Method for producing fusion protein of serum albumin and growth hormone

Abstract

To provide methods for producing fusion proteins of human serum albumin and human growth hormone.SOLUTION: Provided is a method for producing a fusion protein of human serum albumin and human growth hormone, the method comprising: (a) culturing mammalian cells, which produce the protein, in a serum-free medium to secrete the protein into a culture solution; (b) collecting a culture supernatant by removing the mammalian cells from the culture solution obtained in the step (a), and (c) purifying the protein from the culture supernatant obtained in the step (b) using: a column chromatography using as a stationary phase a material to which an antibody having an affinity for the protein is bound; a column chromatography using as a stationary phase a material having an affinity for a phosphoric acid group; a cation exchange column chromatography; and a size exclusion column chromatography.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for producing a fusion protein of serum albumin and growth hormone with a purity that can be directly used as a medicine.

Background Art

[0002] Human growth hormone (hGH) is a protein secreted from the anterior pituitary gland under the control of the hypothalamus. hGH exhibits growth-promoting activities such as promoting chondrogenesis and protein assimilation, and also exhibits effects on improving body composition and lipid metabolism. Children with low hGH secretion develop growth hormone deficiency short stature, which shows shorter stature compared to healthy children.

[0003] A preparation (hGH preparation) containing as an active ingredient hGH with a molecular weight of about 22 KD produced as a recombinant protein using Escherichia coli into which the hGH gene has been introduced is widely clinically applied as a therapeutic agent for growth hormone deficiency short stature, short stature in Turner syndrome, small-for-gestational age (SGA) short stature, short stature in Noonan syndrome, short stature due to chronic renal failure, short stature in Prader-Willi syndrome, and short stature in chondrodystrophy. The hGH preparation is particularly effective when there is no epiphyseal closure in these diseases. The hGH preparation is administered subcutaneously or intramuscularly and circulates in the blood, and exerts an effect of promoting the growth of patients by its growth-promoting activity. In addition, the hGH preparation is also widely clinically applied as a therapeutic agent for adult growth hormone deficiency. In patients with adult growth hormone deficiency, various abnormalities such as lipid metabolism disorders are recognized, but administration of the hGH preparation improves the quality of life (QOL) of patients, such as normalizing the lipid metabolism of patients. Examples of hGH preparations for growth hormone deficiency short stature, adult growth hormone deficiency, etc. include, for example, Glouject (registered trademark).

[0004] The half-life of hGH in plasma is less than 20 minutes, and the hGH administered to patients rapidly disappears from the blood. Therefore, in order to substantially exert the drug effect of hGH in patients, it is necessary to administer hGH to patients intramuscularly three times a week or subcutaneously every day. Such frequent administration is a burden on patients. Therefore, if the stability of hGH in plasma can be increased and the half-life can be extended to reduce the number of administrations of hGH to patients, the burden on patients can be reduced, which is preferable.

[0005] Human serum albumin (HSA) is a protein whose mature form consists of 585 amino acids. When simply referred to as human serum albumin, this mature form is meant. HSA is the most abundant component among plasma proteins and has a long half-life of 14 - 20 days in plasma. HSA contributes to the regulation of plasma osmotic pressure and has the function of binding to and transporting exogenous substances such as cations, fatty acids, hormones, bilirubin and other endogenous substances and drugs in the blood. Generally, substances bound to HSA are less likely to be taken up by organs and can circulate in the blood for a longer time.

[0006] It is known that there are multiple natural variants of human serum albumin (HSA). Human serum albumin Redhill is one of them (Non-Patent Documents 1, 2). Human serum albumin Redhill differs from the amino acid sequence of the above-mentioned normal human serum albumin consisting of 585 amino acids in that the 320th amino acid residue from the N-terminal side is threonine instead of alanine, and one arginine residue is added to its N-terminal, and consists of 586 amino acids. Due to the change of the above-mentioned alanine to threonine, a sequence represented by Asn-Tyr-Thr occurs in the amino acid sequence of albumin Redhill, and the Asn (asparagine) residue in this sequence is N-linked glycosylated. Therefore, albumin Redhill is observed to have a molecular weight approximately 2.5 kDa larger than that of the above-mentioned normal human serum albumin.

[0007] Methods for increasing the stability of hGH in plasma by binding HSA to hGH have been reported (Patent Documents 1 to 8). The protein obtained by binding HSA to hGH is produced as a recombinant protein in a culture medium or in cells by preparing a transformed cell into which an expression vector incorporating DNA in which the gene encoding hGH and the gene encoding HSA are ligated in-frame is introduced and culturing this cell.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Summary of the Invention

Problems to be Solved by the Invention

[0009] Under the above background, an object of the present invention is to provide a method for producing a fusion protein of human albumin and human growth hormone with a purity that can be used directly as a medicine, and a medicine containing the same as an active ingredient.

Means for Solving the Problems

[0010] As a result of repeated studies in research aimed at the above object, the inventors have found a method for efficiently producing a compound (human serum albumin variant-hGH fusion protein) obtained by binding a variant (human serum albumin variant) having an amino acid sequence in which arginine, which is the 320th amino acid residue from the N-terminus, is replaced with threonine, to human growth hormone (hGH), with a purity that can be used as a medicine as it is, and completed the present invention. That is, the present invention includes the following. 1. A method for producing a fusion protein of human serum albumin and human growth hormone, comprising: (a) culturing mammalian cells that produce the protein in a serum-free medium to secrete the protein into the culture solution; (b) recovering the culture supernatant by removing the mammalian cells from the culture solution obtained in step (a); (c) purifying the protein from the culture supernatant obtained in step (b) using column chromatography using a material to which an antibody having an affinity for the protein is bound as a stationary phase, column chromatography using a material having an affinity for a phosphate group as a stationary phase, cation exchange column chromatography, and size exclusion column chromatography; A production method comprising the above steps. 2. The production method according to 1 above, wherein in step (c), column chromatography using a material to which an antibody having an affinity for the protein is bound as a stationary phase, column chromatography using a material having an affinity for a phosphate group as a stationary phase, cation exchange column chromatography, and size exclusion column chromatography are used in this order. 3. The production method according to 1 or 2 above, wherein the antibody having an affinity for the protein has an affinity for human serum albumin or human growth hormone. 4. The production method according to 1 or 2 above, wherein the antibody having an affinity for the fusion protein has an affinity for human growth hormone. 5. The method for production according to any one of 1 to 4 above, wherein the material having an affinity for the phosphate group is either fluoroapatite or hydroxyapatite. 6. The method for production according to any one of 1 to 4 above, wherein the material having an affinity for the phosphate group is hydroxyapatite. 7. The method for production 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 method for production according to 7 above, wherein the weak cation exchanger retains selectivity based on both hydrophobic interaction and hydrophobic bond formation. 9. The method for production according to any one of 1 to 8 above, further comprising a step for inactivating a virus. 10. The method for production according to 9 above, wherein the virus is inactivated with respect to the eluate of the protein obtained by column chromatography using, as a stationary phase, a material to which an antibody having an affinity for the protein is bound. 11. The method for production according to 9 above, wherein the virus is inactivated with respect to the eluate of the protein obtained by the size exclusion column chromatography. 12. The method for production according to any one of 1 to 8 above, comprising two steps for inactivating a virus. 13. The method for production according to 12 above, wherein the virus is inactivated with respect to the eluate of the protein obtained by column chromatography using, as a stationary phase, a material to which an antibody having an affinity for the protein is bound, and the eluate of the protein obtained by the size exclusion column chromatography. 14. The method for production according to 12 or 13 above, wherein one of the steps for inactivating a virus is performed by adding a nonionic surfactant to a solution containing the HSA-hGH fusion protein to inactivate the virus, and the other step is performed by passing the solution containing the HSA-hGH fusion protein through a filtration membrane. 15. The production method according to any one of claims 1 to 14, wherein the protein is one in which human serum albumin is bound by a peptide bond to the C-terminal side of human growth hormone, either via a linker sequence or directly. 16. The production method according to claim 15, 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 amino acid sequences in which these amino acid sequences are continuously repeated 1 to 10 times. 17. The production method according to claim 16, wherein the linker sequence has an amino acid sequence represented by the amino acid sequence (Gly-Ser). 18. The production method according to any one of claims 1 to 14, wherein the amino acid sequence of the protein has 85% or more identity with the amino acid sequence represented by SEQ ID NO: 11. 19. The production method according to any one of claims 1 to 14, wherein the amino acid sequence of the protein has 95% or more identity with the amino acid sequence represented by SEQ ID NO: 11. 20. The production method according to any one of claims 1 to 14, wherein the amino acid sequence of the protein has the amino acid sequence represented by SEQ ID NO: 11.

Advantages of the Invention

[0011] According to the present invention, for example, a fusion protein of serum albumin and growth hormone purified to such an extent that it can be marketed as a medicine can be efficiently produced.

Brief Description of the Drawings

[0012]

Figure 1

Modes for Carrying Out the Invention

[0013] In this specification, when simply referring to "human serum albumin" or "HSA", in addition to the normal wild-type human serum albumin consisting of 585 amino acid residues represented by SEQ ID NO: 1, as long as it has the function of a normal wild-type human serum albumin such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, mutants of HSA corresponding to those in which one or more amino acid residues are substituted, deleted, and / or added (in this specification, "addition" of an amino acid residue means adding a residue to the end or inside of the sequence) to the amino acid sequence represented by SEQ ID NO: 1 are also included without particular distinction. When substituting an amino acid residue with another amino acid residue, the number of amino acid residues to be substituted is preferably 1 to 10, more preferably 1 to 5, and still more preferably 1 to 3. When deleting an amino acid residue, the number of amino acid residues to be deleted is preferably 1 to 10, more preferably 1 to 5, and still more preferably 1 to 3. For example, mutants consisting of 584 amino acid residues with the N-terminal or C-terminal amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 deleted are also included in human serum albumin. Also, combinations of these substitutions and deletions of amino acid residues may be made. Furthermore, one or more amino acid residues may be added to the amino acid sequence of normal wild-type HSA or its mutant, either in the middle of the amino acid sequence or on the N-terminal side or C-terminal side of the amino acid sequence. At this time, the number of amino acid residues to be added is preferably 1 to 10, more preferably 1 to 5, and still more preferably 1 to 3.

