MX2007015893A - Human granulocyte-colony stimulating factor isoforms - Google Patents

Abstract

Disclosed is a novel physiologically active protein, which is a human granulocyte colony stimulating factor isoform, constructed in order to increase the in vivo lifetime of human granulocyte colony sedtimulating factor. The human granulocyte colony stimulating factor isoform comprises a polypeptide and polyethylene glycol (PEG) bound thereto as a non-protein polymer. A specific site of the polypeptide is selected so that polyethylene can be bound to the site while not adversely affecting the activity of the protein. The amino acid of the site is modified with cysteine and polyethylene glycol is bound to the modified site. A pharmaceutical composition comprising the isoforms, genes encoding the isoforms, and a primer for modifying the amino acid sequence are also disclosed.

Description

iSOFQRMAS OF THE HUMAN GRANULOCBT COLONY STIMULATION FACTOR FIELD OF THE INVENTION The present invention relates to an isoform of novel human granulocyte colony stimulation factor, by which the in vivo lifetime (rhG-CSF) is prolonged. More particularly, the present invention relates to an isoform of the novel human granulocyte colony stimulation factor, obtained from the covalent attachment of polyethylene glycol as a non-protein region to an isoform of the stimulation factor of the granulocyte colony. of novel human that has cysteine added to the N-terminal or the C-terminal thereof.

ANTECEDENTS OF THE INVENTION The stimulation factor of the human granulocyte colony has the main biological function of specific stimulation leukocytes, known as neutrophilic granulocytes or neutrophils, APRA to carry out the growth and development thereof in vivo (Welte et al., PNAS, 82, 1526-1530, 1985: Souza et al Science, 232, 61-65, 1986). These neutrophilic granulocytes function to protect a biological species against infection with microorganisms, when they are discharged into the bloodstream.

The amino acid sequence of the stimulation factor of the human granulocyte colony has been reported by Niagata et al (Nature, 319, 415-418, 1986). The stimulation factor of the human granulocyte colony is a protein capable of forming a complex with a receptor thereof in a ratio of 2: 2 via receptor dimerization (Horan et al., Biochemistry, 35, 4886-96 , nineteen ninety six). Aritomi et al. have shown that the X-ray structure of the BN-BC domain complex of the stimulation factor of the human granulocyte colony with a receptor thereof (Nature, 401, 713-717, 1999). It is reported by Aritomi et al. that the amino acids of the human granulocyte colony stimulation factor, which exist in the contact region or region adjacent thereto when the receptor binds to the stimulation factor of the human granulocyte colony, include G4, P5, A6 , S7, S8, L9, P10, Q11, S12, L15, K16, E19, Q20, L108, D109, D112, T1 15, T116, Q119, E122, E123 and L124. Some stimuli of the stimulation factor of the human granulocyte colony, obtained by means of a protein design technique, have been described (USP5581476, USP5214132, USP5362853, USP4904584). Ridehall-Olsen et al., Have reported that Lys40, Val48, Leu49 and Phe144, present in the amino acid sequence of the stimulation factor of the human granulocyte colony, participate in the binding with a receptor of the stimulation factor of the colony. of human granulocytes. In addition, Rayton et al. have reported that Glu46, Leu49 and Phe144 are in contact with an immunoglobulin-like domain (Ig-like domain) of a human granulocyte colony stimulation factor receptor, while Lys40 and Val49 are remote from the domain. Thus, it is not true that Lys40 and Val49 are in contact with the receptor (J. Biol. Chem., 2001, 276, 36779-36787). In addition, Rayton et al., Have shown that Glu19 of the human granulocyte colony stimulation factor interacts with Arg288 from a receptor thereof, thus serving to transfer stimulation signal signals from the human granulocyte colony. (J. Biol. Chem., 1999, 274, 17445-17451). It has been suggested to introduce at least one additional sugar chain artificially into the stimulation factor protein of the human granulocyte colony by means of a genetic design method (USP5218092). Said introduction of the sugar chain is carried out by using an exchange method, deleting and adding amino acids in the amino acid sequence of a polypeptide. In addition, many studies including the binding of polyethylene to the stimulation factor of the human granulocyte colony have been reported (Satake-lshikawa et al., Cell Structure and Function, 17, 157-160, 1992).; USP5824778, USP5824784, USP96 / 11953, W095 / 21629, and WO94 / 20069). The removal in vivo of a polypeptide or a polymer thereof is carried out by means of removal (or elimination) and by means of receptor-mediated degradation of a protein in the kidney, spleen or liver. Said removal is carried out by means of the action of a protease specific to a substrate or not. in general, the in vivo removal of the protein depends on the size of the protein (such as a size that the glomerular filtration can be prevented), the loading of a protein molecule, attachment of a sugar chain, protein receptor in the cell surface, or similar. Particularly, the removal of the protein in the kidney depends on the physical properties of a protein or a polymer thereof, such as size (molecular diameter), symmetry, shape / stiffness or charge, or the like. The degradation mediated by the receptor of a protein is realized when the protein loses its function in the union with a receptor. At the initial moment, leukocytes are insufficient, and binding to a receptor on the surface of primordial hematopoietic stem cells results in differentiation and growth in leukocytes. However, once the number of leukocytes increases to reach a certain level, the stimulation factor of the human granulocyte colony is removed by the receptor present on the surface of leukocytes in order to prevent excessive differentiation and growth. caused by the stimulation factor of the human granulocyte colony. The ratio of the primordial hematopoietic stem cells to the stimulation factor receptors of the human granulocyte colony present on the surface of leukocytes is approximately 1: 5.

