CN112195194A

CN112195194A - Recombinant human follicle stimulating hormone and preparation method thereof

Info

Publication number: CN112195194A
Application number: CN202011192785.9A
Authority: CN
Inventors: 高丽婷; 杨仲璠; 鲍朋; 朱红雨; 张龙章; 许可; 王丹丹; 徐明波
Original assignee: Beijing Sl Lisheng Medical Technology Co ltd; BEIJING SL PHARMACEUTICAL CO LTD; Beijing Shuanglu Biological Technology Co ltd
Current assignee: Beijing Sl Lisheng Medical Technology Co ltd; BEIJING SL PHARMACEUTICAL CO LTD; Beijing Shuanglu Biological Technology Co ltd
Priority date: 2020-10-30
Filing date: 2020-10-30
Publication date: 2021-01-08

Abstract

The invention discloses a recombinant human follicle stimulating hormone and a preparation method thereof, wherein the preparation method comprises the following steps: transfecting the constructed expression vector containing the FSH alpha subunit and the constructed expression vector containing the FSH beta subunit into Chinese hamster ovary cells in a combined manner, screening out a cell strain with stable expression, and purifying and culturing the cell strain to obtain a culture solution to obtain the recombinant human follicle-stimulating hormone; wherein the C-terminus of the FSH β subunit is linked to a CTP. The preparation method of the recombinant human follicle stimulating hormone clones hFSH alpha and hFSH beta sequences including introns from a human embryonic kidney 293 cell genome respectively, a CTP sequence is connected to the tail end of a beta subunit in a primer extension mode to obtain hFSH beta-CTP, the two subunit sequences are transfected to CHO-S cells to obtain the rhFSH-CTP through expression, the rhFSH-CTP obtained through expression has longer half-life in vivo, the half-life at the tail end is 2-3 times of that of the recombinant FSH, and the bioactivity is higher.

Description

Recombinant human follicle stimulating hormone and preparation method thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to recombinant human follicle stimulating hormone and a preparation method thereof.

Background

FSH is a heterodimeric glycoprotein having a molecular weight of about 31KD and consisting of a non-covalently bound α subunit and β subunit, wherein the α subunit has a relative molecular weight of about 14KD, consists of 92 amino acid residues, contains 2N sugar chains and 5 disulfide bonds; the beta subunit has a relative molecular mass of about 17KD, consists of 111 amino acid residues, and contains 2N sugar chains and 6 disulfide bonds. Both the FSH α subunit and FSH β subunit are glycosylated after transcription, and glycosylation of the protein is important for its biological activity and is essential for its active role.

The long-acting recombinant human follicle stimulating hormone is a follicle stimulating hormone with longer half-life. The drug molecule consists of two subunits. Wherein the alpha subunit is completely the same as the alpha subunit of recombinant human follicle stimulating hormone (rhFSH), consists of 92 amino acids, has 2N-linked glycosylation sites and contains 5 disulfide bonds; the beta subunit is formed by connecting rhFSH beta subunit with 28 amino acid residues of the Carboxy Terminal Peptide (CTP) of Human Chorionic Gonadotropin (hCG) beta subunit, the mature beta subunit contains 139 amino acid residues, 2N-linked glycosylation sites and 4O-linked glycosylation sites, and contains 6 disulfide bonds. Therefore, the drug molecule is also called recombinant human follicle stimulating hormone-CTP fusion protein, which is called rhFSH-CTP for short. In the conventional methods for purifying glycoprotein hormone proteins, a wide-spectrum affinity chromatography such as blue gel affinity chromatography is widely used, or a combination of a plurality of methods such as reverse phase chromatography, ion exchange chromatography, molecular exclusion and the like is used. The blue gel affinity chromatography often causes the product safety problem due to the falling of pigment, the reverse chromatography can influence the activity of the product, the size exclusion takes long time, and the purification efficiency is low.