[0014] Among these three types of mutations, substitution, deletion, and addition of amino acids, as mutants of HSA in which at least two types of mutations are combined and introduced, those having an amino acid sequence obtained by performing a deletion of 0 to 10 amino acid residues, a substitution of 0 to 10 amino acid residues with other amino acid residues, and further an addition of 0 to 10 amino acid residues to the amino acid sequence represented by SEQ ID NO: 1 are preferred. More preferably, the number of amino acid residues to be deleted, substituted, and / or added to the amino acid sequence represented by SEQ ID NO: 1 is preferably 5 or less, and still more preferably 3 or less, respectively.

[0015] In the present invention, the term "human serum albumin Redhill" (HSA-Redhill) means a variant of human serum albumin consisting of 586 amino acid residues represented by SEQ ID NO: 2. Human serum albumin Redhill corresponds to the amino acid sequence of wild-type human serum albumin consisting of 585 amino acids represented by SEQ ID NO: 1, in which the 320th amino acid residue from the N-terminus is threonine instead of alanine and one arginine residue is added to the N-terminus. By this substitution of alanine with threonine, a sequence portion represented by Asn-Tyr-Thr occurs in the amino acid sequence of albumin Redhill, and the Asn (asparagine) residue in this sequence portion is N-linked glycosylated. Therefore, albumin Redhill is observed to have a molecular weight approximately 2.5 kDa larger than that of normal wild-type albumin (SEQ ID NO: 1).

[0016] In the present invention, the term "human serum albumin mutant" (HSA mutant) refers to a mutant with respect to normal wild-type HSA (SEQ ID NO: 1), provided that it is other than the variant (HSA-Redhill) represented by SEQ ID NO: 2. Preferred HSA mutants in the present invention include, in addition to the one represented by SEQ ID NO: 3 in which alanine at the 320th position from the N-terminus of the amino acid sequence of wild-type HSA is substituted with threonine, as long as it has the functions of normal wild-type human serum albumin, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, etc., an amino acid sequence in which one or more amino acid residues are substituted, deleted, or added to the amino acid sequence represented by SEQ ID NO: 3, provided that the asparagine residue at the 318th position and the threonine residue at the 320th position from the N-terminus of the amino acid sequence represented by SEQ ID NO: 3 are conserved in a state where they are linked by a peptide bond via a single amino acid residue (X) other than proline between these two residues. When substituting an amino acid residue in the amino acid sequence with another amino acid residue, the number of amino acid residues to be substituted is preferably 1 to 10, more preferably 1 to 5, and still more preferably 1 to 3. When deleting an amino acid residue, the number of amino acid residues to be deleted is preferably 1 to 10, more preferably 1 to 5, and still more preferably 1 to 3. For example, a mutant consisting of 584 amino acid residues in which the amino acid residue at the N-terminus or C-terminus of the amino acid sequence represented by SEQ ID NO: 3 is deleted may be used. Also, a combination of substitution and deletion of these amino acid residues may be used. Further, one or more amino acid residues may be added to the amino acid sequence of these mutants or to the N-terminal side or C-terminal side of the amino acid sequence. That is, with respect to the amino acid sequence represented by SEQ ID NO: 3, at least two types of mutations among the three types of mutations of amino acid substitution, deletion, and addition are introduced in combination, and it can be one in which 0 to 10 amino acid residues are deleted, 0 to 10 amino acid residues are substituted with other amino acid residues, and further 0 to 10 amino acid residues are added.However, the amino acid residues at positions 318 to 320 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 3 must be asparagine-X-threonine (where "X" is an amino acid residue other than proline), preferably asparagine-tyrosine-threonine. The human serum albumin variant is included in human serum albumin as long as it retains the function as 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 still more preferably 95% or more with the amino acid sequence of the normal wild-type HSA shown in SEQ ID NO: 1.

[0017] In the present invention, the position and the form (deletion, substitution, addition) of each mutation when compared with the normal wild-type HSA in various HSA variants can be easily confirmed by the alignment of the amino acid sequences of both HSAs. In the present invention, the identity between the amino acid sequence of the wild-type HSA and the amino acid sequence of the HSA with the mutation added can be easily calculated using a well-known homology calculation algorithm. For example, such algorithms include BLAST (Altschul SF. J Mol. Biol. 215. 403-10, (1990)), the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA. 85. 2444 (1988)), the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2. 482-9(1981)), and the like.

[0018] Examples of substitutions of amino acids in the amino acid sequence of the above-mentioned HSA with other amino acids include amino acids classified into the same group such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids having a hydroxyl group (Ser, Thr), and amino acids with a small side chain (Gly, Ala, Ser, Thr, Met). Substitutions with such similar amino acids are predicted not to cause a significant change in the function of HSA (i.e., conservative amino acid substitutions).

[0019] In the examples described below, a human serum albumin mutant (a typical example of an HSA mutant) bound to human growth hormone differs only in that the 320th amino acid residue from the N-terminus is threonine instead of alanine in the amino acid sequence of wild-type human serum albumin consisting of 585 amino acids (SEQ ID NO: 1) (SEQ ID NO: 3). Due to this difference, in this HSA mutant (referred to as "HSA(A320T)"), a sequence portion represented by Asn-Tyr-Thr occurs in its amino acid sequence, and the Asn (asparagine) residue can be N-glycosylated in this sequence portion.

[0020] In this specification, the term "fusion protein of human serum albumin and human growth hormone" or "human serum albumin-hGH fusion protein (HSA-hGH fusion protein)" refers to a growth hormone to which HSA is bound, which is a compound obtained by binding polypeptides having the respective amino acid sequences of both. Here, "binding" these polypeptides includes not only the case of direct peptide bonding between the N-terminus of one and the C-terminus of the other, but also the case of indirectly binding both polypeptides via a linker. In particular, when human serum albumin is a human serum albumin variant, it is referred to as "fusion protein of human serum albumin variant and human growth hormone" or "human serum albumin variant-hGH fusion protein (HSA variant-hGH fusion protein)". That is, the human serum albumin variant-hGH fusion protein is included in the human serum albumin-hGH fusion protein.

[0021] Here, the "linker" is a structural moiety that is located between the above two polypeptides and covalently binds them, and does not originate from either the HSA (including HSA variants) or the growth hormone that is its binding partner. The linker can be a single amino acid residue that is peptide-bonded to both polypeptides or a peptide chain moiety (peptide linker) consisting of two or more amino acid residues, and the linker consisting of one or more of these amino acid residues is comprehensively referred to as a "peptide linker" in this specification. Also, in this specification, when it is said that HSA and growth hormone are "bound via a peptide bond", it includes both the case where they are directly bound by a peptide bond and the case where they are bound by a bond with a peptide linker. In addition, in this specification, when HSA and growth hormone are directly or indirectly bound via a peptide linker, the compound "human serum albumin-hGH fusion protein (HSA-hGH fusion protein)" can also be referred to as "human serum albumin fused hGH (HSA fused hGH)". The same applies to the human serum albumin variant-hGH fusion protein (HSA variant-hGH fusion protein).

[0022] In the present invention, when HSA and growth hormone are linked via a peptide linker, the linker is preferably composed of 1 to 50, more preferably 1 to 17, still more preferably 1 to 10, and yet more preferably 1 to 6 amino acid residues. For example, it is composed of 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, or 25 to 29 amino acids. Further, for example, it is composed of only 1 amino acid residue, or 2, 3, 5, 6, or 20 amino acid residues. There is no limitation on the amino acid residues or amino acid sequences constituting the peptide linker as long as the HSA moiety linked by the peptide linker retains the function of HSA and the growth hormone moiety can also exhibit the physiological activity of growth hormone under physiological conditions. Preferably, it is composed of glycine and serine. Preferred examples of the peptide linker include those consisting of Gly-Ser, Gly-Gly-Ser, Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 5), Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 6), and those comprising these amino acid sequences. Those having a sequence in which any one of these amino acid sequences is repeated 2 to 10 times, or 2 to 5 times continuously can also be suitably used as the peptide linker. Also, those having a sequence in which any two or more of these amino acid sequences are combined and repeated 1 to 10 times, or 2 to 5 times continuously can be suitably used as the peptide linker. A preferred example of a peptide linker in which any two or more of these amino acid sequences are combined is one containing a 20-amino acid sequence in which the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 5) follows the amino acid sequence Gly-Ser three times continuously.

[0023] As a method for linking two different polypeptides, for example, an expression vector incorporating DNA in which a gene encoding the other polypeptide is ligated in-frame downstream of the gene encoding one polypeptide is prepared, and a host cell transformed with this expression vector is cultured to express it as a recombinant fusion protein. This method is common and can be used in the present invention.

[0024] When producing an HSA-hGH fusion protein by expressing it in a transformed cell as a recombinant, a polypeptide comprising the amino acid sequence of growth hormone is obtained as a fusion protein linked to either the N-terminus or the C-terminus of the polypeptide comprising the amino acid sequence of HSA. The HSA-hGH fusion protein produced using genetic recombination technology is particularly referred to as a recombinant HSA-hGH fusion protein.

[0025] When a polypeptide comprising the amino acid sequence of growth hormone is linked to the N-terminal side of the polypeptide comprising the amino acid sequence of HSA, an expression vector incorporating DNA in which a gene encoding the polypeptide comprising the amino acid sequence of HSA is ligated in-frame downstream of the gene encoding the polypeptide comprising the amino acid sequence of growth hormone is used. When the two polypeptides are indirectly linked via a peptide linker, a DNA sequence encoding the linker is inserted in-frame between the genes encoding the two polypeptides.

[0026] When a polypeptide comprising the amino acid sequence of growth hormone is linked to the C-terminal side of the polypeptide comprising the amino acid sequence of HSA, an expression vector incorporating DNA in which a gene encoding the polypeptide comprising the amino acid sequence of HSA is ligated in-frame upstream of the gene encoding the polypeptide comprising the amino acid sequence of growth hormone is used. When the two polypeptides are indirectly linked via a peptide linker, a DNA sequence encoding the linker is inserted in-frame between the genes encoding the two polypeptides.

