阅读说明：本技术 一种重组人血小板生成因子原液的制备方法 (Preparation method of recombinant human thrombopoietic factor 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：本发明提供了一种重组人血小板生成因子原液的制备方法,所述方法包括以下步骤：1)以批次流加培养模式培养rh-TPO工程细胞；2)将步骤1)培养获得的发酵液采用S/D法灭活；3)将步骤2)灭活后的发酵液依次经过阳离子层析、疏水层析、阴离子层析,得到重组人血小板生成因子原液。采用本发明的方法制备的重组人血小板生成因子原液纯度大于99.5％～100％,蛋白活性为3.0x10<Sup>5</Sup>IU/mg～3.6x10<Sup>5</Sup>IU/mg,收率接近30％。通过采用本发明的方法,能够高产量地获得目的蛋白。(The invention provides a preparation method of recombinant human thrombopoietin stock solution, which comprises the following steps: 1) culturing rh-TPO engineering cells in a batch fed-batch culture mode; 2) inactivating the fermentation liquor obtained by the culture in the step 1) by adopting an S/D method; 3) and (3) sequentially carrying out cation chromatography, hydrophobic chromatography and anion chromatography on the inactivated fermentation liquor obtained in the step 2) to obtain a recombinant human platelet-derived factor stock solution. The purity of the recombinant human thrombopoietin stock solution prepared by the method is more than 99.5-100%, and the protein activity is 3.0x10 5 IU/mg～3.6x10 5 IU/mg, yield close to 30%. By using the method of the present invention, a target protein can be obtained in high yield.)

1. A method for preparing a recombinant human thrombopoietin stock solution, comprising the steps of:

1) culturing the recombinant human platelet-derived factor engineered cells in a batch fed-batch mode by using a bioreactor;

2) inactivating the fermentation liquor obtained by the culture in the step 1) by using an S/D method;

3) and (3) sequentially carrying out cation chromatography, hydrophobic chromatography and anion chromatography on the inactivated fermentation liquor obtained in the step 2) to obtain a recombinant human platelet-derived factor stock solution.

2. The method according to claim 1, wherein the minimal medium used in the culture in step 1) is EX-CELL Advanced^TMCHO Fed-batch culture medium; the fed-batch culture medium is HyClone^TM Cell Boost^TM7a and HyClone^TM Cell Boost^TM 7b。

3. The method according to claim 2, wherein the fed-batch medium HyClone is supplemented every 1 to 3 days^TMCell Boost^TM7a and HyClone^TM Cell Boost^TM7b, the addition amounts of which are respectively 4-6% and 0.4-0.6% of the culture volume;

preferably, the fed-batch medium HyClone is supplemented every 2 days^TM Cell Boost^TM7a and HyClone^TM Cell Boost^TM7b in amounts of 5% and 0.5% of the culture volume, respectively.

4. The production method according to claim 1, wherein the step 1) is achieved by a method comprising the steps of:

recovering recombinant human thrombopoietin engineering CELL and using EX-CELL Advanced^TMCulturing in CHO Fed-batch culture medium until the viable cell density is not lower than 3.0 × 10⁶Per mL, the activity is not lower than 90%;

inoculating to a bioreactor, and continuously culturing until harvesting;

wherein, the inoculated 3 rd, 5 th, 7 th and 9 th days are respectively supplemented with HyClone with the culture volume of 4-6% and the culture volume of 0.4-0.6%^TMCell Boost^TM7a and HyClone^TM Cell Boost^TM 7b；

Wherein the bioreactor is a 100L bioreactor and has the following culture parameters:

the temperature is 35.5 +/-1.5 ℃; pH6.9 + -0.5; dissolved oxygen is 30 to 70 percent;

preferably, the 100L bioreactor also has the following culture parameters:

the rotating speed is 50-80 rpm;

ventilating the surface layer by 10-30%;

deep aeration is carried out for 0-0.1 lpm;

0-0.5lpm of deep oxygen;

0-0.4lpm of deep carbon dioxide;

preferably, the concentration of glucose is 4-7 g/L in the culture process.

