Patent Publication Number: US-2023151078-A1

Title: Recombinant single-chain fviii and chemical conjugate thereof

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a National Stage of International Application No. PCT/KR2017/006633 filed Jun. 23, 2017, claiming priority based on Korean Patent Application No. 10-2016-0079442 filed Jun. 24, 2016. 
    
    
     TECHNICAL FIELD 
     The present invention relates to a preparation technique of recombinant single-chain FVIII protein used for the treatment of hemophilia and its chemical conjugates. 
     BACKGROUND ART 
     Hemophilia is a disease in which bleeding occurs continuously due to lack of coagulation factors. The coagulation factors in the blood are composed of I to XIII. Among them, the factors related to hemophilia are VIII, IX and XI. Hemophilia is caused by the genetic defect of the above factors. Depending on the type of a deficient coagulation factor, hemophilia can be classified into Hemophilia A where Factor VIII is deficient, Hemophilia B where Factor IX is deficient, and Hemophilia C where Factor XI is deficient. Hemophilia A accounts for about 80 to 85% of the hemophilia. 
     The goal of hemophilia therapy is hemostasis. The treatment is conducted in two aspects, i.e., prevention and on demand treatment, using the enzyme replacement therapy (ERT). The current trend of treatment is to shift toward prevention. 
     As for the protein therapeutic agent used for the ERT, FVIII (Hemophilia A) and FIX (Hemophilia B), which were extracted from whole blood and plasma and concentrated, were used in the past. However, there were problems such as side effects and complications arising from using the human-derived raw material. Therefore, recently, various factors produced by recombinant DNA technology have been developed and used. FVIII is also produced by recombinant DNA technology, which reduces the risk of infectious diseases and complications. However, it has a short half-life like blood-derived coagulation FVIII, which requires frequent administrations, and is inconvenient for patients. Thus, continuous efforts have been made to improve the life quality of patients by developing various FVIII having long in vivo half-life. 
     One of them was pegylated FVIII. It was developed to lower the binding of LRP1 (low density lipoprotein receptor related protein), which is known to be responsible for the in vivo clearance of FVIII, with pegylated FVIII. The pegylated FVIII improved the in vivo half-life by preventing the clearance of FVIII. However, the extent of the improvement was below expectations. Since FVIII strongly interacts with vWF, most of FVIII exist in vivo in association with vWF. And thus, the half-life of FVIII is determined by the in vivo half-life of vWF bound to FVIII. This situation also applies to pegylated FVIII. Since the half-life of pegylated FVIII was also controlled by the half-life of vWF after pegylation, there was a limitation that the improvement of the half-life is below the half-life of vWF. 
     In addition, FVIII-Fc fusion protein (FVIII-Fc) in which the Fc domain of the antibody is fused to the C-terminal of FVIII have been developed. FVIII-Fc has a longer in vivo half-life than FVIII due to in vivo recycling by FcRn. However, by the binding with vWF, the in vivo retention time of FVIII-Fc was also controlled by the half-life of vWF. 
     Moreover, for the treatment of Hemophilia A, there are ongoing efforts to replace the function of FVIII as a blood coagulation factor through other pathways other than the present FVIII, and to promote an in vivo sustained effect and patient convenience. 
     As an example, FVIII-mimetic bi-specific antibody, which is a bi-specific antibody in the form of an intact IgG form containing two types of Fabs specific for FIX (or activated FIX) and FX, has been designed to replace the function of FVIII in vivo (Kitazawa et al, Nature medicine 2012 18 (10): 1570-4). 
     Another example is the use of anti-TFPI (Tissue factor pathway inhibitor). This is a substance that inhibits the function of the TFPI and is being developed in the form of an aptamer in addition to antibodies and peptides (Hilden et al., BLOOD 2012, 119 (24): 5871-8). TFPI prevents the activation process of FX by TF (tissue factor)-activated FVII complex to inhibit the production of FXa, thereby inhibiting the production of thrombin by FXa to stop the blood coagulation progress. 
     Anti-thrombin inhibitor has also been proposed. It inhibits the function of thrombin inhibitor in blood to improve the activity of thrombin, thereby facilitating blood coagulation in hemophilia animals or hemophilia patients. 
     However, these approaches do not directly replace or supplement the activity of FVIII and FIX proteins deficient in Hemophilia A or Hemophilia B, but indirectly mimic or bypass the activity of FVIII. Since the modes of action of these approaches are complex, their safety and effectiveness in clinical trials should be sufficiently demonstrated. 
     Korean Patent Laid-Open Publication No. 2014-0114266 discloses an anti-hemophilia Factor VIII having an increased specific activity. 
     The therapeutic agents developed so far have the problems of short intervals of administrations (intravenous injections) in terms of functions, and of frequent bleedings arising from the loss of efficacy due to short half-lives. Also, in terms of productivity, the problems of low productivity and high cost of treatment should also be solved. Thus, it is necessary to develop a FVIII having an improved half-life and high productivity. 
     DISCLOSURE OF INVENTION 
     Technical Problem 
     An object of the present invention is to provide a single-chain FVIII which shows improved in vivo retention time and can be easily produced on mass scale. 
     Solution to Problem 
     According to one embodiment of the present invention, there is provided a single-chain blood coagulation factor VIII (Factor VIII) comprising a heavy chain, a light chain and a partially-deleted B domain fragment of human Factor VIII, wherein the partially-deleted B domain fragment lacks at least 5 amino acids from residue 1648 of SEQ ID NO:1 to the N-terminal direction and at least 5 amino acids from residue 1649 of SEQ ID NO:1 to the C-terminal direction, so as not to comprise a cleavage site by furin protease, and contains 4 to 6 glycosylation sites. 
     According to one embodiment of the present invention, the partially-deleted B domain fragment included in the single-chain blood coagulation factor VIII of the present invention comprises about 15% to 40% of a wild type B domain sequence, but is not limited thereto so long as the effect according to the present invention is achieved. Those skilled in the art can select an appropriate single-chain blood coagulation factor VIII in consideration of the condition of the B domain fragment included in the aforementioned single-chain blood coagulation factor VIII. Herein, the B domain is interpreted to include the a3 region as described later. 
     According to one embodiment of the present invention, a portion deleted in the partially-deleted B domain fragment satisfying the above-mentioned condition may be continuous or non-continuous, and the partially-deleted B domain fragment may be represented by the sequence of (i) amino acid residues 741 to 902 and 1654 to 1689; (ii) amino acid residues 741 to 965 and 1654 to 1689; or (iii) amino acid residues 741 to 902, 1637 to 1642, and 1654 to 1689, based on the sequence of SEQ ID NO: 1. 
     According to one embodiment of the present invention, the single-chain FVIII in the present invention includes the sequences having the amino acid sequences represented by SEQ ID NOS: 2 to 7 or the sequence having about 90% or more homology thereto, which may be easily determined with reference to the homology determination method described later and the disclosure relating to the factor according to the present invention and biological functional equivalence. 
     According to one embodiment of the present invention, the nucleic acid molecules encoding the single-chain FVIII of the present invention having the amino acid sequences represented by SEQ ID NOS: 2 to 7 include the sequences represented by SEQ ID NOS: 10 to 15, codon-optimized sequences therefrom for the expression in a cell, or altered nucleic acid sequences due to a degenerate codon. 
     The single-chain FVIII according to the present invention has a specific activity of 90% or higher than that of a two-chain blood coagulation factor VIII when measured by a CS or APTT method. 
     According to one embodiment of the present invention, the single-chain FVIII is conjugated to a hydrophilic polymer, which includes polyethylene glycol (PEG), polyethylene oxide, dextran, or polisialic, and when PEG is used, it may be conjugated at the conjugation position through acryloyl, sulfone or a maleimide. 
     According to one embodiment of the present invention, the conjugated blood coagulation factor VIII is conjugated to a hydrophilic polymer at some amino acid residues of the A domain and/or B domain fragment; the conjugation position of the B domain fragment is at least one selected from the group consisting of amino acid residues 754, 781,782, 788, 789, 825 and 897 based on the sequence of SEQ ID NO: 1 and the conjugation position of the A domain is at least one selected from the group consisting of amino acid residues 491, 495, 498 and 1806 based on the sequence of SEQ ID NO: 1; and the residue of the conjugation position may be substituted with cysteine for the conjugation with the hydrophilic polymer. 
     According to another embodiment of the present invention, the single-chain FVIII is conjugated to PEG, particularly having an average molecular weight of 20 kDa or more. 
     According to another embodiment of the present invention, there is provided a single-chain FVIII, specifically a single-chain FVIII represented by SEQ ID NOS: 2 to 7, or a single-chain FVIII where amino acid residue of the conjugation positions are substituted with cysteine for conjugation to a hydrophilic polymer, wherein the conjugation positions in the B domain are at least one selected from the group consisting of amino acid residues 754, 781,782, 788, 789, 825 and 897 based on the sequence of SEQ ID NO: 1; and the conjugation positions in the A domain are at least one selected from the group consisting of amino acid residues 491, 495, 498 and 1806 based on the sequence of SEQ ID NO: 1; or a nucleic acid molecule encoding the single-chain FVIII comprising a substituted cysteine group 
     According to another embodiment of the present invention, the residues into which cysteine was introduced are as shown in Table 3, and the nucleic acid molecule in which specific residues are substituted with cysteine for conjugation to a hydrophilic polymer is represented by SEQ ID NOS: 17 to 32, and the amino acid sequences corresponding thereto are represented by SEQ ID NOS: 33 to 48. Further, a codon-optimized sequence for expression in a cell or altered nucleic acid sequence by a degenerate codon is also comprised in the scope of the present invention. 
     According to another embodiment of the present invention, there is also provided a vector comprising the nucleic acid molecule according to the present invention, and a cell comprising the vector. 
     According to another embodiment of the present invention, there is provided a method for producing a single-chain FVIII comprising: transfecting the vector of the present invention into a eukaryotic cell; or alternatively providing the cell transfected with the vector of the present invention; expressing a single-chain FVIII where the free thiol group of the cysteine introduced thereinto is masked with a disulfide bond by cysteine or glutathione through culturing the cells in a culture solution; collecting the single-chain FVIII expressed from the culture solution and treating the single-chain FVIII with a reducing agent to release the masked cysteine or glutathione; and treating the treated culture solution with a pegylation buffer. 
     In another embodiment of the present invention, there is also provided a blood coagulation kit comprising at least one of the single-chain FVIII disclosed in the present invention to be used for a hemophilia patient or a patient requiring blood coagulation, a use of the single-chain FVIII for the treatment of hemophilia patient or a composition, or a blood coagulation method comprising administering the single-chain FVIII to a hemophilia patient or a patient requiring blood coagulation in a therapeutically effective amount. 
     Advantageous Effects of Invention 
     A single-chain FVIII according to the present invention comprises a heavy chain and a light chain which are connected by a B domain where a part of the sequence is deleted. Specifically, the single-chain FVIII comprises the partially-deleted B domain which does not comprise a cleavage site cleaved by furin protease, which is generated during the normal FVIII expression process, and contains at least 4 glycosylation sites. The single-chain Factor VIII according to the present invention or the single-chain FVIII in which some residues of the A or B domain are pegylated not only retains activities but also can be easily produced on mass scale because of its single-chain form. The said single-chain Factor VIII has an increased in vivo half-life through pegylation to enhance convenience for patients as a therapeutic agent for Hemophilia A and to lead a reduction of medical expenses through reduction in production cost. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG.  1    schematically shows a pegylated single-chain FVIII structure according to one embodiment of the present invention, wherein 1648 residue represents the furin cleavage site. 
         FIG.  2    schematically depicts the preparation of a single-chain FVIII according to one embodiment of the present invention from a full length FVIII. The furin cleavage site (1648 th  amino acid residue based on SEQ ID NO: 1) is shown, and the numbers in the Figure indicate the respective amino acid residues corresponding to the designated sites. 
         FIG.  3    demonstrates the result of Western blot analysis of the single-chain FVIII expression according to one embodiment of the present invention. 
         FIG.  4    indicates the pegylation positions tested to determine the optimal pegylation position of the single-chain FVIII according to one embodiment of the present invention. 
         FIG.  5    illustrates the analysis results of FVIII activity after the expression of the mutant single-chain FVIII into which free cysteine was introduced according to one embodiment of the present invention. 
         FIG.  6    shows the results of SDS-PAGE analysis after pegylation of the mutant single-chain FVIII into which free cysteine was introduced according to one embodiment of the present invention, and the lines marked with an asterisk indicate the introduced cysteine that pegylation efficiency is relatively high. 
         FIG.  7    describes the test results of the PK (pharmacokinetics) of scFVIII according to one embodiment of the present invention compared to the commercialized FVIII substance. 
         FIGS.  8   a  and  8   b    are brief illustration and experimental results, respectively, of an experimental method for comparing tail bleeding hemostatic efficacy test of the pegylated scFVIII according to one embodiment of the present invention with the efficacy of commercialized FVIII substance.  FIG.  8   b    shows PEG40 kDa-scFVIII-B3. 
     