[0027] To produce HSA-hGH fusion protein in host cells, an expression vector incorporating DNA encoding any of them is introduced into the host cells. The host cells that can be used for this purpose are not particularly limited as long as they can express HSA-hGH fusion protein by introducing such an expression vector, and can be any of eukaryotic cells such as mammalian cells, yeast, plant cells, insect cells, and prokaryotic cells such as Escherichia coli and Bacillus subtilis, but mammalian cells are particularly preferred. However, when expressing as a glycosylated protein, the host cells are selected from eukaryotic cells such as mammalian cells, yeast, plant cells, and insect cells. Usually, the Asn residue in the sequence portion represented by Asn-Tyr-Thr generated by the 320th amino acid residue of wild-type HSA being threonine, or the Asn residue in the sequence represented by Asn-X-Thr ("X" is an amino acid residue other than proline) is N-linked glycosylated by expressing the HSA-hGH fusion protein in eukaryotic cells.

[0028] When using mammalian cells as host cells, there is no particular limitation on the type of mammalian cells, but cells derived from humans, mice, and Chinese hamsters are preferred, and in particular, CHO cells derived from Chinese hamster ovary cells or NS / 0 cells derived from mouse myeloma are preferred. Also, at this time, 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 can bring about the expression of the gene when introduced into mammalian cells. The gene incorporated into the expression vector is arranged downstream of a DNA sequence (gene expression control site) that can regulate the frequency of gene transcription in mammalian cells. Examples of the gene expression control site that can be used in the present invention include a promoter derived from cytomegalovirus, SV40 early promoter, human elongation factor-1α (EF-1α) promoter, and human ubiquitin C promoter.

[0029] Mammalian cells into which such an expression vector has been introduced will come to express the protein incorporated in the expression vector, but the expression level varies among individual cells and is not uniform. Therefore, in order to efficiently produce the HSA-hGH fusion protein, it is necessary to select cells with high expression levels from mammalian cells into which the expression vector has been introduced. To perform this selection step, a gene that acts as a selection marker is incorporated into the expression vector.

[0030] The most common selection markers are enzymes that decompose drugs such as puromycin and neomycin (drug resistance markers). Mammalian cells usually die in the presence of these drugs at a certain concentration or higher. However, mammalian cells into which an expression vector incorporating a drug resistance marker gene has been introduced can detoxify or attenuate the above drugs by the expressed drug resistance marker, and thus can survive even in the presence of the above drugs. When an expression vector incorporating a drug resistance marker as a selection marker is introduced into mammalian cells and cultured in a selection medium containing the drug corresponding to the drug resistance marker, for example, while gradually increasing the concentration of the drug, cells that can proliferate even in the presence of a higher concentration of the drug can be obtained. In the cells selected in this way, generally, together with the drug resistance marker, the expression level of the gene encoding the target protein incorporated in the expression vector also increases, and as a result, cells with a high expression level of the protein are selected.

[0031] In addition, glutamine synthetase (GS) can also be used as a selection marker. Glutamine synthetase is an enzyme that synthesizes glutamine from glutamate and ammonia. When mammalian cells are cultured in a selection medium containing an inhibitor of glutamine synthetase, such as L-methionine sulfoximine (MSX), and not containing glutamine, the cells usually die. However, when an expression vector incorporating glutamine synthetase as a selection marker is introduced into mammalian cells, the expression level of glutamine synthetase increases in these cells, so that they can proliferate even in the presence of a higher concentration of MSX. At this time, if the culture is continued while gradually increasing the concentration of MSX, cells that can proliferate even in the presence of a higher concentration of MSX can be obtained. In the cells thus selected, generally, the expression level of the gene encoding the target protein incorporated into the expression vector also increases together with glutamine synthetase, and as a result, cells with a high expression level of the protein are selected.

[0032] In addition, dihydrofolate reductase (DHFR) can also be used as a selection marker. When DHFR is used as a selection marker, mammalian cells into which the expression vector has been introduced are cultured in a selection medium containing a DHFR inhibitor such as methotrexate or aminopterin. If the culture is continued while gradually increasing the concentration of the DHFR inhibitor, cells that can proliferate even in the presence of a higher concentration of the DHFR inhibitor can be obtained. The selection medium used at this time preferably does not contain hypoxanthine and thymidine. In the cells thus selected, generally, the expression level of the gene encoding the target protein incorporated into the expression vector also increases together with DHFR, and as a result, cells with a high expression level of the protein are selected.

[0033] An expression vector is known in which a glutamine synthetase (GS) is arranged as a selection marker via an internal ribosome entry site (IRES) downstream of a gene encoding a target protein (International Patent Publication Nos. WO2012 / 063799 and WO2013 / 161958). The expression vectors described in these documents can be particularly preferably used for the production of an HSA-hGH fusion protein.

[0034] For example, an expression vector for expressing a target protein, which contains a first gene expression control site, a gene encoding the protein downstream thereof, an internal ribosome binding site further downstream, and a gene encoding glutamine synthetase further downstream, and further contains a dihydrofolate reductase gene or a drug resistance gene downstream of the first gene expression control site or another second gene expression control site, can be preferably used for the production of an HSA-hGH fusion protein. In this expression vector, as the first gene expression control site or the second gene expression control site, a promoter derived from cytomegalovirus, an SV40 early promoter, a human elongation factor-1α promoter (hEF-1α promoter), or a human ubiquitin C promoter is preferably used, and the hEF-1α promoter is particularly preferred.

[0035] In addition, as the internal ribosome binding site, a genome of a virus selected from the group consisting of picornaviridae viruses, foot-and-mouth disease virus, hepatitis A virus, hepatitis C virus, coronavirus, bovine enterovirus, Theiler's murine encephalomyelitis virus, and Coxsackie B virus, or a 5'untranslated region of a gene selected from the group consisting of a human immunoglobulin heavy chain binding protein gene, a Drosophila antennapedia gene, and a Drosophila ultrabithorax gene is preferably used. However, an internal ribosome binding site derived from the 5'untranslated region of the mouse encephalomyocarditis virus genome is particularly preferred. When using an internal ribosome binding site derived from the 5'untranslated region of the mouse encephalomyocarditis virus genome, in addition to the wild-type one, those in which some of the multiple start codons contained in the wild-type internal ribosome binding site are disrupted can also be preferably used. In this expression vector, the drug resistance gene preferably used is preferably a puromycin or neomycin resistance gene, more preferably a puromycin resistance gene.

[0036] In addition, for example, an expression vector for expressing a target protein, which contains a human elongation factor-1α promoter, a gene encoding the protein downstream thereof, an internal ribosome binding site derived from the 5'untranslated region of the mouse encephalomyocarditis virus genome further downstream, and a gene encoding glutamine synthetase further downstream, and further contains another gene expression control site and a dihydrofolate reductase gene downstream thereof, and the internal ribosome binding site is one in which some of the multiple start codons contained in the wild-type internal ribosome binding site are disrupted, can be preferably used for the production of an HSA-hGH fusion protein. As such an expression vector, the expression vector described in WO2013 / 161958 can be mentioned.

[0037] Also, for example, an expression vector for expressing a target protein, which contains a human elongation factor-1α promoter, a gene encoding the protein downstream thereof, an internal ribosome binding site derived from the 5′ untranslated region of the mouse encephalomyocarditis virus genome further downstream, and a gene encoding glutamine synthetase further downstream, and further contains another gene expression control site and a drug resistance gene downstream thereof, and wherein the internal ribosome binding site has a part of a plurality of start codons contained in the wild-type internal ribosome binding site disrupted, can be suitably used for the production of an HSA-hGH fusion protein. Examples of such expression vectors include pE-mIRES-GS-puro described in WO2012 / 063799 and pE-mIRES-GS-mNeo described in WO2013 / 161958.

[0038] At the 3′ end of the internal ribosome binding site derived from the 5′ untranslated region of the wild-type mouse encephalomyocarditis virus genome, there are three start codons (ATG), and the sequence portion containing the three start codons is represented by SEQ ID NO: 7 (5′-ATGataatATGgccacaaccATG-3′: the ATG of the disclosed codon is shown in capital letters). Examples of those with a part of the start codons in this sequence portion disrupted include those represented by SEQ ID NO: 8 (5′-atgataagcttgccacaaccatg-3′), and the above pE-mIRES-GS-puro and pE-mIRES-GS-mNeo are expression vectors having an IRES containing the sequence represented by SEQ ID NO: 8.

[0039] In the present invention, mammalian cells into which an expression vector incorporating a DNA fragment encoding an HSA-hGH fusion protein has been introduced are selectively cultured in a selection medium in order to select cells with high expression levels.

[0040] In selective culture, when using DHFR as a selection marker, the concentration of the DHFR inhibitor contained in the selective medium is increased stepwise. The maximum concentration is preferably 0.25 to 5 μM, more preferably 0.5 to 1.5 μM, and still more preferably about 1.0 μM when the DHFR inhibitor is methotrexate.

[0041] When using GS as a selection marker, the concentration of the GS inhibitor contained in the selective medium is increased stepwise. The maximum concentration is preferably 10 to 1000 μM etc., for example, 20 to 500 μM, 20 to 80 μM, 20 to 30 μM when the GS inhibitor is MSX. At this time, a medium generally not containing glutamine is used as the selective medium.

[0042] When using an enzyme that degrades puromycin as a selection marker, the maximum concentration of puromycin contained in the selective medium is preferably 3 to 30 μg / mL, more preferably 5 to 20 μg / mL, and still more preferably about 10 μg / mL.

[0043] When using an enzyme that degrades neomycin as a selection marker, the maximum concentration of G418 contained in the selective medium is preferably 0.1 to 2 mg / mL, more preferably 0.5 to 1.5 mg / mL, and still more preferably about 1 mg / mL.

[0044] In addition, as a medium for culturing mammalian cells, for both the medium used in selective culture and the medium used for producing the recombinant protein described later (medium for producing recombinant protein), any medium that can culture and grow mammalian cells can be used without particular limitation, but a serum-free medium is preferably used. Since HSA has the property of adsorbing components contained in serum, when producing HSA using a medium containing serum, HSA adsorbed with impurities in the serum is obtained, and thus it is necessary to remove these impurities in a subsequent step.

