阅读说明：本技术 去除和/或灭活重组人血小板生成因子原液中病毒的方法 (Method for removing and/or inactivating virus in recombinant human thrombopoietin stock solution ) 是由 钟正明 秦小强 王一凡 于 2019-09-05 设计创作，主要内容包括：本发明提供了一种用于去除和/或灭活重组人血小板生成因子原液中病毒的方法,所述方法包括：将重组人血小板生成因子的发酵液使用S/D法灭活病毒；和使用纳米膜过滤纯化后的发酵液以除去病毒。根据本发明所述的方法,其包括以下步骤：1)培养rh-TPO工程细胞,获得发酵液；2)将发酵液使用S/D法灭活病毒；3)纯化使用S/D法灭活后的发酵液；4)使用纳米膜过滤纯化后的发酵液,即得。使用本发明的方法不仅能够去除大量的内源及外源脂胞膜病毒,还可以去除非脂胞膜病毒。使用本发明的方法,不仅对较为复杂的糖修饰细胞因子类蛋白灭活有效,而且能够将重组人血小板生成因子的蛋白活性保持在较高的水平,不影响成品的质量。(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.)

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

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

2. The process according to claim 1, wherein the reagents used in the S/D process 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).

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;

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

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.

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