BR112015028737B1 - Methods for producing and/or purifying hormones - Google Patents

Abstract

METHODS FOR THE PRODUCTION AND/OR PURIFICATION OF HORMONES The present invention relates to methods for the production and/or purification of hormones, particularly follicle stimulating hormone (FSH), of human or animal origin, using a platform of human cells, as well as as products obtained in this way and the uses thereof.

Description

Field of Invention

[001] The present invention relates to methods for the production and/or purification of hormones, particularly follicle stimulating hormone (FSH), of human or animal origin, using a human cell platform, as well as products obtained in this manner and the uses thereof.

Prior Art

[002] Hormones are involved in several stages of mammalian reproduction, in particular, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (CG), as well as thyrotropin or stimulating hormone. of the thyroid (TSH).

[003] Due to their direct or indirect role in reproduction, these hormones have been used in the treatment of infertility and reproductive disorders in mammals, such as humans, cattle, sheep, pigs, goats, buffaloes, horses and male monkeys. or female.

[004] In nature, FSH, LH and TSH are pituitary hormones, while CG is produced during placenta formation.

[005] For pharmaceutical use, hormones were produced by recombinant technology or from mammalian urine, the critical factor being the high degree of purity.

[006] In particular, FSH is used in female patients in ovulation induction and controlled ovarian hyperstimulation for assisted reproduction (eg, assisted reproductive technologies, intrauterine insemination, in vitro fertilization and intracytoplasmic sperm injection). It is also used in male patients suffering from oligospermia to induce and maintain spermatogenesis. Treatment with FSH is based on successive injections of the hormone and requires a product with a high degree of purity and specific activity.

[007] Human FSH (hFSH) is a glycoprotein secreted by the anterior pituitary gland. The molecule is active as a non-covalently linked heterodimer containing alpha and beta subunits. The alpha subunit of hFSH 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 amino acids. There are several isoforms of the hFSH molecule, which differ depending on the glycosylation molecule.

[008] FSH from non-human animals is used to induce superovulation in female donors and to induce follicular growth during IVF programs in anaestrus females.

[009] The FSH currently used in animal breeding programs is purified from porcine pituitary extracts. This product is prepared using a more economical technique, which makes it possible to obtain a cheaper, but less effective, hormonal preparation. Furthermore, commercial preparations depend on the availability of a sufficient number of fresh pituitary glands for the preparation of each batch, leading to great variability in results during superovulation treatments with different batches of purified products.

[010] The bovine FSH hormone (bFSH) is composed of two glycoprotein subunits encoded by CGA genes (α chain, common to all pituitary hormones: FSH, LH and TSH) and bFSH (β chain, specific for this hormone), where CGA is 363 base pairs and bFSH is 389 base pairs.

[011] Recombinant bFSH has also been expressed in several recombinant systems, such as in: yeast, plants, insect cells, mouse epithelioid cells, hamster ovary cells, milk from transgenic mice and transgenic rabbits. However, only bFSH expressed in mammalian cells showed biological activity in vivo. This is explained by the fact that the bFSH hormone is very complex, composed of two polypeptide chains, each of which contains post-translational modifications, that is, glycosylation.

[012] While animal embryo transfer techniques are widespread, treatment-based variability of the response to treatment-based superovulatory gonadotropin persists as a major limitation. This variability results from the lack of reproducibility of commercially available preparations (FSH extracted from porcine pituitary glands). These products often have undesirable immunogenic contaminants and potential vectors for the transmission of viral particles or prions. Furthermore, commercial FSH preparations always contain some LH (luteinizing hormone) contamination, contributing to the inconsistency of superovulatory treatment. Finally, the species-specific hormone is more effective at inducing ovulation and preventing immune responses in animals. For these reasons, from the point of view of biotechnology, biosecurity and economics, the development of recombinant FSH in animal cells is of great interest.

[013] In recent years, new methods for the preparation of hormones, especially with high purity, have been sought by several research groups, mainly in relation to the FSH hormone. From a technical perspective, there are several options in terms of methodologies for the purification of FSH. Several of these methodologies avoid using immunoaffinity chromatography due to high costs and the generation of impurities, such as antibodies immobilized on the resin used to separate the product of interest.

[014] International Patent Application No. WO 98/20039, filed by IBSA Institut Biochimique AS, describes a process for the purification of human urinary FSH from urinary extracts (menopausal gonadotropin) using ion exchange chromatography on DEAE resins of basic anion exchange weekly, followed by affinity chromatography on resin with an anthraquinone derivative as a binder.

[015] International Patent Application No. WO 00/63248, filed by Instituto Massone AS, describes a process for the purification of gonadotropins, including FSH from human urine, using ion exchange chromatography with a strong sulfopropyl-type cationic resin, ion exchange with strong anion resin and hydrophobic interaction chromatography. .

[016] The U.S. document 5,990,288, filed by Musick et al., discloses a method for purifying FSH from biological samples such as human pituitary glands or human postmenopausal urine, EMD SO3-650M, followed by dye affinity on a mimetic Orange 1 resin and interaction hydrophobic acid in Bakerbond wide pore HI-propyl resin.

[017] International patent application WO 88/10270, filed by Instituto di Ricerca Cesare Serono SPA, discloses a method for the purification of human FSH from urine by immunochromatography (using monoclonal antibodies linked to Sepharose 4B) followed by reversed-phase HPLC .

[018] International patent application No. WO 2005/063811, filed by Ares Trading SA, describes the purification of recombinant human FSH comprising the steps of ion exchange chromatography, immobilized metal ion affinity chromatography and hydrophobic interaction chromatography, in any order.

[019] International patent application No. WO 2006/051070, filed by Ares Trading SA, describes the purification of recombinant human FSH comprising the steps of dye affinity chromatography, hydrophobic interaction and reversed-phase chromatography, in any order.

[020] Thus, there is still a need for new methodologies for the production of human and animal hormones, especially those capable of providing high purity products.

Brief Description of Figures

[021] Figure 1 is a flowchart showing the method to produce recombinant bovine FSH (rbFSH) from the construction of recombinant vectors by Genetic Engineering to the in vitro and in vivo biological activity tests, according to example 2.

[022] Figure 2 shows the fractionation of the fragments that correspond to the bFSH α and β chains, and the pCXN2 vector in 1.5% agarose gel stained with ethidium bromide. In this figure, one can observe the size of the fragments in relation to molecular weight (M) markers, ie: β chain (398 bp), α chain (363 bp) and vector (~ 6,000 bp).

[023] Figure 3 shows the fractionation on 1.5% agarose gel stained with ethidium bromide of the plasmid preparations containing the fragments of interest (both subunits of bFSH) and digested with restriction enzyme, together with the same constructs undigested, where M represents molecular weight markers.

[024] Figure 4 illustrates the transfection of both bFSH chains into a mammalian cell for their expression.

[025] Figure 5 shows the autoradiography of the Western blot that corresponds to the rbFSH samples.

[026] Figure 6 shows the in vivo biological activity assay.

[027] Figure 7 shows the general flowchart of the purification method for bovine and human recombinant hormones (steps according to the present invention).

[028] Figure 8 shows the chromatographic profile of the FSH purification procedure observed in the different chromatographic steps.

[029] Figure 9 shows a Western Blot, in which: 1) rhFSH sample after anion exchange chromatography; 2) rhFSH sample after gel filtration.

[030] Figure 10 shows the method for the identification and quantification of FSH using HPLC.

[031] Figure 11 shows representative chromatograms of the method for the identification and quantification of the FSH hormone in cell culture medium (DMEM).

[032] Figure 12 shows the identification and quantification of progesterone performed by HPLC.

[033] Figure 13 shows the in vivo FSH activity assay. A) human and B) bovine.

[034] Figure 14 shows the biological activity of FSH in cattle. The graph correlates the number of ovarian follicles eight or more mm in diameter with the time after cows were treated with the commercial product (Folltropin) or rbFSH.

[035] Figure 15 shows the analysis, in SDS-PAGE, of the levels of rhFSH in conditioned media or culture supernatants. Conditioned culture media obtained from 3D and 6E cell clones grown for 24h were subjected to SDS-PAGE gel electrophoresis under reducing conditions. After electrophoresis, the proteins were stained with Coomassie brilliant blue.

