TWI891671B - Method for producing fusion protein of serum albumin and growth hormone - Google Patents

Abstract

The present invention provides a method for producing a fusion protein of human serum albumin and human growth hormone. The solution provided by the present invention is a method for producing a fusion protein of human serum albumin and human growth hormone, comprising the following steps: (a) culturing mammalian cells that produce the protein in a serum-free culture medium to secrete the protein into the culture medium; (b) removing the mammalian cells from the culture medium obtained in step (a); (c) purifying the protein from the culture supernatant obtained in step (b) above by column chromatography using a material bound to an antibody having an affinity for the protein as a stationary phase, column chromatography using a material having an affinity for phosphate groups as a stationary phase, cation exchange column chromatography, and size selection column chromatography.

Description

Method for producing fusion protein of serum albumin and growth hormone

The present invention relates to a method for producing a fusion protein of serum albumin and growth hormone to a purity sufficient for direct use as a medicine.

Human growth hormone (hGH) is a protein secreted by the anterior pituitary gland under the control of the hypothalamus. In addition to exhibiting growth-promoting activities such as promoting cartilage formation and protein assimilation, hGH also modulates body composition and lipid metabolism. Children with low hGH secretion can develop growth hormone deficiency dwarfism, resulting in shorter height than healthy children.

Preparations containing hGH (hGH preparations) as the active ingredient, produced as a recombinant protein using E. coli with an introduced hGH gene, are widely used clinically as treatments for the following conditions: growth hormone-deficient dwarfism, dwarfism in Turner syndrome, SGA (small-for-gestational age) dwarfism, dwarfism in Noonan syndrome, dwarfism due to chronic renal failure, dwarfism in Prader-Willi syndrome, and dwarfism in achondroplasia. hGH preparations are particularly effective in these conditions when epiphyseal closure is not present. hGH preparations are administered subcutaneously or intramuscularly and circulate in the blood, promoting growth through their growth-promoting activity. They are also widely used clinically as treatments for adult growth hormone deficiency. While various abnormalities, such as abnormal lipid metabolism, are observed in patients with adult growth hormone deficiency, hGH administration can normalize lipid metabolism and improve quality of life. For example, GROWJECT (registered trademark) is an hGH preparation for growth hormone-deficient dwarfism and adult growth hormone deficiency.

The half-life of hGH in plasma is believed to be less than 20 minutes, and hGH administered to patients disappears rapidly from the bloodstream. Therefore, to maximize the efficacy of hGH, patients must receive hGH intramuscularly three times a week or subcutaneously daily. This frequent administration places a burden on patients. Therefore, it would be desirable to increase the stability of hGH in plasma and extend its half-life, thereby reducing the frequency of hGH administration and thus alleviating the burden on patients.

Human serum albumin (HSA), in its mature form, is a protein consisting of 585 amino acids. When referred to simply as human serum albumin, this mature form is the reference. HSA is the most abundant component of plasma proteins, with a half-life in plasma of 14 to 20 days. HSA helps regulate plasma osmotic pressure and also has the ability to bind to and transport endogenous substances such as cations, fatty acids, hormones, and bile rubin, as well as exogenous substances such as pharmaceuticals. Generally speaking, substances bound to HSA become less likely to be absorbed by organs, allowing them to circulate in the blood for longer periods of time.

Human serum albumin (HSA) is known to have multiple naturally occurring variants. Human serum albumin Redhill is one of these variants (non-patent documents 1 and 2). Compared to the amino acid sequence of the standard human serum albumin described above, which contains 585 amino acids, human serum albumin Redhill differs in that the 320th amino acid residue from its N-terminus is threonine instead of alanine, and an arginine residue is added to its N-terminus, resulting in a total of 586 amino acids. This change from alanine to threonine creates an Asn-Tyr-Thr sequence in the amino acid sequence of albumin Redhill, in which the Asn (aspartic acid) residue is N-linked glycosylated. Therefore, it was observed that the molecular weight of Redhill albumin was approximately 2.5 kDa larger than that of the normal human serum albumin mentioned above.

A method for increasing the stability of hGH in plasma by conjugating HSA to hGH has been reported (Patent Documents 1-8). The protein resulting from the conjugation of HSA and hGH is produced by creating a transformed cell into which an expression vector has been introduced. This expression vector incorporates DNA that binds the gene encoding hGH and the gene encoding HSA in frame, and then culturing these cells. The protein is then produced as a recombinant protein in a culture medium or within the cell. [Prior Art Document] [Patent Document]

[Patent Document 1] WO2017/154869 [Patent Document 2] WO2017/159540 [Patent Document 3] Japanese Patent Publication No. 2003-530838 [Patent Document 4] Japanese Patent Publication No. 2005-514060 [Patent Document 5] Japanese Patent Publication No. 2000-502901 [Patent Document 6] Japanese Patent Publication No. 2008-518615 [Patent Document 7] Japanese Patent Publication No. 2013-501036 [Patent Document 8] Japanese Patent Publication No. 2013-518038

[Problem to be solved by the invention]

Against this background, one object of the present invention is to provide a method for producing a fusion protein of human albumin and human growth hormone to a purity sufficient for direct use as a pharmaceutical, and a pharmaceutical containing the fusion protein as an active ingredient. [Means for Solving the Problem]

As a result of repeated research in pursuit of the above-mentioned objectives, the inventors discovered a method for efficiently producing a compound (human serum albumin variant-hGH fusion protein) obtained by conjugating a variant containing the following amino acid sequence (human serum albumin variant) to human growth hormone (hGH) to a purity sufficient for direct use as a pharmaceutical. This led to the completion of the present invention. The amino acid sequence is one in which alanine is substituted for threonine at the 320th amino acid residue from the N-terminus of normal human serum albumin, which contains 585 amino acids. Specifically, the present invention encompasses the following. 1. A method for producing a fusion protein of human serum albumin and human growth hormone, comprising the following steps: (a) culturing mammalian cells that produce the protein in a serum-free culture medium to secrete the protein into the culture medium; (b) removing the mammalian cells from the culture medium obtained in step (a) to recover the culture supernatant; and (c) Purifying the protein from the culture supernatant obtained in step (b) above using column chromatography using as the stationary phase a material that binds to an antibody having an affinity for the protein, column chromatography using as the stationary phase a material that has an affinity for phosphate groups, cation exchange column chromatography, and size selection column chromatography. 2. The production method described in 1 above, wherein in step (c) the following column chromatography methods are used in order: column chromatography using as the stationary phase a material that binds to an antibody having an affinity for the protein, column chromatography using as the stationary phase a material that has an affinity for phosphate groups, cation exchange column chromatography, and size selection column chromatography. 3. The production method according to 1 or 2 above, wherein the antibody having affinity for the protein has affinity for human serum albumin or human growth hormone. 4. The production method according to 1 or 2 above, wherein the antibody having affinity for the fusion protein has affinity for human growth hormone. 5. The production method according to any one of 1 to 4 above, wherein the material having affinity for phosphate groups is fluorapatite or hydroxyapatite. 6. The production method according to any one of 1 to 4 above, wherein the material having affinity for phosphate groups is hydroxyapatite. 7. The production method according to any one of 1 to 6 above, wherein the cation exchanger used in the cation exchange column chromatography is a weak cation exchanger. 8. The production method according to 7 above, wherein the weak cation exchanger maintains selectivity based on both hydrophobic interactions and hydrophobic bond formation. 9. The production method according to any one of 1 to 8 above, further comprising a step for viral inactivation. 10. The production method according to 9 above, wherein viral inactivation is performed on an eluate of the protein obtained by column chromatography using a material bound to an antibody having an affinity for the protein as the stationary phase. 11. The production method of 9 above, wherein the virus is inactivated in the eluate of the protein obtained by the size-selection column chromatography. 12. The production method of any one of 1 to 8 above, comprising a second step for virus inactivation. 13. The production method of 12 above, wherein the virus is inactivated in the eluate of the protein obtained by column chromatography using a material bound to an antibody having affinity for the protein as the stationary phase, and in the eluate of the protein obtained by the size-selection column chromatography. 14. The production method according to 12 or 13 above, wherein one of the virus inactivation steps is performed by adding a nonionic surfactant to a solution containing the HSA-hGH fusion protein, and the other step is performed by passing the solution containing the HSA-hGH fusion protein through a filter membrane. 15. The production method according to 1 to 14 above, wherein the protein is bound to human serum albumin at the C-terminus of human growth hormone via a linker sequence or directly via a peptide bond. 16. The production method described in 15 above, wherein the linker sequence comprises an amino acid sequence selected from the group consisting of one glycine, one serine, the amino acid sequence (Gly-Ser), the amino acid sequence (Gly-Gly-Ser), and 1 to 10 consecutive amino acid sequences of these amino acid sequences. 17. The production method described in 16 above, wherein the linker sequence comprises the amino acid sequence represented by the amino acid sequence (Gly-Ser). 18. The production methods described in 1 to 14 above, wherein the amino acid sequence of the protein is 85% or greater identical to the amino acid sequence represented by SEQ ID NO: 11. 19. The production methods described in 1 to 14 above, wherein the amino acid sequence of the protein is 95% or greater identical to the amino acid sequence represented by SEQ ID NO: 11. 20. The production method according to 1 to 14 above, wherein the amino acid sequence of the protein is the amino acid sequence represented by SEQ ID NO: 11. [Effects of the Invention]

According to the present invention, for example, a fusion protein of serum albumin and growth hormone can be efficiently produced and purified to a level that allows it to be circulated on the market as a pharmaceutical.

[Form used to implement the invention]

In this specification, the term "human serum albumin" or "HSA" refers not only to normal wild-type human serum albumin comprising the 585 amino acid residues set forth in SEQ ID NO: 1, but also to HSA variants, as long as they have the function of normal wild-type human serum albumin, such as the function of binding to and transporting endogenous substances in the blood and exogenous substances such as pharmaceutical agents. Such HSA variants are equivalent to those having one or more substitutions, deletions, and/or additions of amino acid residues to the amino acid sequence set forth in SEQ ID NO: 1 (in this specification, "addition" of amino acid residues means the addition of residues to the end of or within the sequence). When amino acid residues are substituted with other amino acid residues, the number of substituted amino acid residues is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When amino acid residues are deleted, the number of deleted amino acid residues is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. For example, a variant comprising 584 amino acid residues deleted at the N-terminus or C-terminus of the amino acid sequence set forth in SEQ ID NO: 1 is also included in human serum albumin. Furthermore, combinations of these amino acid residue substitutions and deletions are also possible. Furthermore, one or more amino acid residues may be added to the amino acid sequence of normal wild-type HSA or its variants, or to the N-terminus or C-terminus of the amino acid sequence. In this case, the number of added amino acid residues is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.