The removal of the protein caused by the receptor present on the surface of leukocytes is carried out by introducing a polypeptide bound to the receptor in the cells, and by means of the lysosomal degradation of protein in the presence of proteases present in endosomes. Protein degradation by binding of a receptor to stimulation factor of the human granulocyte colony is described in detail by Shaker and Roufenburger (Mol.Pharmacol., 2003, 63, 147-158) and Saker (Nature Biotech, 2002, 20, 908-913). To increase the half-life of a protein in the blood, it is necessary to reduce the removal of the protein in the kidney and degradation of the protein by means of the binding of a receptor. It is possible to reduce protein removal in the kidney and increase the half-life in vivo by binding a polymer capable of increasing the size of the apparent molecule to the protein. In addition, the adhesion of a polymer to a protein can effectively disrupt the proteases and thus prevent the functions of non-specific proteases. Among such polymers, polyethylene glycol (PEG) is one of the polymers widely used to prepare therapeutic protein products. The surface modification of a protein molecule caused by the binding of a protein drug to a synthetic polymer can increase the solubility of the drug to water or organic solvents. Consequently, it is possible to increase the biocompatibility of the drug, to reduce the immunoreactivity, to improve the stability in vivo, and to delay the elimination in the intestinal system, kidney, spleen or liver. The removal of small protein molecules is done by filtration in the intestinal or kidney system. Therefore, when a high molecular weight PEG is bound to said small protein molecules, it is possible to prevent such removal (Knauf, M. J. et al, J. Hiol, Chem. 263: 15064, 1988). Introduction of a high molecular weight polyethylene glycol (PEG) in a protein drug can increase the stability of the protein molecule in a solution. In addition, it is possible to prevent adsorption of a non-specific protein by protecting the characteristics of the intrinsic surface of the protein effectively. In this regard, attempts have been made to increase the in vivo half-life of a protein, to increase the solubility of a protein and reduce immuno-reactivity in vivo by binding PEG to a biologically active peptide or protein. USP4, 179,337 reports the result of these attempts for the first time. Although the binding of PEG to a protein reduces many side effects due to the advantages mentioned above, the PEG binding protein undesirably shows significantly reduced in vivo activity due to the active sites of the protein being blocked by PEG. PEG, widely used to date, binds to the surface of the protein by forming a covalent bond with at least one free lysine residue. Here, if a site is present, which is present on the surface of the protein and is directly related to the activity of the protein, binds to PEG, the activity of the protein decreases. In addition, since the binding between PEG and lysine residues occurs in a random fashion, several types of PEG-linked protein conjugates are present in the form of a mixture, so that the separation of a desired conjugate in a pure state becomes complicated and difficult. For example, EP 0401384 describes a material and method for the production of G-CSF added to polyethylene glycol. EP 0335423 describes a modified polypeptide having a stimulation factor activity of the human granulocyte colony. However, according to the prior art, the polyethylene glycol molecule can not bind to a specific residue predictable but bound to any reactive group present in the protein, such as a lysine residue., or the N-terminus in a specific way, resulting in the formation of an inhomogeneous product. In order to bind a specific protein region with PEG, USP5, 766,897, and WO00 / 42175 describe a method of binding to human growth hormone with PEG-maleimide so that PEG is prevented from reacting with the hormone active region. of human growth. It is required that a free cysteine residue not participating in the disulfide bond is present in the human growth hormone, to allow the use of the above type of PEG. However, all four cysteine residues present in human growth hormone participate in the disulfide bond. Therefore, according to the prior art, pegylation is carried out by using a derivative of human growth hormone, wherein a residue of cysteine is artificially inserted. In addition, USP6,555,660 and USP 6,753,165 and US patent No. 2005/0058621 and KR2002-0079778 open to the public describe the mutation of cysteine at various positions in the amino acid sequence of stimulation factor of the human granulocyte colony. Meanwhile, Bowen et al., Have shown that the molecular weight of polyethylene in stimulation factor of the granulocyte colony of polyethylene-modified human is related to the lifetime of a drug. In an in vitro test, the efficacy of a protein drug has an inverse relationship with the molecular weight of polyethylene bound to the protein. However, the in vivo activity increases as the molecular weight increases. It is thought that a polymer of a physiologically active protein shows a low affinity in the degradation mediated by the receptor, and therefore shows an increased half-life. Therefore, although said polymers of human granulocyte colony stimulation factor achieve the recovery of neutrophils in vivo, they show a lower activity than the stimulation factor of the unpolymerized human granulocyte colony in terms of activity in vitro. Recently, a stimulation factor of the modified human granulocyte colony including polyethylene glycol 20kDa bound to the N-terminus has been developed and marketed (Neulasta). The stimulation factor of the human granulocyte colony polymerized with polyethylene glycol has an increased half-life, and in this way can reduce the frequency of administration.

Particular examples of commercially available human granulocyte colony stimulation factor include filgrastim derived from E. coli (trade name: Gran and Neupogen), lenograstim (trade name: Neutroginand Granocite) produced in cells of animal origin, such as Chinese Hamster ovary (CHO), and nartograstimo derived from E-coli (trade name: Neu-up), where the mutation occurs in the N-terminal regions in the five amino acid sequences in order to increase the efficacy of the stimulation factor protein of the human granulocyte colony.

BRIEF DESCRIPTION OF THE INVENTION Technical problem However, the isoforms of the stimulation factor of the human granulocyte colony according to the prior art still have problems because said isoforms of the stimulation factor of the human granulocyte colony modified with amino acid sequence have a low activity or insufficient half-life in vivo, or inhomogeneous products are generated in the binding of PEG to stimulation factor of the human granulocyte colony.

Technical solution Therefore, the present invention has been made in view of the problems mentioned above. It is an object of the present invention to provide a human granulocyte colony stimulation factor that has excellent activity and a significantly improved half-life by binding polyethylene glycol to a stimulation factor of the modified human granulocyte colony with the sequence of amino acids, wherein the amino acid sequence is modified by substitution with or addition of cysteine at a specific site for the purpose of facilitating the binding to polyethylene glycol to the stimulation factor of the human granulocyte colony, and then the modified site is partially bound to polyethylene glycol. According to the prior art, the stimulation factor of the human granulocyte colony has generally been produced by modifying the sequence of ammonium acids in the polypeptide and performing the pegylation at the position of a suitable amino acid at a specific site to increase the activity in vivo or life time. However, the inventors of the present invention have found that a novel isoform of human granulocyte colony stimulation factor, which shows increased in vivo activity or life time compared to isoforms according to the prior art, they can be obtained by adding amino acids to both terminals of the stimulation factor polypeptide of the human granulocyte colony and by conducting pegylation at the same addition sites. The present invention is based on its finding. According to one aspect of the present invention, an isoform of the stimulation factor of the human granulocyte colony having at least one amino acid added before Thr of the N-terminus of the stimulation factor of the granulocyte colony is provided. of human represented by sequence No. 1 or after Pro of the C-terminal thereof, wherein at least one of the amino acids added to the stimulation factor of the human granulocyte colony and at least one cysteine is attached to polyethylene glycol. It is generally known to those skilled in the art that the N-terminus in sequence No. 1 may include Met. In this case, the cysteine can be added between the Met and the threonine of the N-termini. The above isoform is also included in the scope of the present invention. More particularly, 1-12 amino acids can be added to each terminal of the sequence. Preferably, a cysteine is added. More preferably, the cysteine is 175c, added to the C-terminus. According to another preferred embodiment of the present invention, polyethylene glycol is covalently linked to the cysteine added to human granulocyte colony stimulation factor. Preferably, polyethylene glycol has a molecular weight of 20 kDa ~ 40 kDa. More preferably, polyethylene glycol is branched. According to a preferred embodiment of the present invention, at least one amino acid of the stimulation factor of the human granulocyte colony is replaced with another amino acid before the polyethylene binds to the stimulation factor of the human granulocyte colony. According to another aspect of the present invention, there is provided a pharmaceutical composition comprising the stimulation factor of the human granulocyte colony mentioned above and a pharmaceutically acceptable carrier. Still according to another aspect of the present invention, there are provided genes encoding a stimulus factor protein from the aforementioned human granulocyte colony that has cysteine added to the N-terminus or C-terminus. According to still another aspect of the present invention, an oligodeoxynucleotide used as a primer is provided for modifying the amino acid sequence of a human granulocyte colony stimulation factor in the isoform of the granulocyte colony stimulation factor. human. Preferably, the primer is the oligodeoxynucleotide represented by any of sequence Nos. 2-7.

Suitable effects The isoform of the stimulation factor of the human granulocyte colony according to the present invention shows a significant increase in in vivo activity or life time, and therefore can reduce the frequency of administration.

