Method for clarifying adenovirus harvest liquid

Abstract

The invention provides a method for clarifying an adenovirus harvest liquid, which comprises deep filtration, the deep filtration comprises salt injection treatment on a feed liquid: a positive salt ion solution is added into the virus harvest liquid subjected to pyrolysis and enzymolysis, and the final concentration of the positive salt ion solution is 200-600mmol / L. The deep filtering specifically comprises the following steps: assembling a deep filter and a pipeline: connecting the deep filter to a proper pipeline, and installing a pressure gauge or a pressure sensor at the liquid inlet end of the deep filter; the deep filter is washed with purified water and a Tris-HCl buffer solution; performing salt injection treatment on the feed liquid; filtering the feed liquid, and detecting that the pressure at the liquid inlet end of a deep filter does not exceed 1bar; and carrying out top washing by using a Tris-HCl buffer solution to obtain 1-2 deep filter cavity volumes. According to the method, the recovery rate of the deep filtering process can be increased to 80% or above, and the loading capacity of the deep filter is also increased to some extent.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical purification, and particularly relates to a method for clarifying adenovirus harvest fluid. Background Art

[0002] In the field of biopharmaceuticals, adenovirus, as an important vector tool, its recovery efficiency during the production process has always been one of the key factors restricting product quality and output. In traditional production processes, depth filtration is a commonly used step, which is mainly used to remove impurities in the feed liquid, thereby achieving preliminary purification of the target product. However, there is a significant limitation in the application of existing depth filtration technologies, that is, without increasing the salt concentration of the feed liquid, the recovery rate of adenovirus is extremely low, usually only reaching about 10%.

[0003] This low recovery rate not only causes a large amount of adenovirus to be lost during filtration, increasing production costs, but also may affect the subsequent process, thereby reducing the efficiency and economy of the entire production process. In addition, the low recovery rate may also lead to quality differences between product batches, posing challenges to quality control. The low loading capacity of depth filtration is also an urgent problem to be solved, which limits the processing capacity of filtration equipment, making it necessary to frequently replace the filtration medium in large-scale production, further increasing the operation complexity and production costs.

[0004] Therefore, improving the recovery rate of adenovirus in clarification filtration and the loading capacity of depth filtration has become an important research direction in the current field of biopharmaceuticals. Solving this problem can not only improve the production efficiency of adenovirus, reduce production costs, but also enhance the stability of product quality, which is of great significance for promoting the research and production of adenovirus-related biological products. Based on such a background, the present invention aims to provide a method that can effectively improve the recovery rate of adenovirus to overcome the deficiencies in the prior art and provide technical support for the efficient production and application of adenovirus. Summary of the Invention

[0005] Based on the above description, the present invention provides a method for clarifying adenovirus harvest fluid to solve the problem of low adenovirus recovery rate in related technologies.

[0006] The technical solution of the present invention for solving the above technical problems is as follows: A method for clarifying adenovirus harvest fluid includes depth filtration, and the depth filtration includes performing salt injection treatment on the feed liquid: adding a positive salt ion solution to the virus harvest fluid that has completed lysis and enzymatic hydrolysis, and the final concentration of the positive salt ion solution is 200 - 600 mmol / L, and the pH is 7.5 - 8.5.

[0007] On the basis of the above technical solution, the present invention can also be improved as follows.

[0008] Furthermore, the deep filtration specifically includes: Assembly of the deep filter and the pipeline: Connect the deep filter to a suitable pipeline, and install a pressure gauge or a pressure sensor at the liquid inlet end of the deep filter; Pretreatment of the deep filter: Flush the deep filter with purified water at a flow rate of 150 - 600 LMH for not less than 30 minutes, and then flush it with Tris-HCl buffer solution at a flow rate of 100 - 200 LMH for not less than 10 minutes; Perform salting treatment on the feed liquid; Filtration of the feed liquid: Filter the above-mentioned feed liquid at a flow rate of 100 - 200 LMH, and detect that the pressure at the liquid inlet end of the deep filter does not exceed 1 bar until the feed liquid filtration is completed; Backwashing of the residual feed liquid: Use Tris-HCl buffer solution to backwash out 1 - 2 volumes of the deep filter cavity at a flow rate of 100 - 200 LMH.

