Method for purifying recombinant human thrombopoietin

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a method for purifying recombinant human thrombopoietin. The method comprises the following steps: pretreating milk or cell culture fluid containing rhTPO, wherein the pretreatment comprises dilution and centrifugation; carrying out affinity chromatography on the obtained supernate, combining target protein by using an anti-rhTPO antibody coupling medium, eluting by using a low-pH buffer solution, and immediately neutralizing; carrying out ion exchange chromatography on the neutralized eluent, and carrying out linear gradient elution by adopting strong cation exchange resin and NaCl; finally, the obtained sample is subjected to gel filtration chromatography, high-purity rhTPO is obtained, the method further comprises the steps of concentration, sterilization and freeze-drying of a gel filtration product, the method can effectively treat complex raw materials sourced from a breast bioreactor, the technological process is stable, the purity of the final product is higher than 95%, the activity recovery rate is larger than 80%, and the method is suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a purification method for recombinant human thrombopoietin. Background Technology

[0002] Human thrombopoietin (TPO) is a key cytokine stimulating megakaryocyte growth and differentiation. Recombinant human thrombopoietin (rhTPO) has important applications in the treatment of thrombocytopenia and in biomedical research. Producing rhTPO using animal mammary gland bioreactors offers advantages such as high yield, low cost, and the ability to perform correct post-translational modifications, making it a highly promising production platform.

[0003] However, purifying rhTPO from milk in mammary bioreactors faces significant challenges. Milk has a complex composition, containing large amounts of casein, fat globules, cell debris, and other impurities that severely interfere with the purification process. Existing purification methods often have significant drawbacks: First, they lack adaptability to raw materials; most methods are designed for cell culture media and are inefficient at processing milk. Second, the purification process is cumbersome, often requiring five or more chromatography steps, resulting in long operation times and significant loss of target protein activity. Third, the purification effect is unsatisfactory; for milk-derived rhTPO, the product purity is generally below 90%, and the activity recovery rate is often below 60%. Finally, the process stability and reproducibility are poor, with large batch-to-batch quality fluctuations, making it difficult to meet the needs of industrial production.

[0004] Therefore, there is an urgent need in this field to develop a method that can efficiently and stably purify high-purity, high-activity rhTPO from complex dairy raw materials. This method should have the advantages of simplified steps, controllable cost, and wide applicability. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the prior art, the inventors have proposed the present invention.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for purifying recombinant human thrombopoietin.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for purifying recombinant human thrombopoietin, comprising the following steps:

[0009] (a) Raw material pretreatment: Dilute the milk containing rhTPO with phosphate buffer at pH 7.0-7.5 by 1-5 times, then centrifuge at 4°C and 5000-10000 rpm for 15-30 minutes and collect the supernatant;

[0010] (b) Affinity chromatography: The supernatant obtained in step (a) is loaded onto a chromatography column conjugated with anti-rhTPO antibody, equilibrated and washed with phosphate buffer containing 0.05-0.15M NaCl at pH 7.0-7.5, and then eluted with buffer containing 0.1-0.5M NaCl at pH 2.5-4.0. The eluent is collected and immediately neutralized to pH 6.0-7.0 with 1-2M Tris solution.

[0011] (c) Ion exchange chromatography: The eluent neutralized in step (b) was loaded onto a strong cation exchange chromatography column equilibrated with phosphate buffer at pH 5.0-7.0. After rinsing, the column was eluted with a linear gradient of 0-1M NaCl, and the elution peak containing rhTPO was collected.

[0012] (d) Gel filtration chromatography: After desalting the eluent collected in step (c), the sample was loaded onto a gel filtration chromatography column and eluted with pH 7.0-8.0 Tris-HCl buffer containing 0.1-0.2M NaCl. The high-purity rhTPO elution peak was collected.

[0013] In a preferred embodiment of the purification method for recombinant human thrombopoietin described in this invention, in step (a), the milk is derived from transgenic dairy cows, transgenic goats, or transgenic sheep.

[0014] In a preferred embodiment of the purification method for recombinant human thrombopoietin described in this invention, in step (b), the chromatography medium conjugated with the anti-rhTPO antibody is an antibody-conjugated agarose gel.

[0015] In a preferred embodiment of the purification method for recombinant human thrombopoietin described in this invention, in step (c), the linear gradient elution time is 50-70 minutes.

