Novel multilayer polymer-coated crosslinked alginate gel fiber

Abstract

There has been demand for an additional method for producing antibodies. The present invention provides: a polymer-coated crosslinked alginate gel fiber in which a core layer containing a crosslinked alginate gel and either antibody-producing cells (e.g., antibody-producing CHO cells) or bioactive-substance-producing cells (e.g., MIN6 cells derived from pancreatic β cells) is coated with a cationic polymer; and a method for producing antibodies, a bioactive substance, etc., using the fiber.

Description

Novel multilayer polymer-coated cross-linked alginate gel fibers

[0001] The present invention relates to a polymer-coated cross-linked alginate gel fiber for producing antibodies, physiologically active substances, etc., a method for producing the fiber, and a method for producing antibodies, physiologically active substances, etc. The present invention also relates to a multilayer polymer-coated cross-linked alginate gel fiber for producing antibodies, physiologically active substances, etc., a method for producing the fiber, and a method for producing antibodies, physiologically active substances, etc.

[0002] Various methods for producing antibodies, physiologically active substances (e.g., interferon, erythropoietin, IL-2 (interleukin-2), CSF (colony stimulating factor), TNF (tumor necrosis factor), etc.) by culturing animal cells have been known so far.

[0003] In the case of antibody production, CHO cells (derived from Chinese hamster ovary), Sp2 / 0 cells, NS0 cells, and the like are used as host cells for producing antibodies. In particular, CHO cells are frequently used for antibody production because they can be cultured in suspension, have a high proliferation rate, and can easily be mass-produced by mass culturing CHO cells.

[0004] In recent years, efforts to stabilize and reduce the cost of antibody pharmaceuticals have been required in the development and manufacturing of antibody pharmaceuticals. To achieve these goals, attention has been focused on the development of more productive and efficient production systems (e.g., continuous production methods, novel culture techniques for producing the required amount of antibodies using small-scale production facilities, etc.).

[0005] In the cultivation of antibody-producing cells, antibody-producing cell lines are cultured in spinner flasks or the like, and then expanded while controlling culture conditions such as medium composition, temperature, agitation conditions, gas exchange, and pH, and finally cultured in large-scale production culture tanks on the scale of several thousand to 10,000 liters.

[0006] When antibody-producing cells are continuously cultured at high densities, problems can arise, such as (1) how to separate the cells from the culture medium and (2) how to effectively supply oxygen. Although various improvements have been made regarding (1) and (2), various other problems must be resolved in order to efficiently produce antibodies.

[0007] Alginate gel fibers having a core-shell structure are known, in which the core layer contains various cells and the shell layer is made of alginate gel (Patent Document 1: International Publication No. 2011 / 046105, Patent Document 2: Japanese Patent Laid-Open No. 2016-77229).

[0008] Alginate gel fibers having a core-shell structure in which the core layer contains antibody-producing cells and the shell layer is made of alginate gel are known (Patent Document 3: International Publication No. 2020 / 032221).

[0009] Alginate gel hollow fibers are known, which contain various cells in the hollow portion and have an outer shell layer made of alginate gel (Patent Document 4: International Publication No. 2015 / 178427, Patent Document 5: Japanese Patent No. 6601931, Patent Document 6: Japanese Patent Laid-Open No. 2014-236698).

[0010] Bundles are known in which alginate hydrogel fibers containing cells (specifically, human dermal fibroblasts, HEK293T cells) are bonded using an adhesive containing nanoparticles whose surfaces are coated with a cationic water-soluble polymer (Patent Document 7: WO 2019 / 078251 pamphlet, Patent Document 8: WO 2019 / 123886 pamphlet).

[0011] A nerve bundle for transplantation is known in which neural stem cells or alginate hydrogel fibers containing neural stem cells are bundled with chitosan (Patent Document 9: JP 2014-136128 A).

[0012] A cell structure (e.g., sheet-like (plate-like), fiber-like (fibrous), spherical, etc.) in which a mixture containing adherent cells (specifically, C2C12 cells), microcarriers, and gel-like polysaccharides (specifically, alginate gel) is coated with polyamino acids is known (Patent Document 10: JP 2019-075993 A).

[0013] A tubular gel is known, which is obtained by coating calcium alginate microfibers containing cells (293 / GFP cells) with poly-L-lysine and dissolving them in a sodium citrate solution (Non-Patent Document 1: PA-Lab Chip, 2008, 8, pp. 1255-1257).

[0014] Chemically modified alginic acid derivatives are known in which a cyclic alkyne group or an azide group is introduced into any one or more carboxyl groups of alginic acid via an amide bond and a divalent linker (Patent Document 11: WO 2019 / 240219 Pamphlet, Patent Document 12: WO 2021 / 125255 Pamphlet).

[0015] Alginate gel fibers with a core-shell structure are known, in which the core layer contains antibody-producing cells and the shell layer consists of a cross-linked alginate gel formed from a chemically modified alginate derivative (Patent Document 13: International Publication No. 2021 / 125279).

[0016] Patent Documents 1 to 13 and Non-Patent Document 1 do not disclose or suggest the polymer-coated cross-linked alginate gel fiber or multi-layer polymer-coated cross-linked alginate gel fiber for producing the antibodies, physiologically active substances, etc. of the present invention, the method for producing each gel fiber, or the method for producing antibodies, etc. using each gel fiber.