REVE DESCRIPTION OF THE The foregoing and other objects, features and advantages of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings wherein: Figure 1 illustrates the genetic sequence and protein sequence of the colony stimulation factor of human granulocytes; Figure 2 illustrates the amino acid sequence of an isoform of the stimulation factor of the human granulocyte colony, a modified amino acid at the N-terminal site; Figure 3 illustrates an amino acid sequence of an isoform of human granulocyte colony stimulation factor, modified amino acid at the C-terminal site; Figure 4 illustrates the amino acid sequence of an isoform of human granulocyte colony stimulation factor, modified amino acid in the middle part of the polypeptide; Fig. 5 illustrates the structure of a plasmid vector expressing a stimulus of the stimulation factor of the human granulocyte colony; and Figure 6 is a graph showing the in vivo lifetime of the stimulation factor isoforms of the human granulocyte colony in the rats.

PREFERRED MODALITY OF THE SNVENCflQN Now with reference in detail to the preferred embodiments of the present invention. Figure 1 illustrates the genetic sequence and protein sequence of the stimulation factor of the human granulocyte colony. The amino acid sequence No. 1 is the same as the amino acid sequence shown in Figure 1 with the exception of Met at position -1. Figure 2 illustrates the amino acid sequence of an isoform of the human granulocyte colony stimulation factor, a modified amino acid at the N-terminal site, the modified amino acid at the N-terminal site. AA represents amino acids, and n is the number of amino acids. N is an integer ranging from 1 to 12. The amino acids include at least one cysteine. Figure 3 illustrates the amino acid sequence of an isoform of the stimulation factor of the human granulocyte colony, amino acid modified at the C-terminal site. AA represents amino acids, and n is the number of amino acids. N is an integer ranging from 1 to 12. The amino acids include at least one cysteine. Figure 4 illustrates the amino acid sequence of an isoform of the stimulation factor of the human granulocyte colony, amino acid modified in the part comprising a sugar chain. In the present, 133-threonine is replaced with cysteine and polyethylene glycol is covalently linked with the same cysteine. This is a known isoform according to the prior art (see Korean Patent No. 2002-0079778 open to the public). Figure 5 illustrates the structure of a plasmid vector expressing an isoform of. As a promoter, PR of a bacteriophage lamda is used. To perform E-coi expression, widely used in laboratories, the plasmid vector includes cl 857 genes capable of inducing said expression in the plasmid vector. Figure 6 is a graph showing the in vivo lifetime of isoforms of the stimulation factor of the human granulocyte colony in rats. The PBS carrier is used as a control, and test samples include: T133C20kDa (Br) (G-CSF isoform obtained by means of the substitution of 133-Thr with cysteine and covalently linked PEG branched with 20 kDa to the same cysteine of according to Korean patent open to the public No. 2002-0079778); Neulasta commercially available (Amgene Co.) having 20kDa PEG attached to the N.terminal; N + 20kDa (Br) (G-CSF isoform obtained by adding cysteine between threonine and methionine of the N-terminal (-1 C) of G-CSF and covalently linking PEG branched with 20 kDa to the same cysteine); 175 + 40kDa (Br) (G-CSF isoform obtained by adding cysteine after 174 Pro of the C-terminus of G-CSF and branched PEG binding covalently with 40 kDa to the same cysteine). Each isoform is diluted with PBS, and administered to rats via the tail vein in a dose of 100 μg / kg based on the body weight measured on the day of administration. The administration is carried out only once on the test day. The inventors of the present invention have studied the structure of the stimulation factor protein of the human granulocyte colony and the effect of the amino acids that form the stimulation factor of the human granulocyte colony on its activity. Reference is made to the structure of the stimulation factor of the human granulocyte colony, information about the effect of the amino acids that form the stimulation factor of the human granulocyte colony on its activity, and the prediction results. of secondary structure of GOR4 protein used to determine the effect of amino acid modification on the protein structure (GOR program in the secondary structure prediction, http: us.expasy.org). As used herein, the term "isoforms of the human granulocyte colony stimulation factor" includes analogs, variants, mutants, conjugates, or the like, which maintain the natural activity of the granulocyte colony stimulation factor. of human, although they undergo modification of an amino acid residue in another amino acid residue in the original amino acid sequence of the stimulation factor of the human granulocyte colony. Three letter or one mnemonic letter codes for amino acids used herein are defined as follows according to biological standards: Ala (A): alanine; Asx (B): asparagine or aspartic acid; Cys (C): cysteine; Asp (D): aspartic acid; Glu (E): glutamic acid; Phe (F): phenylalanine; Gly (G): glycine; His (H): Histidine; lie (I): isoleucine; Ls (K): lysine; Leu (L): leucine; Met (M): methionine; Asn (N): aspargin; Pro (P): proline; Gln (Q): glutamine; Arg (R): arginine; Ser (S); serine; Thr (T): threonine; Val (V): valine; Trp (w): triptopane; Tyr (Y): tyrosine; Glx (Z): glutamine or glutamic acid. As described here, "(single letter for an amino acid) (amino acid position) (single letter for another amino acid) "means that the amino acid former at the corresponding amino acid position is substituted with the letter" amino acid. "For example, T133C refers to threonine, amino acid 133 in the sequence of the amino acid. stimulation factor of the human granulocyte colony, is replaced with cysteine.Here, the primer used to induce the modification of an amino acid at the specific site is represented by the expression (single letter for an amino acid) (amino acid position) ( single letter for another amino acid) 1 or 2, where the suffix 1 represents the primer for a single-strand template that progresses in the 5 '? 3' direction in a double-stranded template, while the suffix 2 represents the primer for a single strand template that progresses in the 3 '? 5' direction The genes of the stimulation factor of the human granulocyte colony obtained by means of the proc In the previous edition, at least one codon can be modified in the position. As used herein, "modification" refers to a change in the amino acid sequence of the stimulation factor of the human granulocyte colony caused by substitution or addition of at least one codon in the gene encoding the stimulation factor of the human colony of human granulocytes. More particularly, said modification includes: addition of cysteine before threonine, the first amino acid in the amino acid sequence of the stimulation factor of the human granulocyte colony; addition of cysteine after proline from the C-terminus of the same amino acid sequence; the modification of the sequence in the middle part of the polypeptide sequence in addition to the modification in the terminal sites; or the similar. For example, T133C refers to the substitution of threonine, the amino acid 133 of the stimulation factor of the human granulocyte colony, with cysteine. According to one embodiment of the present invention, a synthetic oligonucleotide including a codon encoding modification of a target amino acid in the stimulation factor of the human granulocyte colony is constructed. In general, an oligonucleotide with a length corresponding to approximately 27-36 nucleotides is used. Although a shorter oligonucleotide can be used, the oligonucleotide preferably has 12-15 nucleotides complementary to the template on both sides of the nucleotide coding modification. Said oligonucleotides can be sufficiently hydrided with the template DNA.

