RU2502798C2 - Cell line hufshik releasing recombinant human follicle-stimulating hormone - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention proposes cell line huFSHIK - producer of recombinant human follicle-stimulating hormone (FSH) released to culture liquid. The line releases FSH in suspension culture in concentration of 18IU/10⁶ cells a day.

EFFECT: invention optimises FSH expression system, which leads to increase of its products and improvement of efficiency and cost effectiveness of FSH production for medical purpose.

4 dwg, 7 ex

Description

The invention relates to biotechnology and can be used to produce recombinant human follicle-stimulating hormone.

Follicle-stimulating hormone (FSH) is a heterodimeric hormone produced and secreted into the bloodstream by the adenohypophysis. FSH plays an important role in reproductive processes such as follicular maturation and ovulation. In combination with luteinizing hormone (LH), FSH regulates egg maturation and spermatogenesis. The protein is included in the group of glycoprotein hormones, which also includes luteinizing hormone (LH), chorionic gonadotropin (CG) and thyroid-stimulating hormone (TSH). The FSH dimer consists of two subunits - α (14 kD) and β (17 kD). The α subunit is common to all hormones of this group, and β is specific for each hormone, and mainly determines its binding to receptors and biological activity. As with most secreted proteins, the primary amino acid sequence of each subunit includes a signal sequence that is subsequently deleted during protein processing and secretion (Pierce JG, Parsons TF (1981), "Glycoprotein hormones: structure and function". Annu. Rev. Biochem. 50: 465-495). Both subunits are synthesized in the same cells, after which they undergo primary N-glycosylation at asparagine residues in the endoplasmic reticulum. The α subunit has 2 carbohydrate chains linked to Asn 52 and Asn 78; The β subunit has 2 carbohydrate chains linked to Asn 7 and Asn 24. Secondary modifications of the carbohydrate chains include the formation of branched structures based on sialic acids. The various forms and degrees of sialylation of the carbohydrate chains result in a number of glycoforms, creating microheterogeneity of the hormone (Fares FA et al. (1992). "Design of a long-acting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin beta subunit to the follitropin beta subunit. "Proc Natl Acad Sci USA; 89 (10): 4304-8). Glycosylated α and β subunits are bound by non-covalent bonds and are already secreted by the cell in the form of a dimer. Glycosylation plays a large role in the formation of dimers, the formation of tertiary and quaternary protein structures and in the secretion of hormone by the cell. The degree of glycosylation and, in particular, sialylation determines the biological activity of the hormone in relation to target cells and the rate of clearance (inactivation and removal of the hormone from the bloodstream) (Ulloa-Aguirre A. et al. (1995). "Follicle-stimulating isohormones: characterization and physiological relevance ". Endocr Rev .; 16 (6): 765-87). The analysis of iso-hormones of recombinant FSH revealed an in vivo dependence of bioactivity on the isoelectric point (pI). Isohormones with pI in the range from 3 to 5 are 100-200 times higher in biological activity than isohormones with pI from 5.5 to 6.0. (Mulders J.W. et al. (1997). "Prediction of the in vivo Biological Activity of Human Recombinant Follicle Stimulating Hormone Using Quantitative Isoelectric Focusing." Biologicals, 25 (3): 269-81)

In the clinic, the hormone is used to correct anovulation, as well as for assisted reproductive techniques. There are 2 classes of pharmaceutical preparations containing FSH: obtained from the urine of postmenopausal women (menotropins, urofollitropins) and obtained recombinantly. Urinary FSH preparations contain both trace amounts of LH and other proteins not associated with gonadotropins. (Bassett R. et al. (2005). "Comparative characterization of a commercial human chorionic gonadotrophin extracted from human urine with a commercial recombinant human chorionic gonadotrophin." Curr Med Res Opin .; 21 (12): 1969-76).

Recombinant human FSH was developed in the mid-90s (Howles S.M. et al. (1996). "Genetic engineering of human FSH." Hum Reprod Update; 2 (2): 172-91) and immediately gained recognition as having advantages over menotropins. It is characterized by greater purity and the absence of various impurities, which leads to high reproducibility in the production of various series of the drug (Horsman G. et al. (2000). "A biological, immunological and physico-chemical comparison of the current clinical batches of the recombinant FSH preparation Gonal -F and Puregon. "Hum Reprod .; 15 (9): 1898-902).

Two main forms of recombinant human FSH are known - Follitropin α and Follitropin β, produced and secreted into the culture medium by genetically modified ovary cells of the Chinese hamster CHO.

