BR102015032660B1 - PROCESS OF PRODUCTION OF A RECOMBINANT EQUINE CHORIONIC GONADOTROFIN (RECG): VETERINARY COMPOSITION AND USE - Google Patents

Abstract

Production process of a recombinant equine chorionic gonadotropin (recg): veterinary composition and use. The present invention relates to the production of a recombinant equine chorionic gonadotropin (recg), a veterinary composition comprising recg, and the use of the recg or composition comprising recg. The recg of the present invention has a glycosylation profile similar to that of commercially available chorionic gonadotropins and exhibits an in vivo activity level corresponding to ecg and similar to that of the fsh and lh hormones. The present invention has application in the field of biotechnology, more specifically in the field of veterinary assisted reproduction and is especially suited for use in the treatment of veterinary conditions related to mammalian reproduction and ovulation, the manufacture of diagnostic kits or as supplement for cell culture.

Description

“PROCESS OF PRODUCTION OF A RECOMBINANT EQUINE CHORONIC GONADOTROFIN (reCG), VETERINARY COMPOSITION AND USE” Technical Field [0001] The present invention relates to the production process of recombinant equine chorionic gonadotropin, referred to herein as reCG or reCGpa, to the reCG produced through of the process of the present invention, to a veterinary composition comprising reCG, and to the use of reCG or the composition comprising reCG.

[0002] The reCG produced has a glycosylation profile similar to that of commercially available chorionic gonadotropins and exhibits a level of in vivo activity similar to that of luteinizing hormone (LH, acronym for Luteinizing Hormone) and follicle stimulating hormone (FSH, acronym in English) for Follicle Stimulating Hormone).

[0003] The present invention has application in the field of biotechnology, more specifically, in the field of veterinary assisted reproduction, being especially suitable to be used in the treatment of veterinary conditions related to the reproduction and ovulation of mammals, for the manufacture of diagnostic kits or as a supplement for cell culture.

History of the Invention [0004] Currently, Brazil is in the privileged position of the world's largest producer and exporter of beef, making cattle raising one of the most important and profitable national economic activities. This data emphasizes the importance of basic and technological research in bovine reproduction, especially in the area of ovulation-stimulating hormones, such as equine chorionic gonadotropin (eCG).

[0005] Several hormones are used in veterinary assisted reproduction techniques and, in recent years, equine chorionic gonadotropin has emerged as the first option in inducing ovulation in cows in gestational anestrus, in the Fixed Time Artificial Insemination (IATF) protocols, aiming improve fertility rates of bovine females reared in an extensive regime (SÁ FILHO et al., Equine chorionic gonadotropin and gonadotropin-releasing hormone enance fertility in a norgestomet-based, timed

Petition 870190032656, of 4/4/2019, p. 16/66 artificial insemination protocol in sucked Nellore (Bos Indicus) cows. Theriogenology 2010; 73 (5): 651-8; FERREIRA et a /., Effect of different doses of equine chorionic gonadotropin on follicular and lutteal dynamics and P / AI of high-producing Holstein cows. Anim Reprod Sci 2013; 140 (1-2) 26-33; CARVALHO et a /. Equine chorionic gonadotropin improves the efficacy of a timed artificial insemination protocol in buffalo during the nonbreeding season. Theriogenology 2013; 79 (3): 423-8; BARREIROS et a /., Dynamics of follicular growth and progesterone concentrations in cyclic and anestrous suckling Nelore cows (Bos indicus) treated with progesterone, equine chorionic gonadotropin, or temporary calf removal. Theriogenology 2014; 81 (5): 651-6).

[0006] eCG is especially used in beef cows raised in extensive management and has also been used in superovulatory treatments of cows and heifers as an effective treatment option in irascible animals (MAPLETOFT et al., Recent advances in the superovulation Reprod Nutr Dev 2002; 42 (6): 601-11), since conventional hormonal treatments in bovine farming adopt the use of eight doses of purified commercial preparations of porcine FSH - Folltropin-V ^© , Bioniche ^© (data obtained in the product insert).

[0007] eCG is secreted by the equine uterus, more specifically by the endometrial chalices, which are formed around the forty-fifth day of pregnancy, remaining until around the eighty-fifth day. The secretion of this hormone, which has a biological action similar to both FSH and LH, stimulates the development of ovarian follicles in the pregnant mare. Some of these follicles ovulate, but most become luteinized follicles due to the action similar to LH. Such accessory luteal bodies produce the progesterone necessary to maintain pregnancy in the mare (HAFEZ AND HAFEZ, Animal Reproduction. Editora Manole 2004; single volume).

[0008] The eCG currently used for animal reproduction is purified from plasma of pregnant mares (CHRISTAKOS E BAHL, Pregnant mare serum gonadotropin. Purification and physicochemical, biological, and immunological characterization. J Biol Chem 1979; 254 (10): 4253 -61; MAPLETOFT et al.,

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3/43

Recent advances in the superovulation in cattle. Reprod Nutr Dev 2002; 42 (6): 601-11), being marketed as Folligon (Intervet); SincroeCG (Ourofino) and Novormon (Coopers), there is no commercially available recombinant eCG.

[0009] This unique gonadotropin has the ability, in heterologous species, to bind to both FSH and LH receptors, and to stimulate folliculogenesis (STEWART et al., Pregnant mare serum gonadotrophin: ratio of follicle-stimulating hormone and luteinizing hormone activities measured by radioreceptor assay. J Endocrinol 1976; 71 (3): 471-82; COMBARNOUS, Original hypothesis on the specificity of the binding of glycoprotein hormones to their receptors. CR Seances Acad Sci III 1981). Its prolonged plasma half-life, due to its high amount of sugars (MCINTOSH et al., Pregnant mare serum gonadotrophin: rate of clearance from the circulation of sheep. J Reprod Fertil 1975; 44 (1): 95-100), allows that a single application of this hormone is efficient to induce ovulation, thus saving the time spent on handling, and also reducing possible stresses of animals submitted to superovulatory treatment (KIMURA et al., Successful superovulation of cattle by a single administration of FSH in aluminum hydroxide gel.Theriogenology 2007; 68 (4): 633-9).

[0010] In addition to gonadotropin extraction being a laborious and expensive task, the lack of biosafety of the resulting material is of great concern, since any purification attempt that preserves glycoprotein hormones demonstrates a limited capacity for the destruction of resistant viruses (THARASANIT et al ., Effects of recombinant human follicle stimulating hormone on follicle development and ovulation in the mare. Theriogenology 2006; 65 (6): 1071-81). In addition, because it is a biological extract, there is a great variability in the activity of glycoprotein between lots of eCG, thus contributing to the variability in the response and quality of the embryos obtained (WILSON et al., Superovulation of cattle with a recombinant-DNA bovine follicle stimulating hormone.

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Reproduction Science 1993; 33 (1-4): 71-82).

[0011] Considering the above, and taking into account the fact that the source of origin of the eCG is limited (blood of pregnant mares), and also that there are bioethical factors related to obtaining this material , we are looking for a source of eCG that is unlimited, ethical, and obtaining more practical and reproducible, ensuring the uninterrupted supply to a growing consumer market of this hormone.

[0012] The recombinant gonadotropins have maximum biosafety, better performance in the results, no interference from other hormones and few contaminants, being, therefore, widely used in human reproductive medicine, representing great interest for veterinary assisted reproduction. Recombinant technology proved to be possible to produce complex proteins in a reproducible way. However, the structure of gonadotropins is difficult to reproduce, since, in addition to the presence of several post-translational modifications, gonadotropins are composed of two transcribed and translated chains, which are subsequently combined (LOUMAGEL et al., Human follicle-stimulating hormone produced by recombinant DNA technology: a rewiew for clinicians (Human Reproduction 1995; v. 1 p. 188-199).

[0013] The process of the present invention can also be applied to other forms of eCG that are not fused, such as, for example, the chains expressed separately, or changes in the protein to increase the circulating time of the molecule, such as, for example, modifications as PEGlation.

[0014] The α subunit of eCG, composed of 96 amino acids, exhibits two complex N-linked oligosaccharide chains, located in the residues of asparagines 56 and 82. However, the carbohydrate chains of the two hormones exhibit differences in their structures: the α subunit eLH has two biantenary N-glycans finalized in sulfated N-acetylgalactosamines (SO4-4-GalNAc), while the N-glycans attached to the α subunit of eCG are finalized in sialic acid at α2,3 and α2,6 (DAMM et al., Structure determination of the major N- and O-linked

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5/43 carbohydrate chains of the beta subunit from equine chorionic gonadotropin. Eur J Biochem. 1990 Apr 20; 189 (1): 175-83.), Possibly extended with lactosamine repetitions (BOUSFIELD et al., Differential effects of alpha subunit Asparagine56 oligosaccharide structure on equine lutropin and follitropin hybrid conformation and receptor-binding activity. Biochemistry. 2004 Aug 24; 43 (33): 10817-33). The β subunit of eCG, composed of 149 amino acid residues (SUGINO et al., Structural studies on equine glycoprotein hormones. Amino acid sequence of equine chorionic gonadotropin beta-subunit. J Biol Chem. 1987 Jun 25; 262 (18): 8603 -9), has a single N-glycan chain located at Asn13, ending with sulfated GalNAc in eLH and a2.3Gal sialized in eCG (SMITH et al., Equine lutropin and chorionic gonadotropin bear oligosaccharides terminating with SO4-4-GalNAc and Sia alpha 2,3Gal, respectively. J Biol Chem. 1993 Jan 15; 268 (2): 795-802; MATSUI et al., Structural analysis of N-linked oligosaccharides of equine chorionic gonadotropin and lutropin beta-subunits. Biochemistry. 1994 Nov 29; 33 (47): 14039-48). Both β subunits have up to 12 O-glycans extended with sialized poly N-acetyl-lactosamine (a2,3) located in the serines or threonines of the terminal carboxy-peptide (CTP) of 28 amino acid residues (HOKKE et al., Structure determination of the disialylated poly- (Nacetyllactosamine) -containing O-linked carbohydrate chains of equine chorionic gonadotropin. Glycoconj J. 1994 Feb; 11 (1): 35-41; BOUSFIELD et al., Identification of twelve O-glycosylation sites in equine chorionic gonadotropin beta and equine luteinizing hormone ss by solid-phase Edman degradation. Biol Reprod. 2001 Jan; 64 (1): 136-47.). As eLH and eCG have identical polypeptide chains, the difference between the structure of N- and O-glycans explains the difference between the molecular weights and the biological activity of these two hormones: eCG has an exceptional circulatory half-life compared to eLH (BOUSFIELD et al., Identification of twelve O-glycosylation sites in equine chorionic gonadotropin beta and equine luteinizing hormone ss by solid-phase Edman degradation. Biol Reprod. 2001 Jan; 64 (1): 136-47). The removal of terminal sialic acid residues from the glycan chains of the

Petition 870190032656, of 4/4/2019, p. 20/66 eCG dramatically decreases the in vivo activity of this glycoprotein, as its half-life is reduced from six days to approximately 1-2 hours (MARTINUK et al., Effects of carbohydrates on the pharmacokinetics and biological activity of equine chorionic gonadotropin in Biol Reprod. 1991 Oct; 45 (4): 598-604).

