CN110590931A

CN110590931A - Method for removing and/or inactivating virus in recombinant human thrombopoietin stock solution

Info

Publication number: CN110590931A
Application number: CN201910837015.6A
Authority: CN
Inventors: 钟正明; 秦小强; 王一凡
Original assignee: JIANGSU KANGHE BIOLOGICAL PHARMACEUTICAL Co Ltd
Current assignee: JIANGSU KANGHE BIOLOGICAL PHARMACEUTICAL Co Ltd
Priority date: 2019-09-05
Filing date: 2019-09-05
Publication date: 2019-12-20
Anticipated expiration: 2039-09-05
Also published as: CN110590931B

Abstract

The invention provides a method for removing and/or inactivating viruses in a recombinant human thrombopoietin stock solution, which comprises the following steps: inactivating virus by using a fermentation liquor of the recombinant human thrombopoietic factor by using an S/D method; and filtering the purified fermentation broth using a nanomembrane to remove viruses. The method according to the invention comprises the following steps: 1) culturing rh-TPO engineering cells to obtain fermentation liquor; 2) inactivating viruses of the fermentation liquor by using an S/D method; 3) purifying the fermentation liquor inactivated by an S/D method; 4) filtering the purified fermentation liquor by using a nano membrane to obtain the nano-composite. The method of the invention can remove not only a large amount of endogenous and exogenous lipid cell membrane viruses, but also non-lipid cell membrane viruses. The method of the invention is not only effective for inactivating more complex carbohydrate-modified cytokine-type proteins, but also can keep the protein activity of the recombinant human thrombopoietin at a higher level without affecting the quality of finished products.

Description

Method for removing and/or inactivating virus in recombinant human thrombopoietin stock solution

Technical Field

The invention belongs to the field of biological preparations, and relates to a method for removing and/or inactivating viruses in recombinant human thrombopoietin (PAF) stock solution.

Background

In the last 50 th century, Kelemen et al discovered and proposed that a humoral factor capable of stimulating the production of platelets in blood existed in vivo, and named Thrombopoietin (TPO), which has a significant effect of promoting the development and maturation of the megakaryocyte system. Recombinant human thrombopoietin (rh-TPO) is a recombinant glycoprotein containing 332 amino acids and expressed by CHO engineering bacteria, and can regulate the growth, differentiation, maturation and division of megakaryocytes to form functional platelets by binding with a specific receptor Mpl, and can be used for treating severe thrombocytopenia and Idiopathic Thrombocytopenic Purpura (ITP) caused by solid tumor and acute leukemia radiotherapy and chemotherapy, bone marrow transplantation, aplasia and other bone marrow insufficiency, HIV and the like.

In the process of preparing the recombinant human thrombopoietin by using CHO engineering bacteria, virus inactivation is required. The existing virus inactivation process mainly has the following modes in blood products and monoclonal antibodies:

1. pasteurization: the method is suitable for a stable albumin inactivation process, and is mature for inactivating HIV and hepatitis viruses.

2. Dry heat method (lyophilized formulation): the virus such as HCV, HBV, HIV and HAV can be inactivated by heating at 80 deg.C for 72 hr, but the water content and content of the product should be considered to influence the inactivation of virus.

3. Low pH inactivation incubation method: several lipofectin viruses can be inactivated by low pH (e.g. pH 4) in immunoglobulin production, and the conditions for inactivation should take into account pH, incubation time, temperature, solute in the solution, etc.

4. The membrane filtration method comprises the following steps: the method is only used for filtering the virus with the effective diameter of the filter membrane smaller than that of the virus, and the method cannot be used alone and needs to be combined with other methods for use.

The virus inactivation processes of the 1 st, the 2 nd and the 4 th methods are mainly suitable for blood preparations, and the monoclonal antibody is mainly suitable for the combined use of the 3 rd and the 4 th methods.