5. The preparation method according to claim 1, wherein in step 2), the reagents used in the S/D process are polyethylene glycol octyl phenyl ether and tributyl phosphate;

preferably, in step 2), the working concentration of polyethylene glycol octylphenyl ether is 1% (V/V); the working concentration of tributyl phosphate was 0.3% (V/V).

6. The production method according to claim 1, wherein the step 2) is achieved by a method comprising the steps of:

adding an S/D reagent into the fermentation liquor while stirring, adjusting the pH value to 6.5 by using an HCl solution, and inactivating the fermentation liquor for 1 to 2 hours at the temperature of 25 +/-2 ℃.

7. The production method according to claim 1, wherein in the step 3), cation chromatography is performed using a composite weak cation medium, MMC Diamond;

preferably, the conditions of the cation chromatography are:

loading conditions 20mM phosphate buffer, 0.1M sodium chloride, pH6.5 ± 0.2, conductance: 11-15 ms/cm;

cleaning conditions are as follows: 1M sodium chloride pH 6.5;

elution conditions: 50mM Tris pH 8.01M NH₄Cl 2M Urea.

8. The production method according to claim 1, wherein in the step 3), hydrophobic chromatography is performed using Butyl HP packing;

preferably, the conditions of the hydrophobic chromatography are:

loading conditions are as follows: 20mM phosphate buffer, 0.7-0.8M (NH)₄)₂SO₄pH7.0 conductance: 115 ms/cm;

the lower bar piece: 3M (NH)₄)₂SO₄Adjusting the difference between the conductance and the sample loading conductance to be +/-2 ms/cm, wherein the sample loading quantity is less than or equal to 6.4mg/mL of medium;

cleaning conditions are as follows: 20mM phosphate buffer, 0.7M (NH)₄)₂SO₄ pH 7.0；

Elution conditions: 10mM phosphate buffer was eluted in one step.

9. The preparation method according to claim 1, wherein in the step 3), anion chromatography is performed using a MixA Mustang ion exchange packing;

preferably, the conditions of the anion chromatography are:

loading conditions are as follows: 20mM Tris 0.1M NaClpH 8.0;

the lower bar piece: 20mM Tris, 1M NaCl, pH 8.0, the adjusted conductance is 12 +/-2 ms/cm, and the sample loading amount is less than or equal to 2.78mg/mL of medium;

cleaning conditions are as follows: 20mM phosphate buffer, 0.1M NaCl, pH7.0 + -0.1;

elution conditions: 20mM phosphate buffer, 0.5M NaCl, pH 7.0.

10. The method of any one of claims 1-9, further comprising the step of subjecting the resulting stock solution to nanomembrane filtration, sterile filtration and/or sub-packaging.

Technical Field

The invention belongs to the field of biological preparation, and relates to a preparation method of recombinant human thrombopoietic factor 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.

Patent ZL00109612.5 discloses specific culture conditions and purification method of recombinant human thrombopoietin. The purification method used by the method is complex, the cell culture solution needs to be subjected to the complicated steps of ultrafiltration, anion, gel filtration, cation, reverse chromatography and gel filtration chromatography, and the final culture solution 40L only obtains 260mg of protein, so that the yield is low, and the method is not suitable for large-scale production.

The invention with the patent publication number CN1276382 discloses a preparation method for large-scale production of recombinant human thrombopoietin. The method comprises culturing transformed mammalian cells carrying a gene sequence encoding human thrombopoietin protein in a specific culture medium under specific culture conditions to obtain a high expression harvest of the protein product. However, the preparation method disclosed by the method has animal and plant source nutrient medium alpha-MEM which is not approved at present, wherein the fetal bovine serum is added in an amount of 5-20%, a relatively complex perfusion process is adopted, the perfusion speed is 0.5-15 liters per day, and the method comprises the steps of sequentially carrying out ultrafiltration, cation exchange chromatography, gel chromatography, anion exchange chromatography, reversed-phase high-performance liquid chromatography, gel filtration chromatography and the like on the final fermentation liquor. The production process is complex and tedious, the yield is low, and the industrialization amplification is not facilitated.