[0016] In a preferred embodiment of the purification method for recombinant human thrombopoietin described in this invention, in step (c), before sample loading, the neutralized eluent is filtered through a 0.45 μm filter membrane.

[0017] As a preferred embodiment of the purification method for recombinant human thrombopoietin according to the present invention, in step (d), the gel filtration chromatography medium is Sephadex G-75 or Sephacryl S-100; the sample loading volume does not exceed 10% of the column volume.

[0018] As a preferred embodiment of the purification method for recombinant human thrombopoietin described in this invention, step (d) is followed by step (e) of subsequent processing: the eluent collected by gel filtration is concentrated by ultrafiltration, and then subjected to sterile filtration through a 0.22 μm filter membrane and freeze-drying.

[0019] As a preferred embodiment of the purification method for recombinant human thrombopoietin described in this invention, the freeze-drying conditions include: pre-freezing temperature -40 to -30°C, sublimation drying temperature -20 to -10°C, and desorption drying temperature 20-30°C.

[0020] In a preferred embodiment of the purification method for recombinant human thrombopoietin described in this invention, the purification process is maintained at a temperature of 4-25°C.

[0021] As a preferred embodiment of the purification method for recombinant human thrombopoietin described in this invention, the purified rhTPO has a purity greater than 95%, an activity recovery rate greater than 80%, and an endotoxin content less than 1.0 EU / μg.

[0022] Beneficial effects of the present invention: The purification method provided by the present invention has the following significant advantages:

[0023] The product boasts high purity and excellent activity recovery: Through precise combination and optimization of three-step chromatography, impurities such as host proteins can be effectively removed, resulting in a final product purity significantly higher than 95%, while the activity recovery rate remains stable at over 80%.

[0024] The process is simplified and the cost is reduced. The method steps are clear and concise, reducing the number of chromatography steps and the consumption of expensive reagents, and reducing the dependence on special equipment and production costs.

[0025] With strong raw material adaptability, the core process can be applied to various raw material sources such as milk from different mammary bioreactors (e.g., cow and goat) and cell culture medium through pretreatment adjustments, demonstrating good versatility.

[0026] The process has high stability and repeatability, and key parameters such as temperature, pH and flow rate are clearly defined, ensuring a high degree of consistency in product quality between different production batches and meeting the requirements of industrial production. This method effectively solves the defects of existing technologies such as poor raw material adaptability, complex steps, difficulty in balancing purity and recovery rate, high cost and insufficient process stability. Detailed Implementation

[0027] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0028] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it a single embodiment or an embodiment selectively excluded from other embodiments.

[0030] Example 1

[0031] This embodiment provides a method for purifying recombinant human thrombopoietin. Specifically, it demonstrates the standard procedure and effectiveness of purifying rhTPO from the complex composition of transgenic cow milk using the method of this invention.

[0032] Materials: Transgenic cow milk (containing rhTPO); phosphate-buffered saline (PBS, pH 7.2); Tris; NaCl; anti-human TPO monoclonal antibody conjugated to agarose gel; SP Sepharose FastFlow strong cation exchange resin; Sephadex G-75 gel; ultrafiltration centrifuge tubes (30 kDa molecular weight cutoff); 0.45 μm and 0.22 μm filter membranes.

[0033] Methods: Raw material pretreatment: 1.0 L of transgenic cow milk was measured and placed at 4°C. 2.0 L of pre-cooled PBS buffer (pH 7.2) was added and slowly stirred until well mixed. The diluted milk was aliquoted into centrifuge bottles and centrifuged at 8000×g for 25 minutes at 4°C. After centrifugation, the middle layer was carefully aspirated, avoiding the aspiration of the upper fat layer and the bottom precipitate, yielding approximately 2.8 L of clear supernatant.

[0034] Affinity chromatography: A chromatography column containing 20 mL of anti-rhTPO antibody agarose gel was mounted on the protein purification system. The column was equilibrated with 5 column volumes (CV) of equilibration buffer (PBS containing 0.1 M NaCl, pH 7.2) at a flow rate of 2.0 mL / min. The pretreated supernatant was loaded at a flow rate of 1.0 mL / min. After loading, the column was washed with equilibration buffer until the absorbance of the UV detector at 280 nm returned to baseline. Elution was then performed with elution buffer (glycine-HCl buffer containing 0.3 M NaCl, pH 3.0) at a flow rate of 1.0 mL / min, and the elution peak was collected using an automated fraction collector. Immediately afterwards, 1.0 M Tris-HCl buffer (pH 8.5) was slowly added dropwise to the collected eluent (approximately 85 mL) while gently stirring to neutralize the pH to 6.8.

