Purification method of long-acting recombinant human follicle-stimulating hormone

Abstract

The invention provides a purification method of long-acting recombinant human follicle-stimulating hormone (FSH-CTP), and belongs to the field of protein separation and purification. Specifically, the invention relates to a method for realizing purification of FSH-CTP through two steps of dye affinity chromatography and hydrophobic chromatography, a decontaminant virus inactivation process is specifically matched between the two steps of chromatography, the purification yield of the FSH-CTP is about 97%, the purity reaches 99.9%, a purified solution hardly contains dissociation subunits, the content of process impurities is low, and the method is suitable for industrial production. The technical problem that the quality control requirement and the cost control requirement cannot be met at the same time in an existing FSH-CTP purification process is solved.

Description

Technical Field

[0001] The invention belongs to the field of protein separation and purification, and specifically relates to a method for purifying long-acting recombinant human follicle-stimulating hormone. Background Art

[0002] Follicle-stimulating hormone (FSH) is an endogenous gonadotropin synthesized and secreted by the pituitary gland, which regulates a series of physiological processes related to human development, growth, puberty, and reproduction. In women, FSH acts on the granulosa cells of the ovaries, which have specific FSH receptors on their surface. When FSH binds to the FSH receptors on the granulosa cells, it activates the downstream signaling pathway, which can promote the growth, development and maturation of follicles at different stages of the female ovulation hormone cycle. At the same time, granulosa cells can produce two steroid hormones, estrogen and progesterone. In men, FSH promotes the maturation of the seminiferous tubules of the testicles and regulates sperm production by acting on the specific FSH receptors on the surface of the Sertoli cells (Sertoli cells). Recombinant human follicle-stimulating hormone (rhFSH) is prepared by genetic recombination technology, has the same biological activity as natural FSH, can simulate the FSH function of FSH in vivo, and can be used for clinical applications such as superovulation in assisted reproductive cycle treatment and promoting sperm production in male infertility.

[0003] Existing rhFSH products mainly include short-acting FSH and long-acting FSH (i.e. rhFSH-CTP). Due to the introduction of the CTP sequence, its half-life in the body is significantly longer than that of rhFSH. One injection of the recommended dose of rhFSH-CTP can replace the daily injection of rhFSH for 7 consecutive days. Whether it is used for female superovulation in the assisted reproductive cycle or male idiopathic hypogonadism, it can significantly reduce the frequency of administration and improve patients' medication compliance.

[0004] Similar to rhFSH, the development of rhFSH-CTP products requires that impurities be removed as much as possible in the downstream purification process with a simple, convenient and cost-effective purification process, so as to reduce protein dissociation and degradation, improve protein purity and purification yield, and meet the quality control and cost control requirements of large-scale production. In the prior art, Liu Xiaohang reported the preparation of FSH-CTP by three-step chromatography of dye affinity chromatography, DEAE anion exchange chromatography and hydrophobic chromatography. The purity of the obtained FSH-CTP sample can reach 98%, but the yield is only about 20%, and the entire purification process is complicated, and the sample needs to be additionally treated between the chromatography steps (Liu Xiaohang, Gao Liting. Research and identification of purification process of recombinant human long-acting follicle-stimulating hormone FSH-CTP [J]. Progress in Biotechnology, 2016, 6 (3): 219-223). The applicant disclosed in the previous application (CN116143901B) that after incubation at low pH to remove viruses, rhFSH-CTP samples were purified by composite anion exchange chromatography. The purity of the obtained rhFSH-CTP can reach 99.8% and the yield can reach 96%. However, this method relies on the introduction of an affinity chromatography filler (Capture Select Follicle Stimulating Hormone (FSH) Affinity Matrix) designed for purifying FSH in the affinity chromatography step. The filler is a unique Capture Matrix of Thermo Fisher Scientific. Select technology is based on specially designed alpaca antibody fragments (VHH fragments) that can specifically bind to FSH molecules. It improves purification efficiency through specific binding with FSH, but the filler is very expensive, 13,983.00 yuan / 5ml (source: Thermo Fisher Scientific official website); and it has poor pressure resistance, is fragile, and the chromatography medium is not alkali-resistant, which is not conducive to cleaning and disinfection during production. This may lead to excessive microbial load and bacterial endotoxins in purified samples, and may also cause cross-contamination between batches or cross-contamination between co-products, increasing the quality control risk and cost of the overall process; and the filler column will have a large amount of broken gel after about 50 cycles, and it is difficult to fill the chromatography column in the later stage. In addition, the purification process also requires continuous detection and control of the ligand shedding of the filler.

