Methods of producing gonadotropin

Abstract

A host cell characterized in that it comprises a sequence encoding the alpha chain of hCG integrated into its genome, and use of said host cell for producing recombinant hCG.

Description

[0001] This application is a divisional application of Chinese patent application No. 201680022637.8, whose application date is April 22, 2016 and invention name is “Method for producing gonadotropin”.

[0002] The present invention relates to gonadotropins for treating infertility, and in particular to human chorionic gonadotrophin (hCG).

[0003] Gonadotropins are a group of heterodimeric glycoprotein hormones that regulate gonadal function in both males and females. They include follicle stimulating hormone (FSH), luteinizing hormone (LH), and chorionic gonadotrophin (CG).

[0004] Human chorionic gonadotropin (hCG) is mainly secreted by the human placenta during pregnancy, and supports the maintenance of the corpus luteum. hCG comprises an α subunit (α chain) of 92 amino acids (which is also common to other glycoprotein hormones LH and FSH) and a β subunit (β chain) of 145 amino acids unique to hCG, which indicates hormone specificity. Each subunit is post-translationally modified by adding complex sugar residues. The α subunit is included in the 2-N-linked glycosylation sites of amino acids 52 and 78, and the β subunit is included in the 2-N-linked glycosylation sites of amino acids 13 and 30 and the four O-linked glycosylation sites of amino acids 121, 127, 132 and 138.

[0005] hCG extracted from the urine of pregnant women [Choragon (Ferring)] has been used for many years in the treatment of infertility. The preparation of hCG extracted from urine involves the collection and processing of large amounts of urine. A recombinant form of hCG, Ovitrelle (Serono), is also used in the treatment of infertility. This recombinant product is expressed in Chinese hamster ovary (CHO) cells and has a different pharmacokinetic profile than hCG produced from human urine.

[0006] There is considerable heterogeneity associated with hCG preparations, which involves differences in the amounts of the various isoforms present. Individual hCG isoforms display identical amino acid sequences, but differ in the extent to which they are post-translationally modified; specific isoforms are characterized by heterogeneity in sugar branching structure and differences in the amount of sialic acid (terminal sugar) attached, both of which appear to affect specific isoform biological activity.

[0007] The glycosylation of recombinant hCG ("rhCG") products reflects the range of glycosyltransferases present in the host cell line. The existing rhCG product Ovitrelle is derived from modified Chinese hamster ovary cells (CHO cells). The range of glycan modifications in CHO-derived rhCG is more limited than those found in natural products derived from urine. Examples of reduced glycan heterogeneity found in CHO-derived rhCG include the lack of bisecting glucosamine and reduced levels of core fucosylation and acetyllactosamine extensions. In addition, CHO cells are only able to add sialic acid using α2,3 linkages (Kagawa et al., 1988, Takeuchi et al., 1988, Svensson et al., 1990).

[0008] Human urine hCG (i.e., hCG extracted from the urine of pregnant women) that has been used for infertility treatment to date is actually placental hCG; hCG produced in the (human) placenta and then excreted in the urine. This hCG is extracted from urine and used as a pharmaceutical product. Bousfield et al. (Rev Endocr Metab Disord (2011) 12: 289-302) state that "while pituitary gonadotropins have both α2,3- and α2,6-linked sialic acids, placental hCG and recombinant gonadotropins produced in Chinese hamster ovary cells have only α2,3-linked sialic acids." Therefore, human urine hCG (also produced in the placenta) contains only α2,3-sialylation (like the recombinant product derived from the CHO cell line). Therefore, known pharmaceutical compositions containing hCG contain only α2,3-linked sialic acids, and not α2,6-linked sialic acids.

[0009] It has been shown that when compared with pituitary, serum or postmenopausal urine FSH, recombinant FSH preparation (Organon) is different in the amount of FSH (considered to be acidic isoform) with an isoelectric point (pI) lower than 4 (Ulloa-Aguirre et al. 1995). Compared with recombinant products Gonal-f (Serono) and Puregon (Organon), the amount of acidic isoform in FSH urine preparation is much higher (Andersen et al. 2004). This must be reflected in the sialic acid of lower molar content in rFSH, because the content of negatively charged polysaccharides modified with sulfate in FSH is low. Compared with natural FSH, lower sialic acid content is the feature of two commercial FSH products, and therefore must reflect the limitation in the preparation method (Bassett and Driebergen, 2005). The cycle life of FSH has been recorded about materials from various sources. Some of these materials have been separated based on total molecular charge, and total molecular charge is as characterized by their pI, and wherein more acid is equivalent to higher negative charge.The main contributor for total molecular charge is the total sialic acid content of each FSH molecule.For example, rFSH (Organon) has the sialic acid content of about 8mol / mol, and the FSH of urine origin has higher sialic acid content (de Leeuw et al. 1996).The corresponding plasma clearance in rats is 0.34 and 0.14ml / min (Ulloa-Aguirre et al. 2003).In another example, wherein recombinant FSH sample is divided into high and low pI fractions, the in vivo efficacy of high pI (lower sialic acid content) fraction reduces and it has shorter plasma half-life (D'Antonio et al. 1999). The applicants have found that, similar to FSH, known recombinant hCG products derived from CHO (e.g., Ovitrelle) also have lower amounts of hCG with an isoelectric point (pI) below 4 (considered to be the acidic isoform) than urinary hCG, which also reflects the lower sialic acid content of known rhCG products than urinary hCG.

[0010] The applicant has developed a recombinant hCG of human origin (i.e., recombinant hCG produced or expressed in a human cell line, e.g., produced by engineering a human cell line) which has a more acidic pattern than the rhCG product Ovitrelle derived from a CHO cell line, and which has a higher sialic acid content. The rhCG is the subject of International Patent Application No. PCT / GB2010 / 001854 (published as WO2011 / 042688). Recombinant hCG having a mixture of both α2,3 and α2,6-linked sialic acids is prepared by engineering a human cell line to express both rhCG and an α2,3 sialyltransferase. The expressed product is highly acidic, has a sialic acid content of, e.g., 19.1 mol / mol [expressed as a ratio of moles of sialic acid to moles of protein], and carries a mixture of both α2,3- and α2,6-linked sialic acids; the latter being provided by endogenous sialyltransferase activity. Applicants' studies have shown that the type of sialic acid linkage, α2,3- or α2,6-, can have a dramatic effect on the bioclearance of hCG. In contrast to CHO cell lines, human cell lines can express recombinant hCG with sialic acids attached by both α2,3 and α2,6 linkages.

[0011] The cell line of WO 2011 / 042688 expresses recombinant hCG of human origin well. However, the cell line also expresses relatively high levels of free β chains (free β subunits) and requires purification (to remove the free β chains from the product hCG).

[0012] Applicants have now developed a novel cell line that expresses both rhCG and α2,3 sialyltransferase and which produces rhCG with reduced amounts of free β chains, thereby increasing yield and reducing the level of product purification required (see Figure 7 ).

