A method for purifying high-purity recombinant follicle-stimulating hormone.

Abstract

This invention provides a method for purifying high-purity recombinant follicle-stimulating hormone (FSH). Specifically, it includes the following steps: (a) providing a raw material solution containing recombinant FSH; (b) sequentially subjecting the raw material solution to: (b1) ammonium sulfate precipitation; (b2) hydrophobic interaction chromatography (HIC); (b3) low pH incubation to inactivate the virus; and (b4) reversed-phase chromatography (RPC); wherein steps (b1), (b2), (b3), and (b4) cannot be performed in any arbitrary order. Compared with existing preparation methods, the recombinant FSH obtained by the method of this invention is simpler and easier to operate, and the purity of the recombinant FSH obtained by the method of this invention can reach up to 99.5% w / w, with a specific activity as high as 16000 IU / mg.

Description

Technical Field

[0001] This invention relates to a method for purifying recombinant follicle-stimulating hormone (FSH), and more particularly to a method for purifying high-purity recombinant FSH. Background Technology

[0002] Follicle-stimulating hormone (FSH) is a heterodimeric glycoprotein with a molecular weight of approximately 31 kDa, composed of non-covalently bound α and β subunits. The α subunit has a relative molecular mass of approximately 14 kDa, and the β subunit has a relative molecular mass of approximately 17 kDa. It is a glycoprotein gonadotropin synthesized and secreted by basophilic cells of the anterior pituitary gland and is controlled by gonadotropin-releasing hormone from the hypothalamus. In women, FSH promotes the proliferation and differentiation of granulosa cells in follicles, and its concentration is crucial for the initiation and cycle of follicle development, as well as the time and number of follicles that subsequently reach maturity. In men, FSH binds to testosterone, which is controlled by LH (luteinizing hormone), and acts on the seminiferous tubules of the testes, thereby activating and maintaining a normal sperm count and quality. FSH is a bioactive molecule that plays an important role in the diagnosis and treatment of infertility. It plays an indispensable role in promoting normal follicle growth and maturation and the production of gonadal steroids.

[0003] Low FSH levels, resulting from insufficient FSH synthesis and secretion, are a major cause of infertility in both women and men. In women, this condition is characterized by anovulation or abnormal ovulation; in men, it can lead to infertility due to insufficient production of motile sperm. FSH is a vital bioactive molecule in the diagnosis and treatment of infertility, playing a crucial role in assisted reproductive technologies and is one of the most commonly used drugs for treating male and female infertility.

[0004] Early urinary gonadotropins (also known as menopausal urinary gonadotropins or menopausal gonadotropins), prepared from the urine of postmenopausal women, contained follicle-stimulating hormone (FSH) and luteinizing hormone (LH). However, they suffered from drawbacks such as low activity, high cost, significant pollution, and potential for pathogenic microbial infection. Recombinant human follicle-stimulating hormone (rhFSH), produced using recombinant DNA technology, has standardized molecular weight and biological activity. This avoids the differences in the biological activity of FSH isomers caused by variations in glycosyl structure between urinary FSH extracts, thus improving product quality and yield. Furthermore, rhFSH exhibits excellent safety characteristics.

[0005] Currently, recombinant human follicle-stimulating hormone (rhFSH) produced using DNA recombination technology has been launched, with the first commercially available products being Gonal-f® from Serono (Switzerland) and Puregon® from Organon (Netherlands). Gonal-f® and Puregon® are recombinant human follicle-stimulating hormones, with Gonal-f® boasting a purity exceeding 99% and stable efficacy, thus offering significant advantages. Currently, there are three main types of recombinant human follicle-stimulating hormones on the Chinese market. and (Produced by Changchun Jinsai Pharmaceutical Co., Ltd.). Domestic pharmaceutical companies producing recombinant FSH products generally have a purity not exceeding 99%. Given the crucial role FSH plays in treating infertility, promoting normal follicle growth and maturation, and in the production of gonadal steroids, providing high-purity and high-specific-activity recombinant FSH is promising. Highly purified FSH preparations can be administered subcutaneously, allowing patients to self-medicate, thus increasing patient comfort and compliance.

[0006] However, due to limitations in technology and funding, recombinant FSH products are significantly more expensive than urinary FSH products, making them financially unaffordable for most patients.

[0007] Therefore, there is an urgent need in this field to develop a low-cost, easy-to-operate FSH purification technology that yields high purity and good biological activity of FSH. Summary of the Invention

[0008] The purpose of this invention is to provide a low-cost, easy-to-operate FSH purification technique that yields high-purity and bioactive FSH.

[0009] This invention provides a method for purifying high-purity recombinant follicle-stimulating hormone, comprising the following steps:

[0010] (a) Provide a raw material solution containing recombinant follicle-stimulating hormone;

[0011] (b) The raw material liquid is subjected to the following steps in sequence:

[0012] (b1) Ammonium sulfate precipitation;

[0013] (b2) Hydrophobic interaction chromatography (HIC);

[0014] (b3) Low pH incubation to inactivate the virus; and

[0015] (b4) Reversed-phase chromatography (RPC);

[0016] Steps (b1), (b2), (b3), and (b4) cannot be performed in any order.

[0017] In another preferred embodiment, the method does not include cation exchange chromatography or anion exchange chromatography.

[0018] In another preferred embodiment, the raw material liquid is a fermentation product.

[0019] In another preferred embodiment, the fermentation is carried out in eukaryotic cells, preferably mammalian cells, and more preferably CHO cells.

[0020] In another preferred embodiment, the follicle-stimulating hormone is human follicle-stimulating hormone.

[0021] In another preferred embodiment, the follicle-stimulating hormone includes wild-type and mutant follicle-stimulating hormone.

[0022] In another preferred embodiment, the follicle-stimulating hormone is composed of an α subunit and a β subunit.

[0023] In another preferred embodiment, the α subunit has the amino acid sequence shown in SEQ ID No. 1, while the β subunit has the amino acid sequence shown in SEQ ID No. 2.

[0024] In another preferred embodiment, the recombinant follicle-stimulating hormone includes PEG-rh FSH, rh FSH-Fc fusion protein, rh FSH-CTP fusion protein, bifunctional antibody containing rh FSH, multifunctional antibody containing rh FSH, conjugate drug containing rh FSH, rhFSH glycosylation modifier, or a combination thereof.

[0025] In another preferred embodiment, the conditions for ammonium sulfate precipitation include a pH of 7.1-8.5, more preferably 7.2-8.0, and most preferably 7.3-7.6.

[0026] In another preferred embodiment, in step (b1), pH is adjusted using an inorganic acid and / or base.

[0027] In another preferred embodiment, in step (b1), the pH is adjusted using ammonia and / or sulfuric acid.

[0028] In another preferred embodiment, the conditions for the ammonium sulfate precipitation include: an ammonium sulfate concentration of 40%-60%, more preferably 42%-49%, even more preferably 43%-47%, and most preferably 44%-45%.

[0029] In another preferred embodiment, in step (b2), the chromatographic medium used in the hydrophobic interaction chromatography is selected from the group consisting of: Capto Phenyl (HS), Capto Butyl, Butyl Sepharose 4FF, Butyl Sepharose 4FF (HS), Octyl Sepharose 4FF, Phenyl Sepharose 6FF (LS), Phenyl Sepharose 6FF (HS), Phenyl Sepharose Big Beads, Phenyl Sepharose 6FF, Phenyl Sepharose HP, Butyl Sepharose HP, Butyl Sepharose 4B, Octyl Sepharose CL-4B, Phenyl Sepharose CL-4B, or combinations thereof.

[0030] In another preferred embodiment, in step (b3), the conditions for incubating the virus at low pH include: pH 2.5-4.5, preferably pH 3-4.

[0031] In another preferred embodiment, in step (b3), pH is adjusted using inorganic and / or organic acids.

