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

Abstract

The present invention describes methods for obtaining mammalian cell lines that express recombinant equine chorionic gonadotropin (reCG) hormone. Cell lines expressing reCG, large-scale methods for producing reCG, reCG with higher biological activity relative to PMSG, formulations containing reCG, nucleic acids encoding reCG, and uses are also described.

Description

[Technical Field]

[0001] The present invention resides in the field of biotechnology, specifically the development of protein hormones through recombinant DNA technology and their production in mammalian cells. [Background technology]

[0002] Equine chorionic gonadotropin (eCG) is a member of the glycoprotein hormone family, along with luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH) (Murphy and Martinuk, 1991). eCG was originally called pregnant mare's serum gonadotropin (PMSG) because it is produced by trophoblast cells in the endometrial cup of pregnant mares. eCG plays an important role in maintaining early pregnancy (the first trimester), indirectly stimulating progesterone production by the corpus luteum until the placenta is able to secrete it itself. The concentration of eCG secreted by trophoblast cells reaches its maximum concentration at approximately day 50 of pregnancy and then begins to gradually decline (Allen and Moor, 1972).

[0003] eCG has two distinct characteristics compared to other glycoprotein hormones. On the one hand, in species other than horses, eCG exhibits high FSH-like and LH-like activity and has high affinity for the receptors of these hormones (Combarnous et al., 1984). On the other hand, it exhibits a high carbohydrate content, accounting for 45% of its total molecular weight. This last characteristic determines eCG's long circulating half-life of approximately 6 days. Both characteristics allow eCG to be used in veterinary medicine to control the reproductive activity of different 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, designated α and β. The α subunit is common to all members of the family and is encoded by a single gene, whereas a different gene encodes the β subunit, which confers specificity to the heterodimer (Stewart and Allen, 1976).

[0005] The α subunit is composed of 96 amino acids and has two N-glycosylation sites located at Asn56 and Asn82, whereas the β subunit is composed 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) that contains 12 O-glycosylation sites at Ser or Thr residues (Bousfield and Butnev, 2001). Both subunits contain multiple intramolecular disulfide bonds, and their assembly occurs primarily in the endoplasmic reticulum, representing the limiting step in the dimeric secretion process (Hoshina and Boime, 1982). In horses, both placental CG and pituitary LH β subunits are encoded by the same gene (Sherman et al., 1992). However, eCG has a higher, more branched carbohydrate content than eLH. Both hormones differ considerably in their N-glycan termination. eCG has glycans capped with N-acetylneuraminic acid (sialic acid), while eLH has glycans capped with sulfated N-acetylgalactosamine (SO4 -4 -GalNAc) glycans. The significant difference in their molecular weights is essentially due to the presence of longer disialylated poly-N-acetylamine O-glycan structures in eCG (Smith et al., 1993). The high sialic acid content of eCG accounts for its exceptional circulating half-life, as this residue reduces both glomerular filtration and hepatic metabolism.

[0006] Currently, commercially available products include partially purified eCG preparations from pregnant mare blood (PMSG), which have many disadvantages. On the one hand, they exhibit batch-to-batch variability due to differences in glycosylation profiles between animals and between serum samples from different stages of pregnancy. On the other hand, PMSG may also contain contaminants that pose potential health risks. This contradicts the current trend among regulatory agencies to obtain safer veterinary products free of viruses, prions, and other contaminating proteins. Last but not least, the practice by which eCG-containing serum is obtained from pregnant mares involves the extraction of 10 liters of blood per week and the subsequent induction of manual abortion by reaching the uterus and rupturing the amniotic sac. The practice to which animals are subjected is completely questionable from a bioethical perspective, undermining animal welfare: it is a cruel process that can cause severe anemia and, in some cases, ends in the animal's death.

[0007] For this reason, the development of recombinant eCG (reCG) as an alternative to PMSG is reasonable. Some efforts to produce recombinant eCG have been reported in different hosts. Meanwhile, Legardinier et al. (2005) described the production of eCG in two insect cell lines, Sf9 and Mimic™, the latter being a cell line derived from the former that has been modified to express a gene different from mammalian glycosyltransferases.

[0008] Nevertheless, 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 caused by the absence of terminal sialic acid in the oligosaccharide chain. Meanwhile, Ubach et al. (2009) and Ingles et al. (2012) described the expression and purification of eCG in Pichia pastoris, but the recombinant hormone nevertheless failed to exhibit bioactivity in an in vivo bioassay in female rats. Similarly, these results correlated with the short circulating half-life of the recombinant hormone, as only 1% of the injected protein was detectable in serum after 90 min. This rapid clearance could be explained by activation of the mannan-binding lectin pathway, which occurs after injection of a protein with a high mannose content. These results highlight the importance of a correct glycosylation profile for eCG to exhibit in vivo bioactivity, and therefore, the importance of selecting the correct host for its recombinant expression.

[0009] The development of recombinant versions of eCG in CHO cells, including the CHO DG44 cell line, has been reported in publications and patents. The WO2017112987A1 patent application describes the use of this CHO cell line and the production of reCG with a glycosylation profile predicted depending on the host cell used. Despite this, commercial versions of reCG are not yet available on the market. This result demonstrates a problem that must be solved, consisting of the fact that large amounts of recombinant eCG (or recombinant gonadotropins in general) cannot be obtained in an "efficient manner" or with a glycosylation profile similar to that of native eCG that would ensure its in vivo bioactivity (Hesser, 2011). Thus, the challenge is to develop a production system that exhibits high productivity to obtain sufficient quantities of reCG to meet the high demand (as the hormone is used in different types of livestock) and therefore results in lower production costs, which are the limiting factor for the commercial success of recombinant hormones. WO2017112987A1_2017 describes the use of a DHFR-MTX gene amplification system, resulting in a production of 18 IU / mL (in serum-free medium), a value that cannot be achieved for a profitable process with current PMSG production processes. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram of the assembly of third generation lentiviral particles to obtain a reCG-producing recombinant CHO-K1 cell line. [Figure 2] The concentration of reCG in the supernatant from the reCG-producing strain is assessed using a specific anti-reCG antibody, SDS-PAGE followed by Western blot is shown. [Figure 3] Shown is an SDS-PAGE followed by Western blot in which the "apparent" productivity of preselected reCG-producing clones is assessed. [Figure 4]Graphs comparing the evolution of viable cell concentration, viability, lactate and glucose concentrations over time for cultures of the P5C3 clone in 1 L bioreactors. Daily temperature and perfusion rate are indicated. (↓) indicates bleeding of the bioreactor. [Figure 5] (A) PMSG molecule after RP-HPLC, (B) reCG RP-HPLC, and (C) structural characterization and purity analysis of reCG HIC by SDS-PAGE, RP-HPLC, and SEC-HPLC. [Figure 6] Isoform profile analysis. (A) IEF followed by colloidal Coomassie blue staining. The isoform profiles of PMSG variants (commercial preparation A, Foli-G) and CaptoB reCG eluates from cell lines and clones were compared. (B) IEF followed by Western blot of PMSG (commercial preparation A, Foli-G) and CaptoB reCG eluates from cell lines and clones were compared. (C) IEF followed by colloidal Coomassie blue staining. The isoform profiles of PMSG (commercial preparation B, Novormon), reCG RP-HPLC, and reCG HIC variants were compared. [Figure 7] Fluorescence emission spectra of PMSG, reCG RP-HPLC, and reCG HIC in sodium phosphate buffer solution are shown. [Figure 8] Notched box plots are shown in which significant differences in Neu5Ac content (mol / mol protein) are indicated. [Figure 9] Analysis of 2-AB-labeled N-glycans of PMSG, reCG RP-HPLC, and reCG HIC by WAX-HPLC. N-glycans were separated according to their charge. [Figure 10] Comparative pregnancy rates.