[0013] According to the present invention, a cell (e.g., a host cell) is provided, characterized in that it comprises a (nucleic acid) sequence encoding the α chain of hCG integrated into its genome, the sequence being selected from: a sequence shown in SEQ ID NO: 1; a sequence having at least 96.5% homology to the sequence of SEQ ID NO: 1 (e.g., a sequence having at least 96.5% sequence identity to the sequence of SEQ ID NO: 1); a sequence having at least 97% homology (e.g., at least 98% or at least 99% homology) to the sequence of SEQ ID NO: 1 (e.g., a sequence having at least 97% sequence identity (e.g., at least 98% or at least 99% sequence identity) to the sequence of SEQ ID NO: 1); a sequence shown in SEQ ID NO.4; a sequence having at least 96.5% homology to the sequence of SEQ ID NO: 4 (e.g., a sequence having at least 96.5% sequence identity to the sequence of SEQ ID NO: 4); a sequence having at least 97% homology (e.g., at least 98% or at least 99% sequence identity) to the sequence of SEQ ID NO: 1; a sequence having at least 97% homology (e.g., at least 98% or at least 99% sequence identity) to the sequence of SEQ ID NO: 1; a sequence having at least 96.5% homology to the sequence of SEQ ID NO: 4 (e.g., a sequence having at least 96.5% sequence identity to the sequence of SEQ ID NO: 4); NO:4 has at least 97% homology (e.g., at least 98% or at least 99% homology) (e.g., a sequence having at least 97% sequence identity (e.g., at least 98% or at least 99% sequence identity) with the sequence of SEQ ID NO:4). Preferably, the (nucleic acid) sequence encoding the α chain of hCG is not the sequence registered as AH007338 (i.e., preferably, the (nucleic acid) sequence encoding the α chain of hCG is not the sequence shown in SEQ ID NO:5). The cell may further comprise a cDNA encoding α-2,3-sialyltransferase integrated into its genome, for example, the sequence described in SEQ ID NO:3. The cell may further comprise a (nucleic acid) sequence encoding the β chain of hCG integrated into its genome, for example, the sequence described in SEQ ID NO:2.

[0014] For example, the host cell can be PER. cells, HT1080 cells, GT-5s cells, etc. Preferably, the cells are PER. Cells, such as PER deposited under ECACC no.96022940. cell.

[0015] According to the present invention, PER is provided. Cells, such as PER deposited under ECACC no.96022940. A cell, characterized in that it further comprises a (nucleic acid) sequence encoding the alpha chain of hCG integrated into its genome. The (nucleic acid) sequence encoding the alpha chain of hCG can be selected from: a sequence shown in SEQ ID NO: 1; a sequence having at least 90% homology to the sequence of SEQ ID NO: 1 (e.g., a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 1); a sequence having at least 97% homology (e.g., at least 98% or at least 99% homology) to the sequence of SEQ ID NO: 1 (e.g., a sequence having at least 97% sequence identity (e.g., at least 98% or at least 99% sequence identity) to the sequence of SEQ ID NO: 1); a sequence shown in SEQ ID NO. 4; a sequence having at least 90% homology to the sequence of SEQ ID NO: 4 (e.g., a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 4); and a sequence having at least 97% homology (e.g., at least 98% or at least 99% homology) to the sequence of SEQ ID NO: 4 (e.g., a sequence having at least 97% sequence identity (e.g., at least 98% or at least 99% sequence identity) to the sequence of SEQ ID NO: 4 (e.g., at least 98% or at least 99% sequence identity) to the sequence of SEQ ID NO: 4). NO:4 has at least 97% sequence identity (e.g., at least 98% or at least 99% sequence identity). Preferably, the (nucleic acid) sequence encoding the α chain of hCG is not the sequence registered as AH007338. The cell further comprises a cDNA encoding α-2,3-sialyltransferase integrated into its genome, such as the sequence described in SEQ ID NO:3. The cell may further comprise a (nucleic acid) sequence encoding the β chain of hCG integrated into its genome, such as the sequence described in SEQ ID NO:2.

[0016] It should be understood that the term "sequence encoding the α chain of hCG" or "(nucleic acid) sequence encoding the α chain of hCG" as used herein also refers to the sequence encoding the α chain of FSH (or the sequence encoding the α chain of LH), since the α chain of hCG is identical to the α chain of FSH (and LH).

[0017] The term "nucleic acid sequence" as used herein refers to any nucleic acid molecule encoding a polypeptide (e.g., peptide, protein, etc.). These nucleic acid molecules can be made of DNA, RNA, or analogs thereof. However, nucleic acid molecules made of DNA are preferred.

[0018] It is well known to those skilled in the art that modification of the starting nucleotide sequence describes the process of optimizing codon usage. By comparing the modified sequence with the starting sequence, the introduced changes can be easily identified. And, the two sequences (i.e., the starting sequence and the optimized sequence) will encode the same amino acid sequence.

[0019] The amino acid sequence of the α-chain of human hCG is disclosed in WO2011 / 042688 (referred to as SEQ ID1 in this document) and described in Fiddes and Goodman 1979. It is registered as AH007388 and is shown as SEQ ID NO.5 below. The amino acid sequence of the β-chain of human hCG is shown as SEQ ID No.2 and is registered as NP_000728. These amino acid sequences correspond to the wild-type amino acid sequences of the α-chain and β-chain of human hCG. The term "wild-type nucleic acid sequence" or "starting nucleic acid sequence" when used in the present invention relates to a nucleic acid sequence intended for (over) expression in a host cell and not suitable for codon usage in the host cell, but the actual wild-type nucleic acid sequence encoding the protein. The term "optimized nucleic acid sequence" when used in the present invention relates to a sequence modified for expression in a host cell by changing the sequence of the unmodified / starting nucleic acid sequence. The optimized nucleic acid sequence encodes the same amino acid sequence as the protein encoded by the unmodified sequence. Applicants have developed optimized sequences encoding the α-chain of hCG (see, for example, SEQ ID Nos. 1 and 4, Figure 1 ).

[0020] According to the present invention, in another aspect, a polynucleotide sequence (e.g., a polynucleotide sequence encoding the α chain of hCG) is provided, which comprises a sequence selected from the following: the nucleic acid sequence shown in SEQ ID NO: 1; the nucleic acid sequence shown in SEQ ID NO: 4; a nucleic acid sequence having at least 97% homology (e.g., at least 98% or at least 99% homology) with the nucleic acid of SEQ ID NO: 1 (e.g., a nucleic acid sequence having at least 97% sequence identity (e.g., at least 98% or at least 99% sequence identity) with the nucleic acid of SEQ ID NO: 1); and a nucleic acid sequence having at least 97% homology (e.g., at least 98% or at least 99% homology) with the nucleic acid of SEQ ID NO: 4 (e.g., a nucleic acid sequence having at least 97% sequence identity (e.g., at least 98% or at least 99% sequence identity) with the nucleic acid of SEQ ID NO: 4).