At present, for recombinant human thrombopoietin type products, glycosylation accounts for a high percentage, accounting for about 30% of the molecular size, and compared with monoclonal antibodies, no mature virus inactivation method exists at present due to complex glycosylation modification sites and glycoforms, unstable proteins and the like. For example, the domestic rh-TPO product mainly has the special otou of sheng yang, sansheng, and is produced and marketed in 2006, and in view of the previous regulations and the condition limitations of industrial cognition, protein complexity and the like, the product cannot inactivate and remove potential viruses and does not meet the requirements of the current stage. Furthermore, the prior art does not mention the inactivation and treatment of potential viruses on cytokine products with complex glycosylation and unstable protein, which causes great risk and hidden danger to the drug safety.

Thus, there is a need for a method for removing and/or inactivating viruses from a stock solution of recombinant human thrombopoietin.

Disclosure of Invention

Based on the deficiencies of the prior art, it is an object of the present invention to provide a method for removing and/or inactivating viruses from a recombinant human thrombopoietin stock solution. The method provided by the invention has simple steps, simultaneously solves the problem that the traditional method can not remove the adipocyte membrane virus and the non-adipocyte membrane virus, such as porcine virus, bovine virus and the like, and has important significance for the production and the use of the recombinant human thrombopoietic factor.

In one aspect, the present invention provides a method for removing and/or inactivating virus from a recombinant human thrombopoietin stock solution, the method comprising:

inactivating virus by using a fermentation liquor of recombinant human thrombopoietin (rh-TPO) by using an S/D method; and

the purified fermentation broth was filtered using a nanomembrane to remove viruses.

The method of the invention, wherein the reagents used in the S/D method are tributyl phosphate and triton-100;

wherein the working concentration of the tributyl phosphate is 0.3% + -0.03% (v/v), and the working concentration of the triton-100 is 1% + -0.1% (v/v).

The method of the invention, wherein the inactivation condition of the S/D method is as follows: treating with S/D reagent at 23-27 deg.C for at least 1 hr.

Preferably, the conditions for inactivation by the S/D method are as follows: at 25 ℃ the treatment was carried out for 4 hours using S/D reagent.

Preferably, the nanomembrane filtration is performed using a ViresolvePro virus removal filtration membrane from Millipore corporation.

The method according to the invention comprises the following steps:

1) culturing engineering cells of recombinant human platelet-derived factor (rh-TPO) to obtain fermentation liquor;

2) inactivating viruses of the fermentation liquor by using an S/D method;

3) purifying the fermentation liquor inactivated by an S/D method;

4) filtering the purified fermentation liquor by using a nano membrane to obtain the nano-composite.

The method according to the present invention, wherein, in step 3), the purification is sequentially cation chromatography, hydrophobic chromatography and anion chromatography.

The method according to the present invention, wherein the method further comprises the step of sterile filtration and/or dispensing.

The applicant finds that in the preparation of the recombinant human thrombopoietin stock solution, the traditional process is used for virus inactivation, so that lipid cell membrane viruses and non-lipid cell membrane viruses, such as porcine viruses, bovine viruses and the like, cannot be removed, the use risk and the medication safety of patients are greatly increased, and the requirements of the current pharmaceutical industry specifications are not met. Therefore, it is necessary to add the inactivation process of lipid enveloped virus and non-lipid enveloped virus in the preparation process, thereby greatly reducing the huge risk caused by incomplete virus inactivation. The inventor actively explores and researches the inactivation mode of the potential virus in the rH-TPO cytokine production process according to related monoclonal antibody inactivation processes and blood product inactivation processes at home and abroad and related legal and regulatory requirements, and finally completes the blank field of virus removal and inactivation with complex glycosylation modification of cytokines and unstable protein activity through a large amount of experimental investigation. The method of the invention can remove a large amount of endogenous and exogenous lipid cell membrane viruses, can also remove non-lipid cell membrane viruses, can simultaneously maintain the protein activity with higher degree and the lowest S/D inactivator residue, and plays a great role in the medication safety of patients.

