CN110627892A - Preparation method of recombinant human thrombopoietic factor stock solution - Google Patents

Abstract

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 3.0x10⁵IU/mg～3.6x10⁵IU/mg, yield close to 30%. By using the method of the present invention, a target protein can be obtained in high yield.

Description

Preparation method of recombinant human thrombopoietic factor stock solution

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.

EXAMPLE 1 culturing of engineered cell-selection Medium

The purpose of this example is to select a suitable medium.

The applicant of the application finds that the culture effect of the rhTPO engineering cells is better than that of 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 without animal-derived serum is free.

Further, the Applicant determined that the fed-batch medium was HyClone, on the basis of the selection of Merck (Sigma) as basal medium^TM Cell Boost^TM 7a，HyClone^TM Cell Boost^TM7b, in Sigma minimal Medium EX-CELL Advanced^TMThe 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^TMCompared with various culture media in Merck (Sigma), Hyclone, Gibco and BD, the culture medium can ensure reasonable collocation of basal culture media, prolong the cell growth cycle, improve the cell expression level, 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 10⁶The cell count/mL, the cell activity is not less than 95%, and the culture time is not more than 16 days; when the shake flask culture is carried out, the harvest time is shortThe protein expression amount of (a) is not less than 80 mg/L.

1.2 design of the experiment

TABLE 2 incubator parameter settings

CO2Concentration 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/L⁶The 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 the 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 10⁶When the cell count/m and the cell activity are not less than 95 percent, taking part of the cells to passage to 40mL/125mL, namely taking 40mL of the cells in a 125mL shake flask to culture, wherein the living cell density is 1.0 multiplied by 10⁶And (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 total/viable cell density, calculating viability, and time as abscissaThe density of the living cells in each day is plotted by a vertical coordinate, and the density change of the living cells is observed; 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 x10 at the highest value⁶The 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 graphs of the activity of three groups of experimental cells along with the change of culture time are shown in figure 2. As can be seen from FIG. 2, the three groups of experiments showed consistent trend of viability, which indicates that the culture process was relatively stable, and the cell viability was maintained for more than 95% of the time up to 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 summary, the recovery subculture of rh-TPO engineering cells in Sigma medium was 16 days oldThe internal culture volume can reach 1200mL, and the viable cell density is not less than 4.0 x10⁶The cell viability is not less 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 taken as a basic culture medium, and the culture medium 7a/7b is taken as an additive 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 the 100L batch mode, and further to solve the problems of complicated 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, using Sigma culture medium to culture and expand to 2.4L in a shake flask, wherein the density of the living cells is not less than 3.0 multiplied by 10⁶seed/mL, activity not less than 90%, inoculating into 10L reactorThe culture is carried out in a fed-Batch (Feed-Batch) mode, the initial volume of the culture is 9L, the culture is carried out for 12 days or the vitality is lower than 75%, and a sample is taken 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 310L culture parameters for 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 CO2And 2mol/L sodium carbonate solution, automatically adjusting
			Dissolved oxygen	40％	The correlation between O2 and deep ventilation regulation requires a fluctuation range of 10%
Rotational speed	100-220rpm	Stepwise regulation according to cell density
			Surface layer aeration	5％	Clean compressed air is introduced
Deep layer ventilation	0.03lpm	Clean compressed air is introduced
			Deep oxygen	15-35ccm	According to the growth condition, gradually regulating
Deep carbon dioxide	25-30ccm	According to the growth condition, gradually regulating
			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 7b
			3	540mL	54mL
5	360mL	36mL
			7	360mL	36mL
9	360mL	36mL

2.2.2 the density of viable cells in three batches is shown in FIG. 4, up to 6.0X 10⁶The expression amount of the target protein per mL is shown in Table 5

TABLE 5 protein expression level of mesh

The expression level of the target protein is obviously higher than 30mg/L of the target protein cultured by using 10L perfusion.

Example 3 culture of engineered cells-verification of three-batch Process for 100L Scale cell culture

3.1 purpose of verification

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 designs

When performing three-batch scale-up verification (denoted as F20161118, F20161119 and 20161220) of 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 densities are shown in FIG. 5, and it is clear from FIG. 5 that the viable cell densities of the three batches were all at 3X 10 when the viable cell densities were the highest⁶cell/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

Through the verification of three batches of 100L production processes, the TPO upstream culture process achieves the preset aims 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 the 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 + -2 ms, 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 type weak cation media MMC Diamond and Mix ADiamond media to purify, the composite type ion exchange media can combine with target protein under certain salt concentration, and because MixA series media can adsorb a large amount of pigments and is not easy to regenerate the media in the later period, the invention emphasizes using MMCDiamond as the first step of chromatography media. In the previous process research of MMC, TPO can be combined under the combination conditions of pH7.0, 0.1M NaCl and 20mM PB, TPO is negatively charged under the purification condition, but TPO has stronger hydrophobicity, MMC can well capture target protein, does not combine pigments and can flow through most of mixed protein, the purification effect is also good, and the following optimization is further carried out on the MMC chromatography condition.

