A method for removing pyrogen and virus of chymotrypsin

Abstract

The present invention relates to a method for removing heat source and virus of chymotrypsin, comprising the following steps: S1 raw material preparation; S2 dissolution of crude cattle chymotrypsin; S3 liquid replacement; S4 column loading; S5 pre-equilibrium; S6 sample loading; S7 post-equilibrium; S8 cleaning and regeneration. The present invention ensures the safety of chymotrypsin products and reduces potential infection risks by removing heat source and virus. The delicate purification step helps to remove impurities and improve the purity and activity of chymotrypsin. The use of automated equipment and precise chemical treatment steps can improve production efficiency and reduce manual operation errors.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for removing heat sources and viruses of chymotrypsin. Background Art

[0002] Chymotrypsin is a protease isolated and purified from bovine or porcine pancreas. It has two main pharmacological and clinical functions: 1. Anti-inflammatory: It has good effects on various inflammations, inflammatory edema, hematoma, ulcers and thrombosis. 2. Anti-cancer: It can promote the penetration of anti-cancer drugs into the lesions and promote the effect of chemotherapy drugs. It has been reported that the efficacy of anti-cancer enhancement is more than 50%, and the effect is stronger when used locally in large doses. Large doses can treat a variety of tumors, such as breast cancer, cervical cancer, gastric cancer, etc., without any toxic side effects or adverse reactions.

[0003] After searching the existing patent literature, the chromatography medium used in the Chinese patent CN106591268A, a method for preparing chymotrypsin capable of removing heat sources and viruses, is already very outdated, namely, DEAE-cellulose medium. The medium is prone to swelling during the production process and has a low DEAE loading capacity. The processing capacity per unit quantity is lower than that of the strong anion chromatography medium Q.

[0004] Therefore, technicians in this field are committed to developing a more economical and efficient chromatography method to remove pyrogens and viruses. Summary of the invention

[0005] (I) Purpose of the invention

[0006] In order to overcome the above shortcomings, the purpose of the present invention is to provide a method for removing heat sources and viruses of chymotrypsin to solve the above technical problems.

[0007] A method for removing pyrogen and virus of chymotrypsin comprises the following steps:

[0008] S1 raw material preparation, weigh the crude minced beef source, add 8 times the amount of 0.01mol / L acetic acid solution to dissolve; S2 crude beef minced beef dissolution, add 20mol / L phosphate buffer to the crude beef minced beef extracted from S1 to dissolve;

[0009] S3: Change the liquid, use the membrane package ultrafiltration system to continuously add water to ultrafilter the dissolved beef chyme, and collect the intercepted liquid;

[0010] S4 column packing: Use an automatic packing machine to evenly fill 100 ml of MaXtar Q filler into the column, and wash it with purified water three times to replace the original ethanol solution;

[0011] S5 pre-equilibration, using phosphate buffer to pre-equilibrate the chromatography column;

[0012] S6 was loaded, and the ultrafiltrate was passed through the column at a linear flow rate of 180-200 cm / h, and the flow-through was collected;

[0013] After S7, balance, use 3-5B volumes of pH 6.5 phosphate buffer for top washing, and collect the feed liquid;

[0014] S8 is cleaned and regenerated by washing with 2 to 3B volumes of 1M salt, and then washing the column with 2 to 3B volumes of 0.5M alkali.

[0015] Preferably, S2 specifically includes:

[0016] The pH value of 20 mol / L phosphate buffer was adjusted to 7.3-7.5 to simulate physiological conditions. A certain amount of crude beef mince was weighed and added to the phosphate buffer with adjusted pH value in batches. The constant temperature stirrer was turned on to fully mix and dissolve the beef mince.

[0017] Preferably, S3 specifically includes:

[0018] Replace the membrane package ultrafiltration system with a 10kDa ultrafiltration membrane, pre-wet the ultrafiltration membrane with an appropriate amount of buffer, set the pressure in front of the membrane to 0.1MPaM, slowly add the dissolved crude bovine chyme to the ultrafiltration membrane, start the pump to allow the crude bovine chyme to pass through the ultrafiltration membrane, monitor the conductivity of the retentate in real time, and collect the retentate when the conductivity is less than 2ms / cm.