[0011] BRIEF DESCRIPTION OF THE INVENTION The present invention provides a method for obtaining a mammalian cell line expressing recombinant equine chorionic gonadotropin (reCG), comprising the steps of: a. providing coding sequences for reCG α and β subunits optimized for their expression in mammalian cells; b. introducing the coding sequence into a lentiviral expression vector; c. Producing a lentivirus containing a reCG coding sequence; d. transducing mammalian cells with the lentivirus; and selecting the most suitable mammalian cell clone for producing e.reCG.

[0012] The recombinant cell line exhibits reCG production of at least 100 IU / mL in serum-free medium.

[0013] In a preferred embodiment of the present invention, step a of the method uses subunit sequences substantially similar to the 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 transient transfection of HEK293 cells with pREV, pVSVG, pMDL, pLV-reCGα, and pLV-reCGβ plasmids using cationic lipids as a vehicle. Step d involves transducing CHO-K1 cells.

[0014] In an alternative method, the method of the invention involves performing two sequential transduction events.

[0015] Another object of the present invention is to provide a mammalian cell line obtained by the aforementioned method and containing a nucleic acid encoding recombinant equine chorionic gonadotropin (reCG) hormone, wherein the coding sequences for the reCG α and β subunits comprise sequences substantially similar to SEQ ID NO: 1 and SEQ ID NO: 2. Preferably, the mammalian cell line is CHO-K1 and exhibits reCG production of at least 100 IU / mL.

[0016] Another object of the present invention is a method for producing recombinant equine chorionic gonadotropin (reCG) hormone, comprising: a. culturing the mammalian cell line in a medium free of fetal bovine serum in a bioreactor for large-scale production of the reCG; b. harvesting the supernatant; and c. a purification step.

[0017] This production method results in a reCG productivity of at least 100 IU / mL in serum-free medium.

[0018] In a preferred embodiment of the present invention, step a. involves culturing in a serum-free medium containing 50% commercially available Excel302 medium and 50% phosphate-buffered saline. In step c., purification involves dye pseudo-affinity chromatography. Preferably, the dye pseudo-affinity chromatography uses a CaptoBlue-Sepharose matrix. Alternatively, purification step c. consists of tangential flow filtration followed by reCG concentration.

[0019] Optionally, the purification method also includes an HPLC purification step using a C4 column.

[0020] The reCG obtained by this production method contains a specific activity (as in vivo potency units related to protein mass as determined by ELISA) of at least 6000 IU / mg.

[0021] Another object of the present invention includes nucleic acids encoding the reCG alpha subunit obtainable by the described production method, which comprise a sequence substantially similar to SEQ ID NO: 1. It also includes nucleic acids encoding the beta subunit of reCG, of a sequence substantially similar to SEQ ID NO: 2.

[0022] Another object of the present invention is the reCG hormone obtainable by the described production method, comprising a glycosylation profile with at least 3% neutral structures and at least 3% tetrasialylated structures. Preferably, said reCG comprises a glycosylation profile with at least 3% neutral structures, 26-30% monosialylated structures, 50-55% bisialylated structures, 8-15% trisialylated structures, and at least 3% tetrasialylated structures.

[0023] Another object of the present invention is a pharmaceutical formulation comprising a therapeutically effective amount of reCG as described herein. In a preferred form, the formulation is liquid and is kept refrigerated, preferably at 5°C, without the need for refrigeration for commercialization. In an alternative form, the formulation is lyophilized. In a preferred form, the formulation further comprises sugar, a preservative, an antioxidant, mannitol, and an anti-aggregating agent. Preferably, the formulation comprises trisodium citrate dihydrate, citric acid monohydrate, arginine, sucrose, mannitol, L-methionine, poloxamer 188, m-cresol, and water.

[0024] Another object of the present invention is a method for inducing ovulation in animals, comprising the administration of at least 140 IU / animal of reCG, the administration of said dose inducing ovulation 48 hours after its application. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention describes a method for obtaining mammalian cell lines (clones) expressing recombinant equine chorionic gonadotropin (reCG) involving the following steps: a. providing coding sequences for reCG α and β subunits optimized for their expression in mammalian cells; b. Introducing the coding sequence into a lentiviral expression vector c. Producing lentivirus containing the reCG coding sequence d. Transducing mammalian cells with the lentivirus e. Select the most suitable mammalian cell clone for producing reCG

[0026] Cell clones that show production of at least 100 IU / mL in serum-free medium in large-scale bioreactors are obtained from the methods described in this invention.

[0027] One of the main features of the method for obtaining a reCG-producing mammalian cell line is the use of an innovative and optimized DNA sequence encoding reCG. The sequence has been modified and optimized for expression in mammalian CHO cells, preferably CHO-K1. reCG contains at least one alpha subunit and one beta subunit, and therefore the coding DNA sequences for both subunits have been optimized. The coding sequences for each subunit are substantially similar to or identical to SEQ ID NO: 1 (α) and SEQ ID NO: 2 (β).

[0028] These coding sequences for each subunit contained minor modifications to their nucleotides to optimize their transcription, translation, and post-translational mechanisms in CHO-K1 cells. After optimization, the recombinant sequence of the eCG beta subunit showed 82.2% homology with the non-optimized beta subunit sequence ("native" eCG beta), but lower homology (78%) was observed with other recombinant sequences published in different patents. Nucleotide positions in our optimized recombinant sequence that differed from both residues in the native sequence and residues in optimized sequences published in other patents were observed to correspond to 4% of the total. This percentage was sufficient to act as a determinant in obtaining higher levels of eCG expression relative to those reported in the state of the art.

[0029] The method for obtaining a reCG-producing mammalian cell line of the present invention achieves a stable reCG-producing cell line by using a third-generation lentiviral vector as a genetic material introduction tool. The lentiviral vector used is pLV, which contains the EF-1α promoter. The vectors carrying the coding sequences for each subunit are called pLV-reCGα and pLV-reCGβ. In addition, pLV contains the coding region of a puromycin resistance gene as a selection marker.

[0030] To produce lentiviral particles, the present invention describes transient transfection of HEK293 cells with the plasmids pREV, pVSVG, pMDL, pLV-reCGα, and pLV-reCGβ using cationic lipids as a vehicle. Once the lentiviral particles are obtained, they are used to transduce mammalian cells (step d). Preferably, the mammalian cells are CHO-K1 cells. Preferably, the process involves two successive transductions.

[0031] Another object of the present invention includes a mammalian cell line having as part of its genome a nucleic acid encoding recombinant equine chorionic gonadotropin hormone (reCG), wherein the coding sequences for the alpha and beta subunits of reCG comprise sequences substantially similar to SEQ ID NO: 1 and SEQ ID NO: 2. These cells are developed by the method of obtaining a reCG-producing mammalian cell line of the present invention. Preferably, the cell line is CHO-K1. This cell line (clone) produces at least 100 IU of reCG / mL in serum-free medium.

[0032] Another object of the present invention is to provide a method for producing recombinant equine chorionic gonadotropin (reCG) hormone, comprising the following steps: d. Cultivation of the mammalian cell line (clone) in a medium free of fetal bovine serum in a bioreactor for large-scale production of the reCG. e. Supernatant collection, and f. Because it involves refining.

[0033] The method for producing recombinant equine chorionic gonadotropin (reCG) of the present invention achieves production of at least 100 IU / mL in serum-free medium. These high levels of production are due to several factors. First, the high reCG productivity of the cell lines transformed with SEQ ID NO:1 and SEQ ID NO:2 of the present invention has been further adapted and optimized for culture in bovine serum-free medium. The medium used for production is MC02 medium, which contains 50% commercially available Excel302 medium and 50% combinations of salts, amino acids, carbohydrates, etc., as described in Table 2.

[0034] Another important factor is the purification step. The state of the art describes complex and expensive purification processes that make the final product more expensive. The method of producing recombinant equine chorionic gonadotropin hormone (reCG) of the present invention incorporates a purification step involving dye-pseudo-affinity chromatography. In a preferred embodiment, the matrix used to perform said chromatography is a CaptoBlue-Sepharose matrix. Optionally, an extra HPLC purification step can be added using a C4 column.

