In vivo sustained-release recombinant blood coagulation factor VIII and preparation method thereof
阅读说明：本技术 体内持续释放重组凝血因子ⅷ及其制备方法 (In vivo sustained-release recombinant blood coagulation factor VIII and preparation method thereof ) 是由 康官烨 金泰润 柳在奂 金然汀 金用栽 于 2019-01-11 设计创作，主要内容包括：本发明提供了其中生物相容性聚合物与至少一个糖链的末端缀合的体内持续释放重组凝血因子VIII及其制备方法。根据本发明的体内持续释放重组凝血因子VIII具有通过比常规糖缀合技术更精细和更安全的方法修饰的蛋白质表面,并且具有免疫原性降低和体内持续性提高的优点。(The present invention provides an in vivo sustained release recombinant factor VIII in which a biocompatible polymer is conjugated to the end of at least one sugar chain, and a method for preparing the same. The in vivo sustained release recombinant factor VIII according to the present invention has a protein surface modified by a finer and safer process than the conventional glycoconjugate technology, and has advantages of reduced immunogenicity and improved in vivo persistence.)
1. An in vivo long-acting recombinant factor VIII comprising a linker linked to a sialic acid derivative of at least one sugar chain terminal of factor VIII, wherein the linker is represented by formula 1:
wherein R is1And R2Each independently is C1-8Alkyl radical, C6-12Aryl radical, C3-6Cycloalkyl, 3-to 10-membered heterocycloalkyl, amino, (amino) C1-6Alkyl, (amino) C3-6Cycloalkyl, (amino) C6-12Aryl, (amino) (3-to 10-membered heterocycloalkyl), or 5-to 12-membered heteroaryl (wherein the heterocycloalkyl and heteroaryl may each independently comprise at least one heteroatom selected from N, S and O, and may each independently be selected from halogen, C1-4Alkyl and CF3Is substituted with at least one substituent group; and said amino group may be selected from H, halogen, CN, C1-6Alkyl, halo C1-3Alkyl or C1-6At least one substituent of the alkoxycarbonyl group being substitutedGeneration) or R1And R2Can be combined with each other to form 8-to 16-membered fused heterorings (wherein the fused heterorings can each independently comprise at least one heteroatom selected from N, S and O, and can each independently be selected from halogen, C1-4Alkyl and CF3Substituted with at least one substituent of (a); and is
The linker comprises a linker with R1、R2Or when R is1And R2At least one linked biocompatible polymer of the rings formed when combined with each other.
2. The in vivo long acting recombinant factor VIII according to claim 1, wherein the biocompatible polymer is selected from the group consisting of polyethylene glycol (PEG), polysialic acid (PSA), polysialic acid
3. The in vivo long acting recombinant factor VIII according to claim 2, wherein the polyethylene glycol has a weight average molecular weight of 5 to 40k Da.
4. The in vivo long acting recombinant factor VIII according to claim 1, wherein the structure of formula 1 is at least one selected from the group consisting of formulae 2 to 18 and isomers thereof:
wherein L may be a single bond, halogen, C1-8Alkyl radical, C6-12Aryl, -CO-, -R3CONR3-、-OCONR3-or-R3NCOR3- (wherein R)3Can be selected from H, C independently1-4Alkyl and C6-12Aryl) and X is a biocompatible polymer.
5. The in vivo long acting recombinant factor VIII according to claim 1, wherein the structure of formula 1 is at least one selected from the group consisting of formulae 19 to 26:
wherein n is an integer of 100 to 3,000.
6. A method for preparing long acting recombinant coagulation factor VIII in vivo comprising the steps of:
(1) introducing an expression vector comprising a factor VIII-encoding nucleotide into a host cell; and
(2) culturing the host cell in a medium supplemented with a sialic acid metabolite derivative having a first functional group linked thereto to obtain the factor VIII with a sialic acid derivative having the first functional group linked thereto introduced at a terminal of at least one sugar chain thereof.
7. The method of claim 6, wherein the first functional group is an azido group or an alkynyl group.
8. The method of claim 6, wherein in step (2), the sialic acid metabolite derivative is at least one compound selected from the group consisting of formulae 27 to 32, and isomers thereof:
9. the method according to claim 6, wherein in step (2), when the concentration of the host cell is 1.0 × 105Or higher, the sialic acid metabolite derivative is supplemented at a concentration of 0.1 to 300 uM.