A synthetic oligonucleotide used for modification of the amino acid according to the present invention is shown in the following Table 1. The oligonucleotide can be prepared by the method known to those skilled in the art. According to a preferred embodiment of the present invention, Isoform DNA from the stimulation factor of the human granulocyte colony, one of those amino acids is modified, constructed. In this case, PCR is performed by using the DNA of the stimulation factor of the human granulocyte colony (Table 1) as a template and a synthetic oligonucleotide encoding the modification as a primer. If the double chain template is separated in the PCR heating step, a complementary primer hybridizes with each single chain template. Does DNA polymerase allow the oligonucleotide complementary to the template to be linked continuously to that of -OH groups of the primer encoding modification along with the 5 'direction? 3'. As a result, the second strand includes the primer encoding the modification, so that the resulting genes encode the target modification. The second chain functions as a template DNA during the repeated replication stages, so that the genes encoding the modification can be amplified continuously. Hereinafter, the modification in the middle part of the polypeptide of the stimulation factor of the human granulocyte colony will be explained in more detail with reference to T133C taken as an example. T133C refers to threonine substitution, amino acid 133 of stimulation factor of human granulocyte colony, with cysteine. To obtain T133C, PCR is performed by using the DNA stimulation factor of the human granulocyte colony (Figure 1) as a template and by introducing each of the pairs of primers formed of N-term with T133C and T133C1 with C- term By doing this, the two DNA fragments, which have a modified codon corresponding to cysteine that replaces threonine, amino acid 133, can be obtained. Then, PCR is also performed by introducing the two DNA fragments and using pairs of primers of N-term and C-term. In this way, it is possible to obtain modified genes of the isoform of the stimulation factor of the human granulocyte colony T133C, where the threonine is replaced with cysteine at the position of the amino acid 133. According to another example of the present invention, The amino acid addition is performed at the N-terminus or the C-terminus. The construction of the stimulation factor of the human granulocyte colony having at least one modified amino acid can be performed at the N-terminus and C-terminus. Said modification includes the sequence of Met (AA) n-Thr at the N-terminus (see Figure 2) and the sequence of -Gln-Pro- (AA) n at the c-terminus (see Figure 3). Here, AA refers to any amino acid and particular examples thereof include an amino acid having a low molecular weight and showing non-specific function, such as glycine, serine or alanine. At least one AA includes cysteine. Suffix n refers to the number of amino acids and varies from 1 to 12. At least about 12 amino acids are required for the in vivo recognition of the stimulation factor of the modified human granulocyte colony, to which amino acids are added, as a protein heterogeneity , followed by the production of an antibody. This is because the maximum number of amino acids is limited to 12. Therefore, with the purpose to construct genes from the human granulocyte colony stimulation factor that have additional amino acids, the N-terminus has the amino acid sequence of Met. (AA) n-Thr- and includes the DNA base sequence of ATG- (NNN) n-ACT, although the C-terminus has the amino acid sequence of -Gln-Pro- (AA) nn- and includes the sequence DNA base of -CAG-CCG- (NNN) n-TAA. Here, NNN represents the base sequence of DNA encoding the corresponding amino acid. Then, the human granulocyte colony stimulation factor genes having at least one modified amino acid are constructed by using the primer that includes the ADSN base sequence obtained as described above and the stimulation factor genes from the colony of Human granulocytes as shown in Figure 1. For example, if a glycine and a cysteine are added to the N-terminus, the amino acid sequence at the N-terminus may include the terminal sequence of Met- (Gly-Cys) Thr-. The primer used here can be designed to have the sequence of GTAATAAATA-ATG- (GGT-TGT-) ACT-. Then, the human granulocyte colony stimulation factor genes having the Met- (Gly-Cys-) Thr- sequence can be constructed from the previous primer, by using the template of the stimulation factor genes of the human granulocyte colony as shown in Figure 1 and the C-term primer as shown in Table 1. The human granulocyte colony stimulation factor genes obtained as described above are further subjected to PCR at use N-term and C-term primers. According to another embodiment of the present invention, it is possible to add an amino acid to the N-terminal or C-terminal and to replace at least one amino acid placed in the middle part of the polypeptide of the stimulation factor of the human granulocyte colony. with another amino acid, before polyethylene joins this. For example, -1 C / T133C includes the addition of cysteine to the N-terminus and substitution of the threonine, amino acid 133 of the stimulation factor polypeptide of the human granulocyte colony with cysteine. To obtain -1C T133C, PCR is performed by using DNA made from the isoform of the human granulocyte colony stimulation factor as a template and by introducing primer pairs of N-term with T122C2 and T133C1 with C-term. By doing this, the two DNA fragments, which have a modified codon corresponding to cysteine that replaces threonine, amino acid 133, can be obtained. Then, PCR is carried out additionally by introducing the two DNA fragments and using pairs of N-term and C-term primers. In this way, it is possible to obtain modified isoform genes from the human granulocyte colony stimulation factor -1 C / T133C, where threonine is replaced with cysteine at amino acid position 133 and cysteine is added before threonine of the C-terminal. The construction of isoforms in a position other than 133 can be performed by using the corresponding DNA primer in the same manner as the isoform -1C / T133C. The amino acid modification in the middle part of the amino acid added isoform polypeptide including at least one cysteine at the N-terminus or the C-terminus can be performed at a site known to those skilled in the art. For example, methods of replacing at least one amino acid in the middle part of the polypeptide of the stimulation factor of the human granulocyte colony with another amino acid are described in Korean Patent Publication No. 0248111 (G-CSF linked to polyethylene glycol). N-terminal or its analogs, and its preparation), the Korean patent open to the public Nos. 2002-0007297 (conjugated G-CSF), 2002-0079778 (conjugated G-CSF) and 2004-0084884 (conjugated G-CSF), U.S. Patent No. 5214132 (polypeptide derivatives of human granulocyte colony stimulation factor) and 5416195 (polypeptide derivatives of human granulocyte colony stimulation factor), US patent open to the public Nos. 2003- 0158375 (G-CSF and conjugated polypeptides), 2004-0214287 and 2005-0058621. more particularly, according to USP5214132, USP5416195 and US2004-0214287, amino acids in position 1, 3, 4, 5 and 17 of the stimulation factor of the human granulocyte colony are replaced with Ala, Thr, Tyr, Arg and Ser , respectively, or the cysteine, amino acid at position 17, is replaced with alanine or serine.

