Methods for obtaining a mammalian cell line expressing recombinant equine chorionic gonadotropin (reCG), recombinant cell lines producing reCG, large scale reCG production methods, reCG, formulations containing reCG, nucleic acids encoding reCG, and uses

Abstract

A method for obtaining a mammalian cell line expressing recombinant equine chorionic gonadotropin (reCG) hormone is described. Also described is a cell line expressing reCG, a method for large scale production of reCG, a reCG having higher bioactivity with respect to PMSG, a formulation containing reCG, a nucleic acid encoding reCG and uses.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the development of protein hormones and their production in mammalian cells using recombinant DNA technology. Background Technology

[0002] Equine chorionic gonadotropin (eCG), along with luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH), is a member of the glycoprotein hormone family (Murphy and Martinuk, 1991). Because eCG is produced by the trophoblast cells of the endometrial cups in pregnant mares, it was originally called pregnant mare serum gonadotropin (PMSG). eCG plays a crucial role in maintaining early pregnancy (the first trimester) by indirectly stimulating progesterone production through progesterone until the placenta can secrete it spontaneously. The concentration of eCG secreted by the trophoblast reaches its peak around day 50 of gestation and then gradually declines (Allen and Moor, 1972).

[0003] Compared to other glycoprotein hormones, eCG has two unique characteristics. Firstly, in species other than horses, eCG exhibits high FSH and LH-like activity and a high affinity for the receptors of these hormones (Combarnous et al., 1984). Secondly, it exhibits a high carbohydrate content, accounting for 45% of its total molecular weight. This last property determines the long circulating half-life of eCG, approximately six days. Due to these two characteristics, eCG is used in veterinary medicine to control reproductive activity in various types of livestock, including cattle, sheep, goats, and pigs (Rensis and Lopez-Gatius, 2014).

[0004] Like other members of the glycoprotein hormone family, eCG is a heterodimeric protein composed of two distinct, non-covalently linked subunits (α and β). The α subunit is common to all members of the family and is encoded by a single gene, while the β subunit is encoded by different genes, giving it heterodimeric specificity (Stewart and Allen, 1976). The α subunit consists of 96 amino acids and has two N-glycosylation sites at Asn56 and Asn82, while the β subunit consists of 149 amino acids and has only one N-glycosylation site at Asn13. Furthermore, the β subunit has a carboxyl-terminal peptide (CTP) of 28 amino acids (122–149) containing 12 O-glycosylation sites at the Ser o Thr residues (Bousfield and Butnev, 2001). Both subunits contain multiple intramolecular disulfide bonds, and their assembly primarily occurs in the endoplasmic reticulum, representing a limiting step in the dimer secretion process (Hoshina and Boime, 1982). In equines, the placental CG and pituitary LHβ subunits are encoded by the same gene (Sherman et al., 1992). However, eCG is composed of a higher and more branched carbohydrate content than eLH. The N-glycan ends of the two hormones differ significantly. The glycan of eCG is capped with N-acetylneuraminic acid (sialic acid), while eLH exhibits sulfated N-acetylgalactosamine (SO42-). -4 -GalNAc) polysaccharides. The significant differences in their molecular weights are mainly due to the long disialylated poly-N-acetamide O-glycan structure in eCG (Smith et al., 1993). The high sialic acid content of eCG is the reason for its excellent circulating half-life, as this residue reduces glomerular filtration and liver metabolism.

[0005] Currently available products on the market are partially purified eCG preparations (PMSG) derived from the blood of pregnant mares, which have several drawbacks. Firstly, they exhibit batch-to-batch variability due to differences in glycosylation profiles between animals and in serum at different stages of pregnancy. Secondly, PMSG may contain contaminants with potential health risks. This contradicts the current regulatory trend towards safer veterinary products free from viruses, prions, and other contaminating proteins. Last but not least, obtaining eCG-containing serum from pregnant mares involves drawing 10 liters of blood weekly and subsequently inducing abortion by touching the uterus and rupturing the amniotic sac. This treatment of the animals is detrimental to animal welfare and is thoroughly questionable from a bioethical perspective: it is a bloody process that can lead to severe anemia and, in some cases, death.

[0006] For this reason, it is reasonable to develop recombinant eCG (reCG) as an alternative to PMSG. Several efforts to produce recombinant eCG in different hosts have been reported. On the one hand, Legardinier et al. (2005) described the production of recombinant eCG in two insect cell lines, Sf9 and Mimic. TM The latter (a cell line derived from the former, modified to express a different gene from mammalian glycosyltransferases) produces eCG. However, the produced hormone did not exhibit FSH / LH-like activity in an in vivo rat model, which the authors attributed to its extremely short circulating half-life due to the absence of terminal sialic acid in the oligosaccharide chain. On the other hand, Ubach et al. (2009) and Ingles et al. (2012) described the expression and purification of eCG in Pichia pastoris; however, the recombinant hormone described did not exhibit biological activity in in vivo bioassays in female rats. Again, these results were associated with the short circulating half-life of the recombinant hormone, as only 1% of the injected protein was detectable in serum after 90 minutes. This rapid clearance could be explained by the activation of the mannan-binding lectin pathway following injection of a protein with a high mannose content. These results demonstrate the importance of the correct glycosylation profile for the in vivo biological activity of eCG, and thus the importance of proper host selection for its recombinant expression.

[0007] The development of recombinant versions of eCG in CHO cells (including the CHO DG44 cell line) has been reported in publications and patents. Patent application WO2017112987A1 describes the use of such a CHO cell line and the acquisition of reCG with the desired glycosylation profile depending on the host used. Nevertheless, commercial versions of reCG are not yet available on the market. This demonstrates an unresolved issue that large quantities of recombinant eCG (or, in general, recombinant gonadotropins) cannot be obtained in an “efficient manner” or with the glycosylation profile that guarantees their bioactivity in vivo, as is the case with natural eCG (Hesser, 2011). Thus, the challenge lies in developing a production system that exhibits high productivity to obtain sufficient quantities of reCG to meet high demand (since the hormone is used in different types of livestock) and thus reduces production costs, a limiting factor for the commercial success of recombinant hormones. WO2017112987A1_2017 describes the use of a DHFR-MTX gene amplification system that yields a production rate of 18 IU / mL (in serum-free medium). This figure is not yet profitable compared to the current PMSG production process. Attached Figure Description

[0008] Figure 1 This is a schematic diagram of assembling third-generation lentiviral particles to obtain reCG-producing recombinant CHO-K1 cell lines.

[0009] Figure 2 The SDS-PAGE and subsequent Western blot are shown, in which the concentration of reCG in the supernatant from the reCG-producing cell line was assessed using a specific anti-reCG antibody.

[0010] Figure 3 The SDS-PAGE and subsequent Western blot are shown, in which the “apparent” productivity of the preselected reCG-producing clones was evaluated.

[0011] Figure 4 This is a graph comparing the changes in viable cell concentration, viability, lactate, and glucose concentrations over time when culturing P5C3 clones in a 1L bioreactor. Daily temperature and perfusion rates are shown. (↓) indicates bleeding from the bioreactor.

[0012] Figure 5 shows the structural characterization and purity analysis of (A) PMSG molecules after RP-HPLC, (B) reCG RP-HPLC, and (C) reCG HIC by SDS-PAGE, RP-HPLC, and SEC-HPLC.

[0013] Figure 6 shows isotype analysis. (A) IEF and subsequent staining with colloidal coomassie blue. Isotype profiles of PMSG variants (commercial formulation A, Foli-G) and CaptoB reCG eluents from cell lines and clones were compared. (B) IEF and subsequent Western blots of PMSG (commercial formulation A, Foli-G) and CaptoB reCG eluents from cell lines and clones were performed. (C) IEF and subsequent staining with colloidal coomassie blue. Isotype profiles of PMSG (commercial formulation B, Novormon), reCG RP-HPLC, and reCG HIC variants were compared.

[0014] Figure 7 The fluorescence emission spectra of PMSG, reCG RP-HPLC, and reCG HIC in sodium phosphate buffer solution are shown.

[0015] Figure 8 A notched box plot is shown, which illustrates the significant differences in Neu5Ac content (mol / mol protein).

[0016] Figure 9 Analysis of 2-AB-labeled N-glycans by WAX-HPLC, reCG RP-HPLC, and reCG HIC. N-glycans were separated according to their charge.

[0017] Figure 10Comparative pregnancy rates. Summary of the Invention

[0018] This invention describes a method for obtaining a mammalian cell line expressing recombinant equine chorionic gonadotropin (reCG), comprising the following steps:

[0019] a. Provide coding sequences for the reCGα and β subunits, which are optimized for their expression in mammalian cells;

[0020] b. Introduce the coding sequence into a lentiviral expression vector;

[0021] c. Producing lentiviruses containing reCG coding sequences;

[0022] d. Transducing mammalian cells using the lentivirus;

[0023] e. Select the most suitable mammalian cell clone to produce reCG.

