RU2694598C1 - Cell line hufshkkc6, producer of recombinant human follicle stimulating hormone (fsh) and method for producing fsh using said line - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to cellular technologies and can be used for recombinant production of human follicle-stimulating hormone (FSH). Cell line huFSHKKc6 is obtained by transforming huFSHIK cells with an expression plasmid of length 7719 base pairs consisting of a PuroR with SV40 polyA resistance gene shortened by 3'LTR HIV-1, Amp resistance gene, an AmpR promoter, SV40 and pBR322 replication origin, a human p53 NP promoter, a pgk constitutive promoter of the phosphoglycerate kinase gene, a synthetic nucleotide sequence coding the full-size alpha-subunit of human FSH, a translation termination signal consisting of 3 base pairs and a consensus Kozak signal sequence. Method of producing recombinant human FSH involves culturing huFSHKKc6 cells in a serum-free medium, recovering from a formed culture fluid of human FSH and chromatographic purification thereof by successive ion-exchange, affinity and hydrophobic chromatography.

EFFECT: invention provides obtaining a cell line producing high-productive and stably secrete FSH.

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Description

Technical field

The invention relates to the field of biotechnology and is intended for the production of recombinant human follicle-stimulating hormone (FSH) - an analogue of human follicle-stimulating hormone.

The level of technology

Follicle-stimulating hormone or follitropin (hereinafter also referred to as FSH) is a heterodimeric protein synthesized and secreted by the anterior pituitary gland that plays a leading role in the regulation of human reproductive function.

The target of the hormone action are FSH receptors located on the surface of granulosa cells or Sertoli cells, when activated, which regulates the growth of ovarian follicles in the female body, as well as the regulation of spermatogenesis in men. The lack of endogenous follitropin in the body is one of the causes of infertility. FSH is used to treat certain pathologies in the genital area, for the correction of anovulation and in assisted reproductive technologies.

FSH belongs to the family of glycoprotein hormones, which also includes the luteinizing and thyrotropic hormones of the anterior pituitary and the hormone of the placenta - chorionic gonadotropin. Hormones of this group consist of two non-covalently bound α- and β-subunits that function only as part of a dimer. The primary structures of the α-subunits of these hormones are almost identical, and the structures of the β-subunits differ more significantly and provide for their specific interaction with membrane receptors of target cells. Amino acid sequences of mature α- and β-subunits of human FSH are represented in sequences SEQ ID NO: 2 (amino acid residues 25-116) and SEQ ID NO: 4 (amino acid residues 19-129), respectively, with underlining in the sequences SEQ ID NO: 2 and SEQ ID NO: 4 are signal peptides, respectively, of α- and β-subunits of FSH (amino acid residues 1-24 and 1-18 of sequences SEQ ID NO: 2 and SEQ ID NO: 4, respectively). Signal peptides are removed during the post-translational processing of the α- and β-subunits of FSH and are absent from the mature heterodimeric FSH protein. The nucleotide sequences of the α and β subunits of FSH (FSH α and FSH β, respectively) are presented in SEQ ID NO: 1 and SEQ ID NO: 3, while underlined in SEQ ID NO: 1 and SEQ ID NO: 3 are marked Nucleotides encoding the signal peptides of the α- and β-subunits of FSH.

Oligosaccharide chains in the FSH molecule are attached by asparagine residues (Asn-52 and Asn-78 in the α-subunit and Asn-7 and Asn-24 in the β-subunit). The assembly of the FSH molecule and its glycosylation are carried out in the endoplasmic reticulum and the Golgi apparatus of the FSH producer cell (Yongna Xing, Rebecca V. Myers, et al. Glycoprotein Hormone Assembly in the Endoplasmic Reticulum: I. The glycosylated end-of-human α-subunit loop 2 is threaded through a β-subunit hole. // "The Journal of Biological Chemistry", volume 279, No. 34, August 20, 2004, pp. 35426-35436). FSH is secreted by the cell in the form of a dimer (34-45 kDa). The process of glycosylation leads to the formation of different isoforms of the hormone (which causes the formation of microheterogeneity), differing in the composition of carbohydrate chains, which is important for biological activity, because affects the binding to FSH receptors and the rate of clearance. The carbohydrate part of recombinant FSH is up to 36% of the total mass. For biological activity, an important role is played by the three-dimensional structure of the dimer and post-translational modifications. The source for obtaining FSH is the urine of women in the postmenopausal period, as well as the cells-producers of recombinant human FSH. Preparations obtained using recombinant technologies are characterized by a high degree of purity, low variability from batch to batch, lack of biological impurities, and their production does not limit the amount of initial raw materials, making them more affordable for medical use. Formulations containing FSH are approved for use in Russia, with two clinically approved varieties: Gonal F (follitropin α), produced by Merk Serono S.A., and Puregon (or follistim / follitropin β), produced by NV Organon.

A method for producing follitropin α is known, based on the creation of two plasmids expressing the α and β subunits of FSH and cotransfection of these plasmids into Chinese hamster ovary cells defective in dihydrofolate reductase (hereinafter CHO dhfr cells), described in Chappel, S.C. et al. (1992). "Expression of human gonadotropins by rDNA methods." In "Humans in Gynecological Endocrinology", Parthenon Press, Carnforth, UK, 179-184 and in US Pat. No. 5,240,832 A, publ. 08/31/1993 (IPC: C07K 14/575, C07K 14/59, C12N 15/00, C12N 15/16, C12N 15/67, C12N 15/79, C12N 15/85, C12P 21/04).

According to the method proposed by Chappel, SC et al. (1992), the first plasmid contains the full-length α-subunit gene (containing all exons and introns) and the dihydrofolate reductase (dhfr) gene as a marker for selection and amplification. The second plasmid contains a partial β-subunit gene. The α and β genes are cloned from the human genomic library. The expression of subunit genes is under the control of the metalteonein promoter (MT), which is not the strongest for the expression of proteins. The plasmid design is based on the premise that introns contain enhancer elements that enhance protein expression. To obtain the product, producer cells are cultured in a bioreactor on microcarriers as an adhesion culture. With this method, the level of secretion of FSH by producer cells averages 1 pg / cell per day, which corresponds to 12.6 IU / 10 ⁶ cells per day. Protein production involves 5 stages of chromatographic purification, including affinity chromatography using expensive FSH-specific monoclonal antibodies. In the patent there is no information about the stability of the production of the target protein, in addition, the cultivation of cells on microcarriers is not the best way to industrial production of proteins.

From Olijve W.et al. (1996). Molecular biology and recombinant follicle stimulating hormone of human. // Molecular Human Reproduction, 2 (5), 371-382, there is a known method for producing recombinant follitropin β, according to which the FSH α-subunit gene, including some introns, and the coding DNA (cDNA) of the FSH β-subunit, is cloned into the pKMS vector, based on the vector pBR327. Gene expression is under the control of the early SV40 promoter and the MT-II metallotheonein promoter, respectively. Selection of clones with a high copy number of FSH genes is carried out using a cotransfection of an auxiliary selection plasmid pAG60 / MT2, containing the metalteonein gene, which provides resistance to heavy metals, and further selection in the presence of Cd ²⁺ . The productivity of the producer line during cultivation in a bioreactor on microcarriers is 50-80 IU / ml per day at a density of 10 ⁷ / ml, which corresponds to 5-8 IU / 10 ⁶ cells per day. Purification is carried out using anion-and cation-exchange, hydrophobic and exclusion chromatography. This method has the same drawbacks as the plasmid creation method according to US Pat. No. 5,240,832 A, since it is characterized by the choice of non-optimal promoters for the expression of a heterologous protein, as well as the presence of possible negative regulatory elements in the present intron elements of FSH subunit genes. Also, the duration of stable expression of the protein is not indicated.