[0032] In another preferred embodiment, in step (b3), the pH is adjusted using citric acid.

[0033] In another preferred embodiment, in step (b4), reversed-phase chromatography (RPC) uses matrix packing materials selected from the group consisting of silica gel, alumina, zirconium oxide, etc., as matrix materials, and polymers such as polystyrene, polyethylstyrene-divinylbenzene, polybutadiene, polyethylene oxide, polysiloxane, agarose, polychloromethylstyrene-diethoxymethylvinylsilane, carbon vapor deposition, or combinations thereof as coatings for inorganic matrix packing materials.

[0034] In another preferred embodiment, in step (b4), the matrix packing material used for reversed-phase chromatography (RPC) is polystyrene / divinylbenzene.

[0035] In another preferred embodiment, in step (b4), reversed-phase chromatography (RPC) is used to ligand polystyrene resin.

[0036] In another preferred embodiment, in step (b4), the organic solvent of the reversed-phase chromatography (RPC) buffer solution is acetonitrile, methanol, ethanol, propanol, or acetone.

[0037] In another preferred embodiment, in step (b4), the organic solvent of the reversed-phase chromatography (RPC) buffer solution is isopropanol.

[0038] In another preferred embodiment, in step (b4), the pH of the reversed-phase chromatography (RPC) buffer solution is between 7 and 8, preferably between 7.2 and 7.8, and most preferably between 7.4 and 7.6.

[0039] In another preferred embodiment, the purity of the obtained recombinant human follicle-stimulating hormone is greater than, more preferably >99.90% w / w, and even more preferably >99.99% w / w.

[0040] In another preferred embodiment, the method further includes (b5) reversed-phase chromatography (RPC).

[0041] In another preferred embodiment, step (b) of the method includes the step:

[0042] (b1) Ammonium sulfate precipitation;

[0043] (b2) Hydrophobic interaction chromatography (HIC);

[0044] (b3) Low pH incubation to inactivate the virus; and

[0045] (b4) Reversed-phase chromatography (RPC).

[0046] (b5) Reversed-phase chromatography (RPC).

[0047] In another preferred embodiment, step (b) of the method includes the step:

[0048] (b1) Ammonium sulfate precipitation;

[0049] (b2) Hydrophobic interaction chromatography (HIC);

[0050] (b3) Low pH incubation to inactivate the virus; and

[0051] (b4) Reversed-phase chromatography (RPC).

[0052] (b5) Gel filtration chromatography (GFC).

[0053] In another preferred embodiment, step (b) of the method includes the step:

[0054] (b1) Ammonium sulfate precipitation;

[0055] (b2) Hydrophobic interaction chromatography (HIC);

[0056] (b3) Low pH incubation to inactivate the virus; and

[0057] (b3a) Hydrophobic Interaction Chromatography (HIC)

[0058] (b4) Reversed-phase chromatography (RPC).

[0059] (b5) Gel filtration chromatography (GFC).

[0060] In another preferred embodiment, the method further includes one or more ultrafiltration and / or nanofiltration steps.

[0061] In another preferred embodiment, the method does not involve immunoaffinity purification.

[0062] In another preferred embodiment, the method does not involve lectin affinity purification.

[0063] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0064] Figure 1 The chromatogram of protein elution during the hydrophobic chromatography step in Example 1 is shown.

[0065] The diagram shows the ultraviolet absorption at 280 nm (UV280).

[0066] Figure 2 The electrophoretic images of the hydrophobic chromatographic components in the hydrophobic chromatography step of Example 1 are shown (non-reducing SDS-PAGE, separating gel concentration 15%).

[0067] Wherein, M is the molecular weight standard (Thermo Scientific, i.e., Fermatas, Unstained Protein MW Marker; molecular weights are 116.0, 66.2, 45.0, 35.0, 25.0, 18.4, 14.4 KD respectively); 1 is the HIC sample; 2 is the HIC flow-through fraction (FT); 3 is the HIC 45% B elution fraction; 4 is the HIC 100% B elution fraction.

[0068] Figure 3 The chromatogram of protein elution during the reverse-phase chromatography step in Example 2 is shown.

[0069] The diagram shows the ultraviolet absorption at 280 nm (UV280).

[0070] Figure 4 The electrophoresis diagrams of the components in the reversed-phase chromatography step of Example 2 are shown (non-reducing SDS-PAGE, separating gel concentration 15%).

[0071] Wherein, M is the molecular weight standard (Thermo Scientific, i.e., Fermatas, Unstained Protein MW Marker; molecular weights are 116.0, 66.2, 45.0, 35.0, 25.0, 18.4, 14.4 KD respectively); 1 is the precipitate after ammonium sulfate treatment (45% saturation); 2 is the component before RPC loading (component eluted with 100% B HIC); 3 is the component eluted with NaOH HIC; 4 is the component eluted with 20% B RPC; 5 is the component eluted with 40% B RPC; 6 is the component eluted with 60% B RPC; and 7 is the component eluted with 100% B RPC. Detailed Implementation

[0072] Through extensive and in-depth research and numerous screenings, the inventors have developed, for the first time, a method for purifying high-purity recombinant follicle-stimulating hormone (FSH). Specifically, in this method, the culture supernatant is purified sequentially through ultrafiltration, ammonium sulfate precipitation, hydrophobic chromatography, low-pH virus inactivation, reverse-phase chromatography, size exclusion chromatography, and nanofiltration. Experimental data show that the final product obtained in this invention is accurate, and the FSH exhibits high bioactivity and purity. This invention was completed based on these findings.

[0073] Purification method of the present invention

[0074] The purpose of this invention is to provide a high-purity purification process for recombinant FSH or recombinant FSH variants, which produces recombinant FSH or recombinant FSH variants, such as human recombinant FSH or human recombinant FSH variants, with economic efficiency, high purity and high specific activity.

[0075] In a preferred embodiment, the recombinant FSH of the present invention has an FSHα subunit with a sequence as shown in SEQ ID NO:1 and an FSHβ subunit with a sequence as shown in SEQ ID NO:2.

[0076] MDYYRKYAAIFLVTLSVFLHVLHSAPDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKS(SEQ ID NO:1)

[0077] MKTLQFFFLFCCWKAICCNSCELTNITIAIEKEECRFCISINTTWCAGYCYTRDLVYKDPARPKIQKTCTFKELVYETVRVPGCAHHADSLYTYPVATQCHCGKCDSDSTDCTVRGLGPSYCSFGEMKE*(SEQ ID NO:2)

[0078] Preferably, the nucleotide sequence of the FSHα subunit gene is shown in SEQ ID NO:3, and the nucleotide sequence of the FSHβ subunit gene is shown in SEQ ID NO:4.

[0079] atggattactacagaaaatatgcagctatctttctggtcacattgtcggtgtttctgcatgttctccattccgctcctgatgtgcaggattgcccagaatgcacgctacaggaaaacccattcttctcccagccgggtgccccaatacttcagtgcatgggctgctgcttctctaga gcatatcccactccactaaggtccaagaagacgatgttggtccaaaagaacgtcacctcagagtccacttgctgtgtagctaaatcatataacagggtcacagtaatggggggtttcaaagtggagaaccacggcgtgccactgcagtacttgttattatcacaaatcttaa(SEQ ID NO:3)

[0080] atgaagacactccagtttttcttccttttctgttgctggaaagcaatctgctgcaatagctgtgagctgaccaacatcaccattgcaatagagaaaga agaatgtcgtttctgcataagcatcaacaccacttggtgtgctggctactgctacaccagggatctggtgtataaggacccagccaggcccaaaatcca gaaaacatgtaccttcaaggaactggtatacgaaacagtgagagtgcccggctgtgctcaccatgcagattccttgtatacatacccagtggccaccc agtgtcactgtggcaagtgtgacagcgacagcactgattgtactgtgcgaggcctggggcccagctactgctcctttggtgaaatgaaagaataa(SEQ ID NO:4)

[0081] Accordingly, the present invention relates to a method for high-purity purification of recombinant FSH or recombinant FSH variants, comprising the following steps on a liquid recombinant FSH or recombinant FSH variant:

[0082] (1) Ammonium sulfate precipitation;

[0083] (2) Hydrophobic interaction chromatography (HIC);

[0084] (3) Low pH incubation inactivates the virus; and

[0085] (4) Reversed-phase chromatography (RPC).