There is a method of creating eukriotic producers of recombinant human heterodimeric hormones, including FSH, to produce follitropin α (Chappel, SC et al. (1992). "Expression of human gonadotropins by rDNA methods." In "Humans in Gynecological Endocrinology", Parthenon Press , Carnforth, UK, 179-184; US patent US 5,240,832), based on the creation of two plasmids expressing the α and β subunits of FSH and cotransfection of these plasmids in CHO dhfr cells. The first plasmid contains the full-sized gene α (containing all exons and introns) and the dhfr gene as a marker for selection and amplification. The second plasmid contains the partial β gene. The α and β genes are cloned from the human genomic library. The expression of subunit genes is controlled by the Metallotoneonein promoter (MT), which is not the strongest for protein expression. The design of plasmids is based on the premise that introns contain enhancer elements that enhance protein expression. To obtain the product, producer cells are cultured in a microcarrier bioreactor as an adhesive culture. With this method, the level of FSH secretion by producer cells averages 1 pcg / cell per day, which corresponds to 12.6 IU / 10 ⁶ cells per day. The method has several disadvantages. It is known that introns can contain regulatory elements both positively (enhancers) and negatively (silencers) affecting the expression of the encoded protein, which can lead to a decrease or complete loss of its expression. The description of this method does not indicate how many passages the production of the target protein is preserved.

There is another way to create a producer of recombinant human FSH based on CHO cells, which is the basis for the creation of Follitropin β (Olijve W. et al. (1996). "Molecular biology and biochemistry of human recombinant follicle stimulating hormone" Molecular Human Reproduction, 2 (5) , 371-382). The α subunit gene, including some introns, and the cDNA (encoding DNA) of the β subunit are cloned into the pKMS vector based on the pBR327 vector. Gene expression is controlled by the early promoter SV40 and MT-II (metalloteonein promoter), respectively. Clones with a high copy number of FSH genes were selected by cotransfection of the auxiliary selection plasmid pAG60 / MT2 containing the metalloteonein gene providing resistance to heavy metals and further selection in the presence of Cd2 +. The productivity of the producer line when cultured in a microcarrier bioreactor is 8 IU / ml per day at a cell density of 10 ⁶ / ml or 50-80 IU / ml per day at a density of 10 ⁷ / ml, which corresponds to 5-8 IU / 10 ⁶ cells in a day. This method has the same drawbacks as the above method of creating plasmids for expression of Follitropin α - the choice of non-optimal promoters for expression of a heterologous protein, as well as the presence of possible negative regulatory elements in the present elements of the introns of FSH subunit genes. The stability of this line is confirmed by the integration of the plasmid into the genome of producer cells using in situ fluorescence hybridization, however, it is not indicated how many FSH products remain for how many passages.

Known methods for increasing the biological activity of recombinant FSH by modifying the sequences of subunits. Modifications include introducing mutations in the amino acid sequence of subunits aimed at increasing glycosylation sites (US patent applications 2008 / 0280832A and 2005/0100289 A1) or the addition of CTP (carboxy terminal peptide) β subunit of human chorotropic hormone (CG), which has additional glycosylation sites, to the β-subunit of FSH (US patents 5,538,835 and 5,585,345).

Known closest in technical solution to the claimed invention, a method of creating a producer of FSH (application for US patent 2009/0291473 A1). In this method, for the expression of FSH cDNA, FSH subunits are cloned into the pE-hygr vector. The cDNA of the α subunit is obtained by PCR from the cDNA of the human placenta library, and the cDNA of the β subunit from the cDNA of the human pituitary library. However, this method has several disadvantages: the EF-1 promoter used to induce the expression of both subunits is a weak promoter for protein expression. The productivity of this line adapted for growth in suspension in serum-free medium does not exceed 12 IU / 10 ⁶ cells per day. The main emphasis in this patent application is made on the method of purification of the final product, and not on creating the 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.

The objective of the invention is to create a stable cell line of a CHO producer of human follicle-stimulating hormone (FSH) with an increased productivity of at least 1.5 μg / 10 ⁶ cells per day (which corresponds to 18 IU / 10 ⁶ cells per day).