    
    
     BEST MODE FOR CARRYING OUT THE PRESENT INVENTION 
     The present invention is based on the development of coagulation factor VIII mutants that can be expressed in the form of single-chain with increased productivity and in vivo stability, and the development of blood coagulation factors with improved compliance in terms of patient&#39;s administration intervals through the pegylation of the mutant of Factor VIII. 
     Blood coagulation factor VIII is composed of A1-A2-B-A3-C1-C2 domains, which is synthesized as a single-chain protein in hepatocytes. After synthesis, it is matured through processing to form a 280 kDa heterodimer composed of a heavy chain and a light chain. Among them, the light chain has molecular weight of 80 kDa and is composed of A3-C1-C2 domains. And the heavy chain is composed of A1-A2-B domains and has a molecular weight of 90-200 kDa, and its molecular weight varies significantly depending on the differences in the length and glycosylation degree of the B domain. The a1 domain between the A1 and the A2 domains, the a2 domain between the A2 and B domains, and the a3 domain are included in the heavy chain. The heterodimer is present in an inactivated state in the blood by binding to vWF, and when exposed to stimuli such as vascular injury, it is cut by thrombin after arginine residue, that is, after the residues 372, 740 and 1689. As a result, it is separated from vWF and activated to form a trimer of A1, A2 and A3-C1-C2. Then the trimer catalyzes the activation of FX by FIXa, and is rapidly deactivated. 
     It has been reported that single-chain FVIII is structurally stable and shows an increase in the expression level (see WO2004/067566). The FVIII in the form of a two-chain has been reported to show differences in the expression levels depending on the length of the B domain (Miao et al., Blood 2004 May 1; 103 (9): 3412-9). 
     In the present invention, it was found that the specific activity and the APTT/CS ratio were changed in addition to the differences in the expression level depending on the length and sequence of the B domain including a3 in the preparation of the single-chain FVIII. A recombinant single-chain FVIII with a specific activity and APTT/CS ratio similar to those of the natural-type of a two-chain FVIII was prepared by optimizing the length and sequence of the B domain. The single-chain FVIII according to the present invention was found to be stable and excellent in the expression level while maintaining the inherent property of wild type FVIII. 
     Thus, according to one embodiment, the present invention provides a single-chain Factor VIII in which a heavy chain region and a light chain region of human Factor VIII are linked by a partially-deleted B domain fragment of human Factor VIII. Specifically, the partially-deleted B domain fragment lacks at least 5 amino acids from residue 1648 of SEQ ID NO:1 to the N-terminal direction and at least 5 amino acids from residue 1649 of SEQ ID NO:1 to the C-terminal direction, so as not to comprise a cleavage site by furin protease, and contains 4 to 6 glycosylation sites. 
     The single-chain FVIII according to the present invention comprises the A 1-A2-partial B-A3-C1-C2 domains. The residue positions and sequences of each domain comprised in the FVIII in the present invention are known in the art, and the human amino acid sequence such as human FVIII can be represented by the sequence of SEQ ID NO: 1. Based on the sequence, the heavy chain is composed of 1-740 residues including the A1 and A2 domains; the B domain is composed of 741-1689 residues; and the light chain is composed of 1690-2332 residues including the A3, C1 and C2 domains ( FIG.  2   ; and Orlova et al., Acta Naturae. 2013 Apr.-Jun.; 5(2): 19-39). The B domain in the present invention is intended to include the a3 domain (1649 to 1689) described in  FIG.  2   . 
     In this respect, the single-chain FVIII according to the present invention can be represented by the formula A1-A2-B′-A3-C1-C2, wherein A1 is the A1 domain, A2 is the A2 domain, B′ is a part of the B domain, A3 is the A3 domain, C1 is the C1 domain, and C2 is the C2 domain. Reference can be made to the above-mentioned descriptions for each domain. 
     The partially-deleted B domain (B′) in the single-chain FVIII according to the present invention lacks a cleavage site of the proteolytic enzyme furin. According to one embodiment, the B′ domain lacks at least 5 amino acids from residue 1648 of SEQ ID NO:1 to the N-terminal direction and at least 5 amino acids from residue 1649 of SEQ ID NO:1 to the C-terminal direction. Since the B domain has several glycosylation sites and the glycosylation of a protein affects the activity and stability of the protein during and after the expression process, the number of glycosylation may be taken into consideration when determining the deletion sites of the B domain. 
     According to one embodiment of the present invention, the deletion site of the B domain is determined so as to include 4 to 6 glycosylation sites. 
     When determining the deletion site of the B domain, the B domain having deletion of the furin cleavage site included in the single-chain FVIII according to the present invention can be determined within a range including about 15 to 40% of the entire wild type B domain. 
     The deletion sites of the partially-deleted B domain comprised in the single-chain FVIII according to the present invention may be continuous or non-continuous. 
     In an embodiment, in case where the single-chain FVIII according to the present invention comprises a B domain having continuous deletions including the furin cleavage site, the B domain may have a sequence represented by (i) amino acid residues 741 to 902 and 1654 to 1689 (i.e., 903 to 1653 residues of the B domain are deleted continuously); (ii) amino acid residues 741 to 965 and 1654 to 1689 (i.e., residues 966 to 1653 of the B domain are deleted); or in case where the single-chain FVIII according to the present invention comprises a B domain having discontinuous deletions, the B domain may be represented by the sequence of (iii) amino acid residues 741 to 902, 1637 to 1642, and 1654 to 1689, based on the sequence of SEQ ID NO: 1. 
     According to another embodiment, the FVIII amino acid sequence according to the present invention, including such deleted B domains, may be represented by SEQ ID NOS: 2 to 7. 
     The recombinant FVIII disclosed herein is not limited to the above sequences, but includes biological equivalents thereof. Biological equivalents mean that they have substantially the same activity as the polypeptides according to the present invention although additional modifications have been made to the amino acid sequences disclosed in the present invention, and such modifications include, for example, deletion, insertion and/or substitution of amino acid sequence residues. 
     According to one embodiment, a recombinant FVIII polypeptide prepared by conducting a conservative amino acid substitution to the FVIII according to the present invention is included in the present invention. 
     Conservative amino acid substitution refers to a substitution that does not substantially affect or diminish the activity of a particular polypeptide, and includes, for example, 1 to 15 conservative substitutions, or 1 to 12, for example, 1, 2, 5, 7, 9, 12, or 15 conservative amino acid substitutions. 
     Conservative amino acid substitutions are known in the art, and reference can be made to, for example, the descriptions in Creighton (1984) Proteins based on Blosum (BLOcks SUBstitution Matrix). W. H. Freeman and Company (Eds); and Henikoff, S.; Henikoff, J. G. (1992). “Amino Acid Substitution Matrices from Protein Blocks”. PNAS 89 (22): 10915-10919. doi: 10.1073/pnas.89.22.10915; WO2009012175 A1, and the like. 
     Such amino acid modifications, especially substitutions, are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. Based on the analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; alanine, glycine and serine have similar sizes; phenylalanine, tryptophan and tyrosine have similar shapes. Thus, considering the above, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are biologically functional equivalents. 
     Also, when introducing amino acid substitutions, the hydropathic index of amino acids can be taken into consideration. Each amino acid is given a hydropathic index depending on its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5). Such hydropathic index is particularly important in order to impart an interactive biological function to the protein. It is known in the art that it is necessary to substitute with an amino acid having a similar hydropathic index to retain a similar biological activity. When a mutation is introduced with reference to the hydropathic index, it is advantageous to conduct substitution between amino acids which shows the hydropathic index difference, preferably within±2, more preferably within±1, even more preferably within±0.5. 
     It is also known in the art that the substitution between amino acids with similar hydrophilicity values results in a protein with an equivalent biological activity. For example, as disclosed in U.S. Pat. No. 4,554,101, the following hydrophilicity values are given to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); and tryptophan (−3.4). 
     Amino acid substitutions that do not entirely alter the activity of a recombinant protein according to the present invention are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). For example, the most commonly performed substitutions are those between the amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly. 
     Thus, variants having biological and functional equivalence as described above have a substantial identity with an amino acid sequence disclosed in the present specification or a nucleic acid molecule encoding the same, and are included within the scope of the present invention 
     Such substantial identity can be determined using sequence comparison methods known in the art. The sequences showing preferably at least 80% homology, specifically 85% or more, more specifically 90% or more homology, even more specifically 95% or more homology means substantial identity when the sequence disclosed herein and any other sequence are aligned for maximum correspondence between them and the aligned sequences are analyzed using algorithms commonly used in the art. Alignment methods for sequence comparison are known in the art. For example, they are disclosed in: Smith and Waterman,  Adv. Appl. Math . (1981) 2:482; Needleman and Wunsch,  J. Mol. Bio . (1970) 48:443; Pearson and Lipman,  Methods in Mol. Biol . (1988) 24: 307-31; Higgins and Sharp,  Gene  (1988) 73:237-44; Higgins and Sharp,  CABIOS  (1989) 5:151-3; Corpet et al.,  Nuc. Acids Res . (1988) 16:10881-90; Huang et al.,  Comp. Appl. BioSci . (1992) 8:155-65 and Pearson et al.,  Meth. Mol. Biol . (1994) 24:307-31. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al.,  J. Mol. Biol.  215: 403-10(1990)) is available from the National Center for Biological Information (NBCI, Bethesda, Md.), etc., for use in connection with the sequence analysis programs such as blast, blastp, blasm, blastx, tblastn and tblastx. BLAST can be accessed at www.ncbi.nlm.nih.gov/BLAST/and a sequence homology comparison method using this program is available at www.ncbi.nlm.nih.gov/BLAST/blast_help.html. 