[036] Figure 16 shows the analysis, by Western Blot, of rhFSH levels in conditioned media from culture supernatant. Conditioned culture media obtained from the parental cell line and from cell clones grown for 24h were prepared, separated by SDS-PAGE, transferred by electrophoresis to nitrocellulose membrane and immunostained using an antibody to the β subunit of human FSH.

[037] Figure 17 shows cell growth and production of recombinant protein (rhFSH) under high and low serum concentration conditions. Cell growth kinetics were quantified in terms of population doubling time. The production of rhFSH was determined along the entire growth curve by quantifying the hFSH present in the conditioned medium using the chemiluminescence assay.

[038] Figure 18 shows the production of rhFSH in serum-containing and serum-free culture media. The production of rhFSH was determined in the conditioned medium by the cells after cultivation for 24, 48 and 72h and quantified using the chemiluminescence assay.

[039] Figure 19 shows the increase in progesterone production by rFSHR-17 cells after stimulation with rhFSH compared to unstimulated cultures. rFSHR-17 cells were stimulated with culture supernatants obtained from recombinant clones after culturing for 24h. The progesterone released into the culture medium was measured by the chemiluminescence method.

[040] Figure 20 shows the dose-effect relationship of recombinant human follicle stimulating hormone (rhFSH) on rat ovary weight.

[041] Figure 21 shows the production of progesterone, in ng per mL, by the GFSHR17 cell line, in response to the rbFSH stimulus, measured by chemiluminescence. 293T: conditioned medium from untransfected 293T cells; 3A: negative control (empty pCXN2 vector without insertion); 6E to 6K: 293T/rbFSH cell clones; cell population (mixture): conditioned medium from the transfected 293T culture prior to cell clone isolation. The experiments were performed in triplicate.

[042] Figure 22 shows Western Blot analysis of rbFSH present in conditioned media or culture supernatants and in a partially purified preparation. 48h conditioned medium from clone 6E 293T/rbFSH, partially purified conditioned medium from the same clone, commercial purified porcine FSH (Folltropin-V®) and conditioned culture medium obtained from negative control/293T (clone 3A) were prepared, separated under 12% SDS-PAGE reducing conditions, electrophoretically transferred to nitrocellulose membrane, incubated with polyclonal antibody to bFSH and detected by chemiluminescence using a radiographic film. 1: Conditioned medium from negative control 293T/3A cells transfected with empty pCXN2 vector without insert; 2 to 4: foltropin preparations (2, 5 and 10 μg, respectively); 5 and 6: conditioned medium concentrated for 48h of 293T/rbFSH clone 6E containing 2 and 10 μg of rbFSH, respectively; 7 and 8: Partially purified rbFSH preparations containing 5 and 50 μg rbFSH, respectively. The upper black arrow indicates the holoprotein, while the middle black arrow indicates the single FSH beta chain and the lower black arrow indicates the single FSH alpha chain.

[043] Figure 23 shows the effect of recombinant bovine follicle stimulating hormone (rbFSH) on rat ovary weight gain.

Description of the Invention

[044] Recombinant proteins generally have maximum biosecurity, better performance in superovulation and no interference with other hormones and/or contaminants due to the purity of the preparation, thus being of great interest for assisted reproduction.

[045] Recombinant technology proved to be viable to generate proteins with different degrees of complexity in a reproducible way. However, the structure of gonadotropins is difficult to reproduce because, in addition to the presence of the post-translational modifications already mentioned, gonadotropins are composed of two chains, whose coding sequences are independently transcribed and translated, and their products are subsequently combined.

[046] The advantage of the recombinant protein expression system in mammalian cells is the possibility to add the glycosidic complex to the newly synthesized chains, which is absolutely essential for the correct tertiary structure of each subunit, being a prerequisite for the bend with the opposite subunit. This method facilitates the formation of disulfide bonds, which is a critical factor in the folding and maturation of the hormone's functional subunits. In addition, glycosylation directly affects the half-life of the molecule in circulation and the recognition of the molecule by the receptor, as well as hormonal exposure to regulatory mechanisms operating in vivo, interference with its solubility, and protection against proteases and thermal inactivation. Therefore, the addition of sugar has a great impact on the biological properties of glycoprotein hormones.

[047] However, even in mammalian cell expression systems, there is great variability in the pattern of sugars added to the expressed recombinant protein, since the structure and composition of the carbohydrates bound to the glycoprotein are determined by the pattern of glycosylation (types and levels of enzymes responsible for the addition of glycans: glycosyltansferases) available in each specific cell type, which can generate specific glycoforms according to their origin. Thus, different cell lines containing different types of glycosyltransferases will catalyze the same protein glycosylation in a cell-specific manner.

[048] Today, one of the most effective ways to produce pharmacologically active glycoproteins uses Chinese hamster ovary (CHO) cell-based expression systems which, despite containing a variety of oligosaccharide processing enzymes, are not capable of to add the penultimate N-acetylgalactosamines and terminal sulfates, being deficient to perform α2,6-type fucolizations and signaling. These sugars are essential to ensure the prolonged half-life of the eCG, as described above.

[049] Therefore, taking into account that the sugars attached to both FSH chains are essential for signal transduction, that protein metabolism and half-life, as well as heterodimer folding and stability are affected by glycosylation, and that the glycosylation profile of the cell line must present the greatest phylogenetic proximity to the species of interest (be it human or animal), to be less immunogenic and better recognizable by hormone receptors, the expression of hormones in 293T cells was developed as a biotechnologically more effective expression system. Furthermore, the HEK cell family, which includes 293T, such as those used in the method according to the present invention, exhibits great transfection capacity, allowing greater influx of copies of the recombinant constructs of the vector of interest into cells.

[050] The expression system in human embryonic kidney (HEK) or cell-derived cell lines (293, 293T) is capable of producing glycoproteins with N-glycan complexes, which are, for the most part, biantennary, relatively homogeneous and partially labeled, with about 60% of the molecules carrying terminal sialic acid residues.

[051] In accordance with the present invention, hormones include, without limitation, follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (CG), thyroid stimulating hormone (TSH). Preferably, the hormone is FSH.

[052] The production strategy using HEK-derived cells such as 293T can be used for expression at high expression levels of any other homo- or multimeric proteins with complex post-translational modifications.

[053] Furthermore, in accordance with the present invention, mammalian species include humans, cattle, sheep, swine, pigs, goats, buffalo, horses, and male or female monkeys. Preferred species are humans and cattle.

[054] Thus, in a first embodiment, the present invention relates to a method of producing and/or purifying hormones that comprises the following steps: 1) Extracting RNA from a tissue sample; 2) cDNA synthesis by reverse transcription of the RNA obtained in step 1; 3) Amplification of cDNA fragments corresponding to the complete α and β chains; 4) Cloning of each of the amplified fragments (α and β chains) into an expression vector for mammalian cells; 5) Co-transfection of vectors containing the α and β chains (ie the recombinant plasmid constructs) obtained in step 4, together with a vector containing the antibiotic resistance gene, into a mammalian cell line of kidney cells human embryonic (HEK) or HEK-derived cells (293, 293T); 6) Selection of recombinant cell clones through their resistance to antibiotics (preferably to hygromycin); 7) Optional detection of recombinant protein expression by Western blotting and testing of biological activity in vitro and in vivo. 8) Optional purification of the obtained hormone.

[055] The vector used in step 4 is a plasmid for the expression of protein chains in mammalian cells, preferably pCXN2. However, other plasmids can be used as long as they comprise the same elements as pCXN2, i.e. one or more strong promoters, multiple cloning sites, antibiotic resistance sequence for cloning in bacteria, and recombination sites.

[056] The way to insert the plasmids into the cell (transfection) can be performed by various systems, such as lipofection, cationic molecules, calcium-mediated transfection, electroporation with polyethyleneimine, since the transfection rate is kept at the same level.

[057] The ratio of antibiotic resistance plasmids versus those used in the co-transfection strategy ranges from 1:1 to 1:5,000.

[058] The concentration of antibiotic in the culture medium (such as hygromycin) used for the selection of stable cell clones ranges from 10μg/mL to 2,000μg/mL.

[059] The cell line used for the expression of the product can 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 they are cultured in vitro (similar replication capacity , expression and transfection skills, etc.).

[060] The cell can be grown in batches in monolayers or in suspension in different containers, such as in culture flasks of various sizes, on plates, T-flasks, in shaker vessels (spinners), in culture bags, bioreactors, etc.

[061] The culture medium can be any medium that meets the metabolic needs of the cell, for example fetal or adult serum from various species.