[0086] Steps (1), (2), (3) and (4) can be performed in any order.

[0087] The method described above avoids cation exchange chromatography and anion exchange chromatography.

[0088] Preferably, ammonium sulfate precipitation or hydrophobic interaction chromatography is performed as the first step in the four steps. In a more preferred embodiment, ammonium sulfate precipitation is performed as the first step in the four steps.

[0089] The purification process may optionally include additional steps, such as affinity chromatography (e.g., dye affinity chromatography or perborate affinity chromatography) and / or a second filtration (e.g., dialysis filtration, ultrafiltration, or nanofiltration).

[0090] In a preferred embodiment, the process of the present invention includes gel filtration chromatography (SEC) as step (5).

[0091] In a preferred embodiment, steps (1), (2), (3), and (4) are performed in the following order.

[0092] (1) Ammonium sulfate precipitation;

[0093] (2) Hydrophobic interaction chromatography (HIC);

[0094] (3) Low pH incubation inactivates the virus; and

[0095] (4) Reversed-phase chromatography (RPC).

[0096] Ammonium sulfate precipitation is preferred as the first step because this embodiment allows for the direct precipitation of relatively "crude" recombinant FSH products, optionally described below, after the clarified sample has undergone cleaning (e.g., filtration), concentration, and / or buffer replacement steps. This embodiment offers the advantage of thoroughly removing any potentially residual nucleic acids, endotoxins, viruses, proteins, and other impurities. Furthermore, the clarified sample can be concentrated more than 15 times, facilitating ammonium sulfate precipitation. Overall, using ammonium sulfate precipitation as the first step reduces the number of preparative steps required before initiating chromatographic purification and allows for the use of large volumes of sample solution.

[0097] In yet another preferred embodiment, a second hydrophobic interaction chromatography (HIC) is performed after incubation at low pH to inactivate the virus (3) and before reversed-phase chromatography (RPC) (4). As described above, additional steps may be performed in addition to the steps described above and between the steps described above.

[0098] In yet another preferred embodiment, a third hydrophobic interaction chromatography (HIC) is performed after reversed-phase chromatography (RPC) (4). As described above, additional steps may be performed in addition to the steps described above and between the steps described above.

[0099] In yet another preferred embodiment, gel filtration chromatography (GFC) is performed after reversed-phase chromatography (RPC) (4). As described above, additional steps may be performed in addition to the steps described above and between the steps described above.

[0100] In yet another preferred embodiment, reversed-phase chromatography (RPC) is performed after reversed-phase chromatography (RPC) (4). As described above, additional steps may be performed in addition to the steps described above and between the steps described above.

[0101] The purification method of the present invention provides high-purity recombinant FSH and recombinant FSH variants, which can then be formulated into pharmaceutical compositions. Purity is typically above 99.50% w / w, preferably >99.80% w / w, more preferably >99.90% w / w, and even more preferably >99.99% w / w, based on total protein. Furthermore, the purification method of the present invention can be easily scaled up, even to an industrial scale, without significant changes to the purification conditions.

[0102] The recombinant FSH and recombinant FSH variants forming the raw materials for the purification process according to the invention can be obtained from liquids of natural origin or by recombinant techniques, such as in cell culture harvests containing recombinant FSH and recombinant FSH variants. Generally, before performing the first step, the raw materials obtained from natural sources or cell harvests, preferably cell harvests, are first cleaned (e.g., filtered), followed by concentration and / or buffer replacement steps.

[0103] In the ammonium sulfate precipitation step, commercially available ammonium sulfate is used, preferably a commercially available analytical grade product that meets national standards.

[0104] In the chromatographic step, commercially available resin fillers are used, preferably polymeric resins or agarose resins.

[0105] The purification process of this invention is described in more detail below.

[0106] Ammonium sulfate precipitation (1) step

[0107] This invention relates to the ammonium sulfate precipitation (1) step. In a preferred embodiment, particularly in the case of recombinant FSH, ammonium sulfate precipitation is performed as the first step. Recombinant FSH is highly hydrophilic, has high solubility in aqueous solution, and is not prone to intermolecular aggregation. To precipitate it, a preferably higher concentration of ammonium sulfate is required. A suitable higher concentration of ammonium sulfate allows more impurities to precipitate while preventing the desired recombinant FSH from precipitating.

[0108] Ammonium sulfate precipitation is generally carried out as follows: ammonium sulfate solid or ammonium sulfate solution is added directly, preferably a saturated ammonium sulfate solution, and even more preferably a 100% saturated ammonium sulfate solution.

[0109] The ammonium sulfate concentration is 40%-60%, preferably 42%-49%, more preferably 43%-47%, and even more preferably or about 44%-45%.

[0110] When using this solution, calculate the required amount of saturated solution based on the concentration to be added, and slowly add it to the stirred sample solution.

[0111] Ammonium sulfate precipitation is preferably carried out in a solution that is weakly alkaline, for example, at or about pH 7.1 to 8.5, more preferably at or about pH 7.2 to 8.0, and even more preferably at or about pH 7.3 to 7.6. In a preferred embodiment, the pH adjuster is an inorganic acid or base, more preferably ammonia or sulfuric acid.

[0112] Preferably, the precipitate is removed by centrifugation or ultrafiltration after step (1).

[0113] Preferably, no virus inactivation step is performed after step (1).

[0114] Hydrophobic interaction chromatography (HIC) (2) steps.

[0115] The process of this invention also involves the hydrophobic interaction chromatography (2) step. Hydrophobic interaction chromatography is typically performed by equilibration, column loading, subsequent washing, and subsequent elution.

[0116] Hydrophobic interaction chromatography (HIC) is a separation method that utilizes the hydrophobic properties of proteins. High ionic strength in solution enhances the hydrophobic interactions between proteins and the hydrophobic chromatographic medium. Utilizing this property, adsorption is strengthened through hydrophobic interactions between the nonpolar regions of the protein and immobilized hydrophobic ligands on a solid support. The sample to be separated is adsorbed onto the hydrophobic chromatographic medium under high ionic strength, and then eluted selectively with a linear or stepwise reduction in ionic strength. The substrate used in HIC is a matrix substituted with hydrophobic ligands such as ethyl, butyl, phenyl, or hexyl groups. Preferred substrates are those substituted with butyl or phenyl ligands.

[0117] Hydrophobic interaction chromatography (HIC) resin packings are known in the art and include resins such as ButylSepharose (GE Healthcare), phenylSepharose (low and high substituted), octylSepharose and alkylSepharose (all from GE Healthcare; other HIC resin sources include Biosepra, France; E. Merck, Germany; BioRad USA).

[0118] In a preferred embodiment, hydrophobic interaction chromatography is performed using a resin such as phenyl Sepharose 6 fast flow (highly substituted) (available from GE Healthcare). It should be understood that step (2) can be performed using alternative resins with similar characteristics. Alternative resins that can be used include: Capto Phenyl (HS); Capto Butyl; Butyl Sepharose 4FF; Butyl Sepharose 4FF (HS); Octyl Sepharose 4FF; Phenyl Sepharose 6FF (LS); Phenyl Sepharose 6FF (HS); Phenyl Sepharose Big Beads; Phenyl Sepharose 6FF; Phenyl Sepharose HP; Butyl Sepharose HP; Butyl Sepharose 4B; Octyl Sepharose CL-4B; Phenyl Sepharose CL-4B. All from GE Biosciences (800) 526-3593. Other resins include: Phenylseplife 6FF (from seplife) and Octyl Focurose 4FF (from Huiyan Biotechnology, see www.biomart.cn).