10. The method of claim 9, wherein in step (2), the temperature is adjusted to 29 ℃ to 35 ℃ when supplementing the sialic acid metabolite derivative.
11. The method of claim 6, further comprising the steps of:
(3) removing the sialic acid metabolite derivative from the culture medium;
(4) adding a compound having a second functional group and a biocompatible polymer attached thereto to the medium to allow a click reaction to proceed; and
(5) collecting the factor VIII having a biocompatible polymer attached to the end of at least one sugar chain thereof.
12. The method of claim 11, wherein the biocompatible polymer is selected from the group consisting of polyethylene glycol (PEG), polysialic acid (PSA), poly (l-y-l-y-
13. The method of claim 11, wherein when the first functional group is an azido group, the second functional group is an alkynyl group or a cycloalkynyl group; and when the first functional group is an alkynyl group, the second functional group is an azido group.
14. The method according to claim 11, wherein in step (4), the compound having the second functional group and the biocompatible polymer attached thereto is at least one compound selected from the group consisting of formulae 33 to 49, and isomers thereof:
wherein L may be a single bond, halogen, C1-8Alkyl radical, C6-12Aryl, -CO-, -R3CONR3-、-OCONR3-or-R3NCOR3- (wherein R)3Can be selected from H, C independently1-4Alkyl and C6-12Aryl) and X is a biocompatible polymer.
15. The method according to claim 6, wherein the in vivo long-acting recombinant factor VIII comprises a linker linked to a sialic acid derivative of at least one sugar chain terminal of factor VIII, wherein the linker is represented by formula 1:
wherein R is1And R2Each independently is C1-8Alkyl radical, C6-12Aryl radical, C3-6Cycloalkyl, 3-to 10-membered heterocycloalkyl, amino, (amino) C1-6Alkyl, (amino) C3-6Cycloalkyl, (amino) C6-12Aryl, (amino) (3-to 10-membered heterocycloalkyl), or 5-to 12-membered heteroaryl (wherein the heterocycloalkyl and heteroaryl may each independently comprise at least one heteroatom selected from N, S and O, and may each independently be selected from halogen, C1-4Alkyl and CF3Is substituted with at least one substituent group; and said amino group may be selected from H, halogen, CN, C1-6Alkyl, halo C1-3Alkyl or C1-6Substituted with at least one substituent of the alkoxycarbonyl group), or R1And R2Can be combined with each other to form 8-to 16-membered fused heterorings (wherein the fused heterorings can each independently comprise at least one heteroatom selected from N, S and O, and can each independently be selected from halogen, C1-4Alkyl and CF3Substituted with at least one substituent of (a); and is
The linker comprises a linker with R1、R2Or when R is1And R2At least one linked biocompatible polymer of the rings formed when combined with each other.
16. The method of claim 15, wherein the structure of formula 1 is at least one selected from formulas 19 to 26:
wherein n is an integer of 100 to 3,000.
Technical Field
The present invention provides an in vivo long-acting recombinant factor VIII having a structure in which a biocompatible polymer is linked to the end of at least one sugar chain, and a method for preparing the same.
Background
A glycoprotein refers to a protein having a sugar chain on the surface, which is linked to a protein residue in the Golgi apparatus during the production of the protein. In glycoproteins, the function of the sugar chain has a significant influence on the protein properties. For example, sugar chains can improve the aqueous solubility of proteins, protect proteins from proteases, reduce the immunogenicity of proteins, determine the directionality and localization of intracellular transport of proteins, or affect intercellular interactions (int.j.mol.sci.2012, 13, 8398-.
Because of the importance of sugar chains, attempts have been made to improve protein properties by glycoengineering. For example, for Erythropoietin (EPO), two additional N-sugar chains are introduced to improve its half-life in vivo (Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins, J PharmSci.200594 (8): 1626-35). In addition, it has been reported that, in the case of cancer cell-targeted antibody therapy, fucose-deficient antibodies produced by glycoengineering can induce better immune responses in the host and have better therapeutic effects (Production of therapeutic antibodies with controlled glycosylation, MAbs.20091 (3): 230-). 236). These methods using glycoengineering have advantages in that the production of by-products caused by side reactions can be minimized, and such methods are effective compared to conventional chemical methods.
Meanwhile, as for a method for improving the characteristics of glycoproteins by modifying sugar chains, it has been attempted to apply metabolic sugar engineering so as to achieve modification of sugar chains in a more complicated manner; however, its specific application and commercial use are not sufficient.