[0019] Preferably, the S4 specifically includes:

[0020] Use an automatic filling machine to evenly fill 100 ml of MaXtar Q filler into the chromatography column to form a stable filler bed. Start the fluid system of the chromatography column and replace the original ethanol solution with purified water at a flow rate of 1-2 BV. Repeat the washing of the filler with purified water for 3 times, using 200 ml of purified water each time, and observe the flow-through until the ethanol is completely washed out.

[0021] Preferably, S5 specifically includes:

[0022] Connect the chromatography column correctly to the fluid system, start the pump, pump pH 6.5 sulfate buffer into the chromatography column, adjust the flow rate, ensure that 5-6 column volumes of buffer are used for balancing, collect the flow-through and test, and stop balancing when the pH value of the flow-through is in the range of 6.7 to 6.9.

[0023] Preferably, S6 specifically includes:

[0024] Adjust the flow rate of the pump so that the ultrafiltrate passes through the chromatography column at a linear flow rate of 180-200 cm / h. Slowly add the ultrafiltrate in S3 to the top of the chromatography column using a syringe or an automatic sampler. Observe the flow of the sample in the chromatography column to ensure that the sample passes through the packing bed evenly. Start the collection system to collect the flow-through from the chromatography column.

[0025] Preferably, S7 specifically includes:

[0026] According to the volume of the chromatography column, 3-5 volumes of pH 6.5 phosphate buffer are pumped into the chromatography column at an appropriate flow rate to elute the analysis that interacts weakly with the filler, and the feed liquid flowing out of the chromatography column is collected.

[0027] Preferably, S8 specifically includes:

[0028] Prepare 2 to 3 volumes of 1M salt solution and 0.5M alkaline solution according to the volume of the chromatography column, start the pump, and pass the 1M salt solution through the chromatography column to remove charged pollutants with high ionic strength. When the waste liquid becomes lighter in color and more transparent, replace it with 0.5M alkaline solution and continue flushing. Monitor the pH value to ensure that the alkaline solution neutralizes the residue and completes the cleaning.

[0029] Preferably, an algorithm is used to determine the volume of purified water required to ensure complete replacement of the ethanol:

[0030]

[0031] Among them, V 置换 is the volume of purified water required, V 柱 is the column volume, n is the number of washes, C 初始 and C 目标 are the initial and target ethanol concentrations, respectively.

[0032] (II) Technical solution

[0033] To achieve the above objectives, the technical solutions provided by this application are as follows:

[0034] Beneficial effects:

[0035] The present invention ensures the safety of chymotrypsin products and reduces potential infection risks by removing heat sources and viruses. Fine purification steps help remove impurities and improve the purity and activity of chymotrypsin. Using automated equipment and precise chemical treatment steps can improve production efficiency and reduce manual operation errors. In the S2 step, by adjusting the pH value to 7.3-7.5, physiological conditions are simulated, which helps to maintain the biological activity of chymotrypsin. In the S3 step, by controlling the pressure before the membrane and monitoring the conductivity, the ultrafiltration process can be accurately controlled to ensure the quality of the retentate. In the S8 step, 1M salt solution and 0.5M alkaline solution are used to clean the chromatography column, which can effectively remove residues and neutralize charged pollutants, and extend the service life of the chromatography column. By using an algorithm to determine the required purified water volume, it can be ensured that ethanol is completely replaced and the purification efficiency is improved. Fine purification steps help to improve the yield of chymotrypsin and reduce the waste of raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a schematic diagram of the process of the present invention;

[0037] Figure 2 It is a graph showing the change in PPV titer in each component of each batch of samples after AEX chromatography treatment in the present invention;

[0038] Figure 3 It is the change of ReoV-3 titer in each component of each batch of samples after AEX chromatography treatment in the present invention. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of the present invention more clear, the following is a detailed description of the present invention in conjunction with the specific implementation methods and with reference to the attached drawings. Figure 1-3 , the present invention is further described in detail. It should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present invention. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the concept of the present invention.

[0040] The present invention provides a method for removing pyrogen and virus of chymotrypsin, comprising the following steps:

[0041] S1 raw material preparation, weigh the crude minced source, add 8 times the amount of 0.01 mol / L acetic acid solution to dissolve;

[0042] S2: Dissolving the crude beef mince. Add 20 mol / L phosphate buffer to the crude beef mince extracted from S1 to dissolve it.

