Method for removing recombinant human thrombopoietin aggregates by means of superose 12 molecular sieve tandem column chromatography

Abstract

The present invention relates to the field of biopharmaceuticals. Disclosed is a method for removing recombinant human thrombopoietin aggregates by means of Superose 12 molecular sieve tandem column chromatography. The method of the present invention comprises the following steps: subjecting a harvest solution of recombinant human thrombopoietin expressed by CHO cells to ultrafiltration and concentration and then sequentially to cation exchange chromatography, first anion exchange chromatography, reversed-phase chromatography, and second anion exchange chromatography, followed by concentration and buffer exchange to obtain a concentrated intermediate; and loading the concentrated intermediate onto two Superose 12 molecular sieve chromatographic columns horizontally connected in tandem, and then using an eluent for elution, and collecting a target protein. The method of the present invention increases the sample loading capacity, reduces the frequency of sample loading, improves the working efficiency, has a degree of sample resolution of 1.5 or more and a yield and purity both higher than those of existing chromatography methods using a single column, and reduces the packing height in a single column and the column pressure, thereby reducing the pressure resistance requirements for the chromatographic column and chromatographic equipment, achieving the scale-up of gel chromatography. The method is suitable for all processes involving separating and purifying macromolecules by means of gel chromatography.

Description

Method for removing recombinant human thrombopoietin polymer by Superose 12 molecular sieve series column chromatography

[0001] The present application claims priority from patent application No. CN202410736902.5 filed on June 7, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of biopharmaceuticals, in particular to a method for removing recombinant human thrombopoietin polymer by Superose 12 molecular sieve series column chromatography. BACKGROUND

[0003] Human thrombopoietin (hTPO) is a cytokine that stimulates the growth, maturation and differentiation of platelet precursors-megakaryocytes, and is a glycoprotein with a molecular weight of 70-100 kilodaltons. As a normal hormone, it can stimulate the proliferation and differentiation of megakaryocyte precursor cells, and promote the maturation of megakaryocytes, making them larger and polyploid, and further producing platelets. Therefore, it can be used clinically to treat platelet reduction caused by various reasons. Traditional purification methods for human thrombopoietin, such as high-performance liquid chromatography and gel chromatography for purifying thrombopoietin in Chinese patent CN1137897C, require multiple times of sample loading on the gel chromatography column, and multiple times of collection of protein peaks for combination as the stock solution. Finally, 260 mg of protein is obtained from 40 L of culture solution, which has the problems of small production scale, limited sample loading, difficult scale-up, low efficiency, and low yield.

[0004] Gel chromatography, as a separation technology, can separate by molecular size. In the large-scale production of human thrombopoietin, gel chromatography cannot follow the linear scale-up principle. Even if the principle of constant column bed height is followed, in order to ensure good separation effect, a relatively high packing height is required, and the increase of packing height will cause the increase of back pressure, which will cause pressure on the chromatography column and chromatography equipment, limit the process flow rate, and prolong the purification process. For a chromatography column with a height of 1 meter, if the compression coefficient is 1.15, the actual height used is 43.5-60.9 cm, which cannot achieve the separation effect of the gel chromatography for separation and purification. If a column filler is used, the overall height of the chromatography column will be increased, and the height of the operation space will be limited. Moreover, the sample loading capacity of the gel chromatography for separation and purification is only 1%~5% of the column volume, and the sample processing capacity is limited. SUMMARY

[0005] To solve the above problems, the purpose of the present application is to provide a method for removing recombinant human thrombopoietin polymer by Superose 12 molecular sieve series column chromatography, which optimizes the purification yield, improves the purification efficiency, and realizes the large-scale preparation of pure samples.

[0006] The present application also aims to provide a recombinant human thrombopoietin purified by the above method.

[0007] To achieve the above object, the present application adopts the technical scheme of:

[0008] The first aspect of the present application provides a method for removing recombinant human thrombopoietin polymers by Superose 12 molecular sieve column chromatography, comprising the following steps:

[0009] (1) After the CHO cell-expressed recombinant human thrombopoietin harvest liquid is ultrafiltrated and concentrated, it is sequentially subjected to cation exchange chromatography, first anion exchange chromatography, reverse phase chromatography, and second anion exchange chromatography to obtain a concentrated intermediate.

[0010] (2) The concentrated intermediate is loaded onto two horizontally connected Superose 12 molecular sieve chromatography columns, then eluted with an eluent and the target protein is collected.

