WO2014183175A1 - Methods for production and/or purification of hormones - Google Patents
Methods for production and/or purification of hormones Download PDFInfo
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- WO2014183175A1 WO2014183175A1 PCT/BR2013/000165 BR2013000165W WO2014183175A1 WO 2014183175 A1 WO2014183175 A1 WO 2014183175A1 BR 2013000165 W BR2013000165 W BR 2013000165W WO 2014183175 A1 WO2014183175 A1 WO 2014183175A1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
Definitions
- the present invention refers to methods for production and / or purification of hormones, particularly the follicle stimulating hormone (FSH) , from human or animal origin, through a human cells platform, as well as products thus obtained and their uses.
- hormones particularly the follicle stimulating hormone (FSH)
- FSH follicle stimulating hormone
- Hormones are involved in various stages of the mammal reproduction, in particular, the gonadotropins follicle stimulating hormone (FSH) , luteinizing hormone (LH) and chorionic gonadotropin (CG) , as well as thyrotropin or thyroid stimulating hormone (TSH) .
- FSH gonadotropins follicle stimulating hormone
- LH luteinizing hormone
- CG chorionic gonadotropin
- TSH thyroid stimulating hormone
- FSH, LH and TSH are pituitary hormones, whereas the CG is produced during the formation of the placenta.
- the hormones have been produced by recombinant technology or from mammalian urine, the critical factor being the high degree of purity.
- FSH is used in female human patients in ovulation induction and controlled ovarian hyperstimulation for assisted reproduction (e.g. assisted reproductive technologies, intra uterine insemination, in vitro fertilization and intracytoplasmatic sperm injection) . It is also used in male human patients suffering from oligospermia in order to induce and maintain spermatogenesis. FSH treatment is based on successive injections of the hormone, and requires a high purity and specific activity product.
- hFSH Human FSH
- the molecule is active as a non-covalently linked heterodimer containing alpha and beta subunits.
- the hFSH alpha subunit is homologous to the alpha subunits of several other glycoprotein hormones (LH, TSH and hCG) and contains 92 amino acids.
- the beta subunit is specific and contains 111 aminoacids.
- isoforms of hFSH molecule which differ among themselves depending on the molecule glycosylation .
- Non-human animal FSH is used to induce super- ovulation in female donors and to induce follicular growth during in vitro fertilization programs in anestrous females .
- the FSH currently used in the animal reproduction programs is purified from porcine pituitary extracts. This product is prepared by a technique, which affords a cheaper, but less effective, hormonal preparation. Additionally, commercial preparations depend on the availability of a sufficient number of fresh pituitaries for the preparation of each batch, leading to great variability in results during superovulation treatments with different purified product batches.
- the bovine hormone FSH (bFSH) is composed of two glycoprotein subunits encoded by genes CGA (a chain, common to all pituitary hormones: FSH, LH and TSH) and bFSH ( ⁇ chain, specific for this hormone), wherein CGA ' has 363 base pairs and bFSH has 389 base pairs.
- the recombinant bFSH has also been expressed in various recombinant systems, such as: yeast, plants, insect cells, mouse epithelioid cells, hamster ovary cells, transgenic mice milk and transgenic rabbits.
- yeast, plants, insect cells, mouse epithelioid cells, hamster ovary cells, transgenic mice milk and transgenic rabbits only the bFSH expressed in mammalian cells showed in vivo biological activity. This is explained by the fact that the bFSH hormone is very complex, composed of two polypeptide chains, each of which containing post-translational modifications, i.e. glycosylation .
- U.S. 5,990,288, filed by Musick et al. discloses a method for purifying FSH from biological samples, such as human pituitary glands or human post-menopausal urine, using cation exchange chromatography on Fractogel EMD S03- 650M, followed by dye affinity in a Mimetic Orange 1 resin and hydrophobic interaction in Bakerbond Wide Pore HI- Propyl resin.
- Figure 1 is a flowchart showing the method for producing the recombinant bovine FSH (rbFSH) , from construction of the recombinant vectors by genetic engineering up to the tests of biological activity in vitro and in vivo, according to example 2.
- rbFSH bovine FSH
- Figure 2 shows the fractionation of the fragments corresponding to the bFSH a and ⁇ chains and of the pCXN2 vector on 1.5% agarose gel stained with ethydium bromide.
- M molecular weight markers
- Figure 3 shows the fractionation on 1.5% agarose gel stained with ethydium bromide of the plasmid preparations containing the fragments of interest (both subunits of bFSH) , digested with restriction enzyme, along with the same undigested constructions, where M represents the molecular weight markers.
- Figure 4 illustrates the transfection of both bFSH chains in a mammalian cell for their expression.
- Figure 5 shows the autoradiography of the Western blot corresponding to rbFSH samples.
- Figure 6 shows the in vivo biological activity assay .
- Figure 7 shows the general flowchart of the purification method for human and bovine recombinant hormones (steps according to the present invention) .
- Figure 8 shows the chromatographic profile of the FSH purification procedure observed at the different chromatographic steps.
- Figure 9 shows a Western Blot, wherein: 1) rhFSH sample after anion exchange chromatography; 2) rhFSH sample after gel filtration.
- Figure 10 shows the method for identification and quantification of FSH using HPLC.
- Figure 11 shows representative chromatograms of the method for identification and quantification of FSH hormone in cell culture medium (DMEM) .
- Figure 12 shows the identification and quantification of progesterone performed by HPLC.
- Figure 13 shows FSH in vivo activity assay.
- Figure 14 shows the biological activity of FSH in bovines.
- the graph correlates the number of ovarian follicles of 8 or more mm diameterover time after treatment of the cows with the commercial product (Folltropin) or the rbFSH.