[0017] International Publication No. WO 2011 / 046105 Pamphlet Japanese Patent Application Laid-Open No. 2016-77229 International Publication No. WO 2020 / 032221 Pamphlet International Publication No. WO 2015 / 178427 Pamphlet Japanese Patent No. 6601931 Japanese Patent Application Laid-Open No. 2014-236698 International Publication No. WO 2019 / 078251 Pamphlet International Publication No. WO 2019 / 123886 Pamphlet Japanese Patent Application Laid-Open No. 2014-136128 Japanese Patent Application Laid-Open No. 2019-075993 International Publication No. WO 2019 / 240219 Pamphlet International Publication No. WO 2021 / 125255 Pamphlet International Publication No. WO 2021 / 125279 Pamphlet

[0018] PA-Lab Chip, 2008, 8, p1255-1257

[0019] There has been a demand for alginate gel fibers containing cells capable of producing antibodies, physiologically active substances, etc., and in particular for more practical alginate gel fibers that allow cell culture for long periods of time (e.g., 7 days or more, 14 days or more, 28 days or more, etc.) without the fibers decomposing.

[0020] As a result of extensive research, the present inventors have discovered a novel polymer-coated cross-linked alginate gel fiber for producing antibodies, physiologically active substances, etc., and a method for producing the same, which is formed by coating a cross-linked alginate gel with a cationic polymer, the cross-linked alginate gel containing cells capable of producing antibodies, physiologically active substances, etc., and obtained by carrying out a cross-linking reaction using chemically modified alginate derivatives represented by formula (I) and formula (II) described in aspect [1] below. Furthermore, when cells producing antibodies, physiologically active substances, etc. were cultured using the polymer-coated cross-linked alginate gel fiber, the inventors found that the polymer-coated cross-linked alginate gel fiber was not decomposed and allowed for continuous long-term production of antibodies, physiologically active substances, etc., which led to the completion of the present invention. Furthermore, the inventors have discovered a novel multilayer polymer-coated crosslinked alginate gel fiber for producing antibodies, physiologically active substances, etc., which is formed by coating with an anionic polymer a crosslinked alginate gel fiber, which is formed by carrying out a crosslinking reaction using chemically modified alginate derivatives represented by formula (I) and formula (II) described in aspect [1B] below, and a method for producing the same. Furthermore, when antibody-producing cells were cultured using the multilayer polymer-coated crosslinked alginate gel fiber, the inventors have found that the multilayer polymer-coated crosslinked alginate gel fiber allows for continuous long-term production of antibodies, physiologically active substances, etc., without degradation.

[0021] The present invention provides novel polymer-coated cross-linked alginate gel fibers and methods for producing antibodies, physiologically active substances, etc. using the gel fibers. In some aspects, a cross-linked alginate gel prepared using a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc., and the chemically modified alginate derivatives represented by formula (I) and formula (II) described in aspect [1] below is coated with a cationic polymer to provide polymer-coated cross-linked alginate gel fibers capable of continuously producing antibodies, physiologically active substances, etc. over a long period of time. The present invention also provides novel multilayer polymer-coated cross-linked alginate gel fibers and methods for producing antibodies, physiologically active substances, etc. using the gel fibers. In some embodiments, a cross-linked alginate gel prepared using a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc., and chemically modified alginate derivatives represented by formula (I) and formula (II) described in embodiment [1B] below, is coated with a cationic polymer to obtain a polymer-coated cross-linked alginate gel fiber, which is then coated with an anionic polymer to provide a multilayer polymer-coated cross-linked alginate gel fiber capable of continuously producing antibodies, physiologically active substances, etc. for a long period of time.

[0022] As shown in the Examples below, a crosslinked alginate gel (also referred to as a core layer) formed using a mixed solution containing antibody-producing cells (anti-GPVI antibody-producing CHO cells, tocilizumab-producing CHO cells) or physiologically active substance-producing cells (MIN6 cells derived from pancreatic β cells), chemically modified alginate derivatives represented by Formula (I) and Formula (II), and the like, can be coated with a cationic polymer (also referred to as a cationic polymer layer) such as poly-L-ornithine, polyallylamine, polyethyleneimine, or polymethylene-co-guanidine (PMCG). It was found that polymer-coated crosslinked alginate gel fibers could be produced by culturing the fibers, and antibodies or insulin could be continuously produced for long periods of time (up to 47 days in the Examples below). The polymer-coated crosslinked alginate gel fiber of the present invention provides an environment suitable for cells capable of producing antibodies, physiologically active substances, and the like. The antibodies, physiologically active substances, and the like produced in the core layer of the fiber are characterized by continuously permeating the core layer and cationic polymer layer and being released to the outside of the fiber.

[0023] Furthermore, the Examples described below have shown that a multilayer polymer-coated cross-linked alginate gel fiber can be produced by coating the outside of the cationic polymer layer of the polymer-coated cross-linked alginate gel fiber, in which tocilizumab-producing CHO cells are contained in the core layer, with alginic acid, a chemically modified alginate derivative represented by formula (I), or a chemically modified alginate derivative represented by formula (II) as an anionic polymer (also referred to as an anionic polymer layer), and that antibodies can be produced continuously for long periods of time (up to 35 days in the Examples described below) by culturing using the fiber. The multilayer polymer-coated cross-linked alginate gel fiber of the present invention, like the polymer-coated cross-linked alginate gel fiber described above, provides an environment suitable for cells capable of producing antibodies, physiologically active substances, etc., and is characterized in that antibodies, physiologically active substances, etc. produced in the core layer of the fiber continuously permeate the core layer, cationic polymer layer, and anionic polymer layer and are released to the outside of the fiber.

[0024] The polymer-coated cross-linked alginate gel fiber and multilayer polymer-coated cross-linked alginate gel fiber of the present invention provide an environment suitable for the production of antibodies, physiologically active substances, etc. The antibody-, physiologically active substance-producing cells encapsulated in the core layer are subject to minimal physical stress, and the encapsulated cells are expected to continue producing antibodies, physiologically active substances, etc. over a long period of time. Therefore, a method for producing antibodies, physiologically active substances, etc. using such fibers is expected to dramatically improve the production efficiency of antibodies, physiologically active substances, etc. For example, in the case of antibody production, unlike antibody suspension culture, which requires large-scale culture tanks, it is expected that antibodies can be produced in small-scale production facilities. It is also expected to serve as a next-generation continuous production technology for antibody pharmaceuticals, suitable for the production of small quantities of a wide variety of antibody pharmaceuticals.