[0005] On this basis, it is very necessary and valuable to develop a FSH-CTP purification method that meets both quality control and cost control requirements, has fewer process steps and is easy to operate, and can maintain high yield and high purity. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for purifying long-acting recombinant human follicle-stimulating hormone (FSH-CTP). Specifically, the present invention relates to a method for purifying FSH-CTP by two steps of dye affinity chromatography and hydrophobic chromatography, and a detergent virus inactivation process is specifically matched between the two chromatography steps.

[0007] The present invention achieves the technical problem that the present invention intends to solve through the following technical solutions.

[0008] The present invention provides a method for purifying long-acting recombinant human follicle-stimulating hormone, which comprises the following steps:

[0009] (1) Dye affinity chromatography

[0010] (2) Hydrophobic chromatography

[0011] The step (1) of dye affinity chromatography and the step (2) of hydrophobic chromatography also includes a virus inactivation step, wherein the virus inactivation step is performed using a detergent.

[0012] In certain specific embodiments, the dye affinity chromatography is performed by loading the cell culture fluid onto a chromatography column filled with Diamond Blue and eluting with a salt-containing Tris solution.

[0013] In some embodiments, the salt contained in the salt-containing Tris solution in the dye affinity chromatography is NaCl, KCl or MgCl 2 , preferably NaCl; the concentration of the salt contained is 1.0M to 3.0M, preferably 2.0M; the Tris concentration in the Tris solution is 10mM to 50mM, and a pH buffer system is formed by adding an acid component, and the acid component is selected from HAc or HCl.

[0014] In certain specific embodiments, the dye affinity chromatography eluent further comprises isopropanol; the volume concentration of the isopropanol is 5% to 30%, preferably 5% to 20%, and more preferably 10%.

[0015] In certain specific embodiments, the pH of the dye affinity chromatography eluent is 6-8.

[0016] In some specific embodiments, the dye affinity chromatography elution conditions are: elution is performed using a Tris-HAc solution containing NaCl, the concentration of the NaCl is 2.0 M, and the concentration of Tris in the Tris-HAc solution is 10 mM to 50 mM; the dye affinity chromatography eluent also contains isopropanol with a volume concentration of 10%; the pH of the dye affinity chromatography eluent is 7.2±0.2.

[0017] In certain specific embodiments, the detergent used for virus inactivation is selected from Tween 80 or Triton X-100.

[0018] In certain specific embodiments, the virus is inactivated by incubation with 1% Tween 80; the incubation time is 4 to 8 hours, preferably 6 hours.

[0019] In certain specific embodiments, the hydrophobic chromatography is to load the virus inactivated liquid onto a chromatography column filled with Generik MC60-HICPhenyl and elute with a Tris solution; the Tris concentration in the Tris solution is 5mM to 20mM, and a pH buffer system is formed by adding an acid component, and the acid component is selected from HAc or HCl.

[0020] In certain specific embodiments, the pH of the hydrophobic chromatography eluent is 6-8.

[0021] In certain specific embodiments, the hydrophobic chromatography elution conditions are: elution is performed using a Tris-HAc solution, wherein the Tris concentration in the Tris-HAc solution is 5 mM to 20 mM, preferably a Tris concentration of 5 mM; and the pH of the hydrophobic chromatography eluent is 7.2±0.2.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. Only two steps of chromatography and a specific virus inactivation method are used. Under the premise of simplifying the chromatography steps, the yield of FSH-CTP is about 97%, and the purity reaches 99.9%. The purified liquid contains almost no dissociated subunits and the content of process impurities is low;

[0024] 2. No additional sample processing steps such as ultrafiltration, fluid exchange, dilution and desalting are required between chromatography steps to obtain FSH-CTP that meets quality standards. The use of more cost-effective dye affinity chromatography fillers also simplifies the purification process, saves costs, and is economical and practical.

