CN110627892B - Preparation method of recombinant human thrombopoietic factor stock solution - Google Patents
Preparation method of recombinant human thrombopoietic factor stock solution Download PDFInfo
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Abstract
The invention provides a preparation method of a 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 platelet production factor stock solution prepared by the method is more than 99.5-100 percent, 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.
Description
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 methods 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 the purification of thrombopoietin by affinity chromatography, which uses affinity chromatography and ion exchange chromatography to purify mammalian thrombopoietin, resulting in TPO purities of 90% or more, respectively. 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-forming factor is complicated and the scale production and amplification cannot be realized, but also solves the problem that the traditional process of recombinant human platelet-forming cytokines cannot remove lipid envelope viruses and non-lipid envelope viruses, such as porcine-derived viruses, bovine-derived viruses and the like, 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 thrombopoietin (rh-TPO) engineering cells in a batch fed-batch mode by using a bioreactor;
2) Inactivating the fermentation liquor obtained by culturing 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 TM CHO Fed-batch culture medium; the fed-batch culture medium is HyClone TM Cell Boost TM 7a and HyClone TM Cell Boost TM 7b；
Preferably, the feeding medium HyClone is supplemented every 1 to 3 days TM Cell Boost TM 7a and HyClone TM Cell Boost TM 7b, the addition amounts of which are respectively 4-6% and 0.4-0.6% of the culture volume; more preferably, the fed-batch medium HyClone is supplemented every 2 days TM Cell Boost TM 7a and HyClone TM Cell Boost TM 7b, 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 number 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:
(1) recovering rh-TPO engineering CELL and using EX-CELL Advanced TM Culturing in CHO Fed-batch culture medium until the viable cell density is not lower than 3.0 × 10 6 Per mL, the vitality is not lower than 90%;
(2) 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 TM 7a 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 50-80rpm; ventilating the surface layer by 10-30%;
deep ventilation 0-0.1lpm;
0-0.5lpm of deep oxygen;
0-0.4lpm of deep carbon dioxide;
preferably, the glucose concentration during the culture is 4-7 g/L.
The preparation method of 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 be 6.5 by using HCl solution, and inactivating for 1-2 h at 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 8.0 1M NH 4 Cl 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 ) 2 SO 4 pH7.0 conductance: 115ms/cm;
the lower bar piece: 3M (NH) 4 ) 2 SO 4 Adjusting 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 ) 2 SO 4 pH 7.0；
Elution conditions: 10mM phosphate buffer was eluted in one step.
The preparation method of the invention is characterized in that in the step 3), anion chromatography is carried out by using MixA Mustang ion exchange packing;
preferably, the conditions of the anion chromatography are:
loading conditions are as follows: 2 mM Tris 0.1M NaCl pH 8.0;
the following sample pieces: 20mM Tris, 1M NaCl, pH 8.0, adjustment conductance of 12 +/-2 ms/cm and sample loading amount of less than or equal to 2.78mg/mL 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 with 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 3.0x10 5 IU/mg～3.6x10 5 IU/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 10L perfusion process which is relatively complex and has higher risk, the process is unstable and easy to pollute, the purification and storage period is long, the difference between batches is large, and the like, and the culture of 100L requires 35 days, 3 g of protein, 20 percent of yield and 10000-15000 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 similar products, has obvious advantages in the aspect of 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 experimental groups of live cells as a function of culture time when rh-TPO engineered cells were subjected to shake flask culture using Sigma medium as a basal medium and 7a/7b medium as a supplement 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 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 strain adopted in the specific embodiment of the invention is a cell strain constructed in patent number 201310491722.7 of Jiangsu cornus biopharmaceuticals Co.
EXAMPLE 1 culturing of engineered cell-selection Medium
The purpose of this example was to select a suitable medium.
The applicant of the application finds that the culture effect of the rh-TPO engineering cells is better than that of the chemical component fixation of Hyclone, gibco and BD international known brands by using Merck (Sigma) as a basic culture medium, and the basic culture medium does not contain animal-derived serum.
Further, the Applicant determined that the fed-batch medium was HyClone based on the selection of Merck (Sigma) as the basal medium TM Cell Boost TM 7a，HyClone TM Cell Boost TM 7b, in Sigma minimal Medium EX-CELL Advanced TM The CHO Fed-batch Medium is used together, and has high expression effect on the growth of cells.