The problem is solved by the huFSHIK cell line, a highly productive stable producer line of the recombinant human afollicul-stimulating hormone, created on the basis of the CHO K1 DXB11 line, the genome of which contains the pcDNA / FSHα expression plasmid, consisting of a 9-bp Kozak sequence, a synthetic sequence encoding a full-length α subunit of human FSH 348 bp in length, including a signal peptide, a 3 bp stop codon, and a 5407 bp pcDNA-TOPO plasmid fragment containing the promoter en cytomegalovirus hanser (CMV), herpes simplex virus thymidine kinase polyadenylation signal (HSV TC), neomycin antibiotic resistance gene (neo), SV40 virus polyadenylation promoter and signal; pOptiVEC / FSHβ expression plasmid consisting of a 9-bp Kozak sequence, a synthetic sequence encoding the full-length β subunit of human FSH, 387 bp in length, including a signal peptide, a 3 bp stop codon, a plasmid fragment pOptiVEC-TOPO with a length of 4402 bp containing the promoter-enhancer of cytomegalovirus (CMV), the gene of resistance factor of transfected cells to methotrexate - dihydrofolate reductase (DHFR) and regulatory elements for DHFR expression - encephalomyocarditis virus IRES sequence (EMCV), which provides a bicistronic expression in animal cells, polyadenylation signal, a resistance gene to an antibiotic ampicillin and the bacterial promoter is the gene for ampicillin resistance.

The technical result of the claimed huFSHIK cell line is high FSH productivity, which is 1.5 times higher than that of a well-known producer, and stably secretes FSH into a culture medium containing no animal products for 30 passages; as well as in optimizing the cultivation of this line both in the conditions of a monolayer and suspension culture. These properties make it economically viable for widespread use in biopharmaceutical manufacturing to obtain a product using various cultivation methods. The huFSHIK line was deposited in the Russian collection of cell cultures at the Institute of Cytology RAS under the number RKKK (P) 744D.

Creation of expression plasmids.

The phenomenon of gene polymorphism can lead to slight variations in the nucleotide sequences of the same gene in different individuals. In this regard, the cloning of therapeutic genes or cDNA from genomic or cDNA libraries does not seem to be optimal. The Consensus CDS project allowed us to collect and synthesize a large amount of data on the cDNA nucleotide sequences of human genes (http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi). Amino acid sequences determined by consensus sequences of cDNA genes most accurately reflect the real amino acid sequences of proteins that can be present in the vast majority of the human population. It seems most appropriate to create recombinant therapeutic proteins based on such consensus amino acid sequences. The synthesized genes are optimally suited to this end. The small molecular weight of FSH subunits allows the synthesis of their cDNA sequences with great accuracy. Another advantage of synthetic genes is the ability to optimize codons for optimal expression of a protein in a certain type of host cell.

In the present invention, expression plasmid DNAs are created using nucleotide sequences encoding the FSH α and β subunits synthesized based on consensus amino acid sequences, taking into account codon optimization for increased expression in mammals.

1. The expression plasmid pcDNA / FSHα (Figure 1) consists of a DNA fragment containing a 9-bp Kozak sequence, a synthetic sequence encoding a full-length 348 bp human FSH α subunit, including a signal peptide, a stop codon length 3 bp, and a fragment of the plasmid pcDNA-TOPO with a length of 5407 bp containing

- regulatory elements providing FSH expression: cytomegalovirus promoter-enhancer (CMV) and herpes simplex virus thymidine kinase polyadenylation signal (HSV TC);

- the antibiotic resistance gene neomycin (neo) for the selection of stable cell lines with geneticin (G418), the promoter and the SV40 polyadenylation signal for its expression;

- the antibiotic resistance gene ampicillin and the bacterial promoter of the ampicillin resistance gene, allowing for the preparative production of plasmids in E. coli.

2. The expression plasmid pOptiVEC / FSHβ (Figure 2) consists of a DNA fragment containing a 9-bp Kozak sequence, a synthetic sequence encoding a full-length β subunit of human FSH, 387 bp, including a signal peptide, stop codon 3 bp in length, and a fragment of the pOptiVEC-TOPO plasmid 4402 bp in length containing

- regulatory elements providing FSH expression: cytomegalovirus promoter-enhancer (CMV);

- gene of resistance factor of transfected cells to the effects of methotrexate - dihydrofolate reductase (DHFR) and regulatory elements for expression of DHFR - IRES sequence of encephalomyocarditis virus (EMCV), providing bicistronic expression in animal cells, and a polyadenylation signal;

- the antibiotic resistance gene ampicillin and the bacterial promoter of the ampicillin resistance gene, allowing for the preparative production of plasmids in E. coli.

Obtaining the huFSHIK cell line expressing human FSH based on the Chinese hamster ovary cell line CHO K1 DXB11.

The starting material for creating a FSH producer is the Chinese hamster ovary cell line CHO K1 DXB11, obtained from the Russian Collection of Cell Cultures (RCSC) at the Institute of Cytology RAS (St. Petersburg, Russia). The baseline was cultured in F12 medium supplemented with 10% FBS (fetal calf serum) as an adhesion culture at 37 ° C with 5% CO ₂ . Reseeding is carried out with a 0.25% trypsin solution every 2-3 days with a sieving ratio of 1:10 or 5 × 10 ⁵ cells per bottle with a surface of 25 cm ² .