The invention solves the problem of inactivation mode of potential viruses in the production process of recombinant cytokine products, finally completes the blank field of ineffective virus inactivation/virus removal in the production process with relatively complex glycosylation modification and unstable protein activity through a large amount of experiments, and has a certain guiding function on virus removal and inactivation in the future production process of cytokine products.

Compared with the prior art, the method provided by the invention has the following advantages:

1. the method of the invention can remove not only a large amount of endogenous and exogenous lipid cell membrane viruses, but also non-lipid cell membrane viruses.

2. The method of the invention is not only effective for inactivating more complex carbohydrate-modified cytokine-type proteins, but also can keep the protein activity of the recombinant human thrombopoietin at a higher level without affecting the quality of finished products.

3. By using the method of the invention, S/D residues can be almost completely removed by carrying out ion exchange chromatography separation subsequently, so that the product meets the standard requirement of no harm to human bodies, and plays a great role in the medication safety of patients.

4. The method of the invention solves the blank that the cytokine products can not inactivate the potential viruses, and has important production and practical significance.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is an HPLC chromatogram of rh-TPO stock after inactivation using the S/D method in example 1 according to the present invention;

FIG. 2 is a SDS PAGE result of rh-TPO stock after inactivation using S/D method in example 1 according to the present invention;

FIG. 3 is a SDS PAGE result of rh-TPO stock after inactivation using low pH incubation in comparative example 1 according to the present invention;

FIG. 4 is an HPLC plot of a sample after inactivation using low pH incubation according to comparative example 1 of the present invention, prior to low pH inactivation, as shown in method 1 under hydrophobic chromatography;

FIG. 5 is an HPLC plot of samples subjected to low pH inactivation by hydrophobic chromatography as described in method 1 after inactivation by low pH incubation in comparative example 1 according to the present invention;

FIG. 6 is an HPLC plot of the sample after inactivation using low pH incubation of comparative example 1 according to the present invention, prior to low pH inactivation of the sample by anion chromatography as described in method 2;

FIG. 7 is an HPLC chromatogram of a sample subjected to low pH inactivation by anion chromatography as shown in method 2 after inactivation by low pH incubation in comparative example 1 according to the present invention;

FIG. 8 is a SDS PAGE result of rh-TPO stock after inactivation of the fermentation broth using low pH incubation in comparative example 2 according to the present invention.

Detailed Description

Example 1 inactivation of Lipocytic Membrane Virus Using the method of the invention

In this example, the lipid membrane of enveloped viruses was disrupted mainly with an organic solvent/detergent mixture (S/D) to achieve an effective inactivation of lipid enveloped viruses.

1.1 Experimental methods

First, the procedure for preparing rh-TPO stock solution in this example is as follows:

1) culturing rh-TPO engineering cells;

2) sequentially carrying out cation chromatography, hydrophobic chromatography and anion chromatography on the fermentation liquor;

wherein, cation chromatography is carried out by using a composite weak cation medium MMC Diamond;

hydrophobic chromatography was performed using Butyl HP packing;

anion chromatography was performed using a MixA Mustang ion exchange packing;

3) sterilizing, filtering and/or packaging.

Based on the fact that S/D reagent is an organic reagent and detergent is a relatively hydrophobic substance, S/D inactivation is not suitable to be placed before the hydrophobic chromatography process, the applicant has studied placing S/D inactivation between the hydrophobic chromatography process and the anion chromatography Mustang process or the fermentation broth step to test.

The method comprises the following steps: inactivation with 0.3% tributyl phosphate 1% Triton-100

Subjecting the fermentation liquor to hydrophobic chromatography, sampling 50mL of the pilot test hydrophobic chromatography, adding 150 μ L of TNBP and 0.5mL of Triton X100 to make the final volume concentration of the fermentation liquor respectively 0.3% and 1%, stirring uniformly, and inactivating for 1 h. The solution was a white turbid solution, which was filtered through a 0.45 μm membrane, and the solution was still turbid.