HyClone TM Cell Boost TM 7a，HyClone TM Cell Boost TM Compared with various culture media in Merck (Sigma), hyclone, gibco and BD, 7b can ensure reasonable collocation of basal culture media, prolong the growth cycle of cells, improve the expression quantity of the cells, is expected to be more than 90mg/mL on average, and is stable in batches.
As shown in Table 1 below, the final selected basal and feed media were used
TABLE 1 basal and feed-through media
1.1 purpose of the experiment
Verifying whether the Sigma culture medium and the 7a/7b culture medium are suitable for the growth and expression of the rh-TPO engineering cells; and simultaneously, whether the 7a/7b culture medium is reasonably supplemented is verified.
The experimental requirements are that when the rh-TPO engineering cells are cultured by using a Sigma culture medium, the cells are recovered until the volume of cell suspension reaches 1.2L and the density of living cells is not less than 3.64 multiplied by 10 6 Cell viability is not lower than 95% per mL, and culture time is not longer than 16 days; when the shake flask culture is carried out, the expression quantity of the target protein is not lower than 80mg/L when the target protein is harvested.
1.2 design of the experiment
TABLE 2 incubator parameter settings
| CO 2 Concentration of | Rotational speed | Temperature of |
| 5.0％ | 120rpm | 37℃ |
1.3 Resuscitation growth passage experiment
Taking a working generation cell, recovering with Sigma culture medium, culturing to 4 cells with density not lower than 3.64 × 10 and 300mL/L 6 The cell viability is not less than 95 percent, and the cell viability is counted as a first group. Sampling every day to detect the total/living cell density, calculating the activity, drawing by taking time as an abscissa and the total cell number every day as an ordinate, and observing the change of the total cell number; the time is plotted on the abscissa and the cell viability per day is plotted on the ordinate, and the change in viability per day is observed.
Three parallel experiments were performed.
1.4 Shake flask culture experiment
On the basis of 1.1.3 experiments, the cells are cultured to 4 cells with the density of 300mL/L and the viable cell density of not less than 3.64 multiplied by 10 6 Cell per m, cell viability not less thanAt 95%, taking part of cells to subculture to 40mL/125mL, namely taking 40mL of cells to culture in a 125mL shake flask, wherein the viable cell density is 1.0X 10 6 And (4) counting the number of cells per mL, starting the shaking culture on the 1 st day, and finishing the culture until the cell viability is lower than 70%. Sampling every day to detect the total/living cell density, calculating the activity, drawing by taking the time as an abscissa and the living cell density every day as an ordinate, and observing the change of the living cell density; the time is plotted on the abscissa and the cell viability per day is plotted on the ordinate, and the change in viability per day is observed. On days 3, 5, 7, and 9, the culture medium was supplemented with 1.6/0.16mL of 7a/7b medium (the supplement ratio of 7a/7b medium was 4%/0.4% of the culture volume). And (4) the target protein expression quantity is checked on days 4, 7, 9 and 11, the check is carried out once on the day of finishing the culture, and the target protein expression quantity of each shake flask is compared.
1.5 Experimental results and analysis
1.5.1 cell Density analysis
When the Sigma culture medium is used as a basic culture medium and the 7a/7b culture medium is used as an additive culture medium to perform shake flask culture on the rh-TPO engineering cells, the curve graph of the density change of the three groups of experimental living cells along with the culture time is shown in figure 1. As can be seen from FIG. 1, the viable cell density of the three experimental groups varied consistently, indicating that the culture process was relatively stable and the viable cell density was maintained at 4.3 × 10 at the highest value 6 The number of viable cells per ml gradually decreased in a consistent manner with the delay of the culture time.
1.5.2 cell viability assay
When rh-TPO engineering cells are subjected to shake flask culture by taking a Sigma culture medium as a basic culture medium and a 7a/7b culture medium as an additive culture medium, the curve graph of the activity of three groups of experimental cells along with the change of culture time is shown in figure 2. As can be seen from FIG. 2, the three groups of experiments showed consistent trend of viability change, which indicates that the culture process was relatively stable, and the cell viability could be maintained for more than 95% for 7 days.
1.5.3 comparison of expression levels of target proteins
When the culture medium Sigma is used as a basic culture medium and the culture medium 7a/7b is used as an additive culture medium to perform shake flask culture on the rh-TPO engineering cells, a graph showing the change of the expression quantity of the target protein of the three groups of experimental cells along with the culture time is shown in figure 3. As can be seen from FIG. 3, the expression level of the target protein can reach more than 90mg/L in the three groups of experiments during harvesting, which meets the experimental requirements.