[0119] In a preferred embodiment, the equilibration, loading, washing, and elution buffers are selected from phosphate or its salts, sulfuric acid or its sodium salts, MES, Bis-Tris, ADA, PIPES, ACES, BES, MOPS, TES, HEPES, TRICINE, BICINE, and preferably ammonium sulfate. Bonding to the HIC resin is typically achieved using a high-conductivity equilibration and loading buffer, such as by adding a salt like NaCl, NH4Ac, (NH4)2SO4, or Na2SO4, preferably ammonium sulfate. A preferred salt concentration is 1.2 to 1.8 M, preferably about 1.5 M (NH4)2SO4. Washing generally uses a loading buffer. The elution step in hydrophobic interaction chromatography is preferably performed by reducing the conductivity of the mobile phase (reducing the salt concentration). This reduction can be achieved linearly or stepwise. Optionally, the buffered solution may also contain additional inorganic salts. In one embodiment, the inorganic salt is selected from sodium chloride, sodium sulfate, potassium chloride, potassium sulfate, sodium citrate, and potassium citrate.

[0120] Preferably, a balancing, loading, washing, and elution buffer is used, having a pH of about 6.5 to about 8.5, more preferably about 7.0 to about 8.0, and most preferably about 7.4. A particularly preferred balancing, loading, and washing buffer system contains sodium phosphate and ammonium sulfate, preferably with a pH of about 7.4. A preferred elution buffer contains ammonium sulfate with a pH of about 7.4.

[0121] Preferably, no buffer exchange is performed after step (2), which facilitates the subsequent step (b) (RPC). Buffer exchange can then be achieved via subsequent RPC by using a preferred buffer for subsequent chromatographic steps, such as AEX or HIC chromatography, as the running buffer.

[0122] Low pH incubation to inactivate the virus (3) step.

[0123] The process of this invention also involves the step of incubating the virus at low pH (3).

[0124] The envelope structure of viral particles is easily disrupted at low pH, thereby rendering the virus inactive. Higher incubation temperatures result in stronger virus inactivation; lower sample pH also leads to stronger virus inactivation; and longer incubation times further enhance virus inactivation.

[0125] For the low-pH incubation method to inactivate viruses, acidic pH adjusters are preferred, such as organic or inorganic acids, with phosphoric acid, hydrochloric acid, and citric acid being the most preferred. Citric acid is even more preferred.

[0126] The low pH incubation method for inactivating viruses has a pH of about 2.5 to about 4.5, more preferably about 3.0 to about 4.0, and most preferably about 3.5 to about 3.7.

[0127] The low pH incubation method for inactivating viruses has an incubation temperature of about 15°C to about 30°C, more preferably about 18°C ​​to about 26°C, and most preferably about 20°C to 25°C.

[0128] The low pH incubation method for inactivating viruses has an incubation temperature of approximately 15°C to approximately 30°C, more preferably approximately 18°C ​​to approximately 26°C, and most preferably approximately 20°C to 25°C. For example, incubation at 3.5 pH and 20°C is possible.

[0129] The incubation time is preferably at least 30 minutes, at least 60 minutes, at least 90 minutes, at least 2 hours, at least 3 hours, or at least 6 hours. For example, it can be incubated at a pH of about 3.5 and at room temperature for about 90 minutes. This virus inactivation step can be performed at any time during the purification process, but is preferably performed before the final chromatographic step.

[0130] Preferably, pH adjustment is performed after step (3) to facilitate the subsequent execution of step (4) (RPC).

[0131] Reversed-phase chromatography (RPC) (4) steps.

[0132] The process involves a reversed-phase chromatography (4) step. In a preferred embodiment, particularly in the case of recombinant FSH, reversed-phase chromatography is used as a capture step, wherein the recombinant FSH is enriched, for example, from a liquid of a natural source or cell culture harvest. Virus inactivation is preferably performed before elution from the RPC column.

[0133] According to the present invention, "reversed-phase chromatography" specifically refers to a chromatographic step in which a nonpolar stationary phase and, preferably, a polar mobile phase are used. In reversed-phase chromatography, polar compounds typically elute first, while nonpolar compounds are retained.

[0134] Reversed-phase chromatography is typically performed as follows: the column is equilibrated and loaded, followed by washing and elution, each using a buffer preferably containing an organic solvent such as ethanol or isopropanol. The organic solvent, such as isopropanol, can be used to inactivate the virus after elution.

[0135] Equilibration, loading, washing, and elution are preferably performed using a mobile phase buffered at a weakly alkaline pH, for example, pH 7 to 8, preferably pH 7.2 to 7.8, more preferably pH 7.4 to 7.6, and most preferably pH 7.5. In a preferred embodiment, the buffering substance is an acetate buffer, preferably ammonium acetate. Alternative buffers suitable for a pH of 7.4 to 7.6 include BES, MOPS, ammonium acetate, TES, and HEPES.

[0136] In a preferred embodiment, the buffer solution used in the RPC step contains an organic solvent, the concentration of which is adjusted for different chromatographic steps (equilibration, loading, washing, and elution). Preferably, the organic solvent is a water-miscible organic solvent such as acetonitrile, methanol, ethanol, propanol, acetone, etc., and more preferably isopropanol.

[0137] The eluent is a linear gradient elution or a stepped gradient elution, preferably a stepped gradient elution. The organic solvent in the eluent preferably includes at least one of acetonitrile, methanol, ethanol, propanol, acetone, etc. The buffer concentration is 20 mmol / L to 60 mmol / L, preferably about 35 mmol / L to 55 mmol / L or 50 mmol / L, such as 50 mmol / L ammonium acetate. The organic solvent concentration is at least 10% (v / v) isopropanol, at least 20% (v / v) isopropanol, at least 30% (v / v) isopropanol, at least 40% (v / v) isopropanol, at least 50% (v / v) isopropanol, at least 60% (v / v) isopropanol, at least 70% (v / v) isopropanol, or at least 80% (v / v) isopropanol. For example, the eluent is a mixture of 35 mmol / L ammonium acetate and 10% isopropanol (v / v), or a mixture of 50 mmol / L ammonium acetate and 20% isopropanol (v / v), or a mixture of 50 mmol / L ammonium acetate and 50% (v / v) isopropanol.

[0138] The reversed-phase column material is composed of hydrophobic inorganic matrix fillers, organic matrix fillers, or composite matrix fillers. Composite matrix fillers, such as silica gel, alumina, and zirconium oxide, are preferred as the matrix material. The inorganic matrix filler is coated with at least one polymer such as polystyrene, polyethylstyrene-divinylbenzene, polybutadiene, polyethylene oxide, polysiloxane, agarose, polychloromethylstyrene-diethoxymethylvinylsilane, and vapor-deposited carbon. Polystyrene / divinylbenzene is more preferred.

[0139] The ligands are generally selected from (but not limited to) aliphatic ligands, such as C2, C4, C6, C8, C10, C12, C14, C16, or C18 ligands or their derivatives such as cyanopropyl (CN-propyl), or branched aliphatic ligands, or benzene-based aromatic ligands such as phenyl, or at least one of other polar or nonpolar ligands. The ligands may be a mixture of two or more of these ligands.

[0140] Suitable polystyrene resins include, without limitation, resins supplied by Rohm Haas (e.g., AmberliteXAD or Amberchrom CG), resins supplied by Polymer Labs (e.g., PLRP-S), resins supplied by GE Healthcare (e.g., Source 30RPC), and resins supplied by Applied Biosystems (e.g., Poros R). A particularly preferred resin is Source 30RPC (GE Healthcare).