Disclosure of Invention
Technical problem
It is an object of the present invention to provide an in vivo long-acting recombinant glycoprotein in which a biocompatible polymer is linked to a sialic acid derivative introduced to the terminal of at least one sugar chain, preferably to provide factor VIII, in which the biocompatible polymer is linked to a sialic acid derivative introduced to the terminal of at least one sugar chain.
It is another object of the present invention to provide a method for preparing a recombinant factor VIII having a long-lasting effect in vivo, wherein sialic acid at the terminal of at least one sugar chain of factor VIII is modified to a sialic acid derivative into which an azide group or an alkyne group is introduced, using metabolic labeling.
It is still another object of the present invention to provide a method for preparing a modified in vivo long-acting recombinant factor VIII, wherein the modification is performed by adding a biocompatible polymer to a sialic acid derivative introduced with a terminal of a sugar chain.
Problem solving scheme
In order to achieve the above object, in one embodiment of the present invention, there is provided an in vivo long-acting recombinant factor VIII comprising a linker linked to a sialic acid derivative of a terminal of at least one sugar chain of factor VIII, wherein the linker is represented by formula 1:
whereinR1And R2Each independently is C1-8Alkyl radical, C6-12Aryl radical, C3-6Cycloalkyl, 3-to 10-membered heterocycloalkyl, amino, (amino) C1-6Alkyl, (amino) C3-6Cycloalkyl, (amino) C6-12Aryl, (amino) (3-to 10-membered heterocycloalkyl), or 5-to 12-membered heteroaryl (wherein the heterocycloalkyl and heteroaryl may each independently comprise at least one heteroatom selected from N, S and O, and may each independently be selected from halogen, C1-4Alkyl and CF3Is substituted with at least one substituent group; and said amino group may be selected from H, halogen, CN, C1-6Alkyl, halo C1-3Alkyl or C1-6Substituted with at least one substituent of the alkoxycarbonyl group), or R1And R2Can be combined with each other to form 8-to 16-membered fused heterorings (wherein the fused heterorings can each independently comprise at least one heteroatom selected from N, S and O, and can each independently be selected from halogen, C1-4Alkyl and CF3Substituted with at least one substituent of (a); and is
The linker comprises a linker with R1、R2Or when R is1And R2At least one linked biocompatible polymer of the rings formed when combined with each other.
In addition, in another embodiment of the present invention, a method for preparing long-acting recombinant factor VIII in vivo is provided, comprising the steps of: (1) introducing an expression vector comprising a factor VIII-encoding nucleotide into a host cell; and (2) culturing the host cell in a medium supplemented with a sialic acid metabolite derivative having a first functional group linked thereto to obtain factor VIII into which a sialic acid derivative having the first functional group linked thereto is introduced at a terminal of at least one sugar chain thereof.
In addition, in another embodiment of the present invention, a method for preparing long-acting recombinant factor VIII in vivo is provided, further comprising the steps of: (3) removing the sialic acid metabolite derivative from the culture medium; (4) adding a compound having a second functional group and a biocompatible polymer attached thereto to the medium to allow a click reaction to proceed; and (5) collecting the factor VIII having a biocompatible polymer attached to the end of at least one sugar chain thereof.
Advantageous effects of the invention
The in vivo long-acting recombinant factor VIII according to the present invention has a biocompatible polymer (e.g., polyethylene glycol) conjugated with sugar chains present on the surface of a protein, so that the immunogenicity of the in vivo glycoprotein factor VIII itself can be reduced and the in vivo long-acting properties thereof can be improved. In addition, the method for preparing long-acting recombinant factor VIII in vivo according to the present invention takes advantage of the normal glycometabolism process of the target host cell in glycoprotein expression by using recombinant DNA technology, thereby having advantages of high efficiency and accuracy in conjugation between a sugar chain and a biocompatible polymer. Therefore, the modified recombinant factor VIII can be provided in a stable and efficient manner, as compared to conventional methods for converting glycoprotein sugar chains using a glycoconjugation technique.
Drawings
Figure 1 shows a diagram of the expression vectors used in transfecting scFVIII G4B3 into a host cell as in example 1.1.
Figure 2 shows the results obtained in example 1.2 by SDS PAGE analysis of the purification process of scFVIII G4_ B3. (lane 1: molecular weight marker, lane 2: cell culture concentrate, lane 3: VIII selection eluent, lane 4: purified scFVIII G4B 3).