[0035] Ion exchange chromatography: The neutralized affinity chromatography eluent was filtered through a 0.45 μm filter membrane. A chromatography column packed with 50 mL of SP Sepharose FastFlow medium was equilibrated with 5 CV equilibration buffer (20 mM sodium phosphate, pH 6.0) at a flow rate of 1.5 mL / min. The filtered sample was loaded at a flow rate of 1.5 mL / min. After loading, the column was washed with equilibration buffer to baseline. Further, a linear gradient elution was used: the proportion of eluent B (20 mM sodium phosphate, 1 M NaCl, pH 6.0) was increased from 0% to 100% over 60 minutes at a flow rate of 1.5 mL / min, and the elution peak was collected. SDS-PAGE electrophoresis analysis was performed, and approximately 65 mL of the eluent containing rhTPO was combined.

[0036] Gel filtration chromatography: The combined ion exchange chromatography solution was replaced with gel filtration buffer (20 mM Tris-HCl, 0.15 M NaCl, pH 7.5) using a desalting column and concentrated to approximately 10 mL. The column was packed with Sephadex G-75 gel (approximately 100 mL column bed volume) and equilibrated with the above buffer at a flow rate of 0.8 mL / min using 2CV. The concentrated sample was loaded at a flow rate of 0.5 mL / min (10% of the column volume). Isocratic elution was performed with equilibration buffer at a flow rate of 0.8 mL / min. The main peak was collected based on the UV absorption spectrum, yielding approximately 40 mL of high-purity rhTPO solution.

[0037] Subsequent processing: The gel filtration purified solution was concentrated to approximately 10 mL using an ultrafiltration centrifuge tube. Further, it was sterilized by sequentially passing through 0.45 μm and 0.22 μm filter membranes. The sterilized solution was aliquoted into glass vials and placed in a freeze dryer. The freeze-drying program was set as follows: pre-freezing at -35°C for 2 hours; main drying at -15°C for 12 hours (vacuum <100 μBar); and desorption drying at 25°C for 4 hours (vacuum <50 μBar). The final product was a white, spongy, lyophilized rhTPO powder.

[0038] Furthermore, the purity of the lyophilized product was determined using SDS-PAGE electrophoresis and high-performance liquid chromatography.

[0039] SDS-PAGE electrophoresis results showed a single, clear band at approximately 70 kDa, with no other obvious contaminants; high-performance liquid chromatography (HPLC) analysis showed a product purity of 96.5%.

[0040] Product activity was detected using a cell proliferation method.

[0041] Take an appropriate amount of lyophilized powder, reconstitute it with RPMI 1640 medium containing 10% fetal bovine serum, and prepare solutions of different concentrations. Seed MO7e cells into 96-well plates, 100 μL per well (cell concentration of 2 × 10 cells / mL), and add 10 μL of recombinant human thrombopoietin solution of different concentrations per well, with 3 replicates. After culturing for 48 hours, add CCK-8 reagent and continue culturing for 4 hours, and measure the absorbance value at 450 nm.

[0042] The calculated ED50 was 1.5 ng / mL, which meets the activity requirements.

[0043] The product's molecular weight, endotoxin levels, and other indicators were tested.

[0044] The molecular weight was determined to be 70 kDa by SDS-PAGE electrophoresis combined with a standard protein marker; the endotoxin was detected using the Limulus amebocyte lysate (LAL) method, and the result was 0.5 EU / μg, which meets the requirements.

[0045] As can be seen, the purification method of this invention can successfully purify recombinant human thrombopoietin from milk produced by a transgenic cow mammary gland bioreactor. The product has high purity and good activity, and the operation process is stable and controllable, fully meeting the needs of production and application. In actual production, the parameters of each step can be appropriately adjusted according to the production scale, such as the volume of the chromatography column, flow rate, and reagent dosage, but the overall purification principle and steps remain consistent. Furthermore, for milk from other mammary gland bioreactor sources, such as goat milk from transgenic goats, only minor adjustments to the pretreatment centrifugation conditions and dilution factor are needed based on the characteristics of the milk to purify it using the method of this invention.