[0024] The recombinant cell line exhibited a reCG production of at least 100 IU / mL in serum-free medium.

[0025] In a preferred embodiment of the invention, step a. of the method uses subunit sequences substantially similar to those α and β sequences of SEQ ID NO:1 and SEQ ID NO:2, respectively. In step b., the lentiviral vector consists of a pLV vector containing an EF-1α promoter. Step c. involves transiently transfecting HEK293 cells with pREV, pVSVG, pMDL, pLV-reCGα, and pLV-reCGβ plasmids using cationic lipids as a medium. Step d. involves transducing CHO-K1 cells.

[0026] In an alternative approach, the method of the present invention involves performing two consecutive transduction events.

[0027] Another object of the present invention is to provide a mammalian cell line obtained by the aforementioned method and containing nucleic acid encoding recombinant equine chorionic gonadotropin (reCG) hormone, wherein the coding sequences of the reCG α and β subunits comprise sequences substantially similar to those in SEQ ID NO:1 and SEQ ID NO:2. Preferably, the mammalian cell line is CHO-K1 and exhibits a reCG yield of at least 100 IU / mL.

[0028] Another object of the present invention is to provide a method for producing recombinant equine chorionic gonadotropin (reCG) hormone, comprising the following steps:

[0029] a. The mammalian cell line is cultured in a bioreactor in serum-free fetal bovine medium to produce the reCG on a large scale.

[0030] b. Harvest the supernatant, and

[0031] c. Purification.

[0032] This production method yielded a reCG production rate of at least 100 IU / mL in serum-free culture medium.

[0033] In a preferred embodiment of the invention, step a. involves incubation in a serum-free medium containing 50% commercial Excel 302 medium and 50% phosphate-buffered saline. In step c., purification comprises dye pseudoaffinity chromatography. Preferably, the dye pseudoaffinity chromatography uses a CaptoBlue-agarose matrix. Alternatively, purification step c. consists of tangential flow filtration and subsequent reCG concentration.

[0034] Optionally, the purification method also includes an HPLC purification step using a C4 column.

[0035] The reCG obtained by the production method comprises a specific activity of at least 6000 IU / mg (as a unit of in vivo potency related to protein quality determined by ELISA).

[0036] Another object of the present invention includes a nucleic acid encoding the reCGα subunit, which can be obtained by the described production method, comprising a sequence substantially similar to SEQ ID NO:1. It also includes a nucleic acid encoding the reCG β subunit having a sequence substantially similar to SEQ ID NO:2.

[0037] Another object of the present invention is a reCG hormone obtainable by the described production method, comprising a glycosylation profile having at least 3% neutral structure and at least 3% tetrasialylated structure. Preferably, the reCG comprises a glycosylation profile having at least 3% neutral structure, 26 to 30% monosialylated structure, 50 to 55% disialylated structure, 8 to 15% trisialylated structure, and at least 3% tetrasialylated structure.

[0038] Another object of the present invention is a pharmaceutical formulation comprising a therapeutically effective amount of reCG described herein. In a preferred form, the formulation is a liquid and is preferably kept refrigerated at 5°C without freezing for commercial use. In an alternative form, the formulation is lyophilized. In a preferred form, the formulation further comprises sugar, preservatives, antioxidants, mannitol, and anti-aggregating agents. Preferably, the formulation comprises trisodium citrate dihydrate, citrate monohydrate, arginine, sucrose, mannitol, L-methionine, poloxamer 188, m-cresol, and water.

[0039] Another object of the present invention is a method for inducing ovulation in animals, comprising administering reCG at a dose of at least 140 IU / animal. Ovulation is induced 48 hours after the administration of said dose. Detailed Implementation

[0040] This invention describes a method for obtaining a mammalian cell line (clone) expressing recombinant equine chorionic gonadotropin (reCG), which involves the following steps:

[0041] a. Provide coding sequences for the reCGα and β subunits, which are optimized for their expression in mammalian cells.

[0042] b. Introduce the coding sequence into a lentiviral expression vector.

[0043] c. Producing lentiviruses containing reCG coding sequences

[0044] d. Transducing mammalian cells with the lentivirus

[0045] e. Select the most suitable mammalian cell clone to produce reCG.

[0046] Cell clones with a concentration of at least 100 IU / mL can be obtained on a large scale in a bioreactor in serum-free culture medium using the methods described in this invention.

[0047] One of the key features of the method for obtaining a mammalian cell line for reCG production is that it utilizes an innovative and optimized DNA sequence encoding reCG. This sequence has been modified and optimized for expression in mammalian CHO cells, preferably CHO-K1. The reCG comprises at least one α subunit and one β subunit, and thus the encoding DNA sequences for both subunits have been optimized. The encoding sequence for each subunit is substantially similar to or identical to sequences SEQ ID NO:1(α) and SEQ ID NO:2(β).

[0048] Each subunit's coding sequence includes minor modifications in its nucleotides to best suit the transcription, translation, and post-translational mechanisms of CHO-K1 cells. After optimization, the recombinant sequence of the eCGβ subunit showed 82.2% homology with the unoptimized β subunit sequence (“natural” eCGβ), compared to lower homology (78%) with other recombinant sequences disclosed in different patents. It was observed that nucleotide positions in our optimized recombinant sequence that differed from residues in the natural sequence and optimized sequences disclosed in other patents accounted for 4% of the total. This percentage is sufficient to be a determinant of achieving higher levels of reCG expression compared to those reported in the prior art.

[0049] The method of the present invention for obtaining reCG-producing mammalian cell lines obtains stable reCG-producing cell lines by using a third-generation lentiviral vector as a genetic material transfer tool. The lentiviral vector used is pLV containing the EF-1α promoter. The vector carrying each coding sequence of each subunit is referred to as pLV-reCGα and pLV-reCGβ. In addition, the pLV contains the coding region of the puromycin resistance gene as a selection marker.

[0050] To produce lentiviral particles, this invention describes the use of cationic lipids as a medium to transiently transfect HEK293 cells with plasmids pREV, pVSVG, pMDL, pLV-reCGα, and pLV-reCGβ. Once lentiviral particles are obtained, they are used to transduce mammalian cells (step d). Preferably, the mammalian cells are CHO-K1 cells. Preferably, the process comprises two consecutive transductions.

[0051] Another object of the present invention includes a mammalian cell line having nucleic acid encoding recombinant equine chorionic gonadotropin (reCG) as part of its genome, wherein the coding sequences for the α and β subunits of said reCG comprise sequences substantially similar to those in SEQ ID NO:1 and SEQ ID NO:2. These cells are developed by the method of the present invention for obtaining a mammalian cell line for reCG production. Preferably, said cell line is CHO-K1. This cell line (clone) produces at least 100 IU reCG / mL in serum-free medium.

[0052] Another object of the present invention is to provide a method for producing recombinant equine chorionic gonadotropin (reCG) hormone, characterized by comprising the following steps:

[0053] d. The mammalian cell line (clone) is cultured in a serum-free medium in a bioreactor to produce the reCG on a large scale.

[0054] e. Harvest the supernatant, and

[0055] f. Purification.

[0056] The method for producing recombinant equine chorionic gonadotropin (reCG) of the present invention achieves a yield of at least 100 IU / mL in serum-free medium. These high production levels are due to several factors. First, the high reCG production rates of the cell lines transformed with the sequences SEQ ID NO:1 and SEQ ID NO:2 of the present invention have been further adapted and optimized for cultivation in bovine serum-free medium. The medium used for production is MC02 medium, which comprises 50% commercial Excel302 medium and 50% of a combination of salts, amino acids, carbohydrates, etc., as described in Table 2.

[0057] Another important factor is the purification step. Existing technologies describe complex and expensive purification processes, which makes the final product even more expensive. The method of producing recombinant equine chorionic gonadotropin (reCG) of the present invention incorporates a purification step including dye pseudoaffinity chromatography. In a preferred embodiment, the matrix used to perform said chromatography is CaptoBlue-agarose matrix. Optionally, an additional HPLC purification step using a C4 column may be added.

[0058] As an alternative to purification via dye pseudoaffinity chromatography, the purification steps include a tangential flow filtration step and a subsequent reCG concentration step.

[0059] The method for producing recombinant equine chorionic gonadotropin (reCG) of the present invention can yield reCG with a specific activity of at least 6000 IU / mg (as an in vivo potency unit related to protein quality determined by ELISA).

[0060] Another object of the present invention includes a nucleic acid encoding a reCG α subunit that can be obtained by the methods described herein. The α subunit of said reCG comprises a sequence substantially similar to SEQ ID NO:1.

[0061] Another object of the present invention includes a nucleic acid encoding a reCG βα subunit obtainable by the methods described herein. The β subunit of said reCG comprises a sequence substantially similar to SEQ ID NO:2.

