阅读说明：本技术 一种高纯度重组促***纯化方法 (method for purifying high-purity recombinant follicle stimulating hormone ) 是由 彭红卫 张玉晶 张磊 王冲 叶凡 俞峻红 赵家跟 李晓鹏 俞亚波 于 2019-07-31 设计创作，主要内容包括：本发明提供了一种高纯度重组促卵泡刺激素的纯化方法。具体地,包括步骤：(a)提供一含有重组促卵泡激素的原料液；(b)对所述原料液依次进行：(b1)硫酸铵沉淀；(b2)疏水相互作用色谱法(HIC)；(b3)低PH孵育灭活病毒；和(b4)反相色谱层析(RPC)；其中,步骤(b1)、(b2)、(b3)和(b4)不可以以任意次序进行。本发明的重组促卵泡刺激素相比已有的制备方法,简单易操作,并且用本发明的方法得到的重组FSH纯度可高达99.5％w/w,比活性高达16000IU/mg。(the invention provides a method for purifying high-purity recombinant follicle stimulating hormone. Specifically, the method comprises the following steps: (a) providing a raw material solution containing recombinant follicle stimulating hormone; (b) sequentially carrying out the following steps on the raw material liquid: (b1) ammonium sulfate precipitation; (b2) hydrophobic Interaction Chromatography (HIC); (b3) incubating and inactivating the virus at low pH; and (b4) Reverse Phase Chromatography (RPC); wherein steps (b1), (b2), (b3), and (b4) may not be performed in any order. Compared with the existing preparation method, the recombinant follicle stimulating hormone of the invention is simple and easy to operate, and the purity of the recombinant FSH obtained by the method of the invention can reach 99.5% w/w, and the specific activity reaches 16000 IU/mg.)

1. A method for purifying high-purity recombinant follicle stimulating hormone, which comprises the steps of:

(a) providing a raw material solution containing recombinant follicle stimulating hormone;

(b) Sequentially carrying out the following steps on the raw material liquid:

(b1) Ammonium sulfate precipitation;

(b2) hydrophobic Interaction Chromatography (HIC);

(b3) Incubating and inactivating the virus at low pH; and

(b4) Reverse Phase Chromatography (RPC);

Wherein steps (b1), (b2), (b3), and (b4) may not be performed in any order.

2. The purification process of claim 1, wherein the feed solution is a fermentation product.

3. The purification process according to claim 2, wherein the fermentation is a fermentation in a eukaryotic cell, preferably a mammalian cell, more preferably a CHO cell.

4. The purification method of claim 1, wherein the recombinant follicle stimulating hormone comprises PEG-rh FSH, rh FSH-Fc fusion protein, rh FSH-CTP fusion protein, bifunctional antibodies comprising rh FSH, multifunctional antibodies comprising rh FSH, conjugated drugs comprising rh FSH, rhFSH glycosylation modifications, or combinations thereof.

5. The purification method of claim 1, further comprising (b5) Reverse Phase Chromatography (RPC).

6. The purification method of claim 5, wherein step (b) of the method comprises the steps of:

(b1) Ammonium sulfate precipitation;

(b2) Hydrophobic Interaction Chromatography (HIC);

(b3) Incubating and inactivating the virus at low pH;

(b4) Reverse Phase Chromatography (RPC); and

(b5) Reverse Phase Chromatography (RPC).

7. The purification method of claim 1, wherein step (b) of the method comprises the steps of:

(b1) ammonium sulfate precipitation;

(b2) hydrophobic Interaction Chromatography (HIC);

(b3) Incubating and inactivating the virus at low pH;

(b4) Reverse Phase Chromatography (RPC); and

(b5) Gel Filtration Chromatography (GFC).

8. The purification method of claim 1, wherein step (b) of the method comprises the steps of:

(b1) ammonium sulfate precipitation;

(b2) Hydrophobic Interaction Chromatography (HIC);

(b3) Incubating and inactivating the virus at low pH;

(b3a) Hydrophobic Interaction Chromatography (HIC);

(b4) Reverse Phase Chromatography (RPC); and

(b5) Gel Filtration Chromatography (GFC).

9. the purification method of claim 1, further comprising one or more ultrafiltration and/or nanofiltration steps.

10. The purification method of claim 1, wherein the method is free of immunoaffinity purification and the method is free of lectin affinity purification.

Technical Field

The invention relates to a purification method of recombinant follicle stimulating hormone, in particular to a purification method of high-purity recombinant follicle stimulating hormone.

background

Follicle Stimulating Hormone (FSH) is a heterodimeric glycoprotein with a molecular weight of about 31kD and consists of non-covalently bound alpha and beta subunits. Wherein the relative molecular mass of the alpha subunit is about 14 KD; the beta subunit has a relative molecular mass of about 17KD, is a glycoprotein gonadotropin synthesized and secreted by anterior pituitary basophilic cells and is controlled by hypothalamic gonadotropin-releasing hormone. In women, FSH promotes proliferative differentiation of the granulosa cells of the follicle at concentrations critical to the initiation and cycle of follicular development and, subsequently, the time to reach follicular maturation and the number of follicles. In males, FSH binds to testosterone, which is controlled by LH (luteinizing hormone), and acts on the seminiferous tubules of the testes, thereby activating and maintaining the number and quality of normal sperm. FSH is a bioactive molecule with important roles in the diagnosis and treatment of infertility, and plays an essential role in promoting normal growth, maturation and gonadal steroid production of follicles.