     The recombinant single-chain FVIII according to the present invention includes those having a molar activity of 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% as compared to the wild type two-chain FVIII. The methods for measuring the wild type two-chain FVIII and the activity thereof are known in the art, and can be conducted, for example, by determining the specific activity values measured by CS and clotting assay (APTT: activated partial thromboplastin time) and (APTT)/CS ratios (Clotting assays Circulation 2005; 112: e53-e60). Those skilled in the art will be able to measure the activities by selecting a suitable method with reference to the techniques in the art and descriptions of reference documents, and determine the recombinant single-chain FVIII included in the scope of the present invention based on the measurement. The two-chain FVIII is processed by furin as described above and is in the form of a dimer composed of a light chain and a heavy chain. In the present invention, comparisons were made with the recombinant protein of wild type FVIII (Advate®), which is expressed in a recombinant form in CHO cells and most of which is in the form of a two-chain containing a B domain of various lengths. The recombinant protein of wild type FVIII has compositions similar to the plasma-derived wild type FVIII. 
     The two-chain FVIII to be compared with the single-chain FVIII according to the present invention comprises or does not comprise the B domain. The two-chain FVIII which does not comprise the B domain can be mainly produced by intentionally removing the B domain through a recombinant method using a protein cleavage process by FXa (an active form of FX) and thrombin during the procedure of wild type FVIII expression. The two-chain FVIII which comprises a portion of or the entire B domain can be produced by a recombinant method, or by a protein cleavage process by FXa (an active form of FX) and thrombin during the procedure of wild type FVIII expression. However, the two-chain FVIII compared with the single-chain FVIII of the present invention was recombinantly expressed from the wild type FVIII comprising the entire B domain. The two-chain FVIIIs are a mixture of the two-chain FVIII comprising the entire B domain produced by the furin processing when secreted from the inside of the cell to the outside and the FVIII which does not comprise the B domain at all or comprises a portion of the B domain produced by removing a portion of or entire B domain by thrombin or FIXa or FXa thereafter, most of which are recombinant FVIIIs similar to the wild type FVIII. 
     According to another embodiment, the present invention provides a nucleic acid molecule or polynucleotide encoding the single-chain FVIII according to the present invention, or a vector comprising the polynucleotide, or a cell (line) transfected with the vector. 
     The nucleic acid molecule encoding the recombinant single-chain FVIII according to the present invention includes codon-optimized one for the type of cells in which the recombinant single-chain FVIII according to the present invention is expressed. According to one embodiment of the present invention, the codon-optimized nucleic acid sequence may be optimized for the CHO cell codon, which is an optimized nucleic acid sequence encoding the protein of SEQ ID NO: 5, and can be represented by SEQ ID NO: 15. 
     A nucleic acid molecule encoding a recombinant single-chain FVIII according to the present invention also includes one encoding FVIII which is substantially identical and biological equivalent, as described above. 
     Also, the nucleic acid molecules encoding the recombinant single-chain FVIII according to the present invention may also be used for cloning various expression vectors for various purposes. The specific constitution of the expression vector may vary depending on the host cell in which the recombinant single-chain FVIII according to the present invention is to be expressed. However, it comprises the sequence which regulates the expression of the nucleic acid molecule of the present invention to mRNA or the expression of the mRNA to a protein, such as, for example, a promoter and/or an enhancer, and the like. Various vectors and regulatory sequences which may be used for the above or various other purposes are known in the art and may be selected appropriately by a person skilled in the art in light of the specific objects and effects of the present invention. Such vectors and sequences may include, for example, those disclosed in the Examples and Figures of the present invention, but are not limited thereto. 
     A vector comprising a nucleic acid molecule encoding a recombinant single-chain FVIII according to the present invention may be prepared by methods known in the art, for example, by operatively linking a nucleic acid molecule encoding a recombinant single-chain FVIII according to the present invention to a promoter and/or an enhancer. According to one embodiment, it is inserted into a recombinant expression vector which is capable of expressing a foreign gene in a cell, and examples of such vectors include, but are not limited to, general protein expression vectors such as pMSGneo and pcDNA3.1(+). The pMSGneo vector is an expression vector containing a MAR (Matrix attachment region) element that binds to a nuclear matrix, and the MAR element plays a role in enhancing gene expression by inducing position-independent expression when used in an expression vector. Thus, when the pMSGneo vector is used, a stable and high expression level can be achieved. In addition, the pcDNA3.1 (+) vector contains a strong CMV promoter and thus it is widely used for protein expression. 
     In addition, the recombinant expression vector comprising a nucleic acid molecule encoding the recombinant single-chain FVIII according to the present invention may be transfected into an appropriate host cell as described below for various purposes. In order to express the nucleic acid molecule contained in the vector as a protein, the coding sequence of the nucleic acid molecule encoding the protein may be suitably optimized for the desired cell. 
     In this respect, the present invention provides a recombinant cell transfected or transformed with a vector according to the present invention. Such cells include both prokaryotic and eukaryotic cells that can be used for the preparation of the desired protein by amplification of the vector according to the present invention and/or expression of the nucleic acid molecule contained in the vector. Various cells which can be used for the above purpose are known in the art, and a person skilled in the art will be able to select appropriate cells in consideration of the specific purposes and effects of the present invention, including those described in the Examples and Figures of the present invention, but is not limited thereto. For example,  E. coli , mammalian cells, yeasts, plant cells, and insect cells can be included. According to one embodiment of the present invention, eukaryotic cells, in particular, mammalian cell lines, are used for the expression of recombinant FVIII. A person skilled in the art will be able to select appropriate cell lines capable of preparing proteins having such property in consideration of the property of the recombinant FVIII according to the present invention. For example, CHO, BHK, COS7, HEK cell lines, etc., which are known in the art, may be used as such mammalian cell lines. Preferably, CHO cell line, specifically CHO-S cell line, CHO-DG44 or CHO-K1 cell line, or HEK cell line, specifically HEK293 cell line, may be used. 
     The vector according to the present invention is transferred to said host cells for expression. Methods for transferring vectors to such host cells are known in the art, which may be implemented by known methods in the art, such as calcium phosphate precipitation, a shotgun method, a method using liposome, a nanoneedle method or electroporation, but are not limited thereto. 
     The single-chain FVIII according to the present invention may be modified at a specific residue by a hydrophilic polymer for the purpose of increasing the half-life. 
     The protein structure of the B domain has not yet been revealed. The B domain is known as a protein having a flexible structure, high degree of glycosylation and abundant negative charges, but its structure is difficult to predict. In other words, it is theoretically possible to predict the structure of the B domain using the molecular modeling technique, but it is difficult to predict the B domain structure when considering the heterogeneity due to the post-translational modification and glycosylation of the B domain. In order to select the modification position in the B domain, the structure information on the surface of the B domain protein structure is required. However, since there is no information on the structure, it is very difficult to select the pegylation position. 
     However, in the present invention, the residue of the A domain and/or the B domain which does not particularly affect the activity of the single-chain FVIII was selected through an unexpected effort and modified with a hydrophilic polymer. As a result, a single-chain FVIII having an improved half-life while retaining its inherent property was obtained. 
     In particular, in the present invention, the positions between −6 and +6 residues based on the residue (0) near the glycosylation of the B domain were selected as pegylation positions. 
     In this regard, the present invention is related to a single-chain FVIII wherein the single-chain FVIII is conjugated to a hydrophilic polymer at some amino acid residues of the A domain and/or B domain fragment; the conjugation position of the B domain fragment is at least one selected from the group consisting of amino acid residues 754, 781,782, 788, 789, 825 and 897 based on the sequence of SEQ ID NO: 1 and the conjugation position of the A domain is at least one selected from the group consisting of amino acid residues 491, 495, 498 and 1806 based on the sequence of SEQ ID NO: 1; and the residue of the conjugation position is substituted with cysteine for the conjugation with the hydrophilic polymer. 
     Various polymers known in the art can be used as the hydrophilic polymer for use in the modification of the single-chain FVIII according to the present invention, which include, for example, polyethylene glycol, polyethylene oxide, dextran, polisialic, etc., but is not limited thereto. These polymers have a biocompatible (non-toxic or low toxicity) hydrophilic flexible structure, and when chemically conjugated to a protein, the polymers play a role in inhibiting the loss of activities of the conjugated protein due to adhesion to the surface of other molecule or material by a non-specific interaction, and inhibiting inactivation by protease in the blood. 
     According to one embodiment of the present invention, PEG is used, and specifically, PEG of 20 kDa or more is used. PEG is conjugated to a protein and provides an effect of wrapping around the protein, which leads to the defense against the loss after binding to a scavenger receptor or degradation by the inactivating protease. PEG is a polymer in a chain form that is linear or branched, and PEG having small molecular weight cannot fully wrap the protein and thus cannot fully protect the protein. Thus, the molecular weight should be the same or above the critical level to sufficiently wrap the protein to be protected. Pegylation of the single-chain FVIII of the present invention can be carried out by substituting the amino acid at the pegylation position with cysteine and then conjugating to the PEG containing an acryloyl, sulfone or maleimide group specific to cysteine at one end. 
     That is, in the single-chain FVIII according to the present invention, specific residue may be substituted for modification to a hydrophilic polymer, and specifically, the residue to be substituted varies depending on the position where the hydrophilic polymer is conjugated in the single-chain FVIII, and a person skilled in the art may be able to modify with a suitable residue for the conjugation of a hydrophilic polymer. 
     According to one embodiment of the present invention, pegylation is used for the modification. In such case, the residue to be modified is substituted by cysteine. 
     According to one embodiment of the present invention, modification is conducted at 495 valine residue of the A2 domain and/or 782 isoleucine residue of the B domain, and specifically the residues are substituted with cysteines for pegylation, and the residues are pegylated. 