[062] The cultivation temperature varies from 30 to 38°C.

[063] The rotation, when the cultivation is carried out in shaker containers, bags and bioreactors, varies from 10 to 200rpm.

[064] The relative proportion of CO2 used in cell culture ranges from 1 to 10%.

[065] The cultivation pH ranges from 5.0 to 7.5.

[066] The recombinant hormone obtained according to the present invention has a high degree of purity, for example, rbFSH is LH free compared to commercial preparations for bovine species which include about 15% LH.

[067] The high degree of purity is ensured by a complementary purification method as required. Therefore, in another embodiment, the present invention relates to a method for the purification of recombinant hormones which comprises subjecting the culture supernatant containing recombinant hormone to the following steps: a) Dye affinity chromatography (Sepharose blue): the hormone does not binds to the matrix, allowing the original sample to be cleaned, removing contaminants that have an affinity for the resin. b) Ion exchange chromatography (DEAE Sepharose): the hormone binds to the matrix, therefore, its elution and isolation from other contaminants is possible. c) Size exclusion chromatography (Sephacryl S-100): separation of aggregates and remaining contaminants.

[068] Steps a) and b) can be performed in any sequence without compromising the result, as the buffer exchange is suitable for subsequent chromatography.

[069] The recombinant hormone-containing supernatant can be pre-prepared prior to the purification step. In that case, 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.

[070] Buffer exchange and/or concentration is performed by tangential filtration on a hollow fiber membrane using 20 mM sodium phosphate buffer, pH 7.0. Alternatively, buffer exchange is performed by gel filtration using Sepharose G25 resin (eg, as specified below) and the same buffer as mentioned above. TABLE I - GEL FILTRATION

[071] The affinity chromatography of step a) is performed on a resin with a dye, such as the immobilized ligand-Blue Cibacron F3G-A. Dye affinity chromatography is performed using 20 mM sodium phosphate pH 7.0 as binding buffer. Sample elution is then performed by applying a gradient between 20 mM sodium phosphate buffer pH 7.0 and 20 mM sodium phosphate buffer pH 7.0 + 2 M NaCl, optionally, in the presence of antioxidant. The fraction that does not interact with the resin is separated and used in the next step, for example, as in Table II below. TABLE II - AFFINITY CHROMATOGRAPHY

[072] Buffer exchange and concentration can also be performed between steps a) and b).

[073] After completing the dye affinity chromatography, the fractions of interest are subjected to tangential filtration on a hollow fiber membrane (5 KDa) for buffer exchange and/or concentration in 0.16 M ammonium acetate buffer pH 7.0.

[074] Ion exchange chromatography is performed using DEAE Sepharose FF, for example, as shown in Table III. Binding to the resin takes place in 0.16 M ammonium acetate buffer pH 7.0, and elution occurs by applying a gradient of 0.16 M ammonium acetate buffer and 0.16 M ammonium acetate buffer + NaCl 2 .0M at pH 7.0. TABLE III — ION EXCHANGE CHROMATOGRAPHY

[075] After the ion exchange chromatography fractions of interest are eluted and subjected to buffer exchange (PBS at pH 7.4) and are separated by size exclusion using Sephacryl S-100 resin (GE Healthcare), for example , as shown in Table IV below. TABLE IV - EXCLUSION CHROMATOGRAPHY

[076] The concentration of the product after the last stage of purification can be performed by ultrafiltration in hollow fiber membrane of 5 or 10 KDa, preferably in membranes of 5 KDa. Alternatively, depending on volume (< or = to 100 mL) Centricon can be used to concentrate the final product.

[077] Purified recombinant hormone can be frozen or lyophilized after concentration for storage. Alternatively, it can be formulated to provide “ready to use” preparations. For example, such preparations include solution of or solution of m-cresol or benzyl alcohol.

[078] In other embodiments, the present invention also relates to purified or unpurified recombinant hormones, their use in the treatment of infertility or as a fertility enhancer for mammalian species, diagnostic kits, pharmaceutical or veterinary compositions or supplements of cell culture.

[079] The following examples serve to illustrate aspects of the present invention without, however, having any limiting character beyond the content of the claims presented below.

EXAMPLES EXAMPLE 1 - PRODUCTION OF RECOMBINANT HUMAN FSH (rhFSH) RNA Isolation

[080] Total RNA was extracted from tissue frozen in liquid nitrogen using the isothioguanidine/CsCl pad method described by Chirgwin et al., 1979. RNA integrity was verified by electrophoresis by ethidium bromide staining and by ethidium bromide staining. Abs260/Abs280 nm absorption ratio, which was > 1.95.

Cloning the cDNA of α and β-hFSH

[081] Approximately 1 μg of total RNA was transcribed using the enzyme SuperScript® II RT (Invitrogen) and random hexamers in a 20 μL reaction, according to the manufacturer's instructions. One μL of the single-stranded cDNA mixture was used as a sample for further PCR reactions.

[082] The cDNA synthesis was confirmed by amplification of the endogenous control GAPDH transcript using the oligonucleotide primers and conditions described in the PCR-Select® Subtraction cDNA Kit from BD Clontech.

[083] PCR amplification of hFSH α and β subunit cDNAs was performed using 25 μL reactions containing 1 x Promega® PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.4 mM oligos and 0 .5 U of Taq DNA polymerase.

[084] For the amplification of the α and β chains of hFSH, the same PCR cycling was used, as follows: the reactions started with a denaturation step at 94 °C for 2 min, followed by another 35 cycles of 94 °C for 1 minute, 60 °C for 30 seconds and 72 °C for 30 seconds, plus a final extension step of 72 °C for 5 min and then by cooling to 4 °C. The primers FSH1 (5'-AGTATCCGCCCTGAACACAT-3') and FSH2 (5'-CTGTAGGATAAGGAGGAAGG-3') were used to amplify the cDNAs that encompass the coding region for the common FSH alpha chain, and the primers FSH4 (5' - GGCCTGAAATGTCCACTGAT-3') and FSH5 (5'-GGCCTGAAATGTCCACTGAT-3') were used for the amplification of the specific FSH β chain.

[085] The PCR products for these two cDNAs (432 and 567 bp, respectively), were gel purified and blunt-end cloned into the SmaI site of pUC18, using the SureClone® PCR cloning kit ( GE Healthcare), according to the manufacturer's protocol. Recombinant clones (pUC18-αhFSH and pUC18-βhFSH) were selected by colony PCR and checked by restriction enzyme digestion and automated DNA sequencing. Recombinant clones, in the correct orientation, were tested and confirmed using the above-mentioned basic molecular cloning techniques.

[086] The mutagenic primers used for re-amplification of α-hFSH cDNA were AF (5'-CCCAGAGAAATTACCGCCAT-3') and AR (5'-TGTCGACTCATCAAGACAGCA-3'), while for β- hFSH, the oligonucleotides used were BF (5'-GTTTTCAAGTGACCGCCATG-3') and BR (5'-GGCCTGAAATGTCGACTGAT-3'). Kozak consensus sequences are highlighted in bold, and SalI restriction sites are underlined. These PCR products were blunt-ended 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 automated DNA sequencing to produce the pUC18-α(Ko)hFSH and pUC18-β(Ko)hFSH vectors. Expression-ready cDNA inserts for α and β hFSH were released from the pUC18-α(Ko)hFSH and pUC18-β(Ko)hFSH vectors by SalI digestion, followed by gel purification, and then subcloned into XhoI site of the mammalian expression vector pCXN2. 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 cytomegalovirus gene promoter.

Incorporation of α and β-hFSH cDNAs into a human cell line

[087] Equimolar amounts of the α and β FSH expression vector (pCXN2-αhFSH and pCXN2-βhFSH) were cotransfected with the pX343 vector, derived from the pY3 vector (Niwa, H., K. Yamamura and J. Miyazaki, Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene, 1991. 108(2): pp. 193-9), containing the bacterial hygromycin B resistance gene, using a higher proportion of the former to the latter, in a well-characterized mammalian cell line, human embryonic kidney cells transformed by the wild-type SV40 T antigen (cell line 293T, ATCC CRL1591) (DuBridge et al., 1987; Pear et al., 1993), using the Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA).

[088] Bacterial endotoxins were measured by the LAL gel-clot assay (GeneScript USA, Piscataway, New Jersey) by mixing equal volumes of a test sample and lysate with a known sensitivity in a test tube.