Low FSH concentrations due to insufficient FSH synthesis and secretion are the main cause of infertility in both women and men. In women, this state is characterized by anovulation or anovulation; in males, infertility may result from the insufficient production of viable sperm. In the diagnosis and treatment of infertility, FSH is a bioactive molecule playing an important role, and FSH plays an important role in human assisted reproduction technology, and is one of the major drugs for treating male and female infertility on the market at present.

Menotropins (also known as menopause gonadotropin, menopause gonadotropin) prepared from urine of menopausal women at an early stage contain Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH), and have the defects of low activity, high cost, large pollution, potential pathogenic microorganism infection and the like. The recombinant human follicle stimulating hormone (rhFSH) produced by applying the DNA recombination technology has standardized molecular weight and biological activity, avoids the difference of FSH isomer biological activity caused by the difference of the glycosyl structure of FSH extracted from a urine source, can improve the quality and the yield of products, and simultaneously, the rhFSH also has excellent safety characteristic.

At present, recombinant human follicle stimulating hormone (rhFSH) produced by DNA recombination technology has been introduced, and the first products to be commercially available are the Gunafen GONAL-f O R of Celannor, Switzerland and the Purenon O R of Ougango, Netherlands. The fructose sodium and the pulicar belong to recombinant human follicle stimulating hormone, wherein the purity of the follicle stimulating hormone exceeds 99 percent, and the effect is stable, so the fructose sodium and the pulicar have great advantages. At present, three recombinant human follicle stimulating hormones are mainly available in the Chinese marketAnd(manufactured by Changchun golden race pharmaceuticals Co., Ltd.). The purity of recombinant FSH produced by domestic pharmaceutical companies is not more than 99%. Due to the important role FSH plays in the treatment of infertility, to promote normal growth, maturation and production of gonadal steroid in the follicles, it is promising to provide recombinant FSH of high purity and high specific activity. Highly purified FSH preparations may be administered by subcutaneous injection and the patient may self-administer them, thus increasing patient comfort and compliance.

However, recombinant FSH products are significantly more expensive than urinary FSH products due to technical strength and cost, and are economically burdensome to the average patient.

Therefore, there is a strong need in the art to develop a purification technique of FSH that is low in cost, easy to handle, high in purity of obtained FSH, and good in bioactivity.

Disclosure of Invention

The invention aims to provide an FSH purification technology which has low cost, easy operation, high purity of obtained FSH and good bioactivity.

In the present invention, there is provided a method for purifying a high-purity recombinant follicle stimulating hormone, comprising the steps of:

(a) providing a raw material solution containing recombinant follicle stimulating hormone;

(b) Sequentially carrying out the following steps on the raw material liquid:

(b1) ammonium sulfate precipitation;

(b2) Hydrophobic Interaction Chromatography (HIC);

(b3) Incubating and inactivating the virus at low pH; and

(b4) Reverse Phase Chromatography (RPC);

Wherein steps (b1), (b2), (b3), and (b4) may not be performed in any order.

In another preferred embodiment, the method does not comprise cation exchange chromatography, anion exchange chromatography.

In another preferred example, the raw material liquid is a fermentation product.

In another preferred embodiment, the fermentation is a fermentation in a eukaryotic cell, preferably a mammalian cell, more preferably a CHO cell.

In another preferred embodiment, the fsh is human fsh.

In another preferred embodiment, the follicle stimulating hormone comprises wild-type and mutant follicle stimulating hormone.

In another preferred embodiment, the follicle stimulating hormone is composed of an alpha subunit and a beta subunit.

in another preferred embodiment, the alpha subunit has the amino acid sequence shown in SEQ ID No.1, and the beta subunit has the amino acid sequence shown in SEQ ID No. 2.

In another preferred embodiment, the recombinant follicle stimulating hormone comprises PEG-rh FSH, rh FSH-Fc fusion protein, rh FSH-CTP fusion protein, bifunctional antibodies comprising rh FSH, multifunctional antibodies comprising rh FSH, conjugated drugs comprising rh FSH, rh FSH glycosylation modifications, or combinations thereof.

In another preferred embodiment, the ammonium sulfate precipitation conditions include: the pH is 7.1-8.5, preferably 7.2-8.0, most preferably 7.3-7.6.

In another preferred example, in step (b1), the pH adjustment is performed with a mineral acid and/or a base.

in another preferred example, in step (b1), the pH is adjusted with ammonia and/or sulfuric acid.

in another preferred embodiment, the ammonium sulfate precipitation conditions include: the ammonium sulfate concentration is 40% -60%, preferably 42% -49%, more preferably 43% -47%, most preferably 44% -45%.

In another preferred example, in step (b2), the hydrophobic interaction chromatography uses a chromatography medium selected from the group consisting of: capto Phenyl (HS), Capto Butyl, Butyl Sepharose4FF, Butyl Sepharose4FF (HS), Octyl Sepharose4FF, Phenyl Sepharose 6FF (LS), Phenyl Sepharose 6FF (HS), Phenyl Sepharose Big Beads, Phenyl Sepharose 6FF, Phenyl Sepharose HP, Butyl Sepharose 4B, Octyl Sepharose CL-4B, Phenyl Sepharose CL-4B, or combinations thereof.

In another preferred embodiment, in step (b3), the conditions for incubating the inactivated virus at low PH comprise: pH2.5-4.5, preferably pH 3-4.

in another preferred example, in step (b3), the pH adjustment is performed with an inorganic acid and/or an organic acid.

In another preferred example, in step (b3), the pH is adjusted with citric acid.