- Figure 15 shows SDS-PAGE analysis of rhFSH levels in culture supernatant-conditioned media.
- Conditioned culture medium obtained from 3D and 6E cell clones upon culturing for 24h were subjected to SDS-PAGE gel electrophoresis under reducing conditions. After the electrophoretic run, the proteins were stained by Comasssie brilliant blue.
- Figure 16 shows Western Blot analysis of rhFSH levels in culture supernatant-conditioned media.
- Conditioned culture medium obtained from the parental cell line and cell clones upon culturing for 24h were prepared, resolved by SDS-PAGE, electrophoretically transferred to nitrocellulose membrane and immunostained using an antibody to human FSH ⁇ subunit.
- Figure 17 shows the cell growth and recombinant protein (rhFSH) production under high and low serum concentration conditions.
- Cell growth kinetics was quantified in terms of population doubling time.
- rhFSH production was determined throughout the growth curve by quantification of hFSH present in the conditioned medium using the chemioluminescence assay.
- Figure 18 shows the production of rhFSH under both serum-containing and serum-free cell culture media. rhFSH production was determined in cell-conditioned media upon culturing for 24, 48 and 72h and quantified using the chemioluminescence assay.
- Figure 19 shows the increment in progesterone production by rFSHR-17 cells upon stimulation with rhFSH compared to nonstimulated cultures.
- rFSHR-17 cells were stimulated with the culture supernatants obtained from the recombinant clones upon culturing for 24h. Progesterone released to the culture medium was measured by the chemiluminescence method.
- Figure 20 shows the dose-effect relationship of recombinant human follicle stimulating hormone (rhFSH) upon rat ovary weight.
- Figure 21 shows progesterone production, in ng per mL, by the GFSHR17 cell line in response to rbFSH stimulus, measured by chemiluminescence.
- 293T conditioned medium from non-transfected 293T cells
- 3A negative control (empty pCXN2 vector without insert)
- 6E through 6K 293T/rbFSH cell clones
- cell pop (mix) conditioned medium of the transfected 293T culture before cell clone isolation. Experiments performed in triplicates.
- Figure 22 shows Western Blot analysis of rbFSH present in culture supernatant-conditioned media and in a partially purified preparation.
- 48h conditioned medium from 293T/rbFSH 6E, partially purified conditioned medium from the same clone, commercial purified porcine FSH ( Folltropin-V®) and conditioned culture medium obtained from the 293T/negative control (3A) were prepared, resolved under reducing 12% SDS-PAGE, electrophoretically transferred to nitrocellulose membrane, incubated with polyclonal antibody to bFSH and chemiluminescently detected using radiographic film.
- Figure 23 shows the effect of recombinant bovine follicle stimulating hormone (rbFSH) in rat ovary weight augmentation .
- rbFSH bovine follicle stimulating hormone
- Recombinant proteins generally exhibit maximum biosafety, better performance in superovulation and no interference with other hormones and/or contaminants due to the purity of the preparation, being, therefore, of great interest for assisted reproduction.
- gonadotropins The recombinant technology proved to be feasible to generate proteins with different complexity degrees in a reproducible manner.
- the structure of gonadotropins is difficult to reproduce because, besides the presence of the already mentioned post-translational modifications, the gonadotropins are composed of two chains, whose coding sequences are independently transcribed and translated, and their products are subsequently combined.
- the advantage of the recombinant protein expression system in mammalian cells is the possibility to add the complex glycosidic to the newly synthesized chains, which is absolutely essential for the correct tertiary structure of each subunit, being a prerequisite for folding with the counter-subunit .
- This method facilitates the formation of disulfide bridges, which is a critical factor in the folding and maturation of functional subunits of the hormone.
- glycosylation directly affects the half-life of the molecule in the circulation and recognition of the molecule by the receptor, as well as hormone exposure to the in vivo regulatory mechanisms, interference in its solubility, and protection against proteases and thermal inactivation . Therefore, sugar addition causes great impact on the biological properties of glycoprotein hormones.
- glycosylation pattern types and levels of enzymes responsible for addition of glycans: the glycosyltransferases
- the glycosyltransferases available in each specific type of cell, which may generate specific glycoforms according to their origin.
- different cell lines containing different kinds of glycosyltransferases will catalyze the same protein glycosylation in a cell- specific manner.
- the HEK cells family which includes 293T, as used in the method according to the present invention, exhibits great transfection capability, allowing a greater influx of copies of the vector constructs of interest in cells.
- the system of expression in cell lines of human embryonic kidney (HEK) cells or derived cells (293, 293T) is capable of producing glycoproteins with N-glycans complexes, which are, in their great majority, biantennaries , relatively homogeneous and partially marked, with approximately 60% of the molecules carrying terminal sialic acid residues.
- hormones include, without limitation, follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (CG), thyroid stimulating hormone (TSH) .
- FSH follicle stimulating hormone
- LH luteinizing hormone
- CG chorionic gonadotropin
- TSH thyroid stimulating hormone
- the hormone is FSH.
- HEK-derived cells such as 293T
- the production strategy using HEK-derived cells, such as 293T may be used for expression at high expression levels of any other homo- or multimeric proteins having complex post-translational modifications.
- the mammalian species include human, bovine, sheep, pig, goat, buffalo, horse, monkey, either male or female.
- the preferred species are human and bovine.
- the present invention relates to a method for production and / or purification of hormones comprising the following steps:
- the vector used in step 4 is a plasmid for expression of the protein chains in mammalian cells, preferably pCXN2.
- plasmids may be used, provided they comprise the same elements of the pCXN2, i.e. one or more strong promoters, multiple cloning sites, antibiotic resistance sequence for bacterial cloning and recombination sites.