U.S. Pat. No.5,986,049 (ZymoGenetics, Seattle, USA) and U.S. Pat. No.5,744,587 (ZymoGenetics, Seattle, USA) disclose methods for purifying thrombopoietin by affinity chromatography, respectively, which uses affinity chromatography and ion exchange chromatography to purify mammalian thrombopoietin, resulting in TPO of 90% or greater purity. The latter prepared human thrombopoietin expressed by BHK cells by adsorption, affinity chromatography and hydrophobic chromatography, and 250 mg of protein was obtained from 700L of harvest medium. The yield and protein purity are too low to meet the requirements.

Therefore, there is a need for an industrial process that can produce recombinant human thrombopoietin on a large scale.

Disclosure of Invention

Based on the defects of the prior art, the invention aims to provide a method for preparing the recombinant human thrombopoietin in a large scale. The method provided by the invention has simple steps and high yield. The method not only solves the problems that the production process of the current recombinant human platelet-derived factor is complicated and large-scale production and amplification cannot be realized, but also solves the problems that lipid envelope viruses and non-lipid envelope viruses, such as porcine-derived viruses and bovine-derived viruses, cannot be removed in the traditional process of the recombinant human platelet-derived cytokine, and has important significance.

In one aspect, the present invention provides a method for preparing a recombinant human thrombopoietin stock solution, comprising the steps of:

1) culturing recombinant human platelet-derived factor (rh-TPO) engineered cells in a batch fed-batch mode using a bioreactor;

2) inactivating the fermentation liquor obtained by the culture in the step 1) by using an S/D method;

3) and (3) sequentially carrying out cation chromatography, hydrophobic chromatography and anion chromatography on the inactivated fermentation liquor obtained in the step 2) to obtain a recombinant human platelet-derived factor stock solution.

The preparation method according to the invention, wherein, in the step 1), the basic culture medium used for the culture is EX-CELL Advanced^TMCHO Fed-batch culture medium; the fed-batch culture medium is HyClone^TM Cell Boost^TM7a and HyClone^TM Cell Boost^TM 7b；

Preferably, the feeding culture medium HyClone is supplemented every 1-3 days^TM Cell Boost^TM7a and HyClone^TMCell Boost^TM7b in the culture volume4% -6% and 0.4% -0.6%; more preferably, the fed-batch medium HyClone is supplemented every 2 days^TM Cell Boost^TM7a and HyClone^TM Cell Boost^TM7b, the addition amounts of which are 5% and 0.5% of the culture volume respectively;

the cell strain adopted by the invention is a cell strain constructed in patent No. 201310491722.7 of Jiangsu Kanghe biological pharmacy Co.

The preparation method according to the present invention, wherein the step 1) is achieved by a method comprising the steps of:

recovering rh-TPO engineering CELL and using EX-CELL Advanced^TMCulturing in CHO Fed-batch culture medium until the viable cell density is not lower than 3.0 × 10⁶Per mL, the activity is not lower than 90%;

inoculating to a bioreactor, and continuously culturing until harvesting;

wherein, the inoculated 3 rd, 5 th, 7 th and 9 th days are respectively supplemented with HyClone with the culture volume of 4-6% and 0.4-0.6%^TM Cell Boost^TM7a and HyClone^TM Cell Boost^TM 7b；

Wherein the bioreactor is a 100L bioreactor and has the following culture parameters:

the temperature is 35.5 +/-1.5 ℃; pH6.9 + -0.5; dissolved oxygen is 30 to 70 percent;

preferably, the 100L bioreactor also has the following culture parameters:

the rotating speed is 50-80 rpm; ventilating the surface layer by 10-30%;

deep aeration is carried out for 0-0.1 lpm;

0-0.5lpm of deep oxygen;

0-0.4lpm of deep carbon dioxide;

preferably, the concentration of glucose is 4-7 g/L in the culture process.