Known from patent RU 2560596 C2, publ. 08/20/2015 (IPC: C12N 15/63, C12N 15/16, C12N 15/79, C12N 5/10, C12P 21/02) a method for creating an FSH producer using Chinese hamster ovary cells transformed with an expression plasmid containing a DNA fragment, coding chains of α- and β-subunits of human follicle-stimulating hormone or an individual chain of β-subunit of human FSH under the control of a promoter and regulatory elements functioning in a eukaryotic cell. For protein purification according to the method according to patent RU 2560596 C2, a two-step scheme is used, including the sequential use of cation-exchange (Capto MMS) and affinity chromatography (Blue Sepharose FF).

However, the main emphasis in the patent RU 2560596 C2 is made on the creation of an expression system to increase the productivity of the line. In this case, the method for producing recombinant FSH proposed in patent RU 2560596 C2 is not optimal because it requires the cultivation of FSH producing cells in Erlenmeyer flasks, which is insufficient for producing FSH in large volumes. It should also be noted that, apart from electrophoretic data, in the patent RU 2560596 C2 there is no other evidence of the purity of the target product, that with the two-stage purification scheme used according to RU 2560596 C2, it is not enough to assess the compliance of the target product with pharmacopoeial requirements assessment of its use for medical purposes.

From published US patent application US 2009/0291473 A1, publ. 11/26/2009 (IPC: C12P 21/06) a method is known for creating a Chinese hamster ovary cell line - a producer of FSH. According to the method of US 2009/0291473 A1, for the expression of FSH, coding DNA (cDNA) of the FSH subunits is cloned into the pEhygr vector, while the α-subunit FSH cDNA is obtained by polymerase chain reaction (PCR) from the cDNA of the human placenta library, and the β-subunit cDNA is from the human pituitary cDNA library. A purification scheme for recombinant human FSH with high specific activity (14000-18000 units / mg) is described.

The method of purification of recombinant human FSH according to US 2009/0291473 A1 includes the sequential use of cation-exchange chromatography, such as CaptoMMC, affinity chromatography, such as Blue Sepharose FF, hydrophobic chromatography, such as Phenyl Sepharose FF, and gel filtration, Superdex 75. Method according to US 2009/0291473 A1 has a number of disadvantages, since the EF-1 promoter used to induce the expression of both FSH subunits is a weak promoter for the expression of proteins. The productivity of the cell line adapted to growth in suspension in serum-free medium according to US 2009/0291473 A1 does not exceed 12 IU / 10 ⁶ cells per day. The main emphasis in US 2009/0291473 A1 is placed on the method of purification of the final product, and not on creating an optimal expression system to increase the productivity of the producer line. The stability of FSH production by the producer line is not mentioned in this application.

From published US patent application US 2007/0129295 A1, publ. 06/07/2007 (IPC: C07K 14/59) there is a known method of producing FSH, including chromatographic purification of FSH on a strong anion exchange resin, metal chelate chromatography, purification on a weak anion exchange resin, hydrophobic chromatography, reverse phase chromatography, ultra- and nanofiltration.

In accordance with the indicated method, a culture fluid with a low concentration of FSH (2.7-5.4 mg / l) is used to isolate the target product. Also the disadvantages of this method include the use of buffer solutions with a high content of isopropyl alcohol, the use of metal chelate chromatography with expensive sorbents. In addition, among the disadvantages of this method should be noted the low activity of FSH preparations obtained by this method, amounting to 4900-8870 U / mg.

From the publication WO 2010/115586 A2, publ. 10/14/2010 (IPC: C07K 1/18, C07K 1/20, C07K 14/59) a method of purification of recombinant FSH is known, which involves purification of recombinant FSH on a strong anion exchange resin (UNOsphere Q), hydrophobic chromatography (Phenyl Sepharose FF), affinity chromatography on Blue Sepharose FF, ultrafiltration / diafiltration, adsorption on a cationic membrane, repeated purification on a strong cation exchanger (Q-Sepharose FF), repeated ultrafiltration, gel chromatography and nanofiltration. The disadvantages of this method include the multistage purification leading to higher production costs, and the use of a cell line with low productivity (the content of the target product in the culture fluid is 8.1 mg / l on the 28th day of cultivation).

From document WO 2007/065918 A2, publ. 06/14/2007 (IPC: C07K 1/36, C07K 14/00, C07K 14/59 known method of purification of recombinant FSH, which includes the following steps: anionic chromatography on strong anion exchanger Q-Sepharose FF, affinity chromatography on Blue Sepharose FF, anion-exchange chromatography on DEAE-Sepharose FF weak anion exchange resin, Butyl Sepharose FF hydrophobic chromatography, ultrafiltration, Fractogel EMD TMAE HICAP strong anion exchange chromatography, and nanofiltration. At all stages of purification of mutant FSH, L-methionine is used as an antioxidant in all stages of purification of mutant FSH as L-methionine as an antioxidant. Method according to WO 2010/11558 6 A2. In addition, although the specific activity of human FSH preparations obtained by the method according to WO 2007/065918 A2 is quite high and amounts to 11000-17000 U / mg, the production of the drug according to this scheme will be very expensive because of its multi-stage character.

длиной 9 пар оснований, синтетической последовательности длиной 348 пар оснований, кодирующей полноразмерную α-субъединицу человеческого ФСГ, включающую сигнальный пептид, стоп-кодон длиной 3 пар оснований, фрагмент плазмиды pcDNA-TOPO длиной 5407 пар оснований, содержащего промотор-энхансер цитомегаловируса (CMV), сигнал полиаденилирования тимидинкиназы вируса простого герпеса (HSV TK), ген устойчивости к антибиотику неомицину (neo), промотор и сигнал полиаденилирования вируса SV40, а экспрессионная плазмида pOptiVEC/FSHβ состоит из последовательности

длиной 9 пар оснований, синтетической последовательности длиной 387 пар оснований, кодирующей полноразмерную β-субъединицу человеческого ФСГ, включающую сигнальный пептид, стоп-кодон длиной 3 пар оснований, фрагмента плазмиды pOptiVEC-TOPO длиной 4402 пар оснований, содержащего промотор-энхансер цитомегаловируса (CMV), ген фактора устойчивости трансфицированных клеток к воздействию метотрексата-дигидрофолатредуктазы (DHFR) и регуляторные элементы для экспрессии DHFR-последовательности IRES вируса энцефаломиокардита (EMCV), сигнал полиаденилирования, ген устойчивости к антибиотику ампициллину и бактериальный промотор гена устойчивости к ампициллину.Closest to the claimed invention is a method of creating a cell line of ovarian Chinese hamster huFSHIK - producer of recombinant FSH, described in the patent RU 2502798 C2, publ. 12/27/2013 (IPC: C12N 5/00). HuFSHIK cells according to RU 2502798 C2 is obtained based on dihydrofolate reductase deficient (dhfr ^-) cells CHO K1 DXB11 (Urlaub G., Chasin L. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity -. "Proceedings of the National Academy of Sciences" , 1980, volume 77, pp. 4216-20) by cotransfection of these cells with the expression plasmids pcDNA / FSHα and pOptiVEC / FSHβ, where the expression plasmid pcDNA / FSHα consists of the sequence