[0045] In the present invention, the HSA-hGH fusion protein is obtained, in particular, by culturing cells expressing these in a serum-free medium. By using a serum-free medium, the amount of impurities adsorbed onto the HSA-hGH fusion protein can be reduced, thus simplifying the subsequent purification process.

[0046] Cells with a high expression level of the HSA-hGH fusion protein selected by selective culture are used for the production of the HSA-hGH fusion protein (HSA-hGH fusion protein-producing cells). The production of the HSA-hGH fusion protein is carried out by culturing the HSA-hGH fusion protein-producing cells in a medium for producing the HSA-hGH fusion protein. This culture is referred to as production culture.

[0047] In the present invention, as the serum-free medium used as the medium for producing the HSA-hGH fusion protein, for example, a medium containing amino acids at 3 to 700 mg / L, vitamins at 0.001 to 50 mg / L, monosaccharides at 0.3 to 10 g / L, inorganic salts at 0.1 to 10000 mg / L, trace elements at 0.001 to 0.1 mg / L, nucleosides at 0.1 to 50 mg / L, fatty acids at 0.001 to 10 mg / L, biotin at 0.01 to 1 mg / L, hydrocortisone at 0.1 to 20 μg / L, insulin at 0.1 to 20 mg / L, vitamin B12 at 0.1 to 10 mg / L, putrescine at 0.01 to 1 mg / L, sodium pyruvate at 10 to 500 mg / L, and a water-soluble iron compound is preferably used. If desired, thymidine, hypoxanthine, conventional pH indicators, antibiotics, etc. may be added to the medium.

[0048] As a serum-free medium used for producing HSA-hGH fusion protein, DMEM / F12 medium (a mixed medium of DMEM and F12) may be used as the basal medium, and each of these media is well-known to those skilled in the art. Furthermore, as a serum-free medium, DMEM(HG)HAM Modified (R5) medium containing sodium bicarbonate, L-glutamine, D-glucose, insulin, sodium selenite, diaminobutane, hydrocortisone, iron(II) sulfate, asparagine, aspartic acid, serine and polyvinyl alcohol may also be used. Furthermore, commercially available serum-free media, for example, CD OptiCHO TM medium, CHO-S-SFM II medium or CD CHO medium (Thermo Fisher Scientific), IS cho-V TM medium (Irvine Scientific), EX-CELL TM 302 medium, EX-CELL TM Advanced medium or EX-CELL TM 325-PF medium (SAFC Biosciences) etc. can also be used as the basal medium.

[0049] To the medium for producing HSA-hGH fusion protein, hydrolyzates derived from plants such as soybean, wheat, rice, etc. can be appropriately added at 1 to 50 g / L (for example, 1 to 5 g / L). The most commonly used one is the protein hydrolyzate derived from soybean. However, the HSA-hGH fusion protein can be produced without adding hydrolyzates derived from plants such as rice, for example, the protein hydrolyzate derived from soybean to the medium for producing HSA-hGH fusion protein.

[0050] The culture broth after the production culture is completed is subjected to a chromatography step for purifying the HSA-hGH fusion protein. However, what is subjected to the chromatography step is the culture supernatant from which cells etc. have been removed from the culture broth. As methods for obtaining the culture supernatant from the culture broth, there are filtration with a membrane filter, centrifugation, etc.

[0051] In the present invention, each of the chromatography steps for purifying the HSA-hGH fusion protein may be carried out in the presence of a nonionic surfactant, if necessary, to prevent non-specific adsorption of the protein. There is no particular limitation on which nonionic surfactant to use, but polysorbate-based surfactants are preferably used, more preferably polysorbate 80 or polysorbate 20. 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), 0.05% (w / v), etc.

[0052] The purification step of the HSA-hGH fusion protein can be carried out at room temperature or at low temperature, but preferably at low temperature, particularly at 1 to 10 °C.

[0053] In one embodiment of the present invention, the purification step of the HSA-hGH fusion protein includes a column chromatography step using a material having an antibody bound thereto that has an affinity for the HSA-hGH fusion protein as a stationary phase, a column chromatography step using a material having an affinity for a phosphate group as a stationary phase, a cation exchange column chromatography step, and a size exclusion column chromatography step. However, one or more chromatography steps can be added to these chromatography steps. Examples of such additional chromatography steps include a column chromatography step using a material having an antibody bound thereto that has an affinity for the HSA-hGH fusion protein as a stationary phase, a column chromatography step using a material having an affinity for a phosphate group as a stationary phase, a cation exchange column chromatography step, an anion exchange column chromatography step, a hydrophobic column chromatography step, a dye affinity column chromatography step, and a size exclusion column chromatography step.

[0054] In one embodiment of the present invention, the purification process of the HSA-hGH fusion protein includes, in this order, a column chromatography step using a material to which an antibody having an affinity for the HSA-hGH fusion protein is bound as a stationary phase, a column chromatography step using a material having an affinity for a phosphate group as a stationary phase, a cation exchange column chromatography step, and a size exclusion column chromatography step. However, one or more chromatography steps can also be added to these chromatography steps. Examples of such additional chromatography steps include, for example, a column chromatography step using a material to which an antibody having an affinity for the HSA-hGH fusion protein is bound as a stationary phase, a column chromatography step using a material having an affinity for a phosphate group as a stationary phase, a cation exchange column chromatography step, an anion exchange column chromatography step, a hydrophobic column chromatography step, a dye ligand column chromatography step, and a size exclusion column chromatography step. The additional chromatography can be added between any adjacent steps, or can be added as the first chromatography step or the last chromatography step.

[0055] The purification process including, in this order, the column chromatography step using a material to which an antibody having an affinity for the HSA-hGH fusion protein is bound as a stationary phase, the column chromatography step using a material having an affinity for a phosphate group as a stationary phase, the cation exchange column chromatography step, and the size exclusion column chromatography step in one embodiment of the present invention will be described in detail below.

[0056] The first column chromatography step uses column chromatography with a material to which an antibody having an affinity for the HSA-hGH fusion protein is bound as the stationary phase. Here, the antibody may have an affinity for either HSA or hGH, but preferably has an affinity for hGH. For example, those containing a carrier to which an antibody against human growth hormone is bound, such as Capture select Human Growth Hormone Affinity Matrix (Thermo Fisher Scientific), can be preferably used.

[0057] When using a column chromatography column with a material to which an antibody having an affinity for hGH is bound as the stationary phase, the HSA-hGH fusion protein is applied to a column pre-equilibrated with a buffer. Here, the type of buffer is not particularly limited, but Tris-HCl buffer is preferred, and its concentration is preferably 5 to 50 mM, more preferably 10 to 30 mM. Also, its pH is preferably adjusted to 6.7 to 7.3, more preferably 6.9 to 7.1, and even more preferably about 7.0. Elution of the HSA-hGH fusion protein from the column is preferably performed using glycine-hydrochloric acid. The concentration of glycine-hydrochloric acid is preferably 20 to 100 mM, more preferably 40 to 60 mM, and even more preferably about 50 mM. Also, its pH is preferably adjusted to 2.0 to 4.0, more preferably 2.8 to 3.2, and even more preferably about 3.0. The pH of the eluate is immediately adjusted to near neutrality, for example, pH 6.4 to 7.0.

[0058] The second column chromatography step uses a material having an affinity for phosphate groups as the stationary phase. Examples of column chromatography suitable for using a material having an affinity for phosphate groups as the stationary phase include hydroxyapatite column chromatography and fluoroapatite column chromatography, with hydroxyapatite column chromatography being particularly preferred. These utilize the interaction of both the metal affinity by calcium ions and the cation exchange by phosphate groups to remove impurities.

[0059] The case of using hydroxyapatite column chromatography will be described in detail below. There is no particular limitation on the carrier used for hydroxyapatite chromatography, and it may be ceramic or crystalline. One particularly preferred carrier is CHT Type II, 40 μm (Bio-Rad Laboratories).

[0060] The eluate obtained in the first column chromatography step is adjusted for pH and conductivity before being applied to the hydroxyapatite chromatography column. At this time, the pH is preferably adjusted to 6.5 - 7.5, more preferably 6.8 - 7.2, and even more preferably 6.9 - 7.1. Also, the conductivity is preferably adjusted to 0.4 - 0.8 S / m, more preferably 0.5 - 0.7 S / m.

[0061] The eluate adjusted for pH and conductivity is applied to a hydroxyapatite chromatography column pre-equilibrated with a buffer solution. Here, there is no particular limitation on the type of buffer solution, but MES buffer solution is preferred. Its concentration is preferably 5 - 50 mM, more preferably 10 - 30 mM, for example, 20 mM. Also, its pH is preferably adjusted to 6.7 - 7.3, more preferably 6.9 - 7.1, and even more preferably about 7.0. Also, the buffer solution contains phosphate ions, and its concentration is preferably 0 - 3 mM, more preferably 0 - 2 mM, for example, 1 mM.

[0062] Elution of the HSA-hGH fusion protein from the hydroxyapatite chromatography column is performed using a buffer with an increased concentration of phosphate ions. The concentration of phosphate ions at this time is preferably 20 to 50 mM, more preferably 25 to 40 mM, still more preferably 25 to 35 mM, and for example, 30 mM.

[0063] The third column chromatography step uses cation exchange column chromatography to remove contaminating proteins. There is no particular limitation on which cation exchange resin to use in cation exchange column chromatography, but a weak cation exchange resin is preferred, and more preferably a weak cation exchange resin having selectivity based on both hydrophobic interaction and hydrogen bond formation. For example, a weak cation exchange resin having a phenyl group, an amide bond, and a carboxyl group and having selectivity based on hydrophobic interaction and hydrogen bond formation, such as Capto MMC (GE Healthcare), can be preferably used.

[0064] The eluate obtained in the second column chromatography step is adjusted in pH and conductivity before being subjected to cation exchange column chromatography. At this time, the pH is preferably adjusted to 5.3 to 6.2, more preferably 5.4 to 6.1, and still more preferably 5.6 to 5.8. Also, the conductivity is preferably adjusted to 0.5 to 0.9 S / m, more preferably 0.6 to 0.8 S / m.