[0011] The present application first obtains a concentrated intermediate by preliminarily purifying the CHO cell-expressed recombinant human thrombopoietin harvest liquid, and then purifies it by using two connected Superose 12 molecular sieve chromatography columns to remove the recombinant human thrombopoietin polymers in the recombinant human thrombopoietin harvest liquid. The Superose 12 molecular sieve filler is a spherical agarose gel prepared by twice cross-linking, and the cross-linking degree is 12%. The high cross-linking structure significantly improves the particle rigidity and the physicochemical stability, the particle size is small and the particle size distribution range is narrow, so it belongs to high-efficiency gel, is pressure-resistant, can be used at high flow rate, and has the characteristics of wide separation range and high resolution. It can effectively remove polymer substances with large molecular differences, and replace the buffer system with a stock solution stable system. Compared with other gel chromatography fillers, the Superose 12 molecular sieve filler has a better separation effect on recombinant human thrombopoietin polymers, and the connected chromatography columns reduce the packing height of a single column, and the filler does not collapse during the separation process, and can be used for a long time.

[0012] Considering the production cost and actual production, the present application uses two Superose 12 molecular sieve chromatography columns connected horizontally, which reduces the packing height of a single chromatography column, reduces the column pressure, reduces the pressure resistance requirement of the chromatography column and the chromatography equipment, and realizes the scale-up of chromatography. By using the method of the present application, the yield of recombinant human thrombopoietin is more than 70%, which is higher than the separation yield of the existing single gel column. The present application improves the resolution of gel chromatography purification to more than 1.5, realizes the separation of polymer impurities and target proteins, and the purity is more than 99%. The present application increases the sample loading volume, reduces the sample loading times, and improves the work efficiency.

[0013] To ensure the separation and purification effect of the chromatography column, preferably, the theoretical plate height of the two Superose 12 molecular sieve chromatography columns is less than or equal to 0.06 cm, and the symmetry is between 0.8 and 2.0. Further preferably, the theoretical plate height of the two Superose 12 molecular sieve chromatography columns is between 0.05 and 0.059 cm, and the symmetry is between 1.3 and 1.6.

[0014] Preferably, the packing height of the packing material in the two Superose 12 molecular sieve chromatography columns is greater than or equal to 50 cm, and the compression factor is 1.15. The packing material is Superose TM 12 prep grade.

[0015] To ensure the packing quality and improve the separation and purification effect, the packing of the Superose 12 molecular sieve chromatography column in the method of the present application comprises the following steps: pouring the packing material suspension into the chromatography column, and pressurizing the column to the gel surface to be stable at a linear flow rate of not greater than 25 cm / h, then continuing to pressurize the column at a constant pressure, the pressure range is between 0.3 and 0.45 MPa, until the gel surface is stable, disconnecting the connection with the chromatography system, manually rotating the column head to 1 cm below the gel surface, and the packing is completed. The concentration of the packing material in the packing material suspension is between 50% and 70%. Preferably, the packing material suspension is obtained by suspending the packing material in a 0.01 M NaOH-0.2 M NaCl mixed solution. Preferably, the linear flow rate is between 20 and 25 cm / h.

[0016] The sample loading speed and the sample loading volume jointly determine the distribution and diffusion of the sample in the chromatography column. To further improve the separation effect, preferably, the sample loading flow rate of the concentrated intermediate is less than or equal to 0.6 cm / min, and the sample loading volume is less than or equal to 5% of the total volume of the series-connected columns.

[0017] Preferably, the eluent is a PBS solution with a pH of 6.0±0.2. The PBS solution contains 0.01 M PB and 0.18 M NaCl. The target protein is collected using the eluent, and the second peak in the chromatogram is the target protein. Specifically: observing the chromatogram, when the baseline of OD 280 falls to about 2 / 5 of the total volume of the series-connected columns, the first peak begins to rise until the peak falls, which is the impurity peak of the elution aggregate, and is not collected; when the baseline of OD 280 rises again and is higher than 2 mAU of the highest peak value of the impurity peak, the target protein begins to be collected, and the collection is ended when the baseline is stable after the peak falls.

[0018] Preferably, before the concentrated intermediate is loaded, the Superose 12 molecular sieve chromatography column is equilibrated with the eluent at a flow rate of less than or equal to 0.6 cm / min for 1 to 2 column volumes. The equilibration method is the conventional method in the prior art.