[0021] According to the present invention, in another aspect, a method for producing recombinant hCG in a cell is provided, the method comprising culturing the cell in a suitable culture medium, and collecting the recombinant protein (hCG) from the cell and / or the culture medium (e.g., collecting the recombinant hCG from the cell culture supernatant), wherein the cell (host cell) comprises a (nucleic acid) sequence encoding the α chain of hCG integrated into its genome, the sequence being selected from: a sequence as shown in SEQ ID NO: 1; a sequence having at least 90% homology to the sequence of SEQ ID NO: 1 (e.g., a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 1); a sequence having at least 97% homology (e.g., at least 98% or at least 99% homology) to the sequence of SEQ ID NO: 1 (e.g., a sequence having at least 97% sequence identity (e.g., at least 98% or at least 99% sequence identity) to the sequence of SEQ ID NO: 1); a sequence as shown in SEQ ID NO.4; a sequence having at least 90% homology to the sequence of SEQ ID NO: 4 (e.g., a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 1); NO:4); and a sequence having at least 97% homology (e.g., at least 98% or at least 99% homology) to the sequence of SEQ ID NO:4 (e.g., a sequence having at least 97% sequence identity (e.g., at least 98% or at least 99% sequence identity) to the sequence of SEQ ID NO:4). Preferably, the (nucleic acid) sequence encoding the α chain of hCG is not the sequence registered as AH007338. The cell may further comprise a cDNA encoding α-2,3-sialyltransferase integrated into its genome, such as the sequence described in SEQ ID NO:3. The cell may further comprise a (nucleic acid) sequence encoding the β chain of hCG integrated into its genome, such as the sequence described in SEQ ID NO:2. The method may comprise one or more additional steps of purifying the recombinant hCG obtained from the cell and / or the culture medium (e.g., purifying the recombinant hCG from the cell culture supernatant).

[0022] For example, the (host) cell may be a PER. cells, HT1080 cells, GT-5s cells, etc. Preferably, the cells are PER. Cells, such as PER deposited under ECACC no.96022940. cell.

[0023] According to the present invention, in another aspect, there is provided a method for producing recombinant hCG in a cell, the method comprising culturing the cell in a suitable culture medium and collecting the recombinant protein (hCG) from the cell and / or the culture medium (e.g., collecting the recombinant hCG from the cell culture supernatant), wherein the cell is a PER comprising a (nucleic acid) sequence encoding the α chain of hCG integrated into its genome. Cells (such as PER. Cell). The (nucleic acid) sequence encoding the α chain of hCG can be selected from: a sequence as shown in SEQ ID NO: 1; a sequence having at least 90% homology to the sequence of SEQ ID NO: 1 (e.g., a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 1); a sequence having at least 97% homology (e.g., at least 98% or at least 99% homology) to the sequence of SEQ ID NO: 1 (e.g., a sequence having at least 97% sequence identity (e.g., at least 98% or at least 99% sequence identity) to the sequence of SEQ ID NO: 1); a sequence as shown in SEQ ID NO.4; a sequence having at least 90% homology to the sequence of SEQ ID NO: 4 (e.g., a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 4); and a sequence having at least 97% homology (e.g., at least 98% or at least 99% homology) to the sequence of SEQ ID NO: 4 (e.g., a sequence having at least 97% homology (e.g., at least 98% or at least 99% homology) to the sequence of SEQ ID NO: 4 (e.g., NO:4 has at least 97% sequence identity (e.g., at least 98% or at least 99% sequence identity). Preferably, the (nucleic acid) sequence encoding the α chain of hCG is not the sequence registered as AH007338. The cell further comprises a cDNA encoding α-2,3-sialyltransferase integrated into its genome, such as the sequence described in SEQ ID NO:3. The cell may further comprise a (nucleic acid) sequence encoding the β chain of hCG integrated into its genome, such as the sequence described in SEQ ID NO:2. The method may comprise one or more additional steps of purifying the recombinant hCG obtained from the cell and / or the culture medium (e.g., purifying the recombinant hCG from the cell culture supernatant).

[0024] The term "host cell" as used herein refers to any cell routinely used for expression, i.e., any cell that transcribes and translates a nucleic acid sequence for production of, for example, a polypeptide. In particular, the term "host cell" or "organism" refers to prokaryotes, lower eukaryotes, plants, insect cells or mammalian cell culture systems. Preferably, the host cell is a mammalian cell, more preferably, the host cell is a human cell, even more preferably a cell.

[0025] The term "recombinant nucleic acid molecule" within the meaning of the present invention is intended to include all types of nucleic acid molecules that can be introduced into a host cell and affect the expression of the nucleic acid sequence contained in the recombinant nucleic acid molecule. For example, the term includes plasmid vectors and viral vectors (e.g., adenovirus, lentivirus and retroviral vectors), which are well known in the art.

[0026] According to the present invention, in another aspect, a recombinant nucleic acid molecule comprising a (first) nucleic acid sequence encoding the α chain of hCG is provided, wherein the (first) nucleic acid sequence is selected from the following: a sequence as shown in SEQ ID NO: 1; a sequence having at least 96.5% homology to the sequence of SEQ ID NO: 1 (e.g., a sequence having at least 96.5% sequence identity to the sequence of SEQ ID NO: 1); a sequence having at least 97% homology (e.g., at least 98% or at least 99% homology) to the sequence of SEQ ID NO: 1 (e.g., a sequence having at least 97% sequence identity (e.g., at least 98% or at least 99% sequence identity) to the sequence of SEQ ID NO: 1); a sequence as shown in SEQ ID NO.4; a sequence having at least 96.5% homology to the sequence of SEQ ID NO: 4 (e.g., a sequence having at least 96.5% sequence identity to the sequence of SEQ ID NO: 14); and a sequence having at least 97% homology (e.g., at least 98% or at least 99% sequence identity) to the sequence of SEQ ID NO: 1. IDNO:4 has at least 97% homology (e.g., at least 98% or at least 99% homology) to the sequence (e.g., a sequence having at least 97% sequence identity (e.g., at least 98% or at least 99% sequence identity) to the sequence of SEQID NO:4). Preferably, the recombinant nucleic acid molecule of this aspect of the invention comprises both an optimized nucleic acid sequence encoding the α-chain of human hCG and a nucleic acid sequence encoding the β-chain of hCG. Preferably, the (first) nucleic acid sequence is under the control of a promoter active in a host cell. Preferably, the recombinant nucleic acid molecule further comprises a second nucleic acid sequence encoding the β-chain of hCG. The second nucleic acid sequence encoding the β-chain of hCG may be the sequence shown in SEQ ID No.2. The second nucleic acid sequence may be under the control of a separate promoter active in a host cell. The first nucleic acid sequence and / or the second nucleic acid sequence may be under the control of a viral promoter (e.g., CMVie promoter).

[0027] According to the present invention, in another aspect, a host cell comprising or including the above-mentioned recombinant nucleic acid molecule is provided (e.g., a host cell comprising a recombinant nucleic acid molecule of a (first) nucleic acid sequence encoding the α chain of hCG, wherein the (first) nucleic acid sequence is selected from: a sequence shown in SEQ ID NO: 1; a sequence having at least 96.5% homology to the sequence of SEQ ID NO: 1 (e.g., a sequence having at least 96.5% sequence identity to the sequence of SEQ ID NO: 1); a sequence having at least 97% homology (e.g., at least 98% or at least 99% homology) to the sequence of SEQ ID NO: 1 (e.g., a sequence having at least 97% sequence identity (e.g., at least 98% or at least 99% sequence identity) to the sequence of SEQ ID NO: 1); a sequence shown in SEQ ID NO.4; a sequence having at least 96.5% homology to the sequence of SEQ ID NO: 4 (e.g., a sequence having at least 96.5% sequence identity to the sequence of SEQ ID NO: 4); a sequence having at least 97% homology (e.g., at least 98% or at least 99% sequence identity) to the sequence of SEQ ID NO: 1); a sequence shown in SEQ ID NO. A sequence having at least 97% homology (e.g., at least 98% or at least 99% homology) to the sequence of SEQ ID NO:4 (e.g., a sequence having at least 97% sequence identity (e.g., at least 98% or at least 99% sequence identity) to the sequence of SEQ ID NO:4).