[0062] In a preferred embodiment of the present invention, a sequence substantially similar to SEQ ID NO:1 means that the sequence has at least 90% identity with the sequence of SEQ ID NO:1.

[0063] In a preferred embodiment of the present invention, a sequence substantially similar to SEQ ID NO:1 means that the sequence has at least 95% identity with the sequence of SEQ ID NO:1.

[0064] In a preferred embodiment of the present invention, a sequence substantially similar to SEQ ID NO:1 means that the sequence has at least 98% identity with the sequence of SEQ ID NO:1.

[0065] In a preferred embodiment of the present invention, a sequence substantially similar to SEQ ID NO:2 means that the sequence has at least 90% identity with the sequence of SEQ ID NO:2.

[0066] In a preferred embodiment of the present invention, a sequence substantially similar to SEQ ID NO:2 means that the sequence has at least 95% identity with the sequence of SEQ ID NO:2.

[0067] In a preferred embodiment of the present invention, a sequence substantially similar to SEQ ID NO:2 means that the sequence has at least 98% identity with the sequence of SEQ ID NO:2.

[0068] The identity percentage was calculated by dividing the number of matching portions in the comparison window by the total number of positions in the comparison window and multiplying by 100. Identity was determined using the BLAST and BLAST 2.0 algorithms (see, for example, Altschul et al., 1990, J.Mol.Biol. 215:403-410 and Altschul et al., 1997, Nucleic Acids Res. 25(17):3389-3402).

[0069] Another object of the present invention includes a reCG hormone obtainable by the method of producing recombinant equine chorionic gonadotropin (reCG) of the present invention. The reCG hormone comprises a glycosylation profile having at least 3% neutral structure and at least 3% tetrasialylated structure. Preferably, the hormone comprises a glycosylation profile having at least 3% neutral structure, 26 to 30% monosialylated structure, 50 to 55% disialylated structure, 8 to 15% trisialylated structure, and at least 3% tetrasialylated structure.

[0070] Another object of the present invention includes a pharmaceutical formulation characterized by comprising a therapeutically effective amount of the reCG of the present invention. In one embodiment, the formulation is lyophilized. In another embodiment, the pharmaceutical formulation is a liquid. The pharmaceutical formulation of the present invention further comprises sugar, preservative, antioxidant, mannitol, and anti-aggregating agent. The liquid pharmaceutical formulation further comprises trisodium citrate dihydrate, citrate monohydrate, arginine, sucrose, mannitol, L-methionine, poloxamer 188, m-cresol, and water.

[0071] Another object of the present invention includes a method for inducing ovulation in animals, which involves administering reCG obtained by the method of producing recombinant equine chorionic gonadotropin (reCG) of the present invention at a dose of at least 140 IU / animal. This method aims to induce ovulation 48 hours after its administration.

[0072] For the production of reCG, this invention addresses all the disadvantages associated with the use of hormones obtained from the blood of pregnant mares (PMSG) and those disadvantages presented by all recombinant options described to date, none of which have entered the veterinary market:

[0073] - The process for producing reCG as described in this invention replaces the use of animals to obtain hormones, thereby eliminating the cruelty to animals that completely violates socially required bioethical and safety standards.

[0074] - The reCG obtained and described in this invention is a higher quality product derived from the cultivation of animal cells in a serum-free medium in a bioreactor, which allows for standardization of the production process. Therefore, the culture parameters and purification procedures are easily controlled and reproduced (unlike when using an animal host), resulting in a product (reCG) with higher batch-to-batch consistency, free from contaminants from animal plasma, and thus safer from a hygiene perspective;

[0075] - The method described in this invention allows for the production of reCG hormones exhibiting in vivo FSH / LH activity, which differs from other techniques described in the literature that use other types of cell hosts and do not allow for the production of recombinant hormones exhibiting in vivo activity.

[0076] - Fewer International Units of reCG obtained and described in this invention are needed to treat animals. Because the reCG of this invention is more effective, only 100 IU / animal is needed for IATF treatment in zebu (Bos indicus) and only 140 IU / animal for cattle (Bos taurus), instead of the 300 IU and 400 IU required respectively when using PMSG obtained from pregnant mares. Furthermore, for SOV treatment, only 1000 IU is needed in zebu and only 2000 IU for cattle (Bos taurus), instead of the 2000 IU and 4000 IU required respectively when using PMSG obtained from mares;

[0077] - After treatment with 140 IU reCG per animal, 85% of cases restored the anestrous cycle in the cattle, compared to a success rate of 65% after PMSG treatment. This is significant because these animals, in very poor physical condition, only enter a cyclical state due to the effects of hormones on the ovaries. Without hormones, the animals remain in a non-cyclical state (anestrous phase) and are therefore considered non-productive animals.

[0078] - This enables more efficient animal synchronization. This greatly facilitates their use in fixed-time artificial insemination (FTAI) and superovulation (SOV) protocols. All cows treated with reCG of this invention for FTAI ovulated 48 hours after administration, while cows treated with PMSG ovulated 50 to 60 hours after administration. This makes the fixed-time insemination-pregnancy program effective and profitable, which is crucial for livestock management where hundreds of cows require treatment. Thus, cows treated "in the morning" can be inseminated at the same time two days later;

[0079] Compared to other PMSG alternatives obtained in animal cells, such as CHO DG44 (which appears to exhibit a satisfactory glycosylation profile), this invention allows for the production of reCG with the highest productivity described to date. Thus, this invention overcomes a major limiting disadvantage that prevents the commercial availability of recombinant forms of the hormone: this invention is a technique capable of producing large quantities of biologically active reCG at low cost. The high productivity and the use of low-cost serum-free culture media contribute to reducing the overall cost of the method of this invention. The reCG obtained using a high-recovery single-step purification method or the product obtained through a single tangential flow filtration and concentration procedure of the initial sample exhibits a satisfactory glycosylation profile and therefore demonstrates in vivo biological activity in rats, cows, and pigs.

[0080] - A reCG liquid formulation was obtained, which is stable over time and is therefore superior to existing commercially available products composed of lyophilized or frozen liquid formulations, which have all the disadvantages associated with them.

[0081] The following will describe examples of measurements performed to achieve and carry out each object of the present invention. It should be noted that these examples are for illustrative purposes and are not intended to limit the scope of the invention.

[0082] Example

[0083] Example 1 - Encoding Sequence Design and Optimization

[0084] The technology developed in this invention primarily relates to the development of mammalian cell lines, particularly suspension CHO-K1 cells (Chinese hamster ovary cells), which produce recombinant equine chorionic gonadotropin (reCG). The coding sequences for the α and β subunits of reCG (reCGα and reCGβ) have been optimized for expression in CHO-K1 cells to obtain high levels of mRNA and thus maximize the expression of the encoded protein. Gene optimization leverages the degeneracy of the genetic code, whereby proteins can be encoded by a variety of alternative genetic sequences. Since codons are used differently in each organism, this can lead to defects in the expression of recombinant proteins in heterologous hosts, resulting in very low expression. Therefore, gene optimization algorithms allow for multi-parameter optimization of DNA sequences, covering multiple aspects of gene expression: mRNA transcription, splicing, translation, and degradation, to achieve the most efficient expression of a given protein.

[0085] After optimization, it was observed that the recombinant eCGβ sequence shared 82.2% homology with the unoptimized β subunit (“natural” eCGβ) sequence, while showing lower homology (78%) with other recombinant sequences mentioned in the background section of this invention. It was observed that 4% of the nucleotide positions in the coding sequence of the optimized recombinant hormone of this invention differed from residues in the natural sequence and residues in optimized sequences disclosed in the prior art; this is sufficient to be a determining factor for obtaining higher levels of reCG expression compared to those reported in other patents.

[0086] The synthesized sequences were obtained as DNA and then cloned into the p-α_eCG(AmpR) and p-β_eCG(AmpR) vectors, respectively. These vectors contain a bacterial origin of replication (Col E1 origin), which allows plasmid amplification into the *E. coli* and ampicillin antibiotic resistance gene (AmpR). The p-eCGα vector is 3000 bp and contains a 360 bp eCGα coding sequence, of which the first 72 bp encodes the natural eCGα signal peptide. The p-eCGβ vector is 3200 bp and contains a 507 bp eCGβ coding sequence, which includes a 60 bp signal peptide at the beginning of the sequence.

[0087] Example 2 - Construction of Expression Vector

[0088] To obtain stable reCG production cell lines, a third-generation lentiviral vector was constructed as a method for genetic material delivery. For this purpose, the coding sequence of each subunit was first cloned into the lentiviral transfer vector, followed by the assembly of lentiviral particles (LPs) and their subsequent titration.