[0019] In the method of the present application, step (1) is the primary purification of the recombinant human thrombopoietin harvest liquid. The primary purification can remove some process impurities of the recombinant human thrombopoietin, such as host proteins, nucleic acids, culture medium components, shear bodies, modified bodies and other impurities. However, some polymers will be produced after the second step of anion concentration and liquid exchange. Preferably, the method comprises the following steps:

[0020] 1) The CHO cell-expressed recombinant human thrombopoietin harvest liquid is concentrated and exchanged into a citric acid buffer at pH 6.0±0.2 through a 30KD ultrafiltration membrane to obtain intermediate A;

[0021] 2) The intermediate A is purified by a cation exchange chromatography column with a filler of carboxymethyl agarose to obtain intermediate B,

[0022] 3) The intermediate B is purified by an anion chromatography column with a filler of quaternary amine agarose to obtain intermediate C;

[0023] 4) The intermediate C is purified by a reverse phase chromatography column with a filler of C4 to obtain intermediate D;

[0024] 5) The intermediate D is concentrated and exchanged by an anion exchange chromatography column with a filler of quaternary amine agarose to obtain a concentrated intermediate.

[0025] Further preferably, in step 2), the chromatography column is equilibrated with a citric acid buffer at pH 6.0±0.2, the impurities are eluted with a citric acid buffer containing 0.1mol / L NaCl at pH 6.0±0.2, and the target protein intermediate B is eluted with a citric acid buffer containing 0.25mol / L NaCl at pH 6.0±0.2. In step 3), the chromatography column is equilibrated with a Tris-HCl buffer at pH 7.0±0.2, the impurities are eluted with a Tris-HCl buffer containing 0.04mol / L NaCl at pH 7.0±0.2, and the target protein intermediate C is eluted with a Tris-HCl buffer containing 0.25mol / L NaCl at pH 7.0±0.2. In step 4), the chromatography column is equilibrated with a Tris-HCl buffer containing 5% isopropanol at pH 6.5±0.2, the impurities are eluted with a Tris-HCl buffer containing 40% isopropanol at pH 6.5±0.2, and the target protein intermediate D is eluted with a Tris-HCl buffer containing 50% isopropanol at pH 6.5±0.2. In step 5), the chromatography column is equilibrated with a Tris-HCl buffer at pH 7.0±0.2, and the target protein intermediate D is eluted with a Tris-HCl buffer containing 0.25mol / L NaCl at pH 7.0±0.2.

[0026] After the preliminary purification of step (1), the recombinant human thrombopoietin polymer impurity content in the concentrated intermediate is greater than 15%, and the protein concentration of the recombinant human thrombopoietin concentrated intermediate is not less than 1.0 mg / mL. Further preferably, the specific impurity content of the recombinant human thrombopoietin is 16.42-24.64%, and the protein concentration is 2.5-3.21 mg / mL.

[0027] The second aspect of the present application provides a method for purifying recombinant human thrombopoietin obtained by removing the polymer of recombinant human thrombopoietin using the above-mentioned Superose 12 molecular sieve column chromatography.

[0028] According to the present application, the recombinant human thrombopoietin (rhTPO) comprises a sequence of 332 amino acids as shown in SEQ ID NO: 1.

[0029] SEQ ID NO: 1

[0030] The rhTPO also includes variants thereof, and the mutation modes include but are not limited to insertion, deletion or substitution, as long as the variants maintain the biological activity of thrombopoietin, such as increasing the number and function of platelets, synergizing with other hematopoietic factors, and promoting the proliferation and differentiation of megakaryocytes. The rhTPO variants include but are not limited to the TPO variant sequences disclosed in CN1076356C and CN105541994B, such as TPO 1-163, TPO 1-232, TPO 1-151, and TPO with 1-6 amino acids deleted from the N-terminus.

[0031] According to the present application, the rhTPO polymer is defined as a molecular complex formed by 2 or more rhTPO monomers through covalent (such as abnormal disulfide bond connection) or non-covalent interaction (such as hydrophobic interaction), which has a molecular weight greater than the monomer form, and can be quantitatively analyzed by SEC-HPLC and the like. Such polymers are impurities generated during production process or storage, and their content needs to be controlled according to ICH guidelines to ensure product safety and effectiveness. Specifically, when the purity of the target protein is detected by SEC-HPLC, the impurities eluted before the target protein at a retention time of 12 min ± 1 min are collectively referred to as polymer macromolecules. According to the purification of human TPO from the CHO cell line producing TPO described in patent CN 105541994 B, the fractionation of TPO activity determined by gel filtration has a wide molecular weight range, wherein F1 mainly contains TPO molecules with a molecular weight of 66KD-100KD, F2 mainly contains TPO molecules with a molecular weight of 32KD-60KD, and F3 mainly contains TPO molecules with a molecular weight of 32KD-42KD, and all TPO molecules have TPO activity. The TPO molecules with a molecular weight of 66-100KD are subjected to N-terminal amino acid sequence analysis, which has the amino acid sequence of the protein encoded by the human TPO gene, and subjected to enzyme digestion experiment, it is found that the expected theoretical molecular weight is consistent, the molecular weight of the polypeptide part of TPO is about 36KD, and it is a glycoprotein with N- and O-linked sugar chains. Due to the difference in culture process, the degree of glycosylation will be different, in addition to the influence of production and storage process, the sugar chain also exists degradation, thus forming proteins of different molecular weight levels. The present application separates and extracts the target protein with a molecular weight of 70-120KD, and defines the macromolecules above this molecular weight as polymer macromolecules, and defines the degradation fragments below this molecular weight as degradation fragments.