1.6 conclusion
In conclusion, when the recovery subculture is carried out on the rh-TPO engineering cells by using the Sigma culture medium, the culture volume can reach 1200mL within 16 days, and the density of the living cells is not lower than 4.0 x10 6 The cell viability is not lower than 95 percent per mL, and the experimental requirements are met; the Sigma culture medium is used as a basic culture medium, the 7a/7b culture medium is used as an adding culture medium to carry out shake flask culture on the rh-TPO engineering cells, the expression quantity of the target protein can reach more than 90mg/L during harvesting, and the experimental requirements are met.
Therefore, the culture medium Sigma is used as a basic culture medium, and the culture medium 7a/7b is used as an additional culture medium, so that the rhTPO engineering cells can be subjected to subculture and expression of target proteins; on days 3, 5, 7 and 9, 7a/7b culture medium with the culture volume of 4-6%/0.4-0.6%, 4-6%/0.4-0.6% and 4-6%/0.4-0.6% is added to meet the culture of the rh-TPO engineering cells and the expression of target proteins.
EXAMPLE 2 culture of engineered cells-10L reactor experiment
The purpose of examples 2 and 3 is to optimize the culture of engineered cells from the original 10L perfusion culture mode to a 100L batch mode, and further to solve the problems of complex perfusion, easy contamination, low yield, and poor operability in the 10L perfusion culture mode. Further, the problems that the stability of a domestic culture medium (containing serum) used in the prior art is poor among batches, more impurities are generated in the culture process, the purification is difficult, the purity is low and the like are solved.
Based on examples 1-3, the expression level of the target protein is increased from 30mg/mL to more than 90mg/mL by changing the culture mode of the engineered cells and selecting a proper culture medium.
2.1 purpose of the experiment
And designing 3 continuous batches of culture experiments of the 10L bioreactor, further verifying the feasibility of a Sigma culture medium and a culture medium adding method, and confirming and optimizing a culture process.
2.2 design of the experiment
2.2.1 first batch 10L reactor experiment
Recovering one cell of the working generation, shake-flask culturing and amplifying to 2.4L with Sigma culture medium, and viable cell density not less than 3.0 × 10 6 The cells/mL and the vitality is not lower than 90 percent, the cells are inoculated into a 10L reactor for fed-Batch (Feed-Batch) mode culture, the initial volume of the culture is 9L, the culture is carried out for 12 days or the vitality is lower than 75 percent, the cells are sampled every day during the culture period to detect the cell density, the vitality and the glucose concentration, and the sugar is supplemented to 4g/L in time. Samples 4, 6, 8, 10 and 12 were taken to determine the expression level of the target protein.
Culture parameters are shown in table 3, three batches of cells were cultured in parallel, as denoted F20160304, F20160405 and F20160406:
TABLE 3 cultivation parameters for 10L reactor experiments
| Control item | Parameter requirements | Mode of adjustment |
| Temperature of | 37±0.2℃ | Automatic control of heating jacket |
| pH | 6.9±0.2 | Associating CO 2 And 2mol/L sodium carbonate solution, automatically adjusting |
| Dissolved oxygen | 40％ | The fluctuation range of the related O2 and deep ventilation regulation is 10 percent |
| Rotational speed | 100-220rpm | Stepwise regulation according to cell density |
| |
5％ | Clean compressed air is introduced |
| Deep layer ventilation | 0.03lpm | Clean compressed air is introduced |
| Deep oxygen | 15-35ccm | Gradually regulating according to growth conditions |
| Deep carbon dioxide | 25-30ccm | Gradually regulating according to growth conditions |
| Foam | Not covering the surface | Defoaming by adding 10% of defoaming agent |
| Glucose concentration | ≥4g/L | Adding 30% glucose solution for regulation |
TABLE 4 supplement of feed Medium
| Days of culture | 7a supplemental volume | Supplemental volume of |
|
| 3 | | 54mL | |
| 5 | | 36mL | |
| 7 | | 36mL | |
| 9 | 360mL | 36mL |
2.2.2 the density of viable cells in three batches, as shown in FIG. 4, was as high as 6.0X 10 6 The expression amount of the target protein per mL is shown in Table 5
Expression level of protein of the order of Table 5
The expression level of the target protein is obviously higher than 30mg/L of the target protein cultured by using 10L perfusion.
EXAMPLE 3 engineering cell culture-validation of three batches of cell culture Process on 100L Scale
3.1 verification purposes
Carrying out amplification culture according to various parameters of culture medium types, feeding modes, culture modes and the like of cell culture in a 10L bioreactor, continuously carrying out three batches of 100L scale cell culture, and finally determining whether the upstream cell culture process can achieve the purpose of expected development or not, wherein the expression level of the target protein is not lower than 90mg/L during harvesting.