In another preferred example, in step (b4), the Reverse Phase Chromatography (RPC) uses a matrix filler selected from the group consisting of: silica gel, alumina, zirconia, etc. as the matrix material, and polymer such as polystyrene, polyethylstyrene-divinylbenzene, polybutadiene, polyethylene oxide, polysiloxane, agarose, polychloromethylstyrene-diethoxymethylvinylsilane, vapor deposited carbon, or a combination thereof, which coats the inorganic matrix filler.

in another preferred example, in step (b4), the matrix filler used in Reverse Phase Chromatography (RPC) is polystyrene/divinylbenzene.

In another preferred embodiment, in step (b4), Reverse Phase Chromatography (RPC) is performed on the ligand polystyrene resin.

In another preferred example, in step (b4), the organic solvent of the buffer solution of Reverse Phase Chromatography (RPC) is acetonitrile, methanol, ethanol, propanol, acetone.

In another preferred example, in step (b4), the organic solvent of the Reverse Phase Chromatography (RPC) buffer solution is isopropanol.

In another preferred embodiment, in step (b4), the buffer solution of Reverse Phase Chromatography (RPC) has a pH of 7 to 8, preferably 7.2 to 7.8, and most preferably 7.4-7.6.

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

In another preferred embodiment, the method further comprises (b5) Reverse Phase Chromatography (RPC).

In another preferred example, step (b) of the method includes the steps of:

(b1) Ammonium sulfate precipitation;

(b2) Hydrophobic Interaction Chromatography (HIC);

(b3) Incubating and inactivating the virus at low pH; and

(b4) Reverse Phase Chromatography (RPC).

(b5) Reverse Phase Chromatography (RPC).

In another preferred example, step (b) of the method includes the steps of:

(b1) Ammonium sulfate precipitation;

(b2) hydrophobic Interaction Chromatography (HIC);

(b3) Incubating and inactivating the virus at low pH; and

(b4) Reverse Phase Chromatography (RPC).

(b5) Gel Filtration Chromatography (GFC).

In another preferred example, step (b) of the method includes the steps of:

(b1) Ammonium sulfate precipitation;

(b2) hydrophobic Interaction Chromatography (HIC);

(b3) Incubating and inactivating the virus at low pH; and

(b3a) Hydrophobic Interaction Chromatography (HIC)

(b4) reverse Phase Chromatography (RPC).

(b5) gel Filtration Chromatography (GFC).

In another preferred embodiment, the method further comprises one or more ultrafiltration and/or nanofiltration steps.

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

in another preferred embodiment, the method does not perform lectin affinity purification.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

drawings

Figure 1 shows the chromatogram for protein elution in the hydrophobic chromatography step of example 1.

The UV absorption diagram at 280nm (UV280) is shown.

FIG. 2 shows the electrophoretogram (non-reduced SDS-PAGE, gel concentration 15%) of the hydrophobic chromatography components in the hydrophobic chromatography step of example 1.

Wherein M is a molecular weight standard (Thermo Scientific, namely Fermantas, Unstanated Protein MW Marker; the molecular weight is 116.0, 66.2, 45.0, 35.0, 25.0, 18.4 and 14.4KD in sequence); 1 is HIC sample; 2 is HIC flow-through component (FT); 3 is HIC 45% fraction eluted with B; 4 is the fraction eluted at 100% B of HIC.

figure 3 shows the chromatogram for protein elution in the reverse phase chromatography step of example 2.

The UV absorption diagram at 280nm (UV280) is shown.

FIG. 4 shows the electrophoretogram (non-reduced SDS-PAGE, gel concentration 15%) of the components of the reverse phase chromatography in the reverse phase chromatography step of example 2.

Wherein M is a molecular weight standard (Thermo Scientific, namely Fermantas, Unstanated Protein MW Marker; the molecular weight is 116.0, 66.2, 45.0, 35.0, 25.0, 18.4 and 14.4KD in sequence); 1 is the precipitated fraction (45% saturation) after ammonium sulphate treatment; 2, RPC pre-sample fraction (fraction eluted with HIC 100% B); 3 is the component eluted by HIC NaOH; 4 is fraction eluted with RPC 20% B; 5 is fraction eluted with RPC 40% B; 6 is the fraction eluted with RPC 60% B; fraction 7 was RPC 100% B eluted.

Detailed Description

The present inventors have made extensive and intensive studies and extensive screening to develop a method for purifying a recombinant follicle stimulating hormone of high purity for the first time. Specifically, the culture supernatant is sequentially subjected to ultrafiltration, ammonium sulfate precipitation, hydrophobic chromatography, low-pH virus inactivation, reverse phase chromatography, size exclusion chromatography and nano-membrane filtration and purification. Experimental data show that the final product obtained by the method is correct, and the FSH has high bioactivity and high purity. The present invention has been completed based on this finding.

Purification method of the invention

It is an object of the present invention to provide a high purity purification process for purifying recombinant FSH or recombinant FSH variants for producing recombinant FSH or recombinant FSH variants, such as human recombinant FSH or human recombinant FSH variants, with economy, high purity and high specific activity.

In a preferred embodiment, the recombinant FSH of the present invention has a FSH α subunit of sequence as shown in SEQ ID No.1 and a FSH β subunit of sequence as shown in SEQ ID No. 2.