[0032] After removing the recombinant human thrombopoietin polymer and other impurities in the recombinant human thrombopoietin harvest liquid expressed by CHO cells by the method of the present application, the target protein collected is subjected to conventional treatment techniques such as nanofiltration and sterilization filtration, and the recombinant human thrombopoietin meeting the market standard is obtained.

[0033] The method of the present application increases the sample load, reduces the number of sample loading, improves the work efficiency, and improves the sample separation degree to more than 1.5. The yield and purity are higher than those of the existing single column chromatography method, the packing height of the single column is reduced, the column pressure is reduced, the pressure resistance requirement of the chromatography column and the chromatography equipment is reduced, the scale of gel chromatography is enlarged, and the method is suitable for all processes of separating and purifying macromolecules by gel chromatography. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly describe the technical solutions of the present application, a brief introduction will be made below in combination with the drawings. These drawings are only used to show the preferred embodiments and are not considered as limiting the present application.

[0035] Figure 1 is a schematic diagram of the connection of the series column;

[0036] Figure 2A is a single column separation and purification diagram in Example 1;

[0037] Figure 2B is a series column separation and purification diagram in Example 1;

[0038] Figure 2C is a single column HPLC purity profile in Example 1;

[0039] Figure 2D is a series column HPLC purity profile in Example 1;

[0040] Figure 3A is a single column separation and purification diagram in Example 2;

[0041] Figure 3B is a series column separation and purification diagram in Example 2;

[0042] Figure 3C is a single column HPLC purity profile in Example 2;

[0043] Figure 3D is a series column HPLC purity profile in Example 2;

[0044] Figure 4A is a series column separation and purification diagram in Example 3;

[0045] Figure 4B is a series column HPLC purity profile in Example 3. DETAILED DESCRIPTION

[0046] In order to further understand the present application, specific examples are described, it must be pointed out that the equipment and reagents used in the following examples are all commercially available conventional products.

[0047] The method for removing recombinant human thrombopoietin polymers by using Superose 12 molecular sieve series column chromatography in the present application comprises the following steps: (1) after the CHO cell-expressed recombinant human thrombopoietin harvest solution is ultrafiltrated and concentrated, it is sequentially subjected to cation exchange chromatography, first anion exchange chromatography, reverse phase chromatography, and second anion exchange chromatography to obtain a concentrated intermediate; (2) the concentrated intermediate is loaded onto two horizontally connected Superose 12 molecular sieve chromatography columns, and then eluted with an eluent and the target protein is collected.

[0048] The two horizontally connected Superose 12 molecular sieve chromatography columns are two Superose 12 molecular sieve chromatography columns connected horizontally, and the specific series connection mode is shown in Figure 1. The two chromatography columns are chromatography column 1 and chromatography column 2. Chromatography column 1 and chromatography column 2 are connected by a pipeline, one end of the pipeline is connected to a four-way valve at the outlet end of chromatography column 1 by a chuck, and the other end is connected to a four-way valve at the inlet end of chromatography column 2 by a chuck.

[0049] In the method for removing recombinant human thrombopoietin polymer according to the present application, the order of chromatographic column 1 and chromatographic column 2 and the packing height can be adjusted according to the actual situation as long as the desired separation effect is achieved. In the specific embodiment of the present application, chromatographic column 1 is in front of chromatographic column 2, and the concentrated intermediate is loaded into chromatographic column 1. After loading, the solution flows in the direction shown by the arrow in FIG. 1. In the specific embodiment of the present application, the packing height of chromatographic column 1 is higher than that of chromatographic column 2, and preferably, the packing height of chromatographic column 1 is 5-6 cm higher than that of chromatographic column 2. The packing height of chromatographic column 1 is 65-90 cm, and the packing height of chromatographic column 2 is 60-85 cm. More preferably, the packing height of chromatographic column 1 is 90 cm, and the packing height of chromatographic column 2 is 85 cm; or the packing height of chromatographic column 1 is 87 cm, and the packing height of chromatographic column 2 is 81 cm; or the packing height of chromatographic column 1 is 65 cm, and the packing height of chromatographic column 2 is 60 cm.