[0065] The eluate with adjusted pH and conductivity is subjected to cation exchange column chromatography pre-equilibrated with a buffer solution. Here, the type of buffer solution is not particularly limited, but an MES buffer solution is preferred. Its concentration is preferably 30 to 70 mM, more preferably 40 to 60 mM, for example, 50 mM. Also, its pH is preferably adjusted to 5.4 to 6.0, more preferably 5.6 to 5.8, for example, 5.7. The buffer solution contains a salt, and when the salt is a neutral salt, its concentration in the buffer solution is preferably 30 to 150 mM, more preferably 50 to 120 mM, still more preferably 80 to 120 mM, for example, 100 mM. The neutral salt at this time is preferably sodium chloride or potassium chloride, and particularly preferably sodium chloride.

[0066] Elution of the HSA-hGH fusion protein from the cation exchange column chromatography is carried out using a buffer solution with an increased concentration of the neutral salt. The concentration of the neutral salt at this time is preferably 400 to 600 mM, more preferably 500 to 600 mM, still more preferably 530 to 570 mM, for example, 550 mM.

[0067] The fourth column chromatography step uses size exclusion chromatography. Size exclusion chromatography is for removing low-molecular-weight impurities such as endotoxin, multimers, and degradation products of the HSA-hGH fusion protein based on molecular size.

[0068] In the size exclusion chromatography step, the column is pre-equilibrated. Here, the type of buffer is not particularly limited, but a phosphate buffer is preferred. Its concentration is preferably 5 to 20 mM, more preferably 8 to 12 mM, for example 10 mM. Its pH is preferably adjusted to 6.6 to 7.4, more preferably 7.0 to 7.4, for example 7.2. Also, the buffer may contain a disaccharide that can be used as a pharmaceutical additive. Such a disaccharide is preferably sucrose, and its concentration is preferably 60 to 90 mg / mL, more preferably 70 to 80 mg / mL, for example 75 mg / mL. By this first to fourth column chromatography step, a substantially pure HSA-hGH fusion protein is obtained.

[0069] In the purification step of the HSA-hGH fusion protein, a virus inactivation step can be added if desired. The virus inactivation step may be carried out between any two chromatography steps, or before the first chromatography step or after the last chromatography step. Also, the virus inactivation step may be carried out once, twice, or three or more times in the purification step of the HSA-hGH fusion protein.

[0070] When the chromatography process includes, in this order, a column chromatography process (the first column chromatography process) using, as a stationary phase, a material to which an antibody having an affinity for the HSA-hGH fusion protein is bound, a column chromatography process (the second column chromatography process) using, as a stationary phase, a material having an affinity for a phosphate group, a cation exchange column chromatography process (the third column chromatography process), and a size exclusion column chromatography process (the fourth column chromatography process), the virus inactivation process is preferably carried out between the first column chromatography process and the second column chromatography process, or / and after the fourth column chromatography process. There is no particular limitation on which virus inactivation process is applied, but the solvent-detergent method or the filter filtration method can be preferably applied. The solvent-detergent method is a method of inactivating a virus by adding an organic solvent and a detergent to a solution to be virus-inactivated. When the solvent-detergent method is applied, an organic solvent and a nonionic detergent are added to and mixed with a solution containing the HSA-hGH fusion protein, and this mixture is incubated, for example, for more than 3 hours. The solution used in the solvent-detergent method is not particularly limited as long as the HSA-hGH fusion protein is stably retained for at least 2 hours, but a glycine buffer, a phosphate buffer, a MES buffer, a Tris-HCl buffer, or a mixture thereof, to which an organic solvent and a non-detergent are mixed, and adjusted to a pH near neutrality, can be preferably used. Also, there is no particular limitation on which nonionic detergent is used, and for example, polysorbate 20, polysorbate 80, and triton X-100 can be used alone or in any combination thereof. Polysorbate 80 is one of the particularly preferred nonionic detergents and can be used alone or in combination with other nonionic detergents. Also, there is no particular limitation on which organic solvent is used, and for example, tri(n-butyl phosphate) can be used.When using a combination of polysorbate 80 and tri(n-butyl phosphate), the concentration of polysorbate 80 in the mixture is 0.3 - 2%, for example 1%, the concentration of tri(n-butyl phosphate) is 0.1 - 0.5%, for example 0.3%, and the mixture is incubated for 2 - 5 hours, for example 3 hours.

[0071] The filter filtration method is a method of removing viruses by filtering a solution to be virus-removed through a filtration membrane having virus-removing performance. The filtration membrane used in the filter filtration method preferably has an average pore size of 17 - 21 nm, and its material is, for example, regenerated cellulose. When the filter filtration method is applied, a solution containing the HSA-hGH fusion protein is passed through a virus-removing membrane. For example, virus inactivation is performed by the solvent-surfactant method between the first column chromatography step and the second column chromatography step, and after the fourth column chromatography step, virus inactivation is performed by the filter filtration method, whereby virus removal can be more reliably performed.

[0072] Note that the method using a virus-removing membrane can also be referred to as a virus-removing step, but in the present invention, it can also be referred to as a method of virus inactivation step.

[0073] In the present invention, the HSA-hGH fusion protein has increased stability in the blood and a longer half-life compared to the original growth hormone without HSA binding. Although it varies depending on the administration route and dose, when subcutaneously administered to cynomolgus monkeys, the blood half-life (t 1 / 2 β) is, for example, 5 hours or more, and it becomes extremely stable in the blood. For example, the blood half-life (t 1 / 2 β) of the HSA mutant-human growth hormone fusion protein is 5 - 40 hours when single-dose subcutaneously administered to male cynomolgus monkeys at a dose of 0.5 - 10 mg / kg. 1 / 2 β) is 5 - 40 hours.

[0074] In the present invention, a medicament containing an HSA mutant-growth hormone fusion protein as an active ingredient can be administered intravenously, intramuscularly, intraperitoneally or subcutaneously as an injection.

[0075] Human growth hormone mainly includes two types with different molecular weights: one with a molecular weight of 22 kDa (22 kDa human growth hormone, 22K human growth hormone in this specification) and one with a molecular weight of 20 kDa (20 kDa human growth hormone, 20K human growth hormone in this specification). The 22K growth hormone is a protein composed of 191 amino acids having the amino acid sequence shown in SEQ ID NO: 9. Usually, when referring to "human growth hormone (or hGH)", it means this 22K growth hormone. However, in this specification, when simply referring to "human growth hormone (or hGH)", it includes both 22K human growth hormone and 20K human growth hormone.

[0076] In this specification, when simply referring to "22K human growth hormone (or 22KhGH)", it includes not only the wild-type 22KhGH having the amino acid sequence shown in SEQ ID NO: 9, but also 22KhGH mutants in which one or more amino acids are substituted, deleted and / or added thereto and which have growth promoting activity. The number of amino acids that may be substituted, deleted and / or added is preferably 1 to 8, more preferably 1 to 4, and still more preferably 1 to 2 for each mutation type. Here, the amino acid sequence of the 22KhGH mutant preferably has an identity of 85% or more, more preferably 90% or more, and still more preferably 95% or more with the amino acid sequence of the wild-type 22KhGH shown in SEQ ID NO: 9.

[0077] Wild-type 20K human growth hormone corresponds to a protein consisting of 176 amino acids with a growth-promoting activity, which lacks 15 amino acids from the 32nd to the 46th amino acids counted from the N-terminus among the 191 amino acids constituting wild-type 22K growth hormone (SEQ ID NO: 9), and has an amino acid sequence (SEQ ID NO: 10). However, in this specification, when simply referring to "20K human growth hormone (or 20KhGH)", in addition to the wild-type 20KhGH represented by SEQ ID NO: 10, it also includes 20KhGH variants in which one or more amino acids are substituted, deleted and / or added to the said sequence and have a growth-promoting activity. The number of amino acids that may be substituted, deleted and / or added is preferably 1 to 8, more preferably 1 to 4, and still more preferably 1 to 2 for each type of mutation. Here, the amino acid sequence of the 20KhGH variant preferably has an identity of 85% or more, more preferably 90% or more, and still more preferably 95% or more with the amino acid sequence of the wild-type 20KhGH represented by SEQ ID NO: 10.

[0078] In the present invention, the position and form (deletion, substitution, addition) of each mutation when comparing various hGH variants with the normal wild-type hGH can be easily confirmed by aligning the amino acid sequences of both hGHs. In the present invention, the identity between the amino acid sequence of wild-type hGH and the amino acid sequence of hGH with mutations added can be easily calculated using well-known homology calculation algorithms. For example, such algorithms include BLAST (Altschul SF. J Mol. Biol. 215. 403-10, (1990)), the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA. 85. 2444 (1988)), the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2. 482-9(1981)), etc.

[0079] Examples of substitutions of amino acids in the amino acid sequence of the above hGH with other amino acids include amino acids classified into the same group such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids having a hydroxyl group (Ser, Thr), and amino acids with small side chains (Gly, Ala, Ser, Thr, Met). Such substitutions with similar amino acids are predicted not to cause a significant change in the function of hGH (i.e., they are conservative amino acid substitutions).

[0080] A preparation (hGH preparation) containing, as an active ingredient, hGH with a molecular weight of approximately 22 KD produced as a recombinant protein using Escherichia coli into which the hGH gene has been introduced is widely used clinically as a therapeutic agent for growth hormone deficiency dwarfism, short stature in Turner syndrome, SGA short stature, short stature in Noonan syndrome, short stature due to chronic renal insufficiency, short stature in Prader-Willi syndrome, and short stature in achondroplasia. The hGH preparation is particularly effective in these diseases when there is no epiphyseal closure. The hGH preparation is administered subcutaneously or intramuscularly, and the hGH component circulates in the blood, exerting the effect of promoting the growth of patients through its growth-promoting activity. In addition, the hGH preparation is also widely used clinically as a therapeutic agent for adult growth hormone deficiency. In patients with adult growth hormone deficiency, abnormal lipid metabolism is observed, but administration of the hGH preparation normalizes the lipid metabolism, etc. of the patients and improves the QOL of the patients. Growth hormone is also clinically applied as a therapeutic agent for wasting due to AIDS. Examples of hGH preparations for growth hormone deficiency dwarfism, adult growth hormone deficiency, etc. include, for example, Glouject (registered trademark).