[0043] S3: Change the liquid, use the membrane package ultrafiltration system to continuously add water to ultrafilter the dissolved beef chyme, and collect the intercepted liquid;

[0044] S4 column packing: Use an automatic packing machine to evenly fill 100 ml of MaXtar Q filler into the column, and wash it with purified water three times to replace the original ethanol solution;

[0045] S5 pre-equilibration, using phosphate buffer to pre-equilibrate the chromatography column;

[0046] S6 was loaded, and the ultrafiltrate was passed through the column at a linear flow rate of 180-200 cm / h, and the flow-through was collected;

[0047] After S7, balance, use 3-5B volumes of pH 6.5 phosphate buffer for top washing, and collect the feed liquid;

[0048] S8 is cleaned and regenerated by washing with 2 to 3B volumes of 1M salt, and then washing the column with 2 to 3B volumes of 0.5M alkali.

[0049] The S2 specifically includes:

[0050] The pH value of 20 mol / L phosphate buffer was adjusted to 7.3-7.5 to simulate physiological conditions. A certain amount of crude beef mince was weighed and added to the phosphate buffer with adjusted pH value in batches. The constant temperature stirrer was turned on to fully mix and dissolve the beef mince.

[0051] The S3 specifically includes:

[0052] Replace the membrane package ultrafiltration system with a 10kDa ultrafiltration membrane, pre-wet the ultrafiltration membrane with an appropriate amount of buffer, set the pressure in front of the membrane to 0.1MPaM, slowly add the dissolved crude bovine chyme to the ultrafiltration membrane, start the pump to allow the crude bovine chyme to pass through the ultrafiltration membrane, monitor the conductivity of the retentate in real time, and collect the retentate when the conductivity is less than 2ms / cm.

[0053] The S4 specifically includes:

[0054] Use an automatic filling machine to evenly fill 100 ml of MaXtar Q filler into the chromatography column to form a stable filler bed. Start the fluid system of the chromatography column and replace the original ethanol solution with purified water at a flow rate of 1-2 BV. Repeat the washing of the filler with purified water for 3 times, using 200 ml of purified water each time, and observe the flow-through until the ethanol is completely washed out.

[0055] S5 specifically includes:

[0056] Connect the chromatography column correctly to the fluid system, start the pump, pump pH 6.5 sulfate buffer into the chromatography column, adjust the flow rate, ensure that 5-6 column volumes of buffer are used for balancing, collect the flow-through and test, and stop balancing when the pH value of the flow-through is in the range of 6.7 to 6.9.

[0057] S6 specifically includes:

[0058] Adjust the flow rate of the pump so that the ultrafiltrate passes through the chromatography column at a linear flow rate of 180-200 cm / h. Slowly add the ultrafiltrate in S3 to the top of the chromatography column using a syringe or an automatic sampler. Observe the flow of the sample in the chromatography column to ensure that the sample passes through the packing bed evenly. Start the collection system to collect the flow-through from the chromatography column.

[0059] S7 specifically includes:

[0060] According to the volume of the chromatography column, 3-5 volumes of pH 6.5 phosphate buffer are pumped into the chromatography column at an appropriate flow rate to elute the analysis that interacts weakly with the filler, and the feed liquid flowing out of the chromatography column is collected.

[0061] S8 specifically includes:

[0062] Prepare 2 to 3 volumes of 1M salt solution and 0.5M alkaline solution according to the volume of the chromatography column, start the pump, and pass the 1M salt solution through the chromatography column to remove charged pollutants with high ionic strength. When the waste liquid becomes lighter in color and more transparent, replace it with 0.5M alkaline solution and continue flushing. Monitor the pH value to ensure that the alkaline solution neutralizes the residue and completes the cleaning.

[0063] Use the algorithm to determine the volume of purified water required to ensure complete replacement of the ethanol:

[0064]

[0065] Among them, V 置换 is the volume of purified water required, V 柱 is the column volume, n is the number of washes, C 初始 and C 目标 are the initial and target ethanol concentrations, respectively.

[0066] As shown in Table 1, the total amount of PPV in the first batch of samples was 9.02log10. After anion chromatography treatment, the PPV virus was completely removed and the virus titer quickly dropped below the detection limit, with a drop value of ≥6.23log 10 .

[0067] Table 1 PPV removal effect of the first batch of samples (KC1-2408001)

[0068]

[0069] Note: The mark "*" indicates that the diluted sample is inoculated into T25 (8mL / bottle*10) for large-volume determination.

[0070] As shown in Table 2, the total PPV in the second batch of samples was 9.04 log 10After anion chromatography treatment, the PPV virus was completely removed and the virus titer dropped rapidly to below the detection limit, with a decrease value of ≥6.18log 10 .