MDYYRKYAAIFLVTLSVFLHVLHSAPDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKS(SEQ ID NO:1)

MKTLQFFFLFCCWKAICCNSCELTNITIAIEKEECRFCISINTTWCAGYCYTRDLVYKDPARPKIQKTCTFKELVYETVRVPGCAHHADSLYTYPVATQCHCGKCDSDSTDCTVRGLGPSYCSFGEMKE*(SEQ ID NO:2)

Preferably, the nucleotide sequence of the FSH alpha subunit gene is shown as SEQ ID NO. 3, and the nucleotide sequence of the FSH beta subunit gene is shown as SEQ ID NO. 4.

atggattactacagaaaatatgcagctatctttctggtcacattgtcggtgtttctgcatgttctccattccgctcctgatgtgcaggattgcccagaatgcacgctacaggaaaacccattcttctcccagccgggtgccccaatacttcagtgcatgggctgctgcttctctagagcatatcccactccactaaggtccaagaagacgatgttggtccaaaagaacgtcacctcagagtccacttgctgtgtagctaaatcatataacagggtcacagtaatggggggtttcaaagtggagaaccacacggcgtgccactgcagtacttgttattatcacaaatcttaa(SEQ ID NO:3)

atgaagacactccagtttttcttccttttctgttgctggaaagcaatctgctgcaatagctgtgagctgaccaacatcaccattgcaatagagaaagaagaatgtcgtttctgcataagcatcaacaccacttggtgtgctggctactgctacaccagggatctggtgtataaggacccagccaggcccaaaatccagaaaacatgtaccttcaaggaactggtatacgaaacagtgagagtgcccggctgtgctcaccatgcagattccttgtatacatacccagtggccacccagtgtcactgtggcaagtgtgacagcgacagcactgattgtactgtgcgaggcctggggcccagctactgctcctttggtgaaatgaaagaataa(SEQ ID NO:4)

Accordingly, the present invention relates to a method for high purity purification of recombinant FSH or recombinant FSH variants comprising the following steps for a liquid of recombinant FSH or recombinant FSH variants:

(1) ammonium sulfate precipitation;

(2) Hydrophobic Interaction Chromatography (HIC);

(3) Incubating and inactivating the virus at low pH; and

(4) Reverse Phase Chromatography (RPC).

Steps (1), (2), (3) and (4) may be performed in any order.

wherein the method avoids cation exchange chromatography and anion exchange chromatography.

Preferably, ammonium sulfate precipitation or hydrophobic interaction chromatography is performed as the first of four steps. In a more preferred embodiment, the ammonium sulfate precipitation process is performed as the first of the steps.

the purification process may optionally comprise additional steps, for example affinity chromatography such as dye affinity chromatography or perborate affinity chromatography. And/or a second filtration such as diafiltration, ultrafiltration or nanofiltration.

In a preferred embodiment, the process of the invention comprises gel filtration chromatography (SEC) as step (5).

in a preferred embodiment, steps (1), (2), (3) and (4) are carried out in this order

(1) Ammonium sulfate precipitation;

(2) Hydrophobic Interaction Chromatography (HIC);

(3) Incubating and inactivating the virus at low pH; and

(4) Reverse Phase Chromatography (RPC).

Ammonium sulfate precipitation is preferably performed as the first step, as this embodiment allows the option of subjecting the rather "crude" recombinant FSH product directly to precipitation, optionally after the clarified sample obtaining cleaning (e.g. filtration), concentration and/or buffer replacement steps as described below. This embodiment provides the advantage that possible residual nucleic acids, endotoxins, viruses, proteins and other impurities can be completely removed, and in addition, the clarified sample can concentrate the harvest by more than 15 times, facilitating ammonium sulfate precipitation. Overall, precipitation with ammonium sulfate as the first step reduces the number of preparation steps necessary before starting chromatographic purification and allows the use of high amounts of sample solution.

In yet another preferred embodiment, a second Hydrophobic Interaction Chromatography (HIC) is performed after incubation of inactivated virus at low PH (3) and before Reverse Phase Chromatography (RPC) (4). As described above, additional steps may be performed in addition to and in between the steps.

In yet another preferred embodiment, a third Hydrophobic Interaction Chromatography (HIC) is performed after Reverse Phase Chromatography (RPC) (4). As described above, additional steps may be performed in addition to and in between the steps.

In a further preferred embodiment, the Gel Filtration Chromatography (GFC) is performed after Reverse Phase Chromatography (RPC) (4). As described above, additional steps may be performed in addition to and in between the steps.

In a further preferred embodiment, the Reverse Phase Chromatography (RPC) (4) is followed by Reverse Phase Chromatography (RPC). As described above, additional steps may be performed in addition to and in between the steps.

The purification method of the present invention provides recombinant FSH and recombinant FSH variants at high purity, which can then be formulated as pharmaceutical compositions. The purity is typically higher than 99.50% w/w, preferably > 99.80% w/w, more preferably > 99.90% w/w, even more preferably > 99.99% w/w, based on total protein. In addition, the purification process of the present invention can be easily scaled up, even to industrial scale, without significant changes in purification conditions.

The recombinant FSH and recombinant FSH variants forming the starting material for the purification process according to the present invention may be obtained from liquids of natural origin or by recombinant techniques such as in cell culture harvests containing recombinant FSH and recombinant FSH variants. Typically, the starting material from a natural source or cell harvest, preferably a cell harvest, is first cleaned (e.g. filtered) before the first step is performed, and then after the concentration and/or buffer exchange step.

In the ammonium sulfate precipitation step, generally commercially available ammonium sulfate, preferably a commercially available analytical pure product meeting national standards, is used.

In the chromatography step, a generally commercially available resin filler, preferably a polymer-based resin or an agaroses-based resin, is used.

The steps of the purification process of the present invention are described in more detail below.

Ammonium sulfate precipitation (1) step

the invention relates to a step of ammonium sulfate precipitation (1). In a preferred embodiment, particularly in the case of recombinant FSH, which is highly hydrophilic, has a high solubility in aqueous solution and is also less prone to intermolecular aggregation, ammonium sulfate precipitation is used as the first step. To precipitate them, it is necessary to use ammonium sulfate, preferably in higher concentrations. Preferably, a suitably high concentration of ammonium sulphate allows more of the hetero-protein to precipitate and also allows the desired recombinant FSH not to precipitate.