In conclusion, although recombinant human follicle stimulating hormone (rhFSH) prepared by using a DNA recombinant technology has appeared, the preparation cost is high, the preparation is more expensive than that of urinary FSH products, the stability is poor, the bioactivity is low, and the purification effect is poor, so that a new recombinant human follicle stimulating hormone and a preparation method thereof are urgently needed to be developed.

Disclosure of Invention

Therefore, the invention provides a recombinant human follicle stimulating hormone (rhFSH) and a preparation method thereof, which aim to solve the problems of poor purification effect and low bioactivity of the recombinant human follicle stimulating hormone (rhFSH) in the prior art.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a preparation method of recombinant human follicle stimulating hormone, which comprises the following steps: transfecting the constructed expression vector containing the FSH alpha subunit and the constructed expression vector containing the FSH beta subunit into Chinese hamster ovary cells in a combined manner, screening out a cell strain with stable expression, and purifying and culturing a culture solution of the cell strain to obtain the recombinant human follicle-stimulating hormone; wherein the C-terminus of the FSH β subunit is linked to a CTP.

Preferably, the gene sequence of the coding region of the FSH alpha subunit is shown as SEQ ID NO: 1, the C terminal of the FSH beta subunit is connected with a coding region gene sequence of CTP, which is shown as SEQ ID NO: 2, respectively.

Preferably, the construction process of the expression vector containing FSH alpha subunit is as follows:

cloning the FSH alpha subunit gene sequence to an expression vector pcDNA3.1(+), carrying out enzyme digestion on the pcDNA3.1 plasmid to obtain the FSH alpha subunit gene, cloning the FSH alpha subunit gene subjected to enzyme digestion to an expression vector cDNA15.6, and transfecting DH5 alpha by the expression vector cDNA15.6.

Preferably, the construction process of the expression vector containing the FSH β subunit is as follows:

performing PCR amplification by using the first primer, the second primer and the FSH beta subunit gene as templates to obtain an FSH beta subunit DNA fragment;

performing PCR amplification by using the first primer, the third primer and the FSH beta subunit gene DNA fragment as a template to obtain a first extension segment of the FSH beta subunit DNA fragment;

performing PCR amplification by using the first primer, the fourth primer and the extended section of the FSH beta subunit gene DNA fragment as a template to obtain a second extended section of the FSH beta subunit DNA fragment;

performing PCR amplification by using a first primer, a fifth primer and the second extension segment of the FSH beta subunit DNA fragment as a template to obtain an FSH beta subunit gene of which the C terminal is connected with CTP;

cloning the FSH beta subunit gene of which the C terminal is connected with the CTP to an expression vector pcDNA3.1(+), carrying out enzyme digestion on the pcDNA3.1 plasmid to obtain the FSH beta subunit gene, cloning the FSH beta subunit gene subjected to enzyme digestion to an expression vector cDNA15.6, and transfecting DH5 alpha through the expression vector cDNA15.6;

wherein the nucleotide sequence of the first primer is shown as SEQ ID NO: 3, and the nucleotide sequence of the second primer is shown as SEQ ID NO: 4 is shown in the specification; the nucleotide sequence of the third primer is shown as SEQ ID NO: 5 is shown in the specification; the nucleotide sequence of the fourth primer is shown as SEQ ID NO: 6 is shown in the specification; the nucleotide sequence of the fifth primer is shown as SEQ ID NO: shown at 7.

Preferably, the combined transfection process comprises:

transforming the expression vector containing the FSH alpha subunit and the expression vector containing the FSH beta subunit into Escherichia coli to obtain Escherichia coli with the FSH alpha subunit gene and the FSH beta subunit gene, and transfecting the Escherichia coli with the FSH alpha subunit gene and the FSH beta subunit gene into CHO-S cells.

Preferably, the process of purifying and culturing the cell strain to obtain a culture solution comprises the following steps:

and (3) subjecting the supernatant of the culture solution to affinity chromatography, hydrophobic chromatography, mixed-mode chromatography and cationic chromatography, and concentrating the obtained chromatographic solution to obtain the recombinant human follicle-stimulating hormone.