[0071] Table 2 PPV removal effect of the second batch of samples (KC1-2409002)

[0072]

[0073]

[0074] Note: The mark "*" indicates that the diluted sample is inoculated into T25 (8mL / bottle*10) for large-volume determination.

[0075] As shown in Table 3, the total PPV in the third batch of samples is 9.00 log 10 After anion chromatography treatment, the PPV virus was completely removed and the virus titer dropped rapidly to below the detection limit, with a decrease value of ≥6.23log 10 .

[0076] Table 3 PPV removal effect of the third batch of samples (KC1-2409003)

[0077]

[0078] Note: The mark "*" indicates that the diluted sample is inoculated into T25 (8mL / bottle*10) for large-volume determination.

[0079] As shown in Table 4, the total amount of ReoV-3 in the first batch of samples was 7.87 log 10 After anion chromatography treatment, the ReoV-3 virus was completely removed, and the virus titer quickly dropped below the detection limit, with a decrease of ≥5.47log 10 .

[0080] Table 4 ReoV-3 removal effect of the first batch of samples (KC1-2408001)

[0081]

[0082] Note: The mark "*" indicates that the diluted sample is inoculated into T25 (8mL / bottle*10) for large-volume determination.

[0083] As shown in Table 5, the total amount of ReoV-3 in the second batch of samples was 8.13 log 10 After anion chromatography treatment, the ReoV-3 virus was completely removed, and the virus titer quickly dropped below the detection limit, with a decrease of ≥5.68log 10 .

[0084] Table 5 ReoV-3 removal effect of the second batch of samples (KC1-2409002)

[0085]

[0086]

[0087] Note: The mark "*" indicates that the diluted sample is inoculated into T25 (8mL / bottle*10) for large-volume determination.

[0088] As shown in Table 6, the total amount of ReoV-3 in the third batch of samples was 7.73 log 10 After anion chromatography treatment, the ReoV-3 virus was completely removed, and the virus titer quickly dropped below the detection limit, with a decrease of ≥5.36log 10 .

[0089] Table 6 ReoV-3 removal effect of the third batch of samples (KC1-2409003)

[0090]

[0091] Note: The mark "*" indicates that the diluted sample is inoculated into T25 (8mL / bottle*10) for large-volume determination.

[0092] Referring to the "General Principles for Technical Review of Viral Safety of Biological Tissue Extract Products and Eukaryotic Cell Expression Products" and "Technical Methods and Verification Guidelines for Removal / Inactivation of Viruses in Blood Products", the anion chromatography treatment step has a good removal effect on non-lipid enveloped viruses (PPV and ReoV-3), among which the PPV titer reduction value is ≥6.18log10, and the ReoV-3 titer reduction value is ≥5.36log10, both greater than 4.0log10, which is an effective process step for removing non-lipid enveloped viruses.

[0093] The present invention ensures the safety of chymotrypsin products and reduces potential infection risks by removing heat sources and viruses. Fine purification steps help remove impurities and improve the purity and activity of chymotrypsin. Using automated equipment and precise chemical treatment steps can improve production efficiency and reduce manual operation errors. In the S2 step, by adjusting the pH value to 7.3-7.5, physiological conditions are simulated, which helps to maintain the biological activity of chymotrypsin. In the S3 step, by controlling the pressure before the membrane and monitoring the conductivity, the ultrafiltration process can be accurately controlled to ensure the quality of the retentate. In the S8 step, 1M salt solution and 0.5M alkaline solution are used to clean the chromatography column, which can effectively remove residues and neutralize charged pollutants, and extend the service life of the chromatography column. By using an algorithm to determine the required purified water volume, it can be ensured that ethanol is completely replaced and the purification efficiency is improved. Fine purification steps help to improve the yield of chymotrypsin and reduce the waste of raw materials.

[0094] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the presence of other identical elements in the process, method, article or device including the elements.