[0009] Furthermore, the formula of the Tris-HCl buffer solution is: 10000 mL of the buffer solution contains 146.1 - 350 g of NaCl, 24.2 g of Tris, 4.1 g of MgCl2, and 10.8 g of polysorbate 80.

[0010] Furthermore, the preparation method of the virus harvest fluid after lysis and enzymatic hydrolysis is as follows: Use Triton X-100 or polysorbate 80 and nuclease to perform lysis and enzymatic hydrolysis on the cells of the adenovirus harvest fluid in the Tris-HCl buffer solution system to release the virus, and obtain the sample after lysis and enzymatic hydrolysis; the sample after lysis and enzymatic hydrolysis is obtained by adding a positive salt ion solution for treatment.

[0011] Furthermore, the enzyme for enzymatic hydrolysis is nuclease; the conditions for lysis and enzymatic hydrolysis are: stir at 37 ± 1 °C and pH = 8.0 ± 0.5 for about 2.5 - 3.5 h; add the virus harvest fluid after lysis and enzymatic hydrolysis to the positive salt ions and mix well, and clarify and filter through a deep filter.

[0012] Furthermore, the process parameters of the deep filter are: the membrane area is 0.08 m 2 , the flow rate is 100 - 200 LMH, and the operating pressure < 1 bar.

[0013] Furthermore, after the deep filtration, it also includes ultrafiltration concentration, Q anion exchange chromatography, molecular sieve chromatography, and sterilizing filtration.

[0014] Furthermore, the process parameters of the ultrafiltration concentration are: the pore size of the membrane package is 300 KD, the process flow rate is 100 - 200 LMH, the transmembrane pressure is below 1 bar, the concentration is 10 - 20 times, and the diafiltration is 5 - 10 DV; And / or, the process parameters of the Q anion exchange chromatography are as follows: the column packing volume is 3000 - 4000 ml, the single sample loading amount: the total protein amount / packing volume ≤ 2.8 mg / ml; the flow rate: ≤ 160 cm / h; elution with an eluent; the ultraviolet absorption value A260nm / A280nm is between 1.2 and 1.4, and the target peak is collected.

[0015] Further, the process parameters of the molecular sieve chromatography are as follows: the sample loading amount ≤ 0.2 CV, the flow rate is 40 - 60 cm / h, and the first ultraviolet absorption peak is collected; And / or, the process parameters of the sterilizing filtration are as follows: 10% glycerol is added to the solution at the final volume concentration, shaken well and then filtered, and the membrane pore size is 0.2 μm.

[0016] Further, the adenovirus yield ≥ 80%, and the pressure at the end of filtration is < 1 bar.

[0017] Compared with the prior art, the technical solution of the present invention has the following beneficial technical effects: The present invention provides a method for clarifying an adenovirus harvest solution, which can improve the adenovirus yield and the loading capacity of depth filtration during clarification filtration. That is, through this method, the recovery rate of the depth filtration process can be increased to more than 80%, and the loading capacity of the depth filter is also increased to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a process flow chart of a method for clarifying an adenovirus harvest solution provided by an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] To facilitate the understanding of the present application, the present application will be described more comprehensively below. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the disclosure of the present application more thorough and comprehensive.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present application belongs. The terms used in the specification of the present application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0021] A method for clarifying an adenovirus harvest solution includes depth filtration, and the depth filtration includes subjecting the feed liquid to salt injection treatment: adding a positive salt ion solution to the virus harvest solution that has completed lysis and enzymatic hydrolysis, and the final concentration of the positive salt ion solution is 200 mmol / L - 600 mmol / L, and the pH is 7.5 - 8.5.