4.1.1 Experimental arrangement:

column: BXK 50/30h 17.5cm volume: 350mL

And (3) buffer solution A:20mM PB pH 6.50.1M NaCl cond12.48

And (3) buffer solution B:20mM PB pH 6.51M NaCl cond87.91

Buffer C50 mM Tris pH 8.01M 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 (pH7.18), 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: 1M NaOH 2M NaCl treatment column 1CV → B liquid balance (promote the target protein and coupling group to combine tightly) → A liquid balance → loading 25mL/min 20CV each collected sample 0.2CV → A liquid washing 3CV → B liquid washing 3CV → C liquid elution, UV > 20mAU begin to collect → A liquid washing 1CV → 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, pH6.5 ± 0.2, Cond: 11-15 ms/cm, and the cleaning conditions are as follows: 1M NaCl pH6.5, elution conditions: 50mM Tris pH 8.01M NH₄Cl 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)₂SO₄(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 pH7.0 Cond 115ms/cm

Buffer B10 mM PB pH7.0

Buffer A2: 12.5% B

Buffer A3: 25% B

Sample preparation: 1) MMC sample 10mL AS 0.2M was added, pH7.0 adjusted, Cond: 116ms/cm

2) MMC sample 10mL AS 0.1M was added, pH7.0 adjusted, Cond: 105ms/cm

3) MMC sample 10mL, pH7.0 adjusted, Cond: 96ms/cm

4.2.1.2 HPLC assay:

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 (%)
			0.6M AS 3-19-21 elution	1132	61.71
0.7M AS 2-20-22 elution	1635	61.90
			0.8M AS 1-25-27 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 was somewhat higher, with 0.7M AS having somewhat flow-through at 8CV, 0.8M AS 8CV having no flow-through, and best binding capacity, so both 0.7M AS and 0.8M AS could be used AS the loading conditions, and 0.6MAS was visibly evident in flow-through, so 0.6M AS was not suitable 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 pH7.0 Cond 115ms/cm

Buffer B10 mM PB pH7.0

Buffer B1: 85% B (25ms/cm)

Buffer B2: 90% B (20ms/cm)

Buffer B3: 95% B (10ms/cm)

Sample preparation: MMC sample 30mL AS 0.1M was added, pH7.0 was adjusted, Cond: 105ms/cm

The process is as follows: buffer A equilibration 10CV → loading 5CV 10mL 0.5mL/min → liquid A Wash 8CV → liquid B Wash 15CV → 100% B elution 10CV

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 combined elution	736	63.69
90% 1-5, 6, 7 tubes combined elution	831	60.63
			95% 1-6, 7, 8 tube combined 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:

the loading condition is 20mM PB 0.7-0.8M (NH)₄)₂SO₄pH7.0, sample MMC 3M (NH)₄)₂SO₄Adjusting 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)₂SO₄pH7.0, elution was performed in one step with 10mM PB.

4.3 Mix A Mustang (produced by Shanghai Bogelong Biotechnology Co., Ltd.) chromatography

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; according to the invention, a composite ionic medium MMC and a composite ionic medium Mix A are selected for comparison, and the comparison result shows that the composite medium Mix A Diamond has an obvious TPO purification effect 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 sample Loading pH value 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 12.70ms/cm

Buffer A2 20mM PB 0.1M NaCl pH7.0 Cond 12.92ms/cm

Buffer A3:20mM Tris 0.1M NaCl pH 8.0Cond:12.45ms/cm

Buffer B1 20mM PB 1M NaCl pH 6.0Cond 88.24ms/cm

Buffer B2 20mM PB 1M NaCl pH7.0 Cond 90.14ms/cm

Buffer B3:20mM Tris 1M NaCl pH 8.0Cond:87.57ms/cm

Sample preparation: 1) butyl HP 10mL, pH 6.0 adjusted with acetic acid

2) Butyl HP 10mL, pH7.0 adjusted

3) Butyl HP 10mL, adjusted pH 8.0 with Tris

The process is as follows: buffer A equilibration 10CV → loading 5CV 10mL 0.5mL/min → liquid A Wash 8CV → liquid B Wash 15CV → 100% B elution 10CV

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 suitable.

2) The pH values are 7.0 and 8.0, SDS PAGE and non-ammonia silver staining show that the purity of elution is higher, 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 pH 8.0 elution in the Loading stage

Column: EzFast 1mL 2Mix A Mustang

Buffer A20 mM Tris 0.1M NaCl pH 8.0Cond 12.45ms/cm

Buffer B20 mM Tris 1M NaCl pH 8.0Cond 87.57ms/cm

Sample preparation: 20160602 prepare a Butyl HP loading, adjust pH 8.0 with 1M Tris;

the process is as follows: liquid A balance → 0.5mL/min of loading, 10mL → liquid A wash → 13% B wash → 20% B elution → 100% B elution → 0.5M NaOH elution

4.3.4 detection result:

SDS PAGE is shown in FIG. 11.

Analysis and results:

1) in the experiment, 13% of B has no target protein, some hybrid protein can be seen, and the cleaning condition is feasible.

2) The elution condition is not well controlled, the concentration of the B solution can be increased, and the B solution is eluted by 0.6M NaCl, namely 50 percent of B, so that macromolecular impurities eluted by 100 percent of B can be avoided, and the target protein can be completely washed

3) The final elution conditions were liquid A equilibration → loading 0.5mL/min, 10mL → liquid A wash → 50% B elution → 100% B elution → 0.5M NaOH elution.

After a series of brand new purification processes, 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 passes through a series of animal and plant-free sources with higher market awareness and recognitionBasic culture medium, feeding culture medium, cell culture shake flask, 10L reactor, and 100L reactor for searching a large number of technological parameters, such as feeding proportion, O₂、CO₂Air, 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 protein cultured in 100L of the present invention:

TABLE 9

Comparative example 1

The experiment was repeated according to the 10L perfusion process of invention No. CN 103555760A, and 10L of perfusion was administered daily. The applicant finds that the average expression amount is 20mg/L, the yield is 15-20%, and the protein with the purity of more than 98% obtained by culturing 100L fermentation broth is 300-400 mg. That is, under the experimental conditions of comparative document 1, the following results cannot be obtained as claimed:

the total separation of 50L culture solution obtained in 5 days to obtain rh-TPO of about 1020mg, protein purity of 98.5%, and activity of 3.6x10⁵U/mg。

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although 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 (10)

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.