HSA variants introduced by combining at least two of these three types of mutations: amino acid substitution, deletion, and addition preferably have an amino acid sequence comprising 0 to 10 amino acid residues deleted, 0 to 10 amino acid residues substituted with other amino acid residues, and 0 to 10 amino acid residues added to the amino acid sequence set forth in SEQ ID NO: 1. More preferably, the number of amino acid residues deleted, substituted, and/or added to the amino acid sequence set forth in SEQ ID NO: 1 is preferably 5 or fewer, and even more preferably 3 or fewer.

In the present invention, the term "human serum albumin Redhill" (HSA-Redhill) refers to a variant of human serum albumin comprising the 586 amino acid residues set forth in SEQ ID NO: 2. Human serum albumin Redhill corresponds to the amino acid sequence of wild-type human serum albumin comprising the 585 amino acids set forth in SEQ ID NO: 1, except that the 320th amino acid residue from the N-terminus is threonine instead of alanine, and an arginine residue is additionally present at the N-terminus. This substitution of threonine with alanine results in a sequence portion represented by Asn-Tyr-Thr in the amino acid sequence of albumin Redhill, in which the Asn (aspartic acid) residue in this sequence portion is N-linked glycosylated. Therefore, it was observed that the molecular weight of Redhill albumin was approximately 2.5 kDa larger than that of normal wild-type albumin (SEQ ID NO: 1).

In the present invention, the term "human serum albumin variant" (HSA variant) refers to the aforementioned variants relative to normal wild-type HSA (SEQ ID NO: 1), except for the variant shown in SEQ ID NO: 2 (HSA-Redhill). Preferred HSA variants in the present invention include, in addition to the variant shown in SEQ ID NO: 3 in which the 320th alanine from the N-terminus of the wild-type HSA amino acid sequence is substituted with threonine, those having the following amino acid sequences as long as they have the function of normal wild-type human serum albumin, such as the function of binding to and transporting endogenous substances and exogenous substances such as drugs in the blood: The amino acid sequence shown has one or more amino acid residues substituted with other amino acid residues, deleted, or added, but the amino acid sequence is a state in which the 318th asparagine residue and the 320th threonine residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 3 are peptide-bonded via a single amino acid residue (X) other than proline between these two residues. When amino acid residues in the amino acid sequence are substituted with other amino acid residues, the number of substituted amino acid residues is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. In the case of amino acid residue deletion, the number of amino acid residues deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. For example, a variant comprising 584 amino acid residues may be obtained by deleting the amino acid residues at the N-terminus or C-terminus of the amino acid sequence set forth in SEQ ID NO: 3. Alternatively, a combination of substitutions and deletions of these amino acid residues may be used. Furthermore, one or more amino acid residues may be added to the amino acid sequence of such variants or to the N-terminus or C-terminus of the amino acid sequence. Specifically, the amino acid sequence set forth in SEQ ID NO: 3 may be introduced by combining at least two of the three types of mutations: amino acid substitution, deletion, and addition. These mutations include deletion of 0 to 10 amino acid residues, substitution of 0 to 10 amino acid residues with other amino acid residues, and addition of 0 to 10 amino acid residues. However, amino acid residues 318 to 320 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 3 must be asparagine-X-threonine ("X" represents an amino acid residue other than proline), preferably asparagine-tyrosine-threonine. Furthermore, human serum albumin variants are included in human serum albumin as long as they retain the function of human serum albumin. Here, the amino acid sequence of the human serum albumin variant preferably has an identity of 85% or more, more preferably 90% or more, and even more preferably 95% or more to the amino acid sequence of the normal wild-type HSA shown in SEQ ID NO: 1.

In the present invention, the position and form (deletion, substitution, addition) of each variant in various HSA variants can be easily determined by aligning the amino acid sequences of the two HSAs when compared with normal wild-type HSA. Furthermore, in the present invention, the identity between the amino acid sequence of wild-type HSA and the amino acid sequence of HSA containing the variant can be easily calculated using a known homology calculation algorithm. For example, such algorithms include BLAST (Altschul SF. J Mol. Biol. 215. 403-10, (1990)), Pearson and Lipman's similarity search method (Proc. Natl. Acad. Sci. USA. 85. 2444 (1988)), and Smith and Waterman's local homology algorithm (Adv. Appl. Math. 2. 482-9 (1981)).

The substitution of amino acids in the amino acid sequence of HSA with other amino acids, for example, occurs within an amino acid family whose amino acids are related in side chains and chemical properties. Such substitutions within the amino acid family are not expected to significantly alter the function of HSA (i.e., they are conservative amino acid substitutions). As examples of such amino acid families, there are the following: (1) Aspartic acid and glutamine, which are acidic amino acids; (2) Histidine, lysine, and arginine, which are basic amino acids; (3) Phenylalanine, tyrosine, and tryptophan, which are aromatic amino acids; (4) Serine and threonine, which are amino acids having a hydroxyl group (hydroxyl amino acids); (5) Methionine, alanine, valine, leucine, and isoleucine, which are hydrophobic amino acids; (6) Cysteine, serine, threonine, aspartic acid, and glutamine, which are neutral hydrophilic amino acids; (7) Glycine and proline, which are amino acids that affect the orientation of the peptide chain; (8) Aspartic acid and glutamine are amide-type amino acids (polar amino acids); (9) Alanine, leucine, isoleucine, and valine are aliphatic amino acids; (10) Alanine, glycine, serine, and threonine are amino acids with small side chains; (11) Alanine and glycine are amino acids with particularly small side chains.

In the examples described below, a human serum albumin variant that binds to human growth hormone (a typical example of an HSA variant) differs from the 585-amino acid sequence of wild-type human serum albumin (SEQ ID NO: 1) only in that the 320th amino acid residue from the N-terminus is threonine instead of alanine (SEQ ID NO: 3). This difference results in a portion of the amino acid sequence of this HSA variant (referred to as "HSA(A320T)") represented by Asn-Tyr-Thr, in which the Asn (aspartic acid) residue is N-linked glycosylated.

In this specification, the term "human serum albumin and human growth hormone fusion protein" or "human serum albumin-hGH fusion protein (HSA-hGH fusion protein)" refers to a protein formed by binding HSA to growth hormone. Here, "binding" these proteins includes, but is not limited to, linking them via a peptide bond. Furthermore, "binding" these proteins includes not only direct peptide bond linkage between the N-terminus of one and the C-terminus of the other, but also indirect binding of the two proteins via a linker. In particular, when the human serum albumin is a human serum albumin variant, the term "fusion protein of a human serum albumin variant and human growth hormone" or "human serum albumin variant-hGH fusion protein (HSA variant-hGH fusion protein)" is used. That is, the human serum albumin variant-hGH fusion protein is contained in the human serum albumin-hGH fusion protein. "Human serum albumin and human growth hormone fusion protein" can also be referred to as HSA-linked hGH.

Here, the term "linker" refers to the structural moiety that covalently binds the two polypeptides and is not derived from either terminus of HSA (including HSA variants) or the growth hormone to which it binds. A linker can be a single amino acid residue that forms a peptide bond with the two polypeptides, or a peptide chain moiety containing two or more amino acid residues (peptide linker). In this specification, these linkers containing one or more amino acid residues are collectively referred to as "peptide linkers." Furthermore, in this specification, references to HSA and growth hormone being bound "via a peptide bond" encompass both direct peptide bond binding and binding through a peptide linker. Furthermore, in this specification, when HSA is conjugated to growth hormone directly or via a peptide linker, the compound referred to as "human serum albumin-hGH fusion protein (HSA-hGH fusion protein)" may also be referred to as "human serum albumin fused to hGH (HSA fused to hGH)." The same applies to human serum albumin variant-hGH fusion protein (HSA variant-hGH fusion protein).

In the present invention, when HSA and growth hormone are conjugated via a peptide linker, the linker is preferably composed of 1-50 amino acid residues, more preferably 1-17, even more preferably 1-10, and even more preferably 1-6 amino acid residues, for example, 2-17, 2-10, 10-40, 20-34, 23-31, or 25-29 amino acids. Furthermore, for example, the linker is composed of only one amino acid residue, or 2, 3, 5, 6, or 20 amino acid residues. As long as the HSA portion bound by the peptide linker retains its function as HSA and the growth hormone portion can exert growth hormone physiological activity under physiological conditions, the amino acid residues or amino acid sequence constituting the peptide linker are not limited. However, a peptide linker composed of glycine and serine is preferred. Preferred examples of peptide linkers include those composed of one glycine, one serine, Gly-Ser, Gly-Gly-Ser, Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 5), and Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 6), as well as peptide linkers comprising these amino acid sequences. A peptide linker comprising any of these amino acid sequences repeated 2 to 10 times, or 2 to 5 times, can also be preferably used as a peptide linker. Furthermore, a peptide linker comprising any combination of two or more of these amino acid sequences repeated 1 to 10 times, or 2 to 5 times, can also be preferably used as a peptide linker. A preferred peptide linker comprising any combination of two or more of these amino acid sequences includes a 20-amino acid sequence consisting of the amino acid sequence Gly-Ser followed by three consecutive amino acid sequences: Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 5).

As a method for combining two different polypeptides, for example, the following method is generally used and can be utilized in the present invention: an expression vector is prepared that incorporates DNA that is incorporated in-frame with a gene encoding one polypeptide downstream of a gene encoding the other polypeptide, and host cells transformed with this expression vector are cultured to express the recombinant fusion protein.

When HSA-hGH fusion proteins are produced by recombinant expression in transformed cells, a fusion protein is obtained in which a polypeptide comprising the amino acid sequence of growth hormone is bound to either the N-terminus or the C-terminus of a polypeptide comprising the amino acid sequence of HSA. HSA-hGH fusion proteins produced using recombinant techniques are specifically referred to as recombinant HSA-hGH fusion proteins.

To conjugate a polypeptide comprising the amino acid sequence of growth hormone to the N-terminus of a polypeptide comprising the amino acid sequence of HSA, an expression vector can be used that incorporates DNA that binds in-frame to a gene encoding a polypeptide comprising the amino acid sequence of HSA, downstream of a gene encoding a polypeptide comprising the amino acid sequence of growth hormone. To indirectly conjugate the two polypeptides via a peptide linker, a DNA sequence encoding the linker is inserted in-frame between the genes encoding the two polypeptides.