Preferably, the affinity chromatography column packing is CaptureSelect packing specific for the whole FSH molecule;

the filler of the hydrophobic chromatographic column is Butyl-650M;

the filler of the mixed-mode chromatographic column is Capto Adhere;

the filler of the cation exchange chromatographic column is CM Sepharose Fast Flow.

Preferably, the preparation method further comprises:

and (3) performing ultrafiltration concentration on the chromatographic solution by using an ultrafiltration membrane with the molecular weight cutoff of 10kD before loading the cation exchange chromatographic column and after passing through the cation exchange chromatographic column.

Preferably, the concentration of the concentrated solution obtained by ultrafiltration concentration is 0.8-1.5 mg/mL.

Preferably, the turbidity of the supernatant is < 10 NTU.

The preparation method of the recombinant human follicle stimulating hormone clones hFSH alpha and hFSH beta sequences including introns from a human embryonic kidney 293 cell genome respectively, a CTP sequence is connected to the tail end of a beta subunit in a primer extension mode to obtain hFSH beta-CTP, the two subunit sequences are transfected to CHO-S cells to obtain the rhFSH-CTP through expression, the rhFSH-CTP obtained through expression has longer half-life in vivo, the half-life at the tail end is 2-3 times of that of the recombinant FSH, and the bioactivity is higher.

The invention adopts immunoaffinity chromatography, hydrophobic chromatography, mixed mode chromatography, cation exchange chromatography and ultrafiltration to purify the recombinant human follicle stimulating hormone. Affinity chromatography adopts specific combination with complete FSH medium to capture target molecules with high efficiency; the hydrophobic chromatography can effectively remove residual dissociated subunits and oxidized subunits, remove part of endotoxin and further improve the purity of the purified protein; the mixed mode chromatography can effectively remove impurities such as polymers, residual DNA, HCP and the like, and can improve the safety of the product; the cation exchange chromatography can remove basic rhFSH-CTP protein with insufficient glycosylation, and effectively improve the sialic acid content of the product; the application of the ultrafiltration method shortens the process flow, is easy for industrial production, the adopted method is simple and easy to implement, the obtained protein has stable quality, and the industrial amplification is easy to carry out.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

FIG. 1 is a map of recombinant expression plasmid pcDNA15.6-FSH α provided by the present invention;

FIG. 2 is an electrophoresis diagram of a recombinant expression plasmid pcDNA15.6-FSH alpha enzyme-digested DNA fragment provided by the present invention, wherein M: marker; 1. 2 are the restriction enzyme DNA fragments of 2 recombinant expression plasmids respectively;

FIG. 3 is a map of recombinant expression plasmid pcDNA16.1-FSH beta CTP provided by the present invention;

FIG. 4 is an electrophoresis diagram of a DNA fragment digested by recombinant expression plasmid pcDNA16.1-FSH β CTP provided by the present invention, wherein M: marker; 1. 2 and 3 are the enzyme-digested DN fragments of 3 recombinant expression plasmids;

FIG. 5 is an SDS-PAGE electrophoresis of purified long-acting recombinant human FSH according to the present invention, wherein lane 1 is rhFSH-CTP, and lane 2 is Marker;

FIG. 6 is a chromatogram of SEC-HPLC of purified recombinant human follicle-stimulating hormone of the invention 4;

FIG. 7 is an isoelectric focusing electrophoresis chart of a purified long-acting recombinant human follicle stimulating hormone flat plate according to the invention 4, wherein lane 1 is a commercially available primary drug Elonva, lane 2 is purified rhFSH-CTP according to the invention, and lane 3 is a Marker;

FIG. 8 is an SDS-PAGE electrophoresis of purified long-acting recombinant human follicle-stimulating hormone impurities (dissociated subunits and polymers) of the present invention, wherein lane 1 is a sample of rhFSH-CTP polymer, lane 2 is a control of rhFSH-CTP polymer, lane 3 is a sample of rhFSH-CTP dissociated subunits, lane 4 is a control of rhFSH-CTP dissociated subunits, and lane 5 is Marker;

FIG. 9 is a comparison graph of a liquid-phase peptide map of the purified long-acting recombinant human follicle-stimulating hormone (rhFSH-CTP) of the invention 4 and a commercial original drug Elonva.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 construction of an expression vector for the alpha subunit gene of rhFSH

The gene sequence of the coding region of the FSH alpha subunit is shown as SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO: shown at 10. The amplification primers of the FSH alpha subunit gene of the invention are designed by adding Nhe I and NotI enzyme cutting sites on the primers at both ends of the FSH-alpha subunit gene, and the primers are shown in Table 1.