Generation of r-hFSH overproducing 293T human strains

[089] Six different experimental conditions were used, as shown in the table below. Table V - Experimental conditions

[090] After transfection with the expression vectors pCXN2-αhFSH, pCXN2-βhFSH and pX343, colonies that were resistant to hygromycin (100 mg/mL) were isolated. Genetically stable cell clones were selected for the highest levels of FSH production and secretion into the cell culture medium, determined by immunoassay using chemiluminescence (CRIESP, 2001). For bioproduction purposes, stable cell lines that expressed the FSH α and β dimer in relatively abundant amounts were selected. This was followed by a very detailed evaluation of a particular cell line that showed promising characteristics (high and stable levels of FSH expression). Culture supernatants collected at different time periods were collected, stored and cryopreserved until tests for biological potency were performed.

Establishment of the Master and Work Cell Bank

[091] After a series of cell expansion and evaluation steps, a 293T cell transfectant (6E cell line) was chosen based on its higher levels of rhFSH production. This cell line was used to establish the Master Cell Bank (MCB), consisting of individual flasks containing samples from the same identical cell preparations of cell clone 6E, which were cryopreserved in liquid nitrogen.

[092] A Working Cell Bank (WBC) was then established by expanding the cells recovered from a single vial of the MCB. Cells were successively expanded and aliquots were placed in flasks and cryopreserved. Cells from one or more flasks are cultured for each cycle of rhFSH production.

[093] Both MCB and WCB were tested for sterility and absence of mycoplasma and bacterial endotoxin contamination, per ECC and FDA guidelines.

Analysis of rhFSH preparations by SDS-PAGE and Western Blot

[094] The r-hFSH preparations were reduced with β-mercaptoethanol, denatured by boiling in the presence of SDS and applied to a 12% polyacrylamide gel. After fractionation, proteins were visualized by Coomassie brilliant blue staining and transferred to nitrocellulose membranes using standard techniques. The membrane was blocked in 5% BSA for 2 h and then incubated overnight in a 1:6250 dilution of an anti-human FSH β-subunit monoclonal antibody. The membrane was subsequently washed three times and then incubated in a 1:1000 dilution of a peroxidase-conjugated polyclonal antibody to mouse Ig (Vector Laboratories) for 1h. Bands were visualized after incubation with a chemiluminescent detection reagent (GE Biosciences) and exposed to an X-ray film. The α and β subunits of rhFSH comigrated under reducing and denaturing conditions, appearing as a single broad band on Coomassie blue stained and immunostained gels. The apparent molecular weight of the dissociated subunits, estimated using suitable molecular weight markers, was 16 kDa.

Growth characteristics of the 6E cell line overexpressing rhFSH

[095] Cell growth kinetics were assessed by automated counting (Celm, São Paulo, SP, BR) and quantified in terms of population doubling time. To this end, cells from conditions 3 (3D cell line) and 6 (6E cell line) were grown in 6-well plates (5 x 10 4 cells per well) in growth medium (DMEM supplemented with 5% FBS or DMEM supplemented with 0.5% FBS) for up to 9 days. RhFSH production was determined along the growth curve by quantifying hFSH in the conditioned medium using the chemiluminescence assay.

In vitro bioassay for FSH

[096] The biological activity of rhFSH was measured using the mouse preovulatory follicle cell line (rFSHR-17), overexpressing the mouse FSH receptor, which responds to human FSH stimulation by producing progesterone. Cell culture supernatants from clones obtained under conditions 3 (3D cell line) and 6 (6E cell line) were chosen for in vitro characterization. The rFSHR-17 cells were cultured in DMEM/F12 medium containing 5% fetal bovine serum (FBS) until 80% confluence and then stimulated with the culture supernatants obtained from the recombinant clones by culture medium for 24 h at 37°C. The progesterone released into the culture medium was measured by the chemiluminescence method.

In vitro bioassay for FSH

[097] The potency of rhFSH preparations was evaluated by determining the ovarian weight gain of female rats in response to FSH administration, in accordance with the standard Steelman-Pohley assay. For this purpose, 45 female Sprague-Dowley rats, 22 days old, were housed (three to five rats per cage) and given food and water ad libitum. The animals were randomly assigned and divided into 8 experimental groups. Each animal received an injection of rhFSH and hCG daily for a period of 3 days. On day 4, the animals were sacrificed and the ovaries were removed and dissected to remove adjacent tissue and weighed. The FSH reference standard was Follitropin α (Gonal-F®, Serono International S.A. Geneva, Switzerland).

Results Construction of a mammalian expression vector of α and β- hFSH

[098] The RNA source for the cloning of cDNAs encoding the α and β polypeptide chains of human FSH was isolated from a surgical specimen of a pituitary tumor, which overexpressed FSH.

[099] PCR amplification of hFSH α and b subunit cDNAs was achieved as described in the Material and Methods section. The PCR products for these two cDNAs (567 and 432 bp, respectively) were cloned into pUC18, resulting in recombinant clones (pUC18-αhFSH and pUC18-βhFSH).

[100] To generate the mammalian expression vectors for hFSH, the inserts obtained from the pUC18-αhFSH and pUC18-βhFSH vectors were reamplified using the mutagenic primers oligonucleotides to incorporate the Kozak consensus sequence (ACCGCC) juxtaposed to the initial ATG codon. , as well as a SalI restriction site at the 3' end of each cDNA. These PCR products were cloned into the pUC18 vector to produce the pUC18-α(Ko)hFSH and pUC18-β(Ko)hFSH vectors. Finally, expression-ready cDNA inserts for the α and β chains of hFSH were released from the pUC18 vectors and then subcloned into the pCXN2 mammalian expression vector, which was chosen for high expression of mRNA subunits.

Expression of human rFSH in 293T cells

[101] Since FSH requires glycosylation for maximum biological activity, rhFSH was produced by genetically engineered mammalian cells into which genes encoding the α and β subunits were inserted.

[102] The human embryonic kidney cell line 293T was selected as a recipient cell, as these cells are easily transfected with exogenous DNA and are capable of synthesizing and secreting glycoproteins. In addition, they can be grown on a large scale.

Tabela VIII - Produção de proteína recombinante (rhFSH) no sobrenadante (meio condicionado) de clones celulares, de acordo com os protocolos CRIESP.

[103] Six different conditions were used for the generation of rhFSH overproducing cells, as described in Table V. After transfection, genetically stable cell clones were isolated for each of the conditions (Table VI) and selected according to levels. production and secretion of rhFSH into the cell culture medium. As expected, cell clones derived from control conditions 2 to 5 showed low production of rhFSH. However, condition 6, which contains the gene sequence that encodes the α and β-FSH cDNAs, showed high values of hFSH production (Table VIII). Table VII - Selection of rhFSH overproducing cells. Number of rhFSH cell transfectants obtained from each experimental condition.

Table VIII - Production of recombinant protein (rhFSH) in the supernatant (conditioned medium) of cell clones, according to CRIESP protocols.

[104] Control and rhFSH-producing cell clones (shown in bold in table VIII) were selected for characterization of protein production and to establish Master (MCB) and Working Cell Banks (WCB) to enable production continuous and reproducible FSH. Cell productivity, cell morphology and cell growth kinetics of MCB and WCB were found to be similar (data not shown).

Characterization of the rhFSH protein

[105] The electrophoretic mobility pattern on SDS-PAGE under reducing conditions of the 6E cell conditioned medium showed a band that likely includes the dissociated α and β subunits, with an apparent molecular weight of 16 kDa, consistent with the predicted relative molecular size. from laser densitometric mass spectrometry (Figure 15). As expected, this band corresponded exactly to the only well-defined band from the reference standard preparation (Puregon®).

[106] Western immunoassay analysis, using the anti-human monoclonal antibody to the β subunit of FSH, and performed under reducing conditions, showed a migrating band with an apparent molecular weight of 16 kDa for the preparation of 6E cell conditioned medium. (Figure 16). As expected, this band also corresponded to the only well-defined band from the reference standard preparation (Puregon®). No cross-reactive material was detected in cells transfected with the empty vector or in the parental cells.

[107] The characterization of the rhFSH-producing cell clone is shown in Figures 17 and 18.

In vitro activity of human rFSH

[108] The in vitro activity of human rhFSH was evaluated using a rat granulosa cell bioassay. This assay measures bioactive FSH by evaluating the induced production of progesterone by rat granulosa cells (cell line FSHR-17). Conditioned medium preparations from cell clone 6E increased progesterone production by FSHR-17 cells in a dose-dependent manner (Figure 19).