The contents of prior patents and patent applications are incorporated herein for reference. Also, methods for producing isoforms of the human granulocyte colony stimulation factor, pegylation methods, or the like as described in the prior art are also incorporated herein for reference. The DNA sequence encoding the isoforms of the human granulocyte colony stimulation factor according to the present invention can be synthesized by conventional methods known to those skilled in the art, for example, by using a synthetic synthesizer. Automatic DNA (available from Blosearch, Applied Biosystem ™, etc.). According to another aspect of the present invention, there is provided a recombinant expression vector that includes the sequence of DNA encoding the isoform of the stimulation factor of the human granulocyte colony and a host cell transformed or transfected with the above expression vector. As used herein, the term "polypeptide" can be used interchangeably with "protein" or "physiologically active protein". As used herein, the term "isoform" refers to a physiologically active protein, which has the primary function of the physiologically active protein but is modified by genetic or other design means. Here, the term "modification" refers to substitution, addition and loss in an amino acid sequence, and includes substitution with cysteine or addition of cysteine to perform the binding with polyethylene glycol. In addition, said modification also includes a covalent bond formed between the cysteine and polyethylene glycol via a chemical reaction. As used herein, the term "in vivo life time" is defined as follows: a physiologically active protein loses its function when administered to humans or animals, because the total amount and activity of the physiologically active protein decreases due to protease-mediated removal and removal in the kidney. Therefore, the term "in vivo lifetime" refers to the retention time of a protein during which the protein can perform its function in vivo, and is represented by the in vivo half-life of the physiologically active protein. As noted herein, the term "vector" refers to a DNA molecule as a carrier capable of delivering foreign genes stably in a host cell. To be a useful vector, it is necessary for a vector that allows replication, to have a medium, through which it can be introduced into a host cell, and to include a means for self-detection. In addition, the term "recombinant expression vector" refers to a cyclic DNA molecule formed by a vector operably linked to foreign genes so that foreign genes can be expressed in a host cell. As a recombinant expression vector, a DNA vector having a stimulation factor of the human granulocyte colony inserted into it can be produced. When constructing a recombinant expression vector, for the purpose of increasing the level of expression of the transfected genes in a host cell, the corresponding genes can be operably linked to a transcriptional and translational expression regulatory sequence that functions in a host cell selected, as is generally known in the art. Preferably, the expression regulatory sequence and the corresponding genes are included in a simple expression vector that also includes a bacterial selection marker and an origin of replication. A suitable vector that includes not only genes encoding the isoforms of the human granulocyte colony stimulation factor but also the above elements (eg, regulatory sequence) can be constructed by means of a basic recombinant DNA technique. In order to form a desired vector, individually separated DNA fragments can be linked together. In this way, each fragment of DNA is divided first by using restriction enzymes, and then the DNA fragments are linked together in a specific order and orientation. DNA can be divided by using a specific restriction enzyme in a suitable pH regulator. In general, about 0.2-1 μg of a plasmid or DNA fragment is used in about 20 μl of a pH buffer together with about 1-2 units of the corresponding restriction enzyme. The appropriate pH regulator, the concentration of DNA, the reaction time and the reaction temperature will be determined by the producer of the restriction enzyme. Generally, the reaction is carried out at 37 ° C for approximately 1-2 hours. However, some enzymes require a higher temperature. After the reaction, the enzymes and other impurities are removed by extracting the digestion solution with a mixture of phenol and chloroform. The DNA can be precipitated with ethanol to recover it from the aqueous layer. At this time, the terminals of DNA fragments that bind to each other must be compatible with each other in order to bind DNA fragments so that a functional vector can be formed. The divided DNA fragments are classified and selected on the basis of size via electrophoresis. DNA electrophoresis can be performed through an agarose or polyacrylamide matrix. The selection of the matrix depends on the size of DNA to be separated. After electrophoresis, the DNA is extracted from the matrix via electro-elution, or extracted directly from the matrix. When using a low melting point agarose matrix, agarose can be melted before DNA extraction. The DNA fragments to be linked together can be added to a solution in an equimolar amount. The solution contains ATP, pH ligase regulator, and approximately 10 units of ligase such as T4 ligase per 0.5 μg of DNA. To link the DNA fragments to a vector, the vector must be divided by means of a suitable restriction enzyme first forming a linear vector. Said linear vector can be used after being treated with alkaline phosphatase or bovine intestinal hydrolase. Said hydrolase treatment prevents the vector from self-ligating. The recombinant expression vector constructed as described above is used to carry out the transformation or transfection of a host cell. The polynucleotides can be introduced into a host cell according to a known method described in the laboratory manuals such as [Davís et al., Basic Methods in Molecular biology (1986)] and [Sambrook, J., et al (1989) "Molecular Cloning" A laboratory manual 2nd edition]. Preferred methods for introducing polynucleotides into a host cell include calcium chloride transformation, electroporation, or the like. According to the present invention, the host cells are incubated in a nutrition medium suitable for the production of polypeptides using a known technique. For example, the host cells can be incubated in a laboratory or industrial fermentation vessel via small scale or large scale fermentation / shake flask incubation with a suitable medium under conditions that allow the expression and / or secretion of polypeptides. Incubation is carried out in a suitable nutrition medium containing a carbon source, a nitrogen source and inorganic salts using a known technique. The medium is generally known to those of skill in the art and is commercially available. On the other hand, a medium prepared directly in a laboratory can be used. If the polypeptides are secreted directly into the nutrition medium, the polypeptides can be separated directly from the medium. If the polypeptides are not secreted into the nutrition medium, they can be separated from the cell lysate. The separation of polypeptides can be carried out in a convenient manner. For example, the polypeptides can be separated from the medium by means of a conventional method including centrifugal separation, filtration, extraction, spray drying, evaporation or precipitation. In addition, polypeptide purification can be performed by various methods known in the art, including chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography or size exclusion chromatography), electrophoresis, fractional solubility separation (by example, precipitation of ammonium sulfate), SDS-PAGE or extraction. Then, the isoform of the stimulation factor of the human granulocyte colony purified as described above is subjected to pegylation in the manner described hereinafter. Suitable "non-protein segments" used for the pegylation according to the present invention include polyethylene glycol, and branched polyethylene glycol is preferred. Other examples of such non-protein segments include, but are not limited to: at least one material selected from the group consisting of water-soluble polymers such as polypropylene glycol (PPG), polyoxyethylene (POE), polytrimethylene glycol, polylactic acid and its derivatives , polyacrylic acid and its derivatives, poly (amino acid), poly (amino acid), poly (vinyl alcohol), polyurethane, polyphosphazenes, poly (L-lysine), polyalkylene oxide (PAO), polysaccharide, or the like, and polymers not immune agents such as dextran, polyvinyl pyrrolidone, polyvinyl alcohol (PVA), polyacryl amide, or the like. The biocompatible polymer used in the preparation of the polymer derivatives according to the present invention preferably has a molecular weight of about 20000-40000. In order to form a link between a biocompatible polymer and G-CSF via the thiol group of G-CSF, the biocompatible polymer must be activated. For this purpose, the biocompatible polymer can be linked with a reactive functional group. The term "reactive polymer group" refers to a group or a radical capable of activating a biocompatible polymer so that it can be bound with a biologically active material. To form a link between a biocompatible polymer and a biologically active material, one of the end groups of the biocompatible polymer is converted to a reactive functional group. This procedure is referred to as "activation." For example, to form a bond between the poly (alkylene oxide) and a biologically active protein, one of the terminal hydroxyl groups can be converted to a reactive group, such as carbonate. As a result, activated poly (alkylene oxide) can be obtained. The reactive group that can be used to activate a biocompatible non-protein segment polymer according to the present invention is selected from the group consisting of maleimide, acetamide, pentenoic amide, butenoic amide, isocyanate, isothiocyanate, cyanuric acid chloride, , 4-benzoquinone, disulfide, or similar.