- transfection The way to insert plasmids in the cell ( transfection) may be carried out by various systems, such as lipofection, cationic molecules, calcium mediated transfection, polyethylenimine electroporation, since the transfection rate is maintained at the same level.
- the proportion of antibiotic resistance plasmids versus the ones used in co-transfection strategy varies from 1:1 to 1:5,000.
- the concentration of antibiotic in culture medium (such as hygromycin) used for selection of stable cell clones varies from lOug/mL to 2.000ug/mL.
- the cell line used for expression of the product may be replaced by any other derived from HEK 293 cells, since such a cell adds similar post-translational modifications and has the same behavior when cultured in vitro (similar replication capacity, protein expression and transfection abilities, etc.).
- the cell may be grown in batches in monolayers or in suspension in different containers, such as culture bottles of various sizes , plates, T flasks, spinners, culture bags, bioreactors, etc.
- the culture medium may be any one which complies with the cell metabolic needs, for instance, adult or fetal serum from several species.
- the temperature of cultivation varies from 30 to
- the rotation when the cultivation is carried out in spinners, bags and bioreactors, varies from 10 to 200rpm.
- the relative proportion of C0 2 used for the cell culture ranges from 1 to 10%.
- the cultivation pH varies from 5.0 to 7.5.
- the recombinant hormone obtained according to the present invention has high purity, for instance, rbFSH is free from LH, when compared with the commercial preparations for bovine species that includes about 15% LH .
- the present invention refers to a method for purification of recombinant hormones comprising subjecting the culture supernatant containing recombinant hormone to the following steps:
- Dye affinity chromatography (Blue Sepharose) : the hormone does not bind to the matrix, which allows cleaning the original sample by removing contaminants that have affinity for the resin.
- Steps a) and b) may be carried out in any sequence without compromising the result since the buffer exchange is appropriate for the subsequent chromatography.
- the recombinant hormone containing supernatant may be previously prepared before the purification step.
- the starting material obtained from the method for production described above is optionally clarified and/or concentrated (by ultrafiltration) and / or subjected to buffer exchange by diafiltration or chromatography before the first chromatographic step.
- the buffer exchange and / or concentration are carried out by tangential filtration in hollow-fiber membrane using 20 mM sodium phosphate buffer pH 7.0.
- the buffer exchange is performed by gel filtration using G25 Sepharose resin (for instance, as specified below) and the same buffer as mentioned above.
- the affinity chromatography of step a) is carried out in a resin having a dye as the immobilized ligand - Cibacron Blue F3G-A.
- the dye affinity chromatography is performed using as the binding buffer 20 mM sodium phosphate pH 7.0.
- the sample elution is then performed by applying a gradient between 20 mM sodium phosphate buffer pH 7.0 and 20mM sodium phosphate buffer pH 7.0 + 2 M NaCl, optionally in the presence of anti-oxidant .
- the fraction which does not interact with the resin is separated and used in the following step, for instance, as in Table II below .
- the buffer exchange and concentration may also be performed between steps a) and b) .
- fractions of interest are subjected to tangential filtration in hollow-fiber membrane (5kDa) for buffer exchange and / or concentration in ammonium acetate buffer 0.16 M pH 7.0.
- Ion exchange chromatography is carried out using DEAE Sepharose FF, for instance, as shown in Table III. Binding to the resin occurs in ammonium acetate buffer 0.16 M pH 7.0 and elution occurs by applying a gradient of ammonium acetate buffer and 0.16 M ammonium acetate buffer 0.16 M + 2.0 NaCl pH 7.0.
- the concentration of the product after the last stage of purification may be performed by 5 or lOkDa hollow fiber membrane ultrafiltration, preferably 5kDa membrane.
- Centricon may be used to concentrate the final product.
- the purified recombinant hormone may be frozen or lyophilized after concentration for storage. Alternatively, it may be formulated to provide "ready for use" preparations. For instance, such preparations include m- cresol solution or benzyl alcohol.
- the present invention also refers to purified or non-purified recombinant hormones, their use in infertility treatment or as a fertility enhancer for mammalian species, diagnostic . ki. s , pharmaceutical or veterinary compositions or cell culture supplements .
- Superscript ® II RT Invitrogen
- PCR amplification of the hFSH a and ⁇ subunits cDNAs was achieved using 25 ⁇ 1, reactions containing 1* PCR Buffer Promega ® , 1.5mM gCl 2 , 0.2 mM dNTPs, 0.4 ⁇ oligos and 0.5U of Taq DNA polymerase.
- PCR products for these two cDNAs (567 and 432 bp, respectively) were gel-purified and blunt-end-cloned into the Smal site of pUC18, using the SureClone ® PCR Cloning Kit (GE Healthcare), according to the manufacturer's protocol.
- Recombinant clones (pUC18-ahFSH and pUC18- hFSH) were screened by colony PCR and checked by restriction digestion and automated DNA sequencing. Recombinant clones, in the correct orientation, were screened and confirmed using the aforementioned basic molecular cloning techniques.
- the mutagenic primers used for a-hFSH cDNA re- amplification were AF ( 5 ' -CCCAGAGAAATTACCGCCAT-3 ' ) and AR (5' -TGTCGACTCATCAAGACAGCA-3 ' )
- the oligos used were BF ( 5 ' -GTTTTCAAGTGACCGCCATG-3 ' ) and BR (5' -GGCCTGAAATGTCGACTGAT-3 ' ) .
- Kozak consensus sequences are highlighted in bold, and Sail restriction sites are underlined.
- PCR products were blunt-end-cloned into the pUC18 vector using the SureClone ® PCR Cloning Kit (GE Healthcare) , and the resulting recombinant clones were screened by colony PCR followed by confirmation by restriction digestion and automatic DNA sequencing, to yield the pUC18-a (Ko) hFSH and pUC18-p (Ko) hFSH vectors.