In the CHO K1 DXB11 cell line, one allele of the dhfr gene is delegated, the other mutates with loss of function. (Urlaub and Chasm 1980; Urlaub et al. 1983). Auxotrophic line for hypoxanthine or adenine, glycine, thymidine and proline.

The enzyme dehydrofolate reductase (DHFR) catalyzes the reduction of 5, 6-dihydrofolate to 5, 6, 7, 8-tetradehydrofolate, which is necessary for DNA synthesis. CHO K1 DXB11 cells do not exhibit DHFR activity and grow only in an environment supplemented with purine precursors of hypoxanthine and thymidine, until they are stably transfected with a vector containing the dhfr gene.

The CHO K1 DXB11 line is adapted for growth in DMEM with the addition of a mixture of antibiotics and antimycotics to a final concentration of: 100 units / ml penicillin, 100 μg / ml streptomycin and 250 ng / ml amphotericin B; 2 mM glutamine; 1: 100 100-fold solution of a mixture of interchangeable amino acids; HT additives (containing hypoxanthine and thymidine) to a final concentration of 100 μM hypoxanthine and 16 μM thymidine. The line is tested for the absence of DHFR (dehydrofolate reductase) activity, i.e. auxotrophy for hypoxanthine and thymidine: the exclusion of HT supplement from the medium leads to a gradual stop of cell division and subsequent cell death. The line is also checked for the absence of mycoplasma contamination by staining with fluorescent dyes DAPI and Hoechst DNA.

The expression plasmids pcDNA / FSHα and pOptiVEC / FSHβ are co-transfected into cells of the CHO K1 DXB11 line by liposomal transfection. After double selection in a selection medium of a certain chemical composition that does not contain hypoxanthine and thymidine with the addition of a selection agent, geneticin (G418, 600 μg / ml), a pool of stably transfected cells is obtained. Further amplification of expression cassettes is performed by culturing cells in the presence of increasing doses (50-100 nM) of methotrexate (MTX). The resulting MTX resistant cell pool is cloned by limiting dilution method. Clones obtained from single cells are analyzed by enzyme immunoassay. Select 5-6 clones with a FSH secretion level of 0.6-1 μg / ml (8-12 IU / ml). Of the selected clones, a clone is selected with a maximum initial productivity of at least 1 μg / ml (12 IU / ml), stable expression of the target protein for 30 passages, and the ability to adapt to the growth and production of FSH in suspension. The clone is propagated in the early passages (3-5) and stored by cryopreservation for the subsequent creation of a master bank.

Cultural properties

The cells of the selected clone are grown in DMEM medium with the addition of 1: 100 100x solution of antibiotics / antimycotics (A / A), 2 mM glutamine, 1: 100 100x solution of essential amino acids (Gibco), 10% dialyzed FBS (Sigma), 100 nM methotrexate ( MTX, Sigma) at 37.5% CO ₂ as an adhesive culture. Reseeding the monolayer is carried out with a 0.25% trypsin solution with a ratio of 1:10 every 3-4 days. The initial K1 phenotype inherent in the parent line is confirmed by the lack of growth in a proline-free medium. Confirm the absence of fungi, bacteria and mycoplasma in the culture. The production of human FSH is determined by ELISA and immunoblot methods.

FSH production is maintained at the initial level for at least 30 passages (3 months) when cultured with MTX (under selective conditions) and for at least 15 passages (1.5 months) without MTX (under non-selective conditions) ), which indicates the high stability of genomic copies of target genes. High stability allows us to conclude on the creation of a line that is potentially applicable in the pharmaceutical industry for the production of FSH dosage forms. The line is called huFSHIK.

The huFSHIK line is adapted for suspension growth in serum-free media. The productivity of the suspension culture is 1.5 μg / 10 ⁶ cells per day (18 IU / 10 ⁶ cells per day), which is 1.5 times higher than the declared productivity of the known FSH analogue producer described above.

Cryopreservation

Cells are frozen in a medium containing 90% FBS and 10% DMSO at a concentration of 2 million / ml and stored in liquid nitrogen. Defrosting by rapid thawing of the tube in a water bath at 37 ° C and subsequent dilution of the cells with 5 ml of heated medium. 24 hours after sowing, the medium is replaced with fresh. Viability after thawing is 80% by trypan blue staining.

The invention is illustrated by graphic materials:

Figure 1. Scheme of the created plasmid pcDNA / FSHα.

Figure 1 shows a schematic map of the expression vector pcDNA / FSHα.