9 base pairs in length, synthetic sequence 348 base pairs in length, encoding a full-length α-subunit of human FSH, including a signal peptide, a stop codon with a length of 3 base pairs, plasmid fragment pcDNA-TOPO with a length of 5407 base pairs, containing a promoter-enhancer of cytomegalovirus (CMV) , the herpes simplex virus thymidine kinase (HSV TK) polyadenylation signal, the neomycin antibiotic resistance gene (neo), the promoter and polyadenylation signal of the SV40 virus, and the expression plasmid pOptiVEC / FSHβ consists of the sequence

a length of 9 base pairs, a synthetic sequence of 387 base pairs in length, encoding a full-length β-subunit of human FSH, including a signal peptide, a stop codon with a length of 3 base pairs, a fragment of the plasmid pOptiVEC-TOPO with a length of 4402 base pairs, containing a promoter-enhancer of cytomegalovirus (CMV) , the gene of resistance factor of transfected cells to the effects of methotrexate dihydrofolate reductase (DHFR) and regulatory elements for the expression of the DHFR-sequence IRES of encephalomyocarditis virus (EMCV), polyadenylation signal, n resistance to the antibiotic ampicillin and the bacterial promoter of the gene for resistance to ampicillin.

The huFSHIK cells according to patent RU 2502798 C2 are deposited in the Russian Cell Culture Collection at the Institute of Cytology of the Russian Academy of Sciences (Institute of Cytology RAS), the deposit number of RACS (P) 744D.

According to RU 2502798 C2, expression plasmid DNAs pcDNA / FSHα and pOptiVEC / FSHβ are created using nucleotide sequences encoding the α and β subunits of FSH, respectively, synthesized on the basis of consensus amino acid sequences for codon optimization for enhanced expression in mammalian cells . The expression of subunit genes is under the control of a cytomegalovirus (CMV) promoter-enhancer. The expression plasmids pcDNA / FSHα and pOptiVEC / FSHβ are co-transfected into CHO K1 DXB11 cells by liposomal transfection. After double selection in a medium that does not contain hypoxanthine and thymidine with the addition of a selection agent for geneticin, a pool of stably transfected cells is obtained. Further amplification of expression cassettes is carried out by culturing cells in the presence of increasing doses of methotrexate (hereinafter referred to as MTX).

The solution proposed in accordance with the patent RU 2502798 C2 has a number of drawbacks, among which the low productivity of the huFSHIK cell line should be noted. Thus, according to Example 6, shown on pp. 13-14 of the description of this patent, the productivity of the huFSHIK line, adapted to growth in suspension in serum-free medium, does not exceed 5 μg of FSH per 1 ml of culture liquid for 3–4 days of cell culture, which, in terms of 10 cells per day, is 2.046 μg / ml and corresponds to about 25 IU / 10 ⁶ cells per day.

It should also be noted that, according to patent RU 2502798 C2, huFHSIK cells are cultured in the presence of 50-100 nM methotrexate, which is insufficient to obtain maximum amplification of expression cassettes in the genome. In addition, due to the design feature of vectors, amplification of expression cassettes can be observed only for the sequence encoding the β-subunit of FSH, which can lead to a significant stoichiometric imbalance in the production of α- and β-subunits of FSH Finally, the main focus in the patent RU 2502798 C2 given the creation of an expression system to increase the productivity of the line-producer of FSH, while other ways to optimize the production of the target product in this patent are not considered.

The objective of the invention is to provide a method for the production of recombinant human follicle-stimulating hormone for use in the pharmaceutical industry (for obtaining FSH on an industrial scale). The problem is solved in the framework of the present invention due to the production of highly productive stable cell lines-producer of recombinant human FSH, which is the object of the present invention, hereinafter referred to as huFSHKKc6. The huFSHKKc6 cells are obtained by inserting into the genome of the huFSHIK cells described in patent RU 2502798 C2 and deposited in the Russian cell culture collection at the Institute of Cytology, Russian Academy of Sciences under the number RKKK (P) 744D, an expression plasmid of size 7719 bp that encodes the α-subunit of FSH, consisting of the following key genetic elements:

(a) PuroR puromycin resistance gene with SV40 polyadenylation signal (SV40 polyA),

(b) shortened 3'-long terminal repeat of the HIV-1 virus (3'LTR),

(c) the antibiotic resistance gene ampicillin (AmpR) and the bacterial promoter of the ampicillin resistance gene (AmpR promoter),

(d) the origin of SV40 virus replication (SV40 ori),

(d) the origin of replication pBR322,

(e) the NP promoter of the human p53 gene,

(g) constitutive promoter of the phosphoglycerate kinase (pgk) gene,

(h) the synthetic nucleotide sequence presented in SEQ ID NO: 1, encoding the full-size α-subunit of human follicle-stimulating hormone, presented in SEQ ID NO: 2 and including the signal peptide (amino acid residues 1-24 of the sequence SEQ ID NO: 2),

(i) a broadcast termination signal consisting of 3 base pairs,

and

состоящую из 9 пар оснований.(k) consensus signal sequence

consisting of 9 base pairs.

The structure of this plasmid, consisting of key genetic elements (a) - (k), is shown in Figure 1.

As mentioned above, for huFSHIK cells according to patent RU 2502798 C2, selected as the closest analogue of the present invention, excessive production of β-subunit and insufficient α-subunit of FSH are characteristic, which leads to disruption of the stoichiometric balance between α and β-subunits of FSH . The stoichiometric balance characteristic for the closest analogue, as will be shown below, is eliminated in the framework of the present invention by transfecting huFSHIK cells, a plasmid DNA (plasmid) of 7,719 bp in length, consisting of the key genetic elements (a) - (k) and presented on Figure 1, which provides additional expression of the α-subunit of FSH in cells transfected with the indicated plasmid DNA.

The amino acid sequence of the α-subunit of human FSH transmitted from the indicated plasmid DNA includes the N-terminal signal peptide (amino acid residues 1-24 of sequence SEQ ID NO: 2) and the mature α-subunit of the FSH molecule (residues 25-116 of sequence SEQ ID NO: 2). The N-terminal signal peptide is separated during post-translational processing and is thus absent in the mature secreted heterodimeric follitropin (Pierce JG, Parsons TF Glycoprotein hormones: structure and function. // "Annual Review of Biochemistry", 1981, volume 50, pp. 465 -495) As described in the present invention, the plasmid disclosed in Figure 1 and consisting of key genetic elements (a) - (k), is used to transfect huFSHIK cells according to the patent RU 2502798 C2, which already produce, among others, the human FSH β-subunit , then transfection of huFSHIK cells with an expression plasmid of 7719 bp in length, consisting of the key genetic elements (a) - (k), the structure of which is disclosed in Figure 1, makes it possible to obtain mature recombinant human FSH in transfected cells with this plasmid, while post-translational ny processing and formation of mature human FSH molecules of separate α- and β-subunits of FSH is done in the endoplasmic reticulum-producing cells. This leads to the restoration of the disturbed stoichiometric balance between the α- and β-subunits of FSH, characteristic of the huFSHIK cells.