[0025] 1 is a cross-sectional view of a polymer-coated cross-linked alginate gel fiber. 2 is a schematic diagram of the core layer and cationic polymer layer of a polymer-coated cross-linked alginate gel fiber. 3 is a schematic diagram illustrating one embodiment of the manufacturing process of a polymer-coated cross-linked alginate gel fiber. 4 is a cross-sectional view of a polymer-coated cross-linked alginate gel fiber. 5 is a schematic diagram illustrating the permeation of the cationic polymer layer by antibodies, physiologically active substances, metabolites, waste products, culture medium (nutrient source), and oxygen produced in the core layer. 6 is a photograph of the polymer-coated cross-linked alginate gel fiber (FB2-A-5-c1) of Example F2-C before culture. 7 is a photograph of the polymer-coated cross-linked alginate gel fiber (FB2-A-5-c1) of Example F2-C after culture. 8 is a fluorescent microscope photograph of the polymer-coated cross-linked alginate gel fiber produced in Example F3. 9 is a cross-sectional view of a multilayer polymer-coated cross-linked alginate gel fiber.

[0023] Figure 1 is a schematic diagram of the core layer, cationic polymer layer, and anionic polymer layer of a multilayer polymer-coated cross-linked alginate gel fiber. Figure 2 is a schematic diagram illustrating one embodiment of the manufacturing process for a multilayer polymer-coated cross-linked alginate gel fiber. Figure 3 is a cross-section of a multilayer polymer-coated cross-linked alginate gel fiber. Figure 4 is a schematic diagram illustrating the permeation of antibodies produced in the core layer, physiologically active substances, metabolites, waste products, culture medium (nutrient source), and oxygen through the cationic polymer layer and the anionic polymer layer. Figure 5 is a photograph of the polymer-coated cross-linked alginate gel fiber (FB9-3-c3) of Example F9 before culture. Figure 6 is a photograph of the polymer-coated cross-linked alginate gel fiber (FB9-3-c3) of Example F9 after culture. Figure 7 is a photograph of the polymer-coated cross-linked alginate gel fiber (FB9-2-c2) of Example F9 before culture. Figure 8 is a photograph of the polymer-coated cross-linked alginate gel fiber (FB9-2-c2) of Example F9 after culture. 10A and 10B are photographs of the multilayer polymer-coated cross-linked alginate gel fiber (FB12-ac1) of Example F12 before and after culture.1 is a photograph of the multilayer polymer-coated cross-linked alginate gel fiber (FB12-ac3) of Example F12 before culture. 2 is a photograph of the multilayer polymer-coated cross-linked alginate gel fiber (FB12-ac3) of Example F12 after culture. 3 is a photograph of the multilayer polymer-coated cross-linked alginate gel fiber (FB12-ac2) of Example F12 before culture. 4 is a photograph of the multilayer polymer-coated cross-linked alginate gel fiber (FB12-ac2) of Example F12 after culture. 5 is a photograph taken with a fluorescence microscope of the fluorescently labeled alginate gel fiber (FB14-c2-2) prepared in Example F14. 6 is a photograph of the fluorescently labeled alginate gel fiber (FB14-c1-1) prepared in Example F14.

[0026] [Specific Aspects] Specific aspects of a polymer-coated cross-linked alginate gel fiber, a method for producing the fiber, and a method for producing antibodies, physiologically active substances, etc. using the fiber will be described here. Specific aspects of a multilayer polymer-coated cross-linked alginate gel fiber, a method for producing the fiber, and a method for producing antibodies, physiologically active substances, etc. using the fiber will also be described. More specifically, these are as described in the following aspects [1] to [7C-2].

[0027] [1] The first aspect is as follows: A polymer-coated cross-linked alginate gel fiber obtained by coating a core layer containing cells capable of producing antibodies, physiologically active substances, etc., and a cross-linked alginate gel obtained by a cross-linking reaction using chemically modified alginate derivatives represented by the following formulas (I) and (II), with a cationic polymer (cationic polymer layer).

[0028] [Chemically modified alginic acid derivative represented by formula (I)] The following formula (I): [In formula (I), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; Akn-L １ -(Akn represents a cyclic alkyne group; -L １ - is a divalent linker connecting to a cyclic alkyne group (Akn)) is shown in the following table: A chemically modified alginic acid derivative represented by the following partial structural formula:

[0029] [Chemically modified alginic acid derivative represented by formula (II)] A chemically modified alginic acid derivative represented by the following formula (II): [In formula (II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L ２ - is the table below: A chemically modified alginic acid derivative represented by the following partial structural formula: (each formula does not include the areas outside the dashed lines at both ends):

[0030] [1A] Aspect 1A is as follows: A polymer-coated cross-linked alginate gel fiber comprising a core layer and a cationic polymer layer disposed on the outside of the core layer, wherein the core layer comprises cells capable of producing antibodies, physiologically active substances, etc., and cross-linked alginate gel formed by cross-linking using chemically modified alginate derivatives represented by formulas (I) and (II), and the cationic polymer layer is a cationic polymer. The chemically modified alginate derivatives represented by formulas (I) and (II) are the same as those defined in Aspect [1] above.

[0031] [1-1-1] In the above aspect [1] or [1A], the chemically modified alginic acid derivative Akn-L represented by formula (I) １ is preferably as shown in the following table: (In each formula, the right side of the dashed line is not included.)