[0095] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for removing pyrogen and virus of chymotrypsin, characterized in that: The following steps are involved: S1 raw material preparation, weigh the crude minced beef source, add 8 times the amount of 0.01mol / L acetic acid solution to dissolve; S2 crude beef minced beef dissolution, add 20mol / L phosphate buffer to the crude beef minced beef extracted from S1 to dissolve; S3: Change the liquid, use the membrane package ultrafiltration system to continuously add water to ultrafilter the dissolved beef chyme, and collect the intercepted liquid; S4 column packing: Use an automatic packing machine to evenly fill 100 ml of MaXtar Q filler into the column, and wash it with purified water three times to replace the original ethanol solution; S5 pre-equilibration, using phosphate buffer to pre-equilibrate the chromatography column; S6 was loaded, and the ultrafiltrate was passed through the column at a linear flow rate of 180-200 cm / h, and the flow-through was collected; After S7, balance, use 3-5B volumes of pH 6.5 phosphate buffer for top washing, and collect the feed liquid; S8 is cleaned and regenerated by washing with 2 to 3B volumes of 1M salt, and then washing the column with 2 to 3B volumes of 0.5M alkali.

2. The method for removing pyrogen and virus of chymotrypsin according to claim 1, characterized in that: The S2 specifically includes: The pH value of 20 mol / L phosphate buffer was adjusted to 7.3-7.5 to simulate physiological conditions. A certain amount of crude beef mince was weighed and added to the phosphate buffer with adjusted pH value in batches. The constant temperature stirrer was turned on to fully mix and dissolve the beef mince.

3. The method for removing pyrogen and virus of chymotrypsin according to claim 1, characterized in that: The S3 specifically includes: Replace the membrane package ultrafiltration system with a 10kDa ultrafiltration membrane, pre-wet the ultrafiltration membrane with an appropriate amount of buffer, set the pressure in front of the membrane to 0.1MPaM, slowly add the dissolved crude bovine chyme to the ultrafiltration membrane, start the pump to allow the crude bovine chyme to pass through the ultrafiltration membrane, monitor the conductivity of the retentate in real time, and collect the retentate when the conductivity is less than 2ms / cm.

4. The method for removing pyrogen and virus of chymotrypsin according to claim 1, characterized in that: The S4 specifically includes: Use an automatic filling machine to evenly fill 100 ml of MaXtar Q filler into the chromatography column to form a stable filler bed. Start the fluid system of the chromatography column and replace the original ethanol solution with purified water at a flow rate of 1-2 BV. Repeat the washing of the filler with purified water for 3 times, using 200 ml of purified water each time, and observe the flow-through until the ethanol is completely washed out.

5. The method for removing pyrogen and virus of chymotrypsin according to claim 1, characterized in that: S5 specifically includes: Connect the chromatography column correctly to the fluid system, start the pump, pump pH 6.5 sulfate buffer into the chromatography column, adjust the flow rate, ensure that 5-6 column volumes of buffer are used for balancing, collect the flow-through and test, and stop balancing when the pH value of the flow-through is in the range of 6.7 to 6.

9.

6. The method for removing pyrogen and virus of chymotrypsin according to claim 1, characterized in that: S6 specifically includes: Adjust the flow rate of the pump so that the ultrafiltrate passes through the chromatography column at a linear flow rate of 180-200 cm / h. Slowly add the ultrafiltrate in S3 to the top of the chromatography column using a syringe or an automatic sampler. Observe the flow of the sample in the chromatography column to ensure that the sample passes through the packing bed evenly. Start the collection system to collect the flow-through from the chromatography column.

7. The method for removing pyrogen and virus of chymotrypsin according to claim 1, characterized in that: S7 specifically includes: According to the volume of the chromatography column, 3-5 volumes of pH 6.5 phosphate buffer are pumped into the chromatography column at an appropriate flow rate to elute the analysis that interacts weakly with the filler, and the feed liquid flowing out of the chromatography column is collected.

8. The method for removing pyrogen and virus of chymotrypsin according to claim 1, characterized in that: S8 specifically includes: Prepare 2 to 3 volumes of 1M salt solution and 0.5M alkaline solution according to the volume of the chromatography column, start the pump, and pass the 1M salt solution through the chromatography column to remove charged pollutants with high ionic strength. When the waste liquid becomes lighter in color and more transparent, replace it with 0.5M alkaline solution and continue flushing. Monitor the pH value to ensure that the alkaline solution neutralizes the residue and completes the cleaning.

9. The method for removing pyrogen and virus of chymotrypsin according to claim 1, characterized in that: Use the algorithm to determine the volume of purified water required to ensure complete replacement of the ethanol: Among them, V 置换 is the volume of purified water required, V 柱 is the column volume, n is the number of washes, C 初始 and C 目标 are the initial and target ethanol concentrations, respectively.

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