Another object of the present invention is a method for producing recombinant human FSH using huFSHKKc6 cells. The method for producing recombinant human FSH according to the present invention involves the cultivation of huFSHKKc6 cells producing recombinant human FSH, preferably in serum-free medium, followed by isolation of the target product — recombinant human FSH — from the resulting culture fluid. In the most preferred embodiment of the present invention, the selection of the target product is carried out using a three-stage chromatographic purification of the protein, including the sequential use of anion-exchange, affinity and hydrophobic chromatography. The technical result of the invention is to provide a recombinant cell line huFSHKKc6 - producer of human FSH, with high productivity, exceeding 8 times the productivity of the original producer - huFSHIK cells, as well as high viability. In the framework of the present invention, it was possible to show that the huFSHKKc6 cells consistently secrete FSH for at least 25 passages into the culture medium not containing animal products.

In addition, in the framework of the present invention, it was possible to optimize the cultivation conditions of the huFSHKKc6 cell line created in the context of the present invention under suspension culture conditions. In the framework of the present invention, additional techniques have been proposed that allow for efficient production of FSH by optimizing the culture conditions of CHO cells and the steps for isolation and purification of FSH.

The proposed method of producing a protein makes it economically viable to obtain a pharmaceutical substance of recombinant FSH and can be widely used in biopharmaceutical production to obtain the desired product.

The present invention is the creation of a cell line of the producer of recombinant human FSH based on optimized genetic structures and the development of a method for the isolation and purification of recombinant human FSH, allowing to significantly increase its yield. Achieved in the framework of the present invention, the technical result is to provide a cell line - producer of recombinant human FSH, characterized by increased productivity and stability, retaining the ability to secrete FSH for a long time (at least for 25 passages).

DISCLOSURE OF INVENTION

Creating an expression plasmid.

In the present invention to optimize the expression of FSH create plasmid DNA (plasmid) with a length of 7719 base pairs (hereinafter - p. O.), providing additional expression of the α-subunit of FSH in cells of the Chinese hamster ovary huFSHIK, transfected with the indicated plasmid. This plasmid, hereinafter referred to as pL4-Puro-αFSH, consists of the following key genetic elements arranged in accordance with Figure 1:

(a) PuroR puromycin resistance gene with SV40 polyadenylation signal (SV40 polyA),

(b) shortened 3'-long terminal repeat of the HIV-1 virus (3'LTR),

(c) the antibiotic resistance gene ampicillin (AmpR) and the bacterial promoter of the ampicillin resistance gene (AmpR promoter, pbla),

(d) the origin of SV40 virus replication (SV40 ori),

(d) the origin of replication pBR322,

(e) the NP promoter of the human p53 gene,

(g) constitutive promoter of the phosphoglycerate kinase (pgk) gene,

(h) the synthetic nucleotide sequence presented in SEQ ID NO: 1, encoding the full-size α-subunit of human follicle-stimulating hormone of the sequence SEQ ID NO: 2, including the signal peptide (amino acid residues 1-24 of SEQ ID NO: 2),

(and) a broadcast termination signal consisting of 3 base pairs,

повышающей эффективность инициации трансляции.(k) consensus signal sequence

increase the efficiency of translation initiation.

In the above plasmid, the structure of which is disclosed in Figure 1 of the present description, the PuroR gene is designed for the selection of stable cell lines. The SV40 polyadenylation signal (SV40 polyA) is designed to express it.

The shortened 3'-long terminal repeat of the HIV-1 virus (3'-LTR HIV-1) in the above plasmid is necessary to increase the stability of the transcript, while the 3'-LTR is used as an additional polyadenylation signal of the FSH α-subunit gene; this additional polyadenylation signal is intended to increase the stability of the transcript (according to Zufferey R., et al. Self-Inactivating Lentivirus Vector Generation. // Journal of Virology, volume 72, No. 12, Dec. 1998 , pp. 9873-9880; see the paragraph connecting pages 9873 and 9874 of this article).

The ampicillin resistance gene Amp ^R and the bacterial promoter of the Amp ^R gene (pbla) in the above plasmid are necessary for the preparation of the preparative plasmid in bacteria of the genus Escherichia. Preferably (without limitation), bacteria of the genus Escherichia are Escherichia coli.

The origin of replication pBR322, created in the 1970s (Bolivar F., Rodriguez RL, et al. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. // "Gene" (1977), volume 2, issue 2 , pp. 95-113), is widely used to create expression vectors.

The translation product of the synthetic sequence SEQ ID NO: 1 is a polypeptide of the sequence SEQ ID NO: 2, comprising a signal peptide (amino acid residues 1-24 of the sequence SEQ ID NO: 2) and a full-length α-subunit of human follicle-stimulating hormone (amino acid residues 25-92 of the sequence SEQ ID NO: 2). Further, during post-translational processing (Pierce J.G., Parsons T.F., 1981), the signaling polypeptide is cleaved in such a way that the mature α-subunit of recombinant FSH obtained in the cell line according to the invention contains only amino acid residues 25-92 of SEQ ID NO: 2.

The broadcast termination signal consists of 3 base pairs. Preferably (without limitation), the translation termination signal is taa, tag, or tga.

необходима для повышения эффективности инициации трансляции. См.: Kozak М. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. // "Nucleic Acids Research", 1987 October, volume 15, №20, pp. 8125-8148. Предпочтительно (без ограничения), консенсусная сигнальная последовательность

представляет собой последовательность gccgccacc.Consensus signal sequence

necessary to increase the efficiency of translation initiation. See: Kozak, M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. // "Nucleic Acids Research", 1987 October, volume 15, No. 20, pp. 8125-8148. Preferably (without limitation), consensus signal sequence

is the sequence gccgccacc.

The structure of the indicated plasmid DNA (plasmid), consisting of key genetic elements (a) - (k), is presented in Figure 1.

The plasmid according to the invention is obtained from the plasmid vector pLenti-puro, described in the prior art in the work of Guan B., Wang, T. L., Shih Ie. M. ARID1A, a SWI / SNF mediated chromatin remover, is a tumor suppressor in gynecologic cancers. // "Cancer Research", 2011 November 1, volume 71, No. 21, pp. 6718-6127 (hereinafter - Guan V., et al., 2011). To obtain the plasmid of the invention, the sequences of the SV40 virus promoter and the polyclonization site are removed from the pLenti-puro vector by the SalI and Pfl23II restriction sites and replaced by the human NP53 promoter NP (according to Tuck SP, Crawford LI Characterization of the human p53 gene promoter. // // "Molecular and cellular biology", 1989 May 1; volume 9, No. 5, pp. 2163-2172), annealing mutually complementary synthetic oligonucleotides PL4_FLAG-forw and PL4_FLAG-rev, obtaining a new polyclonizing section PL4 with different nucleotide sequence, a set of restriction sites and additional sequencing with the FLAG-tag, insert the duplex with “sticky” ends into the plasmid at the SalI restriction site, replace the cytomegalovirus promoter (CMV) with the constitutive promoter of the phosphoglycerate kinase gene (pgk). The nucleotide sequence of the pgk promoter is synthesized by the polymerase chain reaction (PCR) using the oligonucleotide primers Ppgk-forw and Ppgk-rev sequences SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

The huFSHIK cells according to RU 2502798 C2 are created from CHO K1 DXB11 (Urlaub G., Chasin LA, “Proceedings of the United States of America”), July 1980, volume 77, on the basis of Chinese hamster ovary cells known from the prior art. , No. 7, pp. 4216-4220), transfected, including the expression plasmid pOptiVEC / FSHβ, containing a synthetic sequence 387 bp long, encoding the full-length β-subunit of human FSH. Thus, transfection of huFSHIK cells with the plasmid created in the framework of the present invention, characterized in Figure 1, encoding the α-subunit of human FSH and consisting of key genetic elements (a) - (k), allows restoring the stoichiometric balance between α- and β-subunits , thus eliminating the disadvantage characteristic of huFSHIK cells and providing higher and more stable production of full-size human FSH.