TABLE 1

And (3) amplifying the rhFSH alpha subunit gene by using the primer I and the primer II and using human 293 cell genome DNA as a template, and performing PCR amplification by adopting Pfu DNA polymerase.

The PCR amplification conditions were: amplifying for 30 cycles at 95 ℃ for 5min, 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 2min, extending for 5min at 72 ℃, amplifying to obtain a DNA fragment of the FSH alpha subunit gene, recovering an amplification product by using 1% agarose gel, then carrying out enzyme digestion on the DNA fragment of the FSH alpha subunit gene by using Nhe I and Not I, carrying out enzyme digestion on recombination table plasmid pcDNA3.1 by using Nhe I and Not I to obtain a linearized enzyme digestion plasmid, recovering the enzyme digestion DNA fragment by using agarose gel, connecting with the linearized enzyme digestion plasmid to obtain a recombination expression plasmid, and naming the recombination expression plasmid as pcDNA3.1-rhFSH alpha, and carrying out sequencing identification, wherein a sequencing result shows that a cloning sequence is completely consistent with a target sequence.

The recombinant expression plasmid pcDNA3.1-rhFSH alpha is double digested with Nhe I and Not I endonucleases, and the DNA fragment of FSH alpha subunit gene is recovered by 1% agarose gel. Meanwhile, the expression vector pcDNA15.6 is subjected to double enzyme digestion by Nhe I and Not I endonucleases, and the linearized plasmid DNA fragment is recovered by 1% agarose gel digestion. By T₄The DNA Ligase is connected according to the proportion that the proportion of the DNA fragment of the FSH alpha subunit and the DNA fragment of the double-restriction enzyme linearized plasmid is 1:4, after the DNA Ligase is connected at 16 ℃ overnight, the connected recombinant expression plasmid is transformed into DH5 alpha competent cells, the restriction enzyme is identified, as shown in figure 1 and figure 2, the DNA Ligase is respectively a recombinant expression plasmid map and an electrophoresis chart of the double-restriction enzyme recombinant expression plasmid, and the correctly connected plasmid is named pcDNA15.6-FSH alpha.

Example 2 construction of expression vector for rhFSH beta-CTP subunit Gene

The C terminal of the FSH beta subunit is connected with a coding region gene sequence of CTP as shown in SEQ ID NO: 2, the amino acid sequence of the encoded protein is shown as SEQ ID NO: shown at 11. The enzyme cutting sites of Nhe I and Xho I are added on the two-end primers of the rhFSH beta-CTP subunit gene, the primer sequence is designed by the rhFSH beta-CTP subunit gene, and the primer sequences from the primer I to the primer V are shown in a table 2.

TABLE 2

Using the DNA fragment of FSH beta gene as a template, the amplification was carried out using primer I (SEQ ID NO: 3) and primer II (SEQ ID NO: 4), and pfuDNA polymerase from TIANGEN was used as the DNA polymerase.