The method 2 comprises the following steps: inactivation of 0.3% tributyl phosphate 1% Tween 80

Subjecting the fermentation liquor to hydrophobic chromatography, sampling 50mL of the pilot-scale hydrophobic chromatography, adding 150 μ L of tributyl phosphate, 0.5mL of the tributyl phosphate and Tween 80 to make the final volume concentrations of the tributyl phosphate and the Tween 1% respectively, stirring uniformly, and inactivating for 1 h.

Control 3: the blank is not inactivated

Samples from method 1, method 2 and control 3 were subjected to anion chromatography, procedure: flow rate 0.7mL/min liquid a balance → loading → liquid a wash 3CV → 11% B wash 4CV → 50% B elute 5CV, and collect the eluate.

1.2 analysis of results:

1.2.1 HPLC profileThe results of the HPLC experiments of the above two methods and the control are shown in FIG. 1, wherein FIG. 1 is a graph of the sample after the anion chromatography Mustang step.

The method comprises the following steps: inactivating 0.3% TNBP 1% Triton X100, eluting and collecting the volume of 6.4 mL;

the method 2 comprises the following steps: inactivating 0.3% TNBP 1% Tween 80, eluting and collecting the volume of 9.2 mL;

comparison 3: blank was not inactivated and elution was collected in a volume of 6.2 mL.

It can be seen from the figure that the samples without inactivation of method 1 and control 3 blanks have very similar peak appearance time, peak area and purity in the HPLC liquid phase, and the method 1 protocol is feasible.

1.2.2 SDS PAGE results:

the results of SDS PAGE for the above two methods and controls are shown in FIG. 2, in which 1-10 are shown as follows: 1. the loading solution of control 3; 2. flow-through for control 3; 3. eluent of control 3; 4. the sample loading solution of the method 1; 5. flow-through of method 1; 6. the eluent of method 1; 7. the sample loading solution of the method 2; 8. flow-through of method 2; 9. the eluent of method 2; 10. mark-Marker;

as can be seen from the electropherogram,

1) the inactivation by the S/D method has no influence on the sample preparation by the hydrophobic chromatography, the rh-TPO strip is not degraded,

2) the selected S/D inactivation conditions are as follows: inactivating 0.3% tributyl phosphate and 1% triton-100 for 1h at normal temperature; as can be seen from the experimental pattern, the elution of the blank 3 was almost identical to the SDAPAGE band of method 1 (i.e., using 0.3% tributyl phosphate 1% triton-100), and the volume collected by elution was substantially identical; as can also be seen from SDS PAGE, there was no flow-through in both method 1 and control 3, and the eluted rh-TPO band was substantially the same. In method 2, after inactivation by 0.3% tributyl phosphate and tween-80, the target protein showed a lower yield.

Therefore, the inactivation condition adopted by the method is an S/D inactivator, namely 0.3 percent of tributyl phosphate and 1 percent of triton-100 are inactivated for 1 hour, and the method can be suitable for the inactivation of rh-TPO protein at normal temperature.

1.2.3S/D inactivation effect verification:

the S/D inactivation effect is verified by adopting an indicator virus designated by a hospital inspection, two indicator viruses of VSV (vesicular stomatitis virus)/PRV (pseudorabies virus), and the inactivation capacity of the rh-TPO agent stock solution (20161118, 20161119 and 20161220) is basically up to 4log TCID50/0.1mL no matter PRV or VSV through S/D inactivation results, and most of the inactivation can be completely inactivated after being homogenized for 1 hour. As can be seen, inactivation using the S/D method of the present invention completely inactivated adipocyte membrane virus.