To conjugate a polypeptide containing the amino acid sequence of growth hormone to the C-terminus of a polypeptide containing the amino acid sequence of HSA, an expression vector can be used that incorporates DNA that binds in-frame to a gene encoding a polypeptide containing the amino acid sequence of HSA, upstream of the gene encoding the polypeptide containing the amino acid sequence of growth hormone. To indirectly conjugate the two polypeptides via a peptide linker, a DNA sequence encoding the linker is inserted in-frame between the genes encoding the two polypeptides.

To produce HSA-hGH fusion proteins in host cells, an expression vector incorporating DNA encoding one of these proteins is introduced into the host cells. The host cells used for this purpose are not particularly limited, as long as they can express the HSA-hGH fusion protein upon introduction of such an expression vector. They may include eukaryotic cells such as mammalian cells, yeast, plant cells, and insect cells, or prokaryotic cells such as Escherichia coli and Bacillus subtilis. Mammalian cells are particularly preferred. However, for expression of sugar chain-modified proteins, the host cells are selected from eukaryotic cells such as mammalian cells, yeast, plant cells, and insect cells. Asn residues in the sequence represented by Asn-Tyr-Thr, which is generated by converting the 320th amino acid residue of wild-type HSA to threonine, or Asn residues in the sequence represented by Asn-X-Thr ("X" is an amino acid residue other than proline), are N-linked glycosylated as the HSA-hGH fusion protein is expressed in eukaryotic cells.

When mammalian cells are used as host cells, the type of mammalian cell is not particularly limited, but cells derived from humans, mice, or Chinese hamsters are preferred, with CHO cells derived from Chinese hamster ovary cells or NS/0 cells derived from mouse myeloma being particularly preferred. Furthermore, the expression vector used to incorporate and express the DNA fragment encoding the HSA-hGH fusion protein can be used without particular limitation, as long as it results in expression of the gene when introduced into mammalian cells. The gene incorporated into the expression vector is located downstream of a DNA sequence (gene expression control site) that regulates the gene's transcription frequency in mammalian cells. Examples of gene expression control sites that can be used in the present invention include a cytomegalovirus-derived promoter, an SV40 early promoter, a human elongation factor-1α (EF-1α) promoter, and a human ubiquitin C promoter.

Mammalian cells introduced with such expression vectors begin to express the protein incorporated into the vector, but the amount of expression varies from cell to cell. Therefore, to efficiently produce the HSA-hGH fusion protein, it is necessary to select cells expressing the protein at high levels from mammalian cells introduced with the expression vector. To facilitate this selection, a gene that functions as a selectable marker is incorporated into the expression vector.

The most common selective markers are enzymes that break down drugs such as puromycin and neomycin (drug resistance markers). Mammalian cells normally die in the presence of these drugs above a certain concentration. However, mammalian cells transfected with an expression vector containing a drug resistance marker gene can render these drugs non-toxic or attenuated by the resistance marker, allowing them to survive even in the presence of these drugs. By introducing an expression vector containing a drug-resistance marker as a selection marker into mammalian cells and culturing them in a selective medium containing a drug corresponding to the drug-resistance marker while gradually increasing the concentration of the drug, for example, cells can be obtained that can proliferate even in the presence of higher concentrations of the drug. In cells selected in this manner, the expression level of the gene encoding the target protein incorporated into the expression vector generally increases along with the drug-resistance marker, resulting in selection for cells with high expression levels of the target protein.

Glutamine synthetase (GS) can also be used as a selection marker. Glutamine synthetase is an enzyme that synthesizes glutamine from glutamine and ammonia. If mammalian cells are cultured in a selective medium containing a glutamine synthetase inhibitor, such as L-methionine sulfoximine (MSX), and no glutamine, the cells typically die. However, if an expression vector containing glutamine synthetase as a selection marker is introduced into mammalian cells, the expression level of glutamine synthetase in the cells increases, allowing them to proliferate even in the presence of higher concentrations of MSX. By continuing the culture while gradually increasing the MSX concentration, cells capable of proliferating even in the presence of higher MSX concentrations can be obtained. In cells selected in this manner, the expression level of the gene encoding the target protein incorporated into the expression vector generally increases along with glutamine synthase, ultimately selecting for cells with high expression levels of the target protein.

Alternatively, dihydrofolate reductase (DHFR) can be used as a selection marker. When using DHFR as a selection marker, mammalian cells introduced with an expression vector are cultured in a selection medium containing a DHFR inhibitor such as methotrexate or amitriptyline. Continued culture while gradually increasing the concentration of the DHFR inhibitor yields cells that can proliferate even in the presence of higher concentrations of the DHFR inhibitor. The selection medium used in this manner preferably does not contain hypoxanthine or thymidine. In cells selected in this manner, the expression level of the gene encoding the target protein incorporated into the expression vector generally increases along with DHFR, resulting in selection of cells with high expression levels of the protein.

An expression vector is known in which a glutamine synthetase (GS) is positioned downstream of a gene encoding a target protein via an internal ribosome entry site (IRES) as a selection marker (International Patent Publications WO2012/063799 and WO2013/161958). The expression vectors described in these documents are particularly well-suited for producing HSA-hGH fusion proteins.

For example, the following expression vector can be preferably used for producing an HSA-hGH fusion protein: an expression vector for expressing a target protein, comprising a first gene expression control site, a gene encoding the protein downstream thereof, an internal ribosome entry site further downstream, and a gene encoding a glutamine synthetase further downstream, and further comprising a dihydrofolate reductase gene or a drug resistance gene downstream of the first gene expression control site or a second, different gene expression control site. In this expression vector, the first or second gene expression control site can preferably be a cytomegalovirus-derived promoter, the SV40 early promoter, the human elongation factor-1α promoter (hEF-1α promoter), or the human ubiquitin C promoter, with the hEF-1α promoter being particularly preferred.

Furthermore, as the internal ribosome entry site, preferably used is a site derived from the 5' non-translated region of a virus selected from the group consisting of viruses of the Picornaviridae family, foot-and-mouth disease virus, hepatitis A virus, hepatitis C virus, coronavirus, bovine enterovirus, Theileria's murine encephalomyelitis virus, and Coxsackie B virus, or a site derived from the group consisting of the human immunoglobulin heavy chain binding protein gene, the Drosophila Antennapedia gene, and the Drosophila Ultrabithorax gene. Particularly preferred is an internal ribosome entry site derived from the 5' non-translated region of the mouse encephalomyocarditis virus genome. When using an internal ribosome entry site derived from the 5' non-translated region of the mouse encephalomyocarditis virus genome, in addition to the wild-type version, one in which one of the multiple initiation codons contained in the wild-type internal ribosome entry site is partially disrupted can also be preferably used. Furthermore, the drug resistance gene that can be preferably used in this expression vector is preferably a puromycin or neomycin resistance gene, and more preferably a puromycin resistance gene.

For example, the following expression vector can be preferably used to produce HSA-hGH fusion protein: an expression vector for expressing a target protein, comprising the human elongation factor-1α promoter, a gene encoding the protein downstream thereof, an internal ribosome entry site (IRES) derived from the 5' non-translated region of the mouse encephalomyocarditis virus genome further downstream, and a gene encoding a glutamine synthetase further downstream, and further comprising a dihydrofolate reductase gene downstream of and in addition to control sites found in other genes, wherein the IERS is a vector in which one of the multiple initiation codons contained in the wild-type IERS is partially disrupted. Examples of such expression vectors include those described in WO2013/161958.

For example, the following expression vector can be preferably used to produce HSA-hGH fusion protein: it is an expression vector for expressing the target protein, comprising a human elongation factor-1α promoter, a gene encoding the protein downstream thereof, an internal ribosome entry site derived from the 5' non-translated region of the mouse encephalomyocarditis virus genome further downstream, and a gene encoding a glutamine synthase further downstream, and further comprising a drug resistance gene in addition to other gene expression control sites and downstream thereof, wherein the internal ribosome entry site is a vector in which one of the multiple initiation codons contained in the wild-type internal ribosome entry site is partially destroyed. Examples of such expression vectors include pE-mIRES-GS-puro described in WO2012/063799 and pE-mIRES-GS-mNeo described in WO2013/161958.

Three initiation codons (ATGs) are present at the 3' end of the internal ribosome entry site (IRES) within the 5' non-translated region of the wild-type mouse encephalomyocarditis virus genome. The sequence portion containing these three initiation codons is represented by SEQ ID NO: 7 (5'-ATGataatATGgccacaaccATG-3': the ATG sequence of the initiation codon is capitalized). Sequences in which one of the initiation codons within this sequence portion is partially disrupted, such as those represented by SEQ ID NO: 8 (5'-atgataagcttgccacaaccatg-3'). The pE-mIRES-GS-puro and pE-mIRES-GS-mNeo expression vectors contain an IRES containing the sequence shown in SEQ ID NO: 8.

In the present invention, mammalian cells introduced with an expression vector incorporating a DNA fragment encoding an HSA-hGH fusion protein are cultured in a selective medium in order to select cells expressing the protein at a high level.

When DHFR is used as a selection marker in the selection culture, the concentration of the DHFR inhibitor in the selection medium is gradually increased. When the DHFR inhibitor is methotrexate, the maximum concentration is preferably 0.25-5 μM, more preferably 0.5-1.5 μM, and even more preferably about 1.0 μM.

When using GS as the selection marker, the concentration of the GS inhibitor in the selection medium is gradually increased. When the GS inhibitor is MSX, the maximum concentration is preferably 10-1000 μM, for example, 20-500 μM, 20-80 μM, or 20-30 μM. In this case, a medium that does not contain glutamine can generally be used as the selection medium.

When using a puromycin-degrading enzyme as the selection marker, the maximum puromycin concentration in the selection medium is preferably 3-30 μg/mL, more preferably 5-20 μg/mL, and even more preferably approximately 10 μg/mL.

When using an enzyme that degrades neomycin as the selection marker, the maximum concentration of G418 in the selection medium is preferably 0.1-2 mg/mL, more preferably 0.5-1.5 mg/mL, and even more preferably about 1 mg/mL.