[0028] Applicants have discovered that the cells (host cells), cell lines incorporating these host cells, recombinant nucleic acid molecules, polynucleotides and methods of the present invention can produce recombinant hCG in high yield and purity (eg, with little to no free beta chain).

[0029] According to the present invention, in another aspect, there is provided a recombinant hCG ("rhCG" or "rechCG") comprising α2,3- and α2,6-sialylation, wherein the recombinant hCG is produced in a cell as described and claimed herein. The recombinant hCG may have a sialic acid content of 12 mol / mol to 20 mol / mol [expressed as a ratio of moles of sialic acid to moles of protein], such as 12 mol / mol to 15.5 mol / mol, such as 12 mol / mol to 14.9 mol / mol. The rhCG (or rhCG preparation) may have a sialic acid content of 12.5 mol / mol to 14.5 mol / mol, such as a sialic acid content of 12.8 mol / mol to 13.2 mol / mol. The rhCG (or rhCG preparation) of the present invention may have a sialic acid content of 13 mol / mol.

[0030] In examples of the present invention, rhCG may be present as a single isoform or as a mixture of multiple isoforms.

[0031] The recombinant hCG (or rhCG preparation) produced by the methods and cells of the present invention includes α2,3- and α2,6-sialylation. Sialylation refers to the amount of sialic acid residues present on the hCG sugar structure. α2,3-sialylation means sialylation at the 2,3 position (which is well known in the art); α2,6 sialylation means sialylation at the 2,6 position (which is also well known in the art). Therefore, herein, the phrase "% of total sialylation may be α2,3 sialylation" refers to the % of sialylation at the 2,3 position out of the total number of sialic acid residues present in hCG. The term "% of total sialylation may be α2,6-sialylation" refers to the % of sialylation at the 2,6 position out of the total number of sialic acid residues present in hCG. The term "% of total sialylation may be α2,8-sialylation" refers to the % of sialylation at the 2,8 position out of the total number of sialic acid residues present in hCG.

[0032] The rhCG (or rhCG preparation) produced by the methods and cells of the present invention may have 1% to 99% of the total sialylation as α2,3-sialylation. The rhCG (or rhCG preparation) produced by the methods and cells of the present invention may have 1% to 90% of the total sialylation as α2,3-sialylation. The rhCG (or rhCG preparation) of the present invention may have more than 10% of the total sialylation as α2,3-sialylation, for example, 10% to 90% of the total sialylation may be α2,3-sialylation. For example, more than 20, 30, 40, 45, 50, 55, 60, 70, 80 or 90% of the total sialylation may be α2,3-sialylation. 45% to 80% of the total sialylation can be α2,3-sialylation, for example, 50% to 70% of the total sialylation can be α2,3-sialylation, for example, 55 to 65% of the total sialylation can be α2,3-sialylation. The amount of α2,3-sialylation included in the rhCG (or rhCG preparation) can be 65 to 95% of the total sialylation, for example, 70 to 90% of the total sialylation, for example, 85 to 90% of the total sialylation.

[0033] The rhCG (or rhCG preparation) produced by the methods and cells of the present invention may have 1 to 99% of the total sialylation as α2,6-sialylation. The rhCG (or rhCG preparation) of the present invention may have 5 to 50% of the total sialylation as α2,6-sialylation. For example, 5 to 45%, for example, 6 to 40%, for example, 7 to 30%, for example, 8 to 20%, of the total sialylation may be α2,6-sialylation. The amount of α2,6-sialylation contained in the rhCG (or rhCG preparation) may be 20-75% of the total sialylation, for example, 30-60% of the total sialylation, for example, 35-45% of the total sialylation. The amount of α2,6-sialylation contained in the rhCG (or rhCG preparation) may be 5 to 35% of the total sialylation. For example, 10 to 20% of the total sialylation. For example, 11-55% of the total sialylation may be α2,6-sialylation.

[0034] The rhCG or rhCG preparation produced by the method and cell may optionally further comprise α2,8 sialylation. The rhCG (or rhCG preparation) of the present invention may have less than 5% of the total sialylation as α2,8-sialylation, for example, 0 to 4%, for example, 0.1-4%, of the total sialylation as α2,8-sialylation. The rhCG (or rhCG preparation) of the present invention may have no α2,8-sialylation.

[0035] The rhCG (or rhCG preparation) produced by the methods and cells of the present invention may have a sialic acid content (the amount of sialylation per hCG molecule) (based on the mass of the protein, not the mass of the protein plus the carbohydrate) of 6% or more (e.g., 6%-15%, e.g., 7%-13%, e.g., 8%-12%, e.g., 11%-15%, e.g., 12%-14%) by mass.

[0036] The rhCG (or rhCG preparation) may be produced or expressed in a human cell line, for example, in PER. cell line, HT1080 cell line, etc. The rhCG (or rhCG preparation) can be produced or expressed in a cell line derived from a human or a modified human cell line, for example, in a cell line derived from PER. cell lines or modified PER. cell line, a modified HT1080 cell line or a cell line derived from HT1080, etc. In a preferred embodiment, rhCG is produced or expressed in PER. Cell line, derived from PER. cell lines or modified PER. In human cell lines (e.g., PER. rhCG produced or expressed in the cell lines (e.g., HT1080 cell line, GT-5s cell line) contains some α2,6-linked sialic acid (α2,6 sialylation) provided by the endogenous sialyltransferase activity of [the cell line], and will contain some α2,3-linked sialic acid (α2,3 sialylation) provided by the endogenous sialyltransferase activity of [the cell line]. The cell line can be modified with an α2,3-sialyltransferase. Alternatively or additionally, the cell line can be modified with an α2,6-sialyltransferase. This can simplify (and make it more efficient) the production process because, for example, manipulation and control of the cell growth medium to retain sialylation can be less important than using known methods. The method can also be more efficient because little alkaline rhCG is produced compared to the production of known rhCG products; more acidic rhCG is produced, and separation / removal of alkaline hCG is easier.

[0037] rhCG can be produced using an α2,3-sialyltransferase, e.g., using a human cell line modified with an α2,3-sialyltransferase. rhCG can comprise α2,6-linked sialic acid (α2,6 sialylation) provided by endogenous sialyltransferase activity. rhCG can be produced using an α2,3- and / or α2,6-sialyltransferase, e.g., using a human cell line modified with an α2,3-sialyltransferase and / or an α2,6-sialyltransferase.

[0038] According to the present invention, in another aspect, there is provided a recombinant hCG or a recombinant hCG preparation as described herein, which is produced or expressed by the method as described herein.

[0039] The rhCG structure comprises glycan moieties. Branching may occur, with the result that the glycans may have 1, 2, 3, 4 or more terminal sugar residues or "antennae", as is well known in the art. The rhCG described in various aspects of the invention may have glycans with monoantennary and / or biantennary and / or triantennary and / or tetraantennary glycan structures. The rhCG may comprise monoantennary and / or biantennary and / or triantennary and / or tetraantennary glycan structures, for example, in the following relative amounts: 0.1 to 3% monoantennary; 65% to 85% biantennary; 15 to 25% triantennary and 0.5 to 1.5% tetraantennary (e.g., as shown by WAX analysis of charged glycans).