Figure 3 shows the details of the cell culture process and each cycle thereof in example 2.1.
Figures 4A and 4B show growth curves and expression levels of scFVIII G4B3 for cell lines expressing scFVIII G4B3 with Ac4ManNA1(N- (4-pentanoyl) -mannosamine tetraacylation) and scFVIII G4B3 without Ac4ManNA1, respectively.
Figure 5 shows levels of expression of scFVIII G4B3 for cell lines expressing scFVIII G4B3 with Ac4ManNAz and cell lines expressing scFVIII G4B3 without Ac4ManNAz (N-azido acetylmannosamine tetraacylation).
FIG. 6A shows the results obtained by analysis of the saccharide PEGylation reaction products of scFVIII G4B3Az and purified scFVIII G4B3Az by SDS PAGE (lane 1: molecular weight marker, lane 2: reaction product of scFVIII G4B3Az with 20kDa DBCOPEG, lane 3: reaction product of scFVIII G4B3Az with 30kDa DBCO PEG, lane 4: reaction product of scFVIII G4B3Az with 40kDa DBCO PEG, lane 5: purified scFVIII G4B3 Az).
FIG. 6B shows the results obtained by analysis of the sugar PEGylation reaction products of scFVIII G4B3Az and purified scFVIII G4B3Az by SDS PAGE (lane 1: molecular weight marker, lane 2: purified scFVIII G4B3Az, lane 3: reaction product of scFVIII G4B3Az with 10kDa DBCO PEG).
Figure 6C shows the results obtained by analysis of the purified form of scFVIII G4B3Az and the saccharide pegylation reaction product of scFVIII G4B3Az by SDS PAGE (lane 1: molecular weight marker, lane 2: purified scFVIII G4B3Az, lane 3: purified 20kDa DBCO PEG conjugated scFVIII G4B3Az, lane 4: molecular weight marker).
Figure 6D shows the results obtained by analyzing the purified form of the saccharide pegylation reaction product of scFVIII G4B3Az by SDS PAGE (lane 1: molecular weight marker, lane 2: purified 10kDa DBCO PEG conjugated scFVIII G4B3 Az).
FIG. 7 shows analysis in Cu by SDS PAGE2+Results obtained with azide-PEG 40kDa added to the product obtained with a click reaction of scfviig 4B 3Al in the presence.
Figure 8 shows a graph showing the activity of factor VIII remaining in the blood over time after intravenous administration of sugar-pegylated FVIII compared to non-pegylated FVIII.
Figure 9 shows a graph showing the activity of factor VIII remaining in the blood over time after subcutaneous administration of sugar-pegylated FVIII compared to non-pegylated FVIII.
Figure 10A shows a graph showing the activity of factor VIII remaining in the blood over time after subcutaneous administration of sugar pegylated FVIII compared to non-pegylated FVIII.
Figure 10B shows a graph showing the activity of factor VIII remaining in the blood over time after subcutaneous administration of sugar-pegylated FVIII compared to non-pegylated FVIII.
Best mode for carrying out the invention
In one aspect of the present invention, there may be provided an in vivo long-acting recombinant factor VIII (factor VIII, FVIII) comprising a linker linked to a sialic acid derivative of at least one sugar chain terminal of factor VIII, wherein the linker is represented by formula 1:
wherein R is1And R2Each independently is C1-8Alkyl radical, C6-12Aryl radical, C3-6Cycloalkyl, 3-to 10-membered heterocycloalkyl, amino, (amino) C1-6Alkyl, (amino) C3-6Cycloalkyl, (amino) C6-12Aryl, (amino) (3-to 10-membered heterocycloalkyl), or 5-to 12-membered heteroaryl (wherein the heterocycloalkyl and heteroaryl may each independently comprise at least one heteroatom selected from N, S and O, and may each independently be selected from halogen, C1-4Alkyl and CF3Is substituted with at least one substituent group; and said amino group may be selected from H, halogen, CN, C1-6Alkyl, halo C1-3Alkyl or C1-6Substituted with at least one substituent of the alkoxycarbonyl group), or R1And R2Can be combined with each other to form 8-to 16-membered fused heterorings (wherein the fused heterorings can each independently comprise at least one heteroatom selected from N, S and O, and can each independently be selected from halogen, C1-4Alkyl and CF3Substituted with at least one substituent of (a); and is
The linker comprises a linker with R1、R2Or when R is1And R2At least one linked biocompatible polymer of the rings formed when combined with each other.