Furthermore, as a culture medium for culturing mammalian cells, the medium used for culture and the medium for recombinant protein production (recombinant protein production medium) described later can be used without particular limitation as long as it can culture and proliferate mammalian cells. However, it is preferred to use a serum-free medium. Since HSA has the property of adsorbing components contained in serum, when HSA is produced using a serum-containing medium, HSA adsorbed with impurities in the serum is obtained, necessitating the removal of these impurities in a subsequent step.

In the present invention, HSA-hGH fusion protein is obtained by culturing cells expressing the protein in a serum-free medium. Using a serum-free medium reduces the amount of impurities adsorbed to the HSA-hGH fusion protein, thereby simplifying subsequent purification steps.

Cells expressing high levels of HSA-hGH fusion protein selected by selective culture are used to produce HSA-hGH fusion protein (HSA-hGH fusion protein-producing cells). HSA-hGH fusion protein production is achieved by culturing HSA-hGH fusion protein-producing cells in HSA-hGH fusion protein production medium. This culture is referred to as production culture.

In the present invention, a serum-free medium that can be used as a medium for producing HSA-hGH fusion protein can preferably contain, for example, 3-700 mg/L of amino acids, 0.001-50 mg/L of vitamins, 0.3-10 g/L of monosaccharides, 0.1-10000 mg/L of inorganic salts, 0.001-0.1 mg/L of trace elements, 0.1-50 mg/L of nucleosides, 0.001-10 mg/L of fatty acids, 0.01-1 mg/L of biotin, 0.1-20 μg/L of hydrocortisone, 0.1-20 mg/L of insulin, 0.1-10 mg/L of vitamin B12, 0.01-1 mg/L of putrescine, 10-500 μg/L of glutathione, 0.1-20 μg/L of glutathione, 0.1-1 ... The culture medium contains 100 mg/L sodium pyruvate and a water-soluble iron compound. Thymidine, hypoxanthine, a conventional pH indicator, and antibiotics may also be added to the culture medium as desired.

As a serum-free medium that can be used as a medium for producing HSA-hGH fusion protein, DMEM/F12 medium (a mixed medium of DMEM and F12) can be used as a basic medium. These mediums are well known to those skilled in the art. Furthermore, as a serum-free medium, DMEM (HG) HAM modified (R5) medium can be used. This medium contains sodium bicarbonate, L-glutamine, D-glucose, insulin, sodium selenite, diaminobutane, hydrocortisone, iron (II) sulfate, aspartic acid, aspartic acid, serine, and polyvinyl alcohol. Furthermore, commercially available serum-free media, such as CD OptiCHO ^™ Medium, CHO-S-SFM II Medium, CD CHO Medium (Thermo Fisher Scientific), IS CHO-V ^™ Medium (Irvine Scientific), EX-CELL ^™ 302 Medium, EX-CELL ^™ Advanced Medium, or EX-CELL ^™ 325-PF Medium (SAFC Biosciences), can also be used as the basal medium.

1-50 g/L (e.g., 1-5 g/L) of a hydrolyzate derived from plants such as soybeans, wheat, and rice may be added to the HSA-hGH fusion protein production medium. Soybean-derived protein hydrolyzates are most commonly used. However, HSA-hGH fusion protein can be produced without adding a hydrolyzate derived from plants such as rice, such as soybeans, to the HSA-hGH fusion protein production medium.

After production culture, the culture medium is subjected to a chromatography step to purify the HSA-hGH fusion protein. However, the culture medium subjected to the chromatography step is the culture supernatant from which cells and other substances have been removed. Methods for obtaining the culture supernatant from the culture medium include filtration using a membrane filter and centrifugation.

In the present invention, each chromatography step for purifying the HSA-hGH fusion protein may, if necessary, be performed in the presence of a nonionic surfactant to prevent nonspecific adsorption of the protein. The type of nonionic surfactant used is not particularly limited, but polysorbate-based surfactants are preferred, with polysorbate 80 or polysorbate 20 being more preferred. The concentration of such a nonionic surfactant is preferably 0.005% (w/v) to 0.1% (w/v), more preferably 0.005% (w/v) to 0.05% (w/v), for example, 0.01% (w/v) or 0.05% (w/v).

The purification step of the HSA-hGH fusion protein can be carried out at room temperature or low temperature, but is preferably carried out at low temperature, particularly at 1-10°C.

In one embodiment of the present invention, the purification steps for the HSA-hGH fusion protein include: a column chromatography step using a material bound to an antibody having affinity for the HSA-hGH fusion protein as a stationary phase; a column chromatography step using a material having affinity for phosphate groups as a stationary phase; a cation exchange column chromatography step; and a size selection column chromatography step. However, one or more chromatography steps may be added to these chromatography steps. Examples of such additional chromatography steps include column chromatography using as a stationary phase a material bound to an antibody having an affinity for the HSA-hGH fusion protein, column chromatography using as a stationary phase a material having an affinity for phosphate groups, cation exchange column chromatography, anion exchange column chromatography, hydrophobic column chromatography, dye affinity column chromatography, and size selection column chromatography.

In one embodiment of the present invention, the purification steps for the HSA-hGH fusion protein sequentially include: a column chromatography step using as a stationary phase a material bound to an antibody having affinity for the HSA-hGH fusion protein; a column chromatography step using as a stationary phase a material having affinity for phosphate groups; a cation exchange column chromatography step; and a size selection column chromatography step. However, one or more chromatography steps may be added to these chromatography steps. Examples of such additional analytical steps include column chromatography using a material bound to an antibody having affinity for the HSA-hGH fusion protein as a stationary phase, column chromatography using a material having affinity for phosphate groups as a stationary phase, cation exchange column chromatography, anion exchange column chromatography, hydrophobic column chromatography, dye-ligand column chromatography, and size selection column chromatography. Additional analytical steps can be added between any adjacent steps or as the initial or final analytical step.

The purification steps in one embodiment of the present invention are described in detail below. These purification steps sequentially include: a column chromatography step using a material bound to an antibody having affinity for the HSA-hGH fusion protein as a stationary phase; a column chromatography step using a material having affinity for phosphate groups as a stationary phase; a cation exchange column chromatography step; and a size selection column chromatography step.

The first column chromatography step utilizes a material bound to an antibody with affinity for HSA-hGH as the stationary phase. The antibody can have affinity for either HSA or hGH, but preferably has affinity for hGH. For example, Capture Select Human Growth Hormone Affinity Matrix (Thermo Fisher Scientific) is preferably used, as it contains a carrier bound to an antibody against human growth hormone.

When using a column chromatography method using a material bound to an antibody with affinity for hGH as the stationary phase, the HSA-hGH fusion protein is applied to the column pre-equilibrated with a buffer. The type of buffer is not particularly limited, but Tris-HCl buffer is preferred, preferably at a concentration of 5-50 mM, more preferably 10-30 mM. Furthermore, the pH is preferably adjusted to 6.7-7.3, more preferably 6.9-7.1, and even more preferably approximately 7.0. The HSA-hGH fusion protein is preferably eluted from the column using glycine-hydrochloric acid. The concentration of glycine-hydrochloric acid is preferably 20-100 mM, more preferably 40-60 mM, and even more preferably about 50 mM. Furthermore, the pH is preferably adjusted to 2.0-4.0, more preferably 2.8-3.2, and even more preferably about 3.0. The pH of the rinsing solution is immediately adjusted to near neutral, for example, pH 6.4-7.0.

The second column chromatography step uses a material with an affinity for phosphate groups as the stationary phase. Preferred column chromatography methods using materials with an affinity for phosphate groups as the stationary phase include hydroxyapatite column chromatography and fluorapatite column chromatography, with hydroxyapatite column chromatography being particularly preferred. These methods utilize the interaction between the metal affinity of calcium ions and the cation exchange of phosphate groups to remove impurities.

The following details the use of hydroxyapatite column chromatography. The support used in hydroxyapatite chromatography is not particularly limited and can be ceramic or crystalline. However, CHT Type II, 40μm (Bio-Rad Laboratories) is a particularly preferred support.

The pH and conductivity of the eluate obtained in the first column chromatography step are adjusted before application to the hydroxyapatite chromatography column. The pH is preferably adjusted to 6.5-7.5, more preferably 6.8-7.2, and even more preferably 6.9-7.1. Furthermore, the conductivity is preferably adjusted to 0.4-0.8 S/m, more preferably 0.5-0.7 S/m.

The eluent, whose pH and conductivity have been adjusted, is applied to a hydroxyapatite chromatography column that has been pre-equilibrated with a buffer. The type of buffer is not particularly limited, but MES buffer is preferred, with a concentration of 5-50 mM, more preferably 10-30 mM, for example, 20 mM. Furthermore, the pH is preferably adjusted to 6.7-7.3, more preferably 6.9-7.1, and even more preferably approximately 7.0. Furthermore, the buffer contains phosphate ions, preferably at a concentration of 0-3 mM, more preferably 0-2 mM, for example, 1 mM.

The HSA-hGH fusion protein is extracted from the hydroxyapatite chromatography column using a buffer having an elevated phosphate ion concentration. The phosphate ion concentration is preferably 20-50 mM, more preferably 25-40 mM, and even more preferably 25-35 mM, for example, 30 mM.

The third column chromatography step utilizes cation exchange column chromatography to remove contaminating proteins. While the cation exchange resin used in cation exchange column chromatography is not particularly limited, a weak cation exchange resin is preferred, and a weak cation exchange resin with selectivity based on both hydrophobic interactions and hydrogen bond formation is more preferred. For example, a weak cation exchange resin such as Capto MMC (GE Healthcare) that has phenyl groups, amide bonds, and carboxyl groups and exhibits selectivity based on hydrophobic interactions and hydrogen bond formation is preferably used.

The pH and conductivity of the eluate obtained in the second column chromatography step are adjusted before being subjected to cation exchange column chromatography. The pH is preferably adjusted to 5.3-6.2, more preferably 5.4-6.1, and even more preferably 5.6-5.8. Furthermore, the conductivity is preferably adjusted to 0.5-0.9 S/m, more preferably 0.6-0.8 S/m.

The eluent, whose pH and conductivity have been adjusted, is then applied to a cation exchange column equilibrated with a buffer. The type of buffer is not particularly limited, but MES buffer is preferred, with a concentration of 30-70 mM, more preferably 40-60 mM, for example, 50 mM. Furthermore, the pH is preferably adjusted to 5.4-6.0, more preferably 5.6-5.8, for example, 5.7. The buffer contains a salt. When the salt is a neutral salt, the concentration in the buffer is preferably 30 to 150 mM, more preferably 50 to 120 mM, and even more preferably 80 to 120 mM, for example, 100 mM. In this case, the neutral salt is preferably sodium chloride or potassium chloride, with sodium chloride being particularly preferred.