[0022] Based on the above technical solution, the present invention can also be improved as follows.

[0023] Further, for the above method for clarifying the adenovirus harvest fluid, the depth filtration includes: Assembly of the depth filter and the pipeline: Connect the depth filter to a suitable pipeline, and install a pressure gauge or a pressure sensor at the liquid inlet end of the depth filter; Pretreatment of the depth filter: Rinse the depth filter with purified water at a flow rate of 150 - 600 LMH for not less than 30 minutes, and then rinse it with Tris-HCl buffer at a flow rate of 100 - 200 LMH for not less than 10 minutes; Perform salt injection treatment on the feed liquid; Filtration of the feed liquid: Filter the above feed liquid at a flow rate of 100 - 200 LMH, and detect that the pressure at the liquid inlet end of the depth filter does not exceed 1 bar until the filtration of the feed liquid is completed; Backwash the residual feed liquid through the filter: Use Tris-HCl buffer to backwash out 1 - 2 volumes of the depth filter cavity at a flow rate of 100 - 200 LMH.

[0024] Further, for the above method for clarifying the adenovirus harvest fluid, the formula of the Tris-HCl buffer is: 10000 mL contains 146.1 - 350 g of NaCl, 24.2 g of Tris, 4.1 g of MgCl2, and 10.8 g of polysorbate 80.

[0025] Further, for the above method for clarifying the adenovirus harvest fluid, the preparation method of the virus harvest fluid after lysis and enzymatic digestion is: Use Triton X-100 or polysorbate 80 and nuclease to perform lysis and enzymatic digestion on the cells of the adenovirus harvest fluid in a Tris-HCl buffer system to release the virus, and obtain a sample after lysis and enzymatic digestion; Add a positive salt ion solution to the sample after lysis and enzymatic digestion to obtain the product.

[0026] Further, for the above method for clarifying the adenovirus harvest fluid, the enzyme for enzymatic digestion is nuclease; the conditions for lysis and enzymatic digestion are: Stir at 37 ± 1 °C and pH = 8.0 ± 0.5 for about 2.5 - 3.5 h; Mix the virus harvest fluid after lysis and enzymatic digestion with positive salt ions, and clarify and filter it through a depth filter.

[0027] Further, for the above method for clarifying the adenovirus harvest fluid, the process parameters of the depth filter are: the membrane area is 0.08 m 2 , the flow rate is 100 - 200 LMH, and the operating pressure < 1 bar.

[0028] Further, for the above method for clarifying the adenovirus harvest fluid, it further includes ultrafiltration concentration, Q anion exchange chromatography, molecular sieve chromatography, and sterilization filtration.

[0029] Further, for the method for clarifying the adenovirus harvest solution described above, the process parameters of ultrafiltration and concentration are as follows: the pore size of the membrane package is 300 KD, the process flow rate is 100 - 200 LMH, the transmembrane pressure is below 1 bar, the concentration is 10 - 20 times, and the diafiltration is 5 - 10 DV; The process parameters of the Q anion exchange chromatography are as follows: the column packing volume is 3000 - 4000 ml, the single sample loading amount: total protein amount / packing volume ≤ 2.8 mg / ml; the flow rate: ≤ 160 cm / h; elution with an eluent; the ultraviolet absorption value A260nm / A280nm is between 1.2 and 1.4, and the target peak is collected.

[0030] Further, for the method for clarifying the adenovirus harvest solution described above, the process parameters of the molecular sieve chromatography are as follows: the sample loading amount ≤ 0.2 CV, the flow rate is 40 - 60 cm / h, and the first ultraviolet absorption peak is collected; the process parameters of the sterilizing filtration are as follows: 10% glycerol is added to the solution at the final volume concentration, shaken well and then filtered, and the membrane pore size is 0.2 μm.

[0031] For the method for clarifying the adenovirus harvest solution described in any one of the above, the adenovirus recovery rate ≥ 80%, and the pressure at the end of filtration is 8 - 11 psi.