[0025] 3. The inventors deliberately selected a combination of dye affinity chromatography and hydrophobic chromatography from the common purification processes for protein purification, and used a detergent to perform a virus inactivation step. Under such a specific combination of purification methods and purification conditions, they surprisingly found that an excellent purification effect of FSH-CTP was achieved (the yield of FSH-CTP was about 97%, the purity reached 99.9%, the purified liquid contained almost no dissociated subunits, and the content of process impurities was low). Such an excellent effect has not been reported in the prior art and is beyond the expectations of those skilled in the art. DETAILED DESCRIPTION

[0026] Unless otherwise specified, the reagents or instruments described in the present invention can be purchased commercially.

[0027] The Diamond Blue filler in the present invention was purchased from Boglon (Zhejiang) Biotechnology Co., Ltd.

[0028] The concentration of the Tris-HAc solution or Tris-HCl solution involved in the present invention is expressed as the concentration of Tris, such as 10-50mM Tris-HAc solution means that the concentration of Tris is 10mM-50mM; 5mM Tris-HAc solution means that the concentration of Tris is 5mM.

[0029] The Tris-HAc solution or Tris-HCl solution mentioned in the present invention refers to a pH buffer system formed by adding HAc or HCl to Tris.

[0030] In the present invention, "+" means "and", such as "10mM Tris-HAc+2.0M NaCl+10% isopropanol" in the dye affinity chromatography eluent means that the eluent consists of 10mM Tris-HAc solution, 2.0M NaCl solution and 10% isopropanol.

[0031] 10% isopropyl alcohol means that the volume concentration of isopropyl alcohol is 10%.

[0032] 1% Tween 80 or 1% Triton X-100 means that the weight volume ratio of Tween 80 or Triton X-100 is 1% (w / v).

[0033] In the present invention, HCD and HCP are represented as process residual impurities.

[0034] Long-acting follicle-stimulating hormone (FSH-CTP) Source: Chengdu Jingze Biopharmaceutical Co., Ltd. (Batch No. 36X230201-02)

[0035] The FSH-CTP cell culture is prepared according to the conventional fermentation process of the recombinant protein fermentation protein in the art, specifically: the constructed FSH β-subunit and CTP chimera expression vector and the FSH α-subunit expression vector are transfected into CHO host cells to obtain an expression cell strain, and the FSH-CTP cell culture is obtained by cell recovery, seed amplification and reactor culture under appropriate conditions, for example, the FSH expression method disclosed in CN110305903B can be adapted and adjusted to obtain the FSH-CTP cell culture. Then, FSH-CTP is purified from the cell culture according to the method of the present invention.

[0036] The purification process of long-acting follicle stimulating hormone (FSH-CTP) in the present invention is carried out in the following order of steps:

[0037] (1) Dye affinity chromatography

[0038] Load the FSH-CTP cell solution onto a cleaned Diamond Blue (Bogelon) filled chromatography column; elute with "10mM-50mM Tris-HAc+1.0M-3.0M NaCl+5%-30% isopropanol, (pH6-8)", and collect the eluate, with a peak range of 50mAU / mm-Max-50mAU / mm.

[0039] (2) Virus inactivation

[0040] 1% Tween 80 was added to the dye affinity chromatography eluate, and the mixture was incubated with stirring at room temperature for at least 6 hours to inactivate the virus.

[0041] (3) Hydrophobic chromatography

[0042] The collected virus inactivated liquid was loaded onto a cleaned chromatography column filled with Generik MC60-HIC Phenyl (Saifen Technology); after loading, it was eluted with "5mM~20mM Tris-HAc, (pH6~8)" buffer, and the eluate was collected. The peak range was: 25mAU / mm-Max-25mAU / mm, and purified FSH-CTP was obtained.