The HSA-hGH fusion protein is eluted from the cation exchange column using a buffer having an increased neutral salt concentration. The neutral salt concentration is preferably 400-600 mM, more preferably 500-600 mM, and even more preferably 530-570 mM, for example, 550 mM.

The fourth column chromatography step utilizes size-selective chromatography. Size-selective chromatography is used to remove low-molecular-weight impurities such as endotoxins, as well as polymers or degradation products of the HSA-hGH fusion protein, based on molecular size.

During the size-selection chromatography step, the column is pre-equilibrated. The type of buffer is not particularly limited, but a phosphate buffer is preferred, preferably at a concentration of 5-20 mM, more preferably 8-12 mM, for example, 10 mM. The pH is preferably adjusted to 6.6-7.4, more preferably 7.0-7.4, for example, 7.2. Furthermore, the buffer may contain a disaccharide that can be used as a pharmaceutical additive. Such a disaccharide is preferably sucrose, preferably at a concentration of 60-90 mg/mL, more preferably 70-80 mg/mL, for example, 75 mg/mL. Through the first to fourth column chromatography steps, substantially pure HSA-hGH fusion protein can be obtained.

During the purification of the HSA-hGH fusion protein, a viral inactivation step may be added, if desired. The viral inactivation step may be performed between any two analytical steps, before the first analytical step, or after the last analytical step. Furthermore, the viral inactivation step may be performed once, twice, or three or more times during the purification of the HSA-hGH fusion protein.

When the analytical steps sequentially include a column chromatography step using as a stationary phase a material bound to an antibody having affinity for the HSA-hGH fusion protein (first column chromatography step), a column chromatography step using as a stationary phase a material having affinity for phosphate groups (second column chromatography step), a cation exchange column chromatography step (third column chromatography step), and a size selection column chromatography step (fourth column chromatography step), the viral inactivation step is preferably performed between the first and second column chromatography steps, or/and after the fourth column chromatography step. The type of viral inactivation step employed is not particularly limited, but preferably a solvent-surfactant method or a filter filtration method can be employed. The solvent-surfactant method involves adding an organic solvent and a surfactant to a solution to be inactivated. In the solvent-surfactant method, an organic solvent and a nonionic surfactant are added to a solution containing the HSA-hGH fusion protein, mixed, and the mixture is incubated for, for example, more than 3 hours. The solution used in the solvent-surfactant method is not particularly limited as long as it can stably maintain the HSA-hGH fusion protein for at least two hours. However, preferably, an organic solvent and a non-ionic surfactant are mixed in a glycine buffer, phosphate buffer, MES buffer, Tris-HCl buffer, or a mixture thereof adjusted to a pH near neutral. Furthermore, the type of non-ionic surfactant used is not particularly limited, but for example, polysorbate 20, polysorbate 80, and Triton X-100 can be used alone or in any combination. Polysorbate 80 is a particularly suitable non-ionic surfactant and can be used alone or in combination with other non-ionic surfactants. The organic solvent used is not particularly limited, but for example, tri(n-butyl phosphate) can be used. When polysorbate 80 and tri(n-butyl phosphate) are used in combination, the concentration of polysorbate 80 in the mixture is 0.3-2%, for example, 1%, and the concentration of tri(n-butyl phosphate) is 0.1-0.5%, for example, 0.3%. The mixture is incubated for 2-5 hours, for example, 3 hours.

The filter filtration method removes viruses by filtering a solution to be treated with a filter membrane (virus removal membrane) that has virus removal properties. The filter membrane used in the filter filtration method preferably has an average pore size of 17-21 nm, and is made of, for example, regenerated cellulose. In the filter filtration method, a solution containing the HSA-hGH fusion protein is passed through the virus removal membrane. For example, virus inactivation can be performed using a solvent-surfactant method between the first and second column chromatography steps, and after the fourth column chromatography step, virus inactivation can be performed using the filter filtration method to more reliably remove viruses.

In addition, the method using a virus removal membrane can also be called a virus removal step, but in the present invention, it can also be called a method of a virus inactivation step.

In the present invention, the HSA-hGH fusion protein exhibits enhanced blood stability and a prolonged half-life compared to the original growth hormone unbound to HSA. While this stability varies depending on the route of administration and the dose, a blood half-life (t _1/2 β) of 5 hours or longer, for example, is highly stable when administered subcutaneously to cynomolgus macaques. For example, the blood half-life (t _1/2 β) of an HSA variant-human growth hormone fusion protein ranges from 5 to 40 hours when administered subcutaneously to male _cynomolgus macaques at a single dose of 0.5 to 10 mg/kg.

In the present invention, the drug containing the HSA variant-growth hormone fusion protein as an active ingredient can be administered as an injection into the vein, muscle, abdominal cavity or subcutaneously.

Human growth hormone (HGH) mainly consists of two types: one with a molecular weight of 22 kDa (22kDa HGH, referred to as 22K HGH in this specification) and one with a molecular weight of 20 kDa (20kDa HGH, referred to as 20K HGH in this specification). 22K HGH is a protein composed of 191 amino acids with the amino acid sequence shown in SEQ ID NO: 9. Generally, the term "human growth hormone (or hGH)" refers to this 22K HGH. However, in this specification, the term "human growth hormone (or hGH)" encompasses both 22K HGH and 20K HGH.

In this specification, the term "22K human growth hormone (or 22K hGH)" encompasses not only wild-type 22K hGH having the amino acid sequence set forth in SEQ ID NO: 9, but also 22K hGH variants having one or more amino acid substitutions, deletions, and/or additions thereof, which exhibit growth-promoting activity. The number of amino acids that can be substituted, deleted, and/or added per variant is preferably 1 to 8, more preferably 1 to 4, and even more preferably 1 to 2. The amino acid sequence of a 22K hGH variant preferably exhibits at least 85% identity, more preferably at least 90% identity, and even more preferably at least 95% identity to the wild-type 22K hGH sequence set forth in SEQ ID NO: 9.

Wild-type 20K human growth hormone is a protein with growth-promoting activity. It contains an amino acid sequence consisting of 176 amino acids (SEQ ID NO: 10), which corresponds to a deletion of 15 amino acids, from the 32nd to the 46th amino acid sequence, from the N-terminus, of the 191 amino acids that make up wild-type 22K growth hormone (SEQ ID NO: 9). However, in this specification, the term "20K human growth hormone (or 20K hGH)" includes not only wild-type 20K hGH as shown in SEQ ID NO: 10, but also 20K hGH variants that have one or more amino acid substitutions, deletions, and/or additions to the sequence and exhibit growth-promoting activity. The number of amino acids that can be substituted, deleted, and/or added is preferably 1 to 8, more preferably 1 to 4, and even more preferably 1 to 2 per variant. The amino acid sequence of the 20K hGH variant preferably has an identity of 85% or greater, more preferably 90% or greater, and even more preferably 95% or greater to the amino acid sequence of the wild-type 20K hGH set forth in SEQ ID NO: 10.

In the present invention, the position and form (deletion, substitution, addition) of each variant in various hGH variants can be easily determined by comparing the amino acid sequences of the two hGHs. Furthermore, in the present invention, the identity between the amino acid sequence of wild-type hGH and the amino acid sequence of the variant-incorporated hGH can be easily calculated using known homology calculation algorithms. For example, such algorithms include BLAST (Altschul SF. J Mol. Biol. 215. 403-10, (1990)), Pearson and Lipman's similarity search method (Proc. Natl. Acad. Sci. USA. 85. 2444 (1988)), and Smith and Waterman's local homology algorithm (Adv. Appl. Math. 2. 482-9 (1981)).

The substitution of amino acids in the hGH amino acid sequence with other amino acids, for example, occurs within an amino acid family whose amino acids are related in side chains and chemical properties. Such substitutions within the amino acid family are not expected to significantly alter hGH function (i.e., they are conservative amino acid substitutions). As examples of such amino acid families, there are the following: (1) Aspartic acid and glutamine, which are acidic amino acids; (2) Histidine, lysine, and arginine, which are basic amino acids; (3) Phenylalanine, tyrosine, and tryptophan, which are aromatic amino acids; (4) Serine and threonine, which are amino acids having a hydroxyl group (hydroxyl amino acids); (5) Methionine, alanine, valine, leucine, and isoleucine, which are hydrophobic amino acids; (6) Cysteine, serine, threonine, aspartic acid, and glutamine, which are neutral hydrophilic amino acids; (7) Glycine and proline, which are amino acids that affect the orientation of the peptide chain; (8) Aspartic acid and glutamine are amide-type amino acids (polar amino acids); (9) Alanine, leucine, isoleucine, and valine are aliphatic amino acids; (10) Alanine, glycine, serine, and threonine are amino acids with small side chains; (11) Alanine and glycine are amino acids with particularly small side chains.

Preparations containing hGH (hGH preparations) as the active ingredient, produced as a recombinant protein using Escherichia coli with an introduced hGH gene, are widely used clinically as treatments for the following conditions: growth hormone-deficient dwarfism, dwarfism in Turner syndrome, SGA dwarfism, dwarfism in Noonan syndrome, dwarfism due to chronic renal failure, dwarfism in Prader-Willi syndrome, and dwarfism in achondroplasia. hGH preparations are particularly effective in these conditions without epiphyseal closure. hGH preparations are administered subcutaneously or intramuscularly, and the hGH component circulates in the blood, promoting growth through its growth-promoting activity. hGH preparations are also widely used clinically as treatments for adult growth hormone deficiency. Although abnormalities in lipid metabolism are observed in patients with adult growth hormone deficiency, administration of hGH preparations normalizes lipid metabolism and improves patients' quality of life. Growth hormone is also clinically used as a treatment for AIDS-related wasting. For example, GROWJECT (registered trademark) is an hGH preparation for growth hormone-deficient dwarfism and adult growth hormone deficiency.

In the case of producing a fusion protein of an HSA variant and hGH, a specific method for conjugating a polypeptide comprising the amino acid sequence of an HSA variant and a polypeptide comprising the amino acid sequence of hGH is generally, for example, the following method, which can be utilized in the present invention: an expression vector is prepared that incorporates a DNA fragment that binds in-frame the gene encoding one polypeptide downstream of the gene encoding the other polypeptide, and host cells transformed with this expression vector are cultured to express the recombinant proteins.