[0035] As an alternative to purification by dye pseudo-affinity chromatography, the purification step involves a tangential flow filtration step followed by a reCG concentration step.

[0036] Through the method of production of recombinant equine chorionic gonadotropin hormone (reCG) of the present invention, it is possible to obtain reCG with a specific activity (as in vivo potency units related to protein mass as determined by ELISA) of at least 6000 IU / mg.

[0037] Another object of the present invention includes a nucleic acid encoding a reCG alpha subunit obtainable by the method described in the present invention, said alpha subunit of said reCG comprising a sequence substantially similar to SEQ ID NO: 1.

[0038] Another object of the present invention includes a nucleic acid encoding a reCG beta-alpha subunit obtainable by the method described in the present invention, said beta subunit of said reCG comprising a sequence substantially similar to SEQ ID NO: 2.

[0039] In a preferred embodiment of the present invention, the sequence substantially similar to SEQ ID NO:1 means that it has at least 90% identity with the sequence of SEQ ID NO:1.

[0040] In a preferred embodiment of the present invention, the sequence substantially similar to SEQ ID NO:1 means that it has at least 95% identity with the sequence of SEQ ID NO:1.

[0041] In a preferred embodiment of the present invention, the sequence substantially similar to SEQ ID NO:1 means that it has at least 98% identity with the sequence of SEQ ID NO:1.

[0042] In a preferred embodiment of the invention, the sequence substantially similar to SEQ ID NO: 2 is SEQ ID NO: 2 It means that the sequence has at least 90% identity with the sequence of

[0043] In a preferred embodiment of the invention, the sequence substantially similar to SEQ ID NO: 2 is SEQ ID NO: 2 It means that the sequence has at least 95% identity with the sequence of

[0044] In a preferred embodiment of the invention, the sequence substantially similar to SEQ ID NO: 2 is SEQ ID NO: 2 This means that the sequence has at least 98% identity with the sequence of

[0045] The percentage identity is calculated by dividing the number of matches in the comparison window by the total number of positions in the comparison window and multiplying by 100. Identity is performed using the BLAST and BLAST 2.0 algorithms (see, e.g., Altschul et al., 1990, J. Mol. Biol. 215:403-410 and Altschul et al., 1997, Nucleic Acids Res. 25(17):3389-3402).

[0046] Another object of the present invention includes a recombinant equine chorionic gonadotropin hormone (reCG) obtainable by the method of producing reCG of the present invention. The reCG hormone comprises a glycosylation profile having at least 3% neutral structures and at least 3% tetrasialylated structures. Preferably, the hormone comprises a glycosylation profile having at least 3% neutral structures, 26-30% monosialylated structures, 50-55% bisialylated structures, 8-15% trisialylated structures, and at least 3% tetrasialylated structures.

[0047] Another object of the present invention includes a pharmaceutical formulation comprising a therapeutically effective amount of reCG of the present invention. In one embodiment, the formulation is lyophilized. In another embodiment, the pharmaceutical formulation is liquid. The pharmaceutical formulation of the present invention further comprises sugar, a preservative, an antioxidant, mannitol, and an anti-aggregating agent. The liquid pharmaceutical formulation further comprises trisodium citrate dihydrate, citric acid monohydrate, arginine, sucrose, mannitol, L-methionine, poloxamer 188, m-cresol, and water.

[0048] Another object of the present invention comprises a method for inducing ovulation in animals which involves the administration of at least 140 IU / animal of recombinant equine chorionic gonadotropin hormone (reCG) obtainable by the method of production of reCG of the present invention, which method manages to induce ovulation 48 hours after its application.

[0049] The present invention overcomes all the drawbacks associated with the use of hormones obtained from the blood of pregnant mares (PMSG) for the production of reCG, as well as the drawbacks presented by all the recombinant options described so far, none of which have yet reached the veterinary market: - the process for producing reCG described in the present invention replaces the use of animals to obtain hormones, eliminating cruel practices to which they are subjected, which are completely contrary to the bioethical and safety standards required by the society to which they are subjected; The reCG obtained and described in this invention is a higher quality product derived from the culture of animal cells in serum-free medium, which allows standardization of the production process in bioreactors. Thus, the culture parameters and purification procedures are easily controllable and reproducible (unlike when using animal hosts), have better batch-to-batch consistency, and are free of contaminants from animal plasma, thus resulting in a product (reCG) that is safer from a hygienic point of view. The method described in the present invention makes it possible to achieve a reCG hormone that exhibits in vivo FSH / LH activity, unlike the rest of the techniques described in the literature that use other types of cell hosts, which do not make it possible to obtain a recombinant hormone that exhibits in vivo activity. - Fewer international units of reCG obtained and described in the present invention are required to treat animals. Because the reCG of the present invention is more effective, only 100 IU / animal for Bos indicus and 140 IU / animal for Bos taurus are required for IATF, instead of 300 IU and 400 IU, respectively, required when working with PMSG obtained from pregnant mares. Also, only 1000 IU for Bos indicus and 2000 IU for Bos taurus are required for SOV, instead of 2000 IU and 4000 IU, respectively, required when working with PMSG obtained from mares. - After treatment with 140 IU of reCG per animal, cyclicity is restored in anestrous cows in 85% of cases, compared with a success rate of 65% after PMSG treatment. This is very important, since these animals, with very low body condition, enter a cyclic state only through the action of hormones on the ovaries. In the absence of hormones, the animals remain in a non-cycling state (anestrous) and therefore represent unproductive animals. - A more effective synchronization of animals is achieved, which significantly facilitates their use in fixed-time artificial insemination (FTAI) and superovulation (SOV) protocols. All cows treated with the reCG of the present invention for FTAI ovulate 48 hours after administration, while PMSG-treated cows ovulate 50-60 hours after administration. A fixed time for the insemination-pregnancy procedure to be effective and profitable is essential for livestock management where hundreds of cows are processed. In this way, cows processed "in the morning" can be inseminated at the same time two days later, Compared to other PMSG substitutes obtained in animal cells, e.g., CHO DG44, which appear to exhibit an appropriate glycosylation profile, the present invention makes it possible to obtain reCG with the highest productivity ever described. Thus, the present invention overcomes the main limiting disadvantage that has prevented the presence of recombinant versions of hormones on the market: it is the first technology capable of producing large amounts of biologically active reCG at low cost. The high productivity, along with the use of low-cost serum-free culture media, contributes to a reduction in the overall cost of the method of the present invention. Both reCG obtained using the high-recovery single-step purification method or reCG obtained from a single tangential flow filtration and concentration procedure of the initial sample exhibit an appropriate glycosylation profile and, therefore, in vivo biological activity in rats, cows, and pigs. - A liquid formulation of reCG is achieved that is stable over time and therefore superior to existing commercial products consisting of lyophilized or frozen liquid formulations with all the disadvantages that these entail.

[0050] The following describes examples of assays that can be performed to achieve and implement all of the objectives of the present invention. It should be noted that these examples are illustrative without any intention to limit the scope of protection of the present invention. [Example]

[0051] Example 1 - Coding sequence design and optimization The technology developed in this invention first involved the development of a mammalian cell line, specifically suspension CHO-K1 cells (Chinese hamster ovary cells), that produces recombinant equine chorionic gonadotropin (reCG). The coding sequences for the α and β subunits of reCG (reCGα and reCGβ) were optimized for expression in CHO-K1 cells to obtain high levels of mRNA and thereby maximize expression of the encoded protein. Gene optimization takes advantage of the degeneracy of the genetic code, where proteins can be encoded by a variety of alternative gene sequences. Because codon usage differs in each organism, this can create drawbacks in the expression of recombinant proteins in heterologous hosts, resulting in very low expression. Thus, gene optimization algorithms enable multiparameter optimization of DNA sequences, encompassing multiple aspects of gene expression: mRNA transcription, splicing, translation, and degradation, to achieve the most efficient expression of a given protein.