Getting the cell line huFSHKKc6

The starting material for creating a cell line according to the invention, producing recombinant human FSH, is the huFSHIK Chinese hamster ovary cell line according to the patent RU 2502798 C2. The huFSHIK cells are cultured on Eagle's medium in Dulbecco's modification (hereinafter - DMEM medium) with the addition of 10% fetal calf serum (hereinafter - FBS) as an adhesion culture at 37 ° C with 5% CO ₂ content. An expression plasmid of 7719 bp in length, consisting of key genetic elements (a) - (k) located relative to each other as shown in Figure 1, is transfected into cells of the huFSHIK cell line.

Preferably (without limitation), the liposomal transfection method described in Straus S.E., Wilson T., and Raskas H.J. is used to introduce the indicated plasmid into the huFSHIK cells. Transformation of KB Cells by Liposomes Containing Adenovirus Type 2 DNA. // "Journal of Virology", 1981, July, volume 39, No. 1, pp. 290-294, incorporated herein by reference. The skilled person will understand that in other embodiments of the present invention, other transfection methods known from the prior art can be used to introduce the plasmid into the huFSHIK cells, for example, a method using cationic polymers, a calcium phosphate method, an electroporation method, a nucleofecture method, a microinjection method, a transfection using magnetic particles and so on. Also for the purposes of the present invention, for introducing into the huFSHIK cells the plasmid shown in Figure 1 and consisting of the key genetic elements (a) - (k), other transfection methods can be used that are not explicitly mentioned in the present description, which are known at the level of equipment currently or will be created later. When implementing specific embodiments of the present invention, a specialist, based on an existing level of knowledge, can select the most optimal method for transfecting cells.

After selection on the medium with the addition of the puromycin selection agent to a final concentration of puromycin of 4.5 μg / ml, cells resistant to puromycin are cloned by the method of limiting dilutions. Clones obtained from single cells are analyzed by enzyme immunoassay. Clones with the level of secretion of FSH, exceeding by 40-50% the level characteristic of the original cells of the huFSHIK clone in the early passages, is saved by the method of cryoconservation for the subsequent creation of a master bank.

Cultural properties

Cells are grown in DMEM with the addition of 2 mM glutamine, 10% dialyzed FBS (Sigma), 100 nM methotrexate (MTX, Sigma) at 37 ° C, in an environment with a CO ₂ content of 5%, as an adhesion culture. The cell line is resistant to MTX, geneticin (G418) and puromycin. The cells of the selected clones are adapted for suspension growth on serum-free SFM4CHO medium. The absence of contamination is confirmed by culturing cells without antibiotics. The line was tested for the absence of mycoplasma by staining with DAPI and / or Hoechst DNA fluorescent dyes, as well as using the MycoA1ert ™ PLUS Mycoplasma Detection Kit (LONZA).

The identity of human FSH is confirmed by high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. The selected clone, called huFSHKKc6, consistently secretes FSH for 25 passages. When cultivating the producer strain huFSHKKc6 on serum-free SFM4CHO medium, 41 μg / ml of FSH are secreted into the medium at a cell density of 0.90-1.21 million / ml for 4 days, which is 8 times more than the original huFSHIK culture. When cells are cultured without changing the medium for 10–12 days, 118 μg / ml FSH is secreted into the medium.

The high stability and productivity in the cultivation of cells in the bioreactor allows to draw a conclusion on the creation of the huFSHKKc6 line - a producer of recombinant human FSH, applicable in the pharmaceutical industry.

Cell cryopreservation

Cells according to the invention are frozen in growth medium with 10% dimethyl sulfoxide (DMSO) at a concentration of 2 million / ml and stored in liquid nitrogen. Defrost by rapid thawing of the tube in a water bath at 37 ° C, followed by replacing the medium with DMSO with growth, diluting the cells with 5 ml of heated medium. Cell viability after thawing is not less than 80% (assessed by trypan blue staining).

Receipt, isolation and purification of recombinant human FSH

The method of producing recombinant human FSH involves cultivating the huFSHKKc6 cell line obtained by transforming the huFSHIK cells of a plasmid 7719 bp long, consisting of key genetic elements (a) - (k), revealed in Figure 1, in a serum-free medium to obtain a culture fluid, separating the culture fluid from the cells and debris and the selection of recombinant human FSH from the resulting culture fluid.

In the preferred embodiment, but without limitation, the cultivation of huFSHKKc6 cells in serum-free medium is carried out for at least 12 days.

In the preferred embodiment, however, without limitation, the separation of the culture fluid from the cells and debris is carried out by centrifugation.

Finally, in a preferred embodiment, however, also without limitation, the isolation of recombinant FSH from the resulting culture liquid is carried out chromatographically. In more preferred embodiments, but without limiting them, the selection of recombinant FSH from the culture fluid is carried out by the method of three-stage chromatographic purification, consistently applying anion-exchange, affinity and hydrophobic chromatography. In the most preferred embodiment, the chromatographic purification of FSH is carried out by successively applying anion-exchange chromatography on DEAE-Sepharose FF, affinity chromatography on Affi-Gel Blue and hydrophobic chromatography on Phenyl-Sepharose 6 FF. The resulting solution of purified recombinant FSH is desalted, transferred to phosphate buffer and, if necessary, concentrated to 0.4-0.8 mg / ml using ultrafiltration on membranes with a cut-off limit of 5 kDa. The final product is subjected to microfiltration on membranes with a pore diameter of 220 nm and nanofiltration on membranes with a pore diameter of 15 nm.

Characterization of recombinant FSH produced by huFSHKKc6 cells.

The obtained recombinant human FSH fully complies with the requirements of the European Pharmacopoeia (EP) 8.3 to FSH solutions. It can be used for the manufacture of dosage forms. The biological activity of the obtained recombinant human FSH is 13 IU / μg (when tested by the method of biological samples according to Steelman-Pohley in comparison with the standard of recombinant FSH NIBSC 08/282).

The invention is illustrated by the following figures:

Figure 1. Plasmid map; designations:

pBR322 ori - origin of replication pBR322,

AmpRes - ampicillin resistance gene (β-lactamase gene),

pbla - β-lactamase gene promoter,

SV40 ori - origin of replication SV40,

SV40polyA - SV40 polyadenylation signal,

3'-LTR (delta-U3) - shortened 3'-long terminal repeat of HIV-1,

PuroRes - puromycin resistance gene,

NPpromoter - the promoter of the human p53 gene,

FSH alpha - a full-length α-subunit gene of human FSH,

, сайты рестрикции: Cla I (2127), Eco RI (2671), Xho I (3037), Bam HI (3043) и Sal I (3076).Kozak consensus sequence - consensus sequence

, restriction sites: Cla I (2127), Eco RI (2671), Xho I (3037), Bam HI (3043) and Sal I (3076).

Primer sequences for promoter synthesis:

pgk.Ppgk-forw: 5'-AGTTCATCGATTTGGGGTTGCGCCTTTTCCA-3 '(SEQ ID NO: 5),

Ppgk-rev .: 5'-GTTCTCTAGACCCTGGGGAGAGAGGTCGG-3 '(SEQ ID NO: 6).