[0141] Generally, purified samples are loaded onto the column at a concentration of at least about 0.1 mg per mL of resin, for example, at least about 0.3 mg, 0.4 mg, 0.8 mg, 1.5 mg, 4 mg, 8 mg, or 15 mg per mL of resin; or 0.1-180 mg, for example, 0.1-150 mg, 0.5-100 mg, 2-60 mg, or 3-40 mg per mL of resin; preferably, the loading is at least 1 mg per mL of resin. Measurements of the packed resin volume are generally performed in suspension or similar mode.

[0142] Additional steps

[0143] In addition to the three main chromatographic steps (1), (2), (3), and (4), the process of the present invention may optionally include additional steps known to those skilled in the art, such as chromatographic steps, filtration steps, or virus inactivation steps. Preferred additional steps are filtration, such as dialysis filtration, ultrafiltration, or nanofiltration.

[0144] In a preferred embodiment, the process of the present invention includes the following steps in the following order:

[0145] (0) Ultrafiltration; (optionally an additional dialysis filtration step; preferably using a membrane with a cutoff value of 10 kD or about 10 kD)

[0146] (1) Ammonium sulfate precipitation;

[0147] (2) Hydrophobic interaction chromatography (HIC); (preferably using a Phenyl Sepharose 6fastflow (highly substituted) column)

[0148] (3) Incubation at low pH to inactivate the virus; and (preferably pH 3.6)

[0149] (4) Reversed-phase chromatography (RPC). (Source 30 RPC column is preferred)

[0150] Ultrafiltration is performed on recombinant FSH solution samples, particularly as a virus removal step; that is, to reduce the risk of contamination of glycoprotein preparations with viruses or viral particles derived from cell cultures. Ultrafiltration can be performed at any stage of the purification process; however, it is particularly preferred to perform ultrafiltration before the chromatographic procedure. Ultrafiltration can be performed more than once, for example, it can be performed twice. Ultrafiltration (or percolation) is preferably performed using an ultrafiltration membrane of about 10-15 kD, most preferably 10 kD.

[0151] In a preferred embodiment, the process of the present invention includes the following steps in the following order:

[0152] (0) Ultrafiltration; (optionally an additional dialysis filtration step; preferably using a membrane with a cutoff value of 10 kD or about 10 kD)

[0153] (1) Ammonium sulfate precipitation;

[0154] (2) Hydrophobic interaction chromatography (HIC); (preferably using a Phenyl Sepharose 6fastflow (highly substituted) column)

[0155] (3) Incubation at low pH to inactivate the virus; and (preferably pH 3.6)

[0156] (4) Reversed-phase chromatography (RPC). (Source 30 RPC column is preferred)

[0157] (4a) Hydrophobic interaction chromatography (HIC); (preferably using a Phenyl Sepharose 6fast flow (highly substituted) column)

[0158] (5) Nanomembrane filtration; (preferably a 20-nanometer nanomembrane filter)

[0159] The specific purification process described above is preferably performed without any other chromatographic and / or ultrafiltration and / or dialysis steps. However, in particular embodiments, the purification process may also include additional steps, especially one or more of the additional steps described herein, such as those for removing or inactivating undesirable and / or hazardous substances.

[0160] In another preferred embodiment, the process of the present invention includes the following steps in the following order:

[0161] (0) Ultrafiltration; (optionally an additional dialysis filtration step; preferably using a membrane with a cutoff value of 10 kD or about 10 kD)

[0162] (1) Ammonium sulfate precipitation;

[0163] (2) Hydrophobic interaction chromatography (HIC); (preferably using a Phenyl Sepharose 6fastflow (highly substituted) column)

[0164] (3) Incubation at low pH to inactivate the virus; and (preferably pH 3.6)

[0165] (3a) Hydrophobic interaction chromatography (HIC); (preferably using a Phenyl Sepharose 6fastflow (highly substituted) column)

[0166] (4) Reversed-phase chromatography (RPC). (Source 30 RPC column is preferred)

[0167] (5) Nanomembrane filtration; (preferably a 20-nanometer nanomembrane filter)

[0168] The specific purification process described above is preferably performed without any other chromatographic and / or ultrafiltration and / or dialysis steps. However, in particular embodiments, the purification process may also include additional steps, especially one or more of the additional steps described herein, such as those for removing or inactivating undesirable and / or hazardous substances.

[0169] The advantages of this invention lie in its fewer purification steps, higher purification efficiency, and reduction of the number of chromatographic steps to a minimum of two. In particular, the purification process according to the invention avoids costly and problematic purification steps, especially affinity purification steps, particularly immunoaffinity purification steps and lectin affinity purification steps. The method of this invention provides high purity and specific biological activity of recombinant FSH, with a purity >97%, preferably >99%, more preferably >99.5% w / w, each based on, for example, total protein measured by high-performance liquid chromatography. Its biological activity is >59000 IU / ml, preferably >60000 IU / ml, more preferably >61000 IU / ml. Furthermore, the purification process of this invention also achieves a high recovery rate of the target FSH.

[0170] Storage / Freeze-drying

[0171] The liquid composition containing recombinant FSH obtained from the above purification process can be stored as is or frozen after purification. The eluent can be lyophilized ("freeze-dry") to remove the solvent. The resulting liquid or lyophilized product is referred to as "recombinant FSH total".

[0172] Preparations

[0173] The recombinant FSH of the present invention, or purified according to the method of the present invention, can be formulated into preparations for any class of use, preferably for intramuscular or subcutaneous injection, preferably subcutaneous injection. The recombinant FSH preparation can be lyophilized and dissolved in water for injection prior to injection. The recombinant FSH preparation can also be a liquid preparation, in which case it is injected directly without prior dissolution.

[0174] The recombinant FSH formulation may contain known excipients and stabilizers, and may additionally contain antioxidants and / or surfactants. The recombinant FSH of the present invention can be formulated with known excipients and stabilizers such as sucrose and mannitol. It may also contain antioxidants such as methionine. It may also contain surfactants such as Tween (preferably Tween 20), or Pluronic (preferably Pluronic F68).

[0175] Recombinant FSH formulations may be single-dose or multi-dose. If multi-dose, they should preferably contain an antimicrobial agent, such as alkyl benzyl ester, benzyl alcohol, m-cresol, thymol, or phenol, preferably methyl benzyl ester or m-cresol. Single-dose formulations may also contain an antimicrobial agent. Suitable formulations are described, for example, in PCT / EP2011 / 062986, which is incorporated herein by reference.

[0176] In a particularly preferred multi-dose formulation, the recombinant FSH produced by the method of the present invention is prepared by dissolving it together with sucrose, phosphate buffer (pH 6.6 to 7.4), Tween 20, methionine and an antibacterial agent in water for injection.

[0177] Indications

[0178] The recombinant FSH of this invention is applicable to all treatments where recombinant FSH is indicated. It is particularly suitable for subcutaneous administration of ovulation induction and controlled superovulation in assisted reproductive technologies, controlled ovarian hyperstimulation in assisted reproductive technologies, and the treatment of oligospermia. It can be used in combination with other gonadotropins such as LH and hCG. It can also be used with other compounds that enhance the response to FSH, such as clomiphene citrate, clomiphene citrate, and aromatase inhibitors such as anastrozole, letrozole, fazodazole, and YM-511. Additionally, LH and hCG can be used alone in fertility treatments.