In vivo human rFSH activity

[109] The in vivo activity of rhFSH was determined by the Steelman and Pohley assay. The behavior of the biological activity of the FSH preparations was verified by analyzing the dose and effect relationships. The administered dose regimens are summarized in Figure 20. The FSH preparations were diluted in phosphate saline to final concentrations of 1, 2 and 3 IU FSH/mL. Each of the samples was administered to groups of mice that received s.c. of 0.5 ml/mouse per day for 3 consecutive days, yielding final cumulative doses of 1.5, 3 and 6 IU FSH/mouse. A total dose of 60 IU of human chorionic gonadotropin (HCG) was also administered to each animal. The animals were sacrificed 72 h after the first administration, and the ovaries were then removed, dissected without adjacent tissue and weighed. The average cumulative results of two independent experiments are reported.

[110] These results demonstrate that administration of rhFSH increased ovarian weight in a dose-dependent manner.

EXAMPLE 2 — PRODUCTION OF RECOMBINANT BOVINE FSH (rbFSH)

[111] Figure 1 shows a flowchart of the entire method.

Preparation of total RNA

[112] A pituitary gland was obtained from a mature female Bos taurus taurus. The gland was removed, placed in RNAlater® solution (Ambion, Applied Biosystems, Carlsbad, CA), then frozen in liquid nitrogen and manually macerated using a pestle and mortar under liquid nitrogen. Total pituitary RNA was extracted from raw pituitary material by the cesium chloride method. The quality of the RNA preparation was assessed by absorbance (A) at 260 nm and 280 nm (Hitachi High-Technologies U2000 UV/visible spectrophotometer), and only RNA preparations having an A260nm/A280nm ratio above 1.8 were used. for RT-PCR.

Reverse transcription reaction followed by polymerase chain reaction (RT-PCR)

[113] A sample (500 ng) of total pituitary RNA was reverse transcribed using the SuperScript™ II Reverse Transcriptase enzyme (Invitrogen®, Carlsbad, CA) and 125 ng of oligo(dT)18 primer (Fermentas Life Science, Burlington). , Ontario) in a 20 μL reaction, according to the manufacturer's protocol.

Design of Initiator Oligonucleotides

[114] For the amplification of the sequences that encode the α and β chains of bovine FSH, two pairs of mutagenic primers oligonucleotides were designed, namely: BFSHα - forward sense (5'-GCGCGTCGACgccaccATGGATTACTACAGAAAATATGCAGCTGTCA-3') and BFSHα - reverse sense ( 5'—GCGCGTCGAGTTAGGATTTGTGATAATAACAAGTGCTGCAGTGGCACT—3'), for the α-chain of bFSH; and bFSHβ - forward sense (5'-GCGCGTCGACgccaccATGAAGTCTGTCCAGTTCTGTTTCCTTTTCTGT-3') and BFSHβ - reverse sense (5'GCGCGTCGAGTTATTCTTTGATTTCCCTGAAGGAGCAGTAGCT—3') for the bFSH β chain.

[115] Both forward oligonucleotide primers contain: 1) a restriction site spacer (written in italics); 2) a SalI restriction site (underlined); 3) the Kozak consensus sequence (in bold); and 4) the 5' end sequence encoding bFSHα or bFSHβ. Both reverse oligonucleotide primers contain: 1) a restriction site spacer (written in italics); 2) a SalI restriction site (underlined); and 3) the reverse complement of the 3' end sequence encoding bFSHα or bFSHβ. The oligonucleotide primers were designed based on the regions of the sequences published in the NCBI that correspond to the α and β chains of bovine FSH.

Amplification of cDNAs corresponding to the sequences of bFSHα and bFSHβ

[116] Amplification of the cDNA sequences encoding the α and β chains of bFSH was performed using the Triple Master Taq (Eppendorf®, Westbury, NY), according to the manufacturer's manual. The following conditions were used for the polymerase chain reactions: 93 °C for 2 min; 35 cycles: 93°C for 20 seconds and 68°C for 3 min; with final extension at 72°C for 5 min. The amplified products were subjected to agarose gel electrophoresis and the corresponding bands were purified using the QIAquick® Gel Extraction Kit (QIAGEN) and digested with the restriction enzyme SalI (Fermentas Life Sciences - Thermo Fisher Scientific Inc.) at 37 °C. for 2 h and 30 min and inactivated at 65°C for 20 min before cloning into the mammalian expression vector.

Cloning of bFSH α and β cDNA sequences in mammalian expression vector

[117] To clone the sequences corresponding to both subunits of bovine FSH, we used the mammalian expression vector pCXN2, derived from the plasmid pCXN. This vector is composed of a strong promoter derived from cytomegalovirus and beta-actin, which enables an efficient selection - conferred by the gene encoding neomycin phosphotransferase-II - of transfectant cells with a greater number of vector copies, which are, therefore able to express high levels of foreign proteins. The nucleotide sequences of the cloned rbFSH α and β constructs were determined using the fluorescent terminator reaction (BigDye® Terminator v 3.1 Cycle Sequencing Kit- Applied Biosystems™) and analyzed using an automated DNA sequencer (ABI Prism 3700 DNA Analyzer, Applied Biosystems/Hitashi). The chromatograms were assembled and transformed into contigs using Phred/Phrap software, compared to the sequence published by the NCBI for all isoforms. The restriction sites as well as the Kozak fragment were carefully checked and all sequences proved correct. Miniscale plasmid preparations (GFX miniprep Kit, GE Healthcare, Pittsburgh, PA) were obtained according to the manufacturer's protocol, and the purified plasmids were further used for cell transfection.

[118] Figure 3 shows fractionation on 1.5% agarose gel stained with ethidium bromide of plasmid preparations containing the fragments of interest (both bFSH subunits) and restriction enzyme digested, and the same undigested constructs, where M represents the molecular weight marker.

Cell line and culture conditions

[119] 293T/17 cells, the well-characterized human embryonic kidney cells transformed by the SV40 T antigen, were purchased from the American Cell Culture Collection (ATCC™ - ATCC® number: CRL-11268™) and cultured in Dulbecco's Modified Eagle Medium (DME) supplemented with 10% fetal bovine serum (HyClone, Thermo Scientific) under a 2.5% CO 2 atmosphere at 37°C in adherent culture. For serum free culture medium, cells were maintained in HyQ®SFM4HEK293TM medium (HyClone, Thermo Scientific).

Cotransfection of 293T cells with the pCXN2-rbFSHα and pCXN2-rbFSHβ constructs, together with the pX343 selection vector for the generation of cell clones

[120] In order to generate stable cell clones expressing the α and β chains of bFSH, 293T cells were cotransfected with each of the pCXN2-rbFSH constructs and the hygromycin B resistance vector pX343 (Figure 4), using a ratio in DNA mass 40:1 (8 µg pCXN2-rbFSHα plus 8 µg pCXN2-rbFSHb for 200 ng pX343). The experimental control was carried out under the same conditions, but using the empty pCXN2 vector without the insert. The transfection procedure was performed using Lipofectamine™ 2000 reagent (Invitrogen®), according to the manufacturer's protocol. 24 h after the transfection procedure, the cell population of each condition (control and experimental) was plated at five different dilutions (1:5, 1:10, 1:25, 1:50 and 1:100) and submitted to selection by adding 100 μg/mL of the selective antibiotic hygromycin B (Invitrogen, Carlsbad, CA) to the culture medium. This selective medium was renewed every 48 h until the appearance of recombinant colonies, which took approximately two weeks. Colonies of recombinant cells from each condition were isolated from the remaining population using cloning rings and transferred to 48-well plates to establish cell clones.

Protein Quantification by RIA

[121] After hygromycin selection, cell clones were grown in adherent culture and samples of the conditioned medium were collected to assess rbFSH expression levels. For this purpose, 106 cells from each of the hygromycin-resistant cell clones, as well as the negative control, were plated and samples of the conditioned supernatants were collected after culturing for 48 h. These supernatant samples were subsequently subjected to bFSH quantification by radioimmunoassay.