[0040] According to the present invention, in another aspect, a pharmaceutical composition comprising recombinant hCG (rhCG) having α2,3-sialylation and α2,6-sialylation is provided, wherein the recombinant hCG is produced by the methods and / or cells disclosed herein. The rhCG may have a sialic acid content [expressed as a ratio of moles of sialic acid to moles of protein] of 12 mol / mol to 15.5 mol / mol, for example 12 mol / mol to 14.9 mol / mol (e.g., as described above). The rhCG may have a sialic acid content of 12.5 mol / mol to 14.5 mol / mol, for example, a sialic acid content of 12.8 mol / mol to 13.2 mol / mol. Preferably, the rhCG of the present invention has a sialic acid content of 13 mol / mol.

[0041] The pharmaceutical composition may further comprise FSH and / or LH.

[0042] FSH can be obtained by any means known in the art. FSH used herein includes FSH derived from humans and recombinant FSH. FSH derived from humans can be purified from any appropriate source (e.g., urine) by any method known in the art. FSH can be recombinant FSH--e.g., expressed in a human cell line. The method of expressing and purifying recombinant FSH is well known in the art.

[0043] LH can be obtained by any means known in the art. LH used herein includes human-derived and recombinant LH. Human-derived LH can be purified from any appropriate source (e.g., urine) by any method known in the art. Methods for expressing and purifying recombinant LH are well known in the art.

[0044] The pharmaceutical composition can be used to treat infertility, or in applications for infertility treatment, for example, in applications such as assisted reproductive technology (ART), ovulation induction or intrauterine insemination (IUI). The drug can be used to induce final maturation / ovulation and luteinization, stimulate follicular development in women with severe LH and FSH deficiency, and / or luteal support (rhCG / rLH), or in the above-mentioned applications. The pharmaceutical composition can be used to induce monofollicular development in anovulatory WHO type II women (e.g., rhCG with rLH), to enhance multifollicular response to LH sensitization in patients undergoing COH (e.g., rhCG with rLH), and to supplement COH to improve implantation / pregnancy rates (e.g., rhCG with rLH), and / or to increase pregnancy rates in assisted reproductive technology, or in the above-mentioned applications. The pharmaceutical composition can be used to prevent miscarriage and / or premature birth prevention, or in the above-mentioned applications. The pharmaceutical composition can be used for the treatment of endometriosis, or for use in the treatment of endometriosis.

[0045] For example, the pharmaceutical composition may be used for medical indications where known hCG preparations are used. The invention also provides the use of rhCG and / or rhCG preparations described herein (as described in the various aspects of the invention) for or in the preparation of a medicament for the treatment of infertility.

[0046] The pharmaceutical composition of the present invention can be formulated into known compositions for any drug administration route, for example, oral, rectal, parenteral, transdermal (for example, patch technology), intravenous, intramuscular, subcutaneous, intravaginal, intraperitoneal, local (powders, ointments or drops) or as oral or nasal sprays. Typical compositions include pharmaceutical carriers, such as aqueous solutions, nontoxic excipients, including salts and preservatives, buffers, etc., particularly as described in Remington's Pharmaceutical Sciences (Remington's Pharmacopoeia) the 15th edition (Matt Publishing Company, 1975) pages 1405-1412 and pages 1461–87 and national formulary XIV (national prescription XIV) the 14th edition (American Pharmaceutical Association, 1975). Examples of suitable aqueous and non-aqueous pharmaceutical carriers, diluents, solvents or excipients include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and their appropriate mixtures, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). The compositions of the present invention may also include additives such as, but not limited to, preservatives, wetting agents, emulsifiers and dispersants. Antibacterial and antifungal agents may be included to prevent microbial growth, and include, for example, meta-cresol, benzyl alcohol, parabens, chlorobutanol, phenol, sorbic acid, etc. In addition, isotonic agents such as sugars, sodium chloride, etc. may be desirably included.

[0047] In some cases, in order to achieve a prolonged effect, it is necessary to slow down the absorption of hCG (and other active ingredients, if present) from subcutaneous or intramuscular injection. This can be achieved by using a lipid suspension of crystalline or amorphous material with poor water solubility. The absorption rate of hCG then depends on its solubility, which in turn may depend on the crystal size and crystalline form. Alternatively, delayed absorption of parenterally administered hCG combinations is achieved by dissolving or suspending the hCG combination in an oily excipient. Injectable long-acting formulations can be prepared by forming a microcapsule matrix of hCG (and other ingredients, if present) in a biodegradable polymer (such as polylactic acid-polyglycolide). Depending on the ratio of hCG to polymer and the properties of the specific polymer used, the rate of hCG release can be controlled. Other biodegradable polymers include polyvinylpyrrolidone, poly(orthoesters), poly(anhydrides), etc. Long-acting injectable formulations are also prepared by entrapping hCG in liposomes or microemulsions compatible with body tissues. For example, the injectable preparation can be sterilized by filtration through a bacteria-intercepting filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use. The injectable preparation can be provided in any suitable container, for example, a vial, a prefilled syringe, a syringe barrel, etc.

[0048] The preparation (e.g., an injectable preparation) can be provided as a product having a pharmaceutical composition comprising hCG (optionally with FSH, LH, etc.). If there is more than one active ingredient (i.e., hCG, and, for example, FSH or LH), these can be suitable for separate or combined administration. If administered separately, administration can be sequential. The product can be provided in any suitable packaging. For example, the product can comprise a plurality of pre-filled syringes or vials comprising hCG, FSH, or a combination of FSH and hCG. The syringes or vials can be packaged in blister packs or other forms to maintain sterility. The product can optionally include instructions for use of the hCG and FSH preparations.

[0049] The pH and exact concentration of the various ingredients of the pharmaceutical composition are adjusted according to the conventional practice in the art. See GOODMAN and GILMAN's THE PHARMACOLOGICAL BASIS FOR THERAPEUTICS, 7th edition. In a preferred embodiment, the composition of the present invention is provided as a composition for parenteral administration. The general method for preparing parenteral preparations is known in the art and is described in REMINGTON; THE SCIENCE AND PRACTICE OF PHARMACY (same as above) pp. 780-820. Parenteral compositions can be provided as liquid preparations or as solids, which will be mixed with a sterile injection medium just before administration. In one embodiment, parenteral compositions are provided in unit dosage form for ease of administration and uniformity of dosage. DETAILED DESCRIPTION OF THE INVENTION BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The invention will now be described in more detail with reference to the following examples and the accompanying drawings, in which:

[0051] Figure 1 Nucleic acid and derived amino acid sequences showing the optimized hCGα sequence;

[0052] Figure 2 The nucleic acid and derived amino acid sequences showing the hCGβ (wild-type β chain) sequence;

[0053] Figure 3 The nucleic acid and derived amino acid sequences of ST3GAL4 are shown;

[0054] Figure 4The plasmid map of the phCGα / β expression vector is shown (CMV=cytomegalovirus promoter, BGHp(A)=bovine growth hormone polyadenylation sequence, f1 ori=f1 origin of replication, SV40=simian virus 40 promoter, Neo=neomycin resistance marker, Hyg=hygromycin resistance marker, SV40 p(A)=simian virus 40 polyadenylation sequence, hCGα=human chorionic gonadotropin α polypeptide, hCGβ=human chorionic gonadotropin β polypeptide, ST3GAL4=α2,3-sialyltransferase, ST6GAL1=α2,6-sialyltransferase, ColE1=ColE1 origin of replication, Amp=ampicillin resistance marker);