[0052] After optimization, it was observed that the recombinant sequence of eCG beta showed 82.2% homology with the sequence of the non-optimized beta subunit ("native" eCG beta), but less homology (78%) with other recombinant sequences mentioned in the background section of the invention. It was observed that the nucleotide positions of the coding sequence on the optimized recombinant hormone of the invention that differed from both residues of the native sequence and residues of the optimized sequences published in the state of the art corresponded to 4% of the total, which was sufficient to be a determinant in obtaining higher levels of eCG expression relative to those reported in other patents.

[0053] The synthetic sequences were obtained as DNA and then cloned into the p-alpha_eCG(AmpR) and p-beta_eCG(AmpR) vectors, respectively. These vectors contain a bacterial replication origin (Col E1 origin) that allows for plasmid amplification into E. coli and an ampicillin antibiotic resistance gene (AmpR). The vector p-eCGα has a length of 3000 bp and contains 360 bp of eCGα coding sequence, the first 72 bp of which encode the native eCGα signal peptide. The p-eCGβ vector contains 3200 bp and contains 507 bp of eCGβ coding sequence, including a 60 bp-long signal peptide at the beginning of this sequence.

[0054] Example 2 - Expression Vector Construction To obtain stable reCG-producing cell lines, we constructed a third-generation lentiviral vector as a genetic material delivery method. For this purpose, we first cloned the coding sequences of each subunit in a lentiviral transfer vector, and then performed the assembly of lentiviral particles (LPs) and their subsequent titration.

[0055] We constructed lentiviral expression vectors encoding the α and β subunits of reCG. The vector containing the α subunit was digested with XbaI / EcoRV enzymes to release the reCGα coding sequence, which was then cloned into the Nhel / EcoRV site of the lentiviral plasmid vector pLVenhCEF. The vector containing the β subunit was digested with BamHI / EcoRV enzymes to release the reCGβ coding sequence, which was then cloned into the BamHI / SmaI site of the pLVenhCEF vector. This vector, developed in our laboratory, contains the EF-1α promoter as an expression control element, which is characterized by high expression levels in a wide variety of animal cells. It also contains an expression stimulating fragment derived from a CMV enhancer sequence and the coding region of a puromycin resistance gene as selectable markers. The resulting plasmid was amplified in prokaryotic cells (E. coli), cultured under rocking conditions, and purified by organic solvent extraction and precipitation. Sequencing of selected E. coli clones confirmed the identity of the DNA fragments cloned in the plasmids pLVenhCEF-reCGα and pLVenhCEF-reCGβ, which showed 100% homology to the sequences of the synthetic eCGα and eCGβ genes, respectively.

[0056] HEK293 cells (packaging cells) were transiently transfected with the resulting third-generation lentiviral particles using four plasmids: pREV, pGlyco-G, and pMDL (packaging plasmids), as well as the transfer vectors pLVenhCEF-reCGα and pLVenhCEF-reCGβ (encoding each reCG subunit). For this purpose, cationic lipids were used as DNA carriers. The supernatant containing the lentiviral particles was collected 30 hours after transfection, centrifuged at 65,000 g for concentration, and stored at 80°C until use (Figure 1).

[0057] Lentiviral particle titration was performed using the QuickTiter™ Lentiviral Titer Kit (Cell Biolabs Inc.). This kit is designed to detect only lentivirus-associated HIV-1 p24 core protein, so free protein remaining in the supernatant does not interfere with the assay. Therefore, 2.7 × 10 9 LP / mL and 2.1 × 10 9 Physical titers of LP / mL were obtained for reCGα and reCGβ, respectively, which were 5.0 × 10 6 and 3.9 × 10 6 Transduction titers in TU / mL could be approximated, resulting in high titers for transduction of CHO-K1 cells.

[0058] Example 3 - Cell line generation The resulting lentiviral particles were used to generate reCG-producing recombinant CHO-K1 cell lines in suspension. Because cells did not survive the third transduction event, a total of two sequential transduction events (Td1 and Td2) were performed. The transduced cell lines were subjected to selective pressure by incubation with increasing concentrations of puromycin. This strategy allowed the population to be enriched for cells resistant to higher concentrations of the antibiotic, thereby resulting in increased productivity for all cell lines. Figure 2 shows an SDS-PAGE assay followed by Western blot analysis using a specific polyclonal anti-reCG antibody. As can be seen, for the same cell density, the highest reCG concentration was obtained in the supernatant of the sCHO Td2(200) cell line (which was resistant to up to 200 μg of puromycin).

[0059] The reCG productivity of the generated cell lines was then assessed by measuring the accumulated hormone concentration in the supernatant after a certain period of time as well as the initial and final cell densities. ReCG quantification was performed using a competitive ELISA developed in our laboratory. The assay involved competition between a solid-phase immobilized antigen (reCG) and the same antigen in solution (reference reCG or unknown sample) for binding to a specific rabbit anti-reCG antibody (pAb anti-reCG). These antibodies were previously obtained in our laboratory. Subsequently, a peroxidase-conjugated secondary antibody was added to detect any remaining solid-phase-bound complexes. The productivity of the different cell lines is summarized in Table 1. [Table 1]

[0060] Considering these results, the best reCG-producing cell line (reCG Td2 cell line) was selected to be cloned.

[0061] Example 4 - Clone isolation The reCG productivity of the generated cell lines allowed us to select one of them and obtain a single-cell clone with an appropriate growth profile and high reCG productivity.

[0062] To select clones with the highest reCG expression levels, over 400 clones were evaluated in an initial dot blot screen using a specific anti-reCG antibody. Selected clones were cryopreserved. After a preselection step to reduce the number of clones analyzed, the "apparent" productivity of selected clones (determined as the reCG concentration obtained for each clone at the same cell density) was evaluated by SDS-PAGE followed by Western blot (Figure 3). This analysis revealed that clones P5D9 (lane 3) and P5C3 (lane 4) exhibited the highest reCG expression levels. The reCG productivity of selected clones was then assessed by determining the accumulated hormone concentration in the supernatant after a specific period of time, as well as the initial and final cell densities. The reCG concentration was determined by competitive ELISA as described above. The estimated productivity was 0.80 and 0.81 μg × 10 for the P5D9 and P5C3 clones, respectively. 6 cell -1 ×d -1 Finally, the P5C3 clone was selected because it exhibited better growth performance than the P5D9 clone.

[0063] Example 5 - Optimization of culture conditions to achieve optimal glycosylation 5.1 High-density cultivation of P5C3 clones in serum-free medium in 1-liter bioreactors The P5C3 clone was cultured in a 1 liter bioreactor in perfusion mode in culture medium (MC01) without fetal bovine serum for 27 days. The culture contained 7.8 × 10 5 cells.mL -1 Starting at a cell density of 1.6 x 10 7 cells.mL -1 The cells showed exponential growth without a lag phase until a maximum cell density of 1.0 g L was reached. Cell viability was greater than 94%. The perfusion rate was varied from 0.21 to 1.00 reactor volumes per day, starting on the third day of culture. The lactate concentration was maintained at 1.1 g L -1 The specific growth rate of the clone remained below 0.013 h -1 It was.

[0064] A new production medium (MC02) was then formulated. Important: The composition of this culture medium results from combining commercially available media with salts, amino acids, carbohydrates, etc. (Table 2). This was done to optimize the culture medium cost, reducing its value by 50%.