Figure 2. Electrophoretic analysis of recombinant FSH produced by huFSHKKc6 cells.

Restorative electrophoresis conditions:

Gel Tracks 1.3: culture medium from the huFSHKKc6 line;

Tracks gel 2: internal standard FSH;

The gel track 4: Standard CRS FSH;

Gel Tracks 5.9: Molecular Weight Marker PageRuler Plus Prestained Protein Ladder, Thermo, cat. 26619 (250 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 35 kDa, 25 kDa, 10 kDa).

Non-reducing electrophoresis conditions (without the addition of beta-mercaptoethanol and without heating):

The tracks of the gel 6: internal standard FSH;

Gel tracks 7,10: culture medium from the huFSHKKc6 line;

The track of the gel 8: Standard CRS FSH.

Figure 3. Determination of FSH in culture serum-free medium with antibodies against the β-subunit of FSH by the immunoblot method. Electrophoresis is carried out in 12% polyacrylamide gel (PAAG) with sodium dodecyl sulfate (DDS) under native conditions.

1.3 — culture medium from the huFSHKKc6 line;

2 - culture medium from the huFSHIK line;

4 - CRS FSH standard;

5 - Internal standard FSH;

6 - Molecular Weight Marker PageRuler Plus Prestained Protein Ladder, Thermo, cat. 26619 (250 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 35 kDa, 25 kDa, 10 kDa).

Figure 4. Distribution of FSH isoforms by the method of isoelectric protein focusing (IEF) in the pI range of 3.3-5.5

1 - distribution of FSH isoforms from the huFSHKKc6 line;

2 - distribution of the isoforms of the solution of the standard sample (Follitropin, Eur. Ph., # Y0001629) in the range of pI 3-10;

3 - distribution of isoforms of the standard for IEF pI 3-10.

Figure 5. Determining the quality of recombinant FSH by the method of reversed-phase HPLC. The content of oxidized forms. Sample - FSH after final purification, in the amount of 10 μg.

Figure 6. The determination of the amount of FSH in the solution and the impurity content of high-molecular compounds by the method of exclusion chromatography. Sample - FSH after final purification, in the amount of 8 μg.

Figure 7. The results of the evaluation of the biological activity of recombinant FSH. The drug obtained in the huFSHKKc6 cell line was compared with the reference drug, the WHO IS 08/282 standard.

The implementation of the invention

The implementation of the claimed invention is illustrated by the following examples 1-6.

Example 1. Construction of expression plasmids

The plasmid DNA (plasmid) 7719 bp in length created in the framework of the present invention, consisting of the key genetic elements (a) - (k), the structure of which is disclosed in Figure 1, is obtained from the plasmid vector pLenti-puro described in the above-mentioned Guan B., et al., 2011, as follows.

At the first stage, the sequences of the SV40 virus promoter and the polycloning site are removed from the pLenti-puro vector at the SalI and PIl23II restriction sites and replaced with the NP promoter of the human p53 gene according to the procedure proposed by Tuck S.P., Crawford L.I. Characterization of the human p53 gene promoter. // "Molecular and cellular biology", 1989 May 1; volume 9, No. 5, pp. 2163-72. The nucleotide sequence of the NP promoter is obtained by synthesis in accordance with information taken from the database (GenBank: J04238.1, URL: https://www.ncbi.nlm.nih.gov/nuccore/J04238.l) and integrated into the vector pLenti -puro restriction sites SalI and Pƒl23II with obtaining plasmids, hereinafter referred to as pLCMV-pL4-Flag-Puro.

Then, by annealing the mutually complementary synthetic oligonucleotides PL4_FLAG-forw and PL4_FLAG-rev (sequences SEQ ID NO: 7 and SEQ ID NO: 8), a new PL4 polycloning site with different nucleotide sequence, a set of restriction sites and an additional FLAG-tag is obtained. The resulting duplex with sticky ends is inserted into the plasmid pLCMV-pL4-Flag-Puro at the SalI restriction site.

V, Gossen M, Josewski J, Duda A, Polle L, Owens RJ, Wirth D, Heinz DW. Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development. // "PloS one", 2011 Dec 9; volume 6, №12, e27829.In the resulting vector, the cytomegalovirus promoter (CMV) is replaced with the constitutive promoter of the phosphoglycerate kinase (pgk) gene, and the plasmid pFS-sigHis-PGK (GenBank: JF313343, URL: https: //wwww) is used as the source of the nucleotide sequence of the pgk gene promoter. .nlm.nih.gov / nuccore / JF313343), described in Wilke S, Groebe L, Maffenbeier V,

V, Gossen M, Josewski J, Duda A, Polle L, Owens RJ, Wirth D, Heinz DW. Streamlining homogeneous glycoprotein production // "PloS one", 2011 Dec 9; volume 6, №12, e27829.

The synthesis of the nucleotide sequence of the pgk promoter is carried out by the polymerase chain reaction (PCR) using oligonucleotide primers Ppgk-forw and Ppgk-rev, the sequences of which are presented in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The synthesized promoter sequence is integrated into the vector plasmid at the restriction sites ClaI and XbaI.

At the final stage, a nucleotide sequence consisting of 360 bp is synthesized by PCR. The sequence consists of the structural part of the gene of the full-length α-subunit of human FSH, including the signal peptide, the translation termination signal from 3 bp. and the Kozak consensus signal sequence, which increases the efficiency of translation initiation and is located in front of the initiating codon of the FSH gene. The sequence is synthesized using primers fsh-α_EcoRI and fsh-α_XhoI, the sequences of which are presented in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The nucleotide sequence is integrated into the PL4 polylinker of the plasmid through the restriction sites EcoRI and XhoI to obtain plasmid DNA consisting of the key genetic elements (a) - (k), the structure of which is shown in Figure 1.

The primer sequences PL4_FLAG-forw and PL4_FLAG-rev for the synthesis of the PL4 polyclon site are presented in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

Sequences of primers Ppgk-forw and Ppgk-rev for the synthesis of the pgk promoter are presented in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

The primer sequences fsh-α_EcoRI and fsh-α_XhoI for the synthesis of the FSH α-subunit gene are presented in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

Example 2. Getting a stable clone-producer.

To create a producer of FSH, huFSHIK cells according to patent RU 2502798 C2, deposited in the Russian Cell Culture Collection of the Institute of Cytology, Russian Academy of Sciences under the number RKCC (P) 744D, are used.

The huFSHIK cells were cultured in DMEM with 10% FBS as an adherent culture at 37 ° C with 5% CO ₂ and 100 nM MTX. The expression plasmid obtained in Example 1 was transfected into the huFSHIK cell line by liposomal transfection using Lipofectamin 2000 (Invitrogen) according to the manufacturer's instructions. After one day, the medium is replaced with DMEM medium with 10% serum. Selection is carried out using the puromycin selective agent, which is added to the medium to a final concentration of 4.5 μg / ml. The selection of a stable clone-producer is carried out by limiting dilution method, sifting the cells growing after treatment with antibiotic into 96-well plates. Based on the growth rate, viability and productivity, a clone huFSHKKc6 is selected, from the cells of which a cell bank is created.