[0050] After the packing of the two Superose 12 molecular sieve chromatographic columns is completed, 0.01 M NaOH-0.2 M NaCl mixed solution is used to equilibrate the columns to the baseline stability, 1% column volume of 2% acetone sample is loaded, column efficiency determination is performed, the theoretical plate height is ≤0.06 cm, and the symmetry is 0.8-2.0.

[0051] In the specific embodiment of the present application, the two Superose 12 molecular sieve chromatographic columns are equilibrated with the eluent before the concentrated intermediate is loaded. The specific equilibration method comprises the following steps: rotating the four-way valve at the inlet end of chromatographic column 1 to the exhaust state, pumping the eluent into the chromatographic system at a flow rate of ≤0.6 cm / min until there is no bubble in the liquid inlet pipeline of chromatographic column 1; opening the four-way valve at the outlet end of chromatographic column 1 to the liquid outflow state, rotating the four-way valve at the inlet end of chromatographic column 1 to the liquid inflow state until there is no bubble in the liquid inlet pipeline of chromatographic column 2; opening the four-way valve at the outlet end of chromatographic column 2 to the liquid outflow state, rotating the four-way valve at the inlet end of chromatographic column 2 to the liquid inflow state, and ending the exhaust.

[0052] The chromatographic yield is calculated according to the protein amount of the collected target protein divided by the above-mentioned protein amount. The collected target protein peak is detected by HPLC-SEC for purity, and the peak area percentage of each peak is calculated by chromatographic software integration and peak area normalization method. The peak before the retention time of the target protein peak is defined as a polymer, and the peak after the retention time is defined as a degradation peak. The removal and purification effects of the process are compared according to the ratio of each component.

[0053] Example 1

[0054] Separation purification and HPLC purity detection were carried out using a single column (old process) with specification XK26 / 100 and the serial column (new process) of the present application

[0055] 1.1, obtaining of the concentrated intermediate

[0056] The specific process is as follows:

[0057] 1) The cell harvest liquid expressed by CHO cells was concentrated and exchanged to 0.02M CB citric acid buffer with pH 6.0±0.2 through 30KD ultrafiltration membrane, and intermediate A was obtained;

[0058] 2) The cation exchange chromatography column with carboxymethyl agarose as filler was equilibrated with 0.02M CB citric acid buffer with pH 6.0±0.2, and after intermediate A was loaded, it was equilibrated with 0.02M CB citric acid buffer with pH 6.0±0.2, and then eluted with 0.02M CB citric acid buffer with pH 6.0±0.2 containing 0.1mol / L NaCl to remove impurities, and finally eluted with 0.02M CB citric acid buffer with pH 6.0±0.2 containing 0.25mol / L NaCl to obtain the target protein, and intermediate B was obtained;

[0059] 3) The anion chromatography column a with quaternary amine agarose as filler was equilibrated with 0.02M Tris-HCl buffer with pH 7.0±0.2, and after intermediate B was loaded, it was equilibrated with 0.02M Tris-HCl buffer with pH 7.0±0.2, and then eluted with 0.02M Tris-HCl buffer with pH 7.0±0.2 containing 0.04mol / L NaCl to remove impurities, and finally eluted with 0.02M Tris-HCl buffer with pH 7.0±0.2 containing 0.25mol / L NaCl to obtain the target protein, and intermediate C was obtained;

[0060] 4) The reverse phase chromatography column with C4 as filler was equilibrated with 0.01M Tris-HCl buffer with pH 6.5±0.2 containing 5% isopropanol, and after intermediate C was loaded, it was equilibrated with 0.01M Tris-HCl buffer with pH 6.5±0.2 containing 5% isopropanol, and then eluted with 0.01M Tris-HCl buffer with pH 6.5±0.2 containing 40% isopropanol to remove impurities, and finally eluted with 0.01M Tris-HCl buffer with pH 6.5±0.2 containing 50% isopropanol to obtain the target protein, and intermediate D was obtained;

[0061] 5) Equilibrate the agarose anion exchange chromatography column b (filled with quaternary ammonium compounds) with 0.02M Tris·HCl buffer at pH 7.0±0.2. Load intermediate D onto the column and equilibrate with 0.02M Tris·HCl buffer at pH 7.0±0.2. Elute the target protein with 0.02M Tris·HCl buffer containing 0.25 mol / L NaCl at pH 7.0±0.2 to obtain the concentrated intermediate. The concentrated intermediate contained 23.4% recombinant human thrombopoietin polymer impurities and had a protein concentration of 2.50 mg / mL.