[0081] When producing a fusion protein of an HSA variant and hGH, as a specific method for binding a polypeptide containing the amino acid sequence of the HSA variant and a polypeptide containing the amino acid sequence of hGH, for example, a DNA fragment in which a gene encoding the other polypeptide is ligated in-frame downstream of the gene encoding one polypeptide is incorporated into an expression vector, and the host cell transformed with this expression vector is cultured to express it as a recombinant protein. This method is common and can be used in the present invention.

[0082] When producing a fusion protein of an HSA variant and hGH by the method of expressing it in transformed cells as a recombinant protein, the polypeptide containing the amino acid sequence of hGH is directly or indirectly bound either to the N-terminal side or the C-terminal side of the polypeptide containing the amino acid sequence of the HSA variant, either directly or via a linker.

[0083] When binding a polypeptide containing the amino acid sequence of hGH to the N-terminal side of a polypeptide containing the amino acid sequence of an HSA variant, an expression vector incorporating a DNA fragment in which a gene encoding a polypeptide containing the amino acid sequence of the HSA variant is ligated in-frame downstream of the gene encoding the polypeptide containing the amino acid sequence of hGH is used. When the two polypeptides are indirectly bound via a peptide linker, a DNA sequence encoding the linker is arranged in-frame between the genes encoding the two polypeptides.

[0084] When binding a polypeptide containing the amino acid sequence of hGH to the C-terminal side of a polypeptide containing the amino acid sequence of an HSA variant, an expression vector incorporating a DNA fragment in which a gene encoding a polypeptide containing the amino acid sequence of the HSA variant is ligated in-frame upstream of the gene encoding the polypeptide containing the amino acid sequence of hGH is used. When the two polypeptides are indirectly bound via a peptide linker, a DNA sequence encoding the linker is arranged in-frame between the genes encoding the two polypeptides.

[0085] In the present invention, as a preferred example of a fusion protein of an HSA variant and hGH (a kind of HSA-hGH fusion protein), the C-terminus of 22K human growth hormone having the amino acid sequence shown in SEQ ID NO: 9 is peptide-bonded to the N-terminus of HSA (A320T) having the amino acid sequence shown in SEQ ID NO: 3 without a linker, and an HSA-hGH fusion protein having the amino acid sequence shown in SEQ ID NO: 11 can be mentioned. In the present invention, the one in which HSA (A320T) and 22K hGH are bonded in this order is referred to as "22K human growth hormone-mHSA" or "22K hGH-mHSA". Similarly, the one in which the N-terminus of 22K human growth hormone is peptide-bonded to the C-terminus of HSA (A320T) without a linker is referred to as "mHSA-22K human growth hormone" or "mHSA-22K hGH".

[0086] In addition, the C-terminus of 20K human growth hormone having the amino acid sequence shown in SEQ ID NO: 10 is peptide-bonded to the N-terminus of human serum albumin (A320T) having the amino acid sequence shown in SEQ ID NO: 3 without a linker, and an HSA-hGH fusion protein having the amino acid sequence shown in SEQ ID NO: 12 is referred to as "20K human growth hormone-mHSA" or "20K hGH-mHSA". Similarly, the one in which the N-terminus of 20K human growth hormone is peptide-bonded to the C-terminus of human serum albumin (A320T) without a linker is referred to as "mHSA-20K human growth hormone" or "mHSA-22K hGH".

[0087] In the present invention, the HSA-hGH fusion protein is characterized in that its blood half-life (t 1 / 2 β) is extremely stable in the blood, generally being 5 hours or more when administered subcutaneously to cynomolgus monkeys. Although it varies depending on the dosage, for example, when administered subcutaneously once to male cynomolgus monkeys at a dosage of 4 mg / kg, the blood half-lives (t 1 / 2 β) of mHSA-22K hGH and 22K hGH-mHSA are 20 to 35 hours.

[0088] The HSA-hGH fusion protein in the present invention can be used as a medicine. The HSA-hGH fusion protein can cooperate the functions of human growth hormone and HSA in vivo.

[0089] The HSA-hGH fusion protein in the present invention is extremely stable in blood. Therefore, according to the present invention, human growth hormone can be stabilized in blood and remain in blood while maintaining its activity for a long time. Therefore, when the fusion protein is used as a medicine, the administration frequency or / and the dosage can be reduced compared with human growth hormone. For example, human growth hormone needs to be administered daily, but for the fusion protein, the administration frequency can be, for example, every 3 to 30 days. Also, the total dosage of the medicine during the treatment period can be reduced to, for example, 1 / 3 or less (for example, 1 / 3 to 1 / 10) in molar ratio.

[0090] The HSA-hGH fusion protein in the present invention can be used as a medicine for treating growth hormone deficiency short stature, short stature in Turner syndrome, short stature due to chronic renal failure, short stature in Prader-Willi syndrome, short stature in achondroplasia, SGA short stature, or short stature in Noonan syndrome. hGH preparations are particularly effective in these diseases when there is no epiphyseal closure. In addition, the HSA-hGH fusion protein in the present invention can be used as a medicine for treating adult growth hormone deficiency, wasting due to AIDS, and wasting due to anorexia nervosa, but not limited thereto, and can be used as a therapeutic agent for diseases whose symptoms can be improved by acting on the growth promoting activities such as cartilage formation promotion and protein assimilation promotion, and physiological activities such as improvement of body composition and lipid metabolism, which are possessed by growth hormone, over a long period of time.

[0091] When mHSA-22KhGH is administered to humans for therapeutic purposes, there is no particular limitation as long as the drug efficacy of hGH is shown. The usage and dosage of mHSA-22KhGH are exemplified below, but the usage and dosage are not limited thereto.

[0092] When administering mHSA-22KhGH to patients with growth hormone deficiency short stature without epiphyseal closure, the preferred dose per administration is 0.01 - 0.7 mg / Kg body weight. When administering mHSA-22KhGH to patients with short stature in Turner syndrome without epiphyseal closure, the preferred dose per administration is 0.015 - 1.4 mg / Kg body weight. When administering mHSA-22KhGH to patients with short stature due to chronic renal insufficiency without epiphyseal closure, the preferred dose per administration is 0.01 - 1.4 mg / Kg body weight. When administering mHSA-22KhGH to patients with short stature in Prader-Willi syndrome without epiphyseal closure, the preferred dose per administration is 0.012 - 0.98 mg / Kg body weight. When administering mHSA-22KhGH to patients with short stature in achondroplasia without epiphyseal closure, the preferred dose per administration is 0.015 - 1.4 mg / Kg body weight. When administering mHSA-22KhGH to patients with SGA short stature without epiphyseal closure, the preferred dose per administration is 0.012 - 1.9 mg / Kg body weight. When administering mHSA-22KhGH to patients with adult growth hormone deficiency, the preferred dose per administration is 0.001 - 0.34 mg / Kg body weight. When administering mHSA-22KhGH to patients with short stature in Noonan syndrome without epiphyseal closure, the preferred dose per administration is 0.013 - 1.8 mg / Kg body weight. When administering mHSA-22KhGH to patients wasted by AIDS, the preferred dose per administration is 0.005 - 0.4 mg / Kg body weight. However, the dose should be appropriately adjusted according to the patient's test findings and the like. Also, in these diseases, the preferred administration interval of mHSA-22KhGH is once every 7 - 30 days, and should be appropriately changed to once every 7 - 14 days, once every 10 - 20 days, or once every 14 - 21 days according to the patient's test findings and the like. Also, the administration method is preferably subcutaneous injection, intramuscular injection or intravenous injection, and more preferably subcutaneous injection or intramuscular injection.

[0093] The medicament containing the HSA-hGH fusion protein of the present invention as an active ingredient can be administered intravenously, intramuscularly, intraperitoneally, subcutaneously or intracerebroventricularly as an injection. These injections can be supplied as lyophilized preparations or aqueous solutions. When it is an aqueous solution, it may be in a form filled in a vial, or can also be supplied as a prefilled type preparation prefilled in a syringe. In the case of a lyophilized preparation, it is dissolved and restored in an aqueous medium before use.

Example

[0094] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not intended to be limited to the examples.

[0095] 〔Example 1〕Construction of vector for expressing 22KhGH-HSA A protein having the amino acid sequence shown in SEQ ID NO: 15, which is obtained by binding the C-terminal of 22KhGH and the N-terminal of wild-type HSA (SEQ ID NO: 1), was designated as 22KhGH-HSA. In the amino acid sequence shown in SEQ ID NO: 15, the amino acid residues at positions 1 to 191 correspond to the amino acid sequence of 22KhGH, and the amino acid residues at positions 192 to 776 correspond to the amino acid sequence of HSA. DNA having the base sequence shown in SEQ ID NO: 16 containing the gene encoding 22KhGH-HSA (22KhGH-HSA gene) was chemically synthesized. In this sequence, bases 11 to 88 encode the leader peptide of hGH, bases 89 to 661 encode 22KhGH, and bases 662 to 2416 encode HSA, respectively. This DNA was digested with restriction enzymes (MluI and NotI) and incorporated between the MluI site and the NotI site of pE-mIRES-GS-puro to construct pE-mIRES-GS-puro(22KhGH-HSA), a vector for expressing 22KhGH-HSA. Note that the method for preparing pE-mIRES-GS-puro is described in International Patent Publication WO2012 / 063799 etc. and is well-known.

[0096] 〔Example 2〕Construction of vector for expressing 22KhGH-mHSA It is a product of linking the C-terminus of 22KhGH (SEQ ID NO: 9) and the N-terminus of HSA (A320T) (SEQ ID NO: 3). The protein having the amino acid sequence shown in SEQ ID NO: 11 was designated as 22KhGH-mHSA. Using pE-mIRES-GS-puro(22KhGH-HSA) prepared in Example 1 as a template, a DNA fragment containing the gene encoding 22KhGH-mHSA was amplified by PCR using primer YA082 (SEQ ID NO: 13) and primer YA083 (SEQ ID NO: 14). This DNA fragment was self-annealed to construct pE-mIRES-GS-puro(22KhGH-mHSA), a vector for expressing 22KhGH-mHSA.