[0032] [1-1-2] In the above aspect [1] or [1A], the chemically modified alginic acid derivative Akn-L represented by formula (I) １ More preferably, the compound is selected from the group consisting of the compounds shown in the following table: (In each formula, the right side of the dashed line is not included.)

[0033] [1-1-3] In the above aspect [1] or [1A], the chemically modified alginic acid derivative Akn-L represented by formula (I) １ More preferably, - is represented by the following partial structural formula (in each formula, the right side of the dashed line is not included): is a group selected from the group consisting of:

[0034] [1-1-4] In the above aspect [1] or [1A], the chemically modified alginic acid derivative Akn-L represented by formula (I) １ - is particularly preferably represented by the following partial structural formula (in each formula, the right side of the dashed line is not included): is a group selected from

[0035] [1-1-5] In the above aspect [1] or [1A], the chemically modified alginic acid derivative Akn-L represented by formula (I) １ - is the table below: (in each formula, the right side of the dashed line is not included); preferably, (in each formula, the right side of the dashed line is not included); more preferably, (in each formula, the right side of the dashed line is not included); more preferably, it is a group selected from the group consisting of the following partial structural formulas (in each formula, the right side of the dashed line is not included): is a group selected from

[0036] [1-2-1] In the above aspect [1] or [1A], -L of the chemically modified alginic acid derivative represented by formula (II) ２ is preferably as shown in the following table: (each formula does not include the areas outside the dashed lines at both ends).

[0037] [1-2-2] In the above aspect [1] or [1A], -L of the chemically modified alginic acid derivative represented by formula (II) ２ More preferably, the compound is selected from the group consisting of the compounds shown in the following table: (each formula does not include the areas outside the dashed lines at both ends).

[0038] [1-2-3] In the above aspect [1] or [1A], -L of the chemically modified alginic acid derivative represented by formula (II) ２ More preferably, - is a partial structural formula shown below (each formula does not include the areas outside the dashed lines at both ends): is a group selected from the group consisting of:

[0039] [1-2-4] In the above aspect [1] or [1A], -L of the chemically modified alginic acid derivative represented by formula (II) ２ - is particularly preferably a partial structural formula shown below (each formula does not include the areas outside the dashed lines at both ends): is a group selected from the group consisting of:

[0040] [1-2-5] In the above aspect [1] or [1A], -L of the chemically modified alginic acid derivative represented by formula (II) ２ - is the table below: (in each formula, the area outside the dashed lines at both ends is not included); preferably, the following partial structural formula (in each formula, the area to the right of the dashed lines is not included): (in each formula, the area outside the dashed lines at both ends is not included); more preferably, the following partial structural formula (in each formula, the area to the right of the dashed lines is not included): (in each formula, the areas outside the dashed lines at both ends are not included); more preferably, the group represented by the following partial structural formula (in each formula, the areas outside the dashed lines at both ends are not included): is a group selected from the group consisting of:

[0041] [1-2A] Akn, -L described in the above aspects [1] to [1-2-5] １ -, -L ２ By using chemically modified alginic acid derivatives represented by formula (I) and formula (II) in which the definitions of - are appropriately combined, it is possible to arbitrarily form a preferred embodiment of the cross-linked alginic acid gel in the core layer of the polymer-coated cross-linked alginic acid gel fiber of the present invention.

[0042] [1-3] In the above aspect [1] or [1A], the cells capable of producing antibodies, physiologically active substances, etc., contained in the core layer of the polymer-coated cross-linked alginate gel fiber are, for example, cells selected from the group consisting of antibody-producing cells (various monoclonal antibodies such as human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, etc.), physiologically active substance-producing cells, and cells capable of producing various useful substances useful as pharmaceutical raw materials, chemical raw materials, food raw materials, etc.

[0043] [1-3-1] In the above aspect [1] or [1A], the cells capable of producing antibodies (also referred to as antibody-producing cells) contained in the core layer of the polymer-coated cross-linked alginate gel fiber are hybridomas or cultured cells transformed with an antibody expression vector, and the cultured cells used as their hosts (host cells) are, for example, CHO cells, CHO cell sublines, COS cells, Sp2 / 0 cells, NS0 cells, SP2 cells, The cells are selected from the group consisting of PERC6 cells, YB2 / 0 cells, YE2 / 0 cells, 1R983F cells, Namalwa cells, Wil-2 cells, Jurkat cells, Vero cells, Molt-4 cells, HEK293 cells, BHK cells, KGH6 cells, P3X63Ag8.653 cells, C127 cells, JC cells, LA7 cells, ZR-45-30 cells, hTERT cells, NM2C5 cells, UACC-812 cells, and the like.

[0044] [1-3-2] In the above aspect [1] or [1A], the host cells of the antibody-producing cells contained in the core layer of the polymer-coated cross-linked alginate gel fiber are preferably cells selected from the group consisting of CHO cells, CHO cell sublines, COS cells, Sp2 / 0 cells, NS0 cells, SP2 cells, and PERC6 cells; more preferably, cells selected from the group consisting of CHO cells, CHO cell sublines, Sp2 / 0 cells, and NS0 cells; and even more preferably, CHO cells or CHO cell sublines.