Ammonium sulfate precipitation is generally carried out as follows: ammonium sulfate solids or ammonium sulfate solutions, preferably saturated ammonium sulfate solutions, and more preferably 100% saturated ammonium sulfate solutions, are added directly.

Ammonium sulfate concentration is 40% to 60%, preferably 42% to 49%, more preferably 43% to 47%, even more preferably or about 44% to 45%.

Preferably, the solution is added to the sample solution while stirring, in an amount that is converted to a desired saturated solution according to the concentration of the solution to be used.

The ammonium sulfate precipitation is preferably carried out by a solution having a slightly alkaline pH, for example, at or about pH7.1 to 8.5, more preferably at or about 7.2 to 8.0, more preferably at or about 7.3 to 7.6, and in a preferred embodiment, the pH adjusting agent is an inorganic acid or base, more preferably aqueous ammonia or sulfuric acid.

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

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

hydrophobic Interaction Chromatography (HIC) (2) step.

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

Hydrophobic Interaction Chromatography (HIC) is a separation method that exploits the hydrophobic properties of proteins. The high ionic strength in solution may enhance the hydrophobic interaction between the protein and the hydrophobic chromatography medium. By virtue of this property, adsorption is enhanced by hydrophobic interactions between the apolar regions of the protein and the immobilized hydrophobic ligands on the solid support. The sample to be separated is adsorbed on a hydrophobic chromatography medium at high ionic strength and then eluted with linear or stepwise decreasing ionic strength selectivity. Hydrophobic interaction chromatography materials are matrices substituted with hydrophobic ligands such as ethyl, butyl, phenyl or hexyl groups. Preferred materials are matrices substituted with butyl or phenyl ligands.

Hydrophobic Interaction Chromatography (HIC) resin packing is known in the art and includes resins such as Butyl Sepharose (GE Healthcare), phenyl Sepharose (low and high substitution), octyl Sepharose and alkyl Sepharose (all from GE Healthcare; other sources of HIC resins include Biosepra, france; e.merck, germany; BioRad USA).

in a preferred embodiment, hydrophobic interaction chromatography is performed with a resin such as phenyl Sepharose 6fast flow (highly substituted) (available from GE Healthcare). It is understood that step (2) may be performed with alternative resins having similar characteristics. Alternative resins that may be used are the following: capto Phenyl (HS); capto Butyl; butyl Sepharose4 FF; butyl Sepharose4FF (HS); octyl Sepharose4 FF; phenyl Sepharose 6FF (LS); phenyl sepharose 6FF (HS); phenyl Sepharose Big Beads; phenyl Sepharose 6 FF; phenyl sepharose HP; butyl Sepharose HP; butyl Sepharose 4B; octyl Sepharose CL-4B; phenyl Sepharose CL-4B. All from GE Biosciences (800) 526-3593. The other resins were: phenyl section 6 FF; from seplife; octyl Focurose 4FF is from a colleague organism (see www.biomart.cn).

In a preferred embodiment, the equilibration, loading, washing and elution buffers are selected from phosphoric acid or salts thereof, sulfuric acid or sodium salts thereof, MES, Bis-Tris, ADA, PIPES, ACES, BES, MOPS, TES, HEPES, TRICINE, BICINE, preferably ammonium sulfate. Binding on HIC resins is usually achieved by using high conductivity equilibration and loading buffers, for example by adding salts such as NaCI, NH4Ac, (NH4)2SO4 or Na2SO4, preferably ammonium sulphate. The preferred salt concentration is 1.2 to 1.8M, preferably about 1.5M (NH4)2SO 4. Washing typically uses a loading buffer. The elution step of hydrophobic interaction chromatography is preferably performed by decreasing the mobile phase conductivity (decreasing the salt concentration). The reduction can be achieved in a linear manner or in steps. Optionally, the buffered solution may also comprise additional inorganic salts. In one embodiment the inorganic salt is selected from the group consisting of sodium chloride, sodium sulfate, potassium chloride, potassium sulfate, sodium citrate and potassium citrate.

Preferably, equilibration, loading, washing and elution buffers are used which have a pH of from or about 6.5 to or about 8.5, more preferably from or about 7.0 to or about 8.0, most preferably or about 7.4. A particularly preferred equilibration, loading and wash buffer system contains sodium phosphate and ammonium sulfate, preferably at a pH of at or about 7.4. Preferred elution buffers contain ammonium sulfate at a pH of at or about 7.4.

preferably, no buffer exchange is performed after step (2), facilitating the case where step (b) (RPC) is performed subsequently. Then, buffer exchange can be achieved by the following RPC: the preferred buffer by subsequent chromatography steps such as AEX or HIC chromatography is used as the running buffer.

low pH incubation inactivated virus (3).

The process of the invention also involves a low pH incubation step to inactivate the virus (3).

The envelope structure of the virion is easily destroyed at low pH, thereby inactivating the virus for infection. The higher the incubation temperature, the stronger the virus inactivation ability; the lower the pH of the sample, the stronger the ability to inactivate viruses; the longer the incubation time, the stronger the ability to inactivate the virus.

The pH regulator for the low pH incubation inactivated virus method is preferably an acidic regulator, such as an organic acid or an inorganic acid, preferably phosphoric acid, hydrochloric acid, citric acid. Citric acid is more preferred.

a low pH incubation method for inactivating virus having a pH of from at or about 2.5 to at or about 4.5, more preferably from at or about 3.0 to at or about 4.0, and most preferably from or about 3.5 to about 3.7.