The PCR amplification conditions were: amplifying for 30 cycles at 95 ℃ for 4min, 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 100s, extending for 5min at 72 ℃ to obtain an FSH beta subunit DNA fragment, recovering the FSH beta subunit DNA fragment, performing PCR amplification by using the FSH beta subunit DNA fragment without a template and using a primer I (SEQ ID NO: 3) and a primer III (SEQ ID NO: 5) as primers to obtain a first extension segment of the FSH beta subunit DNA fragment, and recovering the first extension segment of the FSH beta subunit DNA fragment by using the same;

performing PCR amplification by using the first extension segment of the FSH beta subunit DNA fragment as a template and using a primer I and a primer IV (SEQ ID NO: 6) as primers to obtain a second extension segment of the FSH beta subunit DNA fragment, and recovering the amplified product;

taking the second extension segment of the FSH beta subunit DNA fragment as a template, taking a primer I and a primer V (SEQ ID NO: 7) as primers, and carrying out PCR amplification to obtain a third extension segment of the FSH beta subunit DNA fragment, namely an FSH beta subunit gene product with the C terminal connected with CTP, namely adding 28 amino acid sequences at the tail end of an HCG beta sequence behind the FSH beta subunit, namely CTP, which is named as FSH beta-CTP.

The third extension segment of the FSH beta subunit DNA fragment of the PCR product is subjected to double enzyme digestion by Nhe I and Xho I, and then is connected with a linearized plasmid pcDNA3.1 which is subjected to double enzyme digestion by Nhe I and Xho I to obtain a recombinant expression vector which is named pcDNA3.1-rhFSH beta-CTP and is sequenced and identified, and the sequencing result shows that the cloning sequence is completely consistent with the designed sequence.

The pcDNA3.1-FSH beta-CTP is double-digested by Nhe I and Xho I endonucleases, and the DNA fragment of the digested FSH beta-CTP is recovered by 1 percent agarose gel. At the same time, the linearized plasmid pcDNA16.1 was digested in a double digestion with Nhe I and Xho I endonucleases, and the gel was recovered in 1% agaroseThe linearized plasmid fragment was collected. By T₄The DNA Ligase is connected according to the ratio of FSH beta-CTP DNA fragment to linearized plasmid being 1:4, after connecting overnight at 16 ℃, the connected recombinant expression plasmid is transformed to DH5 alpha competent cell, the correct recombinant expression plasmid is identified by enzyme digestion, the plasmid map and the double enzyme digestion identification map are respectively shown in figure 3 and figure 4, and named pcDNA16.1-FSH beta-CTP.

Example 3 cell transfection and screening of high expressing cell line rhFSH-CTP

The recombinant expression vectors pcDNA15.6-FSH alpha and pcDNA16.1-FSH beta-CTP are transformed into competent cells of Escherichia coli DH5 alpha, the transformed bacteria are rejuvenated by LB liquid screening culture medium containing ampicillin and expanded to 100mL for mass culture, after shaking culture overnight at 37 ℃, plasmids are extracted and purified, the concentration of the purified plasmid DNA is adjusted to 1 mu g/mu L, and the recombinant expression plasmids pcDNA15.6-FSH alpha and pcDNA16.1-FSH beta-CTP are mixed according to the molar ratio of 1: 1.

1 day before transfection, the host cells CHO-S cells were treated at 0.5X 10⁶cells/mL were passaged to 20mL fresh medium. Three groups of cells in logarithmic growth phase were collected in parallel on the day of electroporation, and the number of cells in each group was 1X 10⁷And then, after centrifuging to remove the culture medium, washing the cells by PBS, centrifuging to remove the supernatant, then re-suspending and uniformly mixing the cells in 0.7mL of PBS and adding the mixture into an electroporation cup, adding 100 mu g of mixed recombinant expression plasmid of pcDNA15.6-FSH alpha and pcDNA16.1-FSH beta-CTP and uniformly mixing, placing the mixture into an electric shock tank after ice bath for 10 minutes, carrying out electric shock for 1 time by 220V and 200 mu S, and carrying out ice bath on the electroporation cup again for 10 minutes until the end of the electric transfection.

The transfected three groups of CHO-S cells were added to 10mL of non-selective CD-CHO medium at 37 ℃ and 5% CO₂Shaking and shaking the culture medium with 75% humidity and 100 rpm. After 24h, the medium was changed to selection medium containing 25. mu.M Methionine Sulfoximine (MSX) and 500. mu.g/mL G418 without L-glutamine.