[0062] 1.2 Gel Chromatography

[0063] Specifically, the following processes are included:

[0064] 1.2.1 Column Installation

[0065] Superose 12 gel (Superose) was used as the filler. TM Two XK26 / 100 chromatography columns (prep grade 12) were packed to a height of 90 cm for column 1 and 85 cm for column 2, with a packing concentration of 70% and a compressibility factor of 1.15. A packing suspension (theoretically 785 ml and 742 ml for the two columns, respectively) was poured into the columns. A two-stage pressure loading method was used, with a constant flow rate of 25 cm / h until the gel surface stabilized. Then, a constant pressure of 0.45 MPa was maintained until the gel surface stabilized. The column was then disconnected from the chromatography system, and the column head was manually rotated to 1 cm below the gel surface. Packing was then complete. The column was equilibrated with 0.01M NaOH-0.2M NaCl solution until the baseline was stable. 1% column volume of 2% acetone sample was loaded, and the column efficiency was determined. The theoretical plate heights of chromatography column 1 and chromatography column 2 were 0.050 cm and 0.052 cm, respectively, and the symmetries were 1.3 and 1.4, respectively.

[0066] After packing, the two chromatography columns were connected in series using tubing, as shown in Figure 1. Then, the connected columns (column 1 and column 2) were equilibrated with a PBS solution of 0.01 MPa-0.18 M NaCl (pH 6.0 ± 0.2) at a flow rate of 0.6 cm / min and an equilibration volume of twice the column volume until the baseline and conductivity stabilized.

[0067] 1.2.2 Sample loading and elution

[0068] use The concentrated intermediate was loaded onto the column 1 of the avant 150 chromatography system according to the column volume of ≤5% at a flow rate of 0.6 cm / min. After the loading was completed, the column was eluted with 0.01 M PB-0.18 M NaCl PBS solution, and the chromatogram was observed (as shown in FIG. 2B). When the baseline of the OD 280 The first peak began to appear when the baseline of the OD 280 The baseline was raised again, and the target protein was collected when the baseline was higher than the highest peak of the impurity protein by 2 mAU. The collection was completed when the baseline was stable after the peak fell. The HPLC purity of the collected target protein was determined, and the test results are shown in FIG. 2D.

[0069] 1.3 Single Column Control (Old Process)

[0070] The chromatography column 1 was packed and equilibrated, and the concentrated intermediate obtained in 1.1 was subjected to gel chromatography according to the loading and elution method of 1.2.2. The chromatogram is shown in FIG. 2A, and the HPLC purity of the collected target protein is shown in FIG. 2C.

[0071] The improved purification results compared with the results before the improvement are shown in the following table:

[0072] Example 2

[0073] Separation and purification and HPLC purity detection using a single column with specifications of INDEX 70 / 950 (old process) and the tandem column of the present application (new process)

[0074] 2.1 Obtaining of the Concentrated Intermediate

[0075] The CHO cell-expressed cell harvest liquid was processed according to the process of 1.1 to obtain a concentrated intermediate with a recombinant human thrombopoietin polymer impurity content of 16.42% and a protein concentration of 3.21 mg / mL.

[0076] 2.2 Gel Chromatography

[0077] Specifically includes the following processes:

[0078] 2.2.1 Column Packing

[0079] The column was packed with Superose 12 gel (Superose TMTwo INDEX70 / 950 chromatography columns (prep grade 12) were packed to a height of 87 cm for column 1 and 81 cm for column 2, with a packing concentration of 70% and a compressibility factor of 1.15. A packing suspension (theoretical volumes of 5498 ml and 5119 ml for the two columns, respectively) was poured into the columns, prepared by mixing the packing material with a 0.01 M NaOH-0.2 M NaCl mixed solution. The column was then pressurized using a two-stage gel pressing method, maintaining a constant flow rate of 25 cm / h until the gel surface stabilized. This was followed by a constant pressure of 0.3 MPa until the gel surface stabilized. The column was then disconnected from the chromatography system, and the column head was manually rotated to 1 cm below the gel surface. Packing was then complete. The column was equilibrated with 0.01M NaOH-0.2M NaCl solution until the baseline was stable. 1% column volume of 2% acetone sample was loaded, and the column efficiency was determined. The theoretical plate heights of chromatography column 1 and chromatography column 2 were 0.057 cm and 0.058 cm, respectively, and the symmetries were 1.5 and 1.5, respectively.