- the expression-ready cDNA inserts for a and ⁇ hFSH were released from the pUC18-a ( Ko ) hFSH and pUC18-p (Ko) hFSH vectors by digestion with Sail, followed by gel purification and then subcloned into the Xhol site of the pCXN2 mammalian expression vector.
- the pCXN2 expression vector was derived from the pCXN vector (Niwa et al . , 1991) containing the neo gene, which confers resistance to Geneticin (G418), and a strong promoter gene from cytomegalovirus .
- a and ⁇ FSH expression vector (pCXN2-ahFSH and pCXN2-phFSH) were co-transfected with the pX343 vector, derived from pY3 vector containing the bacterial hygromicin B resistance gene, using a higher proportion of the former relative to the latter, into a well characterized mammalian cell line, the human embryonic kidney cells transformed by the wild type T antigen of SV40 (293T cell line, ATCC, CRL1591) (DuBridge et al., 1987; Pear et al., 1993), employing the Lipofectamine Plus Reagent (Invitrogen, Carlsbad, CA) .
- Bacterial endotoxins were assayed by the gel-clot LAL assay (GeneScript USA, Piscataway, New Jersey) ) performed by mixing equal volumes of a test sample and lysate with a known sensitivity in a test tube.
- 293T cell transfectant (6E cell line) was chosen on the basis of its higher levels of rhFSH production. This cell line was used to establish the Master Cell Bank (MCB) , consisting of individual vials containing samples of the same culture of identical cell preparations of the 6E cell clone, which were cryopreserved in liquid nitrogen.
- MBC Master Cell Bank
- a Working Cell Bank was then established by expansion of the cells recovered from a single vial of the MCB. The cells were successively expanded and aliquots were placed into vials and cryopreserved. Cells from one or more vials are cultured for each rhFSH production cycle.
- the r-hFSH preparations were reduced with ⁇ - mercaptoethanol, denatured by boiling in the presence of SDS and applied onto a 12% polyacrylamide gel. Following fractionation, the proteins were visualized by Coomassie brilliant blue staining and transferred to nitrocellulose membranes using standard techniques. The membrane was blocked in 5% BSA for 2h and then incubated overnight in a 1:6250 dilution of an anti-human FSH ⁇ subunit monoclonal antibody. The membrane was subsequently washed three times and then incubated in 1:1,000 dilution of a peroxidase- conjugated polyclonal antibody to the mouse Ig (Vector Laboratories) for Ih.
- the bands were visualized upon incubation with a chemiluminescent detection reagent (GE Biosciences) and exposed to X-Ray film.
- a chemiluminescent detection reagent GE Biosciences
- the a and ⁇ rhFSH subunits co-migrated under reducing and denaturing conditions, appearing as a single broad band on both Comassie brilliant blue and immunostained gels.
- Cell growth kinetics was assessed by automated count (Celm, Sao Paulo, SP, BR) , and quantified in terms of population doubling time.
- cells from conditions 3 (3D cell line) and 6 (6E cell line) were cultured in 6 wells plates (5 x 10 4 cells per well) , in growth medium (DMEM plus 5% FCS or DMEM plus 0.5% FCS), for up to 9 days.
- rhFSH production was determined throughout the growth curve by quantification of hFSH in the conditioned medium using the chemioluminescence assay.
- rhFSH The biological activity of the rhFSH was measured using the rat preovulatory follicle cell line (rFSHR-17), overexpressing the rat FSH receptor, which responds to human FSH stimulus by progesterone production.
- rFSH-17 cells were cultured in DMEM/F12 medium containing 5% fetal calf serum (FCS) to 80% of confluence and then stimulated with the culture supernatants obtained from the recombinant clones upon culturing for 24h at 37°C. Progesterone released to the culture medium was measured by the chemiluminescence method .
- the potency of the rhFSH preparations was assessed by determining the rat ovarian weight gain in response to FSH administration, according to the conventional Steelman-Pohley assay. To this end, 45 female Sprague-Dowley rats, aged 22 days were housed (three to five rats per cage) and given standard ad libitum food and water administration. Animals were randomized and divided into 8 experimental groups. Each animal received one daily rhFSH and hCG injection over a period of 3 days. On day 4, the animals were sacrificed and the ovaries were removed and dissected free from the surrounding tissue, and weighed.
- the reference FSH standard was the Folltropin a (Gonal-F®, Serono International S.A. Geneva, Switzerland).
- RNA source for cloning of the cDNAs encoding the human FSH a and ⁇ polypeptide chains was isolated from a pituitary tumor surgical specimen, which overexpressed FSH.
- PCR amplification of the hFSH a and ⁇ subunits cDNAs was achieved as described in Material and Methods.
- the PCR products for these two cDNAs (567 and 432 bp, respectively) were cloned into the pUC18 resulting in recombinant clones (pUC18-cchFSH and pUC18-phFSH) .
- the inserts obtained from the pUC18-ahFSH and pUC18- phFSH vectors were re-amplified using mutagenic primers to incorporate both the Kozak consensus sequence (ACCGCC) juxtaposed to the initial ATG codon as well as a Sail restriction site at the 3' -end of each cDNA.
- PCR products were cloned into the pUC18 vector to yield the pUC18-a(Ko) hFSH and ⁇ 18- ⁇ (Ko) hFSH vectors.
- expression-ready cDNA inserts for a and ⁇ hFSH were released from the pUC18 vectors and then subcloned into the pCXN2 mammalian expression vector, which was chosen for high expression of the subunits mRNAs .