[0089] Lentiviral expression vectors encoding the α and β subunits of reCG were constructed. The vector containing the α subunit was digested with the Xbal / EcoRV enzyme to release the reCGα coding sequence, which was cloned into the Nhel / EcoRV site of the lentiviral plasmid vector pLVenhCEF. The vector containing the β subunit was digested with the BamHI / EcoRV enzyme to release the reCGβ coding sequence, which was cloned into the BamHI / Smal site of the pLVenhCEF vector. This vector, developed in our laboratory, contains the EF-1α promoter as an expression regulatory element, characterized by high expression levels in various animal cells. It also contains an expression-stimulating fragment derived from a CMV enhancer sequence and a coding region for a puromycin resistance gene as a selection marker. The resulting plasmid was amplified in prokaryotic cells (E. coli), cultured under shaking conditions, and purified by organic solvent extraction and precipitation. Sequencing of the selected E. coli clones confirmed the identity of the DNA fragments cloned in plasmids pLVenhCEF-reCGα and pLVenhCEF-reCGβ, indicating 100% homology with the sequences of the synthetic eCGα and eCGβ genes, respectively.

[0090] To produce third-generation lentiviral particles, transient transfection was performed on HEK293 cells (packaging cells) using four plasmids: pREV, pGlyco-G, pMDL (packaging plasmid), and transfer vectors pLVenhCEF-reCGα and pLVenhCEF-reCGβ (encoding each reCG subunit). For this purpose, cationic lipids were used as DNA vectors. The supernatant containing lentiviral particles was harvested 30 hours post-transfection, concentrated by centrifugation at 65,000 g, and stored at 80°C until use. Figure 1 ).

[0091] Using QuickTiter TM Lentiviral particle titers were performed using a lentiviral titer kit (Cell Biolabs Inc.). This kit is designed to detect only the lentiviral-associated HIV-1p24 core protein; therefore, residual free protein in the supernatant does not interfere with the assay. Consequently, the physical titers of reCGα and reCGβ obtained were 2.7 x 10⁻⁶. 9 LP / mL and 2.1x10 9 LP / mL, which can be approximated by a transduction titer of 5.0 x 10⁻⁶.6 and 3.9x10 6 The titer is TU / mL, thus producing a high titer for transducing CHO-K1 cells.

[0092] Example 3 - Cell line generation

[0093] The obtained lentiviral particles were used to generate reCG in a suspension to produce recombinant CHO-K1 cell lines. Two consecutive transduction events (Td1 and Td2) were performed, as cells did not survive to a third transduction event. The transduced cell lines were subjected to selective stress by incubation with increased concentrations of puromycin. This strategy enriched the population with cells resistant to higher concentrations of the antibiotic, thereby improving the overall productivity of the cell lines. Figure 2 The results show an SDS-PAGE assay and subsequent Western blot using a specific polyclonal anti-reCG antibody. It can be observed that, for the same cell density, the highest reCG concentration was obtained in the supernatant of the sCHO Td2(200) cell line (which is resistant to up to 200 μg of puromycin).

[0094] The reCG productivity of the resulting cell lines was then assessed by determining the accumulated hormone concentration in the supernatant and the initial and final cell densities after a period of time. ReCG quantification was performed using a competitive ELISA developed in our laboratory. The assay involved a competition between a solid-phase immobilized antigen (reCG) and a solution of the same antigen (reference reCG or an unknown sample) for binding to a specific rabbit anti-reCG antibody (pAb anti-reCG). These antibodies were previously obtained in our laboratory. A peroxidase-conjugated secondary antibody was then added to detect any remaining solid-phase binding complexes. The productivity of the different cell lines is summarized in Table 1.

[0095] Table 1. reCG specific productivity of laboratory-scale generated cell lines

[0096] cell lines <![CDATA[Productivity (μg x 10 6 cells -1 x days -1 )]]> sCHO Td1 0.37 sCHO Td1(5) 0.32 sCHO Td2 0.96 sCHO Td2(200) 0.78

[0097] Taking these results into account, the cell line with the highest reCG production (reCG Td2 cell line) was selected for cloning.

[0098] Example 4 - Cloning and Separation

[0099] The reCG productivity of the generated cell lines allows for selection of one of them, thereby obtaining single-cell clones with a qualified growth profile and high reCG productivity.

[0100] To select clones with the highest reCG expression levels, more than 400 clones were evaluated in the initial dot blot screening using a specific anti-reCG antibody. The selected clones were cryopreserved. Following a pre-selection step to reduce the number of clones to be analyzed, the “apparent” productivity of the selected clones was assessed by SDS-PAGE and subsequent Western blotting (which was determined as the concentration of reCG obtained at the same cell density for each clone). Figure 3 This analysis showed that clones P5D9 (lane 3) and P5C3 (lane 4) exhibited the highest reCG expression levels. The reCG productivity of the selected clones was then assessed by determining the accumulated hormone concentration in the supernatant and the initial and final cell densities after a period of time. The reCG concentration was determined using the competitive ELISA described above. The estimated productivity of P5D9 and P5C3 clones were 0.80 and 0.81 μg x 10⁻⁶, respectively. 6 individual cells -1 x days - 1 Finally, the P5C3 clone was chosen because it exhibited better growth performance than the P5D9 clone.

[0101] Example 5 - Optimizing culture conditions to achieve optimal glycosylation

[0102] 5.1 High-density culture of P5C3 clones in serum-free medium in a one-liter bioreactor

[0103] P5C3 clones were cultured for 27 days in serum-free fetal bovine medium (MC01) in a 1-liter bioreactor using perfusion mode. Culture was carried out at 7.8 x 10⁻⁶. 5 cells.mL -1 The cell density initially increased and exhibited exponential growth without a hysteresis phase until it reached 1.6 x 10⁻⁶. 7 cells.mL -1 Maximum cell density was achieved. Cell viability was above 94%. Perfusion rate was initiated on day 3 of culture and varied daily between 0.21 and 1.00 reactor volumes. Lactate concentration was maintained at 1.1 g / L. -1 (12.2mM) and below Figure 4 The specific growth rate of the clones was 0.013 hours. 1 .

[0104] Subsequently, a new production medium (MC02) was formulated. Important note: This medium composition was obtained by combining commercial mediums with salts, amino acids, carbohydrates, etc. (Table 2). This was done to optimize the cost of the medium, reducing its value by 50%.

[0105] This new composition does not alter cell productivity, nor does it change any molecular properties.

[0106] Table 2. Culture medium (MC02) composition

[0107] <![CDATA[g.l -1 ]]> Excel 302 10.5 bicarbonate 1.6 glutamine 0.5825 glucose 2.25 NaCl 4.41 KH2PO4 0.304 Na2HPO4 0.32 Polosham 188 0.5

[0108] 5.2 High-density culture of P5C3 clones in serum-free medium in a 50-liter bioreactor

[0109] Production-scale culture. After culturing cells in a 1-liter bioreactor, the process was scaled up to a 50-liter bioreactor to reproduce the cell culture parameters tested at laboratory scale. With a perfusion rate of one bioreactor volume per day, 50 L of harvest was obtained during this fermentation process, each containing 157 IU / mL reCG. Considering that one dose of the reCG of this invention consists of 140 IU and 50,000 mL of supernatant containing 157 IU / mL was harvested, a total of 7,850,000 IU was obtained over 24 hours. Therefore, the production method of this invention produces approximately 56,000 doses of reCG per day at a production scale (50 L). Thus, a harvest exceeding one dose / mL was obtained at a production scale. Comparing these results with current production methods (extracted from pregnant mares), the culture conditions developed in the 50 L bioreactor represent approximately 600 mares. In other words, the technology reported in this invention can produce the same dose as that produced by 600 pregnant mares in 200 days during a 25-30 day biological process (including culture, purification, formulation, and packaging) [assuming that all mares have the same eCG concentration in their blood and that the eCG obtained from each mare has the same quality (which is impossible)].

[0110] Furthermore, reCG productivity of the P5C3 clone was calculated on a production scale (50L bioreactor) in different culture media with varying costs (Table 3). These media included the original commercial media (MC01, EX-CELL 302), the optimized MC02 medium (previously described), and MC05 medium. Higher productivity was obtained in this last medium, showing a greater difference compared to results published by other authors, and at a lower cost.

[0111] Table 3. ReCG production rate of P5C3 clone in different culture media and cost of each culture medium in a 50L bioreactor.

[0112]

[0113] Culture medium 01 (MC01): Ex-Cell CHO 302

[0114] Medium 02 (MC02): MP01 / P2G 50 / 50 (MP 02) optimized medium

[0115] Culture Medium 05 (MC05): BHK-21CD Production Culture Medium

[0116] In patent application WO2017112987, they used the DHFR-MTX gene amplification system. Instead of reporting productivity, they reported the kinetics of reCGβα expression after adaptation to growth in the absence of MTX. By reading the document, approximate cumulative values ​​of reCG in IU / mL were obtained: 10 IU / mL (24 h), 20 IU / mL (48 h), and 28 IU / mL (72 h) for cell lines cultured in the presence of fetal bovine serum, and 5 IU / mL (24 h), 10 IU / mL (48 h), and 18 IU / mL (72 h) for cell lines cultured in the absence of fetal bovine serum.