[0015] In the literature, several authors have attempted to obtain recombinant equine chorionic gonadotropin, which has been cloned and expressed in insect cells (LEGARDINIER et al., Biological activities of recombinant equine luteinizing hormone / chorionic gonadotropin (eLH / CG) expressed in Sf9 and Mimic insect cell lines, Journal of Molecular Endocrinology 2005; 34: 47-60); secreted in milk of transgenic rabbits (GALET et al., Expression of an in vitro biologically active equine LH / CG without C-terminal peptide (CTP) and / or beta26-110 disulphide bridge. J Endocrinol, 2001; 167 (1): 117-24); expressed in mouse cells (GALET et al., Expression of a single betaalpha chain protein of equine LH / CG in milk of transgenic rabbits and its biological activity. Mol Cell Endocrinol, 2001; 174 (1-2): 31-40) ; and modified and expressed as deglycosylated forms in CHO cells (Chinese Hamster ovary cells). Only for the first two systems were assays of biological activity in vivo, resulting in no activity, a fact due to the difference in the glycan chains added to eCG by these expression systems.

[0016] Additionally, the state of the art teaches methods of producing recombinant gonadotropins, but it lacks to propose an effective way to obtain reCG in large quantities, and at the same time, with the glycosylation profile similar to wild eCG that guarantees in vivo reCG activity similar to that observed for wild eCG.

[0017] HESSER (HESSER, A survey of heterologous expression systems for the production of bovine follicle stimulating hormone and luteinizing hormone. All Dissertations 2011; Paper 692) describes the production of gonadotropins, more specifically, follicle-stimulating hormones in CHO cells. [0018] FURUHASHI et al. (FURUHASHI et al., Fusing the carboxyterminal

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7/43 peptide of the chorionic gonadotropin (CG) betasubunit to the common alphasubunit: retention of Olinked glycosylation and enhanced in vivo bioactivity of chimeric human CG. Mol Endocrinol. 1995 Jan; 9 (1): 5463) describes an hCG analog with a CTP-like polypeptide tail (acronym in English for Carboxy Terminal Peptide), and relates the importance of glycosylation to the analog's biological activity.

[0019] SUGAHARA et al. (SUGAHARA et al., Biosynthesis of a biological active single peptide chain containing the human common α and chorionic gonadotropin β subunits in tandem. Proc Natl Acad Sci USA 1995; 92: 20412045) describes the biological activity of a single polypeptide chain with biological activity , containing a β subunit of hCG fused to an α subunit of gonadotrophins.

[0020] Subsequently, SUGAHARA et al. (SUGAHARA et al., Expression of biological active fusion genes finding the common alpha subunit and the follicle-stimulating hormone beta subunit: Role of a linker sequence. J Biol Chem 1996; 271: 10445-10448) described the conversion of a heterodimer to FSH for a single chain structure, and the impact of the absence of the binding sequence on the rate of secretion and binding of said protein. Still, it shows that the alignment of the α and β domains differ from that presented in the form of heterodimer.

[0021] NARAYAN et al. (NARAYAN et al., Functional expression of yoked human chorionic gonadotropin in baculovirus-infected insect cells. Mol Endocrinol. 1995; 9 (12): 1720-6) describes the simultaneous expression of eCG α and β subunits in insect cells, whose expression is controlled by different promoters, in the same recombinant baculovirus, giving rise to proteins that are biologically active in vitro.

[0022] SHIOTA et al. (SHIOTA et al., Production of recombinant eCG with potent FSH-like activity by site-directed mutagenesis. Nippon Yakurigaku Zasshi 1997; 110 (1): 59P-62P) describes recombinant gonadotropins with different structures in view of the coupled oligosaccharides, and relates the biological activity of the mutants or variants.

Petition 870190032656, of 4/4/2019, p. 22/66 [0023] EP0974599 describes a variant of the recombinant eCG and its corresponding DNA sequence, in addition to a pharmaceutical composition of said recombinant eCG for use as a veterinary drug. The variant form of the revealed recombinant eCG has the α and β chains of eCG fused into a single polypeptide chain, having FSH and LH type activity in vitro. The inventors suggest its use as a medicine for animals, but do not demonstrate: a) the similarity between the glycosylation profile of the obtained recombinant form and that of the wild form; and b) the in vivo biological activity of the obtained recombinant eCG form.

[0024] US 5,767,251 describes methods for generating recombinant human gonadotropins, including hCG, hLH and hFSH, in which hormones composed of two different subunits are synthesized in the same cell by at least one expression vector, in which the expression of each subunit is controlled by a different promoter. In this case, the biological activity of hCG is easily differentiated from that of LH, because, unlike what occurs in horses, in humans, the β chains of these two gonadotropins are encoded by distinct genes.

[0025] US 5,047,335 describes a process for controlling the glycosylation of recombinant proteins involving the use of genetically modified CHO cells to produce sialyltransferases, a class of glycosyltransferases. This supplementary production of sialyltransferases allows, for example, recombinant glycoproteins, produced in this genetically modified CHO cell line, to have a glycan structure closer to natural human glycoproteins. However, US 5,047,335 does not describe what combination of glycosyltransferases would be needed to obtain recombinant eCG with biological activity.

[0026] US 6,103,501, US 6,238,890 and US 6,987,172 describe artificial single-chain variants of gonadotropins that are naturally found in the form of heterodimers, such as CG, TSH, LH

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9/43 and FSH, and which may provide effects or functions, or may behave as agonists or antagonists of native hormones. Also in this case, the biological activity of human CG is easily differentiated from that of human LH, because, unlike what occurs in horses, in humans the β chains of these two gonadotropins are encoded by distinct genes. Additionally, these documents do not describe what combination of glycosyltransferases would be needed to obtain recombinant eCG with biological activity.

[0027] US 2010/120677 describes methods for the production of biologically active recombinant eFSH analogs and methods to improve reproduction in mammals using recombinant eFSH analogs.

[0028] CA 2183564 describes methods to increase fertility by reducing the activity or levels of hormones with LH activity, and methods for selecting antibodies to specific portions of certain proteins, including LH and hCG, to reduce their biological activity . [0029] The state of the art mentioned above shows the constant efforts directed towards the development of new recombinant gonadotropins and processes to produce them safely, with quality and in an unlimited way, emphasizing, even more, the need for new solutions and the importance Research & Development in this field.

[0030] There remains, therefore, the need for improvements in the production process of recombinant eCG, so that it is produced in large quantities, and at the same time, has the necessary glycosylation profile for its biological activity in vivo. Surprisingly, the Depositor found that it is possible to combine high productivity of the recombinant eCG and obtain a glycosylation profile similar to that found in wild eCG, thus ensuring that the recombinant eCG thus obtained has in vivo biological activities similar to the FSH hormones and LH. This technical effect is unexpected since, until then, it was unknown which heterologous expression system based on mammalian cells would be capable of

Petition 870190032656, of 4/4/2019, p. 24/66 promote post-translational modifications of the type N-linked and Oligated glycosylation in large quantities of recombinant eCG, necessary to differentiate the biological activity of eCG from that of eLH. As discussed in detail above, it is not possible to accurately anticipate which factors are associated with obtaining such a glycosylation profile compatible with an eCG type activity, highlighting: 1) which heterologous expression system would mimic the combination of enzymes and factors found in the cells of the endometrial cup, which are the cells that naturally produce eCG in a specific way; or even 2) what are the conditions of generation of a heterologous system and what are the conditions of cultivation of the heterologous system associated with obtaining such a glycosylation profile. Thus, from this knowledge, in the present invention it was possible to improve methods for production of recombinant eCG, guaranteeing the production of large quantities of recombinant eCG presenting a glycosylation profile similar to that of the commercially available chorionic gonadotropins, and consequently, presenting biological activity also similar to that of wild eCG. Such recombinant chorionic gonadotropins are produced through a biotechnologically prepared system for the high expression of these structures.

[0031] The cell containing gene expression of the glycosylation enzyme transcripts mentioned below allows the obtaining of reCG with biological activity in the target species: UDP-GlnNAc-2-epimerase, CMP-SA-synthetase, CMPSA-transporter, β -galactosidase α 2,3-sialyltransferase 3 (ST3-GalTI11), βgalactosidase α 2,3-sialyltransferase 4 (ST3-GalTIV), β-galactosidase α 2,3sialyltransferase 6 (ST3-GalTVI), Sialidase I, Sialidase II.

Summary of the Invention [0032] The present invention relates to a process of producing a recombinant equine chorionic gonadotropin, in which said reCG has a glycosylation profile similar to that of commercially available chorionic gonadotropins. Said process comprises the steps of:

(a) insertion of a DNA sequence that encodes an eCG into a

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11/43 expression vector that includes the cDNA encoding the enzyme dihydrofolate reductase (DHFR);

(b) co-transfection of the vector from step (a) with a selection vector in double negative expression cells for the DHFR gene;

(c) Selection of resistant transfected cells by treatment with the antibiotic to which they must be resistant;

(d) Gene amplification of the expression vector and selection of reCG overproducing cells by treatment with increasing concentrations of DHFR inhibitor;

(e) cultivation of reCGa3 super-producing cells with a gene expression profile determined for the transcripts of the enzymes of the glycosylation pathways for expression of reCGa3;

(f) purification of the obtained reCGa3.

[0033] In accordance with a second aspect of the present invention, a recombinant equine chorionic gonadotropin, produced from the process of the present invention, is provided. Said reCG produced from a fused cDNA chain containing both the coding sequences of the equine chorionic gonadotrophin (eCG) chains α and β or cDNA chains containing the coding sequences of the α and β chains separately.

[0034] In a third aspect, the present invention relates to a veterinary composition comprising:

(a) a veterinarily effective amount of the reCG produced in accordance with the present invention;

(b) optionally additives; and (c) a pharmaceutically acceptable carrier.

[0035] In a fourth aspect, the present invention relates to the use of the reCG of the invention or the composition of the invention, comprising said reCG, in the manufacture of a medicament for the treatment of medical conditions related to the reproduction and ovulation of mammalian animals, as an optimizing agent for in vitro fertilization; in the manufacture of diagnostic kits

Petition 870190032656, of 4/4/2019, p. 26/66 veterinary conditions and protocols related to mammal ovulation; and in the manufacture of a supplement for cell culture.

Brief Description of the Figures [0036] Figure 1 shows the illustrative scheme used for the construction of the fused DNA that encodes the reCG by PCR.