[0179] Recombinant FSH

[0180] The term "recombinant" is used to refer to a formulation of FSH produced using recombinant DNA technology. An example of a method for expressing FSH using recombinant technology is the transfection of eukaryotic cells with DNA sequences encoding the FSH α and β subunits. Typically, the expression medium carries a strong promoter driving FSH expression, such as CMV or SV40, as well as a suitable selection marker incorporated into the medium for selecting host cells. Transfection can be stable or transient. Suitable recombinant expression systems are well known in the art and therefore do not need to be detailed. Preferably, the eukaryotic host cells are selected from primate cells, preferably human cells and rodent cells, preferably CHO cells. As described in European patents EP 0 211 894 and EP 0 487 512, there is one subunit, whether in one vector or two vectors, and each subunit has a single promoter. The DNA encoding FSH can be a cDNA or contain introns.

[0181] Another embodiment of FSH generation using recombinant technology involves inserting a heterologous regulatory fragment into the endogenous sequence encoding one or both subunits of FSH via homologous recombination in an efficient ligation manner, as described in European Patent No. EP0505500 (Applied Research Systems ARS Holding NV). The recombinant technology, as disclosed in WO99 / 57263 (Transkaryotic Therapies), involves heterologous insertion of one subunit into the cell, while another subunit undergoes homologous recombination to insert a heterologous regulatory fragment to activate the genomic sequence for expression. The method of this invention can be used to purify FSH expressed by any of the methods described above.

[0182] The expression of “FSH variants” refers to molecules that exhibit FSH activity but differ from human FSH in amino acid sequence, glycosylation pattern, or linkages between subunits. Examples include CTP-FSH, a long-acting modified recombinant FSH composed of a wild-type α subunit and a hybrid β subunit, wherein the carboxyl-terminal peptide of hCG is fused to the C-terminus of the FSH β subunit, as described by LaPolt et al.; Endocrinology; 1992, 131, 2514-2520; or Klein et al.; Development and characterization of a long-acting recombinant hFSH agonist; CN 101087805B Human Reprod. 2003, 18, 50-56. It also includes single-chain CTP-FSH, a single-chain molecule composed of βFSH (FSH β subunit), βhCGCTP (113-145) (hCG carboxyl-terminal peptide), and αFSH (FSH α subunit).

[0183] The FSH variants referred to here also include PEG-rh FSH modification, rh FSH-Fc fusion proteins, rh FSH-CTP fusion proteins, bifunctional antibodies containing rh FSH, conjugates containing rh FSH, and rhFSH glycosylation modification.

[0184] The FSH variants mentioned in this article also include those from different species such as horses (Equus caballus), pigs (Susscrofa), cattle (Bos taurus), cats (Felis catus), and dogs (Canis familiaris).

[0185] In a preferred embodiment, FSH is recombinantly generated in serum or in a serum-free medium. In yet another preferred embodiment, the purified FSH generated according to the method of the invention is suitable for subcutaneous administration, which enables patient self-administration.

[0186] Unprocessed recombinant FSH

[0187] This refers to the cell culture supernatant from recombinant cells expressing FSH prior to any purification steps. This expression includes the supernatant in its unprocessed form (isolated from the cells), as well as concentrated and / or filtered and / or ultrafiltered supernatants.

[0188] Bioactivity

[0189] This refers to the FSH-related biological functions induced by an FSH preparation, such as ovarian weight obtained in the Steelman-Pohley assay [Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotrophin; Endocrinology; 1953, 53, 604-616], or follicular growth in female patients. Follicular growth in female patients can be estimated by ultrasound, for example, based on the number of follicles with an average diameter of approximately 16 mm on day 8 post-stimulation. Bioactivity can be estimated using a standard acceptable for FSH.

[0190] The term "specific activity" for FSH refers to the biological activity of a formulation, expressed in IU, divided by the weight of the protein. IU is the root of the biological activity.

[0191] According to recognized FSH bioassays such as the Steeelman-Pohley bioassay, protein weight is determined by a total protein content assay, such as the Lowry assay [OH Lowry, NJ Rosebrough, A.L. Farr and RJ Randall (1951) J. Biol. Chem. 193: 265; Hartree EE (1972). Anal. Biochem. 48: 422; JR Dulley and PAGrieve (1975) Anal. Biochem. 64: 136], the Bradford assay [Bradford, MM (1976) Anal. Biochem. 72, 248], or by measuring the absorbance at 280 nm.

[0192] The specific activity of FSH in this invention is preferably greater than or about 10,000 IU / mg, more preferably greater than or about 12,000 IU / mg, even more preferably greater than or about 14,000 IU / mg, and most preferably greater than or about 16,000 IU / mg, wherein the bioactivity is determined by the Steelman-Pohley assay and the protein purity is determined by SEC-HPLC.

[0193] The main advantages of this invention include:

[0194] 1) The method provided by this invention can replace existing methods for purifying human FSH.

[0195] 2) The method of the present invention provides high purity and specific biological activity of recombinant FSH, with a purity >97%, preferably >99%, more preferably >99.5% w / w, and even more preferably >99.9%, each based on total protein as measured, for example, by high performance liquid chromatography.

[0196] 3) The recombinant follicle-stimulating hormone of the present invention has a specific activity as high as 16,000 IU / mg. Its biological activity is >59,000 IU / ml, preferably >60,000 IU / ml, more preferably >61,000 IU / ml. In addition, the recovery rate of the target FSH in the purification process of the present invention is also very high.

[0197] 4) Compared with existing preparation methods, the recombinant follicle-stimulating hormone of the present invention is simpler and easier to operate. Its advantages lie in fewer purification steps, better purification effect, and a reduction in the number of chromatographic steps to a minimum of two. In particular, using the purification process according to the present invention avoids costly and problematic purification steps, especially affinity purification steps, particularly immunoaffinity purification steps and lectin affinity purification steps.

[0198] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0199] Example 1: Purification of FSH using Method 1

[0200] Step (0): Ultrafiltration

[0201] The raw material for forming crude FSH comes from the supernatant of cell culture containing recombinant FSH.

[0202] Prior to the ultrafiltration step, the supernatant was cleaned by filtration through a 1 μm membrane at room temperature. Then, ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 10 KD. The ultrafiltrate was concentrated to approximately 8%.

[0203] Step (1): Ammonium sulfate precipitation

[0204] Buffer solution: 100% saturated (NH₄)₂SO₄ solution, pH 7.4

[0205] Ammonium sulfate was precipitated from the ultrafiltrate from step (0) at room temperature by slowly adding a 100% saturated (NH4)2SO4 solution at a ratio of 45% to the ultrafiltrate from step (0) while stirring.

[0206] Centrifuge at 3500×g for 20 min, remove precipitate, and collect supernatant.

[0207] The supernatant was filtered through a 0.65μm membrane.

[0208] Step (2): Hydrophobic interaction chromatography (HIC); (preferably using a Phenyl Sepharose 6 fast flow (highly substituted) column)

[0209] Solution A: "50 mmol / L NH4Ac + 50% saturated (NH4)2SO4, pH 7.4"

[0210] Solution B: "50 mmol / L NH4Ac, pH 7.4"

[0211] 1) Equilibration: Use HIC equilibration solution (50 mmol / L NH4Ac + 50% saturated (NH4)2SO4, pH 7.4) to equilibrate the column, and the equilibration volume should not be less than 2 CV.

[0212] 2) Sample loading: Receive the sample from the previous process and load it. Adjust the loading flow rate to ensure that the retention time is not less than 5 minutes.

[0213] 3) Flat wash: Wash the column with HIC equilibration solution until A280 drops to baseline.

[0214] 4) Rinse with HIC rinse buffer (50 mmol / L NH4Ac + 27.5% saturated (NH4)2SO4, pH 7.4) to remove contaminating proteins until A280 drops to baseline.

[0215] 5) Elute the target protein with HIC elution buffer (50 mmol / L NH4Ac, pH 7.4 ± 0.2) and collect the A280 main peak.

[0216] The eluent proceeds directly to the next step. This step is performed at room temperature.

[0217] Step (3): Incubate at low pH to inactivate the virus (preferably pH 3.6).