- rhFSH was produced by genetically engineered mammalian cells, into which the genes coding for the a and ⁇ FSH subunits were inserted.
- the 293T human embryonic kidney cell line was selected as recipient cell, since these cells are easily transfected with foreign DNA, and are capable of synthesizing and secreting glycoproteins. Furthermore, they may be culturds in a large scale.
- Control and rhFSH producing cell clones (shown in bold in Table VIII) were selected for protein production characterization and for Master (MCB) and Working (WCB) Cell Bank establishment to allow continuous and reproducible production of FSH .
- the cell productivity, cellular morphology and cell growth kinetics of the MCB and WCB were found to be similar (data not shown) .
- the in vitro activity of rhFSH was evaluated using a rat granulose cell bioassay.
- This assay measures bioactive FSH by assessing the induced production of progesterone by rat granulose cells (FSHR-17 cell line) .
- Preparations from the 6E cell clone conditioned medium increased progesterone production by FSHR-17 cells in a dose-dependent manner ( Figure 19).
- the in vivo activity of rhFSH was determined by the Steelman Pohley assay. The behavior of the biological activity of the FSH preparations was verified by checking the dose effect relationships.
- the dose regimens administered are summarized in Figure 20.
- FSH preparations were diluted in phosphate-buffered saline to final concentrations of 1, 2, and 3 IU FSH/ml .
- Samples were each administered to groups of rats given s.c. injections of 0.5ml /rat daily, for. 3 consecutive days, yielding final cumulative doses of 1.5, 3, an 6 IU FSH/rat.
- a total dose of 60IU human chorionic gonadotropin (HCG) was also administered to each animal. Animals were sacrificed 72h after the first administration, the ovaries were then removed, dissected free of surrounding tissue and weighed. The cumulated mean results of two independent experiments are reported.
- Figure 1 shows a flowchart of the complete method .
- RNAlater ® solution (Ambion, Applied Biosystems, Carlsbad, CA) , subsequently frozen in liquid nitrogen and manually masserated using a crucible and pistil, under liquid nitrogen.
- Total pituitary RNA was extracted from the crude pituitary material by the cesium chloride method.
- the quality of the RNA preparation was evaluated by Absorbance (A) at 260nm and 2 80nm (Hitachi High-Technologies UV/visible spectrophotometer, U2000), and only the RNA preparations displaying A 2 60nmA3 ⁇ 480nm ratio above 1.8 were used for RT- PCR.
- RT-PCR Reverse-transcription-polymerase chain reaction
- RNA sample (500ng) of the total pituitary RNA was reverse-transcribed using the SuperscriptTM II Reverse Transcriptase ( Invitrogen ⁇ , Carlsbad, CA) enzyme and 125ng of oligo(dT)18 primer (Fermentas Life Science, Burlington, Ontario) in a 20uL reaction, according to the manufacturer's protocol.
- SuperscriptTM II Reverse Transcriptase Invitrogen ⁇ , Carlsbad, CA
- oligo(dT)18 primer Fermentas Life Science, Burlington, Ontario
- Both Forward primers contain: 1) a restriction site spacer (italic); 2) a Sail restriction site (underlined); 3) the Kozak consensus enhancer sequence (bold); and 4) the 5 ' -end sequence encoding for bFSHa or bFSHp.
- Both the reverse primers contain: 1) a restriction site spacer (italic); 2) a Sail restriction site (underlined); and 3) the reverse-complement of the 3 ' -end sequence encoding for bFSHa or bFSHp.
- the oligonucleotide primers were designed basing on the NCBI posted sequences regions corresponding to the bovine FSH a and ⁇ chains.
- the amplified products were subjected to agarose gel electrophoresis and the corresponding bands were purified using the QIAquick ® Gel Extraction Kit (QIAGEN) and restriction enzyme digested with Sail (Fermentas Life Sciences - Thermo Fisher Scientific Inc.) at 37 °C for 2h and 30min and inactivated at 65 °C for 20min before cloning into the mammalian expression vector.
- QIAquick ® Gel Extraction Kit QIAGEN
- Sail Fermentas Life Sciences - Thermo Fisher Scientific Inc.
- pCXN2 mammalian expression vector derived from the pCXN plasmid.
- This vector is composed of a strong Cytomegalovirus and beta-actin derived promoter, which allows efficient selection - conferred by the neomycin phosphotransferase-II encoding gene - of high vector copy number cell transfectants, which are consequently able of express high levels of foreign proteins.
- the nucleotide sequences of the cloned rbFSH a and ⁇ constructs were determined using the fluorescent dye termination reaction (BigDye ® Terminator v3.1 Cycle Sequencing Kit - Applied BiosystemsTM) and analyzed using an automated DNA sequencer (ABI Prism 3700 DNA Analyzer, Applied Biosystems/Hitashi ) .
- the chromatograms were assembled and transformed into contigs using the Phred/Phrap software, by comparison with the NCBI posted sequence for all isoforms.
- the restriction sites, as well as the Kozak fragment, were carefully checked and all sequences proved to be correct.
- Plasmid mini-scale (GFX miniprep Kit, GE Healthcare, Pittsburgh, PA) preparations were obtained according to the manufacture's protocol and purified plasmids were subsequently used for cellular transfection .
- Figure 3 shows the fractionation on 1.5% agarose gel stained with ethydium bromide plasmid of the preparations containing the fragments of interest (both subunits bFSH) , digested with restriction enzyme, and the same undigested constructions, where M represents the molecular weight marker.