[0037] Figure 2 illustrates SEQ ID NO: 1 in FASTA format.

[0038] Figure 3 illustrates the expression of reCGpa by populations of CHO-DG44 cells transfected with the plasmid pNUO-eCGpa throughout the process of gene amplification of this plasmid by treatment with increasing doses of the chemotherapeutic MTX.

[0039] Figure 4 illustrates the kinetics of reCGpa expression by the 2.5μΜ CHO population after adaptation to growth in the absence of MTX, over three days, in the presence of fetal bovine serum (FCS, fetal calf serum) or in its absence (SFM, serum free media).

[0040] Figure 5 illustrates the in vitro biological activity of eCG assessed from the progesterone response secreted by MLTC-1 cells after treatment with increasing doses of Folligon or reCGpa produced in the presence (A) or absence (B) of fetal serum bovine.

[0041] Figure 6 illustrates the biological activity in vivo assessed from the uterine response of prepubertal rats from 23 to 25 days after treatment with increasing doses of Folligon or reCGpa produced in the absence of fetal bovine serum.

[0042] Figure 7 illustrates the linear regression referring to the data obtained in the in vivo biological activity assay evaluated from the uterine response of prepubertal rats from 23 to 25 days after treatment with increasing doses of Folligon or reCGpa produced in the absence of fetal bovine serum.

[0043] Figure 8 illustrates the in vivo biological activity of the partially purified reCG by the HiTrap ™ Con A. column from the uterine response of pre-pubertal rats 25 days after treatment with Folligon or reCGpa produced in

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13/43 absence of fetal bovine serum and partially purified by the HiTrap ™ Con A column.

[0044JA Figure 9 illustrates structures of N-glycans obtained for the commercial eCG Folligon after digestion performed with combinations of exoglycosities.

[0045J figure 10 illustrates the putative structures of N-glycans obtained in fractions enriched in reCGpoc.

[0046JA figure 11 illustrates the gene expression profile obtained for the transcripts of the enzymes corresponding to the enzymes of the glycosylation pathways in cells cultured in the presence and absence of fetal bovine serum: UDP-GlnNAc-2epimerase, CMP-AS-synthetase, CMP-SA -transporter, β-galactosidase α 2,3sialyltransferase 3 (ST3-GalTI11), β-galactosidase α 2,3-sialyltransferase 4 (ST3GalTIV), β-galactosidase α 2,3-sialyltransferase 6 (ST3-GalTVI), Sialidase I, Sialidase II.

Brief Description of Sequences [0047] SEQ ID NO 1 illustrates a nucleotide sequence encoding the fC chain of the reCG (reCGc ^).

[0048] _ SEQ ID NO 2 illustrates a polypeptide sequence of the reCG fused chain (reCGc ^).

Detailed Description of the Invention [0049] The present invention deals with a process of producing a recombinant equine chorionic gonadotropin (reCG), in which said recombinant equine chorionic gonadotropin has a glycosylation profile similar to that of natural equine chorionic gonadotropin and has the same level of in vivo activity of the hormones FSH and LH.

[0050] According to a first aspect of the invention, said process comprises the steps of:

(a) inserting a DNA sequence encoding an eCG into an expression vector that includes the cDNA encoding the enzyme dihydrofolate reductase (DHFR);

(b) co-transfection of the vector from step (a) with a selection vector in double negative expression cells for the DHFR gene;

Petition 870190032656, of 4/4/2019, p. 28/66 (c) Selection of resistant transfected cells by treatment with the antibiotic to which they must be resistant;

(d) Gene amplification of the expression vector and selection of reCG overproducing cells by treatment with increasing concentrations of DHFR inhibitor;

(e) cultivation of reCGa3 super-producing cells with a gene expression profile determined for the transcripts of the enzymes of the glycosylation pathways for expression of reCGa3;

(f) purification of the obtained reCGa3.

The DNA sequence encoding the eCG according to the present invention preferably comprises a fused cDNA strand SEQ ID NO. 1, containing both sequences coding for the equine chorionic gonadotropin (eCG) α and β chains.

SEQ ID NO.1:

CGGAATTCGCCACCATGGAGACGCTCCAGGGGCTGCTGCTGTGGATGCTGCTGAGTGTTGGCGG GGTCTGGGCATCCAGGGGGCCACTGCGGCCACTGTGCCGGCCCATCAACGCCACTCTGGCTGCT GAGAAGGAGGCCTGCCCCATCTGCATCACCTTCACCACCAGCATCTGTGCCGGCTACTGCCCCA GCATGGTGCGGGTGATGCCAGCTGCCCTGCCGGCCATTCCCCAGCCAGTGTGCACCTACCGTGA GCTGCGCTTTGCTTCCATCCGGCTCCCCGGCTGCCCGCCTGGTGTGGACCCCATGGTCTCCTTC CCCGTGGCCCTCAGTTGTCACTGCGGGCCCTGCCAGATCAAGACCACTGACTGCGGGGTTTTCA GAGACCAGCCCTTGGCCTGTGCCCCCCAGGCCTCCTCTTCCTCTAAGGATCCCCCATCCCAACC TCTCACATCCACATCCACCCCAACTCCTGGGGCCAGCAGACGTTCCTCTCATCCCCTCCCAATA AAGACTTCTTTTCCTGATGGAGAGTTTACAACGCAGGATTGCCCAGAATGCAAGCTAAGGGAAA ACAAGTACTTCTTCAAACTGGGCGTCCCGATTTACCAGTGTAAGGGCTGCTGCTTCTCCAGAGC GTACCCCACTCCAGCAAGGTCCAGGAAGACAATGTTGGTCCCAAAGAACATCACCTCAGAATCC ACATGCTGTGTGGCCAAAGCATTTATCAGGGTCACAGTGATGGGAAACATCAAGTTGGAGAACC ACACCCAGTGCTATTGCAGCACTTGCTATCACCACAAGATTTAAGCGGCCGCGC [0052] The sequence of the gene encoding RecG was designed to contain, preferably, coding sequences of both units of eCG, α and β, a the way that they were expressed in a fused way,

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15/43 as can be seen in Figure 1 and Figure 2. Figure 2 illustrates SEQ ID NO: 1 in FASTA format, obtained after submitting the sequencing chromatograms of one of the bacterial clones containing the pNUOeCGpoc construct to automated sequencing, using the Sanger method.

[0053] In figure 2, the dashed sequence gaattc represents the site for the restriction enzyme EcoRI; the bypassed string

GCCACC represents the kozac fragment; the underlined string

ATGGAGACGCTCCAGGGGCTGCTGCTGTGGATGCTGCTGAGTGTTGGCGGGGTCTGGGC ATCCAGGGGGCCACTGCGGCCACTGTGCCGGCCCATCAACGCCACTCTGGCTGCTGAGA

AGGAGGCCTGCCCCATCTGCATCACCTTCACCACCAGCATCTGTGCCGGCTACTGCCCC AGCATGGTGCGGGTGATGCCAGCTGCCCTGCCGGCCATTCCCCAGCCAGTGTGCACCTA CCGTGAGCTGCGCTTTGCTTCCATCCGGCTCCCCGGCTGCCCGCCTGGTGTGGACCCCA TGGTCTCCTTCCCCGTGGCCCTCAGTTGTCACTGCGGGCCCTGCCAGATCAAGACCACT GACTGCGGGGTTTTCAGAGACCAGCCCTTGGCCTGTGCCCCCCAGGCCTCCTCTTCCTC TAAGGATCCCCCATCCCAACCTCTCACATCCACATCCACCCCAACTCCTGGGGCCAGCA GACGTTCCTCTCATCCCCTCCCAATAAAGACTTCTTTT is the β chain without the stop codon; the highlighted sequence | CCTGATGGAGAGTTTACAACGCAGGATTGCCCAGAATGCAAGCTAAGGGAAAACAAGTA | | cttcttcaaactgggcgtcccgatttaccagtgtaagggctgctgcttctccagagcgt | ^ CcACTACTCCAGCAAGGTCCAG G AAG AC AAT GTTGGTCC C AAAG AAC AT C AC C T CAGAfl | | tccacatgctgtgtggccaaagcatttatcagggtcacagtgatgggaaacatcaagtt | | ggagaaccacacccagtgctattgcagcacttgctatcaccacaagatttaa | represents the a chain without the signal peptide; and the dotted sequence represents the site for the restriction enzyme A / otl. The total length of the construction is 822bp.

[0054] The primers used for the amplification of the cDNA sequences were designed from the complete coding sequences of each

Petition 870190032656, of 4/4/2019, p. 30/66 eCG subunit contained in the NCBI database (http://www.ncbi.nlm.nih.gov; National Center for Biotechnology Information, US NLM - US National Library of Medicine, Bethesda, USA (NM_access number a: 001099763.1, β: 001490342.2), a computer program was not used for the design of oligonucleotides.

[0055] The amplification of the coding chains can be performed through the PCR technique (Polymerase Chain Reaction) and the products obtained are separated and purified by techniques usually known and used in the state of the art. Preferably, the obtained amplicons are detected on an agarose gel, isolated and purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healtcare, Carlsbad, CA, USA).

[0056] In another aspect of the invention, sequences of the coding gene can be designed comprising only one of the coding sequences of the eCG units, α or β.

[0057] More specifically, the construction of the fused gene encoding reCG occurs from a sequence of PCR reactions, divided into 3 stages, with the purified product. Between each round of PCR the product obtained was purified on an agarose gel.

[0058] The first round of PCR is responsible for adding the restriction sites to the end of the eCGa sequence and to the beginning of the eCGe sequence. In the second round, complementary gene fragments are added to the counter-subunit sequence. A complementary fragment was added to the beginning of both the eCGa and eCGe chains, allowing the subunits to join through these fragments. The third round is responsible for joining the mutated eCGa and eCGe chains into a single chain, eCGea. The obtained amplicons were detected on an agarose gel and purified with the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healtcare, Carlsbad, CA, USA).

[0059] After purification of the fused gene on an agarose gel, the gene was digested with the restriction enzymes EcoRI and NotI and subcloned into a plasmid vector.

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17/43 [0060] The gene can be cloned into any plasmid vector that contains one or more strong promoters, including the chicken β-actin promoter and the cytomegalovirus promoter, multiple bacterial cloning site, antibiotic resistance sequence for bacterial cloning, recombination sites, and the cDNA encoding the enzyme dihydrofolate reductase (DHFR) downstream of an internal ribosome entry site (IRES-international ribosomal entry site). Preferably, the plasmidial expression vector suitable for the invention is the vector pNU0, constructed by the depositor itself.