And after 48 hours, detecting the content of recombinant protein in culture supernatant of the 3 groups of transfection clone libraries by ELISA, preferably selecting a mixed clone library with the highest expression, screening and culturing the mixed clone library by a limiting dilution method to obtain a monoclonal cell strain with higher yield, carrying out passage stability research and batch feed culture on the monoclonal cell strain, evaluating the growth condition, the target protein yield and key quality attributes of the cell strain, and obtaining the cell strain with higher yield, higher purity and better quality, namely the high-expression cell strain rhFSH-CTP.

Example 4 expression purification of recombinant human follicle-stimulating hormone

Step one, culturing the expression cell strain rhFSH-CTP prepared by the invention

Culturing a long-acting recombinant human follicle stimulating hormone (rhFSH-CTP) expression cell strain rhFSH-CTP, and collecting a cell culture solution.

Step two, clarifying the cell culture solution

The cell culture broth collected in step 1 was subjected to secondary depth filtration using Millistak + D0HC, X0HC from Mercury to obtain cell culture supernatant with turbidity < 10 NTU.

Step three, affinity chromatography of cell culture supernatant

Loading the cell culture supernatant into an FSH affinity chromatography column at the flow rate of 150cm/h, washing 3-5 column volumes by using the solution A, then washing 3-5 column volumes by using the solution B, finally eluting by using the solution C, and collecting an elution peak to obtain an rhFSH-CTP affinity chromatography elution peak sample chromatographic solution.

The FSH affinity chromatography packing material is a specific CaptureSelect product for FSH whole molecules, supplied by ThermoFisher company, and has a diameter of 10cm, a height of 23cm and a column volume of 1805 mL.

Wherein the solution A is 20mM Tris-HCl +80mM NaCl; the solution B is 20mM Tris-HCl +1M NaCl; solution C was 20mM Tris-HCl +2.0M MgCl₂(ii) a The pH of the solution A, B, C is in the range of pH7.0 to pH 7.5.

Step four, hydrophobic chromatography of affinity chromatography elution peak sample chromatographic solution

And (3) balancing 3-5 column volumes of the hydrophobic chromatography column by using a solution D, adjusting the conductivity of the affinity chromatography elution sample to be consistent with that of the solution D, loading the sample to the hydrophobic chromatography column at the speed of 100cm/h, and washing 3-5 column volumes by using the solution D. And finally, eluting by using the solution E, and collecting an elution peak which is a hydrophobic chromatographic solution of rhFSH-CTP. The packing material for hydrophobic chromatography is Butyl-650M from TOSOH company.

Wherein, the diameter of the hydrophobic chromatography column is 7.5cm, the height is 18cm, and the column volume is 795 mL. The solution D is 20mM Tris-HCl +3M NaCl; solution E was 20mM Tris-HCl +0.5M NaCl, and the pH range of solution D, E was pH 7.0-pH 7.5.

Step five, mixed mode chromatography of hydrophobic chromatography elution peak chromatographic solution

Balancing 3-5 column volumes of the mixed-mode chromatographic column by using the solution F, diluting a hydrophobic chromatography elution sample to adjust the conductivity to be consistent with that of the solution F, loading the mixed-mode chromatographic column at the speed of 100cm/h, collecting flow-through liquid, balancing the solution F to a base line before loading, and combining the solution F with the flow-through liquid to obtain the mixed-mode chromatographic liquid of the rhFSH-CTP.

Wherein the diameter of the mixed mode chromatographic column is 7.5cm, the height is 15cm, the column volume is 660mL, the filler of the mixed mode chromatography is Capto Adhere of GE company, the solution F is 20mM Tris-HCl +0.3M NaCl, and the pH range of the solution F is pH 7.0-pH 7.5.

Step six, mixed mode chromatography elution chromatography liquid ultrafiltration concentration

Concentrating the mixed mode chromatography elution peak chromatographic solution to 0.5mg/mL by adopting an ultrafiltration method, and replacing the buffer solution to be solution G. The ultrafiltration membrane is a Centramate product of PALL company, has a cut-off molecular weight of 10kD and an effective membrane area of 0.1m². Wherein the solution G is 20mM NaAc-HAc +0.01M NaCl, and the pH range of the solution G is pH 5.5 to pH 5.7.