[0065] This new composition did not alter the productivity of the cells or any of the characteristics of the molecule. [Table 2]

[0066] 5.2 High-density cultivation of P5C3 clones in serum-free medium in 50-liter bioreactors Cultivation at Production Scale. Cells were cultured in a 1-liter bioreactor and then scaled to a 50-liter bioreactor to replicate the cell culture parameters tested at laboratory scale. Because the perfusion rate was one reactor volume per day, this fermentation process yielded 50 L harvests, each containing 157 IU / mL of reCG. Considering that one dose of reCG of the present 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 in 24 hours. Therefore, the production method of the present invention produces approximately 56,000 doses of reCG per day at production scale (50 L). This allows for a harvest of more than 1 dose / mL at production scale. Comparing these results with the current production method (extraction from pregnant mares), the culture conditions developed in the 50-liter bioreactor represent approximately 600 mares. In other words, the technology reported in this invention could produce the equivalent of 600 pregnant mares in 200 days with a 25-30 day bioprocess (including cultivation, purification, formulation, and packaging) [assuming that all mares have the same concentration of eCG in their blood and that the eCG obtained from each mare is of the same quality (which is impossible)].

[0067] Furthermore, the reCG productivity of the P5C3 clone at production scale (50 L bioreactor) was calculated in different culture media with different costs (Table 3). These culture media included the original commercial medium (MC01, EX-CELL 302), the optimized MC02 medium (described above), and MC05 medium. This latter medium also yielded higher productivity, which made a larger difference compared to results published by other authors, and was also less expensive. [Table 3]

[0068] Patent application WO2017112987 uses a DHFR-MTX gene amplification system. It does not report productivity, but instead reports the kinetics of reCGβα expression after adaptation to growth in the absence of MTX. From reading the literature, approximate cumulative values ​​of reCGβα (IU / mL) are obtained for cell lines cultured in the presence of 10 IU / mL (24 hours), 20 IU / mL (48 hours), and 28 IU / mL (72 hours) of fetal bovine serum, and for cell lines cultured in the absence of 5 IU / mL (24 hours), 10 IU / mL (48 hours), and 18 IU / mL (72 hours).

[0069] In comparison with these results, the cell clones obtained in the present invention produced 45.6 IU / mL (P5D9) and 50.2 IU / mL (P5C3) in small scale in 72 hours in the absence of fetal bovine serum, and 15 IU x 10 in continuous perfusion mode in a bioreactor. 6 cell -1 ×d -1 A higher productivity level is achieved, corresponding to 157 IU / mL in 24 hours, which represents a significantly higher value than that reported in patent WO2017112987 (5 IU / mL in 24 hours in the absence of fetal bovine serum). This represents a production of more than 7,000,000 IU per day, representing more than 50,000 doses per day of reCG of the present invention.

[0070] Thus, the technology of the present invention made it possible to obtain the highest productivity and production values ​​reported to date, which resulted from a combination of unique factors of our technology: optimization of the sequence for expression in CHO-K1 cells (Cricetulus griseus species), the use of our third-generation lentiviral vector with unique properties, and the use of a CHO-K1 cell line expressing a wide range of glycosyltransferases that are capable of adding bi-, tri-, and tetra-sialylated complex-type N-glycans to polypeptides in addition to generating bi-sialylated mucin-type O-glycans, which are important factors for eCG to exhibit in vivo bioactivity.

[0071] Indeed, the exclusive DNA sequence optimization process was effective in achieving high hormone expression, as evidenced by the 4% of nucleotides that were modified in the protein's native DNA sequence and not altered in the synthetic sequence reported by other patents.

[0072] Example 6 - Purification 6.1 - First purification step After CHO cell culture in a bioreactor in Excell 302 (Sigma) serum-free medium, the harvest material was used to develop the first capture purification step. Therefore, dye-pseudoaffinity chromatography was selected using CaptoBlue-Sepharose resin packed in an XK column (GE, Healthcare) and equilibrated in 20 mM Tris-HCl buffer, pH 7. The clarified harvest, without prior conditioning, was loaded onto the resin using a flow rate of 153.06 cm / h and a total retention time of 5 min. After a wash step with the same equilibration solution, the protein was eluted using an isocratic gradient (Tris-HCl buffer, pH 8, 2 M NaCl, 20% (v / v) ethanol). The loading conditions were sufficient, as no hormone leakage was observed during the loading and wash steps of this first chromatography. The intact hormone was recovered with a purity significantly higher than that obtained from partial purification from pregnant mare serum (PMSG). Therefore, the dye-pseudoaffinity resin was used to optimize the reCG capture step from cell culture supernatant. High yields were achieved with no protein loss, as recovery was 98% (assessed by both ELISA and RP-HPLC).

[0073] 6.2 - Second purification step Because the partially purified hormone (named PostBlue) from the first capture step was eluted under high ionic strength conditions (2 M NaCl), hydrophobic interaction chromatography was selected as the second purification step. To reduce the number of manipulations and the cost of the comprehensive purification process, the following strategies were proposed: 1) load the "crude eluate," i.e., the unconditioned PostBlue fraction, and avoid the diafiltration step (because this fraction is in high ionic strength conditions); 2) screen two types of hydrophobic ligands: phenyl and butyl, both of which are available in our laboratory; 3) evaluate the diafiltered eluate against citric acid / citrate buffer pH 6.0, because this is the condition in which the PostBlue API (named FD1RECG) is formulated; and 4) evaluate purification performance using different salts: first, NaCl (because it is present in the PostBlue buffer), then NaSO, and finally (NH)SO (because this is the salt with the highest hydrophobic effect). Considering the purification performance (recovery and purity) of all strategies, the best conditions were as follows: DF1REG was loaded onto Butyl Sepharose 4FF resin at a flow rate of 15 cm / h and a total retention time of 3 min. To improve the hydrophobic interaction between the protein and the ligand, the resin was equilibrated with 50 mM citric acid / citrate buffer pH 6.0, 2 M (NH4)2SO4, and the sample (DF1REG) was conditioned using the same equilibration buffer. Two washing steps were then performed: the first step used the same buffer as used in the equilibration step, and the second step removed impurities with a lower ionic strength (50 mM citric acid / citrate buffer pH 6.0, 1.5 M (NH4)2SO4). Finally, isocratic elution was performed using 50 mM citric acid / citrate buffer pH 6.0, 0.5 M (NH4)2SO4.

[0074] Example 7 - Formulation 7.1-Liquid formulation of the final product 7.1.1-Development of reCG liquid formulations using QbD tools Through the present invention, a liquid formulation of reCG has been developed, which allows obtaining a stable and therefore active liquid form of the hormone.In this way, it is possible to avoid the freeze-drying procedure, which represents a more extensive and costly unit operation.Therefore, obtaining a liquid formulation instead of a freeze-dried formulation not only ensures lower costs, but also reduces the production process cycle, which avoids the reconstitution step of the freeze-dried product.In addition, when working with multiple doses, it is possible to fractionate the required amount and ensure its long-term stability during use.

[0075] 7.1.1.1 - Pre-formulation assays - to establish critical factors affecting reCG stability in liquid formulations For thermal decomposition studies of reCG purified by CaptoBlue-Sepharose chromatography, a pH range of 3.0 to 8.0 was used, with temperatures varying from 20 to 70 °C. Samples were heated in a thermocycler for 10 min at each condition and stored at -70 °C until analysis. Aliquots of reCG from each condition were then evaluated by non-reducing SDS-PAGE followed by Coomassie Brilliant Blue staining to visualize the degree of reCG dissociation. Differences in mobility profiles in SDS-PAGE indicated that pH had an effect on reCG heterodimer stability. Samples corresponding to the low pH range (pH 3.0-5.0) incubated at high temperatures showed a different pattern of bands compared to those corresponding to the more basic pH range (pH 6.0-8.0). In parallel, emission (fluorescence) profiles were evaluated (λ excitation: 274 nm) to analyze possible conformational changes. A red shift was observed in samples subjected to higher temperatures and lower pH values. A red shift can be correlated with a higher denatured protein pattern or loss of native conformation. Based on these results, we established an optimal pH range of 5-7, aiming to reduce chemical degradation processes (mainly deamination and oxidation), which have a lower effect at neutral pH. Similarly, because the pI (isoelectric point) of the protein was close to 3.5-5.5 (according to IEF assay), working in the pH range of 5-7 ensures that the protein exhibits a net negative charge, reducing physical degradation events such as aggregation (opposite to what can occur at pH values ​​close to the pI of reCG).