[0061] The vector pNU0 is derived from the vector pUC18 (MESSING AND VIEIRA, The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct; 19 (3): 259-68) and has an MCS cloning cassette containing several sites for restriction enzymes under the control of a hybrid promoter containing cytomegalovirus enhancer element and chicken β-actin promoter, as well as the cDNA encoding the enzyme dihydrofolate reductase (DHFR) downstream of a internal site for entry of ribosomes.

[0062] This structure allows the concomitant expression of the cloned insert in the MCS, in this case eCGea, and of DHFR, aiming at the gene complementation of double negative CHO cells for the DHFR gene, which, in turn, allows the selection of cells expressing high amounts of the product of interest after gene amplification, carried out through treatment with increasing doses of DHFR inhibitors.

[0063] The constructed plasmid was then cotransfected to the cells of interest along with a selection vector for the generation of cell clones stably expressing the fused protein eCGea.

[0064] The insertion of the plasmid in the cell of interest (cotransfection) can be performed by several techniques usually employed in the state of the art. Among them, several types of transfection, for example, by means of cationic molecules, calcium-mediated transfection, PEI, electroporation, nucleofection, as long as the transfectability rate is kept in the same order of magnitude.

Petition 870190032656, of 4/4/2019, p. 32/66 [0065] Preferably, cotransfection is performed by lipofection with selection vector. Preferably, the selection vector provides resistance to the antibiotic hygromycin B. More preferably, the appropriate selection vector is the vector pX343 (ARMELIN et al., Functional role for c-myc in mitogenic response to platelet-derived growth factor. Nature. 1984 Aug 23-29; 310 (5979): 655-60). [0066] The cotransfection of the pNUOeCGpa construct in the cells of interest with the pX343 vector was performed in a ratio of pX343 vector to the pNUOeCGpa construction ranging from 1: 1 to 1: 5000. Preferably, cotransfection was performed in a 1: 120 ratio. Therefore, few copies of the resistance vector are necessary to provide resistance to the transfected cells when the process of the present invention is employed. [0067] Still, the transfected cells, selected for being resistant to hygromycin due to the presence of at least one copy of the pX343 vector, have the advantage of containing a much larger amount of the construct of interest and, consequently, a high expression of the reCG protein .

[0068] As eCG is a complex molecule, presenting around 45% of its molecular mass consisting of post-translational modifications (glycosylation), it is necessary to use a robust expression system that allows the production of an eCG recombinant as similar as possible compared to wild-type eCG. The heterologous expression system according to the present invention was chosen for the production of reCG because it allows the correct processing of newly synthesized polypeptide chains, the folding and assembly of its α and β subunits, in addition to the addition of glycosylated chains of the type complex, which is essential for the produced recombinant eCG to have biological activity. However, there is a lot of variability in glycosylation patterns between different mammalian cells due to the fact that the enzymatic machinery for adding oligosaccharides to proteins differs between cell lines, generating glycoforms with different degrees and types of glycosylation and different biological activities.

[0100] The cells used for the expression of reCG are cells of mammalian lineage or insect cells, or any cell line that has

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19/43 gene expression profile determined for the transcripts of the enzymes of the glycosylation pathways. Therefore, any other cell line that is capable of adding post-translational modifications similar to that obtained for the reCG profile defined for the present invention, with the majority of peaks corresponding to di- or tri-galactosylated N-glycans being these later mono -, di- or tri-sialylates, added by the enzymes UDPGlnNAc-2-epimerase, CMP-SA-synthetase, CMP-SA-transporter, β-galactosidase α 2,3-sialyltransferase 3 (ST3-GalTIII), β-galactosidase α 2,3-sialyltransferase 4 (ST3-GalTIV), β-galactosidase α 2,3-sialyltransferase 6 (ST3-GalTVI), Sialidase I, Sialidase II, and which has the same behavior when grown in vitro, such as capacity replication, protein expression capacity, simple transfectability, among others, can be used.

[0069] Preferably, mammalian lineage cells were chosen; more preferably, cells of the CHO lineage, or derived from them. Even more preferably, the CHO-DG44-DHFR ^{- / -} strain is used. However, other cell types, such as cells of the CHO, BHK, 293, Vero lineage or derivatives thereof, are suitable for the invention and can replace cells of the CHO-DG44-DHFR ^{- / -} lineage. .

[0070] The selection step for resistant transfected cells was carried out by treating the transfected cells with the antibiotic to which they must be resistant for two weeks. Preferably, CHO-DG44 cells, according to the invention, were treated with between 10 pg / ml to 2,000 pg / ml hygromycin B (Life Technologies ™).

[0071] After treatment with hygromycin B, the surviving cells were treated with increasing concentrations of DHFR inhibitors in non-nucleoside culture medium for plasmid gene amplification and selection of reCG overproducing cells.

[0072] Various DHFR inhibitors are known in the art and may be suitable for use in the invention. Preferably, the DHFR inhibitor suitable for the invention is Methotrexate (MTX).

Petition 870190032656, of 4/4/2019, p. 34/66 [0073] Cells deficient in the production of the DHFR enzyme, such as the CHO-DG44 DHFR - / - cells, preferably used in the present invention, are suitable because they cannot survive when cultured in the absence of nucleosides, since this enzyme catalyzes the reduction of dihydrofolate to tetrahydrofolate. Thus, when these cells are transfected with constructions obtained from the vector of the invention, which has the gene sequence corresponding to the enzyme DHFR, they become able to grow in the absence of nucleosides.

[0074] The cells were subjected to an initial concentration of the DHFR inhibitor and cultured for approximately 2 or 3 weeks, changing the medium every 48 hours to remove dead cells. When 90% confluence was reached, the concentration of the DHFR inhibitor was increased and the cycle was repeated, always increasing the concentration of the DHFR inhibitor.

[0075] ReCG overproducing cells can be grown in batches of production in monolayers or in suspension. Different reservoirs can be used, such as cultivation bottles of different sizes, plates, T flasks, spinners, disposable culture bags (bags) and other types of bioreactors with propellers or rounded blades, among other configurations. [0076] The culture media employed can vary as long as they are able to supply the metabolic needs of the cells. In addition, reCG overproducing cells can be cultured in the presence or absence of fetal bovine serum (FCS). The proportion of fetal bovine serum can be adapted according to the culture conditions to maintain cell viability and protein expression levels (from 1 to 100 mg / mL) within the same order of magnitude as the original standard. Preferably, the cells of the invention are cultured in medium containing about 2 to 10% FCS. More preferably, the cells of the invention are cultured in medium containing 7% FCS.

[0077] Means chemically defined or free of serum and proteins, containing substitutes for FCS, such as peptide growth factors and cell proliferation of various types, albumin, insulin, among others; or fetal serum or

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21/43 adults of other species, and supplemented with precursors used in the glycosylation pathways, are also suitable for use in the production of reCG. Cultivation should take place under defined environmental conditions, such as temperature between 25 and 40 ° C, relative proportion of CO2 between 1 and 10%, and pH between 5.0 and 8.0.

[0078] When cultivation is carried out in spinners, bags and bioreactors, the rotation used can vary between 10 and 200 rpm.

[0079] To achieve greater mass and purity of the protein of interest, the product obtained can be partially purified using known techniques for protein purification, and the conditioned culture media containing collected reCGpa are subjected to at least one of the following techniques: precipitation proteins using solvents, such as ammonium sulfate; ultrafiltration; tangential filtration; or two different types of chromatography, ion exchange chromatography and affinity chromatography.

[0080] According to a second aspect of the present invention, a recombinant equine chorionic gonadotropin, produced from the process of the present invention, is provided. Unlike the commercially available options, the recombinant equine chorionic gonadotropin of the invention is produced through biotechnological mechanisms on an industrial scale offering an alternative option to those obtained from equine plasma and involving bioethical factors. In addition, the reCG of the invention will not face the problems encountered in the state of the art related to the scarcity of the source of origin, constituting advantageously a new source of uninterrupted supply of reCG.

[0081] By producing a reCG with a glycosylation pattern similar to that of the commercially available chorionic gonadotropins and which presents the same level of in vivo activity as the hormones FSH and LH, we obtain a product with at least similar biological activity. This characteristic is also not shown by the recombinant chorionic gonadotropins known in the art, which did not even have biological activity in vivo.

Petition 870190032656, of 4/4/2019, p. 36/66 [0082] In a third aspect, the present invention relates to a veterinary composition comprising:

a) a veterinarily effective amount of the reCG produced in accordance with the present invention;

(b) optionally additives; and (c) a pharmaceutically acceptable carrier.

[0083] The composition of the present invention can comprise, in addition to the reCG of the invention, adjuvants, preservatives, diluents, emulsifiers, stabilizers and other pharmacologically acceptable ingredients that are used in gonadotropin preparations for animal use.

[0084] In a fourth aspect, the present invention relates to the use (a) of reCG or (b) of the veterinary composition of the invention, comprising said reCG, in the manufacture of a medicament for the treatment of veterinary conditions related to reproduction and ovulation mammalian animals, as an optimizing agent for in vitro fertilization; in the manufacture of kits for diagnosis of veterinary conditions and protocols related to mammal ovulation; and in the manufacture of a supplement for cell culture.

EXAMPLES

Example 1: RNA extraction and cDNA synthesis from an equine pituitary gland [0085] A pituitary gland was obtained by necropsy of a 19 year old male horse (Equus caballus), half-blooded Arabian breed and immediately packaged in RNAholder (Bioagency © Biotechnology), at 4 ° C, until macerated in liquid nitrogen, using a crucible and pistil. Total RNA was extracted from this pituitary macerate using the Illustra RNAspin Mini RNA Isolation Kit (GE Healthcare®, Buckinghamshire, England), following the manufacturer's recommendations. The concentration and purity of the RNA obtained were determined through quantification on a nanospectrophotometer (ND-1000 Spectrophotometer, NanoDrop Technologies Inc., Wilmington, USA) and reading the Abs260 / Abs280 and Abs260 / Abs230 nm absorbance ratio.

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23/43 [0086] A 1.0pg aliquot of total equine pituitary RNA was used as a template for a reverse transcription reaction, using the ImProm ^TM -II RT enzyme kit (Promega Corporation, Madison, USA); Super Script II (Invitrogen), the reactions being carried out according to the manufacturer's recommendations. At the end, the samples were diluted 3 times in deionized water, obtaining a final volume of 60μ_ and stored at - 80 ° C.

Example 2: Amplification of the coding strands for eCG α and β, from this cDNA library, by PCR [0087] The primers used to amplify the cDNA sequences were designed based on the complete coding sequences of each of the two subunits of eCG contained in the NCBI database (http://www.ncbi.nlm.nih.gov; National Center for Biotechnology Information, US N_M - US National Library of Medicine, Bethesda, USA (NM_access number α: 001099763.1, β: 001490342.2) and synthesized by Thermo Fisher Scientific Inc., Waltham, USA ^TM / Thermo Fisher Scientific Inc. The lyophilized primers were reconstituted in 10mM Tris-Cl buffer solution (pH 8.0) to generate a 100μM stock solution and use at 10μM The sequences of the primers used in the amplification of these chains and their intermediates obtained in the Genetic Engineering steps are shown in Table 1.