[0055] Figure 5 α2,3-sialyltransferase (ST3GAL4) expression vector is shown (CMV=cytomegalovirus promoter, BGHp(A)=bovine growth hormone polyadenylation sequence, f1 ori=f1 origin of replication, SV40=simian virus 40 promoter, Neo=neomycin resistance marker, Hyg=hygromycin resistance marker, SV40 p(A)=simian virus 40 polyadenylation sequence, hCGα=human chorionic gonadotropin α polypeptide, hCGβ=human chorionic gonadotropin β polypeptide, ST3GAL4=α2,3-sialyltransferase, ST6GAL1=α2,6-sialyltransferase, ColE1=ColE1 origin of replication, Amp=ampicillin resistance marker);

[0056] Figure 6 Detection of rhCG isoforms in the composition of the invention by IEF with Coomassie blue staining is shown (lanes 4, 5, 6 and 7, 15 μg / lane); a prior art CHO-derived composition, Ovitrelle (lane 2, 15 μg); and Novarel, a human urine product obtained from a pregnant woman (lane 3, 15 μg); IEF marker (lane 1 (pi from top: 7.0, 6.8, 6.5, 6.0, 5.1 and 4.75, respectively));

[0057] Figure 7 The concentrations of hCG ("α-βhCG") and free β chain ("βhCG") produced using cells of the present invention comprising an "optimized" α subunit nucleic acid sequence are compared to the concentrations of hCG ("α-βhCG") and free β chain ("βhCG") produced in comparative cells comprising a WT (wild-type) α subunit.

[0058] Examples 1-7

[0059] Overview of the Cell Line Development Process

[0060] A single plasmid carrying the hCG α and β chains driven by separate CMVie promoters was transfected into the PER by electroporation. in cells.

[0061] This plasmid carries the neomycin resistance gene cassette ( Figure 4 ), which allows selection of transfectants using G418 (Geneticin). The transfectant pool was expanded and then limited dilution cloning was performed in 96-well plates under G418 selection. Clonal lines were expanded and high expressing clones were identified based on hCG titers in cell culture supernatants.

[0062] Based on high productivity, five leading clones were selected and each clone was then transfected with a second plasmid expressing the α2,3-sialyltransferase gene ST3GAL4 and a hygromycin selection marker ( Figure 5 Each of these five transfection pools was expanded and the cell culture supernatants were evaluated for hCG concentration and in vivo pharmacokinetics (PK).

[0063] Promising pools are then subjected to another round of limiting dilution cloning. The resulting clones are expanded and the cell culture supernatants are evaluated for hCG concentration and exposed galactose on the hCG protein (by lectin ELISA). Those with low levels of exposed galactose are subjected to further analysis, including in vivo PK. Clones that produce hCG with the best PK profile, have low exposed galactose, and express protein at high levels are evaluated and selected for productivity and growth characteristics.

[0064] Example 1: Sequence selection and plasmid vector

[0065] The coding region of the gene for hCGα polypeptide is shown in Figure 1 The coding region of the gene for the hCGα polypeptide sequence is referred to herein as SEQ ID NO: 1.

[0066] The coding region of the gene for the hCGβ polypeptide was used according to Fiddes and Goodman (1980). This sequence is registered as NP_000728 and is identical to the protein sequences of CGβ3, CGβ5 and CGβ7. This sequence is referred to herein as SEQ ID NO: 2.

[0067] The coding region of the gene for β-galactoside α-2,3-sialyltransferase 4 (α2,3-sialyltransferase, ST3GAL4) was used according to Kitagawa and Paulson (1994). This sequence is accessioned as L23767 and is referred to herein as SEQ ID NO: 3.

[0068] Two plasmids were used: the first was phCG, co-expressing the hCG α and β chains; the second expressed the sialyltransferase gene ST3GAL4.

[0069] Example 2a: Construction of hCG expression vector

[0070] phCG is a synthetic hCG alpha chain that is optimized for codon usage in mammalian cells and contains the native hCG alpha signal peptide. It can be modified by methods known in the art.

[0071] The coding sequences of hCGα polypeptide (SEQ ID NO: 1) and hCGβ polypeptide (NP_000728, SEQ ID NO: 2) were amplified by PCR using methods known in the art (e.g., see International Patent Application Published as WO2011 / 042688). The phCGαDNA was digested with BamHI and NheI and inserted into the sites BamHI and NheI of a CMV-driven mammalian expression vector (Crucell vector pcDNA3002Neo). This places the gene in the correct direction to drive expression through the CMVie promoter and downstream BGH polyadenylic acid (polyA) signal. The natural hCGβ gene containing the natural signal peptide was digested with restriction enzymes AscI and HpaI and inserted into the AscI and HpaI sites so that expression is driven by a second CMVie promoter and an additional downstream BGH polyadenylic acid (poly A) signal. The vector backbone also contains a neomycin resistance marker and elements required for selection and replication in prokaryotes. The vector was amplified and sequenced using general methods known in the art. The sequences of the optimized hCG α and native (wild-type) β chains are provided in Figure 1 and 2 middle.

[0072] All colonies selected for sequencing contained the correct sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2. Plasmid phCG A+B was selected for transfection ( Figure 4 ).

[0073] Example 2b: Construction of ST3 expression vector

[0074] The ST3GAL4 gene was expressed in pST3. The β-galactoside α-2,3-sialyltransferase 4 (ST3, L23767, SEQ ID NO: 3, Figure 3 ) coding sequence.

[0075]

[0076] The ST3 DNA obtained by amplification was digested with restriction enzymes BamHI and AflII and inserted into the BamHI and AflII sites of a CMV-driven mammalian expression vector (vector pcDNA3002Neo) carrying a hygromycin resistance marker, so that it is located downstream of the CMVie promoter and upstream of the BGH polyadenylic acid (polyA) sequence. The vector backbone also contains the hygromycin resistance marker and the elements required for selection and replication in prokaryotes. The vector was amplified and sequenced as described above. Clone pST3#1 ( Figure 5 ) contains the correct sequence shown in SEQ ID NO:3 and is selected for transfection.

[0077] Example 3 - Plasmid transfection

[0078] Plasmid phCG( Figure 4 ) has a single PvuI site located in the prokaryotic β-lactamase gene, which was linearized with PvuI (New England Biolabs Cat. No. R0150). The linearized plasmid DNA was transfected into PER. in cells.

[0079] Cell cultures were maintained in complete PERMAB medium (CD4PERMAB (Hyclone Cat. No. SH30871.01) supplemented with L-glutamine to a final concentration of 3 mM (Invitrogen Cat. No. 25030-123) and Pluronic F68 (Invitrogen Cat. No. 24040-032) at a final concentration of 1.0 g / L) in 250 ml Erlenmeyer flasks. Cells were maintained in a shaking incubator (Kuhner Climo-shaker ISF1-X) set at 100 rpm, 5% CO2 and 37°C for at least 14 days prior to transfection. 48 hours prior to transfection, cells were incubated at 0.5 x 10 6 Transfer to fresh medium at a density of 10 cells / ml.