The binding of PEG to protein via a thiol group of the protein can be performed by means of a method generally known in the art. For example, PEG-maleimide is used to allow thiol groups to be linked to activate double bonds via the Michael reaction [Ishii et al., Biophys J. 1986, 50: 75-80]. In addition, as is generally known to those with experience in the field of protein chemistry, the PEG-iodoacetamide reagent can be used. The latter method is convenient since a cysteine derivative attached to PEG, ie carboxymethyl cysteine, can be obtained by means of a strong acid hydrolysis, wherein the derivative can be identified and determined by means of standard amino acid analysis [Gard FRN . Carboxymethylation, Method Enzymol 1972; B25: 424-49]. In addition to the above methods, a method of producing a suitable symmetrical disulfide using PEG-ortho-pyriridyldisulfide (Woghiren et al., Bioconjugate Chem 1993, 50: 75-80); a reaction method of an activated PEG, ie 4- (N-maleimidomethyl) cyclohexane-1-sulfosuccinimidyl carboxylate activated with PEG with a thiol group of cysteine to form a covalent bond [USP5, 166,322]; a method of reacting an activated PEG, ie maleimido-6-aminocaproyl ether, activated with PEG 4000 with the mutant IL-2 having a cysteine substituent at a specific site to be conjugated with cysteine residue [USO5,206,344]; and a method for modifying thiol groups of a protein by using gamma-maleimidobutyric acid and a beta-maleimidopropionic acid [Rich et al., J. Med. Chem. 18, 1004, 1975] can be used.

The pegylation method in the following examples is performed by using mPEG20000-maleimide and branched mPEG40000 available from NOF in a molar ratio of 20: 1 to stimulation factor of the human granulocyte colony. The pegylation reaction is carried out in pH 0.1 M phosphate buffer (pH 7-8) at room temperature for 2 hours with stirring. The pharmaceutically acceptable carriers, excipients or stabilizers should be non-toxic to the subjects and be compatible with other substituents of the pharmaceutical preparation under the corresponding administration dose and concentration. For example, the pharmaceutical preparation should not contain any material known to be harmful to the polypeptide, such as oxidants or other materials. Suitable carriers include: suitable pH regulators such as phosphoric acid, citric acid and other organic acids; Antioxidants include ascorbic acid; polypeptides of low molecular weight; proteins such as plasma albumin in blood, gelatin and immunoglobulin; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, arginine or lysine; monosaccharides, disaccharides or other carbohydrates including glucose, mannose or dextrin; chelation factors such as EDTA; metal ions such as zinc, cobalt, or copper; sugar alcohols such as mannitol or sorbitol; counterparts of salt formation such as sodium; and / or nonionic surfactants such as Tween, Pluronic or polyethylene glycol (PEG).

An isoform of the stimulation factor of the human granulocyte colony attached to the sugar chain can be sterilized before being administered for the purpose of treatment. Said sterilization can be carried out easily by means of filtration through a sterilized filtration membrane. In general, an isoform composition of the stimulation factor of the human granulocyte colony attached to the sugar chain is stored in a container equipped with a sterilized access port, for example, an intravenous injection bag or a vial having a septum through which a subcutaneous injection needle can pass. The isoform of the stimulation factor of the human granulocyte colony according to the present invention can be administered directly to the animals by means of suitable routes including routes of parenteral administration, locally or systemically. A particular route of administration will be determined by the patient's history, including recognized or expected side effects in the administration of the isoform of human granulocyte colony stimulation factor. Particular parenteral administration routes include subcutaneous, intramuscular, intravenous, intra-arterial and intraperitoneal routes. More preferably, the administration is carried out by continuous infusion (e.g., mini-pump such as an osmotic pump) or injection, e.g., intravenous injection or subcutaneous injection. A preferred route of administration for the isoform of stimulation factor of the human granulocyte colony is subcutaneous administration. The isoform of the stimulation factor of the human granulocyte colony according to the present invention can be administered to a patient in a therapeutically effective amount. The term "therapeutically effective amount" refers to the amount sufficient to obtain a desired treatment effect by using a given mode of administration under a given condition. The therapeutic composition of the stimulation factor of the human granulocyte colony can be formulated and administered according to the preferred medical practice, under the consideration of the particular condition to be treated, clinical conditions of an individual patient (particularly, side effects during treatment). simple administration of human granulocyte colony stimulation factor), supply sites of the human granulocyte colony stimulation factor isoform composition, modes of administration, administration schedules, and other factors generally known in the art. technique. The therapeutically effective amount of the stimulation factor isoform of the human granulocyte colony is determined considering the above factors. In general, the daily dose of the isoform of the stimulation factor of the human granulocyte colony according to the present invention ranges from about 1 μg / kg to 100 μg / kg. Reference is now made in detail to the preferred embodiments of the present invention. It should be understood that the following examples are illustrative only, and the scope of the present invention is not limited thereto.

EXAMPLE 1 Construction of isoforms of the stimulation factor d® Ba Humamo granulocyte coloma To construct stimulation factor isoforms of the human granulocyte colony, stimulation factor genes from the human granulocyte colony are synthesized as a template. The genes synthesized in this example have base sequences as shown in the following Table 1, and the DNA sequence and amino acid sequence of stimulation factor of the human granulocyte colony, used as a template, are shown in the figure 1. In Table 1, N-term -175C corresponds to the sequence Nos. 2-7, respectively. 1. Isoform construction of human qranulocyte colony stimulation factor -1C PCR is performed to construct the -1C isoform by using DNA from natural human granulocyte colony stimulation factor as a template and primers pairs formed from primer - 1 C and C-term primer. The amplified DNA fragment is further subjected to PCR by using N-term primer and C-term primer so that the restriction enzyme cleavage sites (Xba I and BamH I) can be introduced, and then introduced into a vector expression. By doing this, it is possible to obtain genes from a stimulation factor isoform of the human granulocyte colony that has cysteine added before threonine from the N-terminus. The amino acid sequence of the N-terminal is methionine-cysteine-threonine-proline-leucine-glycine. 2. Construction of isoform of stimulation factor of human granulocyte colony T133C. T133C is constructed in order to provide a control such as a known isoform or to perform amino acid modification in the middle part of the stimulation factor polypeptide of the human granulocyte colony. T133, which has substitution cysteine for threonine, amino acid 133 stimulation factor of the human granulocyte colony is obtained by performing PCR using stimulation factor DNA from the human granulocyte colony as a template and primer pairs formed from N-term primer with T133C2 and T133C1 with primer C-term. As a result, it is possible to obtain the modified DNA fragments with a codon corresponding to cysteine substituent for threonine, amino acid 133. The two DNA fragments are further subjected to PCR by using primer pairs formed from N-term primer and C-primer. -term to obtain modified genes of T133C, a isoform of stimulation factor of the human granulocyte colony having threonine substituent cysteine at the position of amino acid 133. The introduction into an expression vector is carried out in the same way as is described in the stimulation factor isoform of the human granulocyte colony -1C above. 3. Construction of a stimulation factor isoform of the human granulocyte colony -1 C / T133C. -1C / T133C is constructed in order to perform amino acid addition to the N-terminal and amino acid modification in the middle part of the polypeptide of the stimulation factor of the human granulocyte colony. To construct -1C T133C, threonine, amino acid 133 of the stimulation factor isoform of the human granulocyte colony -1 C is replaced with cysteine. For this, PCR is performed using DNA from the human granulocyte colony stimulation factor isoform -1 C as a template and primers pairs formed from the N-term primer with T133C2 and T133C1 with C-term primer. As a result, it is possible to obtain two modified DNA fragments with a codon corresponding to cysteine substituent for threonine, amino acid 133. The two DNA fragments are further subjected to PCR by using primer pairs formed from N-term primer and C primer -term to obtain modified genes of -1 C / T133C, a isoform of stimulation factor of the human granulocyte colony having cysteine substituent for threonine at the position of amino acid 133. Introduction into an expression vector is made of the same as described in the 'stimulation factor isoform of the human granulocyte colony -1 above. 4. Construction of a stimulation factor isoform of the human granulocyte colony 175C. To construct 175C, PCR is performed by using stimulation factor DNA from the human granulocyte colony as a template and primer pairs formed from primer N-term and primer 175C. The amplified DNA fragment is further subjected to PCR using N-term primer and C-term primer so that the restriction enzyme cleavage sites (Xba I and BamH I) can be introduced, and then introduced into a vector of expression by doing this, it is possible to obtain genes from a stimulation factor isoform of the human granulocyte colony that has cysteine added to the N-terminus. The introduction into the expression vector is carried out in the same manner as described in the stimulation factor isoform of the human granulocyte colony-1 C above. The amino acid sequence of the C-terminus is -alanine-glutamine-proline-cysteine.

