours.

[0066]

[0067] As can be seen from the purity test results in Table 1, different pH values ​​have a significant impact on the purity and bioactivity of IL-10 samples. The samples with pH values ​​between 7.5 and 9.0 have a purity greater than 95% and maintain stable bioactivity.

[0068] Example 2

[0069] This embodiment designs an S / D treatment method and analyzes its effects on the physicochemical stability and biological activity of IL-10.

[0070] 1. Sample preparation: Adjust the pH of the sample to 8.5 with a protein solution containing IL-10; take out 30 mL of the solution as a virus-inactivated sample.

[0071] 2. Virus inactivation reagent: Prepare an inactivation reagent containing 3% TNBP and 10% Tween 80 (w / w); prepare an inactivation reagent containing 3% TNBP and 10% Triton X-100 (w / w).

[0072] The 30mL virus-inactivated sample was divided into three 9.45mL portions, and the remaining sample was used for quality testing.

[0073] Virus-inactivated sample preparation is as follows:

[0074] Sample 1: 9.45 mL of virus-inactivated sample + 1.05 mL of water (control)

[0075] Sample 2: 9.45 mL of virus-inactivated sample + 1.05 mL (3% TNBP and 10% Tween 80)

[0076] Sample 3: 9.45 mL of virus-inactivated sample + 1.05 mL (3% TNBP and 10% Triton X-100)

[0077] After being placed in a constant temperature incubator at 24℃ for 4 hours, samples were taken for testing by SDS-PAGE, SEC-HPLC, and biological activity.

[0078] 3. Reducing and Non-reducing SDS-PAGE Detection: 1) Reducing Sample Preparation: Add 4× reducing sample buffer (containing mercaptoethanol) to the sample to be tested, mix well, heat, and prepare for sample loading; Non-reducing Sample Preparation: Add 4× non-reducing sample buffer (without reducing agent) to the sample to be tested, mix well, do not heat, and prepare for sample loading, and prepare protein molecular weight standards; 2) Add 20 μL of the prepared sample to the prepared gel (4.5% stacking gel, 12% separating gel), start with 80V voltage until the sample enters the separating gel, then adjust the voltage to 120V, and stop electrophoresis when the bromophenol blue dye in the sample is 1 cm from the bottom; 3) Staining and Destaining: Stain with Coomassie Brilliant Blue R-250 for 1 h, and destain with a destaining solution containing 10% acetic acid, 10% ethanol, and 70% water until the protein bands are clearly visible.

[0079] 4. The SEC-HPLC method is as described in Example 1.

[0080] 5. The biological detection method is the same as in Example 1.

[0081] By restoring and not restoring SDS-PAGE ( Figure 1The results show that the reduced and non-reduced SDS-PAGE of sample 2 are consistent with that of sample 1, with no aggregation or hydrolysis. However, compared to sample 1, sample 3 shows a significantly narrower and lighter main band, indicating possible hydrolysis. Subsequent SEC-HPLC results (Table 2) show that the purity of sample 3 decreased by 10% after treatment with TNBP and Triton X-100. Impurity peaks appeared after the main IL-10 peak, indicating hydrolysis and significantly reducing IL-10 purity, which is detrimental to the physicochemical stability of IL-10. Finally, the bioactivity results (Table 2) also show that the ED50 value increased after treatment with TNBP and Triton X-100, indicating a significant decrease in bioactivity. This suggests that the TNBP and Triton X-100 formulation is unsuitable for the IL-10 virus inactivation process, and TNBP and Tween-80 were chosen as the formulation for the virus inactivation process.

[0082] Table 2. SEC-HPLC purity and bioactivity

[0083]

[0084] Example 3

[0085] This embodiment verifies the virus inactivation treatment method.

[0086] 1. Sample preparation: Take out 30 mL of the IL-10 protein solution, adjust the pH to 8.5, and use it as a virus-inactivated sample.

[0087] 2. Virus inactivation:

[0088] Experimental group: Prepare inactivation reagent containing 3% TNBP and 10% Tween 80 (w / w); add 1.05 mL of inactivation reagent to 9.45 mL of protein solution, incubate at 24℃ for 6 h, and inoculate with 10% (V / V) indicator virus (PRV, Sinbdis), mix well, and take samples at 0 min, 15 min, 30 min, 1 h, 2 h, 4 h and 6 h. Immediately after sampling, dilute 100 times with serum-free medium to stop the reaction and test immediately.

[0089] Positive control: Take the same batch of virus (PRV, Sindbis), thaw it at the beginning of the experimental group's experiment, and test it immediately.

[0090] The formula for calculating the reduction in logarithmic viral load is as follows:

[0091] LRV = log 10 (V1×T1) / (V2×T2)

[0092] LRV: Virus reduction expressed as a log value; V1: Volume of the sample before treatment; T1: Virus concentration in the sample before treatment; V2: Volume of the sample after treatment; T2: Virus concentration in the sample after treatment.