[0097] [Example 3] Preparation of 22KhGH-mHSA-expressing cell line Cells for expressing 22KhGH-mHSA were prepared as follows. Using the Gene Pulser Xcell electroporation system (Bio Rad), pE-mIRES-GS-puro(22KhGH-mHSA), a vector for expressing 22KhGH-mHSA prepared in Example 2, was introduced into CHO-K1 cells, which are cells derived from Chinese hamster ovaries. The cells into which the expression vector was introduced were CD OptiCHO containing 16 μM thymidine, 100 μM hypoxanthine, 10 mg / L insulin, L-methionine sulfoximine (SIGMA) and puromycin (SIGMA). TMSelection culture was performed using a medium (Thermo Fisher Scientific). During the selection culture, the concentrations of L-methionine sulfoximine and puromycin were gradually increased, and finally, the concentration of L-methionine sulfoximine was set to 300 μM and the concentration of puromycin was set to 10 μg / mL to selectively grow cells showing higher drug resistance. Next, by the limiting dilution method, the cells selected by the selection culture were seeded on a 96-well plate so that 1 or fewer cells were seeded per well, and cultured for about 10 days until colonies derived from single cells were formed to clone the cells. After further culturing and growing the cloned cells, CD OptiCHO containing 16 μM thymidine, 100 μM hypoxanthine, 300 μM L-methionine sulfoximine, and 10% (v / v) DMSO TM was suspended in the medium and then dispensed into cryotubes and stored in liquid nitrogen. These cells were designated as the 22KhGH-mHSA-expressing cell line.

[0098] 〔Example 4〕Preculture of 22KhGH-mHSA-expressing cells The 22KhGH-mHSA-expressing cell line prepared in Example 3 was thawed in a 37°C water bath, suspended in EX-CELL Advanced medium (serum-free medium for preculture, SIGMA) containing 16 μM thymidine, 100 μM hypoxanthine, and 300 μM L-methionine sulfoximine, and centrifuged to precipitate the cells, and the supernatant was removed. The precipitated cells were suspended in the preculture medium at a density of 2×10 5 cells / mL or more and cultured at 37°C in the presence of 5% CO2 for 2 to 4 days. This culture was repeated while expanding the culture scale until the cell number grew to at least 1.0×10 11 cells.

[0099] 〔Example 5〕Production culture of 22KhGH-mHSA-expressing cells The cells grown in the preculture were adjusted to a cell concentration of 4×10 5It was suspended in EX-CELL Advanced medium (production culture medium, SIGMA), a serum-free medium containing 16 μM thymidine and 100 μM hypoxanthine at a density of cells / mL and having a volume of 200 L. This suspension was cultured for 10 days at 37°C while maintaining the dissolved oxygen content at 40% and the pH at 6.9 with stirring at a speed of 100 rpm using an impeller in a single-use culture tank of an Xcellerex 200L culture system (XDR200).

[0100] [Example 6] Purification of 22KhGH-mHSA (Purification step 1: Harvesting step / Concentration step 1) After completion of the production culture, the culture broth was filtered using a Millistak+(registered trademark) HC pod filter grade D0HC (1.1 m 2 ×2, Merck), then filtered using a Millistak+ HC pod filter grade X0HC (1.1 m 2 ×1, Merck) to remove cells, and further filtered with an Opticap SHC XL3 (pore size: 0.5 / 0.2 μm, Merck) to obtain a culture supernatant. The obtained culture supernatant was washed in advance with pure water and then equilibrated with PBS, and concentrated using an ultrafiltration membrane device with a molecular weight cut-off of 30 kDa (Pellicon3 cassette equipped with an Ultracel PLCTK membrane, 1.14 m 2 , Merck). The solution concentrated by the ultrafiltration membrane device was recovered. Further, the inside of the device was washed with PBS, and the washing solution was combined with the previously recovered concentrated solution and adjusted to a weight of about 15 kg to obtain a concentrated culture broth. Next, the concentrated culture broth was filtered using a hydrophilic filter (Opticap SHC XL600, Merck) with a pore size having a maximum of 0.5 μm and a minimum of 0.2 μm. After filtration, it was confirmed that the pH and conductivity of the concentrated culture broth were 7.0±0.3 and 1.2±0.4 S / m, respectively.

[0101] (Purification 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) filled with Capture select Human Growth Hormone Affinity Matrix (Thermo Fisher Scientific) was washed with 1 column volume of 50 mM glycine hydrochloride buffer (pH 3.0) and then 1 column volume of 0.01 M aqueous sodium hydroxide solution, and then equilibrated with 4 column volumes of 20 mM Tris-HCl buffer (pH 7.0). Next, 1 / 4 of the concentrated culture solution obtained in purification step 1 was loaded onto the column to adsorb 22KhGH-mHSA to the column. Then, the column was washed with 5 column volumes of 20 mM Tris-HCl buffer (pH 7.0) containing 200 mM arginine and 0.05% (w / v) polysorbate 80, and further washed with 5 column volumes of 20 mM Tris-HCl buffer (pH 7.0). Next, 5 column volumes of 50 mM glycine hydrochloride buffer (pH 3.0) were supplied to the column to elute 22KhGH-mHSA. The eluate was collected into a container pre-filled with 1 / 5 volume of the eluate amount of 250 mM MES buffer (pH 7.0) and immediately neutralized so that the pH became 6.7 ± 0.3. Purification step 2 was carried out under cold temperature of 4 - 8 °C, and the linear flow rate of the solution supplied to the column throughout the whole of purification step 2 was set to 200 cm / h. Also, the upper limit of 22KhGH-mHSA loaded per 1 L of the affinity column carrier was set to 7 g.

[0102] (Purification step 3: Virus inactivation step) The eluate obtained in purification step 2 was heated to 25 °C, and to this eluate, an aqueous solution of 6.0% (v / v) tri(n-butyl) phosphate containing 20.0% (w / v) polysorbate 80 at 1 / 19 volume of the liquid amount was added, and the mixture was stirred at 25 °C for 3 hours.

[0103] (Purification step 4: Second chromatography step (hydroxyapatite column chromatography step)) The fraction subjected to virus inactivation treatment in purification step 3 was cooled to 8°C, and appropriate amounts of 1 M Tris-HCl buffer (pH 8.8) and 20 mM MES buffer (pH 7.0) containing 4 M NaCl were added to adjust the pH and conductivity to 7.0 ± 0.1 and 0.6 ± 0.1 S / m, respectively, and then filtered using a hydrophilic filter with a pore size of 0.2 μm (Merck). Subsequently, the following chromatography was performed under refrigeration (linear flow rate: 200 cm / hour).

[0104] A QS column (column volume: 8.8 - 10.8 L, bed height: 20.0 ± 2.0 cm, Merck) packed with CHT Type II, a hydroxyapatite carrier, 40 μm (Bio-Rad Laboratories) was washed with 200 mM phosphate buffer (pH 7.0), then 1 M aqueous NaOH solution, and further 200 mM phosphate buffer (pH 7.0), and then equilibrated with 4 volumes of 20 mM MES buffer (pH 7.0) containing 50 mM NaCl and 1 mM sodium dihydrogen phosphate. Subsequently, the above filtrate was loaded onto the column to adsorb 22KhGH-mHSA onto the column. Then, the column was washed with 3 volumes of 20 mM MES buffer (pH 7.0) containing 50 mM NaCl and 1 mM sodium dihydrogen phosphate. Then, 20 mM MES buffer (pH 7.0) containing 50 mM NaCl and 30 mM sodium dihydrogen phosphate was supplied to the column to elute 22KhGH-mHSA. Purification step 4 was carried out under cold temperature of 4 - 8°C, and throughout purification step 4, the linear flow rate of the solution supplied to the column was set to 200 cm / hour. Also, the upper limit of 22KhGH-mHSA loaded per liter of hydroxyapatite carrier was set to 13 g.

[0105] (Purification step 5: The third chromatography step (multimodal weak cation exchange column chromatography step)) To the eluate obtained in purification step 4, 20 mM MES buffer (pH 5.7) containing 50 mM NaCl in the same volume as this was added, and then dilute hydrochloric acid was added to adjust the pH and conductivity to 5.7 ± 0.1 and 0.7 ± 0.1 S / m, respectively. Thereafter, it was filtered using a hydrophilic filter (Merck) with a pore size of 0.5 / 0.2 μm.

[0106] The prepacked column RTP Capto MMC 10 L (column volume: 8.8 - 10.8 L, bed height: 20.0 ± 2.0 cm, Merck) was washed with 1 M aqueous NaOH solution and then 50 mM phosphate buffer (pH 7.0) containing 1 M NaCl, and then equilibrated with 50 mM MES buffer (pH 5.7) containing 4-fold volume of 100 mM NaCl. Then, the above filtrate was loaded onto the column to adsorb 22KhGH-mHSA onto the column. Then, the column was washed with 50 mM MES buffer (pH 5.7) containing 5-fold volume of 100 mM NaCl. Then, 50 mM MES buffer (pH 5.7) containing 550 mM NaCl was supplied to the column to elute 22KhGH-mHSA. Purification step 5 was carried out under cold temperature of 4 - 8°C, and the linear flow rate of the solution supplied to the column throughout the entire purification step 5 was set to 200 cm / h. Also, the upper limit of 22KhGH-mHSA loaded per 1 L of the multimodal weak cation exchange carrier was set to 11.5 g.

[0107] (Purification step 6: Concentration step 2) An ultrafiltration membrane with a molecular weight cut-off of 30 kDa (Pellicon3 cassette, Ultracel PLCTK membrane 1.14m 2 , Merck) was washed thoroughly by passing pure water through it, and then equilibrated with 10 mM phosphate buffer (pH 7.2) containing 75 mg / mL sucrose. The eluate obtained in purification step 5 was concentrated using this ultrafiltration membrane. 10 mM phosphate buffer (pH 7.2) containing 75 mg / mL sucrose was added to the concentrated solution to adjust the absorbance (280 nm) to 23 ± 3. This concentration step was carried out at room temperature.