A low pH incubation inactivated virus method having an incubation temperature of from at or about 15 ℃ to at or about 30 ℃, more preferably from at or about 18 ℃ to at or about 26 ℃, and most preferably from at or about 20 ℃ to about 25 ℃.

Low pH incubation inactivated virus methods, either at or about 15 ℃ to or about 30 ℃, more preferably at or about 18 ℃ to or about 26 ℃, and most preferably at or about 20 ℃ to about 25 ℃. For example, incubation at 3.5pH 20 ℃.

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, the incubation may be at a pH of about 3.5 for about 90 minutes at about room temperature. This virus inactivation step can be performed at any time during the purification process, preferably before the final chromatography step.

preferably, the pH adjustment is performed after step (3), in favor of the case where step (4) (RPC) is performed subsequently.

Reversed Phase Chromatography (RPC) (4).

the process involves a reverse phase chromatography (4) step. In a preferred embodiment, especially in the case of recombinant FSH, reverse phase chromatography is used as the capture step, wherein the recombinant FSH is enriched, for example from a naturally derived liquid or cell culture harvest. Preferably, virus inactivation is performed prior to elution from the RPC column.

"reverse phase chromatography" according to the invention refers in particular to a chromatography step in which a non-polar stationary phase and preferably a polar mobile phase are used. In reverse phase chromatography, generally polar compounds elute first, while non-polar compounds are retained.

Reverse phase chromatography is typically carried out as follows: the column is equilibrated and loaded, followed by washing and then eluted, each using a buffer preferably containing an organic solvent such as ethanol or isopropanol. Organic solvents such as isopropanol can be used for virus inactivation after elution.

Equilibration, loading, washing and elution are preferably carried out by using a mobile phase which is buffered at a slightly alkaline pH, for example at or about pH7 to 8, preferably at or about 7.2 to 7.8, more preferably 7.4 to 7.6, most preferably 7.5. In a preferred embodiment, the buffer substance is an acetic acid buffer, preferably ammonium acetate. Alternative buffers suitable for use at or about pH7.4 to 7.6 include BES, MOPS, ammonium acetate, TES, HEPES.

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

The eluent is linear gradient elution or step gradient elution, preferably step gradient elution. In the elution solution, the organic solvent preferably contains at least one of acetonitrile, methanol, ethanol, propanol, acetone, and the like. The buffer concentration is 20mmol/L to 60mmol/L, preferably about 35mmol/L to 55mmol/L or 50mmol/L, such as 50mmol/L ammonium acetate. The organic solution is at a concentration of 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, at least 80% (v/v) isopropanol. For example, the eluent is a mixture of 35mmol/L ammonium acetate and 10% isopropanol (v/v), or a mixture of 50mmol/L ammonium acetate and 20% isopropanol (v/v), or a mixture of 50mmol/L ammonium acetate and 50% (v/v) isopropanol.

the reverse phase column material is composed of hydrophobic inorganic matrix filler, organic matrix filler or composite matrix filler. Preferably, the matrix material is a composite matrix filler, such as silica gel, alumina, zirconia, etc., and the polymer coating the inorganic matrix filler is at least one of polystyrene, polyethylstyrene-divinylbenzene, polybutadiene, polyethylene oxide, polysiloxane, agarose, polychloromethylstyrene-diethoxymethylethylvinylsilane, and vapor-deposited carbon, etc., and more preferably polystyrene/divinylbenzene.

The ligands are generally chosen from, but not limited to, at least one of aliphatic, such as C2, C4, C6, C8, C10, C12, C14, C16, or C18 or their derivatives, for example cyanopropyl (CN-propyl), or branched aliphatic, or benzene-like aromatic, such as phenyl, or other polar or non-polar ligands, and the like. The ligand may be a mixture of two or more of these ligands.

Suitable polystyrenic resins include, without limitation, resins supplied by Rohm Haas (e.g., Amberlite XAD or Amberchrom CG), Polymer Labs (e.g., PLRP-S), GEHealthcare (e.g., Source RPC), Applied Biosystems (e.g., Poros R). A particularly preferred resin is Source 30RPC (GE Healthcare).

Typically, the sample is purified at least about 0.1mg per ml of resin, e.g., at least about 0.3mg, 0.4mg, 0.8mg, 1.5mg, 4mg, 8, or 15mg per ml of resin; or 0.1-180mg, e.g., 0.1-150mg, 0.5-100mg, 2-60mg, or 3-40mg per mL of resin is loaded onto the column; preferably the loading is at least 1mg per mL resin. Bulk resin volume measurement is typically done in suspension or similar mode.

Additional step

In addition to the three main chromatography steps (1), (2), (3) and (4), the process of the invention may optionally comprise additional steps known to the person skilled in the art, such as a chromatography step or a filtration step virus inactivation step. Preferred additional steps are filtration such as diafiltration, ultrafiltration or nanofiltration.