- 293T/17 cells the well characterized human embryonic kidney cells transformed by the SV40 wild type T antigen were purchased from the American Type Culture Collection (ATCCTM - ATCC® Number: CRL-11268TM) and cultured in Dulbecco's Modified Eagle's (DME) medium supplemented with 10 % bovine calf serum (HyClone, Thermo Scientific) under a 2.5% C0 2 atmosphere, at 37 °C, in adherent culture. For the serum free media culture, cells were maintained in HyQ ® SFM4HEK293TM medium (HyClone, Thermo Scientific) .
- DME Dulbecco's Modified Eagle's
- 293T cells were co-transfected with .. each of the _ pCXN2-rb.FSH. constructs _and- the pX343 Hygromycin B resistance vector (figure 4), using a 40:1 DNA mass ratio (8 iq pCXN2-rbFSHa plus 8 iq pCXN2- rbFSHP to 200 ng pX343) .
- the experimental control was performed under the same conditions, but using the empty pCXN2 vector without any insert.
- the transfection procedure was carried out using LipofectamineTM 2000 Reagent (Invitrogen ® ), according to the manufacturer's protocol.
- the cell population of each condition were plated in five different dilutions (1:5, 1:10, 1:25, 1:50 and 1:100) and submitted to selection by adding lOOug/mL of the Hygromicin B selective antibiotic (Invitrogen, Carlsbad, CA) into the culture medium. This selective medium was renewed every 48h until the emergence of recombinant colonies, which occurred in approximately two weeks. Recombinant cell colonies of each condition were isolated from the remaining population by using cloning rings and transferred to 48-well plates for cell clones establishment .
- the cell clones were cultured in adherent culture and samples of the conditioned media were collected to evaluate the levels of rbFSH expression. To this end, 10 6 cells of each of the Hygro- resistant cellular clones, as well as the negative control, were plated and samples of the conditioned supernatants were collected after culturing for 48h. These supernatant samples were subsequently submitted to bFSH quantification by radioimmunoassay.
- the biological activity of the rbFSH was assayed using the rat pre-ovulatory follicular cell line (GFSHR- 17), which overexpresses the rat FSH receptor, responding to FSH stimulus by increasing the levels of progesterone production, in a dose-dependent manner.
- GFSHR- 17 rat pre-ovulatory follicular cell line
- Cell culture supernatants obtained from chosen cell clones from both control and experimental conditions were used for the in vitro characterization assays.
- GFSH-17 cells were cultured to 80% confluence in DMEM/F12 medium containing 5% FCS, plated onto a 24-weel plate and subjected to in vitro stimuli.
- the stimulatory treatments were performed in serum-free media supplemented with 10% of the conditioned supernatants obtained from 10 s cells of each recombinant or control clone, upon 24h of culturing under the described conditions. Progesterone released to the culture medium was assessed by chemilumine.scence . Protein Analysis by Western Blot
- the rbFSH supernatants of selected clones were collected and concentrated using the Centricon ® filter concentrators (10 kDa cutoff) (Millipore, Billerica, MA) . Concentrated samples were diluted to lx SDS-PAGE reducing sample buffer (60 mM Tris-Cl pH 6.8; 2.0% SDS; 10% glycerol; 0.025% bromophenol blue; 700 mM ⁇ - mercaptoethanol) and boiled for 5min at 95 °C.
- the membrane was subsequently washed three times in PBSA plus 0.01% (v/v) Tween 20 and then incubated in a 1:2000 dilution of a peroxidase-conjugated polyclonal antibody to goat IgG (Vector Laboratories) for lh.
- the bands were visualized upon incubation with the ECL chemiluminescent detection reagent (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and exposed to autoradiography film (Kodak, Rochester, NY) .
- Figure 5 shows the autoradiograph of the Western blot corresponding to rbFSH samples, wherein:
- rbFSH recombinant bovine FSH produced according to the present invention
- the numbers below the keys show the amount (micrograms - ug) applied to each channel of the gel.
- the whitish bands refer to the high consumption of the substrate by the antibody conjugated peroxidase enzyme, demonstrating the high amount of protein in the gel.
- On this autoradiograph one may verify the presence of bFSH dimer ( ⁇ 34kDa) , oligomers ( ⁇ 68kDa) and the beta subunit ( ⁇ 22kDa) .
- the rbFSH physiological activity present in samples of cell clones culture supernatants was assessed by determining the rat ovarian weight gain of sexually immature rats in response to three consecutive exogenous FSH doses, according to the Pharmacopeia FSH assay.
- Sprague-Dawley female rats, aged 22 days, obtained from the University of Sao Paulo Chemistry Institute Animal facility were housed (three to five rats per cage) and submitted to ad libitum standard food and water administration. Animals were randomized and distributed into the different groups. Each animal received one daily rhFSH plus hCG injection over a period of three days.
- the reference FSH preparation used as an experimental control was the commercial porcine FSH preparation, Folltropin ® -V (Bioniche Animal Health, Belleville, Ontario, Canada) . This experiment was performed in biological triplicates and two independent experimental replicates .
- Figure 6 shows the in vivo biological activity assay results.
- the first two pairs of ovaries refer to negative controls of the experiment: the animals received saline (PBSA) or hCG (human chorionic gonadotropin, 10UI - hCG) used to synergize the FSH effect.
- PBSA saline
- hCG human chorionic gonadotropin
- 10UI - hCG human chorionic gonadotropin
- the treatments were independently carried out: Folltropin (commercial product) , the rbFSH over producer recombinant cell clone grown in culture medium without fetal bovine serum (FBS 1), the same culture medium after a previous purification step 1, the rbFSH over producer recombinant cell clone grown in culture medium without FBS 2, the same culture medium after a previous purification 2.
- FBS 1 fetal bovine serum
- FBS 1 fetal bovine serum
- the rbFSH over producer recombinant cell clone grown in culture medium without FBS 2 the same culture medium after a previous purification 2.