The preparation method according to the invention, wherein in the step 2), the reagents used in the S/D method are polyethylene glycol octyl phenyl ether and tributyl phosphate;

preferably, in step 2), the working concentration of polyethylene glycol octylphenyl ether is 1% (V/V); the working concentration of tributyl phosphate was 0.3% (V/V).

The preparation method according to the present invention, wherein the step 2) is achieved by a method comprising the steps of:

adding an S/D reagent into the fermentation liquor while stirring, adjusting the pH value to 6.5 by using an HCl solution, and inactivating the fermentation liquor for 1 to 2 hours at the temperature of 25 +/-2 ℃.

According to the preparation method, in the step 3), cation chromatography is carried out by using a composite weak cation medium MMC Diamond;

preferably, the conditions of the cation chromatography are:

loading conditions 20mM phosphate buffer, 0.1M sodium chloride, pH6.5 ± 0.2, conductance: 11-15 ms/cm;

cleaning conditions are as follows: 1M sodium chloride pH 6.5;

elution conditions: 50mM Tris pH 8.01M NH₄Cl 2M Urea.

The preparation method according to the present invention, wherein, in the step 3), hydrophobic chromatography is performed using Butyl HP packing;

preferably, the conditions of the hydrophobic chromatography are:

loading conditions are as follows: 20mM phosphate buffer, 0.7-0.8M (NH)₄)₂SO₄pH7.0 conductance: 115 ms/cm;

the lower bar piece: 3M (NH)₄)₂SO₄Adjusting the difference between the conductance and the sample loading conductance to be +/-2 ms/cm, wherein the sample loading quantity is less than or equal to 6.4mg/mL of medium;

cleaning conditions are as follows: 20mM phosphate buffer, 0.7M (NH)₄)₂SO₄ pH 7.0；

Elution conditions: 10mM phosphate buffer was eluted in one step.

The preparation method of the invention, wherein in the step 3), anion chromatography is performed by using a MixA Mustang ion exchange filler;

preferably, the conditions of the anion chromatography are:

loading conditions are as follows: 20mM Tris 0.1M NaCl pH 8.0;

the lower bar piece: 20mM Tris, 1M NaCl, pH 8.0, the adjusted conductance is 12 +/-2 ms/cm, and the sample loading amount is less than or equal to 2.78mg/mL of medium;

cleaning conditions are as follows: 20mM phosphate buffer, 0.1M NaCl, pH7.0 + -0.1;

elution conditions: 20mM phosphate buffer, 0.5M NaCl, pH 7.0.

The preparation method further comprises the step of carrying out nano-membrane filtration, aseptic filtration and/or split charging on the obtained stock solution.

The method provided by the invention aims at the current production process of rh-TPO cytokines, and the industrialization amplification of high efficiency, high yield and high purity is carried out, the purity of the recombinant human platelet production factor stock solution prepared by the method is 99.5-100%, and the protein activity is 3.0x10⁵IU/mg～3.6x10⁵IU/mg, yield is close to 30%, and 1800mg of target protein can be obtained with high yield by culturing 100L. Compared with the prior art, the invention has the following advantages:

1. has obvious advantages in the aspect of safety for patients

In the similar products, the culture medium adopted in the prior production is the culture medium containing plant protein source and animal protein source, and the most advanced import culture medium manufacturer which does not contain animal source, hydrolyzed protein and has clear chemical components is adopted in the invention to replace the risk process of potential harm of the old traditional virus.