[0117] Compared to these results, the cell clones obtained in this invention produced 45.6 IU / mL (P5D9) and 50.2 IU / mL (P5C3) on a small scale within 72 hours in the absence of fetal bovine serum, while achieving greater than 15 IU x 10⁻⁶ in continuous perfusion mode in a bioreactor. 6 individual cells -1 x days - 1 The productivity level corresponds to 157 IU / mL over 24 hours, representing a significantly higher value than that reported in patent WO2017112987 (which reported 5 IU / mL over 24 hours in the absence of fetal bovine serum). This implies the production of over 7,000,000 IU per day, representing over 50,000 doses of the reCG of this invention per day.

[0118] Thus, the technology of this invention allows for the highest reported productivity and output value to date, due to a combination of unique factors in our technology: optimization of the sequence for expression in CHO-K1 cells (grey hamster species), the use of our own third-generation lentiviral vector, and the use of the CHO-K1 cell line expressing multiple glycosyltransferases that, in addition to generating monosialylated and disialylated mucin-type O-glycans, can also add disialylated, trisialylated, and tetrasialylated complex N-glycans to peptides. These are key factors in the in vivo bioactivity of eCG.

[0119] In fact, the unique DNA sequence optimization process is effective in achieving high hormone expression, as evidenced by the fact that 4% of the nucleotides in the protein’s natural DNA sequence have been modified and remain unchanged in the synthetic sequences reported in other patents.

[0120] Example 6 - Purification

[0121] 6.1 - First Purification Step

[0122] Following the culture of CHO cells in a bioreactor in serum-free medium of Excell 302 (Sigma), a first capture purification step was developed using harvested material. Dye pseudo-affinity chromatography was therefore chosen, employing CaptoBlue-agarose resin packed in an XK column (GE Healthcare) and equilibrated in 20 mM Tris-HCl buffer at pH 7. The untreated, clarified harvest was loaded onto the resin at a flow rate of 153.06 cm / h for a total retention time of five minutes. After a washing step with the same equilibration solution, proteins were eluted using an isocratic gradient (Tris-HCl buffer pH 8, 2 M NaCl, 20% (v / v) ethanol). No hormone leakage was observed during the loading and washing steps of the first chromatography, indicating that the loading conditions were satisfactory. The purity level of the recovered intact hormone was significantly higher than that obtained from partial purification from pregnant mare serum (PMSG). Therefore, the reCG capture step of the cell culture supernatant was optimized using dye pseudo-affinity resin. With a recovery rate of 98% (assessed by ELISA and RP-HPLC), a high yield was achieved without any protein loss.

[0123] 6.2 - Second purification step

[0124] Hydrophobic interaction chromatography was chosen as the second purification step because the partially purified hormone from the first capture step (named post-Blue) eluted under high ionic strength conditions (2M NaCl). To reduce the number of operating units and lower the overall cost of the purification process, the following strategies were proposed: 1) Loading a “crude eluent,” i.e., the untreated post-Blue fraction, thus avoiding the dialysis step (since this fraction is under high ionic strength conditions); 2) Screening for two types of hydrophobic ligands available in our laboratory: phenyl and butyl; 3) Evaluating the eluent for dialysis against citrate / citrate buffer at pH 6.0, as this is the condition for preparing the post-Blue API (named FD1RECG); and 4) Evaluating purification performance using different salts: first NaCl (because it is a salt present in the post-Blue buffer), then Na2SO4, and finally (NH4)2SO4 (because this is the salt with the highest hydrophobic interaction). Considering the purification performance (recovery and purity) of all strategies, the optimal conditions were as follows: DF1REG was loaded onto butyl agarose 4FF resin at a flow rate of 15 cm / h for a total retention time of three minutes. To improve the hydrophobic interaction between the protein and ligand, the resin was equilibrated with 50 mM citrate / citrate buffer at pH 6.0 and 2 M (NH4)2SO4, and the sample (DF1REG) was treated with the same equilibration buffer. Subsequently, two washing steps were performed: the first washing step used the same buffer as in the equilibration step, while the second washing step used a buffer with lower ionic strength (50 mM citrate / citrate buffer at pH 6.0 and 1.5 M (NH4)2SO4) to remove impurities. Finally, isocratic elution was performed with 50 mM citrate / citrate buffer at pH 6.0 and 0.5 M (NH4)2SO4.

[0125] Example 7 - Blending Materials

[0126] 7.1 - Liquid formulations for the final product

[0127] 7.1.1 - Developing reCG liquid formulations using QbD tools

[0128] This invention develops a reCG liquid formulation that allows for the acquisition of a stable and therefore active liquid form of the hormone. This avoids the lyophilization process, which represents a more complex and costly unit operation. Therefore, obtaining a liquid formulation instead of a lyophilized formulation not only ensures reduced costs but also shortens the production cycle, thereby avoiding the reconstitution step of the lyophilized product. Furthermore, in the case of using multiple doses, the required amount can be split, thus ensuring its long-term stability during use.

[0129] 7.1.1.1 - Pre-blending determination - Identifying key factors affecting the stability of reCG in liquid formulations

[0130] For the thermally forced degradation study of reCG purified by CaptoBlue-agarose chromatography, temperatures were modified between 20 and 70 °C, and pH ranges were employed between 3.0 and 8.0. Samples were heated in a thermal cycler for ten minutes under each condition and stored at -70 °C until analysis. ReCG aliquots under each condition were then evaluated in non-reducing SDS-PAGE followed by Coomassie Brilliant Blue staining to visualize the degree of reCG dissociation. Differences in mobility spectra in the SDS-PAGE indicated the effect of pH on the stability of the reCG heterodimer. Samples incubated at higher temperatures corresponding to the lower pH range (pH 3.0 to 5.0) exhibited different banding patterns compared to samples corresponding to the more alkaline pH range (pH 6.0 to 8.0). In parallel, emission (fluorescence) spectra (excitation: 274 nm) were evaluated to analyze possible conformational changes. A redshift was observed in those samples subjected to higher temperatures and lower pH values. The redshift may be associated with a higher denatured protein pattern or loss of the native conformation. Taking these results into account, and with the aim of reducing chemical degradation processes (primarily deamination and oxidation, which have a smaller impact at neutral pH), the optimal pH range was determined to be between 5 and 7. Similarly, since the protein's p1 (isoelectric point) is close to 3.5 to 5.5 (as determined by IEF), operating within a pH range of 5 to 7 will ensure that the protein exhibits a negative net charge, thereby reducing physical degradation events such as aggregation (the opposite of what might occur at pH values ​​closer to reCG).

[0131] 7.1.1.2 - Determination of blending ingredients

[0132] One of the major challenges in the production of biotherapeutic proteins is obtaining formulations that guarantee high protein quality and stability. By combining experimental design (DoE) with simple analytical techniques and accelerated stability assays, a liquid formulation was obtained that allows 98% of the biopotency of reCG (intact, active reCG) to be maintained for up to six months under accelerated conditions (25°C, 60% RH) (CAMEVET, 2012).

[0133] Placket-Burman design (PBD) was used to determine the effects of several factors on reCG stability under accelerated conditions (25°C, 60% RH, 7 days). Twelve experiments were performed at the center point, in triplicate, to investigate the standard deviation of the effects at N=15 (experiments). The effects of eight real factors and three dummy variables on reCG stability were evaluated. The factors analyzed were: the amounts of stabilizers (sucrose, mannitol, Arg, L-met) and surfactants (poloxam 188), the volumetric molar concentration and pH of the buffer solution, and the concentration of the API (reCG dose). The response was determined by measuring the area under the curve (t) of intact reCG as evaluated by RP-HPLC. R The reCG content (%) was analyzed after seven days of storage at 25°C and 60% RH (13.58 minutes).

[0134] Pareto plots were used to identify influencing factors. ANOVA was then applied to investigate the effects of these factors on the response and to confirm their significance. The obtained model satisfied the assumptions of normality, homoscedasticity, and independence of the variables. Data analysis indicated that the significant factors affecting the stability of reCG under accelerated conditions were the buffer volume molar concentration (p: 0.0013), L-met (p: 0.0171), sucrose (p: 0.0044), and surfactant amount (p: 0.0097). Furthermore, R-squared (0.8929) and adjusted R-squared (0.8393) indicated a good relationship between the experimental and fitted data.

[0135] During the optimization phase of the reCG liquid formulation, a four-factor (buffer volume molar concentration, sucrose content, L-met, and Pluronic F-68) five-level central composite design (CCD) was performed. Twenty-seven runs were executed, and the response was again determined by RP-HPLC analysis of the reCG content (%) after storage at 25°C, 60% RH and 40°C, 75% RH for 0, 6, 12, 60, 90, 125, and 150 days. The key factors analyzed were buffer volume molar concentration, sucrose, surfactant, and antioxidant dosage. Factors that were found to have no significant effect on reCG stability remained at constant concentration levels in all formulation tests.