Designations:

Ampicillin (bla) resistance gene - ampicillin resistance gene

bla promoter - bacterial promoter of ampicillin resistance gene

FSHα is a synthetic sequence encoding the α subunit of FSH

CMV Promoter is a constitutive promoter of cytomegalovirus that regulates the expression of FSHα

Kozak - ribosome binding site - Kozak sequence optimizing FSHα expression

TK polyA signal - herpes simplex virus thymidine kinase polyadenylation signal (HSV TK)

Neomycin resistance gene - the antibiotic resistance gene neomycin (neo) for the selection of stable cell lines with geneticin (G418)

SV40 early promoter, SV40 polyA signal - regulatory elements for neo gene expression

pUC origin - the region of origin of replication (origin) of plasmids of the pUC family.

Figure 2. Scheme of the created plasmid pOptiVEC / FSHβ.

Figure 2 shows a schematic map of the pcDNA / FSHβ expression vector.

Designations:

Ampicillin (bla) resistance gene - ampicillin resistance gene

bla promoter - bacterial promoter of ampicillin resistance gene

FSHβ is a synthetic sequence encoding the β subunit of FSH

CMV Promoter is a constitutive promoter of cytomegalovirus that regulates the expression of FSHβ

Kozak - ribosome binding site - Kozak sequence optimizing FSHβ expression

ECMV IRES - ribosome binding site - IRES sequence

encephalomyocarditis, which provides bicistronic expression in animal cells.

DHFR (dihydrofolate reductase) - sequence of resistance factor of transfected cells to methotrexate

TK polyA signal - polyadenylation signal

pUC origin - the region of origin of replication (origin) of plasmids of the pUC family.

Figure 3. Determination of the stability of FSH secretion by the huFSHIK line in suspension in serum-free medium without MTX by ELISA. Conducted 29 passages: the first 17 with MTX and the next 11 without MTX. The graph shows FSH production during the last 5 passages without MTX (passages 25-29) and the corresponding number of viable cells for 3-4 days of cultivation at an initial inoculum density of 3 × 10 ⁵ / ml.

Figure 4. Determination of FSH in a serum-free culture medium from the huFSHIK cell line by antibodies against the β FSH subunit by immunoblot.

The arrows indicate the position of protein markers with electrophoretic mobility in the regions of 43 and 30 kDa.

Gel tracks 1-3: culture medium from the huFSHIK line at passages 3, 15, 23 (restoring electrophoresis conditions - with the addition of β-mercaptoethanol, with heating).

Gel tracks 4-6: culture medium from the huFSHIK line at passages 3, 15, 23 (non-reducing conditions for electrophoresis - without the addition of beta-mercaptoethanol, without heating).

Gel lane 7: culture medium from the parent line CHO K1 DXB11.

The invention is illustrated by examples:

Example 1. Obtaining plasmid DNA pcDNA / FSHα encoding the α subunit of the human follicle-stimulating hormone.

The nucleotide sequence for the synthesis of the α subunit is determined based on the amino acid sequence (P01215, Gene Bank) obtained by translation of the consensus CDS α - subunit of human glycoprotein hormones (CCDS5007.1, Gene Bank) and designated as SEQ ID3 by optimizing codons for expression in mammals . The resulting nucleotide sequence is designated as SEQ ID1. The oligonucleotide synthesis is carried out on an Applied Biosystems ABI 3900 synthesizer (USA). The resulting oligonucleotide is cloned into the pAL-TA intermediate vector (Eurogen, Russia) using the TA cloning method (http://evrogen.ru/kit-user-manuals/pAL-TA.pdf). a fragment of the resulting vector containing SEQ ID1 is amplified by PCR under the following conditions:

95 ° C - 15``, 60 ° C - 15 '', 72 ° C - 45 '' for 20 cycles using 10 ng pAL-TA plasmid DNA (with target insert) as template, Tersus polymerase (Eurogen, Russia) and the following primers:

Alpha-dir: 5'-GCCGCCACCATGGACTACTATAGGAAGTACGC and

Alpha-rev: 5'-GTTAGGACTTGTGATAGTAGCAGGTGCTAC.

For optimal expression before the initiating ATG codon, the Kozak GCCGCCACC sequence (M.Kozak, 1987) is included in the direct primer. The reverse primer at the end of the sequence includes a TTA stop codon to successfully complete the translation.

The PCR product was subcloned into the TOPO sites of the pcDNA-TOPO and pOptiVEC-TOPO vectors by TA cloning.

The resulting vectors are called A31 and A04, respectively.