When a fusion protein of an HSA variant and hGH is produced by expressing it as a recombinant protein in transformed cells, a polypeptide comprising the amino acid sequence of hGH is bound directly or indirectly via a linker to either the N-terminus or the C-terminus of a polypeptide comprising the amino acid sequence of the HSA variant.

To conjugate a polypeptide comprising the amino acid sequence of hGH to the N-terminus of a polypeptide comprising the amino acid sequence of an HSA variant, an expression vector can be used that incorporates a DNA fragment that binds in-frame to a gene encoding a polypeptide comprising the amino acid sequence of an HSA variant, downstream of a gene encoding a polypeptide comprising the amino acid sequence of hGH. To indirectly conjugate the two polypeptides via a peptide linker, a DNA sequence encoding the linker is placed in-frame between the genes encoding the two polypeptides.

To conjugate a polypeptide comprising the amino acid sequence of hGH to the C-terminus of a polypeptide comprising the amino acid sequence of an HSA variant, an expression vector can be used that incorporates a DNA fragment that binds in-frame to a gene encoding a polypeptide comprising the amino acid sequence of an HSA variant, upstream of the gene encoding the polypeptide comprising the amino acid sequence of hGH. To indirectly conjugate the two polypeptides via a peptide linker, a DNA sequence encoding the linker is placed in-frame between the genes encoding the two polypeptides.

In the present invention, a preferred example of a fusion protein of an HSA variant and hGH (a type of HSA-hGH fusion protein) is an HSA-hGH fusion protein having the amino acid sequence set forth in SEQ ID NO: 11. This fusion protein comprises a 22K human growth hormone having the amino acid sequence set forth in SEQ ID NO: 9 linked to the N-terminus of HSA(A320T) having the amino acid sequence set forth in SEQ ID NO: 3 via a peptide bond, without a linker. In the present invention, a protein in which HSA(A320T) and 22K hGH are linked in this sequence is referred to as "22K human growth hormone-mHSA" or "22K hGH-mHSA." Similarly, the product in which 22K human growth hormone is linked to the N-terminus of HSA (A320T) via a peptide bond without a linker is called "mHSA-22K human growth hormone" or "mHSA-2K hGH."

Furthermore, an HSA-hGH fusion protein having the amino acid sequence set forth in SEQ ID NO: 12 is referred to as "20K human growth hormone-mHSA" or "20KhGH-mHSA." It is a protein in which the C-terminus of 20K human growth hormone having the amino acid sequence set forth in SEQ ID NO: 10 is bound to the N-terminus of human serum albumin (A320T) having the amino acid sequence set forth in SEQ ID NO: 3 via a peptide bond, without a linker. Similarly, a protein in which the N-terminus of 20K human growth hormone is bound to the C-terminus of human serum albumin (A320T) via a peptide bond, without a linker, is referred to as "mHSA-20K human growth hormone" or "mHSA-20KhGH."

The HSA-hGH fusion protein of the present invention is characterized by being very stable in the blood, with a half-life (t _1/2 β) of approximately 5 hours or longer when administered subcutaneously to cynomolgus macaques. While this half-life varies depending on the dose, for example, the blood half-life (t _1/2 β) of mHSA-22KhGH and 22KhGH-mHSA is 20-35 hours after a single subcutaneous dose of 4 mg/kg to male cynomolgus macaques.

The HSA-hGH fusion protein of the present invention can be used as a medicine. The HSA-hGH fusion protein enables the functions of human growth hormone and HSA to cooperate synergistically in vivo.

The HSA-hGH fusion protein of the present invention is very stable in the blood. Therefore, according to the present invention, human growth hormone can be stabilized in the blood and retained in the blood in an active state for a long time. Therefore, when the fusion protein is used as a medicine, the frequency of administration and/or the dosage can be reduced compared to human growth hormone. For example, human growth hormone needs to be administered daily, but with this fusion protein, the administration frequency can be set to, for example, once every 3 to 30 days. In addition, the total dosage of the drug during the treatment period can be reduced to, for example, less than 1/3 (e.g., 1/3 to 1/10) in terms of molar ratio.

The HSA-hGH fusion protein of the present invention can be used as a pharmaceutical for the following diseases: growth hormone deficiency dwarfism, dwarfism in Turner syndrome, dwarfism due to chronic renal failure, dwarfism in Prader-Willi syndrome, dwarfism in achondroplasia, SGA dwarfism, or dwarfism in Noonan syndrome. In these diseases, hGH preparations are particularly effective when they are not accompanied by epiphyseal closure. Furthermore, the HSA-hGH fusion protein of the present invention can be used as a pharmaceutical for, but is not limited to, the following diseases: adult growth hormone deficiency, wasting due to AIDS, and wasting due to anorexia. Furthermore, it can be used as a therapeutic agent for the following diseases: diseases in which symptoms can be ameliorated by the long-term effects of growth hormone's growth-promoting activities, such as promoting cartilage formation and protein assimilation, and its physiological activities, such as its effects on improving body composition and lipid metabolism.

When 22KhGH-mHSA is administered to humans for therapeutic purposes, the dosage and administration are not particularly limited, as long as the hGH efficacy is demonstrated. Examples of the dosage and administration of 22KhGH-mHSA are shown below, but are not limited thereto.

When 22KhGH-mHSA is administered to patients with growth hormone-deficient dwarfism without epiphyseal closure, the optimal dosage is 0.01-0.7 mg/kg body weight. When 22KhGH-mHSA is administered to patients with dwarfism in Turner syndrome without epiphyseal closure, the optimal dosage is 0.015-1.4 mg/kg body weight. When 22KhGH-mHSA is administered to patients with dwarfism due to chronic renal failure without epiphyseal closure, the optimal dosage is 0.01-1.4 mg/kg body weight. When 22KhGH-mHSA is administered to patients with dwarfism in Prader-Willi syndrome without epiphyseal closure, the optimal dosage is 0.012-0.98 mg/kg body weight. When 22KhGH-mHSA is administered to patients with dwarfism in achondroplasia without epiphyseal closure, the optimal dosage is 0.015-1.4 mg/kg body weight. When 22KhGH-mHSA is administered to patients with SGA dwarfism without epiphyseal closure, the optimal dosage is 0.012-1.9 mg/kg body weight. When 22KhGH-mHSA is administered to patients with adult growth hormone deficiency, the optimal dosage is 0.001-0.34 mg/kg body weight. When 22KhGH-mHSA is administered to patients with dwarfism in Noonan syndrome without epiphyseal synostosis, the optimal dosage is 0.013-1.8 mg/kg body weight. When 22KhGH-mHSA is administered to patients with AIDS-related wasting, the optimal dosage is 0.005-0.4 mg/kg body weight. However, the dosage should be adjusted appropriately based on the patient's examination findings. The preferred administration interval for 22KhGH-mHSA in these diseases is once every 7 to 30 days, which can be adjusted to once every 7 to 14 days, every 10 to 20 days, or every 14 to 21 days, depending on the patient's examination findings. Furthermore, the preferred method of administration is subcutaneous, intramuscular, or intravenous injection, with subcutaneous or intramuscular injection being more preferred.

Pharmaceuticals containing the HSA-hGH fusion protein of the present invention as an active ingredient can be administered as an injection intravenously, intramuscularly, intraperitoneally, subcutaneously, or intracerebrally. These injections can be administered as freeze-dried preparations or aqueous liquids. Aqueous liquids can be prepared in vials or pre-filled syringes. Freeze-dried preparations are reconstituted by dissolving in an aqueous medium before use. [Examples]

Hereinafter, the present invention will be described in more detail with reference to the embodiments, but it is not intended to limit the present invention to the embodiments.

[Example 1] Construction of a 22K hGH-HSA Expression Vector A protein having the amino acid sequence shown in SEQ ID NO: 15, which is formed by linking the C-terminus of 22K hGH to the N-terminus of wild-type HSA (SEQ ID NO: 1), was used as 22K hGH-HSA. In the amino acid sequence shown in SEQ ID NO: 15, amino acid residues 1 to 191 correspond to the amino acid sequence of 22K hGH, and amino acid residues 192 to 776 correspond to the amino acid sequence of HSA. A DNA having the base sequence shown in SEQ ID NO: 16, containing the gene encoding 22K hGH-HSA (22K hGH-HSA gene), was chemically synthesized. In this sequence, alkaloids 11-88 encode the hGH leader peptide, alkaloids 89-661 encode 22K hGH, and alkaloids 662-2416 encode HSA. This DNA was digested with restriction enzymes (MluI and NotI) and inserted between the MluI and NotI sites of pE-mIRES-GS-puro to construct pE-mIRES-GS-puro (22K hGH-HSA), a 22K hGH-HSA expression vector. The production method of pE-mIRES-GS-puro is well known and is described in International Patent Publication WO2012/063799.

[Example 2] Construction of a 22K hGH-mHSA Expression Vector A protein having the amino acid sequence shown in SEQ ID NO: 11, formed by binding the C-terminus of 22K hGH (SEQ ID NO: 9) to the N-terminus of HSA (A320T) (SEQ ID NO: 3), was used as 22K hGH-mHSA. Using pE-mIRES-GS-puro (22K hGH-HSA) prepared in Example 1 as a template, primers YA082 (SEQ ID NO: 13) and YA083 (SEQ ID NO: 14) were used to amplify a DNA fragment containing the gene encoding 22K hGH-mHSA by PCR. This DNA fragment was allowed to self-anneal to construct the 22KhGH-mHSA expression vector pE-mIRES-GS-puro (22KhGH-mHSA).

[Example 3] Preparation of a 22K hGH-mHSA-Expressing Cell Line Cells expressing 22K hGH-mHSA were prepared as follows. CHO-K1 cells, derived from Chinese hamster ovaries, were electroporated using the Gene Pulser Xcell electroporation system (Bio-Rad) to introduce the 22K hGH-mHSA-expressing vector pE-mIRES-GS-puro (22K hGH-mHSA) prepared in Example 2. Cells transfected with the expression vector were cultured in CD OptiCHO ^™ medium (Thermo Fisher Scientific) containing 16 μM thymidine, 100 μM hypoxanthine, 10 mg/L insulin, L-methionine sulfonimide (SIGMA), and puromycin (SIGMA). During the selection culture, the concentrations of L-methionine sulfonimide and puromycin were gradually increased, ultimately reaching 300 μM L-methionine sulfonimide and 10 μg/mL puromycin to selectively promote the proliferation of cells exhibiting high drug resistance. Next, cells selected by selective culture were plated onto 96-well plates using the limiting dilution method, seeding no more than one cell per well. Cultured for approximately 10 days until single-cell colonies formed, thus establishing cell lines. After further growth, the cells were suspended in CD OptiCHO™ medium containing 16 μM thymidine, 100 μM hypoxanthine, 300 μM L-methionine sulfonimide, and 10% (v/v) DMSO. The cells were then aliquoted into ^cryotubes and stored in liquid nitrogen. These cells were designated as 22K hGH-mHSA-expressing cell lines.