[0032] Example 1 Prepare Tris-HCl buffer: Prepare Tris-HCl buffer containing 350 mmol / L sodium chloride, and adjust the pH to 8.0 with HCl. Formula: 10000 mL contains 204.5 g NaCl, 24.2 g Tris, 4.1 g MgCl2, 10.8 g polysorbate 80.

[0033] Add Triton X-100 or polysorbate 80 to the virus harvest solution, and then add nuclease to the adenovirus harvest solution, and stir at 37°C and pH 8.0 for about 3 hours.

[0034] Add the lysed and enzymatically digested virus harvest solution to a sodium chloride solution to a final concentration of 300 mmol / L, mix well, and clarify and filter through a depth filter.

[0035] The specific process of depth filtration is as follows: Collect the filtered feed liquid ① Assembly of the depth filter and pipeline: Connect the depth filter to a suitable pipeline, and install a pressure gauge or pressure sensor at the liquid inlet end of the depth filter.

[0036] ② Pretreatment of the depth filter: Rinse the depth filter with purified water at a flow rate of 150 LMH for 30 minutes. Then rinse with the above Tris-HCl buffer at a flow rate of 150 LMH for 10 minutes.

[0037] ③Inject salt into the filtered liquid: Add 5 mol / L sodium chloride solution to the virus harvest solution after lysis and enzymatic hydrolysis until the final concentration of sodium chloride reaches 300 mmol / L.

[0038] ④Filter the liquid: Filter the above-mentioned liquid at a flow rate of 100 LMH, and detect that the pressure at the inlet end of the depth filter does not exceed 1 bar until the liquid filtration is completed.

[0039] ⑤Backwash the residual liquid through the filter: Use the Tris-HCl buffer solution in (1) above to backwash out 2 volumes of the depth filter cavity at a flow rate of 100 LMH.

[0040] Mix and sample the filtrates collected in steps ④ and ⑤ above, and detect the virus titer and the number of virus particles.

[0041] Table 1 shows the titer recovery rate and particle number recovery rate of depth filters from three different manufacturers without adding sodium chloride and with adding sodium chloride in the lysis solution. It can be seen from Table 1 that after adding sodium chloride to the lysis solution and then filtering, the titer recovery rate and particle number recovery rate of depth filters from three different manufacturers have been significantly improved.

[0042] Table 1 Titer recovery rate and particle number recovery rate of depth filters from three different manufacturers without adding sodium chloride and with adding sodium chloride in the lysis solution

[0043] Example 2 Prepare Tris-HCl buffer solution: Prepare Tris-HCl buffer solution containing 300 mmol / L potassium chloride, and adjust the pH to 8.0 with HCl. Formula: 10000 mL contains 223.7 g KCl, 24.2 g Tris, 4.1 g MgCl2, 10.8 g polysorbate 80.

[0044] Add Triton X-100 or polysorbate 80 to the virus harvest solution, and then add nuclease to the adenovirus harvest solution, and stir at 37 °C and pH 8.0 for about 3 hours.

[0045] Add the virus harvest solution after lysis and enzymatic hydrolysis to the potassium chloride solution until the final concentration is 250 mmol / L and mix well, and clarify and filter through a depth filter.

[0046] The specific process of depth filtration is as follows: Collect the filtered liquid ①Assemble the depth filter and pipeline: Connect the depth filter to the appropriate pipeline, and install a pressure gauge or pressure sensor at the inlet end of the depth filter.

[0047] ②Deep filter pretreatment: Use purified water to rinse the deep filter at a flow rate of 150 LMH for 30 minutes. Then use the above Tris-HCl buffer solution to rinse at a flow rate of 150 LMH for no less than 10 minutes.

[0048] ③Salt addition treatment for the filtered feed liquid: Add 3 mol / L potassium chloride solution to the virus harvest liquid after lysis and enzymatic hydrolysis until the final concentration of sodium chloride is 250 mmol / L.