- the size of the ovaries decreases despite increasing doses of rbFSH applied in treatment, which results from a negative feedback due to excessively applied high dosage.
- the highest overexpressing cell clone was adapted to suspension culture in non-adherent T-flasks by replacing the medium for HyQ®SF 4HEK293TM either directly (100% fresh suspension culture medium) or sequentially (50% fresh suspension culture medium, 50% conditioned adherent culture medium) each 3-4 days. Viability was checked by treating cell samples with 50% v/v Trypan Blue (Gibco, Life Technologies, Carlsbad, CA) , with cells being considered totally adapted to suspension culture as soon as they reached 95% of viability in 100% of HyQ®SFM HEK293TM . After adaptation, conditioned medium was collected and submitted to the previously described biological assays.
- the cDNA obtained from a bovine pituitary gland was used for amplification of the cDNA sequences encoding the bovine FSH a and ⁇ chains.
- the one-round amplification and mutation of both chains was performed as described in Experimental Procedures, and the PCR products were fractionated by agarose gel electrophoresis, in which the 389bp and 416bp amplicons, respectively, bearing the Sail restriction site, the digestion spacers and the Kozak fragment were clearly identified.
- These PCR products were gel purified and subsequently digested with Sail (Fermentas, Burlington, Ontario, Canada) before cloning into the Xhol site of the pCXN2 vector, as previously described.
- Sail Fermentas, Burlington, Ontario, Canada
- rbFSH expression of rbFSH in human 293T cells
- the recombinant plasmids bearing both bFSH chains (pCXN2-abFSH and pCXN2-pbFSH) were co-transfected along with the Hygromicin-resistance pX343 plasmid in a 40:1 ratio - into previously plated 293T cells. 24h after transfection, the experimental and control cell populations were submitted to selection in the presence of Hygromicin B. until the emergence of cellular colonies, which were isolated as described in Material and Methods.
- FSH preparations were diluted in phosphate- buffered saline and different doses (0.05; 0.1 and 0.2mg) were injected. Samples were administered, subcutaneously, to groups of five rats in injections of 0.2mL/rat daily, for three consecutive days. A total dose of 10IU human chorionic gonadotropin (hCG) was also administered to each animal, except for the negative control, at day 1, according to the Pharmacopeia. Animals were sacrificed 72h after the first administration; their ovaries were removed, dissected free from surrounding tissue and weighed. The cumulated mean results of three independent experiments are shown.
- hCG human chorionic gonadotropin
- PBSA phosphate-buffered saline
- 2:10 IU of hCG human chorionic gonadotropin
- 3-5 10 IU of hCG plus Folltropin (0.05, 0.1 or 0.2mg, consecutively)
- 6-8 10 IU of hCG plus 48h DMEM serum-free conditioned medium of 2931/rbFSH 6E containing 0.05, 0.1 or 0.2mg, consecutively, of rbFSH
- 9-11 10 IU of hCG plus 0.05, 0.1 or 0.2mg of partially purified rbFSH obtained from previous DMEM serum free conditioned medium
- 12-14 10 IU of hCG plus 48h conditioned medium of 293T/rbFSH 6E adapted to serum-free medium containing 0.05, 0.1 or 0.2mg of rbFSH
- 15-17 10 IU of hCG plus 0.05, 0.1 or 0.2mg of partially purified rbFSH obtained from cells adapted to serum-free medium
- this group displayed a proportional dose- response ovaries weight augmentation, according to the applied rbFSH dose (0.05, 0.1 or 0.2mg).
- the rbFSH produced by 293T/rbFSH 6E cells adapted to SFM medium (12-17) displayed lower biological response, in comparison to the non-adapted cells (6-11) .
- the levels obtained for the non- purified medium (12-14) and the purified one (15-17) were similar, in all doses, with the purified rbFSH produced under this SFM condition displaying a better dose-response profile .
- the coding sequences for a and ⁇ chains of bovine FSH were successfully amplified and cloned into the expression vector pCXN2.
- Adopting a 40:1 ratio in co- transfection of the rbFSH constructs relative to the pX343 hygromycin-resistance selection vector proved to be very efficient to isolate cell colonies derived from single cells, resulting in the establishment of several overexpressing cell clones.
- the pCXN2 expression vector utilized for the expression of the rbFSH is a derivative from the pCXN plasmid. The elements present in the pCXN2 vector allow high copy number and high yield of cell transfectants expressing high levels of the foreign proteins.
- Some of the cell clones expressing the highest amounts of rbFSH were selected for protein characterization.
- the in vitro activity of these overexpressing clones was evaluated by assessing the levels of progesterone produced by the FSH- responsive GFSHRlVcell lineage upon rbFSH stimulus. All cell clones were able to induce progesterone production in this responsive lineage, showing homogenous recombinant protein activity.
- Conditioned media from selected rbFSH clones were used as stimulus, with two cell clones, namely 293T/rbFSH 6E and 6K, displaying the highest in vitro activity.
- Clone 6E the most in vitro biologically active clone, was selected to undergo further assays.
- the method comprises a step of buffer exchange (diafiltration or gel filtration / desalting) and concentration which aims to prepare the sample for the first chromatographic step. After the first chromatographic step, a new buffer exchange and concentration is performed before performing the next step. After the second chromatography step the material is gel filtrated and the product of interest is lyophilized or frozen stored.
- buffer exchange diafiltration or gel filtration / desalting
- a - Step 1 collecting the fraction 3 obtained during buffer exchange by gel filtration / desalination,
- Figure 9 shows Western blotting, wherein: 1) rhFSH sample after anion exchange chromatography; 2) rhFSH sample after gel filtration.