Since the purpose of this example was to optimize the inactivation step, the inventors found that by locating the inactivation lipocytovirus step at the front end of the purification process, tributyl phosphate and triton-100, the introduced impurities, can be removed by both cationic chromatography and hydrophobic chromatography. Meanwhile, certain potential viruses can be further removed by the corresponding chromatographic packing. Thus, S/D inactivation of lipocytoviruses is carried out in cell culture broth.

Example 2 inactivation of non-adipocyte Membrane Virus Using the method of the invention

The purpose of this example is to add a nano-membrane filtration step to remove non-lipid membrane viruses, based on the removal of lipid membrane viruses,the Pro series nanofiltration membrane is made of double-layer polyether sulfone material, can realize high-level virus removal, and the Log removal rate of the virus is more than or equal to 4 Log.

In example 1, after the chromatography step, ViresolvePro virucidal filtration membrane from millipore was selected to perform virucidal filtration pilot protein load experiments.

Purpose of the experiment: protein load was calculated by needle-type virus filtration membrane removal.

The experimental process comprises the following steps: a viral nanofiltration system was assembled using a peristaltic pump and nanofiltration membranes, and the batch (protein concentration 0.425mg/mL) was filtered, nanofiltration membrane model:pro Micro Device; membrane area: 3.1cm²(ii) a Setting the filtering pressure to be 0.15-0.2MPa, weighing the filtrate every minute, calculating the flow rate change according to the weighing result, determining the full loading capacity of the filtering membrane when the filtering flow rate is reduced to 80% of the initial flow rate, calculating the protein loading capacity at the moment, and calculating the filtering membrane specification required by the production scale.

TABLE 1 data weight per minute summary Unit (g)

Analysis of the results, it can be seen from the above table that the nanofiltration rate drops below 80% of the initial rate at 22 minutes of filtration, i.e. protein loading is reached at 22 minutes.

And (3) calculating:

the loading of the filtration membrane is 0.425mg/mL × 88.9 mL/3.1 cm²＝12.19mg/cm²

When the fermentation is carried out in large-scale batch according to 100L, the total protein of the nano-membrane filtration stage is about 1600mg, so the total area of the required nano-membrane is 1600mg/12.19/cm²＝140cm²

EXAMPLE 3 detection analysis of residual Effect of S/D：

The related TNBP and Triton residues need to meet the requirements of the latest pharmacopoeia, for which the applicant carried out three batches (20161118, 20161119 and 20161220) of rh-TPO stock solution for virus inactivation using the method of the present invention, respectively, and examined the residual effect of S/D in the final product, the results are shown in the following table:

TABLE 2 detection of residual amount of S/D inactivator

As can be seen from the table, the method of the present invention has the lowest S/D inactivator residue, and plays a great role in the safe administration of patients.

Comparative example 1 virus was inactivated using low pH incubation and nanomembrane filtration:

low pH incubation: generally, the pH value of the solution is controlled to be below 3.8, and the solution can be inactivated for 2 hours, so that several lipid enveloped viruses can be inactivated. The stability of the target protein pH3.8 was first determined for each chromatography step as follows:

3.1 purpose of the experiment

Comparing the cation chromatography MMC sample, the hydrophobic chromatography Butyl HP sample and the anion MixA sample, adjusting the pH value to 3.8 for inactivation, and confirming the inactivation process steps.

3.2 Experimental methods

First, the procedure for preparing rh-TPO stock solution in this comparative example was as follows:

1) culturing rh-TPO engineering cells;

2) sequentially carrying out cation chromatography, hydrophobic chromatography and anion chromatography on the fermentation liquor

hydrophobic chromatography was performed using Butyl HP packing;

anion chromatography was performed using a MixAMustang ion exchange packing;

3) sterilizing, filtering and/or packaging.