The correctness of the nucleotide sequence of the α FSH subunit in the final vectors A31 and A04 is confirmed by sequencing on an ABI 3730 XL automatic sequencer (USA) using a CMVf primer:

5'-CGCAAATGGGCGGTAGGCGTG-3 '

Subsequently, the A31 vector, which showed the best results (in combination with the B04 vector), in expression of the target protein (70 ng / ml) than the combination of A04 with B31 (40 ng / ml), gets the final name pcDNA / FSHα.

Plasmid DNA for cloning and subsequent transfection is amplified by transformation of E. coli XL I-Blue cells according to the protocol described on the website of Eurogen, Russia (http://evrogen.ru/kit-user-manuals/Comp_Cells.pdf). DNA for subsequent transfection is isolated and purified using the Plasmid Miniprep plasmid DNA isolation kit (Evrogen, Russia) according to the protocol of Eurogen (http://evrogen.ru/products/P-DNA/P-DNA.shtml).

The resulting expression plasmid DNA pcDNA / FSHα encodes the α subunit of human FSH and has a size of 5767 nucleotide base pairs.

Example 2. Obtaining plasmid DNA pOptiVEC / FSHR encoding the β subunit of the human follicle-stimulating hormone.

The nucleotide sequence for the synthesis of the β subunit is determined based on the amino acid sequence (P01225, Gene Bank) obtained by translation of the consensus CDS β - subunit of human FSH (CCDS5007.1, Gene Bank) and designated as SEQ ID4 by optimizing codons for expression in mammals. The resulting nucleotide sequence is designated as SEQ ID2. The oligonucleotides are synthesized using an Applied Biosystems ABI 3900 synthesizer. The obtained oligonucleotide is cloned into the pAL-TA intermediate vector (Eurogen, Russia) using the TA cloning method (http://evrogen.ru/kit-user-manuals/pAL-TA.pdf). A fragment of the resulting vector containing SEQ ID2 is amplified by PCR under the following conditions:

95 ° C - 15``, 60 ° C - 15 '', 72 ° C - 45 '' for 20 cycles using 10 ng pAL-TA plasmid DNA (with target insert) as template, Tersus polymerase and the following primers :

beta-dir 5'-GCCGCCACCATGAAAACCCTGCAGTTCTTTTTCCTG-3 '

beta-rev 5'-GTTACTCCTTCATCTCGCCGAAGCTG-3 '

For optimal expression before the initiating ATG codon, the Kozak GCCGCCACC sequence (M.Kozak, 1987) is included in the direct primer. The reverse primer at the end of the sequence includes a TTA stop codon to successfully complete the translation.

The PCR product was subcloned into the TOPO sites of the pcDNA-TOPO and pOptiVEC-TOPO vectors by TA cloning. The resulting vectors are called B31 and B04, respectively.

The correctness of the nucleotide sequence of the β FSH subunit in the final vectors B31 and B04 is confirmed by sequencing on an ABI 3730 XL automated sequencer using a CMVf primer:

5'-CGCAAATGGGCGGTAGGCGTG-3 '

Subsequently, the B04 vector, which showed the best results (in combination with the A31 vector), in the expression of the target protein (70 ng / ml) than the combination of B31 with A04 (40 ng / ml), gets the final name pOptiVEC / FSHβ.

Plasmid DNA for cloning and subsequent transfection is amplified by transformation of E. coli XL1-Blue cells according to the protocol described on the website of Eurogen, Russia (http://evrogen.ru/kit-user-manuals/Comp_Cells.pdf). DNA for subsequent transfection is isolated and purified using the Plasmid Miniprep plasmid DNA isolation kit (Evrogen, Russia) according to the protocol of Eurogen (http://evrogen.ru/products/P-DNA/P-DNA.shtml).

The resulting expression plasmid DNA pOptiVEC / FSHβ encodes the B subunit of human FSH and has a size of 4801 base pairs.

Example 3. Cotransfection of CHO K1 DXB11 cells with the genetic constructs pcDNA / FSHα and pOptiVEC / FSHβ and obtaining a pool of cells stably expressing FSH using double selection.

Before transfection begins, determine the concentration of geneticin (G418), suitable for selection, that is, at which the non-transfected cells die in 2 weeks. A concentration of 600 μg / ml is chosen as optimal. Two combinations of vectors, each of which contains one α subunit and one β subunit, are cotransfected into the DXB11 line: A31 + B04 and A04 + B31.