3.2 verification of design
When performing scale-up verification for three batches (denoted as F20161118, F20161119 and 20161220) in CHO-K1 cell culture in a 100L bioreactor, sigma culture medium was used as the basal medium and supplemented with 6%/0.6%, 4%/0.4% of 7a/7b on days 3, 5, 7 and 9. The culture parameter settings are shown in table 6 below:
TABLE 6 culture parameters for reactor experiments
3.3 Experimental results and analysis
3.3.1 cell Density analysis
The viable cell density is shown in FIG. 5, and it is clear from FIG. 5 that the three batches of viable cells all had the highest viable cell density of 3X 10 6 cell/mL, lower than 10L cell culture density.
3.3.2 comparison of cell viability
The cell viability is shown in fig. 6, and the cell viability in the three-batch cell culture process is all over 80%, which meets the verification requirements.
3.3.3 comparison of expression levels of target proteins
TABLE 6 expression level of target protein
It can be seen that the expression level of the target protein is more than 100 mug/mL in the three groups of parallel tests, and the development purpose of being more than or equal to 90 mug/mL and far higher than 30mg/L, which is originally defined by the patent, is achieved.
3.4 summary of the experiments
The three-batch 100L production process verifies that the TPO upstream culture process achieves the preset purpose from parameter control, viable cell density, cell activity and target protein. The original 10L perfusion culture mode is changed into a 100L batch culture mode smoothly, and the expected development purpose of the application is achieved.
Example 4 purification of S/D Virus-inactivated protein
4.1 MMC chromatography
In this example, according to the protein characteristics of TPO and the analysis of the culture medium components after the cell culture, considering that the culture medium is large in volume and high in salt concentration, it is not suitable to be diluted or concentrated for liquid exchange, and a large amount of ammonium sulfate and other conventional purification means are added, and after comparative analysis of a large amount of chromatography fillers, MMC Diamond is finally selected as the chromatography medium for the first step of cation chromatography.
Because, the pH of TPO fermentation liquor is 7.0 + -0.5, the conductance is 16 + -2ms, the isoelectric point of TPO is 3.5 + -1.0, and TPO has stronger hydrophobicity. Based on the relatively high conductivity of the fermentation liquid, if a conventional ion exchange medium is used, the sample needs to be diluted or buffer solution is replaced, and if the sample is diluted, the sample volume is further increased under the condition of large sample volume; such as displacement buffers, add process steps, so conventional ion exchange media are not suitable for purification under these conditions; based on that TPO has stronger hydrophobicity, the invention adopts composite weak cation media MMC Diamond and Mix A Diamond media for purification, the composite ion exchange media can combine with target protein under certain salt concentration, and as the Mix A media can adsorb a large amount of pigment, the regeneration of the media in the later period is not easy, the invention laterally reuses MMC Diamond as a first step chromatography media. In the previous process, MMC found that TPO could bind under the binding condition of pH7.0, 0.1M NaCl,20mM PB, and that TPO was negatively charged under this purification condition, but TPO had relatively strong hydrophobicity, MMC could capture target protein well, did not bind pigment and could flow through most of the impure proteins, and the purification effect was also good, so MMC chromatography conditions were further optimized as follows.
4.1.1 Experimental arrangement:
column: BXK 50/30h: 350mL
Buffer A:20mM PB, pH 6.5.0.1M NaCl cond12.48
And (3) buffer solution B:20mM PB, pH6.5 1M NaCl cond87.91
Buffer C50mM Tris pH 8.0 1M NH4Cl 2M Urea
S/D reagent: 11% Triton X100, 3.3% tributyl phosphate
Sample preparation: the fermentation broth was slowly added with stirring with 80mL of S/D reagent (pH 7.18), pH6.5 was adjusted with HCl, and Cond:14.72ms/cm, loading after 1h inactivation at room temperature (25 ℃. + -. 2).