     Expression of a protein substituted with cysteine for modification can be conducted using a method known in the art. For example, it is necessary to increase the pegylation efficacy by stabilizing the introduced free cysteine through masking the introduced free cysteine until the free cysteine introduced in a reduction condition is restored just before pegylation. Usually, the introduced free cysteine of a protein to be pegylated is stabilized by a disulfide bond with the low molecular weight substance containing free thiol, cysteine or glutathione, during the expression inside a cell or after the secretion outside the cell. 
     Pegylation can be conducted using a method known in the art. For example, cysteine or glutathione used for masking free cysteine should be removed prior to the pegylation reaction so that the free thiol of the introduced cysteine can be restored, and said process can be conducted including a step of treating with reduction agents such as TCEP, DTT, beta-mercaptoethanol, cysteine, and glutathione (reduced type), and an oxidation step for restoring the FVIII&#39;s original disulfide bonds cleaved by reduction. 
     Masking of the introduced cysteine of the expressed recombinant protein can be carried out using known methods. For example, the cysteine or glutathione used for masking free cysteine may be further added to the cell culture medium components, or the cysteine or glutathione components normally produced, stored, and secreted by cells during cell growth and maintenance can be used without further addition to the cell culture medium component. 
     In another aspect, the present invention also provides a method for producing a single-chain FVIII comprising transfecting a vector according to the present invention described above to a eukaryotic cell; culturing the cell in a culture solution; collecting the culture solution to purify a cysteine-introduced FVIII; and treating the purified cysteine-introduced FVIII with a pegylation buffer solution; restoring the free thiol group of the introduced cysteine through a reduction process; oxidizing the resultant to recover a separated disulfide bond in the single-chain FVIII during the reduction process; pegylating the cysteine of the single-chain FVIII restored with a reduced thiol group, and isolating the pegylated single-chain FVIII. 
     Reference can be made to those described in the Examples herein regarding specific vectors, cells and processing steps used in the method according to the present invention. 
     The recombinant single-chain FVIII according to the present invention can be useful for blood coagulation of a patient with a disease requiring blood coagulation, i.e., hemophilia, specifically, Hemophilia A, or for the treatment of Hemophilia A. 
     Accordingly, the present invention also provides a blood coagulation kit for use by a hemophilia patient or a patient requiring blood coagulation, comprising any one of the single-chain FVIIIs described in the present invention including the claims, a use of said single-chain FVIII in treating hemophilia or a composition comprising the same, a composition comprising a nucleotide sequence encoding said single-chain FVIII, a use of the composition in genetic treatment of Hemophilia A, a method for hemophilia treatment or blood coagulation comprising administering the composition comprising a nucleotide sequence encoding said single-chain FVIII or administering said single-chain FVIII in a therapeutically effective amount to a hemophilia patient or a patient requiring blood coagulation. 
     The recombinant single-chain FVIII according to the present invention can be provided in the form of a pharmaceutical composition for treating hemophilia which also contains a pharmaceutical acceptable carrier. 
     In addition, the pharmaceutical composition of the present invention can be used alone or in combination with other drug therapies and methods using a biological response modifier. 
     In addition to the above-mentioned active ingredients, the composition of the present invention may be prepared by further comprising at least one pharmaceutically or physiologically acceptable carrier. 
     The term “carrier” as used herein is intended to mean a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to a cell or mammal exposed to the same at a dose and concentration employed. Examples of such carriers include saline, Ringer&#39;s solution, buffered saline, buffers such as phosphate, citrates and other organic acids, antioxidant such as ascorbic acids, polypeptides with low molecular weight (about less than 10 amino acids), proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, carbohydrates such as monosaccharides, disaccharides, and glucose, mannose or dextrin, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, counter ions for salt formation such as sodium, and/or non-ionic surfactant such as Tween®, polyethylene glycol (PEG) and PLURONICS®. 
     If desired, the composition may further comprise other additives known in the art such as antioxidants, buffers, antibacterial agents. The composition may be formulated into injectable formulations such as solution, suspension, emulsion, etc., by adding diluents, dispersing agents, surfactants, binders and lubricants. 
     Further, the composition can be preferably formulated depending on the disease or ingredients, using appropriate methods in the art or as disclosed in Remington&#39;s Pharmaceutical Science (recent edition; Mack Publishing Company, Easton Pa.). 
     The composition of the present invention is preferably administered parenterally (for example, intravenous, subcutaneous, intraperitoneal) according to a desired method. The dosage varies depending on the disease condition and weight of a patient, degree of the disease, the type of a drug, the administration route and time, but can be appropriately selected by a person skilled in the art. 
     The composition according to the present invention is administered in a therapeutically effective amount. In the present invention, “a therapeutically effective amount” refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio which is applicable to a medicinal treatment. The level of effective dose may be determined depending on factors including types of diseases of patients, severity, activity of a drug, sensitivity to a drug, administration time, administration routes, excretion rates, period of treatment, a simultaneously used drug, and other factors known in the medicinal field. The composition of the present invention may be administered as a single therapeutic agent or administered in combination with other therapeutic agents. Also, the composition of the present invention may be administered with a conventional therapeutic agent sequentially or simultaneously, and the composition may be used for single or multiple administrations. It is important to administer the composition at a minimal dose which can lead to a maximum effect without side effects in consideration of all of the factors described above, and the doses may be easily determined by a person skilled in the art. 
     The recombinant single-chain FVIII according to the present invention may be administered as it is or in the form of a composition as described above in a therapeutically effective amount to a subject with hemophilia or in need of blood coagulation and may be provided in the form of a blood coagulation method or a method for treating hemophilia, and reference can be made to the foregoing description. 
     As used herein, the term “treatment” refers to any action that improves or alters the symptoms of a disease in a beneficial way by administration of a composition of the present invention. The subjects for whom the method according to the present invention is used may be primates, including humans, but are not limited thereto. 
     Hereinafter, examples to aid in the understanding of the present invention will be presented. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples. 
     EXAMPLE 
     Example 1. Construction of Single-Chain FVIII 
     In order to prepare a human FVIII in a single-chain form (single-chain FVIII, scFVIII) which has a high expression rate while retaining the inherent property of human FVIII, recombinant scFVIIIs containing various length of the B domain were constructed and expressed by modifying the length of the B domain including a3 of FVIII, and the expression levels and activities (APTT, CS) thereof were analyzed. Detailed methods and results are as follows. 
     Example 1-1. Preparation of scFVIII 
     For the construction of FVIII expressed in a single-chain form, FVIII was constructed in which a portion of the B domain including the furin cleavage site, i.e., the residue 1648 (based on the amino acid sequence of SEQ ID NO: 1 which represents the full length FVIII amino acid sequence), was deleted. Since the length of the B domain connecting the heavy chain and light chain may affect the final activity and property of FVIII, a total of 7 types of FVIII were constructed as shown in Table 1 for the determination of the length of the B domain comprised in the scFVIII. Each scFVIII was constructed such that it comprises the B domain of a specific length counting from the N-terminal (the 741 st  amino acid residue of SEQ ID NO: 1) of the B domain, and the B domain contains the originally present sugar chain as it is. 
     In addition, in order to impart a property similar to the wild type FVIII activity by minimizing the structural interference caused by the B domain connecting the light chain and the heavy chain region, a structure in which a portion of the a3 domain adjacent to the light chain domain is contained more than other scFVIIIs was also prepared (SEQ ID NO: 6). Further, in order to improve the expression in CHO cells, scFVIII optimized with the CHO codon was prepared (SEQ ID NO: 15). 
     Each scFVIII comprising the partially-deleted B domain was synthesized based on the human FVIII nucleic acid sequence (NM000132). In order to prepare the scFVIII gene expression vector, G1 protein sequence (leader-heavy chain-B domain (741-764, 1653-1689)-light chain) was encoded and the entire gene was synthesized by GeneArt to include the NotI-AsisI enzyme cleavage site at the 5′ end and XhoI-PacI enzyme cleavage site at the 3′ end based on the human FVIII nucleic acid sequence (NM000132). The pcDSW-G1 expression vector was constructed by cloning the synthesized gene into the NotI/XhoI site of the pcDSW vector. The accuracy of the expression vector construction was confirmed by size comparison of the bands which were cleaved using the AsisI/PacI enzyme. Then, each gene corresponding to each scFVIII in which the B domain was deleted (G2, G3, G4, G4-flex, G6) was synthesized by GeneArt to include a sequence from the BamHI site (GGATCC) present in the A2 domain of the FVIII heavy chain to the Pad enzyme cleavage site at the end of the light chain. Then, the pre-existing FVIII region was removed from the previously prepared pcDSW-G1 expression vector using the enzyme BamHI/PacI, and the synthesized gene was cleaved with BamHI/PacI enzyme and cloned into the BamHI Pad site of the pcDSW-G1 expression vector to construct the pcDSW-scFVIII expression vector. The accuracy of the expression vector construction was confirmed by size comparison after cleavage with BamHI/PacI enzyme. In order to improve expression, regarding scFVIII (G6_opt), codon optimization and gene synthesis were conducted by GneArt to include NotI-AsisI cleavage site at 5′ and XhoI-PacI cleavage site at 3′, and to be suitable for animal cell expression. The synthesized genes were cloned into the NotI/XhoI site of the pcDSW vector to construct an expression vector. The accuracy of the expression vector construction was confirmed by size comparison after cleavage with BamHI enzyme. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 scFVIIIs comprising B domains of various lengths constructed in the present invention 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 Portion of 
                   