The method comprises the following steps:

and (3) performing MMC and hydrophobic chromatography on the fermentation liquor, wherein the eluent is 42mL, the pH value is 6.94, Cond: 11.81ms/cm, pH3.8 adjusted with 1M HAC, added slowly and added with a magnetic stirrer in an amount of 3.5mL pH3.80, pH7.0 adjusted with 1M Tris-HClpH 9.0 after 2h hold in an amount of 4mL, measured for Cond: 13.10 (anion chromatography Mustang can load directly), determination of HPLC;

the method 2 comprises the following steps:

taking 50mL of fermentation liquor, performing MMC, hydrophobic chromatography and anion chromatography on the fermentation liquor, adjusting the pH value to 6.98 by using 1M HAC to adjust the pH value to 3.8, slowly adding the fermentation liquor and adding the fermentation liquor by using a magnetic stirrer, wherein the using amount of the fermentation liquor is 4mL, the pH value to 3.80, maintaining the fermentation liquor for 2 hours, adjusting the pH value to 7.0 by using 1M Tris-HCl pH value to 9.0, and measuring the using amount of the fermentation liquor to 4.25mL, and determining HPLC;

the method 3 comprises the following steps:

taking the fermentation liquor, carrying out cation chromatography MMC Diamond sample feeding, wherein the pH value is 38mL and 8.16, adjusting the pH value to 3.8 by using 1M HAC, slowly adding the mixture, adding the mixture by using a magnetic stirrer, keeping the pH value to 3.80 by using 6.5mL, and keeping the mixture for 2 hours; 30mL of the sample was subjected to electrophoresis with 6mL of 1M Tris-HCl pH 9.0 to adjust pH 7.0.

3.3 analysis of results:

3.3.1 SDS PAGE results

The results of SDS-PAGE electrophoresis of the above three methods are shown in FIG. 3, wherein 1-9 in the electrophoresis are shown respectively:

1 is MMC sample (not inactivated at 4 ℃ for 48 h);

2 MMC sample (25 ℃ room temperature 20h without inactivation);

3 is MMC sample (after pH inactivation);

4 is butyl HP sample (not inactivated at 4 ℃ for 48 h);

5 is butyl HP load (25 ℃ room temperature 20h without inactivation);

6 is butyl HP sample (after pH inactivation);

7 is Mix A Mustang sample (not inactivated at 4 ℃ for 48 h);

8. mix a Mustang load (25 ℃ room temperature 20h without inactivation;

9. mix a Mustang (after pH inactivation);

wherein, the 3 rd, 6 th and 9 th bands are samples after low pH inactivation, compared with the samples without inactivation, the protein is degraded and does not meet the quality requirement.

3.3.2 HPLC profile

The results of HPLC chromatography of the above three methods are shown in FIGS. 4-7 and tables 3-6, wherein FIGS. 4 and 3 are the HPLC profile and results of the hydrophobic chromatography sample before low pH inactivation as shown in method 1; before inactivation: HPLC purity 85.32%. Wherein FIG. 5 and Table 4 are HPLC profiles and results of hydrophobic chromatography samples after low pH inactivation as shown in method 1; after inactivation: the main peak retention time was from 14.73min to 15.81min, indicating that the target had degraded. FIG. 6 and Table 5 are HPLC profiles and results of anion chromatography samples prior to low pH inactivation as shown in method 2; before inactivation: the purity is 98.97%; FIG. 7 and Table 6 show the HPLC profile and results after low pH inactivation of the anion chromatography sample, pH3.8 inactivation, purity, as shown in method 2: 89.08 percent.

TABLE 3

TABLE 4

TABLE 5

TABLE 6

And (3) analysis:

1) low pH inactivation: MMC and Butyl samples are unstable at low pH inactivation, which is not suitable after MMC and Butyl.

2) The purity of the liquid phase of the sample subjected to low-pH inactivation by anion chromatography is reduced from 98.97% to 89.08%, the purity is reduced by 7%, and polymers are generated, and the sample subjected to anion chromatography is not suitable for low-pH inactivation.