[0043] The present invention is further illustrated by the following examples, which should not be construed as limiting the present invention.

[0044] Example 1 Purification of long-acting follicle-stimulating hormone (FSH-CTP)

[0045] Take the cell culture medium containing FSH-CTP, remove cells and cell debris, then load the sample, perform dye affinity chromatography, virus inactivation, and hydrophobic chromatography respectively, and collect the hydrophobic chromatography fluid to obtain.

[0046] 1. Dye affinity chromatography

[0047] The FSH-CTP cell fluid was loaded onto a cleaned Diamond Blue (Bogelon) filled chromatography column; after loading, it was eluted with "10mM Tris-HAc+2.0M NaCl+10% isopropanol, (pH7.2±0.2)", and the eluate was collected, with a peak range of: 50mAU / mm-Max-50mAU / mm.

[0048]

[0049]

[0050] 2. Virus inactivation

[0051] 1% Tween 80 was added to the dye affinity chromatography eluate, and the mixture was incubated at room temperature for 6 h with stirring to inactivate the virus.

[0052] 3. Hydrophobic chromatography

[0053] The collected virus inactivated liquid was loaded onto the cleaned Generik MC60-HIC Phenyl (Saifen Technology) filled chromatography column; after loading, it was eluted with "5mM Tris-HAc, pH7.2±0.2" buffer, and the eluate was collected, and the peak range was: 25mAU / mm-Max-25mAU / mm.

[0054]

[0055] Example 2 Purification of long-acting follicle-stimulating hormone (FSH-CTP)

[0056] Compared with Example 1, the dye affinity chromatography elution solution was changed to "30 mM Tris-HAc + 2.0 M NaCl + 10% isopropanol, (pH 7.2 ± 0.2)", and the remaining elution steps and elution conditions were the same as in Example 1 for elution.

[0057] Example 3 Purification of long-acting follicle stimulating hormone (FSH-CTP)

[0058] Compared with Example 1, the dye affinity chromatography elution solution was changed to "50 mM Tris-HAc + 2.0 M NaCl + 10% isopropanol, (pH 7.2 ± 0.2)", and the remaining elution steps and elution conditions were the same as in Example 1 for elution.

[0059] The FSH-CTP chromatographic fluids in Examples 1 to 3 were collected and tested, and the results are shown in Table 1 below:

[0060] Table 1

[0061]

[0062] It can be seen that after two steps of dye affinity chromatography and hydrophobic chromatography, the purity of FSH-CTP can reach 99.9%, and it contains almost no dissociated subunits, and the content of HCP and HCD impurities is low, meeting the safety requirements.

[0063] The following experimental examples are used to illustrate the beneficial effects of the present invention.

[0064] Experimental Example 1 Effect of dye affinity chromatography elution conditions on purification results

[0065] The experiment found that the elution conditions of dye affinity chromatography, i.e. the concentration and type of salt in the eluent, whether isopropanol is added, and the concentration of isopropanol, will affect the final separation effect of FSH-CTP. The details are as follows:

[0066] Table 2

[0067]

[0068] Based on comparison groups 1-6, it can be seen that simply increasing the salt concentration in the Tris-HAc solution, changing the salt type or lowering the pH without adding isopropanol in the elution conditions results in low yield and low purity, which is difficult to meet the needs of large-scale production; based on comparison groups 7-8, it can be seen that using Tris-HAc solution without salt and isopropanol as eluents, the elution yield and purity still do not meet the requirements, and the dissociated subunits increase significantly with the increase in isopropanol concentration; based on comparison groups 9-10, it can be seen that when specific concentrations of salt and isopropanol are added to the Tris-HAc solution at the same time, the yield and purity of the target protein are greatly improved, and the residues of related substances are significantly reduced.

[0069] Experimental Example 2 Effect of Virus Inactivation Conditions on Purification Results

[0070] The purification step provided by the present invention needs to be combined with a specific virus inactivation method. At room temperature, after completing the dye affinity chromatography step with reference to Example 1, the dye affinity chromatography eluate is incubated with low pH, 1% Tween 80, and 1% Triton X-100, respectively. The FSH-CTP results are shown in Table 3.