[0218] Buffer solution: 0.5 mol / L citric acid

[0219] Buffer solution: 1.0 mol / L Tris

[0220] Adjust the pH of the substance from step (2) to 3.6 with 0.5 mol / L citric acid. Incubate the sample at 20°C for 2.5 hours. Adjust the pH of the sample to 7.4 with 1 mol / L Tris. Centrifuge at 1400×g for 20 min. Filter through a 0.2 μm membrane.

[0221] The filtrate proceeds directly to the next step. This step is performed at room temperature.

[0222] Step (4): Reversed-phase chromatography (RPC). (Preferably using a Source 30 RPC column)

[0223] RPC equilibration solution: 50 mmol / L NH4AC, pH 7.4

[0224] RPC rinsing solution A: 50 mmol / L NH4Ac + 10% isopropanol, pH 7.4

[0225] RPC eluent: 50 mmol / L NH4Ac + 20% isopropanol, pH 7.4

[0226] RPC rinsing solution B: 50 mmol / L NH4Ac + 50% isopropanol, pH 7.4

[0227] RPC cleaning solution: 0.5 mol / L NaOH

[0228] RPC preservation solution: 0.01 mol / L NaOH

[0229] The eluent from step (3) is directly loaded onto the column. The column is rinsed with RPC equilibration buffer, then with RPC wash buffer A, followed by elution with RPC eluent, and the elution peak is collected. The column is then rinsed with RPC wash buffer B. The column is rinsed with RPC preservation buffer and then preserved.

[0230] Step (5): Nanomembrane filtration; (preferably using a 20 nm nanomembrane filter)

[0231] It uses Millipore's 20nm Virosolve Pro nanomembrane filter.

[0232] The sample from the previous step was filtered sequentially using a 0.1 μm pre-filter and a 20 nm nanometer membrane filter, with the filtration volume of the nanometer membrane filter not exceeding 13 ml / cm². Record the number of nanometer membrane filters used and the filtration volume of each filter.

[0233] Example 2: Purification of FSH using Method 2

[0234] Step (0): Ultrafiltration

[0235] The raw material for forming crude FSH comes from the supernatant of cell culture containing recombinant FSH.

[0236] Prior to the ultrafiltration step, the supernatant was cleaned by filtration through a 1 μm membrane at room temperature. Then, ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 10 KD. The ultrafiltrate was concentrated to approximately 8%.

[0237] Step (1): Ammonium sulfate precipitation

[0238] Buffer solution: 100% saturated (NH₄)₂SO₄ solution, pH 7.4

[0239] Ammonium sulfate was precipitated from the ultrafiltrate from step (0) at room temperature by slowly adding a 100% saturated (NH4)2SO4 solution at a ratio of 45% to the ultrafiltrate from step (0) while stirring.

[0240] Centrifuge at 3500×g for 20 min, remove precipitate, and collect supernatant.

[0241] The supernatant was filtered through a 0.65μm membrane.

[0242] Step (2): Hydrophobic interaction chromatography (HIC); (preferably using a Phenyl Sepharose 6 fast flow (highly substituted) column)

[0243] Solution A: "50 mmol / L NH4Ac + 50% saturated (NH4)2SO4, pH 7.4"

[0244] Solution B: "50 mmol / L NH4Ac, pH 7.4"

[0245] 1) Equilibration: Use HIC equilibration solution (50 mmol / L NH4Ac + 50% saturated (NH4)2SO4, pH 7.4) to equilibrate the column, and the equilibration volume should not be less than 2 CV.

[0246] 2) Sample loading: Receive the sample from the previous process and load it. Adjust the loading flow rate to ensure that the retention time is not less than 5 minutes.

[0247] 3) Flat wash: Wash the column with HIC equilibration solution until A280 drops to baseline.

[0248] 4) Rinse with HIC rinse buffer (50 mmol / L NH4Ac + 27.5% saturated (NH4)2SO4, pH 7.4) to remove contaminating proteins until A280 drops to baseline.

[0249] 5) Elute the target protein with HIC elution buffer (50 mmol / L NH4Ac, pH 7.4 ± 0.2) and collect the A280 main peak.

[0250] The eluent proceeds directly to the next step. This step is performed at room temperature.

[0251] Step (3): Incubate at low pH to inactivate the virus (preferably pH 3.6).

[0252] Buffer solution: 0.5 mol / L citric acid

[0253] Buffer solution: 1.0 mol / L Tris

[0254] Adjust the pH of the substance from step (2) to 3.6 with 0.5 mol / L citric acid. Incubate the sample at 20°C for 2.5 hours. Adjust the pH of the sample to 7.4 with 1 mol / L Tris. Centrifuge at 1400×g for 20 min. Filter through a 0.2 μm membrane.

[0255] The filtrate proceeds directly to the next step. This step is performed at room temperature.

[0256] Step (4): Reversed-phase chromatography (RPC). (Preferably using a Source 30 RPC column)

[0257] RPC equilibration solution: 50 mmol / L NH4AC, pH 7.4

[0258] RPC rinsing solution A: 50 mmol / L NH4Ac + 10% isopropanol, pH 7.4

[0259] RPC eluent: 50 mmol / L NH4Ac + 20% isopropanol, pH 7.4

[0260] RPC rinsing solution B: 50 mmol / L NH4Ac + 50% isopropanol, pH 7.4

[0261] RPC cleaning solution: 0.5 mol / L NaOH

[0262] RPC preservation solution: 0.01 mol / L NaOH

[0263] The eluent from step (3) is directly loaded onto the column. The column is rinsed with RPC equilibration buffer, then with RPC wash buffer A, followed by elution with RPC eluent, and the elution peak is collected. The column is then rinsed with RPC wash buffer B. The column is rinsed with RPC preservation buffer and then preserved.

[0264] Step (5): Gel filtration chromatography (GFC) (preferably using Hiload 26 / 600 Superdex 75 Prep grade)

[0265] GFC equilibration solution: 10 mmol / L PB, pH 7.0

[0266] GFC cleaning solution: 0.5 mol / L NaOH

[0267] GFC preservation solution: 0.01 mol / L NaOH

[0268] The column is equilibrated with GFC equilibration buffer. Samples collected after RPC chromatography are loaded in portions and washed with GFC equilibration buffer. After the first peak is eluted, the column is washed with GFC cleaning buffer and then washed and stored with GFC preservation buffer.

[0269] Step (6): Nanomembrane filtration (preferably using a 20 nm nanomembrane filter)

[0270] It uses Millipore's 20nm Virosolve Pro nanomembrane filter.

[0271] The sample from the previous step was filtered sequentially using a 0.1 μm pre-filter and a 20 nm nanometer membrane filter, with the filtration volume of the nanometer membrane filter not exceeding 13 ml / cm². Record the number of nanometer membrane filters used and the filtration volume of each filter.

[0272] Example 3: Determination of Sample Purity

[0273] The purity of the total rhFSH after these purification steps was determined as follows:

[0274] Table 1. Determination of total rhFSH purity after each step

[0275]

[0276]

[0277] Example 4: Determination of sample bioactivity

[0278] The bioactivity of purified rhFSH was measured using the Steelman-Pohley ovarian weight gain method. Specific activity was calculated by dividing the bioactivity by the FSH content determined by SE-HPLC, as shown in Table 2. The final total specific activity typically ranged from 14,000 to 17,000 IU / mg.

[0279] The final total FSH values ​​of the two embodiments obtained according to the methods of Examples 1 and 2 are shown in Table 1.

[0280] Table 2. Total activity of rhFSH purified in this invention

[0281]

[0282] The purity values ​​of the final total FSH obtained according to the methods of Examples 1 and 2 are shown in Table 3.