[0045] [1-3-3] In the above aspect [1] or [1A], the antibody-producing cells contained in the core layer of the polymer-coated cross-linked alginate gel fiber are, for example, antibody-producing CHO cells whose host cells are CHO cells, such as muromonab-CD3-producing CHO cells, trastuzumab-producing CHO cells, rituximab-producing CHO cells, palivizumab-producing CHO cells, infliximab-producing CHO cells, basiliximab-producing CHO cells, tocilizumab-producing CHO cells, gemtuzumab-producing CHO cells, bevacizumab-producing CHO cells, and the like. Mab-producing CHO cells, ibritumomab-producing CHO cells, adalimumab-producing CHO cells, cetuximab-producing CHO cells, ranibizumab-producing CHO cells, omalizumab-producing CHO cells, eculizumab-producing CHO cells, panitumumab-producing CHO cells, ustekinumab-producing CHO cells, golimumab-producing CHO cells, canakinumab-producing CHO cells, denosumab-producing CHO cells, mogamulizumab-producing CHO cells, certolizumab-producing CHO cells, ofatumumab-producing CHO cells, pertuzumab-producing CHO cells, blepharitis-producing CHO cells, erythropoietin ... CHO cells producing ntuximab, CHO cells producing natalizumab, CHO cells producing nivolumab, CHO cells producing alemtuzumab, CHO cells producing secukinumab, CHO cells producing ramucirumab, CHO cells producing ipilimumab, CHO cells producing evolocumab, CHO cells producing mepolizumab, CHO cells producing alirocumab, CHO cells producing ixekizumab, CHO cells producing brodalumab, CHO cells producing idarucizumab, CHO cells producing elotuzumab, CHO cells producing pembrolizumab, CHO cells producing sarilumab cells, bezlotoxumab-producing CHO cells, belimumab-producing CHO cells, daratumumab-producing CHO cells, avelumab-producing CHO cells, dupilumab-producing CHO cells, atezolizumab-producing CHO cells, benralizumab-producing CHO cells, inotuzumab-producing CHO cells, emicizumab-producing CHO cells, guselkumab-producing CHO cells, durvalumab-producing CHO cells, obinutuzumab-producing CHO cells, vedolizumab-producing CHO cells, anti-GPVI antibody-producing CHO cells, and the like;For example, the CHO cells are selected from the group consisting of trastuzumab-producing CHO cells, rituximab-producing CHO cells, infliximab-producing CHO cells, tocilizumab-producing CHO cells, adalimumab-producing CHO cells, nivolumab-producing CHO cells, and anti-GPVI antibody-producing CHO cells; for example, tocilizumab-producing CHO cells or anti-GPVI antibody-producing CHO cells;

[0046] [1-3-4] In the above aspect [1] or [1A], the cells capable of producing a physiologically active substance (also referred to as physiologically active substance-producing cells) contained in the core layer of the polymer-coated cross-linked alginate gel fiber are, for example, cells selected from the group consisting of insulin-secreting cells, pancreatic islets, pancreatic islet cells, dopamine-secreting cells, pituitary cells, growth hormone-secreting cells, parathyroid cells, nerve growth factor-secreting cells, blood coagulation factor-secreting cells, hepatocytes, parathyroid cells, erythropoietin-secreting cells, norepinephrine-secreting cells, etc.

[0047] [1-3-5] In the above aspect [1] or [1A], the physiologically active substance-producing cells contained in the core layer of the polymer-coated cross-linked alginate gel fiber are preferably cells selected from the group consisting of insulin-secreting cells, pancreatic islets, and pancreatic islet cells; more preferably, MIN6 cells derived from pancreatic β cells.

[0048] [1-4] In the above aspect [1] or [1A], examples of components that can be additionally contained in the core layer of the polymer-coated cross-linked alginate gel fiber include components selected from the group consisting of alginate solution, alginate gel, culture medium, culture fluid, collagen solution, methylcellulose, sucrose solution, etc.

[0049] [1-4-1] In the above aspect [1] or [1A], the component that can be additionally contained in the core layer of the polymer-coated cross-linked alginate gel fiber is preferably a component selected from the group consisting of an alginate solution, an alginate gel, a culture medium, and a culture fluid.

[0050] [1-5] In the above-mentioned aspect [1] or [1A], the weight-average molecular weight of the chemically modified alginic acid derivative represented by formula (I) used to form the cross-linked alginic acid gel contained in the core layer of the polymer-coated cross-linked alginic acid gel fiber, as measured by gel filtration chromatography, is, for example, in the range of about 100,000 Da to about 3,000,000 Da; preferably in the range of about 300,000 Da to about 2,500,000 Da, and more preferably in the range of about 500,000 Da to about 2,000,000 Da.

[0051] [1-6] In the above-mentioned aspect [1] or [1A], the weight-average molecular weight of the chemically modified alginic acid derivative represented by formula (II) used to form the cross-linked alginic acid gel contained in the core layer of the polymer-coated cross-linked alginic acid gel fiber, as measured by gel filtration chromatography, is, for example, in the range of about 100,000 Da to about 3,000,000 Da; preferably in the range of about 300,000 Da to about 2,500,000 Da, and more preferably in the range of about 500,000 Da to about 2,000,000 Da.

[0052] [1-7] In the above aspect [1] or [1A], the reactive group (Akn-L) of the chemically modified alginic acid derivative represented by formula (I) used to form the crosslinked alginic acid gel contained in the core layer of the polymer-coated crosslinked alginic acid gel fiber is １ -NH ２ Base: Akn-L １ The introduction rate of the aryl group (- is the same as that in the embodiment [1]) is, for example, in the range of about 0.1 to about 30 mol %, preferably in the range of about 0.3 to about 20 mol %, and more preferably in the range of about 0.5 to about 10 mol %.

[0053] [1-8] In the above aspect [1] or [1A], the reactive group (N) of the chemically modified alginic acid derivative represented by formula (II) used to form the crosslinked alginic acid gel contained in the core layer of the polymer-coated crosslinked alginic acid gel fiber is ３ -L ２ -NH ２ Base:-L ２The introduction rate of the aryl group (- is the same as that in the embodiment [1]) is, for example, in the range of about 0.1 to about 30 mol %, preferably in the range of about 0.3 to about 20 mol %, and more preferably in the range of about 0.5 to about 15 mol %.