Factor VIII is a glycoprotein in which sugar chains are linked to protein residues by covalent bonds. It is known that modification of sugar chains on the surface of proteins in glycoproteins may affect protein properties, such as improving the water solubility of proteins, protecting proteins from the action of proteases, and determining the directionality of proteins in cell-cell interactions.
Accordingly, the present inventors have conjugated protein sugar chains to biocompatible polymers through linkers shown in
In particular, in the present invention, factor VIII is labeled with a biocompatible polymer using an azide-alkyne cyclization reaction (click reaction) that does not require a catalyst (e.g., copper with cytotoxicity). Unlike other chemical reactions, this chemical reaction can be carried out on the precursor even in vivo, since the reactive group that has previously been introduced to the precursor is an azide or alkyne, which allows the chemical reaction to take place without any interference due to substances present in vivo.
The biocompatible polymer may be selected from polyethylene glycol (PEG), polysialic acid (PSA), and poly (I-PEG)At least one of oxazoline and heparin precursor (heparosan), preferably polyethylene glycol.
Additionally, the polyethylene glycol can have a weight average molecular weight of 5 to 40kDa, 5 to 30kDa, or 5 to 20 kDa. Multiple conjugation of small size polyethylene glycols with molecular weights of 40kDa or less can reduce the likelihood of PEG toxicity occurring upon conjugation of higher molecular weight polyethylene glycols, further improving the half-life of the glycoprotein and improving its bioavailability upon subcutaneous administration.
In addition, the structure of
wherein L may be a single bond, halogen, C1-8Alkyl radical, C6-12Aryl, -CO-, -R3CONR3-、-OCONR3-or-R3NCOR3- (wherein R)3Can be selected from H, C independently1-4Alkyl and C6-12Aryl) and X is a biocompatible polymer.
Specifically, the compound may be any one of compounds represented by formulae 19 to 26, wherein n is an integer of 100 to 3,000.
The wild-type factor VIII protein, consisting of the A1-a1-A2-a2-B-A3-A3-C1-C2 domain, is expressed as a single chain protein and then forms a 280kDa heterodimer comprising a heavy chain and a light chain by purine maturation in hepatocytes. Factor VIII proteins that may be modified in the present invention are proteins that may be represented by SEQ ID NO: 1, and preferably is a single chain form of factor VIII (scFVIII).
In addition, the factor VIII protein comprises a partially deleted B domain region and a partially deleted
In addition, factor VIII proteins may include a variety of modified peptides, i.e., variants. Modification may be made by substitution, deletion or addition of one or more amino acids within a range that does not alter the function of factor VIII. These various peptides can be linked to seq id NO: 2 to 8, or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical.
In addition, the linker-linked sugar chain in the factor III protein may be included in a peptide based on SEQ ID NO: 1, at least one position of amino acid residues 42, 239, 582, 757, 784, 828, 900, 963, 1001, 1005, 1055, 1066, 1185, 1255, 1259, 1282, 1300, 1412, 1442, 1810, and 2118 of the amino acid sequence of seq id No. 1. Thus, factor VIII proteins, including single chain factor VIII (scFVIII), may have many sugar chains on the surface and are therefore suitable for modifying the protein surface by applying metabolic glycoengineering according to the present invention.
In addition, in another aspect of the present invention, a method for preparing long-acting recombinant factor VIII in vivo may be provided, comprising the steps of: (1) introducing an expression vector comprising a nucleotide encoding factor VIII into a host cell, and (2) culturing the host cell in a medium supplemented with a sialic acid metabolite derivative having a first functional group attached thereto.
In step (1), the sequence of coagulation factor VIII may be SEQ ID NO: 2 to 8, and the nucleic acid sequence of nucleotides may be SEQ ID NO: 9 to 15. Furthermore, the nucleotide may additionally comprise a signal sequence or a leader sequence.
As used herein, the term "signal sequence" refers to a nucleic acid encoding a signal peptide that directs secretion of a fusion protein. The signal peptide is translated in the host cell and then cleaved. In particular, the signal sequence of the present invention is a polynucleotide encoding an amino acid sequence that initiates movement of a protein through the Endoplasmic Reticulum (ER) membrane. Signal sequences useful in the present invention include antibody light chain signal sequences, such as antibody 14.18(Gillies et al., J.Immunol. meth 1989.125: 191-202), antibody heavy chain signal sequences, such as MOPC141 antibody heavy chain signal sequence (Sakano et al., Nature, 1980.286: 676-683), and other signal sequences known in the art (e.g., Watson et al., Nucleic Acid Research, 1984.12: 5145-5164).