[0136] Significant differences were observed among the formulations after 90 days. Therefore, the reCG stability of the 27 formulations was fitted to a quadratic model. The obtained hierarchical model satisfied the assumptions of normality, homoscedasticity, and independence of the variables. Furthermore, the adjusted R-squared (0.7238) indicated a good relationship between the experimental and fitted data: R aj : 0.8122; R 2 aj=0.7238; CV% 1.53, lack of fit: 0.1107 (p<0.05 is considered significant).

[0137] A robust design space was obtained, and optimal formulation conditions were selected, consisting of 70 mM citrate / citrate buffer (pH 6.0), 161 mM sucrose, 1.0 mg / mL L-met, and 1.0 mg / mL surfactant (plus 5 mM L-Arg and 5 mg / mL mannitol).

[0138] This predictive reCG liquid formulation was validated by preparing three separate batches and evaluating their stability after storage at 4 °C, 25 °C, 60% RH, and 40 °C, 75% RH for 0, 15, 45, and 90 days. After 90 days, the robustness of the developed liquid formulation was demonstrated by the intact reCG content (as assessed by AUC via RP-HPLC) of 125 ± 3, 125 ± 1, and 94.6 ± 0.2% at 4 °C, 25 °C / 60% RH, and 40 °C / 75% RH, respectively. The validated liquid formulation, after evaluation for up to 150 days, achieved 98 ± 13% intact reCG under accelerated conditions (25 °C / 60% RH).

[0139] 7.2-Blending of freeze-dried products

[0140] A stable solution or formulation is one that ensures that the degree of degradation, alteration, aggregation, or loss of bioactivity is acceptable or manageable. Ideally, the formulation must retain at least 80% of the initial protein titer during a six-month storage period at 2–8°C (US 7,740,884 B2). Thus, a solid excipient formulation has been developed containing a salt as a buffer, such as citric acid / citrate, at a low volumetric molar concentration (10 mM) and a pH of 6.5.

[0141] Furthermore, the success of solid-state freeze-drying involves balancing two competing requirements: the formation of a firm, cake-like substance that does not collapse during primary drying, and the presence of an amorphous state that allows for interaction between the excipient and the protein (Jhonson et al., 2001). Excipients that act as protein stabilizers, such as sucrose or trehalose, behave like amorphous solids, while excipients like mannitol act as crystalline solids that reduce cake collapse. Therefore, in this invention, a mannitol:sucrose ratio of 4:1 is used, with concentrations of 40 g / L and 10 g / L, respectively.

[0142] Proteins like glycoprotein hormones are prone to oxidative degradation; therefore, compounds with antioxidant properties are needed, such as certain amino acids, like methionine, chelating agents (e.g., EDTA), or sodium bisulfite. Furthermore, nonionic surfactants (Pluronic F68 or Poloxamer P188) are used to prevent reCG adsorption on the vial surface and to reduce protein interactions at the air-water interface. In this invention, 0.1 mg / mL of methionine and 0.25 mg / mL of poloxamer P188 are used, respectively.

[0143] Ideally, the amount of reCG in the reconstituted cake should be close to 1,500 IU / mL, with the final volume being the same as the initial volume (3 mL).

[0144] The process consists of the following steps: mixing the formulation excipient with the API, filtering it using a 0.2-mm PES (polyethersulfone) filter, filling it into a properly washed and sterilized borosilicate vial (sealed with a rubber stopper), and lyophilizing it according to existing technology.

[0145] Example 8 - Biochemical and Physicochemical Characterization

[0146] 8.1 Method

[0147] reCG was produced by culturing suspended P5C3 clones in serum-free medium in perfusion mode (Biostat Q Plus, Sartorius) in a 1-liter bioreactor. The clarified supernatant was then purified using CaptoBlue-Agarose chromatography as a capture step (Sartobran-P 0.45 μm, Sartorius). Subsequently, two alternative purification steps were evaluated to obtain protein aliquots with higher purity:

[0148] a) Reversed-phase high-performance liquid chromatography (RP-HPLC)

[0149] b) Hydrophobic interaction chromatography (HIC)

[0150] The reCG molecules purified by RP-HPLC are named reCG RP-HPLC, while the reCG molecules purified by HIC are named reCG HIC.

[0151] Foli-G, Zoovet SA (Argentina), and Novormon, Syntex (Argentina) were purchased from regional veterinary pharmacies and used as internal reference standards. Codes A and B were assigned to Foli-G and Novormon, respectively.

[0152] 8.1.1 - RP-HPLC is used for structural analysis. RP-HPLC is used to evaluate biopotency.

[0153] Qualitative and quantitative studies were conducted using a C4 column with gradient elution and UV detection (210 nm).

[0154] The EJCR (Elliptical Joint Confidence Region) test was used to demonstrate a good correlation between potency determination in rats and intact reCG measured by RP-HPLC techniques such as AUC. Here, the EJCR test and bilinear least squares (BLS) regression method were applied. The method can be assumed to be accurate if the ideal point (1,0) is contained within the ellipse. This elliptical region is described by a mathematical equation plotted in two dimensions. The ellipse size is related to other analytical parameters, such as the precision of the determination.

[0155] 8.1.2-SDS-PAGE

[0156] Throughout this assay, purity and apparent molecular weight were analyzed under non-reducing conditions. Colorimetric detection (Coomassie Brilliant Blue) or immunochemical detection (Western blot) were performed. For Western blot analysis, rabbit polyclonal anti-reCG serum produced in our laboratory was used.

[0157] 8.1.3 - Isoelectric Focusing (IEF)

[0158] To separate protein variant isoforms, a system consisting of an electrophoresis tank (Multiphor II), a cooling bath (Multitemp III), and a voltage source (EPS3500XL) was used. The equipment was subjected to IEF. The pH range was determined using 75% (w / v) 3–5 amphoteric electrolyte and 25% (w / v) 5–7 amphoteric electrolyte (GE Healthcare). Detection was performed by Coomassie blue colloidal staining or Western blot analysis.

[0159] 8.1.4-Size Exclusion Chromatography (SEC)-HPLC

[0160] The purity and identity of the reCG variant and PMSG were determined by size exclusion chromatography (SEC)-HPLC performed on a TSKgel G3000SW with a particle size of 10 μm and UV detection.

[0161] 8.1.5 - Spectrofluorescence Analysis

[0162] Spectro-fluorescence measurements were performed using a Perkin-Elmer LS-55 emission spectrometer equipped with a xenon discharge lamp, a Monk-Gillieson monochromator, and a gated photomultiplier tube connected to an AMD Sempron PC running Windows XP.

[0163] 8.1.6 - High pH Anion Exchange Chromatography and Pulse Amperometric Detection (HPAEC-PAD)

[0164] The sample was acid-hydrolyzed and then used with CarboPac TM Sialic acid content was determined using a DIONEX ICS-5000 system with a PA20 column (Thermo Fisher Scientific Dionex) and high-pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). N-acetylneuraminic acid (Neu5Ac) standard (Calbiochem, France) was used as a reference standard.

[0165] Furthermore, the types and amounts of neutral monosaccharides present in purified reCG and PMSG glycans were determined by acid hydrolysis of the samples followed by HPAEC-PAD using a DIONEX ICS-5000 system equipped with a CarboPac™ PA20 column. A standard solution of the monosaccharide mixture (CM-Mono-Mix-10, Ludger, UK) was processed in the same manner as the sample solutions and used to identify and quantify peaks derived from the glycoprotein samples.

[0166] 8.1.7 N-Glycan Analysis

[0167] 8.1.7.1 - Enzymatic N-deglycosylation under denaturing conditions

[0168] To remove N-glycans from purified samples, enzymatic digestion was performed under denaturing conditions using the PNGAse F kit (Biolabs Inc.).

[0169] 8.1.7.2 - Weak anion exchange chromatography (WAX) for the analysis of charged labeled N-glycans

[0170] The released N-glycans were purified by ethanol precipitation and labeled with a 2-AB fluorophore. Finally, weak anion exchange (WAX) chromatography was performed to analyze the relative amounts of the neutral, monosialotetarian, disialotetarian, trisialotetarian, and tetrasialotetarian structures of the protein.

[0171] 8.2 Results

[0172] 8.2.1 Sample Preparation

[0173] The supernatant of clarified cell culture from P5C3 production cell clones cultured in a one-liter bioreactor (perfusion mode) was purified using dye pseudo-affinity chromatography (CaptoBlue-Agarose, GE Healthcare) as the first capture step. Two alternative chromatographic steps were then performed: RP-HPLC or HIC (as previously indicated).

[0174] Biochemical and physicochemical characterization was performed compared with PMSG formulations from two commercial branches.