                   
                   
                   
               
               
                   
                   
                 B domain 
               
               
                   
                   
                 sequence* 
               
               
                   
                 Heavy 
                 (including 
                 Light 
                 Number of 
               
               
                   
                 chain 
                 a3 
                 chain 
                 Glycosylation 
                 Protein 
                 Nucleic acid 
               
               
                 scFVIII 
                 sequence* 
                 domain) 
                 sequence* 
                 in B domain 
                 SEQ ID NO 
                 SEQ ID NO 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 G1 
                 scF1 
                 1-740 
                 (741-764)— (1653-1689) 
                 1690-2332 
                 2(757, 764) 
                 SEQ ID NO: 8 
                 SEQ ID NO: 16 
               
               
                 G2 
                 scF2 
                 1-740 
                 (741-788)—(1654-1689) 
                 1690-2332 
                 2(757, 784) 
                 SEQ ID NO: 2 
                 SEQ ID NO: 10 
               
               
                 G3 
                 scF3 
                 1-740 
                 (741-832)—(1654-1689) 
                 1690-2332 
                 3(757, 784, 
                 SEQ ID NO: 3 
                 SEQ ID NO: 11 
               
               
                   
                   
                   
                   
                   
                 828) 
               
               
                 G4 
                 scF4 
                 1-740 
                 (741-902)—(1654-1689) 
                 1690-2332 
                 4(757, 784, 
                 SEQ ID NO: 4 
                 SEQ ID NO: 12 
               
               
                   
                   
                   
                   
                   
                 828, 900) 
               
               
                 G6 
                 scF5 
                 1-740 
                 (741-965)— (1654-1689) 
                 1690-2332 
                 6(757, 784, 
                 SEQ ID NO: 5 
                 SEQ ID NO: 13 
               
               
                   
                   
                   
                   
                   
                 828, 900, 
               
               
                   
                   
                   
                   
                   
                 943, 963) 
               
               
                 G4_flex 
                 scF6 
                 1-740 
                 (741-902)—(1637-1642)—(1654-1689) 
                 1690-2332 
                 4(757, 784, 
                 SEQ ID NO: 6 
                 SEQ ID NO: 14 
               
               
                   
                   
                   
                   
                   
                 828, 900) 
               
               
                 G6_opt 
                 scF7** 
                 1-740 
                 (741-965)— (1654-1689) 
                 1690-2332 
                 6(757, 784, 
                 SEQ ID NO: 7 
                 SEQ ID NO: 15 
               
               
                   
                   
                   
                   
                   
                 828, 900, 
               
               
                   
                   
                   
                   
                   
                 943, 963) 
               
               
                   
               
               
                 *Described based on the sequence of SEQ ID NO: 1 
               
               
                 **Codon-Optimization for CHO cells 
               
            
           
         
       
     
     Example 1-2. scFVIII Expression and Activity Measurement 
     scFVIII Expression 
     The scFVIII constructed in Example 1-1 was expressed in cells, and the cells were cultured in a cell culture medium. First, for the expression, the expression vector constructed in Example 1-1 was transfected into Expi293F™ cells using the Expi293F™ Expression System Kit (Thermofisher Co., Catalog Number A14635), a transient expression system. The Expi293F™ cells were subcultured at a concentration of 2.0×10 6  cells/mL using the Expi 293 culture medium according to the expected required amount 24 hours prior to transfection, and on the day of transfection, the cell number and cell viability were measured, and if the cell viability was 95% or higher, transformation was carried out. 7.5×10 −7  cells were added to a 125 mL flask with the Expi293 culture medium to a final volume of 25.5 mL (based on 30 mL). 30 μg of the expression vector constructed in Example 1-1 was mixed with Opti-MEM to obtain the total volume of 1500 μl. 80 μl of a transfection reagent was mixed with Opti-MEM to a total volume of 1500 μl and incubated at room temperature for 5 minutes. Five minutes later, the Opti-MEM containing the transfection reagent was added to the Opti-MEM containing DNA and the resultant was gently mixed. The reaction was carried out at room temperature for 20 to 30 minutes. 3 mL of DNA: transfection reagent complex was added dropwise to 125 mL flask of Expi293F™ cells (total volume: 28.5 mL) prepared beforehand and the cells were incubated at 37° C. in a 5% CO 2  shaking incubator at 125 rpm. After 16˜20 hours, 150 μl and 1.5 ml of Enhancer 1 and Enhancer 2 were added thereto, respectively, and the cells were cultured in a shaking incubator of 34° C. and 5% CO 2  at 125 rpm. On the third day of culture, the medium was harvested and the activity of scFVIII was measured as follows. 
     Measurement of Expressed scFVIII Activity 
     Currently, two types of recombinant FVIII, a full length FVIII and FVIII having a deleted B domain, are used clinically. However, the issue of whether these two recombinant FVIIIs are clinically equivalent in activity and efficacy has been raised (GRUPPO, R. A. et al., 2003. Comparative effectiveness of full-length and B domain deleted Factor VIII for prophylaxis-a metaanalysis. Haemophilia, 9, 251-60; LOLLAR, P. 2003. The Factor VIII assay problem: neither rhyme nor reason. J Thromb Haemost, 1, 2275-9; MIKAELSSON, M. et al., 2001. Measurement of Factor VIII activity of B domain deleted recombinant Factor VIII. Semin Hematol, 38, 13-23). 
     The activity of BDD-FVIII measured by clotting assay (APTT) was found to be lower than that measured by chromogenic assay (CS) by up to 50%, while the activities of the FVIII of a full length form was similar in both measurements (BARROWCLIFFE et al., SEMIN THROMB HEMOST, vol. 28 (3), 2002: 47-56). The low activity of BDD in the clotting assay has been suggested as a reason that BDD-FVIII is less effective in preventing hemorrhage in patients with Hemophilia A as compared to full length FVIII in actaul long-term prophylaxis (GRUPPO, R. A., et al., 2003. Comparative effectiveness of full-length and B domain deleted Factor VIII for prophylaxis—a metaanalysis. Haemophilia, 9, 251-60; GRUPPO, R. A. et al., 2004. Meta-analytic evidence of increased breakthrough bleeding during prophylaxis with B domain deleted Factor VIII. Haemophilia, 10, 747-50; GRUPPO, R. A. et al., 2004. Increased breakthrough bleeding during prophylaxis with B domain deleted Factor VIII-a robust metaanalytic finding. Haemophilia, 10, 449-51). 
     Thus, in the present invention, in order to select scFVIII having specific activities (CS, APTT) and an specific activity ratio (APTT/CS) similar to those of the full length recombinant FVIII, the specific activities and specific activity ratio (APTT/CS) measured by CS and clotting assay (APTT) were analyzed regarding each of the scFVIII constructed in Example 1-1 and the result was used as criteria for scFVIII selection. As a comparative group, recombinant FVIII (ADVATE®, Baxalta) products in a full length, and two-chain forms, similar to the actual FVIII in vivo, were used as a control for comparison. 
     Specifically, the FVIII activity measurement test methods used here were the one-stage method and chromogenic method. The one-stage method is a method of measuring the FVIII activity of a sample by mixing the FVIII deficient plasma, activator, phospholipids and FVIII sample, and then measuring the blood coagulation time. The chromogenic method is a method in which FIXa, FX, thrombin, calcium, phospholipids and FVIII samples are mixed, and then a chromogenic substrate which forms color when cleaved by FXa is added to measure the amount of FXa activated by the FVIII sample, and the activity of FVIII is measured according to the degree of color formation of the chromogenic substrate. The difference between these two methods is that the one-stage method uses FVIII deficient plasma and measures the clotting time while the chromogenic method does not use the FVIII deficient plasma but measure the degree of color formation of a substrate after adding the factors necessary for activation of FVIII and the substrate for measuring the activated F VIII only. 
     Activity measurements by the one-stage method were conducted as follows using the automated assay method prepared before in the ACL TOP CTS 500 instrument. Samples were diluted to 1 IU/mL using FVIII deficient plasma, and the standard sample was put into the NIBSC FVIII standard vial and dissolved by adding 1 mL of distilled water thereto, and then diluted to 1 IU/mL using FVIII deficient plasma. Consumption reagents and measurement samples were prepared according to the rack type of the experimental equipment. The calcium chloride vials were serially put into the R rack of the ACL TOP CTS 500 equipment such that the barcodes of the vials could be recognized, and a magnetic stirring bar was put into the APTT-SP vial, which was then put into the R rack such that the barcodes of the vials could be recognized. The FVIII deficient plasma was prepared according to the number of samples ×400 μL (per each sample), and tape was placed around their barcodes so that the barcodes cannot be recognized, and then put into the R rack. Into the DA rack, a vial containing the G. O. buffer (Imidazole 3.4 g, NaCl 5.8 g, 1 L, pH 7.4) added with 2.63 mL of albumin was placed. After putting 400 μL or more of the samples and the standard sample prepared before into the sample cup, which was then placed in in the sample rack. In the ACL TOP program, information of unrecognized reagents and measurement samples were inputted, and the automated assay method prepared before was carried out. All reactions in the automated assay method were proceeded as follows at 37° C. First, the measurement samples were pre-diluted by 10-fold with the G. O. buffer, and then the samples were further diluted by 1, 3, and 10-fold to prepare 100%, 33,33%, and 10% samples, respectively. 50 μL each of the diluted samples was allowed to stand for 30 to 45 seconds, mixed with 50 μL of FVIII deficient plasma, and then allowed to stand for 60 to 70 seconds. After addition of 50 μL of the intermediate reagent APTT-SP, the resultant was allowed to stand for 300-340 seconds, and 50 μL of the start reagent (APTT-SP CaCl 2 ) was added thereto finally, and then the clotting time was measured. 
     As for the activity measurement by the chromogenic method, the chromogenic assay kit sold by CHROMOGENIX Co., was used as the endpoint method, and the test was conducted by modifying the test method provided by the manufacturer to be suitable for the present test as follows. Specifically, samples are diluted to be in the standard range (0.25-1 IU/mL) using 1× dilution buffer. Calibration plasmas were prepared using 1×dilution buffer at the concentrations of 0, 0.25, 0.5, and 1 IU/mL. The prepared samples and 10 μL of the calibration plasma were diluted by 790 μL of 1× dilution buffer, and 50 μL of the diluted samples were dispensed into a 96-well plate and allowed to stand at 37° C. for 5 minutes. 50 μL of a factor reagent was dispensed into each well using an automatic dispenser pipette, which was then reacted for 2 minutes at 37° C. 50 μL of a substrate was dispensed into each well, which was then allowed to stand for 2 minutes at 37° C. for color formation. The reaction was stopped by adding 50 mL of 2% citric acid to the samples showing color formation. The absorbance was measured at a wavelength of 405 nm to draw a linear calibration curve, and the absorbance of the sample was substituted into the calibration curve to measure the activity of the sample. 
     The measured results are shown in Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Results of measurement of the activities 
               
               
                 of the expressed recombinant scFVIIIs 
               
            
           
           
               
               
               
            
               
                   
                 CS activity (IU/mL) 
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Expression 
                   
                 Specific 
                   
               
               
                   
                 level 
                 Activity 
                 activity 
                 APTT/CS 
               
            
           
           
               
               
               
               
               
            
               
                 Single chain FVIII 
                 (ng/mL) 
                 (IU/mL) 
                 (IU/mg) 
                 ratio 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 G1 
                 scF1 
                 268 
                 0.56 
                 2090 
                 0.88 
               