[0093] The inactivated virus kinetic curves and validation results are shown in Table 3.

[0094] Table 3 Virus inactivation biomechanics curves

[0095] Virus Dynamic curve LRV(log) PRV Figure 5 ≥5.04 Sinbdis Figure 6 ≥5.45

[0096] Verification showed that after treatment with 3% TNBP and 1% Tween-80, the inactivation index of the two added indicator viruses was greater than or equal to 5log. The inactivation kinetic curves also showed that PRV virus achieved inactivation at 15 min and remained stable; Sinbdis also achieved inactivation at 2 h and remained stable, which can also shorten the sample inactivation time. This indicates that the inactivation reagents of 0.3% TNBP and 1% Tween-80 (w / w) can effectively inactivate the virus in IL-10 protein.

[0097] Example 4

[0098] This embodiment designs a removal method for TNBP and Tween-80, and selects the type of packing material used (strong cation exchange packing material, weak cation exchange packing material, and composite strong anion exchange packing material).

[0099] The sample preparation and corresponding filler types are shown in Table 4.

[0100] Table 4

[0101]

[0102] Chromatography column type: 16mm × 250mm; packing height H = 5cm;

[0103] Buffer type: For weak cation and strong cation exchange packings, the equilibration buffer is 25mM PB, pH 6.7±0.1, and the elution buffer is 50mM PB, 1M NaCl, pH 6.7±0.1; for composite strong anion exchange packings, the equilibration buffer is 50mM Tris-HCl, pH 8.5±0.1, and the elution buffer is 50mM Tris-HCl, 1M NaCl, pH 8.5±0.1.

[0104] The chromatography process is shown in Table 5.

[0105] Table 5

[0106]

[0107] The purity of the samples was determined using the SEC-HPLC method, as described in Example 1.

[0108] Tween-80 and TNBP residue detection: The method is the same as the Tween-80 and TNBP residue detection method in the 2020 edition of the Pharmacopoeia.

[0109] The results are shown in Table 6. The data on the residual contents of TNBP and Tween-80 show that all three ion exchange columns effectively removed TNBP and Tween-80, with significant removal effects that meet international and domestic standards. However, the SEC-HPLC results indicate that the weak cation exchange column effectively removed IL-10 monomeric impurities generated by hydrolysis from the IL-10 protein solution, increasing the purity of the IL-10 sample by more than 95%. Therefore, the weak cation exchange column was selected as the packing material for removing TNBP and Tween-80.

[0110] Table 6. Purity Results of TNBP / Tween-80 / SEC-HPLC

[0111]

[0112]

[0113] Further design of weak cation exchange chromatography conditions and optimization of the equilibration buffer for the weak cation exchange column were conducted: The sample processing method, column type, and chromatography conditions were consistent with those for the weak cation exchange column in the packing material screening, except for the concentration of the equilibration buffer. The equilibration buffer was set at 10 mM PB, 25 mM PB, and 50 mM PB. The SDS-PAGE detection method was performed according to Example 2.

[0114] The results at 25mM PB and 50mM PB equilibration buffer concentrations are shown in Table 7. (From the SDS-PAGE plot...) Figure 7 and Figure 8 Lanes 1 and 2 are for sample flow-through, lane 3 is for 3% washing, and lane 4 is for 20% elution. It can be seen that as the concentration of the buffer increases to 50 mM, the target component in the sample flow-through increases slightly, resulting in a smaller packing material loading. 10 mM PB has a weaker buffering capacity. Although both can achieve the purpose of adsorbing the target component with the packing material, from the perspective of economy and buffering capacity, 25 mM PB is preferred as the equilibrium buffer.

[0115] Table 7 Optimization results for different equilibrium buffers

[0116] Experiment number Balanced buffer Restored SDS-PAGE results 1 25 mMPB, pH 6.7 ± 0.1 Figure 7 2 50 mMPB, pH 6.7 ± 0.1 Figure 8

[0117] Optimization of washing / elution conditions for weak cation exchange columns: Sample preparation method, column type, and chromatographic conditions, except for the washing gradient, are consistent with the conditions for weak cation exchange columns in the packing material screening; the experimental washing / elution conditions are set to 3% elution buffer, 5% elution buffer, 10% elution buffer, and 12% elution buffer; the SDS-PAGE detection method is as described in Example 2.