[0108] (Purification Step 7: Fourth Chromatography Step (Size Exclusion Column Chromatography Step)) The concentrated solution obtained in Purification Step 6 was filtered using a hydrophilic filter (Merck) with a pore size of 0.5 / 0.2 μm. Fractogel, a resin for size exclusion chromatography TM A QS column (column volume: 25.4 - 31.1 L, bed height: 40.0 ± 4.0 cm, Merck) packed with BioSEC resin (Merck) was washed with an aqueous 0.5 M NaOH solution and then equilibrated with 10 mM phosphate buffer (pH 7.2) containing 75 mg / mL sucrose. Subsequently, the above filtrate was loaded onto the column, and then 10 mM phosphate buffer (pH 7.2) containing 75 mg / mL sucrose was supplied. At this time, an absorptiometer for continuously measuring the absorbance of the eluate was placed in the flow path of the eluate from the size exclusion column to monitor the absorbance at 280 nm, and the fraction showing an absorption peak at 280 nm was collected as the fraction containing 22KhGH-mHSA, which was used as the purified 22KhGH-mHSA product. Purification Step 7 was carried out at room temperature, and throughout Purification Step 7, the linear flow rate of the solution supplied to the column was set to 30 cm / h or less. Also, the volume of the filtrate containing 22KhGH-mHSA loaded onto the multimodal weak cation exchange carrier was set to 8% or less of the volume of the carrier.

[0109] (Purification Step 8: Concentration Step 3) A hollow fiber membrane (ReadyToProcess Hollow Fiber Cartridge, molecular weight cut-off 30 kDa, GE Healthcare) was equilibrated with 10 mM phosphate buffer (pH 7.2) containing 75 mg / mL sucrose. Using this hollow fiber membrane, the purified 22KhGH-mHSA product obtained in Purification Step 7 was concentrated to adjust the concentration of 22KhGH-mHSA to 85 mg / mL. To the resulting concentrated solution, 10 mM phosphate buffer (pH 7.2) containing 90 mg / mL poloxamer 188 and 75 mg / mL sucrose in a volume of 1 / 29 of the volume of the solution was added and mixed, and then filtered using a hydrophilic filter (Merck) with a pore size of 0.5 / 0.2 μm. This concentration step was carried out at room temperature.

[0110] (Purification Step 9: Virus Removal Step) A 10 mM phosphate buffer (pH 7.2) containing 3 mg / mL poloxamer 188 and 75 mg / mL sucrose was prepared. Using this buffer, a virus removal membrane (Planova 20N, membrane area: 0.12 m 2 , material: regenerated cellulose, Asahi Kasei Medical Co., Ltd.) was equilibrated. The filtrate obtained in Purification Step 8 was filtered through this virus removal membrane at a pressure of 98 kPa or less. This virus removal step was carried out at room temperature.

[0111] (Purification Step 10: API Formation Step) At room temperature, the filtrate obtained in Purification Step 9 was filtered through a hydrophilic filter with a pore size of 0.2 μm (Merck) to obtain 22KhGH-mHSA as the API.

[0112] [Example 7] Quantification of 22KhGH-mHSA in Each Purification Step The amount of 22KhGH-mHSA after each of the above purification steps was quantified using the method described in Example 8 below. As shown in Table 1, in the above purification process, 111.08 g of 22KhGH-mHSA was subjected to the affinity column chromatography step, which is the first chromatography step, and finally 65.87 g of purified 22KhGH-mHSA was obtained. That is, the recovery rate of 22KhGH-mHSA in the above purification method was 59%, indicating that this purification method is extremely efficient as a purification method for 22KhGH-mHSA. In Table 1, "recovery rate / step" means the ratio of the amount of 22KhGH-mHSA recovered to the amount of 22KhGH-mHSA loaded in each chromatography step (step), and "recovery rate / total" means the ratio of the amount of 22KhGH-mHSA recovered in each step to the initial amount of 22KhGH-mHSA subjected to the chromatography step.

[0113]

Table 1

[0114] 〔Example 8〕Quantification method of 22KhGH-mHSA by Bradford method The purified product of 22KhGH-mHSA produced by the present invention and quantified by amino acid analysis was diluted with water so that the theoretical protein concentration was 1.0 mg / mL to obtain Standard Solution 1. Note that the volume of the prepared solution was made 800 μL or more. Next, 200 μL of Standard Solution 1 and 50 μL of pure water were mixed to obtain Standard Solution 2 so that the theoretical protein concentration was 0.8 mg / mL. In this way, in order, 120 μL, 150 μL, and 400 μL of water were mixed with 180 μL, 100 μL, and 100 μL of Standard Solution 1 so that the theoretical protein concentrations were 0.6 mg / mL, 0.4 mg / mL, and 0.2 mg / mL, respectively, to obtain Standard Solution 3, Standard Solution 4, and Standard Solution 5, respectively. The test substance was taken in an amount of 50 μL or more and diluted with water so that the protein concentration was in the range of 0.4 to 0.8 mg / mL to obtain a sample solution. The standard solution and the sample solution were prepared immediately before use.

[0115] Coomassie reagent (Pierce TM 5 mL each of Pierce Coomasie Assay Reagent (included in Coomassie (Bradford) Protein Assay Kit, Thermo Fisher SCIENTIFIC) was collected in a 15 mL centrifuge tube. 100 μL each of water (for blank), Standard Solutions 1 to 5, and the sample solution was added to the Coomassie reagent collected in the centrifuge tube, and immediately after the addition of each solution, it was gently inverted and mixed. It was allowed to stand at room temperature for 10 minutes to react. After the reaction, the absorbance at 595 nm was immediately measured with a spectrophotometer (UV-2600, Shimadzu Corporation). The reaction operation with the above Coomassie reagent was performed within 2 hours after the preparation of each solution. A calibration curve was created from the measured values of the standard solutions, and the concentration of 22KhGH-mHSA contained in each test substance was calculated by interpolating the measured values of each test substance into this calibration curve.

[0116] 〔Example 9〕Evaluation of the purity of the 22KhGH-mHSA purified product by SE-HPLC analysis SE-HPLC analysis was performed using an LC-20A system (system controller, online degassing unit, liquid delivery unit, autosampler, column oven, and ultraviolet-visible detector) manufactured by Shimadzu Corporation. TSKgel G3000SW XL A column XL (inner diameter 7.8 mm, length 30 cm, manufactured by TOSOH Corporation) was set in the LC-20A system, and after equilibration of the column by flowing a phosphate buffer (200 mM sodium phosphate containing 200 mM NaCl) at a flow rate of 0.7 mL / min, 10 μL of a solution containing 22KhGH-mHSA at a concentration of 2.0 mg / mL was loaded onto this column. Similarly, a sample diluent (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 created by monitoring the absorbance at 215 nm. The purified product of 22KhGH-mHSA obtained by the above production method showed only a single peak corresponding generally to the monomer of 22KhGH-mHSA in the SE-HPLC analysis (Figure 1).

[0117] The results of the evaluation of the purity of the above 22KhGH-mHSA indicate that 22KhGH-mHSA purified by the above purification process has a high purity that can be used as it is as a therapeutic agent for growth hormone deficiency dwarfism and the like.

Industrial Applicability

[0118] According to the present invention, for example, a prodrug of a fusion protein of serum albumin and growth hormone that can be used as a therapeutic agent for growth hormone deficiency dwarfism can be provided.

Sequence Listing Free-Text

[0119] 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 mutant (A320T) SEQ ID NO: 4: Example 1 of the amino acid sequence of the linker Accession No. 5: Example 2 of Amino Acid Sequence of Linker Accession No. 6: Example 3 of Amino Acid Sequence of Linker Accession No. 7: Partial Base Sequence of Internal Ribosome Binding Site Derived from Wild-Type Mouse Encephalomyocarditis Virus Accession No. 8: Partial Base Sequence of Internal Ribosome Binding Site Derived from Mutant Mouse Encephalomyocarditis Virus, Synthetic Accession No. 9: Amino Acid Sequence of 22K Human Growth Hormone Accession No. 10: Amino Acid Sequence of 20K Human Growth Hormone Accession No. 11: Amino Acid Sequence of 22KhGH-mHSA Accession No. 12: Amino Acid Sequence of 20KhGH-mHSA Accession No. 13: Primer YA082, Synthetic Sequence Accession No. 14: Primer YA083, Synthetic Sequence Accession No. 15: Amino Acid Sequence of 22KhGH-HSA Accession No. 16: Base Sequence Containing 22KhGH-HSA Gene, Synthetic Sequence

Claims

Claim 1 A method for producing a fusion protein of human serum albumin and human growth hormone consisting of the amino acid sequence represented by SEQ ID NO: 11, comprising: (a) culturing mammalian cells producing the fusion protein in a serum-free medium to secrete the fusion protein into the culture broth; (b) recovering the culture supernatant by removing the mammalian cells from the culture broth obtained in step (a); (c) purifying the fusion protein by using, in this order, column chromatography using as a stationary phase a material obtained by binding an antibody against human growth hormone in the presence of 0.005% (w / v) to 0.1% (w / v) nonionic surfactant, column chromatography using as a stationary phase a material having an affinity for phosphate groups, cation exchange column chromatography, and size exclusion column chromatography; The method for production, comprising: The material having an affinity for phosphate groups is hydroxyapatite; The cation exchanger used in the cation exchange column chromatography is a weak cation exchanger, and The weak cation exchanger retains selectivity based on both hydrophobic interaction and hydrophobic bond formation. Claim 2 The method for production according to claim 1, wherein the eluate of the fusion protein obtained by column chromatography using as a stationary phase a material bound with an antibody against human growth hormone and the eluate of the fusion protein obtained by size exclusion column chromatography are inactivated for viruses. Claim 3 The method for production according to claim 2, wherein one time of the step for inactivating viruses is performed by adding a nonionic surfactant to the solution containing the fusion protein to inactivate the viruses, and the other time is performed by passing the solution containing the fusion protein through a filtration membrane.

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