The day before transfection, cells were dispersed into a concentration of 10 ^5/500 .mu.l of growth medium DMEM containing 10% fetal calf serum, 2 mM glutamine, 1: 100 100X solution of non-essential amino acid mixture solution of hypoxanthine and thymidine (HT) in 24-well culture plates (additives from Invitrogen). The next day, transfected cells that reached 90% confluency were transfected with the transfection agent Lipofectamine ™ 2000 (Invitrogen). 0.4 μg of the first vector and 0.4 μg of the second vector are diluted in 100 μl of transfection medium OPTIMEM (Invitrogen); separately, 50 μl of lipofectamine is diluted in 100 μl of OPTIMEM transfection medium. Then the complexes are mixed and after 20 minutes incubation at room temperature they are added to the cells. The next day, the cells are dispersed into culture vials in DMEM growth medium supplemented with a 1: 100 100x antibiotic / antimycotic solution (Gibco). The next day (48 hours after transfection), the medium is replaced by breeding: DMEM with the addition of 10% dialyzed FBS, 1: 100 100x antibiotic / antimycotics (A / A) solution, 2 mM glutamine, 1: 100 100x solution of interchangeable amino acids, 600 μg / ml G418. The addition of HT is excluded from the selection medium. Double selection is carried out for two weeks with a change in the selection medium every 3 days. As a negative control, false transfection of cells with a vector-free transfection mixture is used. 14 days after the start of selection, negative control cells maintained in the selection medium die. Cells transfected with both combinations of vectors one week after the start of selection form many colonies, which by the 17th day will grow to a monolayer. The expression of FSH in a pool of stably transfected cells is determined by the ELISA method described in example 4.

Example 4. Determination of secreted FSH by enzyme immunoassay.

The concentration of FSH secreted into the culture medium is determined by the enzyme-linked immunosorbent assay (ELISA) using a kit company Diatech-EM (Russia). In this kit, monoclonal antibodies against the β subunit of human FSH are adsorbed on the surface of the plate wells. The test samples are added together with a conjugate of monoclonal antibodies against the α subunit of human glycoprotein hormones. Thus, only FSH heterodimers give a positive reaction. A pool of cells stably expressing a combination of A31 (pcDNA / FSHα) and B04 (pOptiVEC / FSHβ) vectors is secreted into FSH medium at a concentration of 70 ng / ml. In untransfected cells and pools with stable expression of one pcDNA / FSHα vector or one pOptiVEC / FSHβ vector, FSH is not detected.

Example 5. Gene amplification of target sequences by methotrexate.

To increase the productivity of producer cells, gene amplification of sequences encoding the FSH α and β subunits is carried out by selective cultivation of a stably FSH cell pool in the presence of increasing concentrations of MTX (50 nM, 100 nM). Each selection step is carried out for 4 weeks, and after each amplification step, cells are propagated and cryopreserved.

A pool of cells resistant to MTX at a concentration of 100 nM is obtained. Pool cells are subcloned by limiting dilution in 96-well culture plates. For further analysis, only those wells are selected in which only one colony from one cell has grown.

5-6 clones with a productivity of at least 12 IU / ml are selected. Clones are tested for FSH production stability with regular cell reseeding and product sampling every 3-4 days. The FSH secreted is measured by ELISA. Clones are selected in which the level of FSH production remains unchanged for at least 30 passages (3 months) when cultured with MTX and for at least 15 passages (1.5 months) without MTX.

Example 6. Selection of cultivation conditions and FSH production by stable clones-producers in serum-free media.

The conditions for culturing and production of FSH by stable clones in serum-free media are selected both in a monolayer culture and after adaptation of the cells to growth in suspension.

The monolayer is grown in DMEM growth medium with 10% dialyzed FBS under non-selection conditions (without MTX). Upon reaching 95% confluency, the medium is replaced by one of serum-free media: ExCell ACF (Sigma, USA), CD CHO (Sigma), CD OPTI CHO (Gibco), CDM4, SFM4, SFM4utility (Hyclone, USA) containing 15 mM HEPES and not containing MTX, and then the monolayer is incubated in tightly closed vials without air. The medium is replaced every 3 days for 2.5 months. During this period, FSH production is maintained at a level of 2-4 μg / ml (24-48 IU / ml). This level of stability of the target protein production under non-selective conditions creates the potential for the use of these crops in biopharmaceutical production in roller bottles under conditions with minimal requirements, namely in a closed system without maintaining a certain level of CO ₂ .