Step seven, cation exchange chromatography of ultrafiltration concentrated solution

And balancing 3-5 column volumes of the cation exchange chromatography column by using the solution G, wherein the diameter of the cation exchange chromatography column is 5cm, the height of the cation exchange chromatography column is 22cm, and the column volume is 430 mL. Loading the ultrafiltration concentrate into a cation exchange chromatography column at a speed of 100cm/h, collecting flow-through liquid, balancing solution G to the baseline before loading, and combining with the flow-through liquid. Immediately adjusting the pH of the collected sample liquid to pH7.0 +/-0.1, wherein the sample liquid is cation exchange chromatography liquid of rhFSH-CTP. Wherein the filler of the cation exchange chromatography column is CM Sepharose Fast Flow of GE company.

Step eight, ultrafiltration and concentration of cation exchange chromatography liquid

Concentrating the cation chromatographic solution to 0.8-1.5 mg/mL by adopting an ultrafiltration method, and replacing a buffer solution to obtain a purified long-acting recombinant human follicle stimulating hormone (rhFSH-CTP) protein.

Wherein the ultrafiltration membrane is a Centramate product of PALL company, and the cut-off molecular weight is 10 kD; solution H was 8.375mg/mL sodium citrate dihydrate, with the pH range of solution H being pH 6.8-pH 7.5.

Example 5 identification of purified Long-acting recombinant human follicle stimulating hormone (rhFSH-CTP)

As shown in FIG. 5, SDS-PAGE of purified long-acting recombinant human follicle stimulating hormone (rhFSH-CTP) of the present invention is shown, wherein lane 1 shows purified rhFSH-CTP of the present invention 4, lane 2 shows Marker, and the purity is about 100%.

As shown in FIG. 6, the SEC-HPLC chromatogram of the purified long-acting recombinant human follicle stimulating hormone (rhFSH-CTP) of the invention 4 is shown, wherein the ordinate mAU is A_214nmThe ultraviolet absorption value and the abscissa is time, the long-acting recombinant human follicle stimulating hormone protein has a peak at 7.319min, and the purity is 99.904%.

As shown in FIG. 7, the detection result shows that the isoelectric point of the purified rhFSH-CTP obtained by the purification method is distributed in the range of 3.50-6.55, the isoelectric point of the main zone is distributed in the range of 3.50-4.00, and the detection result of the rhFSH-CTP is consistent with that of Elonva.

As shown in fig. 8, SDS-PAGE electrophoresis charts for detecting impurities (dissociated subunits and polymers) of purified long-acting recombinant human follicle stimulating hormone (rhFSH-CTP) are shown, wherein lane 1 is a rhFSH-CTP polymer sample, lane 2 is a rhFSH-CTP polymer control sample purified according to the present invention 4, lane 3 is a rhFSH-CTP dissociated subunit sample, lane 4 is a rhFSH-CTP dissociated subunit control sample purified according to the present invention 4, lane 5 is Marker, and the detection result shows that the polymer content is less than 7.5% and the dissociated subunit content is less than 2%.

As shown in FIG. 9, the detection result shows that the detection result of the purified rhFSH-CTP of the present invention is consistent with that of Elonva, which is a comparison between the liquid phase peptide map of the purified long-acting recombinant human follicle stimulating hormone (rhFSH-CTP) and the Elonva which is a commercially available original drug.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Beijing Erlu biotech Co., Ltd, Beijing Erlu pharmaceutical industry Co., Ltd, Beijing Erlu Lisheng pharmaceutical technology Co., Ltd

<120> recombinant human follicle stimulating hormone and preparation method thereof