[0080] After packing, the two chromatography columns were connected in series using tubing, as shown in Figure 1. Then, the connected columns (column 1 and column 2) were equilibrated with a PBS solution of 0.01 MPa-0.18 M NaCl (pH 6.0 ± 0.2) at a flow rate of 0.6 cm / min and an equilibration volume of twice the column volume until the baseline and conductivity stabilized.

[0081] 2.2.2 Sample loading and elution

[0082] use The Pilot 400 chromatography system was used. The concentrated intermediate was loaded onto column 1 at a flow rate of 0.6 cm / min, with a column volume of ≤5%. After loading, equilibration was performed using PBS solution, and the chromatogram was observed (as shown in Figure 3B). The OD was then calculated. 280 When the baseline reaches approximately 2 / 5 of the total volume of the tandem column, the first peak begins to appear and continues until it subsides. This peak represents the elution of contaminating proteins from the aggregates and is not collected. Wait for the OD... 280 After the baseline rose again, exceeding the peak value of the contaminating protein peak by 2 mAU, the target protein was collected. Collection was stopped once the baseline stabilized after the peak receded. The collected target protein was then subjected to HPLC purity determination, and the results are shown in Figure 3D.

[0083] 2.3 Single column comparison (old process)

[0084] Gel chromatography was performed using a single column 1 packed and balanced as described in 2.2. The concentrated intermediate obtained in 2.1 was subjected to gel chromatography according to the loading and elution method in 2.2.2. The chromatographic pattern is shown in Figure 3A, and the HPLC purity chromatogram of the collected target protein is shown in Figure 3C.

[0085] The improved purification results are compared with those before the improvement, as shown in the table below:

[0086] Example 3

[0087] Separation and purification were performed using a SAC152 / 950 tandem column, and HPLC purity was determined.

[0088] 3.1 Obtaining the concentrated intermediate

[0089] The cell harvest fluid of CHO cells expressed according to the process in 1.1 was processed to obtain a concentrated intermediate with a recombinant human thrombopoietin polymer content of 24.64% and a protein concentration of 3.13 mg / mL.

[0090] 3.2 Gel Chromatography

[0091] Specifically, the following processes are included:

[0092] 3.2.1 Column Installation

[0093] Superose 12 gel (Superose™) filler was used. 12 Two SAC152 / 950 chromatography columns (prep grade) were packed. Column 1 was packed to a height of 65 cm, and column 2 to a height of 60 cm, with a packing concentration of 65% and a compressibility factor of 1.15. A packing suspension (theoretical volumes of 20858 ml and 19253 ml for the two columns, respectively) was poured into the columns, prepared by mixing the packing material with a 0.01 M NaOH-0.2 M NaCl mixed solution. The column was then pressurized using a two-stage gel pressing method, maintaining a constant flow rate of 20 cm / h until the gel surface stabilized. This was followed by a constant pressure of 0.4 MPa until the gel surface stabilized. The column was then disconnected from the chromatography system, and the column head was manually rotated to 1 cm below the gel surface, completing the packing process. The column was equilibrated with 0.01M NaOH-0.2M NaCl solution until the baseline was stable. 1% column volume of 2% acetone sample was loaded, and the column efficiency was determined. The theoretical plate heights of chromatography column 1 and chromatography column 2 were 0.058 cm and 0.059 cm, respectively, and the symmetries were 1.5 and 1.6, respectively.

[0094] After packing, the two chromatography columns were connected in series using tubing, as shown in Figure 1. Then, the connected columns (column 1 and column 2) were equilibrated with a PBS solution of 0.01 MPa-0.18 M NaCl (pH 6.0 ± 0.2) at a flow rate of 0.4 cm / min and an equilibration volume of twice the column volume until the baseline and conductivity stabilized.