               
                 G2 
                 scF2 
                 103 
                 0.46 
                 4437 
                 NA 
               
               
                 G3 
                 scF3 
                 143 
                 1.01 
                 7082 
                 NA 
               
               
                 G4 
                 scF4 
                 677 
                 5.22 
                 7714 
                 1.05 
               
               
                 G6 
                 scF5 
                 980 
                 6.57 
                 6704 
                 NA 
               
               
                 G4_flex 
                 scF6** 
                 801 
                 5.64 
                 7041 
                 1.12 
               
               
                 G6_opt 
                 scF7* 
                 619 
                 4.83 
                 7803 
                 NA 
               
               
                 — 
                 Advate 
                   
                   
                 6000-7000 
                 1.13 
               
               
                   
               
            
           
         
       
     
     As a result of expression, it was found that the scFVIII expression level in the culture fluid increased as the length of the B domain increased, and the expression levels of scF4, scF5, scF6, and scF7 having the B domain of 198 amino acids or more were significantly increased. In addition, scF3, scF4, scF5, scF6, and scF7, which had the B domain of 92 amino acids or more in length, showed the specific activities similar to that of the full length FVIII in a recombinant form (Advate®, Baxalta product) used as a control for comparison, and also showed APTT/CS ratios similar to the ADVATE® used as a control. On the other hand, scF1, scF2 and scF3 of the above Example, which had the B domain shorter than the 128 amino acid protein sequence, showed significantly lower expression levels and scF1 and scF2 of the above Example, which had B domain shorter than the 84 amino acid protein sequence showed significantly lower specific activities than the full length FVIII, Advate®, used as a control for comparison. 
     The single-chain FVIII is activated by thrombin cleavage at the site containing the B domain connecting the heavy and light chains in the activation step. When the length of the B domain, which is the linkage site between the heavy chain and the light chain, is short, it is considered that single-chain FVIII is not normally converted to an active form FVIII due to the structural restriction from the short linkage, which can affect its activity. However, in the case of two-chain FVIII, it is considered that there is no such structural restriction, and thus thrombin-induced activation is smooth regardless of the length of the B domain, which is evident from the finding that the activity of the two-chain FVIII in which pre-existing B domain had been completely removed shows an activity similar to the full length FVIII. In the present invention, through unexpected efforts, the length and sequence of the B domain of the single-chain FVIII were selected so as not to affect thrombin activation, and the thrombin activation site between the heavy and light chains (1689-1690 and 740-741; based on the sequence of SEQ ID NO: 1) was activated in a way similar to the natural-type (i.e., wild type) FVIII, to obtain the single-chain FVIII showing the specific activity similar to the full length FVIII used for comparison. 
     Example 1-3. Analysis of Expression Pattern of Constructed scFVIII 
     The stability of the single-chain FVIII constructed herein in the culture was confirmed by Western blot analysis. 
     For this, the culture solution and the LDS sample buffer were mixed at a ratio of 3:1, which was mixed with 4-12% bis-tris (BT) gel to prepare a sample, and 30 μL of the sample was loaded and run at 150 volts for about 1 hour. After the transferring the gel which completed the running to the Nitrocellulose (NC) membrane, the gel was left in a blocking buffer (Thermo Scientific) for 1 hour. The primary antibodies recognizing the heavy chain (GMA-012, Green Mountain Antibodies) and the light chain (ab41188, Abeam) of FVIII were prepared by dilution in the TBST buffer and then added to the NC membrane which completed blocking, and reacted for 1 hour. After washing the membrane three times with the TBST buffer at an interval of 5 minutes, secondary antibodies (Goat anti-mouse IgG-HRP conjugate) diluted in the TBST buffer were reacted with the NC membrane for 10 minutes. And then the membrane was washed 5 times at an interval of 5 minutes using the TBST buffer. After scattering the ECL Prime Western Blotting Detection Reagent to the NC membrane for the membrane to form colors, the membrane was developed in Hyperfilm ECL (GE healthcare) in a dark room. As a result, as shown in  FIG.  3   , it was found that all of the FVIIIs produced in a single-chain form were expressed in a single-chain form. In addition, it was found that the single-chain FVIII secreted to the culture medium which had shorter length of the B domain showed less decomposition in the culture medium and more stability. The scF1, scF2, and scF3, in which the B domains are relatively short, showed superior stability to scF4, scF5, scF6, and scF7, but showed a relatively low expression level or low specific activity. 
     In the present invention, the single-chain FVIIIs having the B domains of various length derived from natural-type two-chain FVIII was constructed and their properties were analyzed. The effects of the length of B domain on the expression level, specific activity and stability were evaluated, thereby preparing the recombinant single-chain FVIII having the activity characteristics similar to the natural-type FVIII and showing high expression level. 
     Example 2. Pegylation of scFVIII 
     Selection of Pegylization Position 
     As the sequence and length of the B domain in the single-chain FVIII including a portion of the B domain affects the activity and characteristics of the recombinant blood coagulation FVIII by affecting the activity characteristics of FVIII, in this Example, the pegylation positions were selected based on the scFVIII prepared in Example 1 comprising the B domain with a length and sequence capable of retaining the activity characteristics of a natural-type two-chain FVIII in order to provide in vivo persistence. 
     In order to optimize the pegylation reaction efficiency in the B domain, pegylation was conducted at various positions of the B domain. As the pegylation targets the surface residues of the protein, a three-dimensional structure of the B domain is required to select the pegylation position, but the structure of the B domain is not known, making it difficult to select the pegylation position. The pegylation position of the B domain selected in the present invention was selectively pegylated around the sugar chain of the B domain. That is, the sugar chain is located outside the protein structure, and the residues around the sugar chain in the B domain are likely to be always directing outward, which is considered to be advantageous for pegylation. However, there was a problem that the closer to the sugar chain, the more difficult pegylation can be owing to the steric hindrance by the sugar chain&#39;s interfere with the access of PEG. In the present invention, the residues near the sugar chain positioned at an appropriate distance from the sugar chain, which are directing outward in the protein structure of the B domain and free from steric hindrance by the sugar chain thereby ensuring the high pegylation efficiency were selected through unexpected efforts. 
     In this Example, as shown in Table 3 and  FIG.  4   , recombinant scFVIIIs to which cysteine was introduced in various positions near the sugar chain of the B domain based on the G4 constructed in Example 1, that is, between the +6 and −6 residues based on the N-sugar chain, were produced, and pegylation was selectively conducted to the introduced cysteine. Such pegylation has been described with regard to the G4 and single-chain FVIII, but can also be applied to pegylation of other scFVIII including a sugar chain constructed herein with the same logic. 
     Example 2-1. Construction of scFVIII Expression Vector in which Specific Site is Substituted with Cysteine 
     The gene was synthesized by GeneArt to include a sequence from the BamHI site (GGATCC) present in the A2 domain of the FVIII heavy chain including the cysteine substitution site to the light chain and the Pad enzyme cleavage site. Then, the pre-existing corresponding FVIII region was removed from the pcDSW-scFVIII G4 expression vector produced in Example 1-1 using the enzyme BamHI/PacI, and the synthesized gene was cloned into the BamHI/PacI site to construct the pcDSW-scFVIII G4 (B1, B2, B3, B4, B5, B6, B7, A3-1 or 4L) expression vector substituted with cysteine. The accuracy of the expression vector construction was confirmed by sequence analysis. In addition, regarding the scFVIII G4 (A2-1, A2-2, or A2-3), the gene was synthesized by GeneArt to include a sequence from the AsisI enzyme cleavage site present in the A 1 domain of the FVIII heavy chain including the cysteine substitution site to the KpnI site (GGTACC) present in the A2 domain of the FVIII heavy chain. Then, the pre-existing corresponding FVIII region was removed from the expression vector pcDSW-scFVIII G4 produced in the Example 1-1 using the enzyme AsisI/KpnI, and the synthesized gene was cloned into the AsisI/KpnI site to construct pcDSW-scFVIII G4 (A2-1, A2-2, or A2-3) expression vectors substituted with cysteine. The accuracy of the expression vector construction was confirmed by sequence analysis. 
     Transient expression was conducted by the same method as in Example 1-2 using the constructed mscFVIII expression vector into which cysteine was introduced. Each of the expression levels of the cysteine-introduced FVIIIs is shown in Table 3 below. As shown in Table 3, the scFVIII mutant into which cysteine is introduced around the glycan chain of the B domain was found to be expressed normally in the culture solution. Its expression level was somewhat lower than scFVIII prior to the introduction of cysteine, suggesting that the cysteine introduced into the scFVIII affects the expression process and the stability of scFVIII in the culture solution. 
     Example 2-2. Pegylation 
     For the screening of peglyation positions, pegylation of cysteine-introduced scFVIII mutants as in Example 2-1 was conducted as follows. 50 ml of the cell culture solution was concentrated by 20-fold with 5 kDa MWCO (molecular weight cutoff), and 5 M NaCl conc solution was added thereto to the final concentration of 1 M NaCl. 0.1 mL volume of V8 select resin (GE) equilibrated with the equilibrium buffer (20 mM Histidine, 500 mM NaCl, 0.1% (w/v) Tween® 80, pH 7.0) and a concentrated sample were thoroughly stirred together, washed with the equilibration buffer, and eluted with the elution buffer (20 mM Histidine, 500 mM NaCl, 0.1% (w/v) Tween® 80, pH 7.0, 50% (v/v) propylene glycol). The eluted samples were exchanged with the pegylation buffer (20 mM Histidine, 500 mM NaCl, 0.1% (w/v) Tween® 80, pH 7.0, 5% (w/v) sucrose) using a 5 kDa MWCO membrane, and then the TCEP was treated at 0.2 mM in final and reacted at 4° C. for 1 hour. Then, in order to remove TCEP of the reactant, the sample was desalted with the pegylation buffer (20 mM Histidine, 500 mM NaCl, 0.1% (w/v) Tween® 80, pH 7.0, 5% (w/v) sucrose), and then the maleimide PEG 40 kDa (NOF) was added at a molar ratio of 1:20 (scF VIII: maleimide PEG 40 kDa) and the resultant was reacted at 4° C. for 1 hour. 
     In the scFVIII mutants into which cysteine was introduced, cysteine is masked by cysteine or glutathione with a disulfide bond when expressed through cell culture. Thus, the masking cysteine and glutathione should be removed for pegylation. In the present invention, TCEP (tris (2-carboxyethyl) phosphine) was added to remove cysteine or glutathione which is masking the free thiol group of cysteine, and the TCEP was removed through a desalting column (PD10). And then, the resultant was treated with maleimide-PEG (40 kDa) and pegylated. 
     SDS PAGE was conducted for the pegylated reactants to analyze the degrees of pegylation. 
     The results are shown in  FIG.  6   . It was found that the G4scFVIII mutant into which cysteine is introduced was expressed in an active form in the culture solution and the pegylation was proceeded easily. There was a difference in the expression level and pegylation rate depending on the introduction site of cysteine, which is considered to be due to the difference in the stability of FVIII and pegylation reaction depending on the site of introduced cysteine. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Expression levels and pegylation rates of the mutants in which 
               