The process is as follows: 1CV → B liquid balance (promoting the target protein and the coupling group to be tightly combined) → A liquid balance → 25mL/min of loading sample 20CV each collected sample 0.2CV → 3CV of A liquid cleaning → 3CV of B liquid cleaning → C liquid elution, and UV & gt 20mAu start collection → 1CV of A liquid cleaning → 1.5CV of 1M NaOH 2M NaCl regeneration 1.5CV
The results are shown in FIG. 7;
4.1.2 analysis and conclusion:
MMC Diamond condition: loading conditions 20mM PB 0.1M NaCl, pH 6.5. + -. 0.2, cond: 11-15 ms/cm, cleaning conditions: 1M NaCl pH6.5, elution conditions: 50mM Tris pH 8.0 1M NH 4 Cl 2M Urea. The MMC chromatography of the first step of the invention has good effect of capturing the target protein.
4.2 Butyl HP chromatography
The MMC Diamond eluent has high salt concentration, cond reaches 97ms/cm, and TPO-based hydrophobicity is strong, so that a hydrophobic filler is selected in the second step, and when TPO is captured by Butyl HP and Phenyl HP in the early stage, butyl HP has high resolution and good impurity removal effect on TPO, the hydrophobic group of Butyl HP is weaker than that of Phenyl HP, and target protein is easy to elute, so that Butyl HP is selected as a medium in the second step as a medium purification step, most impurities are removed, and the purification conditions of Butyl HP are optimized as follows.
4.2.1 Butyl HP loading salt concentration determination
The purpose is as follows: comparison of 0.6M, 0.7M, 0.8M (NH) 4 ) 2 SO 4 (hereinafter abbreviated AS AS), the sample application conditions, and the sample application conditions for Butyl HP were confirmed
4.2.1.1 experiment:
column: ezFast 1mL 2butyl HP
Buffer A1:20mM PB 0.8M AS pH 7.0Cond
Buffer B, 10mM PB, pH7.0
Buffer A2:12.5%
Buffer A3:25%
Sample preparation: 1) MMC sample 10mL was added AS 0.2M, pH7.0 was adjusted, cond:116ms/cm
2) MMC sample 10mL was added AS 0.1M, pH7.0 was adjusted, cond:105ms/cm
3) MMC sample 10mL, pH7.0 adjusted, cond:96ms/cm
4.2.1.2 And (3) HPLC detection result:
the TPO Butyl HP 0.6M/0.7M/0.8M AS elution results are shown in Table 7 below:
TABLE 7 elution results for different salt concentrations for the sample salt concentrations
| Sample information | Peak area (mAu) | Purity (C:％) |
| 0.6M AS 3-19. About.21 elution | 1132 | 61.71 |
| 0.7M AS 2-20-22 elution | 1635 | 61.90 |
| 0.8M AS 1-25 to 27 elution | 1557 | 59.32 |
4.2.1.3 HPLC analysis:
it was shown that the purity and peak area of the 0.7M AS elution were somewhat higher, with some flow-through at 8CV for 0.7M AS, no flow-through at 0.8M AS 8CV, and the best binding capacity, so both 0.7M AS and 0.8M AS could be used AS the loading conditions, and 0.6M AS was visibly evident in flow-through, so 0.6M AS was not suitable for use AS the eluent.
4.2.2 Butyl HP optimized elution conditions
4.2.2.1 purpose:
selecting elution conditions optimized by elution with different salt concentrations, selecting Cond:25ms/cm,20ms/cm,10ms/cm conductance.
4.2.2.2 Experimental procedures:
column: ezFast 1mL 2butyl HP
Buffer A1:20mM PB 0.8M AS pH 7.0Cond
Buffer B, 10mM PB, pH7.0
Buffer B1:85% by volume B (25 ms/cm)
Buffer B2:90% by volume B (20 ms/cm)
Buffer B3:95% by volume B (10 ms/cm)
Sample preparation: MMC sample 30mL was added AS 0.1M, pH adjusted 7.0, cond:105ms/cm
The process is as follows: buffer A equilibrate 10CV → Loading 5CV 10mL 0.5mL/min → liquid A Wash 8CV → liquid B elute 15CV → 100%
4.2.2.3 HPLC results:
the results are shown in FIG. 8 and in Table 8 below:
TABLE 8 comparison of results for different eluent concentrations
| Sample information | Peak area (mAu) | Purity (%) |
| 85%1-6, 7, 8 tube pooled elution | 736 | 63.69 |
| 90%1-5, 6, 7 tube pooled elution | 831 | 60.63 |
| 95%1-6, 7, 8 tube combination elution | 898 | 61.66 |
| 10mM PB (100%) control | 1635 | 61.90 |
4.2.2.4 analysis of results
The 85%, 90%, 95% elution did not differ significantly from HPLC, electrophoretic purity, and at the same time did not differ from 100%, so we considered the recovery of the elution, the 10mM PB (100%) control peak area was the largest, and we eluted with 10mM PB in one step.