[0080] On the day of transfection, cells were counted in a Beckman Coulter ViCell XR to determine cell density and ensure viability >90%. Cells were collected by centrifugation and resuspended in fresh PERMAB medium and then mixed with linearized phCG DNA. The cell / DNA mixture was electroporated for 5 msec in the chamber of an electroporator set to 250 V and then quickly transferred to 10 ml of pre-warmed PERMAB medium. This process was repeated 6 times in total and all 6 transfections were combined into a single T-175 cm 2The flask was placed in a static incubator set at 37°C, 5% CO2. After 48 hours, the cells were resuspended in an appropriate volume of selective PERMAB (complete PERMAB medium + G418 (125 μg / ml)) to provide 0.5x10 6 / ml of viable cell density.

[0081] The combined cultures were passaged twice a week and the cells were maintained at 0.3 x 10 6 Cells were cultured at a density of 10 cells / ml until cell viability increased to >50%. At this point, cells were transferred to 250 ml Erlenmeyer flasks for shaking culture. After several weeks of shaking culture, the pooled supernatant was sampled and assayed for hCG concentration. Once the pool was determined to be positive for hCG expression, the cells were prepared for limiting dilution cloning.

[0082] Prepare a cell suspension of 0.3 viable cells / ml in PERMAB medium supplemented with 125 μg / ml of G-418. Disperse the cell suspension into a 96-well flat-bottom tissue culture plate at 200 μl / well and culture in a humid atmosphere at 37°C, 5% CO2 (Binder CB150). Use Genetix clone selection imager to regularly scan the flat plate to track the growth of cells in each well.

[0083] After two weeks, 535 wells containing actively growing cell colonies were identified. The supernatants from these wells were sampled and hCG was determined using a commercial kit (DRG diagnostics HCG ELISA Cat.No.EIA1469). Based on the results of these assays, a total of 162 colonies were transferred to a 24-well plate containing 0.5 ml / well selective PERMAB culture medium. When the cells in the wells were close to confluence, the supernatant from each of the 162 wells was sampled and hCG levels were determined. Based on these results, 91 best expressing cell lines were selected for expansion in T-25 flasks. These cell lines were grown again to confluence, at which point the supernatant was sampled and hCG was determined as described above. Based on these results, 58 best expressing cell lines were expanded into T-75 flasks. For each of these 58 cell lines, specific production rate (SPR) analysis was performed by methods known in the art, and specific production rate was expressed in pg / cell / day.

[0084] Thus, in this example, a plasmid incorporating hCG α and β chains was transfected into PER. The cells were incubated with G418-containing medium to select transfectants. The supernatants of growing cell colonies were screened for hCG concentrations, and those colonies expressing the highest levels were further amplified. In subsequent rounds of amplification and screening, 20 clones expressing hCG were selected for growth and productivity studies. Based on the results of these studies, 5 clones were selected for transfection with a second plasmid incorporating the ST3GAL4 gene.

[0085] Example 4 - Transfection with ST3GAL4 plasmid pST3

[0086] Stable clones were generated as previously described in Example 3.

[0087] The five best clones generated by the method described in Example 3 were selected for use with the ST3GAL4 plasmid pST3 (see Example 2, Figure 3 and 5 ) transfection. Transfection was performed by electroporation using the same method as described in Example 3, except that transformants were selected in complete PERMAB medium supplemented with 0.5 μg / ml hygromycin instead of G418. Using this method, five pools expressing hCG were obtained.

[0088] Supernatant samples from the transfected pools were assayed in a rat pharmacokinetic model and based on this data as well as data from the RCA-lectin assay (measuring exposed galactose on hCG chains), individual pools were selected for further limiting dilution cloning by methods known in the art.

[0089] This dilution cloning produced >600 clones, each of which was also screened for hCG expression and the extent of exposed galactose as determined by RCA-lectin binding. Those clones with the lowest levels of exposed galactose were further expanded and samples were subjected to in vivo PK and validation lectin binding data. Five clones derived from a single pool were further assayed for growth and productivity characteristics.

[0090] Each of these clones was expanded and seed stock cell banks were prepared following standard cryopreservation methods. Stocks from the seed stock cell banks were thawed and found to be viable.

[0091] Example 5 - Analysis by isoelectric focusing

[0092] Electrophoresis is defined as the transport of charged molecules through a solvent by an electric field. The mobility of biomolecules through an electric field depends on the field strength, the net charge on the molecule, the size and shape of the molecule, and the ionic strength and properties of the medium in which the molecule migrates.

[0093] Isoelectric focusing (IEF) is an electrophoretic technique that separates proteins based on their pi. The pi is the pH at which a protein has no net charge and does not migrate in an electric field. The sialic acid content of hCG isoforms subtly alters the pi point of each isoform, which can be studied using this technique to visualize the Per.C6 hCG isoform from each clone.

[0094] The isoelectric point of hCG isoforms produced by Per.C6 was analyzed using isoelectric focusing. Per.C6 hCG was produced as described in Example 6.

[0095] The Per.C6 hCG samples were placed in a 5% polyacrylamide Separation was performed on an IEF gel under non-denaturing conditions in an ampholyte solution with a pH gradient of 3.0-7.0. Proteins were visualized by Coomassie blue staining using methods well known in the art.

[0096] Figure 6 The rhCG isoforms in the composition of the present invention produced by the clonal cell lines prepared by the method described in Example 6 (lanes 4, 5, 6 and 7, 15 μg / lane); the prior art CHO-derived composition, Ovitrelle (lane 2, 15 μg); and the human urine product Novarel obtained from pregnant women (lane 3, 15 μg) are shown by IEF with Coomassie blue staining. The bands represent hCG isoforms containing different numbers of sialic acid molecules. Figure 6 It was shown that recombinant hCG derived from a human cell line engineered with α2,3-sialyltransferase (the composition of the present invention) was more acidic compared to Ovitrelle and urinary hCG from pregnant women.

[0097] Figure 7 The concentrations of hCG ("α-βhCG") and free β chain ("βhCG") produced using cells of the present invention comprising an "optimized" α subunit (i.e., using a cell line prepared by the methods described in Examples 1-4) were compared to the concentrations of hCG ("α-βhCG") and free β chain ("βhCG") produced in comparative cells comprising a WT (wild-type) α subunit. Figure 7 It was shown that the cells of the present invention express a large amount of hCG and a low excess of free β subunit. In other words, the yield and purity of recombinant hCG produced by the cells and methods of the present invention are significantly improved.

[0098] Table A shows the percentage of free β-hCG subunit in semi-purified samples of batches of hCG produced using cells of the present invention (i.e., using a cell line prepared by the methods described in Examples 1-4) comprising an "optimized" α subunit (Optimized αhCG) as compared to the percentage of free β-hCG subunit in semi-purified samples of hCG produced in Comparative Example cells comprising WT (wild-type) α subunit ("WT αhCG"), as determined by hydrophobic phenyl-5PW HPLC chromatography.

[0099] Table A shows that the cells of the present invention express a large amount of hCG and a relatively low excess of free β subunit (5.7-10.6%) compared to cells containing WT α subunit (64-66%). In other words, the yield and purity of recombinant hCG produced by the cells and methods of the present invention are significantly improved.

[0100] Table A

[0101]

[0102]

[0103] Example 6 - Production and Purification Overview

[0104] A method was developed to produce recombinant hCG in PER.C6 cells cultured in suspension in serum-free medium. The method is described below and is applicable to several hCG-producing PER.C6 cell lines.

[0105] Recombinant hCG was prepared from clones transfected with α2,3-sialyltransferase using the methods of Examples 1-4 described above.