Stable clones also adapt to suspension growth in serum-free media. For this, the monolayer is removed with HyQTase reagent (Hyclone) and the cells are scattered at 5 × 10 ⁵ / ml in a mixture of DMEM containing 10% FBS with serum-free medium in a 1: 1 ratio. Cells are cultured in culture bottles for suspension cultures with constant stirring on a shaker at 37 ° in the conditions of 5% CO ₂ . Every 3 days, the medium is replaced by centrifuging the cells at 1500 rpm for 5 minutes and resuspending the cell pellet in fresh medium. Adaptation to growth in serum-free media is carried out by gradually reducing the percentage of DMEM containing 10% FBS from 50% to 25% and then to 12.5%, after which the cells are cultured in pure serum-free medium. Cells are considered adapted to suspension when their viability reaches 90%, and the number of cells increases from 5 × 10 ⁵ / ml on average to 2 × 10 ⁶ / ml in 3-4 days; that is, the doubling time is 36-48 hours. A clone is selected that can transfer into suspension within 6 passages with a doubling time of 36 hours and FSH production of at least 1.5 mg / 10 ⁶ cells per day, which corresponds to 18 IU / 10 ⁶ cells per day. After adaptation to the suspension, the seeding density is reduced to 3 × 10 ⁵ / ml. The stability of FSH secretion in suspension during the first 17 passages with MTX and the next 12 passages without MTX is confirmed by ELISA of the samples. The results of ELISA of samples taken in the last 5 passages without MTX (passages 25-29) are shown in Figure 3. On average, the FSH level is about 5 μg / ml for 3-4 days of cultivation, which, when converted to 10 cells per day, is 2.046 μg / ml and corresponds to about 25 IU / 10 ⁶ cells per day. Thus, a highly productive cell line is obtained that stably secretes recombinant human FSH into a culture medium that does not contain animal products, both in a monolayer, in a suspension and in suspension. The line is called huFSHIK. The high growth potential of the line both in conditions of adhesive and suspension culture makes it possible to widely use it in biopharmaceutical production to obtain a product using various cultivation methods.

Example 7. Determination of the identity of recombinant FSH immunoblot method.

The identity of FSH secreted by the huFSHIK line and quantitatively determined by ELISA is confirmed by immunoblot.

Electrophoretic separation of proteins from the culture fluid from the huFSHIK line is carried out according to the standard Laemmli technique (Laemmli U.K., 1970). To 40 μl containing 200 ng FSH according to the results of ELISA, add 5-fold Laemmli buffer. Non-reducing conditions are used to determine the heterodimeric form of FSH: the samples are diluted in Laemmli buffer without the addition of β-mercaptoethanol, and the samples are not heated. To identify the α and β subunits, reducing conditions are used: the samples are diluted in Laemmli buffer with the addition of 500 mM β-mercaptoethanol and subjected to heat treatment at 95 ° C for 5 min. Separation is carried out in 12% PAG according to the standard procedure at 60 V for protein concentration and at 100 V for their further separation. Protein transfer to the nitrocellulose membrane (0.22 μm, Millipore, USA) is carried out by the method of semi-dry transfer at a constant voltage of 20 V for 30 min. The membrane is incubated with monoclonal antibodies to the α or β subunit of human FSH (Sigma, USA), and then with secondary anti-mouse antibodies conjugated to peroxidase (Jackson ImmunoResearch Laboratories, USA). Protein detection is carried out by enhanced chemiluminescence using the ECL + system (Amersham Pharmacia Biotech, UK). As a negative control, an air-conditioned culture medium from parent CHO K1 DXB11 cells not expressing FSH is used. The results are presented in FIG. 4.

Claims (1)

The huFSHIK cell line is a highly productive stable producer line of the recombinant human follicle-stimulating hormone, created on the basis of the CHO K1 DXB11 line, the genome of which contains the pcDNA / FSHα expression plasmid, consisting of a 9-bp Kozak sequence, a synthetic sequence encoding full-length α subunit of human FSH, 348 bp long, including a signal peptide, 3 bp stop codon, pcDNA-TOPO plasmid fragment 5407 bp long, containing cytomegalovirus promoter-enhancer (CMV), poly signal adenylation of herpes simplex virus thymidine kinase (HSV TK), antibiotic resistance gene neomycin (neo), SV40 virus polyadenylation promoter and signal; pOptiVEC / FSHβ expression plasmid, consisting of a 9 kb Kozak sequence, a synthetic sequence encoding the full-length β subunit of human FSH, 387 bp long, including a signal peptide, a 3 bp stop codon, a plasmid fragment pOptiVEC-TOPO, 4402 bp long, containing the cytomegalovirus promoter-enhancer (CMV), transfected cell resistance factor gene for methotrexate dihydrofolate reductase (DHFR) and regulatory elements for expression of the encephalomyocarditis virus IRES DHFR sequence (EM CV), which provides bicistronic expression in animal cells, a polyadenylation signal, the antibiotic resistance gene ampicillin and the bacterial promoter of the ampicillin resistance gene.

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