In a preferred embodiment, the process of the invention comprises the following steps in the following order:

(0) Ultrafiltration; (optionally an additional diafiltration step; preferably using a membrane with a cut-off of at or about 10 kD)

(1) Ammonium sulfate precipitation;

(2) Hydrophobic Interaction Chromatography (HIC); (preferably with Phenyl Sepharose 6fast flow (high substitution) column)

(3) Incubating and inactivating the virus at low pH; and (preferably with pH3.6)

(4) Reverse Phase Chromatography (RPC). (preferably with Source 30RPC column)

Subjecting a sample of the recombinant FSH solution to an ultrafiltration step, in particular a virus removal step; i.e. to reduce the risk of contamination of the glycoprotein preparation with viruses or virus-like particles originating from cell cultures. Ultrafiltration may be performed at any stage of the purification process, however, it is particularly preferred to perform ultrafiltration prior to the chromatography procedure. The ultrafiltration may be performed more than once, for example it may be performed twice. Preferably, ultrafiltration (or diafiltration) is carried out using an ultrafiltration membrane of about 10-15kD, preferably about 10 kD.

in a preferred embodiment, the process of the invention comprises the following steps in the following order:

(0) Ultrafiltration; (optionally an additional diafiltration step; preferably using a membrane with a cut-off of at or about 10 kD)

(1) Ammonium sulfate precipitation;

(2) Hydrophobic Interaction Chromatography (HIC); (preferably with Phenyl Sepharose 6fast flow (high substitution) column)

(3) Incubating and inactivating the virus at low pH; and (preferably with pH3.6)

(4) Reverse Phase Chromatography (RPC). (preferably with Source 30RPC column)

(4a) Hydrophobic Interaction Chromatography (HIC); (preferably with Phenyl Sepharose 6fast flow (high substitution) column)

(5) filtering by using a nano membrane; (preferably using a 20nm nano-membrane filter)

The above-mentioned specific purification process is preferably carried out without any further chromatography step and/or ultrafiltration step and/or diafiltration step. However, in particular embodiments, the above-described purification process may further comprise additional steps, in particular one or more of the additional steps described herein, such as those for removing or inactivating undesired or and/or hazardous substances.

In a further preferred embodiment, the process of the invention comprises the following steps in the following order:

(0) Ultrafiltration; (optionally an additional diafiltration step; preferably using a membrane with a cut-off of at or about 10 kD)

(1) Ammonium sulfate precipitation;

(2) Hydrophobic Interaction Chromatography (HIC); (preferably with Phenyl Sepharose 6fast flow (high substitution) column)

(3) Incubating and inactivating the virus at low pH; and (preferably with pH3.6)

(3a) Hydrophobic Interaction Chromatography (HIC); (preferably with Phenyl Sepharose 6fast flow (high substitution) column)

(4) Reverse Phase Chromatography (RPC). (preferably with Source 30RPC column)

(5) Filtering by using a nano membrane; (preferably using a 20nm nano-membrane filter)

The above-mentioned specific purification process is preferably carried out without any further chromatography step and/or ultrafiltration step and/or diafiltration step. However, in particular embodiments, the above-described purification process may further comprise additional steps, in particular one or more of the additional steps described herein, such as those for removing or inactivating undesired or and/or hazardous substances.

The method has the advantages of less steps in the purification process, good purification effect and reduction of the number of chromatographic steps to at least 2 chromatographic steps. In particular, using the purification process according to the invention costly and problematic purification steps such as in particular affinity purification steps, in particular immunoaffinity purification steps, lectin affinity purification steps are avoided. The method of the invention provides a high degree of purity and specific biological activity of recombinant FSH with a purity of > 97%, preferably > 99%, more preferably > 99.5% w/w, each based on total protein, as measured, for example, by high performance liquid chromatography. Its biological activity is >59000IU/ml, preferably >60000IU/ml, more preferably >61000 IU/ml. In addition, the recovery rate of the objective FSH in the purification process of the present invention is also high.

Storage/lyophilization

The liquid composition obtained by the above purification process and containing recombinant FSH may be stored frozen as such or after purification, and the eluate may be lyophilized ("freeze-dried") to remove the solvent. The resulting liquid or lyophilized product is referred to as "recombinant FSH stock".

Formulation

The recombinant FSH of the invention or purified according to the method of the invention may be formulated for any type of formulation, preferably for intramuscular or subcutaneous injection, preferably subcutaneous injection. The recombinant FSH preparation may be lyophilized and dissolved in water for injection prior to injection. The recombinant FSH formulation may also be a liquid formulation, in which case it is injected directly without prior solubilization.

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 may be formulated with known excipients and stabilizers such as sucrose and mannitol. It may also contain antioxidants such as methionine. It may also contain a surfactant such as a tween (preferably tween 20), or Pluronic (Pluronic) (preferably Pluronic F68).

Recombinant FSH formulations may be in single or multiple doses. If multiple doses are used, they should preferably contain bacteriostatic agents, for example alkyl benzyl esters, benzyl alcohol, m-cresol, thymol or phenol, preferably methyl benzyl ester or m-cresol. Single dose formulations may also contain bacteriostatic agents. Suitable formulations are described, for example, in PCT/EP2011/062986, incorporated herein by reference.

In a particularly preferred multi-dose formulation, the recombinant FSH produced by the method of the present invention is formulated as follows: it was dissolved in water for injection together with sucrose, phosphate buffer (ph6.6 to 7.4), tween 20, methionine and bacteriostatic.

Indications of

The recombinant FSH of the invention is suitable for use in all treatments where recombinant FSH is indicated. Is especially suitable for the subcutaneous administration of ovulation induction and controlled superovulation in the assisted reproduction technology, the controlled ovarian hyperstimulation in the assisted reproduction technology and the treatment of oligospermia. It can be used in combination with other gonadotropins such as LH and hCG. It may also be used with other compounds that increase the response to FSH, such as clomiphene, clomiphene citrate, aromatase inhibitors such as anastrozole, letrozole, fadrozole and YM-511. In addition, LH and hCG can also be used separately in fertility treatments.