[0054] [1-9] In the above-mentioned embodiment [1] or [1A], the weight-average molecular weight of the alginic acid solution that can be additionally contained in the core layer of the polymer-coated cross-linked alginic acid gel fiber, or the alginic acid (e.g., sodium alginate) used to prepare the alginic acid solution used to form the alginic acid gel, as measured by gel permeation chromatography (GPC) is, for example, in the range of about 150,000 Da to about 2,500,000 Da; preferably in the range of about 300,000 Da to about 2,000,000 Da, and more preferably in the range of about 700,000 Da to about 2,000,000 Da.

[0055] [1-9-1] In the above-mentioned embodiment [1] or [1A], the weight-average molecular weight, as measured by gel permeation chromatography (GPC), of the alginic acid solution that can be additionally contained in the core layer of the polymer-coated cross-linked alginic acid gel fiber, or of the alginic acid (e.g., sodium alginate) used to prepare the alginic acid solution used to form the alginic acid gel, is, for example, in the range of about 150,000 Da to about 2,500,000 Da; preferably, in the range of about 300,000 Da to about 2,500,000 Da; and more preferably, in the range selected from about 700,000 Da to about 1,400,000 Da, about 800,000 Da to about 1,500,000 Da, about 1,400,000 Da to about 2,000,000 Da, or about 1,500,000 Da to about 2,500,000 Da.

[0056] [1-10-1] In the above-mentioned embodiment [1] or [1A], the concentration of the solution of the chemically modified alginic acid derivative represented by formula (I) used to form the cross-linked alginic acid gel contained in the core layer of the polymer-coated cross-linked alginic acid gel fiber is, for example, in the range of about 0.01 to about 1.5 wt %; preferably, in the range of about 0.05 to about 1.0 wt %; and more preferably, in the range of about 0.08 to about 0.75 wt %.

[0057] [1-10-2] In the above-mentioned embodiment [1] or [1A], the concentration of the solution of the chemically modified alginic acid derivative represented by formula (II) used to form the cross-linked alginic acid gel contained in the core layer of the polymer-coated cross-linked alginic acid gel fiber is, for example, in the range of about 0.01 to about 1.5 wt %; preferably, in the range of about 0.05 to about 1.0 wt %; and more preferably, in the range of about 0.08 to about 0.75 wt %.

[0058] [1-10-3] In the above-mentioned aspect [1] or [1A], the concentration of the mixed solution of the chemically modified alginic acid derivative represented by formula (I) and the chemically modified alginic acid derivative represented by formula (II) used to form the cross-linked alginic acid gel contained in the core layer of the polymer-coated cross-linked alginic acid gel fiber is, for example, in the range of about 0.02 to about 2.0 wt %, preferably in the range of about 0.1 to about 2.0 wt %, and more preferably in the range of about 0.15 to about 1.5 wt %.

[0059] [1-10-4] In the above-mentioned embodiment [1] or [1A], the concentration of the alginate solution that can be additionally contained in the core layer of the polymer-coated cross-linked alginate gel fiber, or the alginate solution used to form the alginate gel, is, for example, in the range of 0 to about 1.98 wt %, preferably 0 to about 1.8 wt %, and more preferably 0 to about 1.7 wt %.

[0060] [1-11-1] In the above-mentioned aspect [1] or [1A], when the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber contains an alginate solution or an alginate gel formed from an alginate solution, the total concentration of the mixed solution containing the chemically modified alginate derivatives represented by formula (I) and formula (II) used to form the core layer and the alginate solution is preferably about 0.5 to about 2.0 wt %; more preferably, it is a concentration selected from about 1.0 wt %, about 1.5 wt %, and about 2.0 wt %.

[0061] [1-11-2] In the above-mentioned aspect [1] or [1A], when the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber includes an alginate solution or an alginate gel formed from an alginate solution, the combination of the concentration (C1 (wt %)) of the mixed solution containing the chemically modified alginic acid derivatives represented by formula (I) and formula (II) used to form the core layer and the concentration (C2 (wt %)) of the alginate solution is preferably a combination selected from the group consisting of (C1:C2) = (approximately 0.2:approximately 1.3), (approximately 0.5:approximately 1.0), (approximately 1.0:approximately 0.5), (approximately 1.5:0), (approximately 0.66:approximately 1.34), and (approximately 0.34:approximately 0.66).

[0062] [1-11-3] In the above-mentioned aspect [1] or [1A], when the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber contains an alginate solution or an alginate gel formed from an alginate solution, the concentration of the solution of the chemically modified alginate derivative represented by formula (I) used to form the core layer (C1A (wt%)), the concentration of the solution of the chemically modified alginate derivative represented by formula (II) The combination of the concentration of the alginic acid solution (C1A (wt%)) and the concentration of the alginic acid solution (C2 (wt%)) is preferably a combination selected from the group consisting of (C1A:C1N:C2) = (about 0.1:about 0.1:about 1.3), (about 0.25:about 0.25:about 1.0), (about 0.5:about 0.5:about 0.5), (about 0.75:about 0.75:0), (about 0.33:about 0.33:about 1.34), and (about 0.17:about 0.17:about 0.66).

[0063] [1-11-4] In the above aspect [1] or [1A], in the mixed solution of the solution of the chemically modified alginic acid derivative represented by formula (I) and the solution of the chemically modified alginic acid derivative represented by formula (II) used to form the cross-linked alginic acid gel contained in the core layer of the polymer-coated cross-linked alginic acid gel fiber, the volume ratio (v1, v2) of the solutions of each derivative is, for example, a ratio of v1 + v2 = 15, e.g., (v1:v2) = (7.5:7.5).