4.2.1 Butyl HP final conditions:
loading conditions 20mM PB 0.7-0.8M (NH) 4 ) 2 SO 4 pH7.0, sample MMC 3M (NH) 4 ) 2 SO 4 Adjusting the difference between Cond and A liquid phase to + -2 ms/cm, loading amount to less than or equal to 6.4mg/mL medium, and washing with 20mM PB 0.7M (NH) 4 ) 2 SO 4 pH7.0, elution was performed in one step with 10mM PB.
4.3 Chromatography by Mix A Mustang (produced by Shanghai Bogelong Biotechnology Ltd.)
Based on the isoelectric point and hydrophobicity of TPO, the pH value of the purification condition is controlled to be larger than the isoelectric point, the invention emphatically screens the anion medium, and the anion medium has obvious effect on removing endotoxin. The inventor of the invention finds that the purifying effect of the conventional DEAE 6HP and Q Bestarose Fast Flow is not obvious, and most impurities cannot be removed; the invention selects the composite ionic medium MMC and the Mix A to compare, and the comparison result shows that the composite medium Mix A Diamond has obvious purification effect on TPO and can remove macromolecular impurities and micromolecular impurities. To increase the resolution of Mix a, we chose a matrix Mustang with smaller particles, with an average particle of 34 μm, so the latter phase of the invention focuses on optimizing the purification conditions for Mix a Mustang.
4.3.1 Loading pH screening
The purpose is as follows: screening binding conditions pH 6.0, 7.0, 8.0, cond:0.1M NaCl
The experimental process comprises the following steps:
column: ezFast 1mL 2mix A Mustang (same column)
Buffer A1:20mM PB 0.1M NaCl pH 6.0Cond
Buffer A2:20mM PB 0.1M NaCl pH 7.0Cond
Buffer A3:20mM Tris 0.1M NaCl pH 8.0Cond
Buffer B1:20mM PB 1M NaCl pH 6.0Cond
Buffer B2:20mM PB 1M NaCl pH 7.0Cond
Buffer B3:20mM Tris 1M NaCl pH 8.0Cond
Sample preparation: 1) Butyl HP 10mL, pH 6.0 adjusted with acetic acid
2) Butyl HP 10mL, pH7.0
3) Butyl HP 10mL, adjusted pH 8.0 with Tris
The process is as follows: buffer A equilibrate 10CV → Loading 5CV 10mL 0.5mL/min → liquid A Wash 8CV → liquid B elute 15CV → 100%
The HPLC of the detection result is shown in FIG. 9, and the SDS PAGE of the obtained protein is shown in FIG. 10.
4.3.2 analysis and results:
1) pH 6.0, SDS PAGE, HPLC showed significant impurities in the elution, pH not being suitable.
2) The purity of the elution is higher as shown by SDS PAGE and non-ammonia silver staining at pH7.0 and 8.0, no obvious impurity band is seen, and the HPLC purity is qualified.
3) pH 8.0 was chosen as the binding condition because pH 8.0 binds more readily to Mix A Mustang than pH7.0, and the resolution of gradient elution and impurities would be higher.
4.3.3 Elution at pH 8.0 Loading stage
Column: ezFast 1mL 2mix A Mustang
Buffer A20mM Tris 0.1M NaCl pH 8.0Cond
Buffer B20mM Tris 1M NaCl pH 8.0Cond
Sample preparation: 20160602 prepare Butyl HP loading, adjust pH 8.0 with 1M Tris;
the process is as follows: liquid A balance → loading 0.5mL/min,10mL → liquid A washing → 13% by weight B washing → 20% by weight B elution → 100%
4.3.4 detection results:
SDS PAGE is shown in FIG. 11.
Analysis and results:
1) Experimental cleaning 13% No target protein was observed in B, some hetero-protein was observed, and the cleaning conditions were feasible.
2) The elution conditions were not controlled, and the concentration of the solution B was increased by 50% by elution with 0.6M NaCl, whereby the macromolecular impurities eluted at 100% were avoided, and the target protein was washed completely
3) The final elution conditions were liquid A balance → loading 0.5mL/min,10mL → liquid A washing → 50% B elution → 100% B elution → 0.5M NaOH elution.
After a series of brand-new purification processes are searched, the finally confirmed protein purification conditions are as follows:
S/D inactivation → MMC Diamond → Butyl HP → Mix A Mustang → nano filtration → degerming filtration → stock solution preparation is completed.