In vitro biological activity

[122] The biological activity of rbFSH was analyzed using the mouse preovulatory follicular cell line (GFSHR-17), which overexpresses the mouse FSH receptor, responding to FSH stimulation by increasing levels of progesterone production, in a dose-like manner. -dependent. Cell culture supernatants obtained from cell clones chosen from control and experimental conditions were used for in vitro characterization assays. GFSH-17 cells were cultured to 80% confluence in DMEM/F12 medium containing 5% FBS, plated in a 24-well plate and subjected to in vitro stimulation. Stimulating treatments were performed in serum-free medium supplemented with 10% of conditioned supernatants obtained from 106 cells of each recombinant or control clone, after 24h of cultivation under the conditions described. The progesterone released into the culture medium was evaluated by chemiluminescence.

Protein Analysis by Western Blot

[123] The rbFSH supernatants from selected clones were collected and concentrated using Centricon® concentrator filters (10 kDa cut-off) (Millipore, Billerica, MA). Concentrated samples were diluted 1x in reducing sample buffer for SDS-PAGE (60 mM Tris-Cl pH 6.8; 2.0% SDS; 10% glycerol; 0.025% bromophenol blue; 700 mM β- mercaptoethanol) and boiled for 5 min at 95°C. Samples were applied to 12% polyacrylamide minigels at 100V, 100W and 350mA for approximately 2 h and then transferred to Hybond-ECL nitrocellulose membrane (GE HealthCare, Little Chalfont, Buckinghamshire, UK), blocked in the Starting solution. Block™ (Pierce) plus 0.05% (v/v) Tween 20 for 12 h at 4 °C and then incubated for two hours in a 1:1000 dilution of a polyclonal anti-FSHβ antibody (Santa Cruz Biotechnology , Santa Cruz, CA). The membrane was subsequently washed three times in PBSA with 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 1 h. Bands were visualized after incubation with ECL chemiluminescent detection reagent (GE HealthCare, Little Chalfont, Buckinghamshire, UK) and exposed to autoradiography film (Kodak, Rochester, NY).

[124] Figure 5 shows the autoradiography of the Western blot corresponding to the rbFSH samples, in which: - Folltropin: commercial preparation of FSH obtained from pig pituitary extracts; - rbFSH: recombinant bovine FSH produced according to the present invention; - rbFSH: the same product after a preliminary purification stage.

[125] In this figure, the numbers below the legends show the amount (micrograms - μg) applied to each channel of the gel. The whitish bands refer to the high consumption of the substrate by the peroxidase enzyme conjugated with the antibody, demonstrating the high amount of proteins in the gel. In this autoradiography, the presence of the bFSH dimer (~ 34 KDa), the oligomers (~ 68 KDa) and the beta subunit (~ 22 KDa) can be verified.

In vivo biological activity

[126] The physiological activity of rbFSH present in samples of cell clone culture supernatants was evaluated by determining the ovarian weight gain of sexually immature rats in response to three consecutive exogenous doses of FSH, according to the FSH assay of Pharmacopoeia. For this purpose, 22-day-old female Sprague-Dawley rats obtained from the animal facilities of the Instituto de Química da Universidade de São Paulo were housed (three to five rats per cage) and subjected to standard administration of food and water. ad libitum. The animals were randomly assigned to different groups. Each animal received a daily injection of rhFSH plus hCG over a three-day period. On day 4, the animals were sacrificed and the ovaries were removed and dissected without adjacent tissue, and then weighed. The reference FSH preparation used as an experimental control was the commercial pig FSH preparation, Folltropin®-V (Bioniche Animal Health, Belleville, Ontario, Canada). This experiment was carried out in biological triplicates and in two independent experimental replicates.

[127] Figure 6 shows the results of the in vivo biological activity assay. The first two pairs of ovaries refer to the negative controls of the experiment: the animals received saline (PBSA) or hCG (human chorionic gonadotropin, 10 IU - hCG) used to synergize the FSH effect. The numbers (0.05 mg, 0.1 mg and 0.2 mg) below the dividing line reflect the dose applied in each experimental condition. The treatments were performed independently: Folltropin (commercial product), rbFSH overexpressed by a recombinant cell clone grown in culture medium without fetal bovine serum (FBS 1), the same culture medium after a purification step 1 above, rbFSH overexpressed by a recombinant cell clone grown in culture medium without FBS 2 and the same culture medium after a previous purification 2. In rbFSH medium 1, the size of the ovaries decreases despite increasing doses of rbFSH applied in the treatment, resulting in a negative feedback due to the high dosage applied excessively.

Adaptation of cell clones to suspension culture and stability of overexpression.

[128] The cell clone with the highest overexpression was adapted to suspension culture in non-adherent T-flasks, replacing the medium for HyQ®SFM4HEK293TM either directly (100% fresh suspension culture medium) or sequentially (50% fresh culture medium). for fresh suspension and 50% conditioned adherent culture medium) every 3 to 4 days. Viability was verified by treating cell samples with 50% v/v triptan blue (Gibco, Life Technologies, Carlsbad, CA), with cells being considered fully adapted to suspension culture once they reached 95% viability in 100% HyQ®SFM4HEK293TM. After adaptation, the conditioned medium was collected and submitted to the previously described biological assays.

Results Construction of pCXN2-bFSHα and pCXN2-bFSHβ mammalian expression vectors

[129] RNA obtained from a bovine pituitary gland was used for amplification of cDNA sequences encoding bovine FSH α and β chains. The amplification and mutation of both strands was performed as described in the Experimental Procedures, and the PCR products were fractionated by agarose gel electrophoresis, in which the 389 bp and 416 bp amplicons, with the SalI restriction site, the digestion spacers and the Kozak fragment were clearly identified. These PCR products were gel purified and then digested with SalI (Fermentas, Burlington, Ontario, Canada) before being cloned into the XhoI site of the pCXN2 vector as described above. These steps generated the pCXN2-αbFSH and pCXN2-βbFSH recombinant constructs, which were verified for integrity by DNA sequencing.

Expression of rbFSH in human 293T cells

[130] Recombinant plasmids with both bFSH chains (pCXN2-αbFSH and pCXN2-βbFSH) were cotransfected together with the hygromycin-resistant plasmid pX343 in a 40:1 ratio - into previously plated 293T cells. 24 h after transfection, the experimental and control cell populations were subjected to selection in the presence of hygromycin B, until cell colonies appeared, which were isolated as described in the Material and Methods section.

[131] All recombinant cell clone supernatants were subjected to RIA to assess the rbFSH expression levels obtained from each clone. Values ranging from 1.2 to 20 µg of rbFSH were obtained per mL of conditioned cell medium, and seven cell clones expressing high levels of rbFSH were selected for further characterization as described below.

In vitro biological activity

[132] The in vitro activity of rbFSH was evaluated as described in the Material and Methods section. Conditioned media obtained from seven selected rbFSH-producing cell clones, as well as the original rbFSH transfected population, were able to increase progesterone production by GFSHR-17 cells in a dose-dependent manner (Figure 21), with the highest in vitro biological activity being achieved by clone 293T/rbFSH 6E. This clone was selected to be subjected to molecular characterization by Western blot.

Overexpression of rbFSH revealed by Western blotting

[133] In order to determine the expression pattern of rbFSH by clone 293T 6E, a Western blot analysis was performed. Supernatants from 293T 6E cells cultured for 48 h were concentrated and compared to commercial bFSH (Folltropin®-V) and partially purified rbFSH (data not shown) on a 12% SDS-PAGE under reducing conditions. After fractionation, the proteins were transferred to nitrocellulose membranes, which were incubated with the polyclonal anti-FSH β antibody. Figure 22 shows the pattern obtained for each preparation by means of autoradiography. A band of an apparent molecular mass of 19 kDa can be seen in the conditioned medium preparation of 6E cells (Figure 22, wells 5 and 6) consistent with the single α-chain subunit of bFSH, which is also present in commercial FSH (wells 2 to 4) and partially purified rbFSH (lanes 7 and 8), but absent from the negative control (lane 1). The autoradiograph also shows other bands of 37 and 60 kDa, which correspond to the single β chain of bFSH and the αβ dimer, respectively, in rbFSH and partially purified rbFSH conditions (lanes 5 and 6 and 7 and 8), which are absent. of the negative control condition (well 2). This last band of 60 KDa appears weaker, but it is clearly present in the commercial FSH band, but the band of 37 KDa does not appear in this condition. The black arrows, shown in Figure 22, point to the FSH subunits - the top arrow indicates the holoprotein, while the middle one indicates the FSH a β subunit, and the bottom arrow indicates the FSH α subunit.