[0049] ④Filtration of the feed liquid: Filter the above feed liquid at a flow rate of 100 LMH, and detect that the pressure at the inlet end of the deep filter does not exceed 1 bar until the feed liquid filtration is completed.

[0050] ⑤Top filtration of the residual feed liquid: Use the Tris-HCl buffer solution in (1) above to backwash out 2 volumes of the deep filter cavity at a flow rate of 100 LMH.

[0051] Mix and sample the filtrates collected in ④ and ⑤ above, and detect the virus titer and the number of virus particles.

[0052] Table 2 shows the pressure of the filtered feed liquid and the turbidity after clarification at different salt concentrations. It can be seen from the table that as the salt concentration increases, the pressure of the deep filter is lower when filtering the same volume of feed liquid.

[0053] Table 2 Pressure of the filtered feed liquid and turbidity after clarification at different salt concentrations

[0054] Example 3 On the basis of Example 1, this example, in addition to deep filtration, as Figure 1 shown, the following steps are also included: ultrafiltration concentration, Q anion exchange chromatography, molecular sieve chromatography, and sterile filtration.

[0055] Among them, the process parameters of ultrafiltration concentration are: the pore size of the membrane package is 300 KD, the process flow rate is 100 LMH - 200 LMH, the transmembrane pressure is below 1 bar, concentrated 10 - 20 times, and washed and filtered 5 - 10 DV.

[0056] The process parameters of Q anion exchange chromatography are: the column packing volume is 3000 - 4000 ml, the single sample loading amount: total protein amount / packing volume ≤ 2.8 mg / ml; the flow rate: ≤ 160 cm / h; eluted with the eluent; the ultraviolet absorption value A 260nm / A 280nm is between 1.2 and 1.4, and the target peak is collected. Among them, the components of the eluent (content per 10000 mL): 24.2 g Tris, 4.1 g MgCl2, 10.8 g polysorbate 80, 467.5 g sodium chloride.

[0057] The process parameters of molecular sieve chromatography are as follows: the sample loading amount is ≤ 0.2 CV, the flow rate is 40 - 60 cm / h, and the first ultraviolet absorption peak is collected. Among them, the components of the buffer solution used for the molecular sieve (content per 10,000 mL) are: 24.2 g of Tris, 4.1 g of MgCl₂, 10.0 g of poloxamer 188, and 233.7 g of sodium chloride.

[0058] The process parameters of sterile filtration are as follows: 10% glycerol is added to the solution by final volume concentration, and after shaking evenly, it is filtered through a membrane with a pore size of 0.2 μm.

[0059] The adenovirus recovery rate is ≥ 80%.

[0060] The corresponding effect verification is as follows: Table 3 Results of virus particles (HPLC method) in the whole purification process of different samples

[0061] Table 4 Results of virus titers in the whole purification process of different samples

[0062] It can be seen that the recovery rates of each process and the total recovery rate all have good effects.

[0063] Comparative verification regarding the ultrafiltration concentration process In the embodiment of the present invention, a membrane package is used, and hollow fibers are used as a comparative example. The comparison results are shown in Table 5: Table 5 Ultrafiltration concentration: Comparison between membrane package and hollow fiber

[0064] It can be seen from the comparison that the present application uses a membrane package, which has a higher flux and a shorter process time compared to hollow fibers.

[0065] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for clarifying an adenovirus harvest solution, characterized in that, Including depth filtration, which includes salting treatment of the feed liquid: adding a positive salt ion solution to the virus harvest liquid that has completed lysis and enzymatic hydrolysis, with the final concentration of the positive salt ion solution being 200 - 600 mmol / L and the pH being 7.5 - 8.

5.