[0175] The purity of different molecules was analyzed throughout the SEC-HPLC process. RP-HPLC yielded 90% purity for reCG, with the remaining percentage likely corresponding to the α and β subunits of the heterodimer dissociated during the RP-HPLC purification process. Conversely, reCG molecules purified by HIC exhibited 55% purity, with the main impurity being excess free α subunits (43%). The PMSG formulation purified by RP-HPLC showed a purity of 73% (Figure 5).

[0176] Apparent molecular weights and isoform profiles of different formulations were determined throughout SDS-PAGE, SEC-HPLC (Figure 5), and IEF (Figure 6). SDS-PAGE and SEC-HPLC analyses revealed molecular weights of 67 and 66 kDa for the commercial PMSG, respectively; while the recombinant variants showed molecular weights of 45 kDa and 46 kDa, respectively. Both recombinant and commercial PMSG formulations exhibited complex isoelectric focusing patterns with multiple glycoforms at low isoelectric points. However, although the two hormones shared a considerable number of isoforms, PMSG showed a higher proportion of glycoforms concentrated in the more acidic pH region (this effect was more pronounced for Novormon), while the recombinant variants showed a wider isoform distribution across the entire pH range.

[0177] The purified PMSG formulation exhibits t R Equal to 12.954 minutes, while for reCG purified by RP-HPLC and HIC, the purified recombinant variant showed t R These values ​​are 13.583 and 13.747 minutes, respectively (Figure 5). These differences in the hydrophobicity of the three molecules may be related to differences in glycan structures, such as sialic acid content, as these are among the main structures that impart charge to proteins.

[0178] 8.2.2 - Spectrofluorescence analysis

[0179] Because fluorescence spectroscopy is extremely sensitive to perturbations in the local structural environment, it provides simple and strong evidence supporting a high degree of structural similarity between different batches of a given protein. Furthermore, it can provide useful insights into product comparability and biosimilarity (Houde et al., 2015). Structural conformations assessed by emission spectra of different formulations reveal differences, not only at their maximal peaks but also along their spectral curves. These results should indicate conformational differences between different formulations. Figure 7 ).

[0180] 8.2.3-HPAEC-PAD

[0181] Sialic acid (Neu5Ac) content was evaluated using the HPAEC-PAD (High Performance Anion Exchange Chromatography and Pulse Amperometric Detection) system from a DIONX system. The Neu5Ac contents of PMSG (A, Foli-G), PMSG (B, Novormon), reCG RP-HPLC, and reCG-HIC were 9.4 (n=1), 18±4 (n=12), 7±1 (n=11), and 7±2 (n=11) Neu5Ac mol / protein mol, respectively. These results correlated with those obtained by RP-HPLC and IEF assays. Nonparametric statistical tests (median mood) allowed for the identification of significant differences in sialic acid content between different formulations (p: 0.00048). Figure 8 As can be seen in the notched box plot, a significant difference was observed between recombinant hormones and PMSG(B, Novormon) at the 95% confidence level.

[0182] Although the PMSG formulation exhibited higher sialic acid content than the recombinant variant, the sialic acid:galactose ratio was nearly identical for both PMSG and the recombinant form, as the ratio of sialic acid to galactose residues was similar for the three hormones (Gal content of PMSG(B,Novormon), reCG RP-HPLC, and reCG-HIC: 18 ± 0.9 (n = 2); 4.7 ± 0.1 (n = 2); 6 ± 0.6 (n = 2) Gal mol / protein mol, respectively). This is one of the properties that could potentially benefit from reduced reCG removal from circulation, reduced glomerular filtration, and thus allow reCG to exert its biological effects in the target animal species. Furthermore, the amount of mannose residues was similar among the hormones, but less than expected, since the amount of mannose (mol / mol protein) should be closer to 9 mol / mol if the heterodimer had three N-glycosylation sites. Therefore, this lower content should be attributed to experimental error. The fucose content was similar between PMSG and the recombinant molecule.

[0183] 8.2.4 - Weak anion exchange chromatography (WAX) for the analysis of charged labeled N-glycans

[0184] To evaluate the sialylation patterns of the purified PMSG commercial formulation (B) compared to recombinant variants, N-glycans from each hormone were isolated and labeled with 2-AB. The 2-AB-labeled glycans were then applied to a WAX-HPLC column and separated according to their charge and, to some extent, according to the N-glycan structure. Using appropriate criteria, the glycans were identified as neutral (desialylated), monosialylated, disialylated, trisialylated, and tetrasialylated structures.

[0185] like Figure 9 As can be seen, the N-glycan profiles of different formulations are similar. However, compared to PMSG, the recombinant formulations exhibit higher contents of neutral glycans and tetrasialylated glycans. Furthermore, unlike the recombinant variants that exhibit incomplete bissialylation structures (potentially more complex), the PMSG formulations exhibit higher amounts of bissialylated glycans that are completely substituted with sialic acid residues (peaks corresponding to the last position from left to right in each group).

[0186] The reCG RP-HPLC molecule exhibited 3.09% neutral structure, 30.19% monosialotetarian structure, 54.22% disialotetarian structure, 8.65% trisialotetarian structure, and 3.86% tetrasialotetarian structure. The reCG HIC contained 3.65% neutral structure, 26.76% monosialotetarian structure, 52.91% disialotetarian structure, 13.53% trisialotetarian structure, and 3.86% tetrasialotetarian structure. Finally, the PMSG formulation exhibited the following percentages: 0.72% neutral structure, 29.02% monosialotetarian structure, 57.99% disialotetarian structure, and 12.20% trisialotetarian structure (Table 4).

[0187] Table 4. Relative amounts of charged N-glycans in purified reCG molecules and PMSG.

[0188]

[0189] Example 9 - Efficacy determination in target animals

[0190] 9.1- reCG-induced superovulation in heifers

[0191] The efficacy of single-dose eCG in superovulation and embryogenesis protocols has been shown to be similar to that obtained by applying multiple doses of FSH. The aim of this study was to evaluate the efficacy of reCG in inducing superovulation. Eighteen heifers weighing between 350 and 370 kg were synchronized using the following protocol: Day -10, 150 μg PGF2α (Ciclar) was injected into the heifers; Day 0, they received an intravaginal device containing 1200 mg P4 (IVD, Diprogest 1200, Zocir), and 2 mg EB injection (estradiol benzoate, Zocir); Day 4, the animals were randomly assigned to four groups and injected: Group 1 (n=4): 1000 IU reCG; Group 2 (n=4): 1500 IU reCG; Group 3 (n=5): 2000 IU reCG; and Group 4 (n=4): 2500 IU PMSG; Day 6, 150 μg reCG was injected into the heifers. PGF2α (Ciclar, Zocir); on day 7, IVD was withdrawn and the PGF2α dose was repeated; on day 8, 0.02 mg of busherin acetate (Zocir) was administered. Ultrasound (US) was performed on days -10 and 0 to determine the stage of the estrous cycle at the start of the protocol. Additionally, on day 8, US was performed to assess the number of follicles larger than and smaller than 8 mm (FOL<8; FOL>8, respectively) and the number of corpora lutea (CL), and US Doppler (Mindray Z6Vet) was performed to assess flushing of follicles >8 mm. All data obtained were analyzed by ANOVA and subsequent Duncan post-hoc tests. The variables flushing, FOL>8, and CL were then correlated using Pearson correlation tests (SPSS Statistics 23, IBM). Results are summarized in Table 5. Statistically significant differences were observed between groups 1 and 3 regarding the amounts of FOL>8 and CL (P<0.05). Follicular flushing was positively correlated with FOL>8 and CL (P<0.05). The results suggest that reCG exhibits a dose-response effect on both pre-ovulatory follicle and CL production with increasing dosage. A 2000 IU dose of reCG was demonstrated to be as effective as PMSG in inducing superovulation in heifers. Finally, higher follicular flushing was confirmed to be associated with a greater number of pre-ovulatory follicles and a greater number of CL.

[0192] Table 5. Mean and standard error of mean for FOL<8, FOL>8, CL and flushing in heifers undergoing superovulation with reCG and PMSG (n=18).

[0193]

[0194] Values ​​with different superscript letters in the same column showed significant differences (P<0.05).

[0195] 9.2 - Pregnancy testing using reCG

[0196] 9.2.1-TFAI: Traditional Solution

[0197] The traditional approach involves releasing the P4 intravaginal device over 8 consecutive days.

[0198] Day 0: US diagnosis of ovarian status and conformation of the test group in the animals. An intravaginal device containing 750 mg progesterone (Prociclar) and 2 mg ES injection (Prociclar) was used.

[0199] Day 8: Remove the device, and then administer 150 μg D+ cloprostenol (Ciclar), 1 mg estradiol cyclopentadiene (Ciclar), and 140 IU reCG or 400 IU PMSG to each group, depending on their individual needs. Prior to timed artificial insemination (FTAI), apply heat detector coating to the base of the tail of all cows to determine if a heat spike has occurred.