               
                 the scFVIII conjugation site is substituted with cys. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 Expression 
                 Protein 
                   
                   
                   
               
               
                   
                 Cys introduction position* 
                 level 
                 stability 
                 Pegylation 
                 Nucleic acid 
                 Amino acid 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 ScFVIII mutant 
                 Position 
                 Domain 
                 (IU/mL) 
                 (western) 
                 efficiency 
                 sequence 
                 sequence 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 G4_A2_1 
                 L/491/C 
                 A2 
                 1.53 
                 +++ 
                 ++ 
                 SEQ ID NO: 17 
                 33 
               
               
                 G4_A2_2 
                 V/495/C 
                 A2 
                 1.68 
                 +++ 
                 ++ 
                 SEQ ID NO: 18 
                 34 
               
               
                 G4_A2_3 
                 L/498/C 
                 A2 
                 1.35 
                 +++ 
                 + 
                 SEQ ID NO: 19 
                 35 
               
               
                 G4_A3_1 
                 F/1806/C 
                 A3 
                 2.47 
                 +++ 
                 + 
                 SEQ ID NO: 20 
                 36 
               
               
                 G4_B1 
                 K/754/C 
                 B 
                 2.94 
                 +++ 
                 ++ 
                 SEQ ID NO: 21 
                 37 
               
               
                 G4_1D 
                 D/765/C 
                 B 
                 0.97 
                 + 
                 − 
                 SEQ ID NO: 22 
                 38 
               
               
                 G4_2D 
                 D/770/C 
                 B 
                 0.51 
                 + 
                 − 
                 SEQ ID NO: 23 
                 39 
               
               
                 G4_3R 
                 R/776/C 
                 B 
                 0.41 
                 + 
                 − 
                 SEQ ID NO: 24 
                 40 
               
               
                 G4_B2 
                 K/781/C 
                 B 
                 6.33 
                 +++ 
                 ++ 
                 SEQ ID NO: 25 
                 41 
               
               
                 G4_B3 
                 I/782/C 
                 B 
                 9.07 
                 +++ 
                 ++ 
                 SEQ ID NO: 26 
                 42 
               
               
                 G4_B4 
                 S/788/C 
                 B 
                 4.74 
                 +++ 
                 ++ 
                 SEQ ID NO: 27 
                 43 
               
               
                 G4_B5 
                 D/789/C 
                 B 
                 5.07 
                 +++ 
                 ++ 
                 SEQ ID NO: 28 
                 44 
               
               
                 G4_4L 
                 L/790/C 
                 B 
                 4.13 
                 +++ 
                 ++ 
                 SEQ ID NO: 29 
                 45 
               
               
                 G4_B6 
                 I/825/C 
                 B 
                 6.64 
                 +++ 
                 ++ 
                 SEQ ID NO: 30 
                 46 
               
               
                 G4_B7 
                 G/897/C 
                 B 
                 10.9 
                 +++ 
                 + 
                 SEQ ID NO: 31 
                 47 
               
               
                 G4_1I1D 
                 D/765/C.I/ 
                 B and A3 
                 0.13 
                 − 
                 − 
                 SEO ID NO: 32 
                 48 
               
               
                   
                 1668/C 
               
               
                   
               
               
                 *The residues in Table 3 above are based on the sequence of SEQ ID NO: 1 
               
            
           
         
       
     
     Example 3. Production of Pegylated scFVIII 
     In this Example, the scFVIIIs pegylated at the sites selected in Example 2 were produced as follows. In addition, the pegylated scFVIIIs were produced and their activities as long-acting type blood coagulation FVIII in vivo and the effect of pegylation on the activity of blood coagulation FVIII were evaluated. Site-specific conjugates of G4 and B3, which is scFVIII in which the isoleucine at 782 in the B domain of the scFVIII was substituted with cysteine, were produced and their activities were measured by the CS and OS (APTT) methods, and compared with ADVATE®, are combinant blood coagulation FVIII of a two-chain form. 
     Example 3-1. Cell Culture 
     The scFVIII G4 (B3) and scFVIII G4 (A2_1) to which cysteine was introduced and constructed in Example 2-1 were expressed and cultured in each cell. Specifically, for expression, the expression vector constructed in Example 2-1 was transfected into Expi293F™ cells using the Expi293F™ Expression System Kit (Thermofisher, Catalog Number A14635) according to the manufacturer&#39;s method. In summary, cell numbers and viability were measured at the time of transformation and transformation was carried out when the cell viability was 95% or higher. The Expi293 culture medium was added to a 125 mL flask to obtain the concentration of 7.5×10 7  cells, which was adjusted to 25.5 mL. 30 ug of the expression vector constructed in Example 2-2 was mixed with Opti-MEM to a total volume of 1500 ul. 80 ul of the transfection reagent was mixed with Opti-MEM to a total volume of 1500 ul and incubated at room temperature for 5 minutes. Five minutes later, the Opti-MEM containing the transfection reagent was added to the Opti-MEM containing DNA and the resultant was gently mixed. The reaction was carried out at room temperature for 20 to 30 minutes. 3 mL of DNA: transfection reagent complex was added dropwise to 125 mL flask of Expi293F™ cells (Total volume: 28.5 mL) prepared beforehand and the cells were incubated at 37° C. in a 5% CO 2  shaking incubator at 125 rpm. After 16˜20 hours, 150 μl and 1.5 ml of Enhancer 1 and Enhancer 2 were added thereto respectively, and the cells were cultured in a shaking incubator of 34° C. and 5% CO 2  at 125 rpm. On the second day of culture, all cells were centrifuged and the pre-existing culture medium was completely removed. Cells were completely resuspended in 30 mL of new culture medium and cultured at 34° C. in a 5% CO 2  shaking incubator at 125 rpm. On the third day from the transfection, scFVIII-expressing cells were all centrifuged and the culture solution was recovered. The cells remaining after the recovery of the medium were resuspended in a new culture medium of the same volume and cultured at 34° C. for 1 day. This process was repeated until the 5th day of transfection, and the culture solutions on the 3rd, 4th, and 5th days from the transfection were recovered to conduct the activity analysis and purification of cysteine-introduced scFVIII. 
     Example 3-2. Pegylation and Purification 
     The pegylation and purification process is composed of five steps. The culture solutions on the 3rd, 4th, and 5th days of culture medium in Example 3-1 were pooled to carry out purification. 
     In the first step, the process of separating and purifying FVIII from the culture medium was conducted using the VIIISelect resin developed by GE Inc. for purification. The VIIIS elect resin was packed into a column and the column was washed with 2% citric acid. It was equilibrated by flowing an equilibrium buffer (20 mM Histidine, 5 mM CaCl 2 , 1 M NaCl, 0.02% Tween® 80, pH 7.0). 5 M NaCl buffer was added to the culture solution to a final concentration of 1 M NaCl, which was then titrated to pH 7.0, and loaded onto the column. After loading the culture solution, it was equilibrated by flowing the equilibrium buffer until the UV fell to the baseline. ScFVIII was rapidly eluted by flowing an elution buffer (20 mM Histidine, 5 mM CaCl 2 , 0.9 M Arginine, 45% propylene glycol, 0.02% Tween® 80, pH 6.5). 
     And, in the second step, a process of rapidly removing the propylene glycol mixed in the eluent in the first step was conducted using SP fast flow resin from GE Inc. After the SP fast flow resin was packed into a column, the column was washed by flowing a washing buffer (0.5 M NaOH, 1 M NaCl). It was equilibrated by flowing an equilibrium buffer (20 mM Histidine, 5 mM CaCl 2 , 0.02% Tween® 80, pH 7.0). The eluent in the VIIISelect process was diluted 10-fold by an equilibration buffer, titrated to pH 7.0, and loaded onto the column. After the loading, it was equilibrated by flowing an equilibrium buffer until the UV fell to the baseline. ScFVIII was rapidly eluted by flowing an elution buffer (20 mM Histidine, 5 mM CaCl 2 , 400 mM NaCl, 0.02% Tween® 80, pH 6.5). 
     In the third step, a process of conjugating PEG to the purified scFVIII was conducted. TCEP was added to the purified scFVIII solution to a final concentration of 0.1 mM, and allowed to stand at 4° C. for 1 hour to reduce the inserted cysteine. Residual TCEP was removed using a PD-10 column (GE healthcare) equilibrated with pegylated buffer (20 mM Histidine, 5 mM CaCl 2 , 200 mM NaCl, 0.02% Tween® 80, pH 7.0). It was allowed to stand at 4° C. for 2 hours to oxidize the disulfide bond of the reduced FVIII itself. For the PEG conjugation to the scFVIII G4 (B3), a 40-kDa branched-type methoxy maleimide PEG solution (50 mg/mL) dissolved in DMSO at the PEG to protein ratio of 1:20 was added, and then the resultant was allowed to stand at 4° C. for 12-16 hours. The scFVIII G4 (A2_1) PEG conjunction was carried out using 60 kDa of methoxy maleimide PEG in a branched form. 
     And, in the fourth step, a process of removing scFVIII without PEG and scF VIII with two or more PEGS, which are impurities formed during the pegylation process, was conducted using SP fast flow resin of GE Inc. The SP fast flow resin was packed into the column and washed with the washing buffer (0.5 M NaOH, 1 M NaCl) to wash the column. The equilibration buffer (20 mM Histidine, 5 mM CaCl 2 , 0.02% Tween®80, pH 7.0) was added to reach an equilibrium. The pegylated FVIII was diluted by 10-fold with an equilibration buffer, titrated to pH 7.0, and loaded onto the column. After loading, the equilibrium buffer was flowed until UV fell to the baseline, to reach an equilibrium. The PEG-scF VIII was selectively eluted while the NaCl concentration of the elution buffer (20 mM Histidine, 5 mM CaCl 2 , 50-400 mM NaCl, 0.02% Tween® 80, pH 6.5) was increased stepwise. 
     Finally, in the fifth step, the PEG-scFVIII eluted in the fourth step was concentrated. To this end, the PEG-scF VIII was concentrated to 25 IU/mL using amicon 30 kDa of Millipore Co. 
     Example 3-3. Activity Measurement 
     The activities of the purified pegylated scFVIIIs (PEG-scFVIII-B3, and PEG-scFVIII-A2-2) used in Example 3-2 were measured by the CS and APTT methods. The CS test was carried out by the same method as described in Example 1-2, and the APTT test was carried out by modifying the method described in Example 1-2. It is known that, when measuring the activity of the pegylated FVIII by APTT method, activity values lower than original values are obtained upon analysis if APTT-SP of colloidal silica series is used as an activator, whereas normal activity is measured if synthAFax (IL) of the ellagic acid series is used (Gu. J. M. et al., 2014. Evaluation of the activated partial thromboplastin time assay for clinical monitoring of PEGylated recombinant Factor VIII (BAY 94-9027) for haemophilia A. Haemophilia, 20, 593-600). SynthAFax was used as an activator in the APTT test in consideration of the property of the pegylated scFVIII. The same method as in Example 1-2 was used for the other test methods. The results are shown in Table 4. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 4 
               