[Example 4] Pre-culture of 22K hGH-mHSA-Expressing Cells: The 22K hGH-mHSA-expressing cell line prepared in Example 3 was thawed in a 37°C water bath and suspended in serum-free EX-CELL Advanced medium (pre-culture medium, SIGMA) containing 16 μM thymidine, 100 μM hypoxanthine, and 300 μM L-methionine sulfonimide. The cells were centrifuged to pellet, and the supernatant was removed. The pelleted cells were resuspended in the pre-culture medium at a density of 2×10 ⁵ cells/mL or higher and cultured at 37°C in the presence of 5% CO ₂ for 2-4 days. This culture was repeated while expanding the culture scale until the cell number reached at least 1.0 × 10 ¹¹ cells.

[Example 5] Production Culture of 22K hGH-mHSA-Expressing Cells: Cells grown in the pre-culture were suspended in 200 L of serum-free EX-CELL Advanced medium (production culture medium, SIGMA) containing 16 μM thymidine and 100 μM hypoxanthine at a cell concentration of 4 × 10 ⁵ cells/mL. This suspension was cultured for 10 days in a disposable culture tank of an Xcellerex 200L culture system (XDR200) while being stirred at 100 rpm by an impeller while maintaining a temperature of 37°C, a dissolved oxygen content of 40%, and a pH of 6.9.

[Example 6] Purification of 22K hGH-mHSA (Purification Step 1: Collection Step/Concentration Step 1) After completion of production culture, the culture medium was filtered using a Millistak+ (registered trademark) HC pod filter, grade DOHC (1.1 ^m² × 2, Merck), followed by filtration using a Millistak+ HC pod filter, grade X0HC (1.1 ^m² × 1, Merck) to remove cells. The culture supernatant was then obtained by filtration using an Opticap SHC XL3 (pore size: 0.5/0.2 μm, Merck). The resulting culture supernatant was concentrated using a 30 kDa molecular weight cutoff superfiltration device (Pellicon 3 cassette equipped with Ultracel PLCTK membrane, 1.14 ^m² , Merck), which had been previously washed with pure water and equilibrated with PBS. The concentrated solution was recovered from the superfiltration device. The device was then rinsed with PBS, and this rinse solution was combined with the previously recovered concentrated solution to adjust the weight to approximately 15 kg as the concentrated culture solution. The concentrated culture medium was then filtered through a hydrophilic filter (Opticap SHC XL600, Merck) with a pore size of 0.5 μm at the maximum and 0.2 μm at the minimum. After filtration, the pH and conductivity of the concentrated culture medium were confirmed to be 7.0 ± 0.3 and 1.2 ± 0.4 S/m, respectively.

(Cleanup Step 2: First Chromatography Step (Affinity Column Chromatography Step)) A QS column (column volume: 4.2-5.2 L, bed height: 15.0 ± 1.5 cm, Merck) packed with Capture Select Human Growth Hormone Affinity Matrix (Thermo Fisher Scientific) was washed with 1 volume of 50 mM glycine hydrochloride buffer (pH 3.0), followed by 1 volume of 0.01 M sodium hydroxide solution. The column was then equilibrated with 4 volumes of 20 mM Tris-HCl buffer (pH 7.0). Next, load 1/4 of the concentrated culture medium from purification step 1 onto the column to allow 22K hGH-mHSA to adsorb onto the column. Next, wash the column with 20 mM Tris-HCl buffer (pH 7.0) containing 200 mM arginine and 0.05% (w/v) polysorbate 80 (5 volumes equal to the column volume), followed by another 5 volumes of 20 mM Tris-HCl buffer (pH 7.0) equal to the column volume. Next, apply 5 volumes of 50 mM glycine hydrochloride buffer (pH 3.0) equal to the column volume to elute 22K hGH-mHSA. The eluate was collected in a container pre-filled with 250 mM MES buffer (pH 7.0) at 1/5 the volume of the eluate and immediately neutralized to a pH of 6.7 ± 0.3. Purification Step 2 was performed at a cold temperature of 4-8°C. The linear flow rate of the solution supplied to the column throughout Purification Step 2 was set at 200 cm/hour. Furthermore, the upper limit of the amount of 22K hGH-mHSA that could be loaded per 1 L of affinity column support was set to 7 g.

(Purification Step 3: Virus Inactivation Step) Warm the extract from Purification Step 2 to 25°C. Add 1/19 the volume of a 6.0% (v/v) aqueous solution of tri(n-butyl) phosphate containing 20.0% (w/v) polysorbate 80 to the extract. Stir at 25°C for 3 hours.

(Purification Step 4: Second Chromatography Step (Hydroxyapatite Column Chromatography Step)) The viral inactivation fraction from Purification Step 3 was cooled to 8°C. Appropriate amounts of 1 M Tris-HCl buffer (pH 8.8) and 20 mM MES buffer containing 4 M NaCl (pH 7.0) were added to the fraction to adjust the pH and conductivity to 7.0 ± 0.1 and 0.6 ± 0.1 S/m, respectively. The fraction was then filtered through a 0.2 μm pore size hydrophilic filter (Merck). The following chromatography procedures were then performed under refrigeration (linear flow rate: 200 cm/h).

A QS column (column volume: 8.8-10.8 L, bed height: 20.0 ± 2.0 cm, Merck) packed with CHT Type II, 40 μm (Bio-Rad Laboratories) using hydroxyapatite as a support was washed with 200 mM phosphate buffer (pH 7.0), followed by 1 M NaOH aqueous solution, and further with 200 mM phosphate buffer (pH 7.0). The column was then equilibrated with four volumes of 20 mM MES buffer (pH 7.0) containing 50 mM NaCl and 1 mM sodium dihydrogen phosphate. The filtrate was then loaded onto the column, allowing 22K hGH-mHSA to adsorb onto the column. The column was then washed with three volumes of 20 mM MES buffer (pH 7.0) containing 50 mM NaCl and 1 mM sodium dihydrogen phosphate. 22K hGH-mHSA was then extracted by applying 20 mM MES buffer (pH 7.0) containing 50 mM NaCl and 30 mM sodium dihydrogen phosphate to the column. Purification step 4 was performed at a cold temperature of 4-8°C. The linear flow rate of the solution supplied to the column throughout purification step 4 was set at 200 cm/hour. The upper limit of the amount of 22K hGH-mHSA that could be loaded per 1 L of the hydroxyapatite matrix was set to 13 g.

(Purification Step 5: Third Layer Chromatography Step (Multimodal Weak Cation Exchange Column Chromatography Step)) To the eluate from Purification Step 4, add an equal volume of 20 mM MES buffer (pH 5.7) containing 50 mM NaCl. Then, add dilute hydrochloric acid to adjust the pH and conductivity to 5.7 ± 0.1 and 0.7 ± 0.1 S/m, respectively. Filter through a 0.5/0.2 μm pore size hydrophilic filter (Merck).

A prepacked RTP Capto MMC 10 L column (column volume: 8.8-10.8 L, bed height: 20.0 ± 2.0 cm, Merck) was washed with 1 M NaOH aqueous solution followed by 50 mM phosphate buffer (pH 7.0) containing 1 M NaCl. The column was then equilibrated with four volumes of 50 mM MES buffer (pH 5.7) containing 100 mM NaCl. The filtrate was then loaded onto the column to allow 22K hGH-mHSA to adsorb. The column was then washed with five volumes of 50 mM MES buffer (pH 5.7) containing 100 mM NaCl. Next, 50 mM MES buffer (pH 5.7) containing 550 mM NaCl was supplied to the column to elute 22K hGH-mHSA. Purification Step 5 was performed at a cold temperature of 4-8°C. The linear flow rate of the solution supplied to the column throughout Purification Step 5 was set at 200 cm/hour. Furthermore, the upper limit of 22K hGH-mHSA loading per 1 L of the multiple weak cation exchange medium was set at 11.5 g.

(Purification Step 6: Concentration Step 2) After thorough washing with pure water through a 30 kDa molecular weight cutoff superfilter (Pellicon 3 cassette Ultracel PLCTK membrane, 1.14 ^m² , Merck), equilibrate with 10 mM phosphate buffer (pH 7.2) containing 75 mg/mL sucrose. Concentrate the extract from Purification Step 5 using this superfilter. Add 10 mM phosphate buffer (pH 7.2) containing 75 mg/mL sucrose to the concentrated solution, adjusting the absorbance (280 nm) to 23 ± 3. This concentration step is performed at room temperature.

(Purification Step 7: Fourth Layer Chromatography Step (Size Sizing Column Chromatography Step)) The concentrate from Purification Step 6 was filtered using a 0.5/0.2 μm hydrophilic filter (Merck). A QS column (column volume: 25.4-31.1 L, bed height: 40.0 ± 4.0 cm, Merck) packed with Fractogel ^™ BioSEC resin (Merck), a size-sizing chromatography resin, was washed with 0.5 M NaOH aqueous solution and then equilibrated with 10 mM phosphate buffer (pH 7.2) containing 75 mg/mL sucrose. Next, the filtrate was loaded onto the column, followed by a 10 mM phosphate buffer (pH 7.2) solution containing 75 mg/mL sucrose. An absorptiophotometer was placed in the flow path of the eluate from the size screening column to continuously measure the absorbance of the eluate. The absorbance at 280 nm was monitored, and the fraction showing an absorbance peak at 280 nm was recovered as the fraction containing 22K hGH-mHSA and used as the purified 22K hGH-mHSA product. Purification Step 7 was performed at room temperature, and the linear flow rate of the solution supplied to the column throughout Purification Step 7 was set to 30 cm/hour or less. Furthermore, the volume of the filtrate containing 22K hGH-mHSA loaded into the multiple weak cation exchange medium was set to be less than 8% of the volume of the medium.