[0071] Table 3

[0072]

[0073] From the results in the above table, it can be seen that when low pH is used for virus inactivation incubation, the purity of the target protein will be significantly reduced and the dissociated subunits will be significantly increased; while when Tween 80 or Triton X-100 is used for detergent virus inactivation for an appropriate time, the purity and dissociated subunit results of the experimental group are more ideal.

[0074] Experimental Example 3 Effect of hydrophobic chromatography conditions on purification results

[0075] The purification process of the present invention also needs to integrate a specific hydrophobic chromatography process to achieve the effect of obtaining high-purity FSH-CTP protein with high yield in two-step chromatography. After completing the dye affinity chromatography and virus inactivation steps with reference to Example 1, the FSH-CTP virus inactivation solution is purified under different hydrophobic chromatography conditions. The results are as follows.

[0076] Table 4

[0077]

[0078] As can be seen from the table above, the concentration of the Tris solution and whether additional salt (such as sodium chloride or magnesium chloride) is added have a significant effect on the final FSH-CTP yield, HCP and HCD residues. Only by selecting specific elution conditions without the addition of salt components can the high-quality acquisition of the final target protein be achieved.

Claims

1. A method for purifying long-acting recombinant human follicle-stimulating hormone, the purification method comprising the following steps: (1) Dye affinity chromatography (2) Hydrophobic chromatography The step (1) of dye affinity chromatography and the step (2) of hydrophobic chromatography also includes a virus inactivation step, wherein the virus inactivation step is performed using a detergent.

2. The purification method according to claim 1, wherein the dye affinity chromatography is performed by loading the cell culture fluid onto a chromatography column filled with DiamondBlue and eluting with a salt-containing Tris salt solution; the salt contained in the salt-containing Tris solution is NaCl, KCl or MgCl2, preferably NaCl; the concentration of the salt contained is 1.0M to 3.0M, preferably 2.0M; the Tris concentration in the Tris solution is 10mM to 50mM, and a pH buffer system is formed by adding an acid component, and the acid component is selected from HAc or HCl.

3. The purification method according to any one of claims 1 or 2, wherein the dye affinity chromatography eluent further comprises isopropanol, and the volume concentration of the isopropanol is 5% to 30%, preferably 5% to 20%, and more preferably 10%.

4. The purification method according to claim 3, wherein the pH of the dye affinity chromatography eluent is 6 to 8.

5. The purification method according to claim 4, wherein the dye affinity chromatography elution conditions are: using a Tris-HAc salt solution containing NaCl for elution, the concentration of NaCl is 2.0 M, and the concentration of Tris in the Tris-HAc solution is 10 mM to 50 mM; the dye affinity chromatography eluent also contains 10% isopropanol; and the pH of the dye affinity chromatography eluent is 7.2±0.

2.

6. The purification method according to claim 5, wherein the detergent used for virus inactivation is selected from Tween 80 or Triton X-100.

7. The purification method according to claim 6, wherein the virus is inactivated by incubation with 1% Tween 80; the incubation time is 4 to 8 hours, preferably 6 hours.

8. The purification method according to claim 7, wherein the hydrophobic chromatography is performed by loading the virus inactivated liquid onto a chromatography column filled with Generik MC60-HIC Phenyl and eluting with a Tris solution; the Tris concentration in the Tris solution is 5 mM to 20 mM, and a pH buffer system is formed by adding an acid component, wherein the acid component is selected from HAc or HCl.

9. The purification method according to claim 8, wherein the pH of the hydrophobic chromatography eluent is 6 to 8.

10. The purification method according to claim 9, wherein the hydrophobic chromatography elution conditions are: using a Tris-HAc solution for elution, wherein the Tris concentration in the Tris-HAc solution is 5 mM to 20 mM, preferably 5 mM; and the pH of the hydrophobic affinity chromatography eluent is 7.2±0.2.

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