. Construction of the stimulation factor isoform of the human granulocyte colony 175C / T133C. To construct 175C / T133C, threonine, amino acid 133 of the stimulation factor isoform of the human granulocyte colony of -1 C is replaced with cysteine. For this, PCR is performed by using DNA from the stimulation factor isoform of the human granulocyte colony 175C as a template and primer pairs formed from N-terminus primer with T133C2 and T133C1 with C-term primer. As a result, it is possible to obtain two DNA fragments modified with a codon corresponding to cysteine substitution for threonine, amino acid 133. The two DNA fragments are further subjected to PCR by using pairs of primers formed from N-term primer and primer C -term to obtain modified genes of 175C / T133C, a isoform of stimulation factor of the human granulocyte colony having cysteine substituent for threonine at the position of amino acid 133. Introduction in an expression vector is performed in the same manner as is described in the 'isoform construction of stimulation factor of human granulocyte colony -1C above. The genes of the stimulation factor isoforms of the human granulocyte colony constructed as described above are introduced into an expression vector to be expressed by a PR lamda promoter. Constitutional elements of the expression vector are shown in the following table 2.

TABLE 2 EXAMPLE 2 Expression and purification of isoforms of the sphimulation factor colony of human ranouiocytes Transformation of plasmid vectors containing stimulation factor isoform genes from the human granulocyte colony in E. coli is performed to construct strains that produce stimulation factor proteins from the human granulocyte colony. To perform the transformation, the calcium chloride method generally known to those skilled in the art is used. In the present E. coli used as a host cell, E. coli (HB101, series of NM strains, etc.) is currently available. The transformed E. coli is inoculated into 5 ml of a Luria Broth medium (LB) in a 15 ml tube, and inoculated into a 500 ml fear of Luria Broth (LB) in a 3000 ml triangular flask and incubated at 30 ° C. When the abosrbancy at 600 nm reaches 0.8.1.2, the incubator is heated to 42 ° C and the cells are further incubated for 4 hours to induce the expression of isoforms of the stimulation factor of the human granulocyte colony. After the termination of the culture, the culture solution is subjected to centrifugal separation to collect the cells, and the cells are washed with distilled water. The washed cells are lysed in a microfluidizer (available from Microfluidics Co.) under 816.48 atmospheres four times, and inclusion bodies are recovered via centrifugal separation, followed by washing three times with distilled water. The inclusion bodies are completely suspended and dissolved in 10 ml of 8 M urea, 50 nM glycine (pH 11.0) at room temperature for at least 5 hours, and then the suspension is subjected to centrifugal separation at 10000 rpm for 30 minutes. The supernatant is collected to obtain a stimulation factor isoform protein solution from the human granulocyte colony. To the solution of protein, glycine and distilled water are added to control the concentration of glycine and the concentration of urea at 50 mM and 2 M, respectively. The resulting protein solution is titrated to pH 9.0 and refolded overnight at 4 ° C. After completion of the protein refolding, the solution is adjusted to pH 4.0, diluted with distilled water three times or more, and loaded onto a column packed with 10 ml of CM Sepharose FF (available from Amersham Biosciences), which is they have previously equilibrated by using 2mM HCl in an amount of at least three times the volume of the column. After completion of the loading, the column is equilibrated by using 2 mM HCl in an amount of at least three times the volume of the column and washed by using pH buffer of sodium acetate chloride (pH 5.4) in a amount of at least three times the volume of the column. Then, the stimulation factor isoforms of the human granulocyte colony are eluted by using 50 mM sodium chloride chloride pH buffer (pH 5.4) containing 35 mM sodium chloride in an amount of at least twenty times the volume from the column and divided into fractions of 5 ml. Fractions are collected after HPLC analysis. The human granulocyte colony stimulation factor sofmas, purified by CM Sepharose FF, are concentrated by using a Plus-80 Centricon filter unit with a molecular weight of 10000Da at 3000 rpm for 25 minutes to obtain protein concentrate that it has a final protein concentration of about 5 mg / ml. The concentrated protein is loaded onto a column packed with 300 ml of Sephacryl S-100 (Amersham Biosciences), which have been previously equilibrated by using 10 mM sodium acetate chloride pH buffer (pH 5.4) in an amount of at least three times of the volume of the column. The pH buffer is eluted at a rate of 1 ml / minute, to remove multimers from the stimulation factor isoforms of the human granulocyte colony contained in the protein solution and to separate purified monomers, which, in turn, it is used in the subsequent polyethylene glycol addition reaction.

EXAMPLE 3 Binding of polyethylene glycol to stimulation factor isoforms of the human granulocyte colony 1. Preparation of mPEG (20,000) -maleimide-isoform of branched G-CSF. 10 mg of each of the stimulation factor isoforms of the human granulocyte colony according to example 2 are introduced in pH buffer phosphate 0.1 M (pH 7.0-7.8) and 200 mg mPEG (20000) -maleimide branched (available from NOF) is added to this. The reaction is carried out at room temperature for 2 hours with stirring. The reaction mixture is quenched by lowering the pH to 3.0 with 1 N HCl solution. 2. Preparation of mPEG (40000) -maleimide-isoform of branched G-CSF. 10 mg of each of the stimulation factor isoforms of the human granulocyte colony according to example 2 are introduced in 0.1 M pH phosphate buffer (pH 7.0-7.8) and 400 mg of mPEG (40000) - branchedmaleimide (available from NOF) is added to this. The reaction is carried out at room temperature for 2 hours with stirring. The reaction mixture is quenched by lowering the pH to 3.0 with 1 N HCl solution.