<130> GG19713058A

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 351

<212> DNA

<213> Artificial Sequence

<400> 1

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> 2

<211> 474

<212> DNA

<213> Artificial Sequence

<400> 2

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 gaaagaatcc tcttcctcaa aggcccctcc ccccagcctt 420

ccaagtccat cccgactccc ggggccctcg gacaccccga tcctcccaca ataa 474

<210> 3

<211> 56

<212> DNA

<213> Artificial Sequence

<400> 3

atagctagcc cgccgaccat gaagacactc cagtttttct tccttttctg ttgctg 56

<210> 4

<211> 58

<212> DNA

<213> Artificial Sequence

<400> 4

ggaggggcct ttgaggaaga ggattctttc atttcaccaa aggagcagta gctgggcc 58

<210> 5

<211> 58

<212> DNA

<213> Artificial Sequence

<400> 5

ccgggagtcg ggggaatctt ggaaggctgg ggggaggggt ttccgaggaa gaggattc 58

<210> 6

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 6

ttattgtggg aggatcgggg tgtccgaggg ccccgggagt cgggatggct taggaaggc 59

<210> 7

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 7

gcctcgagtt attgtgggag gatcggggtg tagccggg 38

<210> 8

<211> 57

<212> DNA

<213> Artificial Sequence

<400> 8

atagctagcg ccgccaccat gtagtactac agaaaatacg tagctatctt tctggtc 57

<210> 9

<211> 49

<212> DNA

<213> Artificial Sequence

<400> 9

atagcggccg caattgattt gtgataacaa taagtactgc agtggcacg 49

<210> 10

<211> 92

<212> PRT

<213> Artificial Sequence

<400> 10

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210> 11

<211> 138

<212> PRT

<213> Artificial Sequence

<400> 11

Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys Glu Glu

1 5 10 15

Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly Tyr Cys

20 25 30

Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys Ile Gln

35 40 45

Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Arg Val Pro

50 55 60

Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr

65 70 75 80

Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val

85 90 95

Arg Gly Leu Gly Pro Ser Cys Ser Phe Gly Glu Met Lys Glu Ser Ser

100 105 110

Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro

115 120 125

Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

130 135

Claims

1. A method for preparing recombinant human follicle stimulating hormone, the method comprising:

transfecting the constructed expression vector containing the FSH alpha subunit and the constructed expression vector containing the FSH beta subunit into Chinese hamster ovary cells in a combined manner, screening out a cell strain with stable expression, and purifying and culturing a culture solution of the cell strain to obtain the recombinant human follicle-stimulating hormone; wherein the C-terminus of the FSH β subunit is linked to a CTP.

2. The method for producing recombinant human follicle stimulating hormone according to claim 1,

the gene sequence of the coding region of the FSH alpha subunit is shown as SEQ ID NO: 1, the C terminal of the FSH beta subunit is connected with a coding region gene sequence of CTP, which is shown as SEQ ID NO: 2, respectively.

3. The method for producing recombinant human follicle stimulating hormone according to claim 1,

the construction process of the expression vector containing the FSH alpha subunit comprises the following steps:

4. The method for producing recombinant human follicle stimulating hormone according to claim 1,

the construction process of the expression vector containing the FSH beta subunit comprises the following steps:

5. The method for producing recombinant human follicle stimulating hormone according to claim 1,

the combined transfection process comprises:

6. The method for producing recombinant human follicle stimulating hormone according to claim 1,

the process of purifying and culturing the cell strain to obtain the culture solution comprises the following steps:

7. The method for producing recombinant human follicle stimulating hormone according to claim 6,

the filler of the affinity chromatography column is a specific CaptureSelect filler aiming at FSH whole molecules;

the filler of the hydrophobic chromatographic column is Butyl-650M;

the filler of the mixed-mode chromatographic column is Capto Adhere;

8. The method for producing recombinant human follicle stimulating hormone according to claim 6,

the preparation method further comprises the following steps:

9. The method for producing recombinant human follicle stimulating hormone according to claim 8,

and the concentration of the concentrated solution obtained by ultrafiltration concentration is 0.8-1.5 mg/mL.

10. The method for producing recombinant human follicle stimulating hormone according to claim 6,

the turbidity of the supernatant was < 10 NTU.