Recombinant FSH

The term "recombinant" is used to refer to a preparation of FSH produced by the use of recombinant DNA techniques. An example of a method for expressing FSH using recombinant technology is the transfection of eukaryotic cells with DNA sequences encoding the α and β subunits of FSH, typically an expression vector carrying a strong promoter such as CMV or SV40 driving FSH expression, and a suitable selection marker for selection of the host cell into which the vector has been incorporated. Transfection may be stable or transient. Suitable recombinant expression systems are well known in the art and need not be described in detail. Preferably, the eukaryotic host cell is selected from primate cells, preferably human cells and rodent cells, preferably CHO cells. As described in European patents EP 0211894 and EP 0487512, there is a subunit on either one or both vectors, each subunit having a single promoter. The DNA encoding FSH may be a cDNA or it may contain introns.

Recombinant technology in order to generate FSH another example is to use homologous recombination technology to insert a heterologous regulatory fragment into the endogenous sequence of one or both of the subunits encoding FSH in an operably linked manner, as described in European patent No. EP0505500(Applied research systems ARS Holding NV). Recombinant technology is for example the method disclosed in WO99/57263(Transkaryotic therapeutics), wherein one subunit is inserted heterologously into a cell and the other subunit is inserted by homologous recombination into a heterologous regulatory fragment to activate the genomic sequence for expression. The method of the invention may be used to purify FSH expressed by any of the above methods.

The expression "FSH variant" is intended to include molecules which differ from human FSH in their amino acid sequence, glycosylation pattern or linkage between subunits, but which exhibit FSH activity. Examples include CTP-FSH, a long-acting modified recombinant FSH consisting of a wild-type α subunit and a hybrid β subunit, wherein the carboxy-terminal peptide of hCG is fused at the C-terminus of the FSH β subunit, e.g., lapart et al; endocrinology; 1992, 131, 2514-2520; or Klein et al; development and characterization of a long-acting recombinant hFSH aginst; CN 101087805BHuman repeat.2003, 18, 50-56 ]. Also included is single chain CTP-FSH, a single chain molecule consisting of β FSH (FSH β subunit), β hCGCTP (113-145) (hCG carboxy terminal peptide), α FSH (FSH α subunit).

The FSH variants referred to herein also include PEG-rh FSH, rh FSH-Fc fusion protein, rh FSH-CTP fusion protein, bifunctional antibodies comprising rh FSH, conjugate drugs comprising rh FSH, and glycosylation modifications of rhFSH.

Reference herein to FSH variants also includes FSH from different species such as equine (Equus caballus), porcine (suscrofa), bovine (Bos taurus), feline (Felis catus), canine (Canis familiaris).

In a preferred embodiment, the FSH is produced recombinantly in serum or in a serum-free medium. In a further preferred embodiment, the purified FSH produced according to the method of the present invention is suitable for subcutaneous administration, which makes self-administration to a patient possible.

unprocessed recombinant FSH

Refers to the cell culture supernatant in recombinant cells expressing FSH before any purification steps are taken. The expression includes the crude form of the supernatant (isolated from the cells) as well as the concentrated and/or filtered and/or ultrafiltered supernatant.

Biological activity

Refers to an FSH preparation that elicits biological functions associated with FSH, such as ovarian weight obtained in a Steelman-Pohley Assay [ Assay of the folicline stimulating hormone based on the evaluation with human chorinic gonadotrophin; endocrinology; 1953, 53, 604-616], or follicular growth in female patients. Follicle growth in female patients can be estimated by ultrasound, e.g., based on the number of follicles after stimulation at 8 th scale with a mean diameter of approximately 16 mm. Biological activity may be estimated by a standard acceptable for FSH.

The term "specific activity" with respect to FSH refers to the biological activity of the preparation in IU divided by the weight of protein, IU root

it is recognized that FSH bioassays are measured as Steeelman Pohley bioassays and protein weight is determined according to an assay for total protein content, such as the Lowry assay [ o.h. Lowry, n.j.rosebrough, a.l.farr and r.j.randall (1951) j.biol.chem.193: 265 of a nitrogen-containing gas; hartree E.E, (1972), anal. biochem.48: 422; j.r.dulley and p.a.grieve (1975) anal.biochem.64: 136], Bradford assay [ Bradford, M.M, (1976) anal. biochem.72, 248], or absorbance at 280 nm.

Preferably, the specific activity of the FSH of the invention is greater than or about 10000IU/mg, more preferably greater than or about 12000IU/mg, still more preferably greater than or about 14000IU/mg, and most preferably greater than or about 16000IU/mg, wherein the biological activity is determined by the Steelman-Pohley assay and the protein purity is determined by SEC-HPLC.

the main advantages of the invention include:

1) The method provided by the invention can replace the existing purification method of human FSH.

2) The method of the invention provides a high degree of purity and specific biological activity of recombinant FSH of > 97%, preferably > 99%, more preferably > 99.5% w/w, more preferably > 99.9%, each based on total protein, e.g. as measured by high performance liquid chromatography.

3) the recombinant follicle stimulating hormone of the invention has specific activity as high as 16000 IU/mg. Its biological activity is >59000IU/ml, preferably >60000IU/ml, more preferably >61000 IU/ml. In addition, the recovery rate of the objective FSH in the purification process of the present invention is also high.

4) Compared with the existing preparation method, the recombinant follicle stimulating hormone of the invention is simple and easy to operate, and has the advantages of less steps in the purification process, good purification effect and reduction of the number of chromatographic steps to at least 2 chromatographic steps. In particular, using the purification process according to the invention costly and problematic purification steps such as in particular affinity purification steps, in particular immunoaffinity purification steps, lectin affinity purification steps are avoided.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.