EXAMPLE 4 Purification of isoform polymer from Ca stimulation colony of human qranulocytes with polyethylene gBücol The PEG-G-CSF polymer obtained from example 4 is purified as follows. 1. Chromatography FF CM sepharose. After completion of the reaction, each mPEG-G-CSF polymer is titrated to pH 3.0, diluted with distilled water for at least 10 times, and loaded onto a column packed with 10 ml of CM Sepharose FF (Amersham Bioscience ), which have previously been balanced by using 2 mM HCl in an amount of at least three times the volume of the column. After loading, the column is equilibrated by using 2 mM HCl in an amount of at least three times the volume of the column, and washed by using 50 mM sodium acetate chloride pH buffer (pH 5.0) in a amount of three times the volume of the column. In the cases of mPEG (20,000) -maleimide-G-isoform CSF and mPEG (20,000) -maleimide-G-isoform branched CSF, mPEG-G-CSF polymers are eluted by using pH buffer of 50 mM sodium acetate chloride (pH 5.4) containing 40 mM sodium chloride in an amount at least twenty times of the column volume. In the case of mPEG (40000) -maleimide-G-isoform branched CSF, mPEG-G-CSF polymer is eluted by using 50 mM sodium acetate chloride pH buffer (pH 5.4) containing 20 mM sodium chloride in a amount of at least twenty times the volume of the column. The eluted solution is fractionated into a 5 ml fraction and each fraction collected after the HPLC analysis. 2. Gel impregnation chromatography mPEG-G-CSF polymers are purified by CM sepharose FF, are concentrated by using a Plus-80 Centricon filter unit with a molecular weight of 10000 Da at 30,000 rpm for 25 minutes to obtain protein concentrate having a final protein concentration of about 5 mg / ml. The concentrated protein is loaded onto a column packed with 300 ml of Sephacryl S-100 (Amersham Biosciences), which have been previously equilibrated by using 50 mM sodium acetate chloride pH buffer (pH 5.4) in an amount of at least three times the volume of the column. The pH buffer is eluted at a rate of 1 ml / min to remove mPEG-G-CSF polymers contained in the protein solution. The proteins are eluted in series for the purpose of G-CSF multimer, PEG-G-CSF, G-CSF dimer and G-CSF monomer.

EXAMPLE 5 Evaluation of activity and lifetime of polymers of ippi? G-G-CSF in rats The mPEG-G-CSF polymers prepared and purified according to examples 4 and 5 are evaluated for activity and time of travel in vivo when using 8-week-old rats (SD rats, male, five rats per test sample) . Test samples include: PBS carrier as a carrier; G-CSF T133C20kDa (Br) prepared and purified according to examples 1 to 4 (G-CSF isoform obtained by substituting 133-Trhr with cysteine and covalent binding of branched PEG with 20 kDa to the same cysteine according to open Korean patent to the public No. 2002-0079778); Neulasta commercially available (Amgene Co) having PEG 20kDa bound to the N-terminus; and the isoforms according to the present invention, prepared and purified according to examples 1 to 4, ie N + 20 kDa (Br), (G-CSF isoform obtained by adding cysteine between threonine and methionine of the N-terminal (-1 C) of G-CSF and covalently binding branched PEG with 20 kDa to the same cysteine); 175C + 40 kDa (Br) (G-CSF isoform obtained by adding cysteine after 174-Pro of the C-terminus of G-CSF and covalently attaching branched PEG with 40 kDa to the same cysteine). Each isoform is diluted with PBS and administered to rats through the tail veins in a dose of 100 μg / kg based on the body weight measured during the day of administration. Administration is only done during the test day. To provide analytical samples, 200 μl of blood is collected from the tail veins at a pre-determined time depending on the constitution of a test group. In the case of the target, blood sampling is done before administration. Sampling for PD / PK analysis is performed 7, 24, 48 and 72 hours after administration. Neutrophils were counted in each blood sample to determine in vivo activity. In addition, each blood sample is placed in an EDTA anticoagulant tube and subjected to centrifugal separation to separate blood plasma. The concentration of G-CSF in plasma is measured by using a human G-CSF ELISA kit (Quantokine, R &D Systems). Each sample is diluted in series to the linear zone that allows the detection of concentration in the equipment. The measurement is made twice per sample. The results are shown in the following table 3. As can be seen from table 3, the isoforms of the stimulation factor of the human granulocyte colony bound to polyethylene glycol having cysteine added to the N-terminal or C-terminator according to The present invention shows an increased in vivo activity and life time in vivo. In particular, N + 20kDa (Br) and 175 + 20kDa (Br) shows a significant increase in activity from the point of 24 hours after administration, when compared to the control. In addition, 175C + 40kDa (Br) shows an increase in activity from the point of 24 hours after administration and a similar appearance of neutrophil production, when compared to the control. However, 175C + 40kDa (Br) can maintain its activity up to 72 hours after administration, in particular, 175C + 40kDa (Br) provides a significantly improved lifetime, while 175C + 20kDa (Br) and N + 20kDa (Br) provide significantly improved activity.

TABLE 3 INDUSTRIAL APPLICABILITY As can be seen from the foregoing, the human granulocyte colony stimulation factor isoforms according to the present invention provide a significantly increased in vivo lifetime and can be used for the treatment or prevention of various diseases related to the stimulation factor of human granulocyte colony. Although this invention has been described in conjunction with what is considered herein to be more practical and preferred embodiment, it should be understood that the invention is not limited to the embodiment described and the accompanying drawings. On the contrary, it is intended to cover several modifications and variations within the essence and scope of the appended claims.

Claims (10)

NOVELTY OF THE INVENTION CLAIMS

1. - An isoform of the stimulation factor of the granulocyte colony, which includes at least one amino acid added before N-terminal Thr or after C-terminal Pro in the stimulation factor of the human granulocyte colony represented by the sequence No. 1 of amino acids, wherein at least one of the added amino acids is cysteine, and polyethylene glycol is linked to at least one of the added cysteine.

2. The isoform of stimulation factor of the granulocyte colony according to claim 1, further characterized in that 1-12 amino acids are added to each terminal of the granulocyte colony stimulation factor.

3. The isoform of stimulation factor of the granulocyte colony according to claim 1, further characterized in that a cysteine residue is added to a stimulation factor of human granulocyte colony.

4. The isoform of stimulation factor of the granulocyte colony according to claim 3, further characterized in that the polyethylene glycol is a branched polyethylene glycol and has a molecular weight of 20kDa ~ 40kDa.

5. The isoform of stimulation factor of the granulocyte colony according to claim 1, further characterized in that the cysteine is 175C added to the C-terminus.

6. The isoform of stimulation factor of the granulocyte colony according to claim 4, further characterized in that at least one amino acid stimulation factor of the human granulocyte colony is replaced with another amino acid before it is added to this polyethylene glycol.

7. A pharmaceutical composition comprising the stimulation factor of the granulocyte colony of human according to any of claims 1 to 6 and pharmaceutically acceptable carriers.

8. Genes encoding proteins of the stimulation factor isoform of the human granulocyte colony having cysteine added to the N-terminal or the C-terminal thereof as defined in any of claims 1 to 6.

9. An oligodeoxynucleotide used as a primer to modify the amino acid sequence of stimulation factor of the human granulocyte colony in the stimulation factor isoform of the granulocyte colony according to any of the claims 1 to 6.

10. The oligodeoxynucleotide according to claim 9, further characterized in that the primer is an oligogodeoxynucleotide represented by any of sequences Nos. 2 to 7.