Table 1. Sequences of primers for amplification of coding sequences for reCGa and reCGe and genetic engineering for generation of

fused eCGea sequence. Oligonucleotides Strings (5’- 3 ’) 1 -eCGb F 5'-ATGGAGACGCTCCAGGGGCT- 3 ' 2 -eCGb R 5'-AGAAGTCTTTATTGGGAGGGGATGAGAG- 3 ' 3 - eCGaF 5'-TTTCCTGATGGAGAGTTTACAACGCAG- 3 ' 4 - eCGa R 5'-TTAAATCTTGTGGTGATAGCACGTGCTG-3 '. 5 - Mut eCGa R Notl 5 '- G CGCGGCCGCTTAAATCTTGTTGTGGTG-3' 6 - Mut eCGb F EcoRI 5 '-C GGAATTCGCCACCATGGAGACGCTC-3' 7 - union F 5'-CCCAATAAAGACTTCTTTTCCTGATGGAGAG-3 '

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- union R

5'- CTCTCCATCAGGAAAAGAAGTCTTTATTGGG-3 '.

Bold: restriction sites for EcoRI (primer forward) and Notl (reverse primer) enzymes; Underline: Kozak consensus sequence.

[0088] The total equine pituitary tissue cDNA was used for the amplification of the coding sequences of both subunits of eCG. A single coding chain for both eCG subunits was created (JABLONKA-SHARIFF et al., Expression and bioactivity of a single chain recombinant equine luteinizing hormone (reLH). Theriogenology. 2007 Jan 15; 67 (2): 311-20) so that the subunits were expressed in a fused way. Primers (1) and (2) were used to amplify the eCGp coding sequence without the stop codon and the eCGa coding sequence was amplified without the signal peptide, using primers (3) and (4).

[0089] For amplification, polymerase chain reactions (PCR) were performed using the enzyme High Fidelity Platinum Taq DNA Polymerase (Thermo Fisher Scientific Inc., Waltham, USA ^TM ). To 100 ng of equine pituitary cDNA samples were added 1X High Fidelity PCR Buffer buffer (Thermo Fisher Scientific Inc., Waltham, USA), 0.2 mM dNTPs (Thermo Fisher Scientific Inc., Waltham, USA), 2 mM MgSO4 (Thermo Fisher Scientific Inc., Waltham, USA), 400 nM Forward primer (Thermo Fisher

Scientific Inc., Waltham, USA), 400 nM of Reverse primer (Thermo Fisher

Scientific Inc., Waltham, USA), 1.0 U of the DNA polymerase enzyme described above and deionized water to complete 50.0pL of reaction volume. The reaction was carried out under the following conditions: 94 ° C for 1 minute; 35 cycles of: 94 ° C for 30 seconds, 55 ° C (eCGa) or 58 ° C (eCGP) for 30 seconds and 68 ° C for 1 min; final extension at 68 ° C for 2 min.

[0090] Amplicons were detected using 2.0% agarose gels, stained with 0.5pg / mL ethidium bromide, and the expected size product was isolated with a scalpel and purified using the GFX DNA and Band Purification Kit (GE Healtcare, Carlsbad, CA, USA), according to the manufacturer's recommendations.

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25/43

Example 3: Obtaining a fused cDNA chain containing both sequences coding for eCG (eCGea) [0091] The purified products were used as templates for obtaining the eCGpa sequence, through three stages of subsequent mutagenic PCR reactions, as outlined in Figure 1, in which (A) amplification of the eCGa coding sequence without the signal peptide (α: 288 bp) and the eCGp coding sequence without the stop codon (β: 510 bp) / (B) intermediate constructions, ab and cd, obtained, respectively, in the first and second rounds of mutagenic PCRs / (C) Schematic diagram representing the Genetic Engineering steps performed to obtain the final eCGea insert - (1): representation of Figure 1A; (2): representation of Figure 1B (a and b); (3): representation of Figure 1B (c and d); (4): representation of the final product eCGpa / (D) Digestion of the plasmid pNU0-eCGea with the restriction enzymes EcoRI and NotI, where it is possible to visualize the vector pNU0 (7.6 Kbp) and the insert (822 bp), present in clones distinct bacterial (SP: signal peptide. TAA: stop codon. EcoRI and NotI: site for the restriction enzymes EcoRI and Notl).

[0092] The first PCR reaction step (Figure 1A) adds restriction sites to the end of the eCGa sequence (Notl), using the primers (3) and (5), and to the beginning of the eCGe sequence (EcoRI), using the primers (2) and (6). The PCR reactions were performed using the Long Range enzyme (Thermo Fisher Scientific Inc., Waltham, USA), in which 100X of the plasmideal preparations, containing the sequences eCGe or eCGa, were added 1X High Fidelity PCR Buffer (Thermo Fisher Scientific Inc., Waltham, USA), 0.2mM dNTPs (Thermo Fisher Scientific Inc., Waltham, USA), 2mM MgSO4 (Thermo Fisher Scientific Inc., Waltham, USA), 400nM Forward primer (Thermo Fisher Scientific Inc., Waltham, USA), 400nM Reverse primer (Thermo Fisher Scientific Inc., Waltham, USA), 1.0 U of the DNA polymerase enzyme described above and deionized water to complete 50.0 μΙ of reaction volume. The reaction was conducted under the following conditions: 94 ° C

Petition 870190032656, of 4/4/2019, p. 40/66 for 1 min; 35 cycles of: 94 ° C for 30 seconds, 55 ° C (eCGa) or 58 ° C (eCGP) for 30 seconds and 68 ° C for 1 min; final extension at 68 ° C for 2 min. The products obtained were fractionated on an agarose gel and then, in a procedure similar to that described in the paragraph above, purified from the agarose gel to serve as a template in the next stage of mutagenic reactions. [0093] In the second PCR reaction step, Figure 1B, complementary gene fragments were added to both amplicons to the counter-subunit sequence, so that both subunits could be joined through these fragments. The initial complementary fragment of the eCGa chain was added to the end of the modified eCGp chain in a reaction that used primers (6) and (7), while the final complementary fragment of the eCGp chain was added to the beginning of the eCGa chain and the beginning of eCGp sequence (EcoRI), using primers (5) and (8). For this second stage of mutagenic PCR reactions, the same reagents and concentrations as the first stage were used, under the following cycling conditions: 94 ° C for 1 min; 35 cycles of 94 ° C for 30 seconds, 62 ° C for 30 seconds and 68 ° C for 1 min; final extension at 68 ° C for 2 min. Again, the products of these reactions were purified from the agarose gel using the Illustra GFX PCR DNA and Gel Band Purification Kit and used as a template for the last mutation step. [0094] The third stage of mutagenic PCRs, Figure 1B and 1C, aimed to join the two previously mutated chains into a single chain, eCGpa. The purified templates from the previous step were added in a PCR reaction performed with the primers (5) and (6) and the same reagents and concentrations described, under the following conditions: 94 ° C for 1 minute; 35 cycles of 94 ° C for 30 seconds, 65 ° C for 30 seconds and 68 ° C for 1 minute; final extension at 68 ° C for 2 minutes.

[0095] Amplicons were detected using 2.0% agarose gels stained with ethidium bromide, and the expected size product was isolated with a scalpel and purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, Carlsbad, CA, USA). Then, they were cloned into the pGEM®-T vector, using the reagents provided by Promega

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27/43 pGEM®-T Easy System (Promega Corporation, Madison, USA) in Escherichia coli bacteria, strain XL-1 Blue (genotype: recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relAI lac [F 'proAB lacIqZÁM15 Tn10 (Tetr)] , Agilent Technologies, Santa Clara, USA).

[0096] The sequences of interest were checked on bacterial clones by automated sequencing (BigDye Sequencing Kit, Thermo Fisher Scientific Inc., Waltham, USA, on the ABI3700 I377 model sequencer (Perkin Elmer)). The sequences obtained in the sequencing were analyzed and aligned using the PhredPhrap programs (EWING, HILLIER et al., Basecalling of automated sequencer traces using Phred I. Accuracy assessment. Genome Res. 1998 Mar; 8 (3): 175-85) and BLAST (http://www.ncbi.nlm.nih.gov/BLAST/), respectively.

[0097] The bacterial clones were then expanded in LB lysogenic medium (Becton, Dickinson and Company) and stored at -80 ° C in a freezing medium with 15% glycerol.

Example 4: Cloning of eCG8a in the expression vector for mammalian cells [0098] The plasmid pGEM®-eCGp «was isolated from the bacterial clone selected using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific Inc, Waltham, USA.), Digested with restriction enzymes EcoRI and NotI (Thermo Fisher Scientific Inc.). The digested insert was purified from the agarose gel and then subcloned into a plasmid vector specially constructed by our group called pNU0.

[0099] For the subcloning, the vector was previously linearized by the action of the enzymes EcoRI and NotI, generating 5 'and 3' ends cohesive and complementary to the eCGpa insert, using the insert molar ratio: 3: 1 vector (MI = 3 x MV x TI / TV, where MI = insert mass, MV = vector mass, TI = insert size and TV = vector size). For the ligation reaction of the DNA fragments, the enzyme T4 DNA Ligase (New England Biolabs® Inc.) was used, as recommended by the manufacturer. The reaction took place at 16 ° C for 18h and gave rise to the vector called pNU0-eCGp «. Obtaining

Petition 870190032656, of 4/4/2019, p. 42/66 of positive bacterial clones of Escherichia coli, strain XL-1 Blue, containing this vector, were kept in glycerin solutions at -80 ° C, as previously described. Medium-scale plasmid preparations of a positive clone were obtained using the QIAGEN Plasmid Midi Kit (QIAGEN), following the manufacturer's guidelines, with the concentration of the samples determined by quantification in a nanospectrophotometer at 260 nm.

Example 5: Transfection of the construct of interest in CHO-DG44 cells by lipofection [00100] Cells of the CHO-DG44-DHFR ^{- / - line} , from Chinese Hamster ovary, were purchased as a donation and cultured in HamF12 medium (Thermo Fisher Scientific Inc ., Waltham, USA ^TM ) supplemented with 10% (v / v) FCS (Vitrocell Embriolife, Campinas, SP, Brazil) in adherent culture. The cells were always incubated in a humid atmosphere at 37 ° C and 2% CO2 / 98% air and kept in T flasks or treated culture plates, suitable for the cultivation of cells in adhesion (Thermo Fisher Scientific Inc .; Becton, Dickinson and Company; Corning Inc., Corning, USA; or TPP Techno Plastic Products, Trasadingen, Switzerland). The culture medium was changed every two or three days. When reaching confluence greater than or equal to 80%, the cells were subcultured, being washed with PBSA, followed by the addition of 0.1% trypsin solution and inactivation of it with culture medium containing FCS. The cell stock was maintained by freezing in culture medium supplemented with 10% DMSO (Sigma-Aldrich Co., St. Louis, MO, USA) and storage in liquid nitrogen.