Adaptation of 6E 293T/rbFSH clone to suspension culture

[134] The switch from adherent to suspension culture is a critical step in scaling up the production of recombinant proteins in mammalian cells, as well as improving the purification procedure. Therefore, we sought to adapt the 6E 293T/rbFSH cell clone to such a condition, both directly and sequentially to remove FBS from the culture. Our results revealed that sequential adaptation results in greater cell viability compared to direct adaptation, and therefore, suspension-adapted 6E 293T/rbFSH clone was selected to be subjected to the activity assay.

In vivo biological activity

[135] To assess expression levels of the rbFSH constructs, we used an in vivo biological activity assay based on the Pharmacopoeia Steelman & Pohley assay, as described in Material and Methods. The response of immature female rats to stimuli from different FSH preparations was evaluated by the increase in ovarian weight after three consecutive daily doses of hormone preparations, in a dose-dependent manner. Conditioned medium from recombinant cell clone 6E 293T/rbFSH (1) maintained in DMEM with 10% FBS and collected after 48 h in DMEM without FBS and (2) after adaptation to serum-free medium. In this experiment, HPLC-purified rbFSH was also used. The experimental groups, as well as the response obtained after each treatment, are summarized in Figure 23.

[136] FSH preparations were diluted in phosphate saline and different doses (0.05, 0.1, and 0.2 mg) were injected. Samples were administered subcutaneously in groups of five rats at injections of 0.2 ml/mouse daily for three consecutive days. A total dose of 10 IU of human chorionic gonadotropin (hCG) was also administered to each animal, except for the negative control, on day 1, according to the Pharmacopeia. Animals were sacrificed 72 h after the first administration; her ovaries were removed, dissected to remove adjacent tissue, and weighed. The average cumulative results of three independent experiments are shown. 1: PBSA (phosphate-saline solution); 2:10 IU hCG (human chorionic gonadotropin); 3 to 5:10 IU hCG plus Folltropin (0.05, 0.1 or 0.2 mg, consecutively); 6 to 8: 10 IU hCG plus 48 h serum-free 6E 293T/rbFSH conditioned DMEM medium containing 0.05, 0.1 or 0.2 mg, consecutively, of rbFSH; 9 to 11:10 IU hCG plus 0.05, 0.1 or 0.2 mg partially purified rbFSH obtained from serum-free conditioned DMEM medium; 12 to 14:10 IU hCG plus 48 h conditioned medium of 6E 293T/rbFSH adapted for serum-free medium containing 0.05, 0.1 or 0.2 mg rbFSH; 15 to 17:10 IU hCG plus 0.05, 0.1 or 0.2 mg of partially purified rbFSH obtained from cells adapted to serum-free medium.

[137] These results demonstrate that the commercial product Folligon (3 to 5) exhibited a discrete response by the animals that was very similar to administration of 10 IU hCG (2). It is also possible to note that the rbFSH produced by cells grown regularly in DMEM with 10% FBS before switching to DMEM without FBS (6 to 8) showed the highest biological response among all groups. It is important to note that the 0.05 mg group (6) caused a more pronounced increase in ovarian weight compared to higher doses (0.1 and 0.2 mg) of the same preparation (7 and 8), as a proportionally inverse response. The second best response group was obtained by rbFSH produced under the same conditions as groups 6 to 8, but being purified by HPLC (9 to 11). However, this group showed an increase in ovarian weight proportional to the dose-response, according to the dose of rbFSH applied (0.05, 0.1 or 0.2 mg). The rbFSH produced by 6E 293T/rbFSH cells adapted to SFM medium (12 to 17) showed a low biological response compared to non-adapted cells (6 to 11). The levels obtained for the unpurified (12 to 14) and the purified (15 to 17) medium were similar at all doses, with the purified rbFSH produced under this SFM condition showing a better dose-response profile.

[138] In this study, high levels of expression of a biologically active rbFSH in vitro and in vivo, produced in the modified human embryonic kidney cell line 293T, were demonstrated.

[139] Coding sequences for bovine FSH α and β chains were successfully amplified and cloned into the pCXN2 expression vector. The adoption of a 40:1 ratio in the cotransfection of the rbFSH constructs against the hygromycin resistance selection vector pX343 proved to be very efficient for isolating cell colonies derived from individual cells, resulting in the establishment of multiple overproducing cell clones. The pCXN2 expression vector used for the expression of rbFSH is a derivative of the pCXN plasmid (Niwa, H., K. Yamamura and J. Miyazaki, Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene, 1991, 108( 2): pp. 193-9.). The elements present in the pCXN2 vector allow a high number of copies and a high yield of cellular transfectants expressing high levels of foreign proteins. After transfection and selection with hygromycin of the cell populations plated at low density, we isolated 12 rbFSH cell clones. The production of rbFSH by these cell clones, evaluated by RIA, revealed that the expression levels ranged from 1.2 to 20 μg of rbFSH per mL of culture medium conditioned by these cell clones.

[140] Some of the cell clones expressing the highest amounts of rbFSH (from 5 μg to 20 μg rbFSH/mL) were selected for protein characterization. The in vitro activity of these overexpressing clones was evaluated by evaluating the levels of progesterone produced by the FSH-responsive cell line GFSHR17 through rbFSH stimulation. All cell clones were able to induce progesterone production in this responsive strain, demonstrating the homogeneous activity of the recombinant protein. Conditioned media from selected rbFSH clones were used as a stimulus, with two cell clones, ie, 6E and 6K 293T/rbFSH, showing the highest in vitro activity. Clone 6E, the most biologically active clone in vitro, was selected for further testing.

[141] Western blot analysis (Figure 22) revealed that the α and β chains have a similar molecular mass to the mass of highly purified commercial FSH (foltropin), and it is also possible to identify not only the isolated α and β bands (MM of 19 and 37 kDa, respectively), but also the bFSH dimer (MM about 60 kDa), which remained partially folded even under reducing conditions and after denaturation. Since bFSH is N-linked glycosylated, the band pattern observed is in agreement with the expected size of the fully glycosylated bFSH chains and the dimer 2, which can be demonstrated by treatment with PNGase F. signal of the FSH β chain in Folltropin (lines 2 to 4) can be explained by the low recognition of primary antibody of this polypeptide chain, since Folltropin is purified from pig tissue and, although it has similar molecular mass and activity, when compared to rbFSH, it has different amino acid residue composition.

[142] Another critical assessment is the biological activity of rbFSH in vivo. Using the Pharmacopeia Steelman & Pohley in vivo FSH bioactivity assay, results were similar between animals in the same group, especially between purified conditions (3 to 5; 9 to 11 and 15 to 17). For this assay, conditioned medium from the recombinant cell clone 6E 293T/rbFSH was used under two different conditions, that is: (1) cells grown in DMEM with 10% FBS and conditioned medium collected after 48 h in the presence of DMEM without FBS and (2) after adaptation of cells to serum-free medium. Both conditions were able to induce the biological response in vivo, but conditioned medium from cells cultured in the presence of FBS elicited a significantly greater response compared to those adapted to serum-free medium. Considering the response of groups 6 to 8 described above, the greatest response was obtained at the lowest dosage (0.05 mg - group 6) compared to the lowest levels of ovarian weight gain found at the highest doses ( 0.1 and 0.2 mg - groups 7 and 8), and the proportionally inverse response occurred, we assume that this effect was probably due to a negative feedback mechanism. Since this was the most active preparation, the maximum dose limit was quickly reached and, despite a known amount of rbFSH being applied, the observed response was greater than expected, probably due to a higher specific activity of this preparation. It is important to compare this result with the response presented by the second most active group (9 to 11), which presented a dose-response proportional increase in ovarian weight, according to the dose of rbFSH applied (0.05, 0.1 or 0.2 mg).

[143] A protocol was developed to purify rbFSH from the conditioned medium, and despite a loss of biological activity for groups 9 to 11 (DMEM), the results showed that biological activity was maintained for groups 14 to 17 (medium serum free). Furthermore, for both conditions, the biological activity of purified rbFSH was higher than that of commercial FSH (Folltropin - groups 3 to 5), demonstrating that our expression and purification protocol generates a fully biologically active and stable preparation.

[144] Biological activities of rhFSH or rbFSH are also shown in Figure 13 and Figure 14.