[0076] 7.1.1.2-Formulation assays One of the main challenges in the biotherapeutic protein production process is achieving a formulation that ensures high protein quality and stability. By combining design of experiments (DoE) with simple analytical techniques and accelerated stability assays, a liquid formulation was achieved that maintained 98% of the biological potency of reCG (intact, active reCG) for up to 6 months under accelerated conditions (25°C, 60% relative humidity, RH) (CAMEVET, 2012).

[0077] A Placket-Burman design (PBD) was used to determine the effect of several factors on reCG stability under accelerated conditions (25°C, 60% RH, 7 days). Twelve experiments were performed in triplicate at the center point to test the standard deviation of the effect for N = 15 (number of experiments). The effects of eight real factors and three dummy variables (fictitious variables) on reCG stability were evaluated. The analyzed factors were the amount of stabilizer (sucrose, mannitol, Arg, L-met) and surfactant (Poloxamer 188), buffer molarity and pH, and API concentration (reCG dose). The response was to analyze the reCG content (%) after 7 days of storage at 25°C and 60% RH by determining the area under the curve of intact reCG assessed by RP-HPLC technique (t R :13.58 minutes).

[0078] A Pareto chart was used to determine the influential factors. ANOVA tests were then applied to test the effect of the factors on the response and confirm significant effects of the factors. The resulting model satisfied the assumptions of normality, homoscedasticity, and independence of variables. Data analysis revealed that the significant factors for reCG stability under accelerated conditions were buffer molarity (p: 0.0013), L-met (p: 0.0171), sucrose (p: 0.0044), and surfactant (p: 0.0097) amounts. Furthermore, the R-squared (0.8929) and adjusted R-squared (0.8393) indicated a good relationship between the experimental and fitted data.

[0079] During the optimization phase of the reCG liquid formulation, a four-factor (buffer molarity, amount of sucrose, L-met, and Pluronic F-68), five-level central composite design (CCD) was performed. 27 runs were performed, and the response was to analyze the reCG content (%) after 0, 6, 12, 60, 90, 125, and 150 days of storage at 25°C, 60% RH and 40°C, 75% RH, again by RP-HPLC. The key factors analyzed were buffer molarity, sucrose, surfactant, and antioxidant amount. Factors that were found not to have a significant effect on reCG stability were maintained at constant concentration levels in all formulation studies.

[0080] After 90 days, significant differences between formulations were observed. Therefore, reCG stability in the 27 formulations was fitted with a quadratic model. The resulting hierarchical model met the assumptions of normality, homoscedasticity, and independence of 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 significant).

[0081] A robust design space was obtained and optimal formulation conditions were selected, consisting of 70 mM citric acid / 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).

[0082] This predicted reCG liquid formulation was validated by preparing three separate formulation batches and evaluating their stability after 0, 15, 45, and 90 days of storage at 4°C, 25°C / 60% RH, and 40°C / 75% RH. After 90 days, intact reCG contents (assessed as auc by RP-HPLC) of 125±3, 125±1, and 94.6±0.2% were obtained at 4°C, 25°C / 60% RH, and 40°C / 75% RH, respectively, demonstrating the robustness of the developed liquid formulation. The validated liquid formulation was evaluated for up to 150 days, yielding 98±13% intact reCG under accelerated conditions (25°C / 60% RH).

[0083] 7.2 - Preparation of lyophilized products A stable solution or formulation ensures that degradation, modification, aggregation, or loss of biological activity is acceptable or manageable. Ideally, a formulation should retain at least 80% of the protein's initial potency during a 6-month storage period at 2-8°C (US 7,740,884 B2). Thus, we developed solid-state excipient formulations containing salts as buffers, such as citric acid / citrate salts, at low molarity (10 mM) and pH 6.5.

[0084] Furthermore, successful solid-state freeze-drying involves balancing two competing requirements: the production of a robust cake that does not collapse during primary drying, and the presence of an amorphous state that allows interactions between excipients and proteins (Johnson 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. Thus, in the present invention, a 4:1 mannitol:sucrose ratio was used at concentrations of 40 g / L and 10 g / L, respectively.

[0085] Proteins, such as glycoprotein hormones, are susceptible to oxidative degradation. Therefore, the use of compounds with antioxidant properties, such as methionine, chelating agents (e.g., EDTA), or some amino acids such as sodium bisulfite, is desirable. Furthermore, nonionic surfactants (Pluronic F68 or Poloxamer P188) were used to prevent reCG adsorption onto the vial surface and reduce protein interactions at the air-water interface. In this study, 0.1 mg / mL and 0.25 mg / mL of methionine and Poloxamer P188 were used, respectively.

[0086] Ideally, the amount of reCG in the reconstituted cake should be close to 1,500 IU / mL, the same final volume as the initial one (3 mL).

[0087] The process consisted of mixing the formulation excipients with the API, filtering using a 0.2 mm PES (polyethersulfone) filter, filling into properly washed and sterilized borosilicate vials (sealed with rubber stoppers), and lyophilization according to state of the art technology.

[0088] Example 8 - Biochemical and Physicochemical Characterization 8.1 Methodology reCG was produced by culturing suspension P5C3 clones in serum-free medium in a 1-liter bioreactor in perfusion mode (Biostat Q Plus, Sartorius). The clarified supernatant was then purified using CaptoBlue-Sepharose chromatography (Sartobran-P 0.45pm, Sartorius) as a capture step. Two alternative purification steps were then evaluated to obtain aliquots of protein with higher purity: a) Reverse-phase high-performance liquid chromatography (RP-HPLC) b) Hydrophobic Interaction Chromatography (HIC)

[0089] The reCG molecule purified by RP-HPLC was named reCG RP-HPLC, whereas the reCG molecule purified by HIC was named reCG HIC.

[0090] Commercial preparations of eCG from Foli-G, Zoovet SA. (Argentina) and Novormon, Syntex (Argentina) were purchased from local veterinary drug stores and used as internal reference standards. Codes A and B were assigned to Foli-G and Novormon, respectively.

[0091] 8.1.1- RP-HPLC for structural analysis. RP-HPLC for assessing biological potency Qualitative and quantitative tests were performed using a C4 column with gradient elution and UV detection (210 nm).

[0092] The EJCR (Ellipse Joint Confidence Region) test was applied to demonstrate a good correlation between the potency assay in rats and intact reCG measured as auc by RP-HPLC technique. Here, the EJCR test and bilinear least squares (BLS) regression method were applied. If the ideal point (1,0) was contained within the ellipsoid, it could be assumed that the method was accurate. This ellipse area is described by a mathematical equation, which is plotted in two-dimensional graphics. The ellipse size is related to other analytical parameters as the precision of the assay.

[0093] 8.1.2-SDS-PAGE Throughout this assay, purity and apparent molecular weight were analyzed under non-reducing conditions. Colorimetric (Coomassie Brilliant Blue) or immunochemical (Western blot) detection was performed. For Western blot analysis, we used a rabbit polyclonal anti-reCG serum produced in our laboratory.

[0094] 8.1.3-Isoelectric focusing (IEF) To separate protein variant isoforms, IEF was performed using a Pharmacia® instrument consisting of an electrophoresis chamber (Multiphor II), a cooling bath (Multitemp III), and a voltage source (EPS3500XL). The pH range was established using 75% (w / v) 3-5 zwitterionic and 25% (w / v) 5-7 zwitterionic (GE Healthcare). Detection was performed by Coomassie colloid staining or Western blot analysis.

[0095] 8.1.4 - Size Exclusion Chromatography (SEC) - HPLC The purity and identity of the reCG variants and PMSG were determined by size exclusion chromatography (SEC)-HPLC performed on a TSKgel G3000SW with a particle size of 10 pm and UV detection.

[0096] 8.1.5-Spectrofluorimetric analysis Spectrofluorometry was performed using a Perkin-Elmer LS-55 luminescence spectrophotometer equipped with a Xenon discharge lamp, a Monk-Gillieson type monochromator, and a gated photomultiplier tube connected to an AMD Sempron PC running Windows XP.