The characteristics of signal peptides are well known in the art. The signal peptide typically comprises 16 to 30 amino acid residues, and may comprise more or fewer amino acid residues. A typical signal peptide consists of three regions, a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region.
The central hydrophobic region contains 4 to 12 hydrophobic residues that immobilize the signal sequence on the membrane lipid bilayer during movement of the immature polypeptide. After initiation, the signal sequence is cleaved by cellular enzymes (commonly referred to as signal peptidases) within the ER lumen. The signal sequence can be the signal sequence of tissue plasminogen activator (tPa), the signal sequence of herpes simplex virus glycoprotein d (hsvgd) or the secretion signal sequence of growth hormone. Preferably, a secretory signal sequence used in higher eukaryotic cells including mammals and the like can be used. In addition, for the secretion signal sequence of the present invention, a signal sequence contained in wild-type IL-7 may be used, or a codon having a high expression frequency in a host cell may be substituted and used.
In addition, an expression vector is a vector that can be introduced into a host cell and can recombine with and be inserted into the genome of the host cell. Vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and the like. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses. In addition, the plasmid may contain a selectable marker, such as an antibiotic resistance gene, and the host cell containing the plasmid may be cultured under selective conditions.
In addition, as used herein, the term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. As used herein, the terms "transduction," "transformation," and "transfection" refer to the introduction of a nucleic acid (e.g., a vector) into a cell using techniques known in the art. Suitable host cells can be used to express and/or secrete factor VIII proteins by transduction or transfection with the nucleotide sequences of the present invention. Preferred host cells useful in the present invention include E.coli, immortal hybridoma cells, NS/0 myeloma cells, 293 cells, Chinese hamster ovary cells (CHO cells), HeLa cells, human amniotic fluid derived cells (CapT cells), or COS cells. For example, the most preferred host strain for expressing factor VIII protein in the present invention is CHO-S, and its genes and methods of using the strain are known in the art.
In addition, in step (2), the sialic acid metabolite derivative having the first functional group linked thereto is supplemented upon culturing the host cell, so that the sialic acid derivative having the first functional group linked thereto is introduced into the sugar chain terminal of factor VIII through the sialic acid metabolic pathway of the host cell protein. That is, in step (2), the factor VIII may be modified with a sialic acid derivative to which a first functional group is attached.
Here, the first functional group may be an azide group or an alkyne group. Furthermore, sialic acid metabolite derivatives may comprise acetyl or alkyl groups, such as ethyl and propyl groups, for efficient delivery into cells. The alkyl group forms an ester bond with a hydroxyl group of a sialic acid metabolite having a first functional group attached thereto to facilitate cell penetration thereof; and after cell infiltration, dealkylation by intracellular esterases. Thus, alkyl groups do not prevent the replacement of a portion of the sugar chain by a supplemental sialic acid metabolite.
In addition, in step (2), the sialic acid metabolite derivative that may replace the sialic acid metabolite (an intermediate in the sialic acid biosynthetic pathway of factor VIII) may be at least one compound selected from the group consisting of formulae 27 to 32, and isomers thereof:
meanwhile, in the step (2), when the cell concentration of the host cell derived from the animal cell is 1.0 × 105Since the method of the present invention utilizes the carbohydrate metabolism process of the cell, the sialic acid metabolite having the first functional group attached thereto may replace not only a part of the target coagulation factor VIII but also a part of metabolically produced metabolites related to all glycoproteins in the host cell5Or higher, it is preferable to supplement the sialic acid metabolite derivative.
The host cell is preferably cultured as follows. During the growth phase of the host cell, its culture is carried out without supplementation with sialic acid metabolite derivatives; after a certain level of cell growth is reached, changing the cell culture conditions to conditions that reduce the rate of cell growth and maintain cell survival; the host cell is then cultured in a medium supplemented with a sialic acid metabolite derivative having the first functional group. The supplemented sialic acid metabolite can be introduced into sialic acid on the sugar chain of the target protein at a higher rate when the growth of the host cell is reduced than when the cell is grown.