Example 5 quality standards and quality control results
The invention is researched by a series of animal and plant source-free basal culture media with higher market awareness and recognition and fed-batch culture media, and a large amount of process parameters are researched by a cell culture shake flask, a 10L reactor and a 100L reactor, such as fed-batch proportion and O 2 、CO 2 Air, dissolved oxygen, stirring, pH, glucose concentration and the like, finally determines a proper cell culture process, and explores a set of brand-new protein purification process with high yield, high purity and low impurity, including virus inactivation and removal, through protein characteristics and combination with current industry regulations and industrialization amplification requirements.
The following Table 9 shows the results of quality control of the 100L proteins cultured according to the present invention:
TABLE 9
Comparative example 1
The experiment was repeated according to the 10L perfusion process of CN 103555760A, and 10L of perfusion was performed every day. The applicant finds that the average expression quantity is 20mg/L, the yield is 15-20%, and the protein with the purity of more than 98% obtained by culturing 100L of fermentation liquor is 300-400mg. That is, under the experimental conditions of comparative document 1, the purported following results cannot be obtained:
the obtained 50L culture solution in 5 days is co-separated to obtain rh-TPO of about 1020mg, the protein purity is 98.5%, and the activity is 3.6x10 5 U/mg。
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, while the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (14)
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) Sequentially carrying out cation chromatography, hydrophobic chromatography and anion chromatography on the inactivated fermentation liquor in the step 2) to obtain a recombinant human platelet-derived factor stock solution;
wherein, in the step 1), the basic culture medium used for the culture is EX-CELL Advanced TM CHO Fed-batch culture medium; the fed-batch culture medium is HyClone TM Cell Boost TM 7a and HyClone TM Cell Boost TM 7b；
In the step 2), reagents used in the S/D method are polyethylene glycol octyl phenyl ether and tributyl phosphate;
the method also comprises the steps of nano-membrane filtration, aseptic filtration and/or split charging of the obtained stock solution.
2. The method according to claim 1, wherein the fed-batch medium HyClone is supplemented every 1 to 3 days TM Cell Boost TM 7a and HyClone TM Cell Boost TM 7b, the addition amounts of which are respectively 4-6% and 0.4-0.6% of the culture volume.
3. The preparation method according to claim 2, wherein the fed-batch medium HyClone is supplemented every 2 days TM Cell Boost TM 7a and HyClone TM Cell Boost TM 7b 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:
(1) recovering recombinant human thrombopoietin engineered CELLs and using EX-CELL Advanced TM Culturing in CHO Fed-batch culture medium until the viable cell density is not lower than 3.0 × 10 6 Per mL, the activity is not lower than 90%;
(2) 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% TM Cell Boost TM 7a 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; 30 to 70 percent of dissolved oxygen.
5. The method of claim 4, wherein the 100L bioreactor further has the following culture parameters:
the rotating speed is 50-80rpm;
ventilating the surface layer by 10-30%;
deep ventilation 0-0.1lpm;
0-0.5lpm of deep oxygen;
0-0.4lpm of deep carbon dioxide.
6. The process according to claim 4, wherein the glucose concentration during the culture is 4 to 7g/L.
7. The preparation method according to claim 1, wherein, in step 2), the working concentration of polyethylene glycol octyl phenyl ether is 1% (V/V); the working concentration of tributyl phosphate was 0.3% (V/V).
8. 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 ℃.
9. The production method according to claim 1, wherein in the step 3), cation chromatography is performed using a composite weak cation medium MMC Diamond.
10. The production method according to claim 1, wherein 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 8.0 1M NH 4 Cl 2M Urea.
11. The preparation method according to claim 1, wherein in the step 3), hydrophobic chromatography is performed using Butyl HP packing.
12. The preparation method according to claim 1, wherein the conditions of the hydrophobic chromatography are:
sample loading conditions are as follows: 20mM phosphate buffer, 0.7-0.8M (NH) 4 ) 2 SO 4 pH7.0 conductance: 115ms/cm;
the lower bar piece: 3M (NH) 4 ) 2 SO 4 Adjusting 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 ) 2 SO 4 pH 7.0；
Elution conditions: 10mM phosphate buffer was eluted in one step.
13. The preparation process according to claim 1, wherein in the step 3), anion chromatography is performed using a MixA Mustang ion exchange packing.
14. The preparation method according to claim 1, wherein the conditions of the anion chromatography are:
loading conditions are as follows: 2 mM 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.