[0106] The cells were cultured in 6GRO medium (SAFC) in a shake flask until the cell density reached 1x10 6 -3x10 6 cells / ml. The cells were cultured at a rate of approximately 1x10 6 The cells were transferred to a 5 L glass stirred tank bioreactor at a density of 10 cells / ml. The bioreactor was operated in perfusion mode using Proper1 medium (Lonza).

[0107] Purification of the product rhCG is then performed by methods well known in the art using multiple ultrafiltration steps, anion and cation exchange capture chromatography, hydrophobic chromatography and pseudoaffinity chromatography.

[0108] During all chromatographic procedures the presence of immunoreactive recombinant hCG was confirmed by ELISA (DRG EIA 1469) and IEF (Example 5).

[0109] Example 7 - Sialic acid content

[0110] Sialic acid is a protein-bound sugar that is considered a monosaccharide and is present in combination with other monosaccharides such as galactose, mannose, glucosamine, galactosamine, and fucose. The total sialic acid on the purified rhCG of the present invention was determined using a method based on the method of Stanton et al. (J. Biochem. Biophys. Methods. 30 (1995), 37-48).

[0111] The total sialic acid content of the Per.C6 recombinant hCG samples modified with α2,3-sialyltransferase (produced by the methods of Examples 4 and 6) was determined, and the results are shown in Table 1 [expressed as the ratio of sialic acid moles to protein moles].

[0112] Example 8 - hCG Bioassay According to USP

[0113] Each sample in Table 1 below was subjected to an hCG bioassay to determine the hCG specific activity. The activity was determined according to USP (USPMonographs: Chorionic Gonadotropin, USPC Official 8 / 1 / 09-11 / 30 / 09) using Ovitrelle as a standard. Ovitrelle has a biological activity of 26,000 IU / mg (Curr Med Res Opin. 2005 December; 21(12): 1969–76). The acceptance limit is >21,000 IU hCG / mg. The biological activity of samples of recombinant hCG modified with α2,3-sialyltransferase for hCG derived from a human cell line is shown in Table 1.

[0114] Table 1

[0115]

[0116] As can be seen above, the efficacy is similar to Ovitrelle and may be greater than Ovitrelle (eg, see Sample 7).

[0117] References

[0118] Andersen CY, Westergaard LG, and van Wely M. (2004). FSH isoform composition of commercial gonadotrophin preparations: a neglected aspect? Reprod Biomed Online. 9(2) , 231-236.

[0119] Bassett RM, and Driebergen R. (2005). Continued improvements in the quality and consistency of follitropin alfa, recombinant human FSH. Reprod Biomed Online. 10(2)， 169-177.

[0120] D'Antonio M., Borrelli F., Datola A., Bucci R., Mascia M., Polletta P., Piscitelli D., and Papoian R. (1999) Biological characterization of recombinant human follicle stimulating hormone isoforms. Human Reproduction 14 , 1160-1167

[0121] Fiddes, JC and Goodman, HM (1979) Isolation, cloning and sequence analysis of the cDNA for the alpha-subunit of human chorionic gonadotropin. Nature, 281, 351-356.

[0122] Fiddes, JC and Goodman, HM (1980) The cDNA for the beta-subunit of human chorionic gonadotropin suggests evolution of a gene by readthrough into the 3'-untranslated region. Nature, 286, 684-387.

[0123] Kagawa Y, Takasaki S, Utsumi J, Hosoi K, Shimizu H, Kochibe N, and Kobata A. (1988). Comparative study of the asparagine-linked sugar chains of natural human interferon-beta 1 and recombinant human interferon-beta 1 produced by three different mammalian cells. J Biol Chem. 263(33)， 17508-17515.

[0124] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem. 193 (1) , 265-75.

[0125] Lowry, PJ, McLean, C, Jones RL, and Satgunasingam N. (1976) Purification of anterior pituitary and hypothalamic hormones. Clin Pathol Suppl (Assoc Clin Pathol). 7 , 16–21.

[0126] Royle L, Radcliffe CM, Dwek RA and Rudd PM (2006) Methods in Molecular Biology, ed I Brockhausen-Schutzbach (Humana Press), 347: Glycobiology protocols, 125-144.

[0127] Steelman SL, and Pohley FM. (1953) Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotropin. Endocrinology. 53(6) , 604-616.

[0128] Svensson EC, Soreghan B, and Paulson JC. (1990) Organization of the beta-galactoside alpha 2,6-sialyltransferase gene. Evidence for the transcriptional regulation of terminal glycosylation. J Biol Chem. 265(34): 20863-20868.

[0129] Takeuchi M, Takasaki S, Miyazaki H, Kato T, Hoshi S, Kochibe N, and Kobata A (1988). Comparative study of the asparagine-linked sugar chains of humanerythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells. J Biol Chem. 263(8) , 3657-3663.

[0130] Ulloa-Aguirre A, Midgley AR Jr, Beitins IZ, and Padmanabhan V. (1995). Follicle-stimulating isohormones: characterization and physiological relevance. Endocr Rev. 16(6)， 765-787.

[0131] Ulloa-Aguirre A, Timossi C, Barrios-de-Tomasi J, Maldonado A, and Nayudu P. (2003). Impact of carbohydrate heterogeneity in function of follicle-stimulating hormone: studies derived from in vitro and in vivo models. Biol Reprod. 69(2)， 379-389.

[0132] Optimized hCGα

[0133] Optimized nucleotide sequence of hCGα (SEQ ID NO: 1)

[0134]

[0135] hCG optimized α protein sequence

[0136]

[0137] Human chorionic gonadotropin beta polypeptide

[0138] Accession number NP_000728

[0139] Nucleotide sequence of hCGβ (SEQ ID NO: 2)

[0140] Nucleotide sequence

[0141]

[0142] Protein sequence of hCGβ

[0143]

[0144] β-galactoside α-2,3-sialyltransferase 4

[0145] Registration Number L23767

[0146] Nucleotide sequence of ST3GAL4 (SEQ ID NO: 3)

[0147]

[0148] Protein sequence of ST3GAL4

[0149] Optimized hCG α chain

[0150] Optimized nucleotide sequence of hCG α chain (SEQ ID NO: 4)

[0151]

[0152] Protein sequence of hCG optimized α chain

[0153]

[0154] hCGα peptide

[0155] Registration Number AH007338

[0156] Nucleotide sequence of hCGα (SEQ ID NO: 5)

[0157]

[0158] Protein sequence of hCGα

Claims

1. A PER.C6® cell line comprising a sequence encoding the hCG alpha chain integrated into its genome, characterized in that The sequence encoding the hCG α chain is selected from the sequence according to SEQ ID NO: 1; wherein the PER.C6® cell line is deposited as ECACC no.96022940; The PER.C6® cell line further comprises a cDNA encoding α-2,3-sialyltransferase integrated into its genome, the cDNA being selected from the sequence according to SEQ ID NO: 3; and The PER.C6® cell line also comprises a nucleic acid sequence encoding the beta chain of hCG integrated into its genome, the nucleic acid sequence being selected from the sequence according to SEQ ID NO:

2.

2. A method for producing a recombinant protein in a cell line, comprising culturing the cell line according to claim 1 in a suitable culture medium and harvesting the recombinant protein from the cells and / or the culture medium; The recombinant protein is recombinant hCG.

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