[0118] The results are shown in Table 8, based on the SDS-PAGE plot ( Figure 9 Lane 1 was washed with 3% impurities, lane 2 with 20% elution, lanes 3 and 4 with 5% impurities, and lanes 5 and 6 with 20% elution. Figure 10 Lanes 1, 2, 3, and 4 were washed with 10% impurities; lane 5 was washed with 20% impurities; and lanes 6, 7, and 8 were washed with 12% impurities. Figure 11 (Lane 1 and 2 are eluted with 3% elution, and lanes 3 and 4 are eluted with 50% elution.) It can be seen that the target component is eluted in elution gradients of 5%, 10%, and 12%, and the proportion of the target component eluted increases with the increase of the volume ratio, which greatly reduces the recovery rate of the target component. Therefore, according to the SDS-PAGE results, elution gradients of 3% and 5% are both acceptable, with 3% elution gradient being preferred, as it can ensure both high protein recovery rate and high protein purity. The target component can be eluted in large quantities at a elution gradient greater than 12%, but it is not completely eluted. When the elution gradient is 20%, the target component is completely eluted; however, as the elution gradient increases, some other proteins will also be eluted. Considering all factors, 20% elution gradient is preferred, as it can ensure both high protein recovery rate and high protein purity.

[0119] Table 8. Experimental Results of Different Washing / Elution Gradients

[0120]

[0121] This invention addresses the need for virus inactivation in the preparation of recombinant human interleukin-10 products by designing a novel virus inactivation method. This method controls the sample pH range to 7.5–9.0 and ensures that specific inactivating agents do not affect the physicochemical stability and biological activity of IL-10. The virus inactivation index is greater than or equal to 5log, and inactivation can be achieved in 2 hours, shortening the sample inactivation time. The use of a weak cation exchange column effectively removes organic reagents TNBP and Tween-80, with residual amounts below international standards. It also removes IL-10 monomer impurities generated during the purification process, improving the purity of the IL-10 sample.

[0122] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for virus inactivation during the purification process of recombinant human interleukin-10, characterized in that, The virus inactivation method includes: The pH of the sample containing recombinant human interleukin-10 was adjusted to 8.0-8.5, and it was mixed with a virus inactivating agent containing butyl triphosphate and Tween-80. The inactivation treatment was carried out at 22-26℃ for no less than 2 h. The inactivated sample was then subjected to chromatography using UniGel-30CM weak cation exchange packing material. The mass percentage of butyl triphosphate in the virus inactivating agent is 0.1% to 5%. The mass percentage of Tween-80 in the virus inactivating agent is 0.1% to 5%; The loading conditions for the chromatography process are: pH 6.5~6.8, conductivity 3~5 mS / cm; The purity of the inactivated recombinant human interleukin-10 virus is greater than 97%.

2. The method for virus inactivation during the purification process of recombinant human interleukin-10 according to claim 1, characterized in that, The equilibration buffer for the chromatography process includes any one of PB buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, disodium hydrogen phosphate-citrate buffer, or Bis-Tris buffer.

3. The method for virus inactivation during the purification process of recombinant human interleukin-10 according to claim 2, characterized in that, The equilibration buffer is PB buffer.

4. The method for virus inactivation during the purification process of recombinant human interleukin-10 according to claim 3, characterized in that, The concentration of the PB buffer solution is 10~50mM.

5. The method for virus inactivation during the purification process of recombinant human interleukin-10 according to claim 3, characterized in that, The pH of the PB buffer solution is 6.6~6.

8.

6. The method for virus inactivation during the purification process of recombinant human interleukin-10 according to claim 1, characterized in that, The elution buffer for the chromatography process includes any one of PB buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, disodium hydrogen phosphate-citrate buffer, or Bis-Tris buffer.

7. The method for virus inactivation during the purification process of recombinant human interleukin-10 according to claim 6, characterized in that, The elution buffer is PB buffer.

8. The method for virus inactivation during the purification process of recombinant human interleukin-10 according to claim 7, characterized in that, The concentration of the PB buffer solution is 10~50mM.

9. The method for virus inactivation during the purification process of recombinant human interleukin-10 according to claim 7, characterized in that, The PB buffer solution also contains NaCl, and the solution conductivity is 85~92 mS / cm.

10. The method for virus inactivation during the purification process of recombinant human interleukin-10 according to claim 7, characterized in that, The pH of the PB buffer solution is 6.6~6.

8.

11. The method for virus inactivation during the purification process of recombinant human interleukin-10 according to claim 1, characterized in that, The elution conditions for the chromatography process are elution with 3-5% elution buffer at a volume percentage of ≥15 CV.

12. The method for virus inactivation during the purification process of recombinant human interleukin-10 according to claim 1, characterized in that, The elution conditions for the chromatography process are elution with 15-25% elution buffer for ≥5 CV, and the pH of the eluted sample is adjusted to 7.2-7.

4.

13. The application of the virus inactivation method in the purification process of recombinant human interleukin-10 according to any one of claims 1-12 in the preparation of recombinant human interleukin-10.

14. A method for preparing recombinant human interleukin-10, characterized in that, The method described herein employs the virus inactivation method described in any one of claims 1-12 during the purification process of recombinant human interleukin-10 to inactivate the virus in the sample containing recombinant human interleukin-10, thereby obtaining the final product.

Images