(Purification Step 8: Concentration Step 3) A hollow fiber membrane (ReadyToProcess Hollow Fiber Cartridge, 30 kDa molecular weight cutoff, GE Healthcare) was equilibrated with 10 mM phosphate buffer (pH 7.2) containing 75 mg/mL sucrose. The purified 22K hGH-mHSA obtained in Purification Step 7 was concentrated using this hollow fiber membrane to a concentration of 85 mg/mL. To the resulting concentrated solution, 1/29 of the volume of the solution was added to 10 mM phosphate buffer (pH 7.2) containing 90 mg/mL Poloxamer 188 and 75 mg/mL sucrose, mixed, and filtered through a 0.5/0.2 μm hydrophilic filter (Merck). This concentration step was performed at room temperature.

(Purification Step 9: Virus Removal Step) Prepare a 10 mM phosphate buffer (pH 7.2) containing 3 mg/mL poloxamer 188 and 75 mg/mL sucrose. Use this buffer to equilibrate a virus removal membrane (Planova 20N, membrane area: 0.12 ^m² , material: regenerated cellulose, Asahi Kasei Medical). Filter the filtrate from Purification Step 8 through this virus removal membrane at a pressure of no more than 98 kPa. This virus removal step is performed at room temperature.

(Purification Step 10: Drug Substance Production Step) The filtrate from Purification Step 9 was filtered at room temperature through a 0.2 μm pore size hydrophilic filter (Merck) to obtain the 22K hGH-mHSA drug substance.

[Example 7] Quantification of 22K hGH-mHSA in Each Purification Step The amount of 22K hGH-mHSA after each of the purification steps described above was quantified using the method described in Example 8 below. As shown in Table 1, 111.08 g of 22K hGH-mHSA was supplied to the affinity column chromatography step (the first chromatography step) in the purification steps described above, resulting in 65.87 g of purified 22K hGH-mHSA. This indicates that the recovery rate of 22K hGH-mHSA in this purification method was 59%, demonstrating that this purification method is highly efficient for purifying 22K hGH-mHSA. In Table 1, "Recovery/Step" refers to the ratio of the amount of 22KhGH-mHSA recovered in each chromatography step (step) to the amount of 22KhGH-mHSA loaded, and "Recovery/Overall" refers to the ratio of the amount of 22KhGH-mHSA recovered in each step to the initial amount of 22KhGH-mHSA supplied to the chromatography step.

[Table 1] Table 1 Recovery of 22KhGH-mHSA in each purification step Analytical steps 22KhGH-mHSA Filling volume (g) Recycling amount (g) Recovery rate/step (%) Recovery rate/overall (%) Affinity columns 111.08 122.31 110 110 Hydroxyapatite column 100.31 103.75 103 93 Multiple weak cation exchange column 103.12 76.26 74 69 Size screening column 78.70 65.87 84 59

[Example 8] Quantification of 22K hGH-mHSA Using the Bradford Method The purified 22K hGH-mHSA produced by the present invention and quantified by amino acid analysis was diluted with water to a theoretical protein concentration of 1.0 mg/mL to prepare Standard Solution 1. The prepared solution volume was adjusted to at least 800 μL. Next, 200 μL of Standard Solution 1 was mixed with 50 μL of pure water to a theoretical protein concentration of 0.8 mg/mL to prepare Standard Solution 2. In this order, 180 μL, 100 μL, and 100 μL of standard solution 1 were mixed with 120 μL, 150 μL, and 400 μL of water, respectively, to create standard solutions 3, 4, and 5, respectively, to achieve theoretical protein concentrations of 0.6 mg/mL, 0.4 mg/mL, and 0.2 mg/mL. At least 50 μL of the test substance was diluted with water to a protein concentration between 0.4 and 0.8 mg/mL to create the sample solution. The standard and sample solutions were prepared immediately prior to use.

Place 5 mL of Coomassie reagent (Pierce Coomassie Assay Reagent, included in the Pierce ^™ Coomassie (Bradford) Protein Assay Kit, Thermo Fisher Scientific) in a 15 mL centrifuge tube. Add 100 μL of water (for the blank), standard solutions 1 to 5, and the sample solution to the Coomassie reagent in the centrifuge tube. Immediately after adding each solution, gently invert and mix. Allow the mixture to react at room temperature for 10 minutes. After the reaction, quickly measure the absorbance at 595 nm using a spectrophotometer (UV-2600, Shimadzu Corporation). The above reaction using the Coomassie reagent should be performed within 2 hours of preparing each solution. A calibration curve was prepared from the measured values of the standard solution. The measured values of each test substance were interpolated onto this calibration curve to calculate the 22KhGH-mHSA concentration in each test substance.

[Example 9] Purity Evaluation of Purified 22K hGH-mHSA by SE-HPLC Analysis SE-HPLC analysis was performed using a Shimadzu LC-20A system (system controller, online degassing unit, liquid delivery unit, autosampler, column oven, and UV-visible detector). A TSKgel G3000SW _XL column (7.8 mm inner diameter, 30 cm length, TOSOH) was installed in the LC-20A system. After equilibration with phosphate buffer (200 mM sodium phosphate containing 200 mM NaCl) at a flow rate of 0.7 mL/min, the column was loaded with 10 μL of a solution containing 22K hGH-mHSA at a concentration of 2.0 mg/mL. Similarly, a sample dilution solution (10 mM phosphate buffer containing 75 mg/mL sucrose and 3 mg/mL poloxamer) was loaded at the same flow rate, and an elution profile was generated by monitoring the absorbance at 215 nm. The purified 22K hGH-mHSA obtained by the above production method showed only a single peak, roughly corresponding to the 22K hGH-mHSA monomer, in SE-HPLC analysis (Figure 1).

The purity evaluation results of 22KhGH-mHSA above indicate that the 22KhGH-mHSA purified through the above purification steps is of high purity and can be used directly as a treatment for growth hormone-deficient dwarfism, etc. [Possible Industrial Applications]

According to the present invention, for example, a raw material pharmaceutical of a fusion protein of serum albumin and growth hormone can be provided, which can be used as a therapeutic agent for growth hormone-deficient dwarfism. [Sequence Listing Non-Keyword Text]

SEQ ID NO: 1: Amino acid sequence of wild-type human serum albumin SEQ ID NO: 2: Amino acid sequence of human serum albumin Redhill SEQ ID NO: 3: Amino acid sequence of human serum albumin variant (A320T) SEQ ID NO: 4: Example 1 of the amino acid sequence of a linker SEQ ID NO: 5: Example 2 of the amino acid sequence of a linker SEQ ID NO: 6: Example 3 of the amino acid sequence of a linker SEQ ID NO: 7: Partial base sequence of the internal ribosome entry site from wild-type mouse encephalomyocarditis virus SEQ ID NO: 8: Partial base sequence of the internal ribosome entry site from a variant mouse encephalomyocarditis virus SEQ ID NO: 9: Amino acid sequence of 22K human growth hormone SEQ ID NO: 10: Amino acid sequence of 20K human growth hormone SEQ ID NO: 11: Amino acid sequence of 22K hGH-mHSA SEQ ID NO: 12: Amino acid sequence of 20K hGH-mHSA SEQ ID NO: 13: Primer YA082, synthetic sequence SEQ ID NO: 14: Primer YA083, synthetic sequence SEQ ID NO: 15: Amino acid sequence of 22K hGH-HSA SEQ ID NO: 16: Base sequence of the 22K hGH-HSA gene, synthetic sequence

without.

Figure 1 shows the SE-HPLC profile of purified 22K hGH-mHSA. The vertical axis represents absorbance at 215 nm (arbitrary units), and the horizontal axis represents retention time (minutes). Peak 1 corresponds to the monomer of purified 22K hGH-mHSA, and Peak 2 corresponds to the aggregate of purified 22K hGH-mHSA.

Claims (15)

A method for producing a fusion protein of human serum albumin and human growth hormone comprises the following steps: (a) culturing mammalian cells producing the fusion protein of human serum albumin and human growth hormone in a serum-free culture medium to secrete the fusion protein into the culture medium; (b) removing the mammalian cells from the culture medium obtained in step (a) to recover the culture supernatant; and (c) purifying the supernatant from the culture medium obtained in step (a). b) The resulting culture supernatant is subjected to a step of purifying the fusion protein by sequentially performing column chromatography using a material bound to an antibody having an affinity for the fusion protein as a stationary phase, column chromatography using a material having an affinity for phosphate groups as a stationary phase, cation exchange column chromatography, and size selection column chromatography; the amino acid sequence of the fusion protein is one having an identity of at least 85% to the amino acid sequence set forth in SEQ ID NO: 11. The production method of claim 1, wherein the antibody having affinity for the fusion protein has affinity for human serum albumin or human growth hormone. The production method of claim 1, wherein the antibody having affinity for the fusion protein has affinity for human growth hormone. The manufacturing method of claim 1 or 2, wherein the material having affinity for phosphate groups is either fluorapatite or hydroxyapatite. The manufacturing method of claim 1 or 2, wherein the material having affinity for phosphate groups is hydroxyapatite. In the production method of claim 1 or 2, the cation exchanger used in the cation exchange column chromatography is a weak cation exchanger. The manufacturing method of claim 6, wherein the weak cation exchanger maintains selectivity based on both hydrophobic interaction and hydrophobic bond formation. The production method of claim 1 or 2 further comprises a step for inactivating the virus. The production method of claim 8 is a method for inactivating viruses in an eluate of the fusion protein obtained by column chromatography using a material bound to an antibody having affinity for the fusion protein as a stationary phase. The manufacturing method of claim 8, wherein the virus is inactivated in the eluate of the fusion protein obtained by the size screening column chromatography method. The manufacturing method of claim 1 or 2, which includes a second step for inactivating the virus. The production method of claim 11, wherein the virus is inactivated in an eluate of the fusion protein obtained by column chromatography using a material bound to an antibody having affinity for the fusion protein as a stationary phase, and in an eluate of the fusion protein obtained by size-selection column chromatography. The production method of claim 11, wherein one of the steps for inactivating the virus is performed by adding a non-ionic surfactant to a solution containing the fusion protein to inactivate the virus, and the other step is performed by passing the solution containing the fusion protein through a filter membrane. The production method of claim 1 or 2, wherein the amino acid sequence of the fusion protein is 95% or more identical to the amino acid sequence shown in SEQ ID No. 11. The production method of claim 1 or 2, wherein the amino acid sequence of the fusion protein is composed of the amino acid sequence represented by SEQ ID No. 11.