2. Has obvious advantages in the aspects of industrialization scale and yield

The similar products adopt a relatively complex and relatively high-risk 10L perfusion process, the process has the defects of instability, easy pollution, long purification and storage period, large batch difference and the like, and the culture of 100L requires 35 days, 3 g of protein, 20 percent of yield and 10000 plus 15000 per batch; the process has the advantages of stable and reliable production, small pollution, short period and the like, only 10-11 days are needed for culturing 100L, 10 g of protein is needed, the yield is 30%, each batch is about 28000, the yield is 8 times that of the traditional process in the same time, the problem of difficult industrialization is solved, and the stable industrial amplification can be realized.

3. Compared with the like products, has obvious advantages in removing the risk of potential virus infection

The invention relates to a method for inactivating lipid envelope and non-lipid cell membrane viruses, which is characterized in that lipid cell membrane and non-lipid cell membrane viruses, such as porcine viruses, bovine viruses and the like, can not be removed in the traditional process, so that the use risk and the medication safety of patients are greatly increased, and the requirements of the current pharmaceutical industry specifications are not met.

4. Has obvious advantages in environmental health compared with similar products

The similar products adopt a complicated five-step chromatography process, wherein two steps respectively use a reverse chromatography of isopropanol and acetonitrile, and have residual risks in the final products, and the substances have potential hazards to human occupational health and are not suitable for purification and separation of modern biological products. The invention adopts more advanced, harmless and simple three-step chromatography to make the protein purity reach more than 98%.

5. Has obvious advantages in product quality compared with similar products

Compared with similar products, indexes influencing key quality attributes of the products, such as endotoxin, HCP, HDNA and the like, have obvious advantages compared with the similar products, exogenous impurity DNA of the similar products is 1.6 pg/mu g, the foreign impurity DNA of the similar products is 0.1 pg/mu g, the foreign impurity HCP of the similar products is 0.5 percent, the foreign impurity DNA of the similar products is 0.1 percent, the foreign impurity endotoxin of the similar products is 10 EU/agent, and the foreign impurity DNA of the similar products is 2 EU/agent.

Drawings

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

FIG. 1 is a graph showing the cell density of three groups of experimental living cells as a function of the culture time when rh-TPO engineering cells are subjected to shake flask culture by using a Sigma culture medium as a basic culture medium and a 7a/7b culture medium as an additive culture medium according to example 1 of the present invention;

FIG. 2 is a graph showing the cell viability of three groups of experimental living cells as a function of culture time when rh-TPO engineering cells are subjected to shake flask culture by using Sigma culture medium as a basic culture medium and 7a/7b culture medium as a culture medium according to example 1 of the present invention;

FIG. 3 is a bar graph of the expression level of target proteins in three groups of live experimental cells in shake flask culture of rh-TPO engineering cells with Sigma culture medium as basic culture medium and 7a/7b culture medium as additive culture medium according to example 1 of the present invention;

FIG. 4 is a graph showing the cell density of three batches of experimental living cells as a function of the culture time when cultured using a 10L reactor experiment according to example 2 of the present invention;

FIG. 5 is a graph of cell density versus culture time for three batches of experimental living cells using 100L scale cell culture according to example 3 of the present invention;

FIG. 6 is a graph of cell viability versus culture time for three batches of experimental live cells using 100L scale cell culture in accordance with example 3 of the present invention;

FIG. 7 is a HPLC plot after chromatography using MMC Diamond according to example 4 of the present invention;

FIG. 8 is a HPLC plot eluted with different salt concentrations using Butyl HP chromatography according to example 4 of the present invention;

FIG. 9 is an HPLC plot of samples at different pH using Mix AMustang chromatography according to example 4 of the present invention;

FIG. 10 is an SDS PAGE pattern of samples of different pH values using Mix A Mustang chromatography according to example 4 of the present invention;

FIG. 11 is a SDS PAGE pattern of different eluate eluted samples using Mix AMustang chromatography according to example 4 of the present invention.

Detailed Description

The cell line used in the embodiment of the present invention is a cell line constructed in Jiangsu Kanghe biological pharmacy Co.Ltd.No. 201310491722.7.