             
            
               
                   
                   
               
               
                   
                 Activity IU/mL 
                   
               
            
           
           
               
               
               
               
            
               
                   
                 APPT 
                 CS 
                   
               
               
                   
                 (clotting assay), 
                 (chromogenic 
                 APPT/CS 
               
               
                   
                 synthAFax 
                 assay) 
                 ratio 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                 PEG-scFVIII -B3 
                 19.34 
                 20.21 
                 0.96 
               
               
                 (40K PEG) 
               
               
                 PEG-scFVIII-A2-2 
                 18.62 
                 18.04 
                 1.03 
               
               
                 (60K PEG) 
               
               
                 Advate 
                 25.87 
                 23.77 
                 1.09 
               
               
                 Xyntha 
                 25.32 
                 33.88 
                 0.75 
               
               
                   
               
            
           
         
       
     
     As a result of the experiment, it was found that the APTT/CS ratios (APTT/CS) of PEG-scFVIII-B3, PEG-scFVIII-A2-2 were similar to that of ADVATE®, which is a two-chain FVIII containing the B domain of full length used as a comparative substance, but were different from that of Xyntha, which is a two-chain FVIII from which the B domain was removed. Considering that the activities of the natural-type FVIII are almost the same in CS and APTT, the result indicated that the scFVIII protein produced by the present invention did not show changes in the activity characteristics of blood coagulation FVIII due to single-chain formation and pegylation. Xyntha is a recombinant FVIII from which the B domain is completely removed, and in this Example, its blood coagulation activity (APTT) was decreased as compared with that in CS, which is consistent with the previous report that the coagulation activity is decreased when the B domain is completely removed. Considering that PEG-scFVIII-B3 and PEG-scFVIII-A2-2 maintain blood coagulation activity even after single-chain formation and pegylation, these two substances according to the present invention are considered to have advantages as hemophilia therapeutic agents. 
     Example 4. Animal PK Test 
     The half-life of the pegylated scFVIII was compared with ADVATE®, a comparison substance, in the Hemophilia A mouse model (B6; 129S4-F8tm1Kaz/J (FVIII-knock-out)) of 11-12 weeks of age, which was distributed from Pennsylvania University, bred, and confirmed by genotyping. The test substances were G4, which is scFVIII, PEG40 kDa-G4scFVIII-B3, which was position-specifically pegylated with 40 kDa branched PEG maleimide after the 782 isoleucine of the B domain of G4 was substituted with cysteine (see Example 3), PEG60 kda-G4scFVIII-A2-2, which was position-specific pegylated with a 60 kDa branched PEG maleimide after the 495 valine in the A2 domain of the G4 were substituted with cysteine, and Advate®, which is a two-chain recombinant FVIII. 
     Test animals used in drug pharmacokinetics were hemophilia mice (females), and each sample was administered to 3 animals into the tails veins at a level of 125 IU/kg. Blood samples were collected from the orbit at each time point after the administration (5 min, 4 hours, 8 hours, 16 hours, 24 hours, 32 hours, 40 hours, 48 hours, 56 hours, and 72 hours), which were treated with an anticoagulant (3.2% sodium citrate) by 10% and centrifuged to remove plasma. The activity of FVIII present in the plasma was measured using the CS assay reagent as described in Example 1-2. The profile of FVIII activity average in plasma collected from the mice in each sample group at each time is shown in  FIG.  7   . 
     As for the PK parameter analysis, the mean and standard deviation of FVIII CS activity measurement values in plasma were calculated by the NCA (Non Compartmental Analysis) method using the WinNonlin software, and the result is shown in Table 5. The half-lives of the drugs were found to be in the following order: B3≈A2-2&gt;G4&gt;Advate® (Table 5). When evaluated based on the mean data, the half-lives of the drugs B3, A2-2, and G4 were respectively increased by about 1.9-fold, about 1.8-fold, and about 1.2-fold, based on Advate® (Table 5). 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 PK Results 
               
            
           
           
               
               
               
               
               
            
               
                   
                 PEG60k-G4 
                 PEG40k-G4 
                 G4 
                   
               
               
                   
                 scFVIII-A2-2 
                 scFVIII-B3 
                 scFVIII 
                 Advate ® 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 T 1/2  (hr) 
                 19.3 
                 19.6 
                 13.0 
                 10.5 
               
               
                 MRT INF (hr) 
                 15.8 
                 16.0 
                 9.7 
                 7.2 
               
               
                 AUC (hr*IU/mL) 
                 34.5 
                 38.6 
                 20.6 
                 15.5 
               
               
                 T 1/2  fold-change 
                 1.8 
                 1.9 
                 1.2 
                 1.0 
               
               
                 (vs Advate) 
               
               
                   
               
            
           
         
       
     
     Example 5. Animal PD Test (Long-Term) 
     The Coagulation Effect Regarding Tail Bleeding in Mice was Measured as Follows. 
     The experiment outline is shown in  FIG.  8 A . Specifically, male C57BL/6 mice (provided by Orient Biotech) at 11-12 weeks of age and Hemophilia A mice (B6; 129S4-F8 tm1Kaz /J (FVIII-knock-out), FVIII KO mice) which was distributed from Pennsylvania University, bred, and confirmed by genotyping were used for the test. C57BL/6 mice were subjected to the acclimation process for 7 days. On the day of the test, the saline was filled in a 15 mL conical tube up to 14 mL (×1/rat), and the tube was soaked in a heated water bath to adjust the temperature of the saline to 37° C. The weights of the mice were measured and recorded before administration of the test substance. Entobar (pentobarbital sodium) was administered intraperitoneally at a concentration of 60 mg/kg to anesthetize the mice. After the doses of the test substance based on the weights of the mice, which were 19-25 g, were calculated, the test substance was administered into the jugular vein. 5 minutes after the administration, the tails of the mice were cut at the point which was 4 mm from the end. About 2 cm of mouse tail cut was immediately soaked in conical tubes containing saline and the blood was collected for 30 minutes. 
     The test substance was PEG-scFVIII-B3 prepared in Example 3, which was compared with Advate®. The doses (doses and dose volumes) and the distribution of mice included in each test substance group are shown in Table 6 below. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                   
                   
                   
                 Adminis- 
                 Adminis- 
                   
               
               
                   
                   
                   
                 tration 
                 tration 
               
               
                 Test 
                 Animal 
                 Test 
                 concentration 
                 dose 
                 Number of 
               
               
                 group 
                 species 
                 substance 
                 (IU/kg) 
                 (mL/kg) 
                 animals 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 G1 
                 C57BL/6 
                 PBS 
                 0 
                 5 
                 13 
               
               
                 G2 
                 FVIII KO 
                 PBS 
                 0 
                 5 
                 12 
               
               
                 G3 
                 mice 
                 Advate ® 
                 50 
                 5 
                 10 
               
               
                 G4 
                   
                   
                 100 
                 5 
                 10 
               
               
                 G5 
                   
                   
                 200 
                 5 
                 10 
               
               
                 G6 
                   
                 40kDaPEG- 
                 50 
                 5 
                 5 
               
               
                 G7 
                   
                 G4scFVIII- 
                 100 
                 5 
                 6 
               
               
                 G8 
                   
                 B3 
                 200 
                 5 
                 4 
               
               
                   
               
            
           
         
       
     
     The blood collected from each mouse for 30 minutes was centrifuged at 1500×g for 5 minutes and the supernatant was removed, then, tertiary distilled water was added to a conical tube up to 10 mL using a 10 mL pipet, and then, the blood was subjected to complete hemolysis using a vortex. The blood loss measurement was conducted according to the manual provided by Hemoglobin assay kit (Sigma, MAK115-1KT). In summary, 0.9 ml of tertiary distilled water and 0.1 ml of a hemolysis sample were put into a 1.5 mL eppendorf tube and mixed well for 10-fold dilution. 50 μl of tertiary distilled water and 50 μl of a calibrator were put into a 96 well plate (duplication), and 200 μl of tertiary distilled water was added on top of it. 50 μl of each mouse hemolysis sample was put into the well (duplication) and 200 μl of the reagent was added on top of it. The 96 well plate was lightly tapped so that the inputted substances could be mixed well, and the absorbance was measured at 400 nm using a Multimode plate reader. 
     The absorbance values were statistically compared with the blood loss values (Hb nmol) using the One way ANOVA with Dunnett&#39;s multiple comparison test of GraphPad Prism ver 5.01. A p-value greater than 0.05 was considered to be a statistically not significant difference, and a p-value less than 0.05 was considered to be a statistically significant difference. 
     Test results indicated that the groups administered with 40kDaPEG-G4scFVIII-B3 and Advate® 50, 100, and 200 IU/kg all showed statistically significant differences when compared with the blood loss value of the HA group. The HA mice were administered with 40 kDa PEG-G4scFVIII-B3 and Advate®, and then acute tail-clipping test was conducted. As a result, it was found that both of the two test substances decreased the amount of blood losses in a concentration dependent way (see  FIG.  8   b   ). 
     In summary, in the present invention, in order to select the scFVIIIs which are excellent in expression, have maximum natural FVIII activities, and are stable such that its decomposition in the culture medium can be prevented, the scFVIIIs containing B domains of various lengths and sequences were prepared and expressed comparing their activities with each other and then, the scFVIIIs having useful properties as Hemophilia A type therapeutic agents were developed through confirming their stability. In addition, based on the scFVIIIs developed in the present invention, various pegylated conjugates were prepared, thereby developing FVIII pegylated conjugates with improved half-lives, and thus improved usefulness as therapeutic agents. 
     While the present invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes using the basic concepts of the present application as defined in the claims which may be made by those skilled in the art also fall within the scope of the present invention. 
     All the technical terms that were used in the present invention, have the same meanings that are understood by a skilled person in this art, unless otherwise specified. The contents of all the publications disclosed in the present description as a reference document are incorporated in the present invention.