[0046] Example 2

[0047] This embodiment provides a purification method for recombinant human thrombopoietin. Specifically, the universality of this method for mammary gland bioreactor milk from different species lies in the adaptive adjustment of pretreatment parameters, while the core chromatography steps remain unchanged.

[0048] Materials: Genetically modified goat milk (containing rhTPO); other materials are the same as in Example 1.

[0049] Method: The purification process was exactly the same as in Example 1, except that the pretreatment steps were adjusted according to the characteristics of goat milk:

[0050] Raw material pretreatment: Measure 1.0 L of transgenic goat milk and place it at 4°C. Add 3.0 L of pre-cooled PBS buffer (pH 7.2) (dilution ratio adjusted to 1:3) and mix slowly. Aliquot the diluted goat milk and centrifuge at 4°C and 10000×g for 20 minutes (slightly increase centrifugal force) to more thoroughly separate and remove smaller fat globules from the goat milk. Carefully collect the middle layer supernatant.

[0051] All parameters for subsequent affinity chromatography, ion exchange chromatography, gel filtration chromatography, and subsequent processing, including buffer formulation, column volume, flow rate, elution conditions, etc., were strictly kept consistent with those in Example 1.

[0052] result:

[0053] SDS-PAGE showed the product as a single main band with a purity of approximately 95.8%.

[0054] Cell viability assays showed an activity recovery rate of 81%.

[0055] The endotoxin content was 0.7 EU / μg.

[0056] The results confirmed that, without changing the core purification process, the method of the present invention can efficiently process another milk source (goat milk) and obtain a high-quality product comparable to that of Example 1 by simply fine-tuning the pretreatment parameters, fully demonstrating the robustness and adaptability of the method.

[0057] Example 3

[0058] This embodiment provides a purification method for recombinant human thrombopoietin, specifically demonstrating the applicability of the method to cell culture medium raw materials, including key parameter ranges such as centrifugation conditions, dilution factor, and gradient elution time.

[0059] Materials: CHO cell culture supernatant expressing rhTPO (preliminarily clarified by centrifugation after harvest), other materials are the same as in Example 1.

[0060] Methods: Raw material pretreatment: Take 2.0 L of cell culture supernatant, centrifuge at 4℃ and 5000×g for 30 minutes, and carefully collect the supernatant, avoiding aspirating the precipitate.

[0061] Affinity chromatography: The procedure was the same as in Example 1. The equilibration buffer was PBS (pH 7.5) containing 0.05 M NaCl. Elution was performed using sodium citrate buffer (pH 3.5) containing 0.4 M NaCl.

[0062] Ion exchange chromatography: The procedure is the same as in Example 1. The linear gradient elution time is 65 minutes.

[0063] Gel filtration chromatography: Sephacryl S-100HR gel was used. The elution buffer was Tris-HCl (pH 8.0) containing 0.1 M NaCl.

[0064] The sample loading volume is 8% of the column volume.

[0065] Subsequent processing: Same as in Example 1. The freeze-drying stage was set to 30°C.

[0066] Results: SEC-HPLC analysis showed that the product purity was greater than 98.5%.

[0067] The activity recovery rate reached 85%.

[0068] Endotoxin levels were below 0.3 EU / μg.

[0069] The results show that the method of the present invention is also applicable to the purification of rhTPO from cell culture medium, and can obtain extremely high purity.

[0070] The above embodiments collectively demonstrate that the rhTPO purification method provided by the present invention has outstanding technical effects and wide applicability.

[0071] First, the core advantage of this method lies in its fixed framework of "pretreatment + three-step chromatography". This framework is carefully designed, with the three steps of chromatography (affinity, ion exchange, and gel filtration) separating based on biological specificity, charge properties, and molecular size, respectively. These complementary principles enable the systematic and in-depth removal of various impurities. Examples demonstrate that this framework is applicable to cow's milk, goat's milk, and cell culture media with vastly different compositions. Only minor adjustments are needed in the pretreatment stage; the media, buffer system, and elution mode of the core chromatography steps can remain unchanged, greatly simplifying the process scale-up.