[0097] 8.1.6 - High pH Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) Sialic acid content was determined by acid hydrolysis of the samples followed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a DIONEX ICS-5000 system equipped with a CarboPac™ PA20 column (Thermo Fisher Scientific Dionex). N-acetylneuraminic (Neu5Ac) acid standard (Calbiochem, France) was used as the reference standard.

[0098] Furthermore, the types and amounts of neutral monosaccharides present in the 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 monosaccharide mix standard solution (CM-Mono-Mix-10, Ludger, UK) was treated like the sample solution and used to identify and quantify peaks derived from glycoprotein samples.

[0099] 8.1.7-N-Glycan Analysis 8.1.7.1 - Enzymatic N-deglycosylation under denaturing conditions To remove N-glycans from the purified samples, enzymatic digestion was performed under denaturing conditions using a PNGase F kit (Biolabs Inc.).

[0100] 8.1.7.2 - Weak Anion Exchange Chromatography (WAX) for Charge-Labeled N-Glycan Analysis The released N-glycans were purified by ethanol precipitation and labeled with the 2-AB fluorophore. Finally, weak anion exchange (WAX) chromatography was performed to analyze the relative amounts of neutral, mono-, bi-, tri-, and tetrasialylated structures of the protein.

[0101] 8.2-Results 8.2.1-Sample preparation Clarified cell culture supernatant of P5C3-producing cell clones cultivated in 1-liter bioreactors (perfusion mode) was purified using dye-pseudo-affinity chromatography (CaptoBlue-Sepharose, GE Healthcare) as a first capture step, followed by two alternative chromatographic steps: RP-HPLC or HIC (as previously shown).

[0102] Biochemical and physicochemical characterization was performed in comparison with two commercially available branched PMSG preparations.

[0103] The purity of the different molecules was analyzed throughout SEC-HPLC. A purity of 90% was obtained for reCG RP-HPLC, with the remaining percentage likely corresponding to the α and β subunits of the heterodimer that dissociate during the RP-HPLC purification method. Conversely, the reCG molecule purified by HIC presented a purity of 55%, with the main impurity being excess free alpha subunit (43%). The PMSG preparation purified by RP-HPLC showed a purity of 73% (Figure 5).

[0104] The apparent molecular weights and isoform profiles of the different preparations could be determined through SDS-PAGE, SEC-HPLC (Figure 5), and IEF (Figure 6). The molecular weights of commercial PMSG were approximately 67 and 66 kDa, respectively, as analyzed by SDS-PAGE and SEC-HPLC, whereas the recombinant variants exhibited molecular weights of 45 and 46 kDa, respectively, as analyzed by SDS-PAGE and SEC-HPLC. Both recombinant and commercial PMSG preparations exhibited complex isoelectric focusing patterns, with a wide variety of glycoforms at low isoelectric points. However, while both hormones shared a significant number of isoforms, PMSG exhibited a higher proportion of glycoforms concentrated in the more acidic pH zone (this effect was more pronounced for Novormon), whereas the recombinant variants exhibited a broader spectrum of isoforms distributed throughout the pH range.

[0105] The purified PMSG preparation had a t equal to 12.954 min R presented, whereas the purified recombinant variants showed t equal to 13.583 min and 13.747 min for reCG purified by RP-HPLC and HIC, respectively. R These differences in hydrophobicity of the three molecules may be related to differences in glycan structure, such as sialic acid content, since this is one of the main structures that confers charge to the protein.

[0106] 8.2.2-Spectrofluorimetric analysis Fluorescence profiles are extremely sensitive to perturbations in the local structural environment and therefore provide simple and powerful evidence supporting a high degree of structural similarity between different batches of a given protein. In addition, they can provide useful insights into product comparability and biosimilarity (Houde et al., 2015). Structural conformations assessed through the emission profiles of different preparations revealed differences not only in their peak maxima but also in their spectral profiles. These results should indicate conformational differences between different preparations (Figure 7).

[0107] 8.2.3-HPAEC-PAD The sialic acid content (Neu5Ac) was assessed via HPAEC-PAD (high-performance anion-exchange chromatography with pulsed amperometric detection) using a DIONEX system. PMSG (A, Foli-G), PMSG (B, Novormon), reCG RP-HPLC, and reCG-HIC showed Neu5Ac contents of 9.4 (n = 1), 18 ± 4 (n = 12), 7 ± 1 (n = 11), and 7 ± 2 (n = 11) Neu5Ac mol / mol protein, respectively. These results correlate with those obtained via RP-HPLC and IEF assays. A nonparametric statistical test (mean median) allowed us to determine significant differences between the sialic acid content of the different preparations (p: 0.00048). As can be seen in Figure 8, the notched box plots indicated significant differences between the recombinant hormone and PMSG (B, Novormon) at the 95% confidence level.

[0108] Although the PMSG preparation showed a higher sialic acid content than the recombinant variant, both PMSG and the recombinant forms showed approximately the same ratio of sialic acid:galactose, due to the similar proportions of sialic acid and galactose residues in the three hormones (PMSG (B, Novormon), reCG RP-HPLC, and reCG-HIC showed Gal contents of 18 ± 0.9 (n = 2), 4.7 ± 0.1 (n = 2), and 6 ± 0.6 (n = 2) Gal mol / mol protein, respectively). Therefore, this likely supports the lower clearance of reCG from the circulation, reducing renal glomerular filtration and thus one of the attributes that allows reCG to exert its biological effects in target animal species. Moreover, the amount of mannose residues between the hormones was smaller than expected, as the heterodimer has three N-glycosylation sites, but was similar, and the amount of Man (mol per mol of protein) should be approximately 9 mol / mol. Therefore, this smaller content must be due to experimental error. The fucose content was similar between PMSG and the recombinant molecule.

[0109] 8.2.4 - Weak Anion Exchange Chromatography (WAX) for the Analysis of Charged Labeled N-Glycans To evaluate the sialylation pattern of purified PMSG commercial preparations (B) in comparison with 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 N-glycan structure. Glycans were identified as neutral (asialo), mono-, bi-, tri-, and tetra-sialylated structures using appropriate standards.

[0110] As can be seen in Figure 9, the N-glycan profiles were similar for the different preparations. However, the recombinant preparations showed a higher content of neutral and tetrasialylated glycans compared to PMSG. Moreover, the PMSG preparations showed a higher amount of bisialylated glycans fully substituted with sialic acid residues (peaks corresponding to the last positions, from left to right, in each group), unlike the (presumably more complex) recombinant variants, which showed incompletely bisialylated structures.

[0111] The reCG RP-HPLC molecule showed 3.09% neutral, 30.19% monosialylated, 54.22% bisialylated, 8.65% trisialylated, and 3.86% tetrasialylated structures. The reCG HIC contained 3.65% neutral, 26.76% monosialylated, 52.91% bisialylated, 13.53% trisialylated, and 3.86% tetrasialylated structures. Finally, the PMSG preparation showed the following percentages: 0.72% neutral, 29.02% monosialylated, 57.99% bisialylated, and 12.20% trisialylated structures (Table 4). [Table 4]