[0101] For the generation of cell clones stably expressing the fused eCGpa protein, CHO-DG44 cells cultured in 60 mm diameter plates (P60) were cotransfected with the pNU0-eCGp «construct and with the selection vector pX343, which confers antibiotic resistance hygromycin B, using a DNA mass ratio of 120: 1 (8pg of the plasmid of interest to 66.7ng of the hygromycin resistance plasmid), as previously described (ARMELIN et al., Functional role for cmyc in mitogenic response to platelet-derived growth factor.Nature. 1984 Aug

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23-29; 310 (5979): 655-60). The plasmids were first mixed with 16μΙ_ of the transfection reagent FuGENE HD Transfection Reagent (Hoffmann-La Roche Ltd.) in culture medium in the absence of FCS to form the DNA-liposome complexes. The cells were incubated with the complexes and the culture medium was changed after 24h.

Example 6: Selection of a mixed population of transfected CHO-DG44 cells [0102] 48h after transfection, CHO-DG44 cells were treated with 200 μg / mL hygromycin B (Life TehnologiesTM) for selection of resistant cells in the midst of transfected population, for two weeks, until selection of a resistant sub-population.

[0103] The cell surviving hygromycin treatment must have integrated at least one copy of the resistance plasmid (in this case, pX343), therefore, it must have a much larger number of copies of the plasmid of interest (pNUO) -eCGpa.

Example 7: Gene amplification of the pNU-0- eCGgq construct in CHO-DG44 cells through treatment with increasing doses of methotrexate (MTX) [0104] The gene amplification occurred through treatment with methotrexate, started with 30nM of this drug added to the medium of culture without nucleosides and 7% dialysed FCS (dFCS).

[0105] The cells were cultured for approximately two weeks, with a medium change every 48 hours to remove dead cells, until the culture reached 90% confluence, when, then, the MTX concentration was increased. The treatment continued for approximately six months, with a gradual increase in the concentration of MTX each time the culture reached 90% confluence, with each treatment phase lasting two to three weeks. Example 8: Analysis of the expression of reCG in conditioned media by specific ELISA [0106] 10 ⁶ cells from the CHO-DG44 populations producing reCGpa were plated in 1mL of specific culture medium, in the presence of dFCS, in trays of six wells (B6). Plating was performed in

Petition 870190032656, of 4/4/2019, p. 44/66 duplicate, and the trays were maintained in the culture conditions described in Example 5. 24h after plating, the culture medium was discarded, the adherent cultures were washed with PBSA and received fresh culture medium. The medium of each clone / population was collected after 48 hours of conditioning and used to measure reCGpa through a specific ELISA (PMSG ELISA - ALPCO Diagnostics), following the manual provided by the manufacturer. Some dilutions of the collected conditioned medium (1:10, 1:25, 1:50 and 1: 100) were carried out, in order to obtain reCGpa values within the calibration curve of the kit. After the procedure, the absorbance values were determined at 460nm by reading on a plate spectrophotometer (SpectraMax M2 - Molecular Devices). A standard curve, provided by the kit, was used as a reference for a 4-parameter logistical analysis, performed by the GraphPad Prisma 5.00 for Windows software (GraphPad Software, San Diego California USA, www.graphpad.com). The values obtained for each population were considered in the selection of a candidate for the continuation of the experiments related to this project.

[0107] The level of reCGpa expression in the culture medium during treatment with increasing doses of MTX can be seen in Figure 3. Figure 3 shows in its abscissa the values related to the populations of CHO-DG44 cells overexpressing reCGpa which they are resistant to increasing doses of methotrexate (MTX); and in its ordinate, the values relative to the concentration of reCGpa (Uls / mL) present in the conditioned culture medium for 48 hours by each cell population.

[0108] Through the graph, it is possible to verify that the expression of reCGpa remains at similar levels until treatment with 500nM MTX, increasing dramatically to 23.1UIs and 41.3UIs of reCGpa being expressed in the CHO-DG44 cell populations transfectants subjected to growth in the presence of 1μΜ and 2.5μΜ MTX, respectively.

[0109] The CHO-DG44 population with the highest reCGpa expression was subjected to a reCGpa expression dynamic in which 10 ⁶ cells

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31/43 of these cells were plated in B6 trays, in the same manner as described in the previous procedure, for 10 additional passages, with the addition of culture medium containing or not FCS (fetal bovine serum). The conditioned media were collected 24, 48 and 72 hours after the exchange, diluted (1:10, 1:25 and 1:50) and submitted to the ELISA for PMSG, in exactly the same way as in the previous procedure. The level of expression of reCGpa over time was determined again by a 4-parameter logistic analysis, performed by the GraphPad Prism 5.0 software.

[0110] Figure 4 illustrates the kinetics of reCGpa expression by the CHO 2.5liM population after adaptation to growth in the absence of MTX, over three days, in the presence or absence of fetal bovine serum. The graph presents in its abscissa the values relative to the points (24h, 48h or 72h) of determination of the concentration of reCGpa present in the conditioned medium, with or without FCS (fetal calf serum) and in its ordinate the values related to the concentration of reCGpa UIs / mL) present in the conditioned culture medium 48h for each condition, with FCS (fetal calf serum) being the conditioned culture medium in the presence of fetal bovine serum, and SFM (serum free media) the conditioned culture medium in the absence of dFCS.

[0111] Through the data provided by Figures 3 and 4, we can verify that the expression of reCGpa by the population resistant to 2.5 lM of MTX decreased after 10 passages of removing the selective pressure exerted by MTX. Taking the 48h point as a comparison parameter, this decrease was in the order of 2X, from ~ 40UIs / mL with MTX (Figure 3) to ~ 20UIs without MTX (Figure 4) when the cells were cultured in the FCS condition, and in the order of 4X (from ~ 40UIs with MTX to ~ 10UIs without MTX) when the cells were grown in the SFM condition. Comparing the level of expression of the protein of interest between the FCS and SFM conditions, it is possible to state that the FCS condition showed a higher expression of reCGpa that can be determined by ELISA. A statistical analysis of the data, performed by Two-Way ANOVA (GraphPad Prism), followed by post-Hoc Bonferroni test, showed significance

Petition 870190032656, of 4/4/2019, p. 46/66 statistic (p <0.05) for this phenomenon at the 24h point and a difference with more stringent significance (p <0.01) between the FCS and SFM conditions was observed at the 48h and 72h points of this population.

Example 9: In vitro biological activity assays using the MLTC-1 responsive cell line [0112] To determine the in vitro biological activity of the reCGpa produced by the overproducing cells, the MLTC-1 cell line (Mouse Leydig Tumoral Cell - ATCC) was used -CRL2065), obtained from ATCC ©, which overexpresses gonadotropin receptors (hCG and hFSH), and a protocol adapted from LEGARDINIER et al. (LEGARDINIER et al., Involvement of equine chorionic gonadotropin (eCG) carbohydrate side chains in its bioactivity; lessons from recombinant hormone expressed in insect cells. Reprod Nutr Dev. 2005 MayJun; 45 (3): 255-9). This cell responds to eCG treatment by producing and secreting progesterone in a dose-dependent manner.

[0113] 10 ⁵ MLTC-1 cells were plated in 24-well trays, using 500pL of RPMI culture medium (Gibco®) supplemented with 10% FCS. When reaching 80% of confluence, these cultures were washed 2X with PBSA and lacking FCS for 2h, to then be treated for another 2h with several dilutions of conditioned culture media containing increasing doses of reCGpa and commercial eCG (Folligon - Intervet®). After treatments, these culture media conditioned by MLTC-1 cells were collected and stored at -20 ° C until they were sent for progesterone measurement by HPLC. Dosing can also be performed using ELISA kits.

[0114] Preparations containing increasing doses of reCGpa from CHO-DG44 cells cultured under conditions in the presence (FCS) or absence (SFM) of fetal bovine serum were used as experimental conditions. As a negative control, we used culture medium conditioned by CHODG44 cells not producing reCGpa, and as a positive control we used the commercial eCG Folligon (Intervet).

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33/43 [0115] The biological activity in vitro was determined by the amount of progesterone contained in the samples, which was measured by the high performance chromatography technique, performed by the company Nanocore Biotecnologia S / A. Two independent experiments were carried out for each sample. The two-way ANOVA variance test was used, with post-Hoc Bonferroni analysis, and the analysis was performed using the GraphPad Prism version 5.0 program. The in vitro biological activity obtained for each tested condition can be seen in Figure 5.

[0116] Figure 5 illustrates the in vitro biological activity of eCG assessed from the progesterone response secreted by MLTC-1 cells after treatment with increasing doses of Folligon or reCG of the present invention produced in the presence (A) or absence (B) of fetal bovine serum. The abscissa axis presents the values related to the treatments performed, which consisted of increasing doses (0.25 IU; 0.5 IU; 1 IU; 2 IU and 4 IU) of reCG of the present invention or Folligon; and the ordinate axis shows the values related to the concentration of Progesterone (ng / mL) secreted in the culture medium conditioned by MLTC-1 cells.

[0117] The results shown in Figure 5 show that there is no difference in response to the same dose of eCG from different preparations (FCS or SFM; CHO-DG44 or Folligon). However, by carefully observing the graphs, it is possible to notice a tendency towards a less robust response in conditions without serum (SFM condition), when compared to the same doses in the presence of serum (FCS condition). This result is very positive, as it indicates that the reCGpa produced by the overproductive reCG transfection cells has biological activity in vitro similar to the commercially available product, Folligon, even when produced in the absence of fetal bovine serum. All points showed a statistical difference (p <0.001) in relation to the negative control.

Example 10: In vivo biological activity assays using the Cole & Erway assay

Petition 870190032656, of 4/4/2019, p. 48/66 [0118] All experiments involving animals, as well as methods adopted for breeding and forms of maintenance and experimentation, followed the rules of the Animal Experimentation Ethics Committee (CEUA) of USP's Institute of Chemistry. The study was conducted through the test recommended by the British Pharmacopoeia, described by Cole & Erway (Cole and Erway, 1941). [0119] 60 25-day-old Sprague-Dawley rats, obtained at the Animal Experimentation vivarium of the Institute of Chemistry-Faculty of Pharmaceutical Sciences of the University of São Paulo, were organized in groups of 5 to 10 animals per box, receiving water and ration at libitum and maintained in periods of 12 hours of light and 12 hours of darkness, at constant 22 ° C.