[0072] Secondly, this invention achieves a highly efficient balance between purity and activity through precise definition and combination of key operating parameters. For example, the immediate neutralization operation after affinity chromatography (Example 1) effectively prevents damage to protein activity caused by low pH; the linear gradient elution of ion exchange (Examples 1, 3) provides higher resolution and more thorough impurity removal compared to conventional step elution; and the strict control of sample loading volume in gel filtration (Examples 1, 3) ensures effective separation. The combined effect of these details is key to achieving high activity recovery rates (all >80%) and high purity.

[0073] Finally, the method exhibits excellent process stability and repeatability. Under similar operating parameters, the key quality attributes of the final product (purity, activity recovery, endotoxin) in the three embodiments remained within a narrow high-level range with minimal batch-to-batch variation. This demonstrates that the method is not only effective but also reliable, fully meeting the process robustness requirements of industrial production.

[0074] In summary, this invention successfully provides an efficient, stable, economical, and widely applicable rhTPO purification solution, effectively overcoming many shortcomings of existing technologies and possessing promising industrialization prospects.

[0075] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for purifying recombinant human thrombopoietin, characterized in that: Includes the following steps: (a) Raw material pretreatment: Dilute the milk containing rhTPO with phosphate buffer at pH 7.0-7.5 by 1-5 times, then centrifuge at 4°C and 5000-10000 rpm for 15-30 minutes and collect the supernatant; (b) Affinity chromatography: The supernatant obtained in step (a) is loaded onto a chromatography column conjugated with anti-rhTPO antibody, equilibrated and washed with phosphate buffer containing 0.05-0.15M NaCl at pH 7.0-7.5, and then eluted with buffer containing 0.1-0.5M NaCl at pH 2.5-4.

0. The eluent is collected and immediately neutralized to pH 6.0-7.0 with 1-2M Tris solution. (c) Ion exchange chromatography: The eluent neutralized in step (b) was loaded onto a strong cation exchange chromatography column equilibrated with phosphate buffer at pH 5.0-7.

0. After rinsing, the column was eluted with a linear gradient of 0-1M NaCl, and the elution peak containing rhTPO was collected. (d) Gel filtration chromatography: After desalting the eluent collected in step (c), the sample was loaded onto a gel filtration chromatography column and eluted with pH 7.0-8.0 Tris-HCl buffer containing 0.1-0.2M NaCl. The high-purity rhTPO elution peak was collected.

2. The purification method for recombinant human thrombopoietin as described in claim 1, characterized in that: In step (a), the milk is derived from genetically modified dairy cows, genetically modified goats, or genetically modified sheep.

3. The purification method for recombinant human thrombopoietin as described in claim 1, characterized in that: In step (b), the chromatography medium conjugated with the anti-rhTPO antibody is an antibody-conjugated agarose gel.

4. The purification method for recombinant human thrombopoietin as described in claim 1, characterized in that: In step (c), the linear gradient elution time is 50-70 minutes.

5. The purification method for recombinant human thrombopoietin as described in claim 1, characterized in that: In step (c), before loading the sample, the neutralized eluent is filtered through a 0.45 μm filter membrane.

6. The purification method for recombinant human thrombopoietin as described in claim 1, characterized in that: In step (d), the gel filtration chromatography medium is Sephadex G-75 or Sephacryl S-100; the sample loading volume does not exceed 10% of the column volume.

7. The purification method for recombinant human thrombopoietin as described in claim 1, characterized in that: Step (d) is followed by step (e) of subsequent processing: the eluent collected by gel filtration is concentrated by ultrafiltration, and then subjected to sterile filtration through a 0.22 μm filter membrane and freeze-drying.

8. The purification method for recombinant human thrombopoietin as described in claim 7, characterized in that: The freeze-drying conditions include: pre-freezing temperature -40 to -30°C, sublimation drying temperature -20 to -10°C, and desorption drying temperature 20-30°C.

9. A purification method for recombinant human thrombopoietin as described in any one of claims 1-8, characterized in that: The purification process is maintained at a temperature of 4-25℃.

10. A method for purifying recombinant human thrombopoietin as described in any one of claims 1-8, characterized in that: The purified rhTPO had a purity greater than 95%, an activity recovery rate greater than 80%, and an endotoxin content less than 1.0 EU / μg.