Here, as a condition for decreasing the growth rate of the cells, a method of adjusting the temperature to a low temperature or limiting the nutrients in the medium. Specifically, when the culture temperature is changed from 37 ℃ in the cell growth stage to 29 ℃ to 35 ℃ in the case of supplementation with the sialic acid metabolite derivative, the cell growth may be slowed, and the expression of the target protein (coagulation factor VIII) may last for 10 days or longer. In addition, the concentration of nutrients (e.g., biological growth factors, vitamins, and essential amino acids) in the cell culture medium can be reduced. In addition, in the case of metabolic labeling of the sugar chain of factor VIII, addition of a substance such as butyric acid affects the cell cycle, thereby preventing cell growth and maintaining the factor VIII expression as it is, thereby increasing the ratio of introduction of the sialic acid derivative having the first functional group into the sugar chain of factor VIII.
In addition, the method may further include the steps of: (3) removing the sialic acid metabolite derivative from the medium after purifying the blood coagulation factor VIII having a sialic acid derivative with a first functional group to which the sugar chain is attached from step (2); (4) adding a compound having a second functional group and a biocompatible polymer attached thereto to the medium to allow a click reaction to proceed; and (5) collecting the factor VIII having a biocompatible polymer attached to the end of at least one sugar chain thereof.
In order to react the first functional group with the second functional group of the sialic acid derivative in factor VIII, the sialic acid metabolite derivative that is present in excess in the culture medium must be removed.
Here, the first functional group may be an azide group or an alkyne group. In addition, when the first functional group is azido, the second functional group is alkynyl or cycloalkynyl; and when the first functional group is an alkynyl group, the second functional group may be an azido group.
Specifically, in step (4), the sialic acid derivative having an azido group or an alkynyl group introduced to the sugar chain of the target protein of step (2) in the method according to the present invention may be subjected to biocompatible polymer conjugation using a click reaction. For example, when a sialic acid metabolite derivative having an azido group attached thereto is used in step (2), a compound having an alkynyl group or cycloalkynyl group and a biocompatible polymer attached thereto may be used in step (4); and when a sialic acid metabolite derivative having an alkynyl group attached thereto is used in step (2), a compound having an azido group and a biocompatible polymer attached thereto may be used in step (4).
In step (4), the compound having the second functional group and the biocompatible polymer attached thereto is preferably at least one compound selected from the group consisting of formulae 33 to 49 and isomers thereof, and isomers thereof:
wherein L may be a single bond, halogen, C1-8Alkyl radical, C6-12Aryl, -CO-, -R3CONR3-、-OCONR3-or-R3NCOR3- (wherein R)3Can be selected from H, C independently1-4Alkyl and C6-12Aryl) and X is a biocompatible polymer.
In addition, the factor VIII collected in step (4) comprises a linker represented by
wherein R is1And R2Each independently is C1-8Alkyl radical, C6-12Aryl radical, C3-6Cycloalkyl radicals3-to 10-membered heterocycloalkyl, amino, (amino) C1-6Alkyl, (amino) C3-6Cycloalkyl, (amino) C6-12Aryl, (amino) (3-to 10-membered heterocycloalkyl), or 5-to 12-membered heteroaryl (wherein the heterocycloalkyl and heteroaryl may each independently comprise at least one heteroatom selected from N, S and O, and may each independently be selected from halogen, C1-4Alkyl and CF3Is substituted with at least one substituent group; and said amino group may be selected from H, halogen, CN, C1-6Alkyl, halo C1-3Alkyl or C1-6Substituted with at least one substituent of the alkoxycarbonyl group), or R1And R2Can be combined with each other to form 8-to 16-membered fused heterorings (wherein the fused heterorings can each independently comprise at least one heteroatom selected from N, S and O, and can each independently be selected from halogen, C1-4Alkyl and CF3Substituted with at least one substituent of (a); and is
The linker comprises a linker with R1、R2Or when R is1And R2At least one linked biocompatible polymer of the rings formed when combined with each other.
Detailed Description
Hereinafter, the present invention will be described in more detail by examples. However, the following examples are merely to illustrate the present invention, and the scope of the present invention is not limited thereto. In particular, the techniques of the invention will be described for scFVIII G4B3, scFVIII G4B3 being one of the scfviiis produced by the present inventors in the patent application filed by the present inventors prior to this patent application (PCT/KR 2017/006633). However, the reason for selecting a fviii in describing the technology of the present invention is not because the selected fviii has specific advantages, but only as an embodiment makes the description of the technology to be presented more clear.