[0120] The animals were divided into negative control groups (which received PBSA or conditioned culture medium by non-reCGpa producing cells), positive control group (which received Folligon®) and experimental groups, the latter containing the FCS conditions (media culture cells conditioned by reCG overproducing cells of the present invention containing 7% dFCS) or SFM (culture media conditioned by reCGpa overproducing cells in the absence of FCS).

[0121] Each experimental group, as well as the positive control, comprises five points (2.5UIs; 5.0UIS; 10UIs; 20UIs and 40UIs) with five animals. The experiment was repeated three times. Each animal received a single dose of 200pL, via subcutaneous injection, of a filtered preparation, through filters with 0.22pm pores, containing any of the treatments described above.

[0122] Three days after the injection, the animals were euthanized, using a CO2 chamber, and the ovaries and uteri of each animal were removed, dissected and weighed. The increase in the weight of these organs, compared to negative controls, corresponds to the animal's biological response to the stimulus with eCG.

[0123] The final answer for each dose was obtained through the arithmetic mean of the weight of the organs (ovaries or uteri) of the five animals at each point. The comparison of the response between the doses of the positive control (Folligon®) and the reCG of the present invention allowed us to draw a parallel between the powers

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35/43 of the first and the last. One-way ANOVA variance test was used, with post-Hoc Tukey analysis, and the analysis was performed using the GraphPad Prism version 5.0 program. The data were also analyzed by linear regression.

[0124] Figure 6 illustrates the biological activity in vivo assessed from the uterine response of prepubertal rats from 23 to 25 days after treatment with increasing doses of Folligon or reCG of the present invention produced in the absence of fetal bovine serum. In the experiment illustrated by Figure 6, the increase in uterine weight of the treated animals was evaluated, consisting of a direct response of the animals to the stimulus. The values related to the treatments performed, which consisted of increasing doses (2.5 IU; 5 IU; 10 IU; 20 IU and 40 IU) of reCG of the present invention or Folligon, as well as the negative controls PBSA (phosphate buffer in saline, without Ca ²⁺ and Mg ²⁺ ) and non-βα-producing CHO-DG44 are represented in the abscissa axis, and the values related to the weight (mg) of the reproductive organs dissected on the fourth day after treatment are presented in the ordinate axis. The uterine response observed during treatment with reCGea produced by CHO-DG44 cells, according to the present invention, was, in fact, quite similar to that observed for Folligon.

[0125] In Figure 7, there was a linear regression regarding the uterine response to the hormonal treatments administered. The abscissas axis shows the values related to injected doses (2.5UI; 5UI; 10UI; 20UI and 40UI) of reCG of the present invention or Folligon, or negative controls PBSA and CHO-DG44 not producing βα and the ordinate axis presents the values related to the weight (mg) of the reproductive organs dissected on the fourth day after treatment. In this analysis, the slope of the curve obtained for Folligon, 0.7263 ± 0.3767 (p = 0.0718) was not considered significant, whereas the slope obtained for the response to reCG of the present invention produced by CHO-DG44 cells was more consistent, 2.415 ± 0.4049 (p <0.0001). The controls did not show significant slopes. It was observed that the recombinant hormone produced

Petition 870190032656, of 4/4/2019, p. 50/66 by CHO-DG44 cells showed a better dose-response trend than the commercial product, Folligon. The last dose (40UI) of reCG of the present invention showed a similar response to Folligon.

Step 11: Purification of the reCG of the present invention by chromatography [0126] Two strategies were used for the purification of the reCG of the present invention contained in the conditioned culture media of overproducing cells, namely: a) Concanavalin A affinity chromatography (HiTrap ^TM ConA 4B, GE Healthcare); b) anion exchange chromatography (HiTrap ™ Capto Q, GE Healthcare). The Akta Purifier UPC-100 system (GE Healthcare), of high performance liquid chromatography (HPLC, High Performance Liquid Chromatography), associated with each of the columns at a time, was used to optimize the purification of the reCG of the present invention, in independent procedures. The constant flow of 1 VC / min was used throughout the process, for both columns.

[0127] For the purifications performed with the HiTrap ^TM ConA 4B column, the column was equilibrated with 3VC of buffer A (500 mM NaCl, 20 mM tris-HCl, 1mM MgCl2, 1mM CaCl2, pH 7.4). Then, 50 VC of conditioned medium containing reCGpa diluted 2X in dilution buffer (890mM NaCl, 40mM Tris-HCl, 2mM MgCl2, 0.2mM CaCl2, pH 7.4), filtered through 0.45pm pore filters were applied to the column, followed by washing the column (5 VC) with buffer A. For elution, a linear gradient of methyl-aD-glucopyranoside concentration (Sigma-Aldrich®) was applied, starting at 20 mM and ending up to 300 mM (diluted in buffer A), over an interval of 8 VC. The permeate and wash fractions were collected, as well as the eluate fractions, and kept at 4 ° C.

[0128] For the purifications performed with the HiTrap ^TM Capto Q column, the column was equilibrated with 3VC of buffer A (10 mM NaCl, 100 mM Sodium Acetate, pH 4.5). Then, 20VC of conditioned medium containing reCGpa was diluted 10X in buffer A, filtered through 0.45pm filters and applied to the column, followed by washing the column (5VC) with buffer A. For elution, different isocratic regimes of buffer B (1M NaCl, 100mM acetate

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Sodium, pH 4.5) were applied: 0-11mM NaCI; 11-19mM NaCI; 19-40mM NaCI and finished in 1M NaCl, in an interval of 12 VC. The permeate and wash fractions were collected, as well as the eluate fractions, the pH was adjusted to 7.4 and kept at 4 ° C.

[0129] For the two types of chromatography performed, the fractions, permeate, washes and eluates were diluted and subjected to detection of reCGpa by ELISA (PMSG ELISA Kit, ALPCO), following the manufacturer's protocol. The active fractions were concentrated using 10kDa cut filters (CentriconTM - Millipore®) and used for the following analyzes: analysis of the purification process in polyacrylamide gels, using the Coomassie Brilliant Blue dye, and in vivo testing (Figure 8).

[0130] Figure 8 illustrates the in vivo biological activity of the reCG of the invention partially purified by the HiTrap ^TM Con A column, from the uterine response of pre-pubertal rats 25 days after treatment with Folligon or reCGpa produced in the absence of fetal serum bovine and partially purified by the HiTrap ^TM Con A column. The abscissa axis presents the values related to the treatments performed, which consisted of doses (5, 10, 20 and 40 IU) of the Folligon or control reCG of the invention produced by CHO cells -DG44 and partially purified, and the PBSA negative control; the ordinate axis shows the values related to the weight (mg) of the reproductive organs dissected on the fourth day after treatment.

Example 12: N-glycosylation study by NP-HPLC of N-glycans labeled with 2-AB and digestion with exoglycosidases [0131] The fractions enriched in reCGpa obtained in Example 11 were concentrated around 100X using the Centricon device with 10kDa cut (Millipore®) and applied to SDS-PAGE gels, as described in Example 11. Samples of 20UIs of Folligon and 20UIs of the international standard of eCG were also applied to SDS-PAGE gels in order to perform the comparison proposal. The gels containing the samples to be analyzed were stained by Comassie Blue (1.25g of Coomassie R-250 Brilliant Blue diluted in 250mL of methanol, 50mL of glacial acetic acid and

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200mL of distilled water) for 16h. These gels were washed and bleached with a solution containing 7.5% acetic acid and 15% methanol in distilled water. After discoloration, the bands present in the eCG size range were excised from the gel, cut individually with a scalpel, dried for 8 hours in a vacuum centrifuge at 30 ° C (SpeedVac) and processed through a protocol adapted from ROYLE et al. (ROYLE et al., Determining the structure of oligosaccharides N- and O-linked to glycoproteins. Curr Protoc Protein Sci. 2006 Mar; Chapter 12: Unit 12.6).

[0132] The protocol consists of the release of glycans with a gel digestion carried out with PNGaseF and labeling them with 2-aminobenzene (Glyko Signal 2-AB labeling kit, Prozyme®). These samples of labeled glycans were then applied to an amide column (Waters X-Bridge 3.5um 4.6 x 250mm amide column), coupled to a pump (Waters Binary pump 1525u) and subjected to normal phase chromatography using the system Waters 2475, being detected by a fluorescence detector (Waters multi wavelength fluorescent detector, excitation at 330nm and emission at 420nm). All marked samples to be analyzed, as well as the glucose units standard (2-AB glucose ladder - Prozyme®) and the white (Ultrafiltered Water, Milli-Q - Millipore®) were diluted 5X in Acetonitrile before application to the column of amide. The program used is summarized in Table 2.

Table 2 - NP-HPLC program used to analyze recombinant eCGpa glycans of the invention and commercial eCG.

Time (minutes) Flow (mL / min) Buffer A Buffer B 0 0.86 20 80 48 0.86 50 50 49 0.86 100 0 53 0.86 100 0 55 0.86 20 80 63 0.86 20 80 64 0 20 80

Buffer A: 50mM Ammonium Formate, pH 4.4.

Buffer B: Acetonitrile. Column volume: 1mL.

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39/43 [0133] The calculation of glucose units (GU) corresponding to each peak obtained was performed by comparing their retention time against the glucose pattern curve (nine points), in a polynomial fifth order distribution. In this way, it was possible to determine GUs for each retention time obtained corresponding to each peak of each sample.

[0134] Aliquots from the pool of glycans obtained for each sample were dried by vacuum centrifugation and resuspended in 2μ_ of the 5X glycosidase buffer (Prozyme®), added with 1 μ_ of an array of glycosidases and water up to 10μ_. The following combination of glycosidases, in an incubation at 37 ° C for 16h, was used to digest the samples of glycans to their basal structures:

1. Sialidase (Glyko Sialidase A TM- A. Urefaciens - Prozyme);

2. Sialidase + Fucosidase (Glyko β-Fucosidase ™ - Bovine Testis - Prozyme);

3. Sialidase + Fucosidase + Galactosidase (Glyko β-Fucosidase ™ - Bovine Testis - Prozyme) and

4. Sialidase + Fucosidase + Galactosidase + Hexosaminidase (Glyko Hexosaminidase ™ - Jack Bean - Prozyme).

[0135] After digestion, these samples were purified in a glycan purification cartridge (Glyko clean S - Prozyme) and submitted again to HP_C, under the same conditions described above.

[0136] Comparisons between putative structures of glycans present in eCG described in the literature, pre-characterized structures available in an online bank (Glycobase 3.2) and the GUs obtained by HP_C allowed a quantitative and qualitative analysis of the glycans released by the successive digestions performed.