[0064] [1-11-5] In the above-mentioned aspect [1] or [1A], when the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber includes an alginate solution or an alginate gel formed from an alginate solution, the volume ratio of the volumes of each solution (v1, v2, v3) in the mixed solution of the solution of the chemically modified alginate derivative represented by formula (I), the solution of the chemically modified alginate derivative represented by formula (II), and the alginate solution used to form the core layer is, for example, v1 + v2 + v3 = 15, and combinations such as (v1:v2:v3) = (5:5:5), (2.5:2.5:10), and (1:1:13) are possible.

[0065] [1-12] In the above aspect [1] or [1A], the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber is represented by the following formula (III-L): [In formula (III-L), —CONH— and —NHCO— at both ends represent an amide bond via any carboxyl group of alginic acid; —X— represents a group shown in the following table: -L is a cyclic group selected from the group of partial structural formulas shown in the following formulas (in each formula, the areas outside the dashed lines at both ends are not included); １ When -X- is (CL-1) or (CL-1-r), the following table: -L is a divalent linker selected from the group of partial structural formulas shown in the following formulas (in each formula, the areas outside the dashed lines at both ends are not included); １ When -X- is (CL-2) or (CL-2-r), the following table: -L is a divalent linker selected from the group of partial structural formulas shown in the following formulas (in each formula, the areas outside the dashed lines at both ends are not included); ２ - is the same as the definition of formula (II) in the above embodiment [1]].

[0066] [1-12-1] In the formula (III-L) according to the above aspect [1-12], -L １ - represents the above embodiment [1-12], and preferably, when -X- is (CL-1) or (CL-1-r), the following table: (in each formula, the areas outside the dashed lines at both ends are not included); when -X- is (CL-2) or (CL-2-r), a divalent linker selected from the group of partial structural formulas shown in the following table: (In each formula, the areas outside the dashed lines at both ends are not included); More preferably, when -X- is (CL-1) or (CL-1-r), it is a divalent linker selected from the group of partial structural formulas shown in the following table: (in each formula, the areas outside the dashed lines at both ends are not included); when -X- is (CL-2) or (CL-2-r), a divalent linker selected from the group of partial structural formulas shown in the following table: (In each formula, the areas outside the dashed lines at both ends are not included); more preferably, when -X- is (CL-1) or (CL-1-r), it is a divalent linker selected from the group of partial structural formulas shown below: (In each formula, the areas outside the dashed lines at both ends are not included); When -X- is (CL-2) or (CL-2-r), it is a divalent linker selected from the group consisting of the following partial structural formulas: (In each formula, the areas outside the dashed lines at both ends are not included.) Particularly preferably, when —X— is (CL-1) or (CL-1-r), it is a divalent linker selected from the following partial structural formula: (in each formula, the areas outside the dashed lines at both ends are not included); when —X— is (CL-2) or (CL-2-r), it is a divalent linker represented by the following partial structural formula: (In each formula, the areas outside the dashed lines at both ends are not included.)

[0067] [1-12-2] In the formula (III-L) according to the above aspect [1-12], preferred, more preferred, further preferred, and particularly preferred -L ２ - are the same as those defined in the above embodiments [1-2-1] to [1-2-4].

[0068] [1-12-3] In the above aspect [1-12], -L of the group represented by formula (III-L) ２ -XL １ Preferred combinations of - are those of the formulae in the table below: (-L in the table)１ - is the preferred -L described in the above aspect [1-12-1] １ - is the same as the definition of -; ２ - is the preferred -L described in the above aspect [1-2-1] ２ -X- is as defined in the above embodiment [1-12]); more preferably, -L ２ -XL １ - The combinations are as shown in the following table: (-L in the table) １ - is more preferred -L described in the above aspect [1-12-1] １ - is the same as the definition of -; ２ - is more preferably -L described in the above aspect [1-2-2] ２ - is the same as the definition of -; -X- is as defined in the above embodiment [1-12];

[0069] More preferably, -L ２ -XL １ - The combinations are as shown in the following table: (-L in the table) １ - is a more preferred -L according to the above aspect [1-12-1] １ - is the same as the definition of -; ２ - is a more preferred -L according to the above aspect [1-2-3] ２ - is the same as the definition of -; -X- is as defined in the above embodiment [1-12];

[0070] Particularly preferably, -L ２ -XL １ - The combinations are as shown in the following table: (-L in the table) １ - is a particularly preferred -L according to the above embodiment [1-12-1]. １ - is the same as the definition of -; ２ - is a particularly preferred -L according to the above embodiment [1-2-4] ２ - is the same as the definition of -; -X- is as defined in the above embodiment [1-12]).

[0071] [1-12A] In the above aspect [1] or [1A], the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber is represented by the formula (III-L) described in the above aspect [1-12] [in formula (III-L), -CONH- and -NHCO- at both ends, and -X- are the same as defined in the above aspect [1-12]; -L １ - is the same as the group represented by partial structural formula (LK-1a) described in the above aspect [1-12] when -X- is (CL-1) or (CL-1-r); - is the same as the group represented by partial structural formula (LK-2-1) described in the above aspect [1-12] when -X- is (CL-2) or (CL-2-r); -L ２ - is the same as a group selected from partial structural formulas (LN-1), (LN-3), and (LN-5) described in the above aspect [1].