[0283] Table 3 Purity of the total rhFSH purified in this invention

[0284]

[0285]

[0286] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Jiangsu Jingze Biomedical Co., Ltd. Shanghai Jingze Biotechnology Co., Ltd. Chengdu Zeyan Biotechnology Co., Ltd. <120> Recombinant human follicle-stimulating hormone and its preparation method <130> P2019-0847 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 116 <212> PRT <213> Artificial sequence <400> 1 Met Asp Tyr Tyr Arg Lys Tyr Ala Ala Ile Phe Leu Val Thr Leu Ser 1 5 10 15 Val Phe Leu His Val Leu His Ser Ala Pro Asp Val Gln Asp Cys Pro 20 25 30 Glu Cys Thr Leu Gln Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro 35 40 45 Ile Leu Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro 50 55 60 Leu Arg Ser Lys Lys Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu 65 70 75 80 Ser Thr Cys Cys Val Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly 85 90 95 Gly Phe Lys Val Glu Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr 100 105 110 Tyr His Lys Ser 115 <210> 2 <211> 129 <212> PRT <213> artificial sequence <400> 2 Met Lys Thr Leu Gln Phe Phe Phe Leu Phe Cys Cys Trp Lys Ala Ile 1 5 10 15 Cys Cys Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys 20 25 30 Glu Glu Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly 35 40 45 Tyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys 50 55 60 Ile Gln Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Arg 65 70 75 80 Val Pro Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val 85 90 95 Ala Thr Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys 100 105 110 Thr Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met Lys 115 120 125 Glu <210> 3 <211> 351 <212> DNA <213> artificial sequence <400> 3 atggattact acagaaaata tgcagctatc tttctggtca cattgtcggt gtttctgcat 60 gttctccatt ccgctcctga tgtgcaggat tgcccagaat gcacgctaca ggaaaaccca 120 ttcttctccc agccgggtgc cccaatactt cagtgcatgg gctgctgctt ctctagagca 180 tatcccactc cactaaggtc caagaagacg atgttggtcc aaaagaacgt cacctcagag 240 tccacttgct gtgtagctaa atcatataac agggtcacag taatgggggg tttcaaagtg 300 gagaaccaca cggcgtgcca ctgcagtact tgttattatc acaaatctta a 351 <210> 4 <211> 390 <212> DNA <213> Artificial Sequence <400> 4 atgaagacac tccagttttt cttccttttc tgttgctgga aagcaatctg ctgcaatagc 60 tgtgagctga ccaacatcac cattgcaata gagaaagaag aatgtcgttt ctgcataagc 120 atcaacacca cttggtgtgc tggctactgc tacaccaggg atctggtgta taaggaccca 180 gccaggccca aaatccagaa aacatgtacc ttcaaggaac tggtatacga aacagtgaga 240 gtgcccggct gtgctcacca tgcagattcc ttgtatacat acccagtggc cacccagtgt 300 cactgtggca agtgtgacag cgacagcact gattgtactg tgcgaggcct ggggcccagc 360 tactgctcct ttggtgaaat gaaagaataa 390

Claims

1. A method for purifying high-purity recombinant follicle-stimulating hormone, characterized by comprising the following steps: (a) Provide a raw material solution containing recombinant follicle-stimulating hormone; (b) The raw material liquid is subjected to the following steps in sequence: Step (0): Ultrafiltration, The raw material for forming crude FSH comes from the supernatant of cell culture containing recombinant FSH; Prior to the ultrafiltration step, the supernatant was cleaned by filtration through a 1 μm membrane at room temperature; then ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 10 KD; the ultrafiltrate was concentrated to approximately 8%. Step (1): Ammonium sulfate precipitation, Buffer solution: 100% saturated (NH₄)₂SO₄ solution, pH 7.

4. Ammonium sulfate was precipitated from the ultrafiltrate from step (0) at room temperature by slowly adding a 100% saturated (NH4)2SO4 solution at a ratio of 45% to the ultrafiltrate from step (0) while stirring. Centrifuge at 3500g for 20 minutes, remove precipitate, and collect supernatant; The supernatant was filtered through a 0.65μm membrane; Step (2): Hydrophobic interaction chromatography (HIC); a Phenyl Sepharose 6fastflow column was used. Solution A: 50 mmol / L NH4Ac + 50% saturated (NH4)2SO4, pH 7.4 Solution B: 50 mmol / L NH4Ac, pH 7.4 1) Equilibration: Equilibrate the column with HIC equilibration solution of 50 mmol / L NH4Ac + 50% saturated (NH4)2SO4, pH 7.4, and the equilibration volume should not be less than 2 CV; 2) Sample loading: Receive the sample from the previous process and load it, adjusting the loading flow rate to ensure a retention time of not less than 5 minutes; 3) Flat wash: Wash the column with HIC equilibration buffer until A280 drops to baseline; 4) Rinse for contaminating proteins with 50 mmol / L NH4Ac + 27.5% saturated (NH4)2SO4, pH 7.4 HIC rinse buffer until A280 drops to baseline; 5) Elute the target protein with 50 mmol / L NH4Ac, pH 7.4 ± 0.2 HIC elution buffer, and collect the A280 main peak; The eluent proceeds directly to the next step; this step is performed at room temperature. Step (3): Incubate at low pH to inactivate the virus. Buffer solution: 0.5 mol / L citric acid. Buffer solution: 1.0 mol / L Tris, Adjust the pH of the substance in step (2) to 3.6 with 0.5 mol / L citric acid; incubate the sample at 20°C for 2.5 hours; adjust the pH of the sample to 7.4 with 1 mol / L Tris; centrifuge at 1400g for 20 min; filter through a 0.2 μm membrane; The filtrate proceeds directly to the next step; this step is performed at room temperature. Step (4): Reversed-phase chromatography (RPC); Source 30 RPC column was selected. RPC equilibration solution: 50 mmol / L NH4Ac, pH 7.4 RPC rinsing solution A: 50 mmol / L NH4Ac + 10% isopropanol, pH 7.

4. RPC eluent: 50 mmol / L NH4Ac + 20% isopropanol, pH 7.

4. RPC rinsing solution B: 50 mmol / L NH4Ac + 50% isopropanol, pH 7.

4. RPC cleaning solution: 0.5 mol / L NaOH RPC preservation solution: 0.01 mol / L NaOH The eluent from step (3) is directly loaded onto the sample; Rinse the column with RPC equilibration buffer, rinse with RPC wash buffer A, elute with RPC elution buffer, and collect the elution peak; rinse with RPC wash buffer B; rinse with RPC preservation buffer and preserve the column. Step (5): Gel filtration chromatography (GFC), using Hiload 26 / 600 Superdex 75 Prep grade. GFC equilibrium solution: 10 mmol / L PB, pH 7.0 GFC cleaning solution: 0.5 mol / L NaOH GFC preservation solution: 0.01 mol / L NaOH The column is equilibrated with GFC equilibration buffer. Samples collected after RPC chromatography are loaded in portions. The column is washed with GFC equilibration buffer. After the first peak is eluted, the column is washed with GFC cleaning buffer and then washed with GFC preservation buffer to preserve the column. Step (6): Nanomembrane filtration, It uses Millipore's 20nm Virosolve Pro nanomembrane filter; The sample from the previous step was filtered sequentially using a 0.1μm pre-filter and a 20nm nanometer membrane filter, with the nanometer membrane filter filtration capacity not exceeding 13ml / cm³. 2 Record the number of nanomembrane filters used and the filtration volume of each filter; The recombinant follicle-stimulating hormone is rhFSH.

2. The purification method according to claim 1, characterized in that, The method does not perform immunoaffinity purification, nor does it perform lectin affinity purification.

3. The purification method as described in claim 1, characterized in that, The recombinant follicle-stimulating hormone is composed of an α subunit and a β subunit.

4. The purification method according to claim 3, characterized in that, The α subunit has the amino acid sequence shown in SEQ ID No. 1, while the β subunit has the amino acid sequence shown in SEQ ID No. 2.

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