[0137] Figure 9 shows the putative structures selected for each peak, after comparison with the pattern of digestion performed with exoglycosities. On the abscissa axis there are the relative values of glucose units (GU) obtained for each chromatography, while on the ordinate axis are the intensities of the eluted peaks (EU). In panel A,

Petition 870190032656, of 4/4/2019, p. 54/66 are the undigested structures, and in panels B, C and D are the structures obtained after digestion with sialidase, sialidase + galactosity + fucosidase and sialidase + galactosidase + fucosidase + hexosaminidase, respectively.

[0138] Figure 10 shows the putative structures of N-glycans obtained in fractions enriched in reCG of the invention. On the abscissas axis are the relative values of glucose units (GU) obtained for each chromatography, while on the ordinates axis are the intensities of the eluted peaks (EU). The arrows indicate possible structures of N-glycans attributed to each peak obtained for reCGpa expressed in the CHO-DG44 strain. After analyzing the N-glycosylation profile of reCG, it was possible to verify the majority of peaks corresponding to di- or tri-galactosylated N-glycans, which were subsequently mono-, di- or tri-sialylated. This is in line with the N-glycan profile observed for the commercial eCG Folligon (Figure 9).

[0139] Figure 11 shows the results of gene expression obtained for the transcripts corresponding to the glycosylation pathway enzymes in control cells (non-eCG-producing CHO cells, called Crtl) and eCG-producing cells (called eCG), both cultured in the presence of fetal bovine serum (FCS) or in its absence (SFM - serum-free medium). Gene expression was analyzed using real-time quantitative PCR (Polymerase Chain Reaction) (qRT-PCR), using the GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) gene as a normalizer. The analyzed targets were: UDP-GlnNAc-2-epimerase, CMP-AS-synthetase, CMP-SAtransporter, β-galactosidase α 2,3-sialyltransferase 3 (ST3-GalTIII), βgalactosidase α 2,3-sialyltransferase 4 (ST3- GalTIV), β-galactosidase α 2,3sialyltransferase 6 (ST3-GalTVI), Sialidase I, Sialidase II. It was possible to obtain increased expression of UDP-GlnNAc-2-epimerase transcripts, CMP-AS synthetase, CMP-SA-transporter, β-galactosidase α 2,3-sialyltransferase 3 (ST3GalTIII), β-galactosidase α 2,3- sialyltransferase 4 (ST3-GalTIV), β-galactosidase α 2,3-sialyltransferase 6 (ST3-GalTVI) and Sialidase I.

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Example 12: Clinical trial performed on anesthetized cows [0140] A clinical trial was performed on anesthetized cows with the reCGpa product. The study involved 45 clinically healthy Bos indicus bovine females, weighing between 350 and 600 kg and at reproductive age between 3 and 10 years. The animals were kept in a paddock of Bracharia brizantha with a capacity of 1 AU / ha in continuous grazing system during the experimental period and had free access to water and mineral salt.

[0141] Animals that did not have reproductive diseases were included in the study; they did not receive any type of reproductive procedure (insemination, covering or embryo collection) 20 days before the beginning of the experiment; were clinically healthy at the beginning of the trial period and did not receive any disease treatments in the 10 days prior to the study; they had not received any hormonal product in the 30 days preceding the study; had a body condition score above 2.0 (scale from 1 to 5, AYRES et. al., Validation of body condition score as a predictor of subcutaneous fat in Nelore (Bos indicus) cows. Livest. Sci. 2009; 123: 175-179). To be included in the study, one of the animal's ovaries must have a dominant follicle.

[0142] The 45 females recruited, divided into three treatment groups (reCGp «-CHO-DG44, Negative Control and Folligon Positive Control), so that each group contained 15 animals. The treatment was distributed at random by randomized block design (DBC), within each category of follicular diameter (<8mm, between 8 and 10mm, between 10 and 12mm and> 12mm). The animals in the groups treated with CB050 formulations, prepared by concentrating the protein contained in the cell culture supernatant by ultrafiltration, formulating the concentrate with lactose, mannitol and methyl paraben; and lyophilization, and Positive control received a single dose of 300 IU / animal, intramuscularly, 8 days after the start of the ovulation synchronization protocol. While the animals in the Negative Control group received a single dose of 1.5mL of sterile saline.

Petition 870190032656, of 4/4/2019, p. 56/66 [0143] On the day the study started (D0), the animals were assessed for body condition score, general health status and reproductive evaluation via ultrasound. On that same day (D0), the females were previously synchronized. The synchronization consisted of the intramuscular administration of 2mg of estradiol benzoate and insertion of an intravaginal progesterone device. Eight days later (D8), the intravaginal progesterone device was removed and all animals received intramuscularly, 500pg of Cloprostenol (PGF) and 1 mg of estradiol cypionate. At that moment, the animals were distributed in groups, where they received the treatments (300UI of reCG of the invention; 300UI of Folligon or 1.5 mL of negative control).

[0144] Ultrasound evaluations (Kai Xin, KX 5100, Xuzhou KaiXin Electronic Instrument Company Ltd, China; Figure 6) were performed on all animals in the study, at the moments: animal selection (D0), on D8 for follicle evaluation dominant and separation of treatment groups; on D10 to assess follicular growth between D8 and D10; and at D18 to assess the ovulation rate and diameter of the corpus luteum.

[0145]

Table 3: Clinical study of reCG of the invention in Nellore cows in anestrus.

CLINICAL TRIAL Control CHO reCGPa Folligon P Number of animals 15 15 15 Body Condition score 2.6 ± 0.1 2.7 ± 0.1 2.6 ± 0.1 0.94 Weight (Kg) 443.7 ± 3.4 439.3 ± 7.8 434.0 ± 6.0 0.67 Age (months) 75.2 ± 2.2 76.8 ± 3.1 78.4 ± 2.6 0.88 0 follicular at D8 (mm) 10.4 ± 0.9 10.4 ± 0.9 9.6 ± 0.6 0.91 0 follicular in D10 (mm) 10.7 ± 1.0 12.6 ± 0.7 12.5 ± 0.8 0.27 Follicular growth from D8 to D10 (mm) 0.4 ± 0.1 ^b 1.4 ± 0.3 ^a 1.5 ± 0.3 ^a 0.01 0 of CL in D18 (mm) 20.6 ± 0.8 21.1 ± 0.6 20.9 ± 0.6 0.86 Ovulation rate (%) 60.0 (9/15) 86.7 (13/15) 86.7 (13/15) 0.07

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43/43 [0146] We can observe that the diameter of the dominant follicle of the different experimental groups was, on average, similar in size (P = 0.91), reinforcing the homogeneity of distribution of the different sizes of follicles between treatments. It was found that the cows in the Folligon and reCGpa groups achieved greater final growth of the dominant follicle (P = 0.01) than the control group. Similar results were observed by SALES et al. (SALES et al., Fixed-time AI protocols replacing eCG with a single dose of FSH were less effective in stimulating follicular growth, ovulation, and fertility in suckled-anestrus Nelore beef cows. Anim Reprod Sci. 2011 Mar; 124 (1 - 2): 128), in which treatment with eCG promoted greater follicular growth (eCG - 1.40mm / day vs No eCG - 0.95mm / day). There was no difference between the experimental groups in the diameter of the dominant follicle at D10 (P = 0.27), in the ovulation rate (P = 0.07) and in the diameter of the corpus luteum (P = 0.86).

Claims (16)

1. PROCESS OF PRODUCTION OF A RECOMBINANT EQUINE CHORONIC GONADOTROPHINE, characterized by the steps of: (a) inserting a DNA sequence of SEQ ID No. 1, which encodes an eCG into an expression vector that includes the cDNA encoding the dihydrofolate reductase (DHFR) enzyme; (b) co-transfection of the vector from step (a) with a selection vector in double negative expression CHO-DG44 cells for the DHFR gene; (c) Selection of resistant transfected cells by treatment with the antibiotic to which they must be resistant; (d) Gene amplification of the expression vector and selection of reCG overproducing cells by treatment with increasing concentrations of DHFR inhibitor; (e) cultivation of reCGa3 super-producing cells in serum-free medium; (f) purification of the obtained reCGa3. 2. PROCESS, according to claim 1, characterized by the fact that the DNA sequence encoding an eCG comprises a fused cDNA chain containing both the coding sequences of the equine chorionic gonadotrophin (eCG) chains. 3. PROCESS, according to claim 1, characterized by the fact that the expression vector is the vector pNU0. 4. PROCESS, according to claim 1, characterized by the selection vector being the vector pX343. 5. PROCESS, according to claim 1, characterized by the fact that the ratio of the expression vector to selection vector in the co-transfection stage varies between 1: 1 and 1: 5000. 6. PROCESS, according to claim 5, characterized by the fact that said ratio varies between 1:40 and 1: 120. 7. PROCESS, according to claim 6, characterized by the fact that said ratio is 1:40. Petition 870190032656, of 4/4/2019, p. 63/66 2/3 8. PROCESS, according to claim 1, characterized by the fact that the DHFR inhibitor is methotrexate. 9. PROCESS, according to claim 1, characterized by the fact that, under conditions of cell culture in the absence of supplementation with fetal bovine serum, the gene expression profile for the transcripts of the enzymes of the glycosylation pathways is defined by the increase, at least one and a half times, of the relative levels of the transcripts corresponding to the βgalactosidase α 2,3-sialyltransferase 3 (ST3-GalTIII), β-galactosidase α 2,3sylyltransferase 4 (ST3-GalTIV) and Sialidase I genes. PROCESS according to any one of claims 1 to 9, characterized in that it allows a rate of expression of reCG in the range of 1 to 100 mg / L. 11. PROCESS according to claim 1, characterized by the fact that the purification step is carried out by at least one among ion and affinity chromatography, protein precipitation, ultrafiltration or tangential filtration. 12. RECOMBINANT EQUINE CHORONIC GONADOTROPHINE, characterized by the fact that it is produced by the process defined in claims 1 to 11. 13. VETERINARY COMPOSITION, characterized by comprising: (a) a veterinarily effective amount of the reCG defined by claim 12; (b) optionally additives; and (c) a pharmaceutically acceptable carrier. 14. USE OF THE RECOMBINANT EQUINE CHORONIC GONADOTROFIN, defined in claim 12, or of the veterinary composition containing said gonadotropin, characterized by the fact that it is in the manufacture of a medicine for the treatment of veterinary conditions and protocols related to the reproduction and ovulation of mammals. 15. USE OF RECOMBINANT EQUINE CHORONIC GONADOTROFIN, Petition 870190032656, of 4/4/2019, p. 64/66 3/3 defined in claim 12, or of the composition containing said gonadotropin, characterized by the fact that it is in the manufacture of a diagnostic kit for veterinary conditions and protocols related to the reproduction and ovulation of mammals. 16. USE OF THE RECOMBINANT EQUINE CHORONIC GONADOTROFIN, defined in claim 12, or of the composition containing said gonadotropin, characterized by the fact that it is in the manufacture of a supplement for the cultivation of cells.