2. The method for clarifying an adenovirus harvest solution according to claim 1, characterized in that, The depth filtration specifically includes: Assembly of the depth filter and pipeline: Connect the depth filter to a suitable pipeline, and install a pressure gauge or pressure sensor at the liquid inlet end of the depth filter; Pretreatment of the depth filter: Rinse the depth filter with purified water at a flow rate of 150 - 600 LMH for no less than 30 minutes, and then rinse it with Tris-HCl buffer solution at a flow rate of 100 - 200 LMH for no less than 10 minutes; Perform salting treatment on the feed liquid; Filtration of the feed liquid: Filter the above-mentioned feed liquid at a flow rate of 100 - 200 LMH, and detect that the pressure at the liquid inlet end of the depth filter does not exceed 1 bar until the feed liquid filtration is completed; Backwashing of the residual feed liquid: Use Tris-HCl buffer solution to backwash out 1 - 2 volumes of the depth filter cavity at a flow rate of 100 - 200 LMH.

3. The method for clarifying an adenovirus harvest solution according to claim 2, wherein, The formula of the Tris-HCl buffer solution is: 10000 mL of the buffer solution contains 146.1 - 350.0 g of NaCl, 24.2 g of Tris, 4.1 g of MgCl2, and 10.8 g of polysorbate 80.

4. The method for clarifying an adenovirus harvest solution according to claim 2, characterized in that, The preparation method of the virus harvest liquid that has completed lysis and enzymatic hydrolysis is: Use Triton X-100 or polysorbate 80 and nuclease to perform lysis and enzymatic hydrolysis treatment on the cells of the adenovirus harvest liquid in a Tris-HCl buffer solution system to release the virus, obtaining a sample after lysis and enzymatic hydrolysis treatment; the sample after lysis and enzymatic hydrolysis treatment is obtained by adding a positive salt ion solution for treatment.

5. The method for clarifying an adenovirus harvest solution according to claim 4, wherein, The enzyme for enzymatic hydrolysis is nuclease; the conditions for lysis and enzymatic hydrolysis are: Stir at 37 ± 1 °C and pH = 8.0 ± 0.5 for about 2.5 - 3.5 h; Mix the virus harvest liquid after lysis and enzymatic hydrolysis with a positive salt ion, and clarify and filter it through a depth filter.

6. The method for clarifying an adenovirus harvest solution according to claim 1, wherein The process parameters of the deep filter are as follows: the membrane area is 0.08 m 2 , the flow rate is 100 - 200 LMH, and the operating pressure is < 1 bar.

7. The method for clarifying an adenovirus harvest solution according to claim 1, wherein After the depth filtration, it also includes ultrafiltration concentration, Q anion exchange chromatography, molecular sieve chromatography, and sterilizing filtration.

8. The method for clarifying an adenovirus harvest solution according to claim 7, wherein, The process parameters of the ultrafiltration concentration are: the pore size of the membrane package is 300 KD, the process flow rate is 100 - 200 LMH, the transmembrane pressure is below 1 bar, concentrate 10 - 20 times, and wash and filter 5 - 10 DV; And / or, the process parameters of the Q anion exchange chromatography are: the column packing volume is 3000 - 4000 ml, the single sample loading amount: total protein amount / packing volume ≤ 2.8 mg / ml; the flow rate: ≤ 160 cm / h; elute with an eluent; the ultraviolet absorption value A260nm / A280nm is between 1.2 - 1.4, and collect the target peak.

9. The method for clarifying an adenovirus harvest solution according to claim 7, wherein, The process parameters of the molecular sieve chromatography are: the sample loading amount ≤ 0.2 CV, the flow rate is 40 - 60 cm / h, and collect the first ultraviolet absorption peak; And / or, the process parameters of the sterilizing filtration are: Add glycerol with a final volume concentration of 10% to the solution, shake well and then filter, and the membrane pore size is 0.2 μm.

10. The method for clarifying an adenovirus harvest solution according to any one of claims 1 to 9, characterized in that, The adenovirus recovery rate ≥ 80%, and the pressure at the end of filtration is < 1 bar.

Images