The cultural properties of cells huFSHKKc6

Cells of the selected clones were adapted to suspension growth on serum-free media SFM4CHO, CD4CHO (HyClone), CellVento CHO-200 (Merck Millipore), ChoMaster CD (Macs), ProCHO5 (Lonza), Ex-Cell DHFR, CD (Sigma Aldrich). Reseeding carried out with a multiplicity of 1: 5 every 3-4 days. The identity and production of human FSH will be determined by HPLC, ELISA and immunoblot. The selected clone huFSHKKc6 consistently secretes FSH in the amount of 36-41 mg / l for 4 days. For 10-12 days of cultivation without changing the medium, the content of the secreted protein is 118 mg / l. The absence of contamination was confirmed by culturing cells without antibiotics. The line was tested for the absence of mycoplasma by staining with DAPI and / or Hoechst DNA fluorescent dyes, as well as using the MycoAlert ™ PLUS Mycoplasma Detection Kit (LONZA).

The inoculum is expanded in a bioreactor (50 ml, TPP) at 37 ° C, 5% CO ₂ , 150 rpm, followed by cultivation in the CELLIGEN 310 (New Brunswick) bioreactor with an automated process control system according to specified cultivation parameters. The duration of cultivation is 264-288 hours. To optimize the cultivation parameters in the bioreactor, the factor experiment method with fractional replicas was used, which allowed taking into account the effect of several parameters on each other. As a result of optimization, productivity increased by 1.5 times. The developed method allows the cultivation in periodic mode, with water or perfusion.

Depending on the cultivation method for the isolation and purification of the target protein, the cells are removed from the medium by centrifugation or using a tangential filtration module with hollow fibers.

Example 3. Isolation and purification of recombinant FSH from the culture fluid of the producer of FSH huFSHKKc6

Preparation of recombinant human FSH involves cultivating the producer cell line, separating the culture medium from the cells and debris, and chromatographic purification of FSH from the resulting culture fluid using anion-exchange chromatography on DEAE-Sepharose FF, affine chromatography on Affi-Gel Blue and hydrophobic chromatography on Phenyl-Sepharose 6 Ff. All chromatographic purification steps are carried out in 20 mM Tris-HC1 with a pH of 7.9, containing 10 mM L-methionine, in which the salt and organic components used at a certain stage of the process are added as necessary. Purification involves microfiltration of solutions using a Stericup filtration system with 0.45 and 0.22 micron filters (Merck Millipore) and nanofiltration using Planova 15N filters (Asahi Kasei Medical Co.). All chromatographic processes are carried out at a temperature of (6 ± 2) ° C using a Bio-Rad LP chromatographic system. FSH solutions are stable under storage conditions at 5 ° C during the day. The process of isolation and purification of protein does not imply storage and freezing of samples at intermediate stages of work. However, FSH solutions can be frozen once and stored at -20 ° C. Determining the concentration of FSH in the process of selection is carried out by the method of enzyme-linked immunosorbent assay (ELISA). The quality of the final and intermediate products is determined by electrophoresis in 12% polyacrylamide gel with sodium dodecyl sulfate, as well as by the methods of reversed-phase (RP) HPLC and size exclusion chromatography. Total protein is determined using the method of Lowry and UV spectrophotometry.

After microfiltration on a 450 nm membrane with a pore size of 450 nm, a diluted culture solution of fluid culture (hereinafter - QOL) containing 145 mg of recombinant FSH is diluted five times with a buffer solution of 20 mM Tris-HCl with a pH of 7.9 and containing 10 mM L- methionine, applied to a chromatographic column with DEAE-sepharose FF (GE Healthcare), pre-equilibrated with the same buffer solution, and washed with an equilibration buffer solution. FSH is eluted from the sorbent with sodium chloride in a concentration gradient from 0 to 0.2 M. The fractions containing FSH are combined and transferred to the next purification stage. The output of FSH at the stage is 123 mg.

To the protein solution obtained after purification on DEAE-Sepharose FF, add sodium chloride to a concentration of 0.3M, Tween-20 to a concentration of 0.5% and adjust the pH value of the solution to 7.9. The resulting protein solution after microfiltration is applied to a Affi-Gel Blue (Bio-Rad) affinity sorbent chromatographic column equilibrated with a buffer solution containing 20 mM Tris-HCl, 10 mM L-methionine, 0.3 M NaCl, 0.5% Tween -20 with a pH of 7.9. After applying the sample, the sorbent is washed with a buffer solution of the composition: 20 mM Tris-HCl, 10 mM L-methionine, 0.5 M KCl, 0.01% Tween-20, pH 7.9, and then eluted with FSH with potassium chloride in a concentration gradient from 0.5 to 2.0 M. Fractions containing FSH are combined and transferred to the next stage. The output of FSH at the stage is 96 mg.

Ammonium sulfate is added to the protein solution obtained after purification on Affi-Gel Blue sorbent to a concentration of 1.0 M and the pH value of the solution is adjusted to 7.9. The resulting protein solution after microfiltration is applied to a Phenyl-Sepharose 6 FF (Sigma Aldrich) chromatographic column equilibrated with a buffer solution of 20 mM Tris-HCl, 10 mM L-methionine, 0.01% Tween-20, 1 M (NH ₄ ) SO ₄ with a pH of 7.9. The elution of the target protein is carried out under conditions of a linearly decreasing concentration of ammonium sulfate from 1.0 M to 0 M. The fractions containing FSH of the required quality are combined and transferred to diafiltration. The output of FSH at the stage is 72 mg (exclusion chromatography data), the total protein determined by the Lowry method is 73 mg and 72.6 mg according to UV spectrometry at a wavelength of 280 nm.

The FSH solution obtained in the last stage of chromatographic purification is desalted, transferred to a buffer solution of the composition: 10 mM NaH ₂ PO ₄ * H ₂ O, 10 mM L-methionine, pH 7.4 and, if necessary, concentrated to 0.4- 0.6 mg / ml using ultrafiltration on membranes with a cut-off limit of 5 kDa. The final product is subjected to microfiltration on membranes with a pore diameter of 0.22 μm and nanofiltration on membranes with a pore diameter of 15 nm. The yield of the target product after all stages of purification is 69 mg (according to exclusion chromatography), which is about 48% of the FSH content in the original QOL.

The output of FSH according to the claimed method is about 55 mg in terms of 1 l of culture fluid.

Example 4. Evaluation of the amount of recombinant human FSH produced by huFSHKKc6 cells.

4.1 enzyme-linked immunosorbent assay (ELISA)

For carrying out solid-phase ELISA analysis, the “FSH ELISA” kit (“DRG”, Germany) is used. The FSH ELISA kit is designed for solid-phase analysis of samples containing FSH using monoclonal antibodies to different epitopes of a dimeric protein molecule. The intensity of staining is directly proportional to the concentration of FSH in the sample, which is estimated in mMED / ml relative to a standard calibrated against Internetional Standart for Follicle Stimulation Hormone (FSH), human recombinant for immunoassay NIBSC code 92/510.

4.2. ELISA analysis with monoclonal antibodies.

Enzyme immunoassay with monoclonal antibodies (Leinco Tecnologies) to the α-subunit of FSH for processing 96 well plates and antibodies to the β-subunit of FSH conjugated with horseradish peroxidase (HRP) are also used to confirm the secretion of the dimeric FSH molecule and quantitative determinations. A solution of 3,3 ', 5,5'-tetramethylbenzidine (TMB) is used as a substrate.