[0095] 3.2.2 Sample loading and elution

[0096] use The pilot 400 chromatography system was used to load the concentrated intermediate into the chromatography column 1 at a column volume of 2.2% and a loading flow rate of 0.6 cm / min. After the loading was completed, the column was equilibrated with 0.01 M PB-0.18 M NaCl and the chromatogram was observed (as shown in FIG. 4A). When the baseline reached about 2 / 5 of the total volume of the tandem column, the first peak began to appear and ended until the peak fell. This was the impurity peak of the eluted polymer, and it was not collected. When the baseline rose again, and was higher than the highest peak of the impurity peak by 2 mAU, the collection of the target protein began. When the peak fell and the baseline was stable, the collection ended. The collected target protein was subjected to HPLC purity determination, and the test results are shown in FIG. 4B. 280 The baseline rose again, and was higher than the highest peak of the impurity peak by 2 mAU, the collection of the target protein began. When the peak fell and the baseline was stable, the collection ended. The collected target protein was subjected to HPLC purity determination, and the test results are shown in FIG. 4B. 280 The baseline rose again, and was higher than the highest peak of the impurity peak by 2 mAU, the collection of the target protein began. When the peak fell and the baseline was stable, the collection ended. The collected target protein was subjected to HPLC purity determination, and the test results are shown in FIG. 4B.

[0097] The concentrated intermediate was subjected to scale-up tandem column purification according to Example 3. The purity of the sample after the final purification was 99.18%, and the sample yield was 72.4%. The scale-up was achieved from the chromatography scale XK26 / 100 to SAC152 / 950, and the scale-up was 34 times.

[0098] The above description is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any changes or replacements within the technical scope disclosed by the present application can be easily thought of by those skilled in the art, and should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for removing recombinant human thrombopoietin aggregates by Superose 12 size exclusion column chromatography, characterized in that, It comprises the following steps: (1) the CHO cell-expressed recombinant human thrombopoietin harvest liquid is concentrated by ultrafiltration, and then concentrated and exchanged by cation exchange chromatography, first anion exchange chromatography, reverse phase chromatography, and second anion exchange chromatography to obtain a concentrated intermediate; (2) the concentrated intermediate is loaded onto two horizontally connected Superose 12 molecular sieve chromatography columns, and then eluted with an eluent and the target protein is collected.

2. The method of claim 1, wherein, The theoretical column plate height of the two Superose 12 molecular sieve chromatography columns is ≤0.06 cm, and the symmetry is 0.8-2.

0.

3. The method of claim 1, wherein, The packing height of the packing in the two Superose 12 molecular sieve chromatography columns is ≥50 cm, and the compression factor is 1.

15.

4. The method according to any one of claims 1 to 3, characterized in that, The packing of the packing in the Superose 12 molecular sieve chromatography column comprises the following steps: the packing suspension is poured into the chromatography column, the column is packed to a gel surface at a linear flow rate of not more than 25 cm / h, and then the column is continuously packed at a constant pressure, the pressure range is 0.3-0.45 MPa, until the gel surface is stable, the connection with the chromatography system is disconnected, the column head is manually rotated to 1 cm below the gel surface, and the packing is completed; the concentration of the packing in the packing suspension is 50%-70%.

5. The method of claim 1, wherein, The loading flow rate of the concentrated intermediate is ≤0.6 cm / min, and the loading volume is ≤5% of the total volume of the connected columns.

6. The method of claim 1, wherein, The eluent is a PBS solution with pH 6.0±0.

2.

7. The method according to claim 1 or 6, characterized in that, Before loading the concentrated intermediate, the Superose 12 molecular sieve chromatography column is equilibrated with the eluent for 1-2 column volumes at a flow rate of ≤0.6 cm / min.

8. The method of claim 1, wherein, Step (1) specifically comprises the following steps: 1) the CHO cell-expressed recombinant human thrombopoietin harvest liquid is concentrated and exchanged into a citric acid buffer with pH 6.0±0.2 by a 30KD ultrafiltration membrane to obtain an intermediate A; 2) the intermediate A is purified by a cation exchange chromatography column with packing of carboxymethyl-containing agarose to obtain an intermediate B, 3) the intermediate B is purified by an anion chromatography column a with packing of quaternary amine-containing agarose to obtain an intermediate C; 4) the intermediate C is purified by a reverse phase chromatography column with packing of C4 to obtain an intermediate D; 5) the intermediate D is concentrated and exchanged by an anion exchange chromatography column b with packing of quaternary amine-containing agarose to obtain a concentrated intermediate.

9. The method according to claim 1 or 8, characterized in that, The recombinant human thrombopoietin in the concentrated intermediate has a content of more than 15% of recombinant human thrombopoietin polymer impurities, and the protein concentration of the recombinant human thrombopoietin protein concentrated intermediate is not less than 1.0 mg / mL.

10. The method of claim 1, wherein, The recombinant human thrombopoietin comprises an amino acid sequence as shown in SEQ ID NO:1, or a variant thereof.

11. The recombinant human thrombopoietin purified by the method according to any one of claims 1-10.

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