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1186119A (en) * | 1996-12-25 | 1998-07-01 | 吴海燕 | Recombinative human thrombocytopoietic factor and production thereof |
| CN1276382A (en) * | 2000-06-19 | 2000-12-13 | 沈阳三生制药股份有限公司 | Process for preparing recombined human thrombopoietin preparation |
| CN1408861A (en) * | 2001-09-19 | 2003-04-09 | 上海中信国健药业有限公司 | Recombination human platelet producing factor cDNA sequence and its use |
| WO2005082937A2 (en) * | 2004-02-27 | 2005-09-09 | Octapharma Ag | A method of providing a purified, virus safe antibody preparation |
| CN1752102A (en) * | 2005-09-05 | 2006-03-29 | 武汉海特生物制药股份有限公司 | Technology of preparing rat nerve growth factor using organic solvent virus deactivation method |
| KR100791502B1 (en) * | 2006-09-29 | 2008-01-03 | 한스바이오메드 주식회사 | Production methods of virus inactivated and cell-free body implant |
| CN103555760A (en) * | 2013-10-18 | 2014-02-05 | 江苏康禾生物制药有限公司 | Preparation method and preparation of recombinant human thrombopoietin |
| CN109943554A (en) * | 2017-12-21 | 2019-06-28 | 舒泰神（北京）生物制药股份有限公司 | A method of extracting factor X activator from snake venom |
| CN110590931A (en) * | 2019-09-05 | 2019-12-20 | 江苏康禾生物制药有限公司 | Method for removing and/or inactivating virus in recombinant human thrombopoietin stock solution |
-
2019
- 2019-09-05 CN CN201910837025.XA patent/CN110627892B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1186119A (en) * | 1996-12-25 | 1998-07-01 | 吴海燕 | Recombinative human thrombocytopoietic factor and production thereof |
| CN1276382A (en) * | 2000-06-19 | 2000-12-13 | 沈阳三生制药股份有限公司 | Process for preparing recombined human thrombopoietin preparation |
| CN1408861A (en) * | 2001-09-19 | 2003-04-09 | 上海中信国健药业有限公司 | Recombination human platelet producing factor cDNA sequence and its use |
| WO2005082937A2 (en) * | 2004-02-27 | 2005-09-09 | Octapharma Ag | A method of providing a purified, virus safe antibody preparation |
| CN1752102A (en) * | 2005-09-05 | 2006-03-29 | 武汉海特生物制药股份有限公司 | Technology of preparing rat nerve growth factor using organic solvent virus deactivation method |
| KR100791502B1 (en) * | 2006-09-29 | 2008-01-03 | 한스바이오메드 주식회사 | Production methods of virus inactivated and cell-free body implant |
| CN103555760A (en) * | 2013-10-18 | 2014-02-05 | 江苏康禾生物制药有限公司 | Preparation method and preparation of recombinant human thrombopoietin |
| CN109943554A (en) * | 2017-12-21 | 2019-06-28 | 舒泰神（北京）生物制药股份有限公司 | A method of extracting factor X activator from snake venom |
| CN110590931A (en) * | 2019-09-05 | 2019-12-20 | 江苏康禾生物制药有限公司 | Method for removing and/or inactivating virus in recombinant human thrombopoietin stock solution |
Non-Patent Citations (6)
| Title |
|---|
| "Production of thrombopoietin (TPO) by rat hepatocytes and hepatoma cell lines";Shimada Y 等;《Experimental Hematology》;19951201;第23卷(第13期);第1388-1396页 * |
| "rTMP-GH融合蛋白在大肠杆菌中的可溶性表达与纯化及其促血小板生成作用研究";许杨;《中国优秀硕士学位论文全文数据库 (医药卫生科技辑)》;20090315(第3期);E062-112 * |
| "Virus inactivation by solvent/detergent treatment using Triton X-100 in a high purity factor VIII";Peter L.Roberts;《Biologicals》;20080801;第36卷(第5期);第330-335页 * |
| "新型促血小板生成因子Tmax的中试表达、纯化及生物学活性检测";郎永江;《第七届全国医学生物化学与分子生物学和第四届全国临床应用生物化学与分子生物学联合学术研讨会暨医学生化分会会员代表大会论文集》;20110831;第249页 * |
| "血液制剂中的TNBP/去污剂处理";韩建军;《国外医学.输血及血液学分册》;19880229;第11卷(第1期);第86-87页 * |
| "重组人血小板生成素的分离与纯化";李文辉 等;《中国生化药物杂志》;20010430(第4期);第187-188页 * |
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