[0200] Day 10 (48 hours after device removal): Artificial insemination at a fixed time.

[0201] Day 40 (30 days after AI): Perform a diagnostic ultrasound examination for pregnancy or cyclical changes.

[0202] Ultrasound examination schedule: A transrectal ultrasound will be performed at time 0 to determine the ovarian status of the female being examined. Then, an ultrasound will be performed on day 40 (30 days after fertilization) to obtain a diagnosis of pregnancy.

[0203] Semen and insemination apparatus: To avoid potential fertility differences beyond the protocol that could affect the outcome, all females were inseminated using the same semen from the same bull and the same batch of pipettes, and the insemination was performed by the same professional.

[0204] Results: The results obtained during the procedure and a summary of ultrasound results on day 30 after fixed-time artificial insemination (FTAI) are reported.

[0205] 9.2.2 - J-Synch FTAI Solution

[0206] Day 0: US diagnosis of ovarian status and conformation of the test group in the animals. An intravaginal device containing 600 mg progesterone (Diprogest) and 2 mg estradiol benzoate (EB) injection (Diprogest) was used.

[0207] Day 6: Remove the device, and then administer 150 μg D+ cloprostenol (Ciclar) and 105 IU reCG or 300 IU PMSG to each group, depending on their individual needs. Before timed artificial insemination (FTAI), apply heat detector coating to the base of the tail of all cows to determine if a (heat) spike has occurred.

[0208] Day 9 (72 hours after device removal): All animals were injected with 0.010 mg of buserelline acetate (Zouvi) and then artificial insemination was performed at fixed times.

[0209] Day 43 (34 days after TFAI): Ultrasound examination for pregnancy diagnosis

[0210] Ultrasound examination schedule: A transrectal ultrasound was performed at day 0 to determine the ovarian status of the female being examined. Then, an ultrasound was performed on day 43 (day 34 post-fertilization) to obtain a diagnosis of pregnancy.

[0211] Semen and insemination apparatus: To avoid potential fertility differences beyond the protocol that could affect the outcome, all females were inseminated using the same semen from the same bull and the same batch of pipettes, and the insemination was performed by the same professional.

[0212] Table 6: Pregnancy outcomes in heifers following device removal with supplemental PMSG and reCG in the TFAI program

[0213]

[0214] Table 7: Pregnancy outcomes in calving and dry cows after device removal supplementation with PMSG (400 IU) and reCG (140 IU) in the TFAI program.

[0215]

[0216] sequence list <110> Litoral State University National Science and Technology Research Council (CONICET) biotechnology company N. Sealyo <120> Methods for obtaining mammalian cell lines expressing recombinant equine chorionic gonadotropin (reCG), recombinant cell lines for reCG production, large-scale reCG production methods, reCG, reCG-containing formulations, nucleic acids encoding reCG, and applications. <130> - <160> 2 <170> PatentIn Version 3.5 <210> 1 <211> 366 <212> DNA <213> ‑ <400> 1 atggactact acagaaagca cgccgccgtg atcctggcta ccctgtccgt gttcctgcac 60 atcctgcata gcttccccga cggcgagttc acaacccagg actgccctga gtgcaagctg 120 agagagaaca agtacttctt caagctgggc gtgcccatct accagtgcaa gggctgctgc 180 ttctcccggg cctatcctac ccctgcccgg tccagaaaga ccatgctggt gcccaagaac 240 atcacctccg agtctacctg ctgcgtggcc aaggccttca tcagagtgac cgtgatgggc 300 aacatcaagc tggaaaacca cacccagtgc tactgctcca cctgttacca ccacaagatc 360 tgatga 366 <210> 2 <211> 513 <212> DNA <213> ‑ <400> 2 atggaaacac tgcagggcct gctgctgtgg atgctgctgt ctgtgggcgg cgtgtgggct 60 tctagaggac ctctgaggcc cctgtgccgg cctatcaatg ctaccctggc cgccgagaaa 120 gaggcctgcc ctatctgcat caccttcacc acctccatct gcgccggcta ctgcccctcc 180 atggtgcgag tgatgccagc cgctctgcct gccattcctc agcccgtgtg cacctacaga 240 gagctgagat tcgcctccat ccggctgcct ggatgtcctc ctggcgtgga ccctatggtg 300 tccttccctg tggccctgtc ttgccactgc ggcccctgtc agatcaagac caccgactgc 360 ggcgtgttcc gggatcagcc tctggcatgt gcacctcagg cctccagctc ctccaaggac 420 cctccatctc agcccctgac ctccacctct acccctacac ctggcgcctc tcggagatcc 480 tctcaccccc tgcctatcaa gacctcctga tga 513

Claims

1. A method for obtaining a mammalian cell line expressing recombinant equine chorionic gonadotropin (reCG), comprising the following steps: a. Provide coding sequences for the reCGα and β subunits, SEQ ID NO:1 and SEQ ID NO:2, respectively, which are optimized for their expression in mammalian cells; b. Introduce the coding sequence into a lentiviral expression vector; c. Producing lentiviruses containing reCG coding sequences; d. Transduce mammalian cells using the lentiviruses described above; e. Select the most suitable mammalian cell clone to produce reCG.

2. The method of claim 1, wherein the cell line comprises a reCG yield of at least 100 IU / mL in serum-free culture medium.

3. The method according to claim 1, wherein in step b, the lentiviral vector comprises a pLV lentiviral vector containing an EF-1α promoter.

4. The method according to claim 1, wherein step c comprises using cationic lipids as a medium to transiently transfect HEK293 cells with pREV, pVSVG, pMDL, pLV-reCGα, and pLV-reCGβ plasmids.

5. The method according to claim 1, wherein step d comprises transducing CHO-K1 cells.

6. The method of claim 5, wherein the method comprises two consecutive transductions.

7. A mammalian CHO cell line obtained by the method according to claim 1, wherein the cell line comprises a nucleic acid encoding recombinant equine chorionic gonadotropin (reCG) hormone, wherein the coding sequences of the α and β subunits of the reCG comprise the sequences of SEQ ID NO:1 and SEQ ID NO:2, respectively.

8. The cell line according to claim 7, wherein the cell line is the CHO-K1 cell line.

9. The cell line of claim 7, wherein the cell line produces at least 100 IU / ml reCG.

10. A method for producing recombinant equine chorionic gonadotropin (reCG) hormone, comprising the following steps: a. Cultivation in serum-free medium in a bioreactor The mammalian cell line according to claim 7, for large-scale production of reCG. b. Harvest the supernatant, and c. Purification.

11. The method of claim 10, wherein the method comprises a production rate of at least 100 IU / mL reCG in a serum-free culture medium.

12. The method of claim 10, wherein step "a" comprises incubation in a serum-free medium containing 50% commercial Excel302 medium and 50% phosphate-buffered saline.

13. The method of claim 10, wherein the purification step c. comprises dye pseudoaffinity chromatography.

14. The method of claim 13, wherein the dye pseudo-affinity chromatography uses a CaptoBlue-agarose matrix.

15. The method of claim 10, further comprising an HPLC purification step using a C4 column.

16. The method of claim 10, wherein the purification step c. comprises tangential flow filtration and subsequent reCG concentration.

17. The method of claim 10, wherein the reCG comprises a specific activity of at least 6000 IU / mg (as an in vivo potency unit related to protein quality determined by ELISA).

18. A nucleic acid encoding the α subunit of reCG obtained by the method of claim 10, wherein the nucleic acid comprises the sequence of SEQ ID NO:

1.

19. A nucleic acid encoding the β subunit of reCG obtained by the method of claim 10, wherein the nucleic acid comprises the sequence of SEQ ID NO:

2.

20. A reCG hormone obtained by the method of claim 10, wherein the hormone comprises a glycosylation profile having at least 3% neutral structure and at least 3% tetrasialylated structure.

21. A reCG obtained by the method according to claim 10, wherein the reCG comprises a glycosylation profile having at least 3% neutral structure, 26 to 30% monosialylated structure, 50 to 55% disialylated structure, 8 to 15% trisialylated structure and at least 3% tetrasialylated structure.

22. A pharmaceutical formulation comprising a therapeutically effective amount of reCG according to claim 20.

23. The pharmaceutical formulation according to claim 22, wherein the pharmaceutical formulation is lyophilized.

24. The pharmaceutical formulation according to claim 22, wherein the pharmaceutical formulation is a liquid.

25. The pharmaceutical formulation of claim 24, wherein the pharmaceutical formulation further comprises sugar, preservative, antioxidant, mannitol and anti-aggregating agent, and is kept refrigerated at 5°C without freezing.

26. The pharmaceutical formulation according to claim 24, wherein the pharmaceutical formulation comprises trisodium citrate dihydrate, citrate monohydrate, arginine, sucrose, mannitol, L-methionine, poloxamer 188, m-cresol, and water.

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