[0112] Example 9 - Efficacy Assay in Target Animals 9.1-reCG-induced superovulation in heifers The use of a single dose of eCG in superovulation and embryo production protocols has shown efficacy similar to that obtained by applying multiple doses of FSH. The purpose of this study was to evaluate the effectiveness of eCG in inducing superovulation. Eighteen heifers weighing 350–370 kg were synchronized using the following protocol: On day -10, heifers were injected with 150 μg of PGF2α (Ciclar, ZOOVET). On day 0, they received an intravaginal device with 1200 mg of P4 (IVD, Diprogest 1200, ZOOVET) and 2 mg of EB injection (estradiol benzoate, ZOOVET). On day 4, animals were randomly divided into four groups: group 1 (n = 4): 1000 IU of reCG, group 2 (n = 4): 1500 IU of reCG, group 3 (n = 5): 2000 IU of reCG, and group 4 (n = 4): 2500 IU of PMSG. On day 6, they were injected with 150 μg of PGF2α (Ciclar, ZOOVET). On day 7, the IVD was removed and the dose of PGF2α was repeated. On day 8, 0.02 mg of buserelin acetate (ZOOVET) was applied. Ultrasound examinations (US) were performed on days -10 and 0 to determine the estrous cycle stage at the start of the protocol. Additionally, on day 8, US was performed to assess the number of follicles greater than or equal to 8 mm (FOL<8 and FOL>8, respectively) and the number of corpora lutea (CL), and US Doppler (Mindray Z6Vet) was performed to assess perfusion of follicles greater than 8 mm. All data were analyzed by ANOVA followed by Duncan's post-hoc test. The variables perfusion, FOL>8, and CL were then correlated using a Pearson correlation test (SPSS Statistics 23, IBM). The results are summarized in Table 5. A statistically significant difference was observed between Group 1 and Group 3 with respect to the amount of FOL>8 and CL (P<0.05). Follicular perfusion was positively correlated with FOL>8 and CL (P<0.05). As can be seen from the results, reCG showed a dose-response effect on the production of preovulatory follicles and CL with increasing dose. A 2000 IU dose of reCG proved to be as effective as PMSG in inducing superovulation in heifers. Finally, higher-order follicular perfusion was shown to correlate with a greater number of preovulatory follicles and a greater number of CLs. [Table 5]

[0113] 9.2-Pregnancy test assay using reCG 9.2.1-TFAI: Legacy Protocol Conventional protocol for 8 consecutive days of P4-releasing intravaginal devices.

[0114] Day 0: Diagnostic US of the ovarian status of the animals and the conformation of the group under test. Application of an intravaginal device with 750 mg of progesterone (Prociclar, Zoovet) and 2 mg of ES injection (Zoovet).

[0115] Day 8: Device removal followed by injection of 150ug D+cloprostenol (Ciclar, Zoovet), 1mg estradiol cypionate (Zoovet), and 140IU reCG or 400IU PMSG depending on group. Heat-sensitive paint was applied to the base of the tail of all cows to determine the occurrence of mounting (heat) prior to fixed-time artificial insemination (FTAI).

[0116] Day 10 (48 hours after device removal): Fixed-time artificial insemination

[0117] Day 40 (day 30 from A1): A diagnostic ultrasound examination for pregnancy or periodicity was performed.

[0118] Ultrasound examination time schedule: Transrectal ultrasound was performed at time 0 to determine the ovarian status of the females under study. Ultrasound was then performed on day 40 (30 days post-insemination) to obtain a pregnancy diagnosis.

[0119] Semen and inseminators: To avoid possible fertility differences outside the protocol that could affect the results, all females were inseminated with the same semen from the same bull and the same batch of straw, and inseminations were performed by the same professional.

[0120] Results: We report a summary of the results and ultrasound examinations obtained during the process on day 30 after fixed-time artificial insemination (FTAI).

[0121] 9.2.2- J-Synch FTAI Protocol Day 0: Diagnostic US of the ovarian status of the animals and the conformation of the group under test. Application of an intravaginal device with 600 mg of progesterone (Diprogest, Zoovet) and 2 mg of estradiol benzoate (EB) injection (Zoovet).

[0122] Day 6: Device removal followed by injection of 150ug D+cloprostenol (Ciclar, Zoovet) and 105IU reCG or 300IU PMSG depending on group. Heat sensitive paint was applied to the base of the tail of all cows to determine the occurrence of mounting (heat) prior to fixed time artificial insemination (FTAI).

[0123] Day 9 (72 hours after device removal): All animals were injected with 0.010 mg of buserelin acetate (Zoovet) followed by fixed-time artificial insemination.

[0124] Day 43 (34 days after TFAI): Pregnancy diagnostic ultrasound

[0125] Ultrasound examination time schedule: Transrectal ultrasound was performed at time 0 to determine the ovarian status of the females under study. Ultrasound was then performed on day 43 (day 34 post-insemination) to obtain a pregnancy diagnosis.

[0126] Semen and inseminators: To avoid possible fertility differences outside the protocol that could affect the results, all females were inseminated with the same semen from the same bull and the same batch of straw, and inseminations were performed by the same professional. [Table 6] [Table 7]

Claims

1. 1. A method for obtaining a mammalian cell line expressing recombinant equine chorionic gonadotropin (reCG), comprising: a. providing coding sequences for the reCG alpha and beta subunits of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, optimized for expression in mammalian cells; b. Introducing the coding sequence into a lentiviral expression vector; c. Producing a lentivirus containing the reCG coding sequence; d. Transducing mammalian cells with the lentivirus produced in step c; e. selecting the most suitable mammalian cell clone for producing reCG.

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

3. 2. The method of claim 1, wherein the lentiviral expression vector in step b is a pLV lentiviral vector containing an EF-1α promoter.

4. 2. The method of claim 1, wherein step c comprises transient transfection of HEK293 cells with pREV, pVSVG, pMDL, pLV-reCGα, and pLV-reCGβ plasmids using cationic lipids as a vehicle.

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

6. The method of claim 5, comprising two successive transductions.

7. 2. A mammalian CHO cell line obtained by the method of claim 1, comprising a nucleic acid encoding recombinant equine chorionic gonadotropin (reCG), wherein the coding sequences for the alpha and beta subunits of reCG consist of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

8. The cell line of claim 7, which is a CHO-K1 cell line.

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

10. 1. A method for obtaining recombinant equine chorionic gonadotropin (reCG), comprising: a. Culturing the mammalian CHO cell line of claim 7 in a serum-free medium in a bioreactor for large-scale production of reCG; b. harvesting the supernatant; c. a purification step.

11. The method of claim 10, comprising a reCG productivity of at least 100 IU / mL in serum-free medium.

12. 11. The method of claim 10, wherein step c comprises dye pseudo-affinity chromatography.

13. The method of claim 12, wherein the dye pseudo-affinity chromatography uses a CaptoBlue-Sepharose matrix.

14. 11. The method of claim 10, also comprising an HPLC purification step using a C4 column.

15. 11. The method of claim 10, wherein step c consists of tangential flow filtration followed by reCG concentration.

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

17. A nucleic acid encoding the alpha subunit of reCG obtained by the method of claim 10, the nucleic acid consisting of SEQ ID NO:

1.

18. A nucleic acid encoding the beta subunit of reCG obtained by the method of claim 10, the nucleic acid consisting of SEQ ID NO:

2.

19. 11. A reCG obtained by the method of claim 10, comprising a glycosylation profile having at least 3% neutral structures and at least 3% tetrasialylated structures.

20. 11. The reCG obtained by the method of claim 10, comprising a glycosylation profile having at least 3% neutral structures, 26-30% monosialylated structures, 50-55% bisialylated structures, 8-15% trisialylated structures, and at least 3% tetrasialylated structures.

21. A pharmaceutical formulation comprising a therapeutically effective amount of the reCG of claim 19.

22. 22. The pharmaceutical formulation of claim 21, which is lyophilized.

23. 22. The pharmaceutical formulation of claim 21, which is a liquid.

24. 24. The pharmaceutical formulation of claim 23, further comprising sugar, a preservative, an antioxidant, mannitol, and an anti-aggregating agent, and is kept refrigerated at 5°C without the need for freezing.

25. 24. The pharmaceutical formulation of claim 23, comprising trisodium citrate dihydrate, citric acid monohydrate, arginine, sucrose, mannitol, L-methionine, poloxamer 188, m-cresol, and water.

26. 11. A method for inducing ovulation in a non-human animal, comprising administering the reCG obtained according to claim 10 in a dose of at least 140 IU / animal.

27. 27. The method of claim 26, wherein said administration of said dose induces ovulation 48 hours after its application.

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