4.3. High performance size exclusion chromatography

Quantitative determination of FSH and evaluation of the content of high-molecular compounds in solutions of the target product is carried out using size exclusion chromatography using columns (300–7.8 mm) containing hydrophilic silica gel for chromatography of a class suitable for separating globular proteins in the molecular weight range from 10 to 500 kDa (5 μm). The mobile phase contains 6.74 ml of phosphoric acid, 14.2 g of anhydrous sodium sulfate, pH 6.7. Detection is carried out using a spectrophotometric detector at a wavelength of 214 nm in isocratic mode. The flow rate is 1 ml / min. As a reference, a certified standard of follitropin produced by Sigma-Aldrich (Follitropin, Eur. Ph., # Y0001629) is used. The results are presented in FIG. 6

Example 5. Evaluation of the quality of solutions of FSH

5.1. Electrophoresis.

Electrophoresis (ESP) of QL protein and / or purified preparation is carried out in 12% PAAG with DDS under non-reducing and reducing conditions according to the standard Laemmli technique in a 12% gel stained with Ponce S. Stain. The results are shown in FIG. 2

5.2. Immunoblotting

QOL proteins and / or preparations containing FSH, separated by DDS-PAG electrophoresis, are transferred to a nitrocellulose membrane with a pore diameter of 0.45 μm (Bio-Rad, USA) for wet transfer Mini Trans-Blot Electrophoretic Transfer Cell (170-3930 , Bio-Rad, USA). Murine antibodies to α and β subunits conjugated with horseradish peroxidase (H116 and H101, respectively, Leinco Technologies, USA) are used sequentially for staining the membrane. European standards (Follitropin, Eur. Ph., # Y0001629) and FSH, isolated during the cultivation of the huFSHIK cell line (IBH, Russia), are used as standards. The intensity of the bands on the gels and membranes is calculated using the Imagine system VisionCapt v16.11a program, Vilber Lourmat, Germany. The results are presented in FIG. 3

Example 5.3. Isoelectric focusing.

The distribution of recombinant human FSH isoforms obtained in huFSKKc6 cells is carried out by the method of isoelectric protein focusing (IEF) using a PROTEAN i12 IEF System instrument (No. 166-6000, Bio-Rad) and disposable strip strips on which a polyacryamide gel with a gradient is immobilized pI 3-10 (7 cm long strip - №163-2000, 11 cm - №163-2014, 17 cm - №163-2007, Bio-Rad). A sample of FSH is adjusted with a buffer for isofocusing to a final volume of 125 μl - in case of using a strip strip of 7 cm length, while the amount of FSH in the sample is 10 μg. For the IEF cell, the entire volume obtained is 125 μl. The standard solution (CRS, Follitropin, Eur. Ph., # Y0001629) and protein marker for IEF with a pI range of 3-10, No. 17-0472-01 (Amersham) are similarly diluted in 125 μl of sample buffer to the final protein concentration 15 mcg per strip strip. Separation parameters: strip strips (7 cm), pH 3-10 G, gradient voltage to reach 15,000 volt-hours, with preliminary active rehydration of the sample for 15 hours at 50 V. Staining is carried out with 0.25% Kumassi brilliant blue G-250 at room temperature for 10-15 minutes. Bleach the gel in a wash solution containing 10% acetic acid and 7.5% methanol at room temperature, shaking until the background of the gel is clear. The results are presented in FIG. four.

Example 5.4. Determination of the quality of recombinant FSH by the method of reverse-phase high-performance liquid chromatography. The impurity content of oxidized forms of FSH.

The impurity content of the oxidized forms in solutions of the target product and intermediates is estimated using reverse phase high performance liquid chromatography (RP HPLC) using columns (250 * 4.6 mm) containing butylsilylated silica gel (5 μm).

Mobile phase A contains 1.19 percent by volume (vol.%) Of phosphoric acid, pH 2.5. Mobile phase B is a mixture of water for chromatography with acetonitrile (in a volume ratio of 40:60). Mobile phase C is water for chromatography. Detection is carried out using a spectrophotometric detector at a wavelength of 210 nm. The flow rate is 1 ml / min. The injection volume is 25 µl of a solution with an FSH concentration of 0.4 mg / ml.

A certified standard of follitropin (Follitropin, Eur. Ph., # Y0001629) is used as a reference sample. The results are shown in FIG. 5.

Example 6. Evaluation of the biological activity of recombinant FSH obtained in the cells according to the invention.

Follicle-stimulating activity of the obtained recombinant FSH was evaluated by comparing the degree of increase in the ovaries of immature rats when administered to them SP and a standard sample for biological tests WHO IS FSH (WHO International Standard, 2 ^{nd International Standard for Follicle-Stimulating Hormone} , human, recombinant, for bioassay. NIBSC code: 08/282) with the simultaneous introduction of a standard sample of chorionic gonadotropin WHO IS CG (WHO International Standard, 5 ^th WHO IS, Chorionic Gonadotrophin. NIBSC code: 07/364).

To determine the biological activity, immature female rats of the same line at the age of 19-28 days are used, with a difference in age of not more than 3 days, in weight - not more than 10 g. In the experiment, 30 female rats are taken, divided into 6 groups randomly.

3 doses of the comparison drug and 3 doses of the studied drug are selected in such a way that the smallest dose causes a positive response in at least some rats, and the highest dose does not give the maximum response in any rat. 72 hours after drug administration, each rat is subjected to euthanasia. The ovaries removed are weighed immediately. The expected activity should be at least 80% and not more than 125% of the established activity. The results are presented in FIG. 7

Claims (17)

1. The huFSHKKc6 cell line is a highly productive stable line-producer of recombinant human follicle-stimulating hormone, obtained from the huFSHIK Chinese Hamster Ovary Cell Line deposited in the Russian Cell Culture Collection of the Institute of Cytology RAS (deposition number RKCC (P) 744D) by transfection of these cells, by transferring these cells. presented in Figure 1, having a length of 7719 base pairs and consisting of the following key genetic elements: (a) PuroR puromycin resistance gene with SV40 polyadenylation signal (SV40 polyA), (b) shortened 3 '- long terminal repeat of the HIV-1 virus (3'LTR), (c) the antibiotic resistance gene ampicillin (AmpR) and the bacterial promoter of the ampicillin resistance gene (AmpR promoter), (d) the origin of SV40 virus replication (SV40 ori), (d) the origin of replication pBR322, (e) the NP promoter of the human p53 gene, (g) constitutive promoter of the phosphoglycerate kinase (pgk) gene, (h) the synthetic nucleotide sequence presented in SEQ ID NO: 1, encoding the full-size α-subunit of human follicle-stimulating hormone, presented in SEQ ID NO: 2 and including the signal peptide (amino acid residues 1-24 of the sequence SEQ ID NO: 2), (and) a broadcast termination signal consisting of 3 base pairs, and (k) Kozak consensus signal sequence, which increases the efficiency of translation initiation. 2. The method of obtaining recombinant follicle-stimulating hormone of a person, comprising the following stages: (a) cultivation of cells according to claim 1 in serum-free medium to obtain culture fluid; (b) the selection of recombinant human follicle-stimulating hormone from the culture fluid obtained in stage (a). 3. The method according to p. 2, characterized in that the cultivation of cells is carried out in a bioreactor. 4. Method according to any one of claims. 2-3, characterized in that the selection of recombinant human follicle-stimulating hormone is carried out by the method of three-stage chromatographic purification with the sequential use of ion-exchange, affinity and hydrophobic chromatography.

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