Comparative example 2 virus inactivation using low pH incubation at fermentation broth stage:

after the fermentation broth is inactivated by low pH, purification is started to prepare a sample for analysis:

a cation chromatography step:

column: BXK 50/30h 18cm volume: 360mL N/m: 4913 AS: 1.86

Buffer A：20mM PB pH 6.50.1M NaCl Buffer B：20mM PB pH 6.5 1M NaCl

Buffer C:50mM Tris pH 8.01M NH4Cl 2M Urea

Sample treatment: adjusting pH of 2L fermentation liquid to 3.8 with 1M HAC, adjusting pH to 6.5 with 1M Tris after 2h at room temperature, and filtering with 1 μ M filter to obtain 3.3L sample.

The process is as follows: 0.5M NaOH treatment → purified water → liquid a equilibrium → loading 25mL/min, 2.9L (1.75L fermentation broth) → liquid a washing → liquid B washing → liquid C elution → purified water → 0.5M NaOH regeneration;

a hydrophobic chromatography step:

column: BXK 26/20 h: 14cm

Buffer A20 mM PB 0.8M AS pH7.0 Cond 115ms/cm

Buffer B20 mM PB pH7.0

Sample preparation: 450mL of the sample was subjected to cation chromatography, adjusted to pH7.0 with HCl and Cond with 3M ammonium sulfate: and 100ms/cm is the sample.

The process is as follows: 0.5M NaOH treatment → purified water → liquid A balance → sample application 8mL/min → liquid A washing → liquid B elution → purified water → 0.5M NaOH regeneration

3.5 SDS PAGE result analysis:

the result of SDS-PAGE electrophoresis in this method is shown in FIG. 8, in which 1-10 of the electrophoresis are shown separately

1. Fermentation liquor

2. MMC flow-through is finished

3. MMC flow-through liquid mixed sample 4 and MMC impurity washing first peak

5. Second peak of MMC washing

6. MMC eluted protein peaks of interest

7. butyl HP flow peak-crossing

8. butyl HP Wash miscellaneous peaks

9. Butyl HP eluted the peaks of interest

10、Marker

Analysis and conclusion:

1) compared with the prior purification process, the cation-E3 has the peak height of an AKTA preparation map which is twice lower than that of the prior purification process; hydrophobic chromatography E1 was also more than two times lower than the pilot process.

2) SDS PAGE detection results: no significant TPO band was observed for MMC elution, nor was hydrophobic chromatography elution observed, and all protein degradation.

3) Low pH inactivation is also not suitable when placed on fermentation broth.

In conclusion, the conventional low pH incubation method is not suitable for virus inactivation in the rhTPO recombinant protein production process.

Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.

Claims

1. A method for removing and/or inactivating virus from a recombinant human thrombopoietin stock solution, the method comprising:

inactivating virus by using a fermentation liquor of the recombinant human thrombopoietic factor by using an S/D method; and

2. The process according to claim 1, wherein the reagents used in the S/D process are tributyl phosphate and triton-100;

3. The method of claim 1, wherein the S/D method inactivation is: treating with S/D reagent at 23-27 deg.C for at least 1 hr.

4. The method of claim 3, wherein the conditions for S/D inactivation are: at 25 ℃ the treatment was carried out for 4 hours using S/D reagent.

5. The method of claim 1, wherein the nanomembrane is a Viresolve Pro virus removal filtration membrane.

6. The method according to any one of claims 1-5, comprising the steps of:

1) culturing engineering cells of the recombinant human platelet-derived factor to obtain fermentation liquor;

2) inactivating viruses of the fermentation liquor by using an S/D method;

3) purifying the fermentation liquor inactivated by an S/D method;

7. The method according to claim 6, wherein in step 3), the purification is sequentially cationic chromatography, hydrophobic chromatography and anionic chromatography.

8. The method of claim 6, wherein the method further comprises the step of sterile filtering and/or dispensing.