- Figure 10 shows the method for identification and quantification of FSH on HPLC. SDS-PAGE gel stained with Colloidal Coomassie Blue. 1) material passed through the column without binding to it (Flow Through); 2) representative fraction of the column washing material (Wash-out); 3) Standard Molecular Weight; 4) Fraction 1 eluted from the column; 5) Fraction 2 eluted from the column; 6) Fraction 3 eluted from the column, 7) Fraction 4 eluted from the column, 8) Puregon 200 ng;. 9) culture supernatant before purification.
- the method may be used for process control because it is able to assess the presence of the biomolecule in the culture medium (culture supernatant) as well as to characterize the purified product.
- rhFSH it is possible to identify the molecule by mass spectrometry.
- Two peptides of the alpha chain of hFSH were identified in the 15 kDa band with the rate of error of 5% by culture supernatant producing cells in the absence of fetal serum.
- the beta chain hFSH features 2 glycosylation sites predicted by UNIPROT.
- ELISA was performed as a quantification method using a commercial kit.
- the cell culture supernatant serum free media
- qPCR By using qPCR it is also possible to determine the number of mRNA copies, which has a great relevance in the design of production methods. It is possible to detect the expression of mRNA encoding the human FSH a and ⁇ chains by qRT-PCR to determine and quantify the number of mRNA copies of a and ⁇ hormone chains transcribed in the hFSH producing cells, compared to the GAPDH endogenous control gene. Syber Green fluorophore is used to establish a Melting curve to verify the specificity of amplification. The relative expression of the FSH alpha chain was 50 times greater than the host cell, which was not transfected with the FSH gene and the relative expression of FSH beta chain was 400 times greater than the one used in the qRT-PCR assay.
- rhFSH The biological activity of rhFSH is analyzed based on progesterone production by cells GFSHR-17. These cells overexpress the FSH receptor and respond to the stimulation of FSH secreting progesterone to the culture medium. Thus, supernatants of the production method or already purified FSH are evaluated by applying to GFSHR-17 cells. After incubation with the culture conditioned medium supernatants, the 17-GFSHR cells which were stimulated with different concentrations of rhFSH were collected and the amount of progesterone produced by these cells was determined by electrochemiluminescence.
- the activity of purified human recombinant FSH was higher than 9UI/ug (for bovine recombinant IU activity unit is not used and the market product is presented in milligrams ) .
- A-D representative chromatograms of the selectivity and specificity assay of the method
- A progesterone in methanol
- B culture media evaluated as biological matrices
- C culture media with FSH ( Folltropin)
- D culture media with progesterone (lOug / ml) and FSH
- E Calibration / Linearity of the method
- F Initial evaluation of the accuracy (Kruskal- allis p> 0.05)
- G Specificity and Selectivity
- A-D culture media evaluated as biological matrices
- MeOH methanol as matrix for analysis of progesterone.
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Cited By (6)
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WO2017112987A1 (en) * | 2015-12-28 | 2017-07-06 | Ouro Fino Saúde Animal Participações S.A. | Process of producing a recombinant equine chorionic gonadotropin (recg), veterinary composition and use thereof |
CN108531508A (en) * | 2018-03-20 | 2018-09-14 | 西北民族大学 | A kind of sheep follicle-stimulating hormone (FSH) DNA recombinant expression preparation method |
CN109970871A (en) * | 2019-04-28 | 2019-07-05 | 广州威生医药科技有限公司 | Long-acting reorganization FSH fusion protein, preparation method and its application in sow timing semen deposition |
WO2019218096A1 (en) | 2018-05-18 | 2019-11-21 | Centro De Biotecnología Y Biomedicina Spa | Recombinant bovine follicle-stimulating hormone, composition comprising the same, and method to induce superovulation and estrus synchronization in bovines using said hormone |
WO2020172728A1 (en) * | 2019-02-28 | 2020-09-03 | Ouro Fino Saúde Animal Participações S.A. | Biologically active recombinant equine chorionic gonadotropin (recg) and process for obtaining same, veterinary composition and use |
CN114591414A (en) * | 2022-03-24 | 2022-06-07 | 江西浩然生物制药有限公司 | Preparation method of human chorionic gonadotropin |
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- 2013-05-16 WO PCT/BR2013/000165 patent/WO2014183175A1/en active Application Filing
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Cited By (7)
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WO2017112987A1 (en) * | 2015-12-28 | 2017-07-06 | Ouro Fino Saúde Animal Participações S.A. | Process of producing a recombinant equine chorionic gonadotropin (recg), veterinary composition and use thereof |
CN108531508A (en) * | 2018-03-20 | 2018-09-14 | 西北民族大学 | A kind of sheep follicle-stimulating hormone (FSH) DNA recombinant expression preparation method |
CN108531508B (en) * | 2018-03-20 | 2021-07-16 | 西北民族大学 | Preparation method for sheep follicle-stimulating hormone gene recombinant expression |
WO2019218096A1 (en) | 2018-05-18 | 2019-11-21 | Centro De Biotecnología Y Biomedicina Spa | Recombinant bovine follicle-stimulating hormone, composition comprising the same, and method to induce superovulation and estrus synchronization in bovines using said hormone |
WO2020172728A1 (en) * | 2019-02-28 | 2020-09-03 | Ouro Fino Saúde Animal Participações S.A. | Biologically active recombinant equine chorionic gonadotropin (recg) and process for obtaining same, veterinary composition and use |
CN109970871A (en) * | 2019-04-28 | 2019-07-05 | 广州威生医药科技有限公司 | Long-acting reorganization FSH fusion protein, preparation method and its application in sow timing semen deposition |
CN114591414A (en) * | 2022-03-24 | 2022-06-07 | 江西浩然生物制药有限公司 | Preparation method of human chorionic gonadotropin |
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