EXAMPLE 3 - PURIFICATION TESTS

[145] Recombinant hormone samples obtained according to examples 1 or 2 were purified according to the flowchart shown in Figure 7. As can be seen, the method comprises a buffer exchange step (diafiltration or gel filtration/desalination ) and concentration to prepare the sample for the first chromatographic step. After the first chromatographic step, a new buffer and concentration exchange is performed before carrying out the next step. After the second chromatographic step, the material is subjected to gel filtration and the product of interest is lyophilized or stored frozen.

[146] The chromatographic profile of the purification method in the different chromatographic steps is shown in Figure 8. The arrows indicate the fractions collected and used in the subsequent chromatographic steps: A - Step 1, collect fraction 3 obtained during the gel buffer exchange filtration / desalination, B - Step 2, collection of flow through (fraction not bound to the column) obtained during dye affinity chromatography, C - Step 3, collection of peak eluted during ion exchange chromatography. This fraction is subjected to gel filtration for final purification. The concentration and buffer exchange between steps B and C are not shown. The identification and characterization of the biomolecule (rbFSH or rhFSH) was performed by Western blotting and electrophoresis (SDS-PAGE), respectively. The quantification of biomolecules is performed by radioimmunoassay or assay by colorimetric method.

[147] Figure 9 shows Western blotting, in which: 1) rhFSH sample after anion exchange chromatography; 2) rhFSH sample after gel filtration.

[148] Figure 10 shows the method for the identification and quantification of FSH by HPLC. SDS-PAGE gel stained with colloidal Coomassie blue. 1) the material passed through the column without connecting to it (Flow Through); 2) representative fraction of the column wash material (washing removal); 3) Molecular Weight Standard; 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 prior to purification.

[149] As an alternative to colorimetric or radioimmunoassay methods and protein quantification by gel densitometry, we have also developed a method for the identification and quantification of FSH on HPLC, as shown in Figure 11, where A = chromatogram obtained for Folltropin (10 μg/ml) in methanol, indicating the retention time of the solution used as standard; B = analysis of the representative chromatogram of the culture medium (in different situations); C = chromatogram obtained for Folltropin (10 µg/mL) in DMEM, indicating maintenance of the retention time of the solution (3.4 minutes) used as standard.

[150] The method can 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.

[151] Regarding rhFSH, it is possible to identify the molecule by mass spectrometry. Two hFSH alpha chain peptides were identified in the 15 kDa band with a 5% error rate by culture supernatant producing cells in the absence of fetal serum. The beta chain of hFSH has 2 glycosylation sites predicted by UNIPROT. Furthermore, it is possible to perform estimates based on the densitometry of the separated bands in the gel using the IMAGEJ analysis software and also through approximations obtained by counting the identified peptides (spectral counting approach). From these analyses, it is possible to estimate that FSH represents 0.1 to 2% of the total mass present in the samples of cell culture supernatant, which represents concentrations between 0.2 and 4 μg/mL.

[152] ELISA was performed as a method of quantification, using a commercial kit. Cell culture supernatant (serum-free media) indicated a productivity of 0.14 IU hFSH/cell (equivalent to 100,000 IU/L).

[153] Using qPCR, it is also possible to determine the mRNA copy number, which is of great relevance in the design of production methods. It is possible to detect the expression of mRNA encoding human FSH α and β chains by qRT-PCR to determine and quantify the number of mRNA copies of hormone α and β chains transcribed in hFSH-producing cells compared to endogenous control gene GAPDH. Sybr green fluorophore is used to establish a melting curve to verify amplification specificity. The relative expression of the FSH alpha chain was 50-fold higher than that of the host cell, which was not transfected with the FSH gene, and the relative expression of the FSH beta chain was 400-fold higher than that used in the qRT-PCR assay. .

[154] The biological activity of rhFSH is analyzed based on the production of progesterone by GFSHR-17 cells. These cells overexpress the FSH receptor and respond to FSH stimulation by secreting progesterone into the culture medium. Thus, production method supernatants or already purified FSH are evaluated by application to GFSHR-17 cells. After incubation with the conditioned culture medium supernatants, 17-GFSHR cells that were stimulated with different concentrations of rhFSH were collected and the amount of progesterone produced by these cells was determined by electrochemiluminescence.

[155] The activity of purified human recombinant FSH was greater than 9 IU/μg (for the bovine recombinant, the activity in IU is not used and the marketed product is presented in milligrams).

[156] As an alternative to the FSH detection activity (measured by progesterone production) by electrochemiluminescence, we also developed a methodology for the detection of progesterone by HPLC, as illustrated in Figure 12. In this figure, A to D: representative selectivity chromatograms and method specificity assay; A = progesterone in methanol, B = culture media evaluated as biological matrices, C = culture media with FSH (Folltropin); D = culture media with progesterone (10 μg/mL) and FSH; E: method calibration/linearity; F: initial precision assessment (Kruskal-Wallis p > 0.05); G: specificity and selectivity, A to D = culture media evaluated as biological matrices; MeOH = methanol as a matrix for progesterone analysis.

[157] In summary, this methodology was developed to directly analyze FSH and progesterone in cell culture medium samples. Liquid chromatography with ultraviolet detection (254 nm) and a C18 column were used for the analysis of the samples. Analyzes were performed with a mobile phase of methanol, ethanol and water. The flow was equal to 1 mL/min, with an incubation temperature of 40 °C and an injection volume of the sample equal to 20 μL.

[158] It should be understood that the embodiments described above are illustrative only and that modification throughout the text may occur to the person skilled in the art. Therefore, the invention should not be considered limited to the embodiments described in this document.

Claims (15)

1. Method for the production of hormones that consists of the steps of: 1) Total RNA extraction from a tissue sample (pituitary gland); 2) cDNA synthesis by reverse transcription of the RNA obtained in step 1; 3) Amplification of cDNA fragments corresponding to the complete α and β chains; 4) Cloning of each of the amplified fragments (α and β) in an expression vector for mammalian cells, characterized by the cotransfection of human embryonic kidney (HEK) cell lines or derived from them (293 or 293T), following the following steps: 5) Cotransfection of both vectors containing the α and β chains together with a vector containing the antibiotic resistance gene into a mammalian cell line of human embryonic kidney (HEK) cells or cells derived therefrom; 6) Selection of recombinant cell clones through their antibiotic resistance; 7) Optional detection of recombinant protein expression by Western blotting and in vitro and in vivo biological activity testing; 8) Optional purification of the obtained hormone. 2. Method according to claim 1, characterized in that the HEK cell derivative is 293T or 293. 3. Method according to claim 1, characterized in that the antibiotic is hygromycin. 4. Method according to one of claims 1 or 3, characterized in that the amount of antibiotic used for the selection of stable cell clones in culture medium varies from 10μg/mL to 2,000μg/mL. 5. Method according to claim 1, characterized in that the vector is pX343 and/or pCXN2. 6. Method according to claim 1, characterized in that the proportion of antibiotic resistance plasmids versus those used in cotransfection varies from 1:1 to 1:5,000. 7. Method for the purification of hormones, characterized by the fact that it comprises the submission of the culture supernatant, containing the recombinant hormone, to the following steps: a) dye affinity chromatography, b) ion exchange chromatography, c) exclusion chromatography by size. 8. Method according to claim 7, characterized in that steps a) and b) can be performed in any sequence. 9. Method according to claim 7, characterized in that the samples (before, between or after each chromatographic step) are prepared by clarification and/or concentration by ultrafiltration and/or subjected to buffer exchange by diafiltration or chromatography. 10. Method according to claim 7, characterized in that the affinity chromatography of step a) is performed on a resin having a Blue Sepharose dye as the immobilized ligand. 11. Method according to claim 7, characterized in that the ion exchange chromatography is performed using DEAE Sepharose. 12. Method according to claim 7, characterized in that the size exclusion chromatography is performed using Sephacryl S-100 resin. 13. Method according to one of claims 1 or 7, characterized in that the hormone is selected from follicle-stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (CG), and/or thyroid (TSH). 14. Method according to one of claims 1 or 7, characterized in that the hormone is follicle stimulating hormone (FSH). 15. Method for analyzing hormones directly in a cell culture medium, characterized by the fact that it comprises a liquid chromatography step with ultraviolet detection (254 nm) and a C18 column, in which the analyzes are performed with a mobile phase of methanol, ethanol and water, flow rate of 1mL/min, oven temperature of 40 °C and sample injection volume of 20μL.

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