[0072] [1-12A-1] In the above embodiment [1-12A], when -X- is (CL-1) or (CL-1-r), preferred, more preferred, and even more preferred -L １ - is the same as the group represented by the partial structural formula (LK-1a-1), (LK-1a-2), or (LK-1a-3) described in the above aspect [1-12]; when -X- is (CL-2) or (CL-2-r), preferred, more preferred, and even more preferred -L １ - is the same as the group represented by the partial structural formula (LK-2-1), (LK-2-2), or (LK-2-3) described in the above aspect [1-12]; -L ２ - is preferably the same as the groups represented by the partial structural formulas (LN-1-1), (LN-3-1), and (LN-5-1) described in the above aspect [1-2-1], more preferably the same as the groups represented by the partial structural formulas (LN-1-2), (LN-3-2), and (LN-5-2) described in the above aspect [1-2-2], and even more preferably the same as the groups represented by the partial structural formulas (LN-1-3), (LN-3-3a), and (LN-5-3a) described in the above aspect [1-2-3].

[0073] [1-12A-2] In the above embodiment [1-12A], -L of the group represented by formula (III-L) ２ -XL １Preferred, more preferred and even more preferred combinations of - are those of the formulae in the table below: (-L in the table) １ -, -L ２ - is as defined in the above embodiment [1-12A-1]; -X- is as defined in the above embodiment [1-12]).

[0074] [1-13] In the above aspect [1] or [1A], the crosslinked alginate gel contained in the core layer comprises chemical crosslinking via a group represented by formula (III-L) of the above aspect [1-12] [in formula (III-L), each definition is the same as that in aspect [1-12]] or formula (III-L) of the above aspect [1-12A] [in formula (III-L), each definition is the same as that in aspect [1-12A]] and ionic crosslinking via a divalent metal ion.

[0075] [1-13A] In the above aspect [1] or [1A], the divalent metal ion used to form ionic crosslinks in the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber is preferably a divalent metal ion selected from the group consisting of calcium ions, magnesium ions, barium ions, strontium ions, and zinc ions; more preferably, calcium ions, barium ions, or strontium ions; and even more preferably, calcium ions or barium ions.

[0076] [1-14] In the above aspect [1] or [1A], the aqueous solution containing divalent metal ions used for forming ionic crosslinks in the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber can be supplied from an aqueous solution containing divalent metal ions selected from the group consisting of calcium chloride aqueous solution, calcium carbonate aqueous solution, calcium gluconate aqueous solution, barium chloride aqueous solution, strontium chloride aqueous solution, etc.; preferably, it is a calcium chloride aqueous solution or a barium chloride aqueous solution.

[0077] [1-15-1] In the above aspect [1] or [1A], the cationic polymer in the cationic polymer layer of the polymer-coated cross-linked alginate gel fiber is a cationic polymer selected from the group consisting of polyamino acids, basic polysaccharides, basic polymers, etc.

[0078] [1-15-2] In the above aspect [1] or [1A], the cationic polymer of the cationic polymer layer of the polymer-coated cross-linked alginate gel fiber is preferably a cationic polymer selected from the group consisting of polyamino acids, such as poly-L-ornithine (PLO), poly-D-ornithine (PDO), poly-DL-ornithine, poly-D-lysine (PDL), poly-L-lysine (PLL), poly-DL-lysine, poly-L-arginine (PLA), poly-D-arginine (PDA), poly-DL-arginine, poly-L-homoarginine (PLHA), poly-D-homoarginine (PDHA), poly-DL-homoarginine, poly-L-histidine (PLH), poly-D-histidine (PDH), and poly-DL-histidine; more preferably, poly-L-ornithine or poly-L-lysine; and even more preferably, poly-L-ornithine.

[0079] [1-15-3] In the above-mentioned embodiment [1] or [1A], the cationic polymer in the cationic polymer layer of the polymer-coated cross-linked alginate gel fiber is chitosan.

[0080] [1-15-4] In the above aspect [1] or [1A], the cationic polymer of the cationic polymer layer of the polymer-coated cross-linked alginate gel fiber is a cationic polymer selected from the group consisting of polymethylene-co-guanidine (PMCG), polyallylamine (PAA), polyvinylamine (PVA), polyethyleneimine, allylamine-diallylamine copolymer, and allylamine-maleic acid copolymer; preferably, it is polyallylamine (PAA), polyethyleneimine, or polymethylene-co-guanidine (PMCG).

[0081] [1-16] In the above aspect [1] or [1A], the outer diameter of the polymer-coated cross-linked alginate gel fiber is, for example, in the range of about 0.1 to about 2000 μm.

[0082] Akn, -L as described in the above embodiment １ -, -L ２ By using chemically modified alginic acid derivatives represented by formula (I) and formula (II) in which the definitions of - and X are appropriately combined, it is possible to arbitrarily form a preferred embodiment of the cross-linked alginic acid gel in the core layer of the polymer-coated cross-linked alginic acid gel fiber of the above embodiment.

[0083] [1-17-1] The polymer-coated cross-linked alginate gel fiber of the above aspect [1] or [1A] is preferably a polymer-coated cross-linked alginate gel fiber in which the alginate derivative represented by formula (I) used to form the cross-linked alginate gel is selected from the alginate derivatives described in the above aspect [1-1-1] or the preferred alginate derivatives described in the above aspect [1-1-5], the alginate derivative represented by formula (II) is selected from the alginate derivatives described in the above aspect [1-2-1] or the preferred alginate derivatives described in the above aspect [1-2-5]; the antibody-producing cells are selected from the cells described in the above aspects [1-3-1] to [1-3-3]; and the cationic polymer layer is selected from the cationic polymers described in the above aspect [1-15-1].

[0084] [1-17-1B] The polymer-coated cross-linked alginate gel fiber of the above aspect [1] or [1A] is preferably a polymer-coated cross-linked alginate gel fiber in which the alginate derivative represented by formula (I) used to form the cross-linked alginate gel is selected from the alginate derivatives described in the above aspect [1-1-1] or the preferred alginate derivatives described in the above aspect [1-1-5], the alginate derivative represented by formula (II) is selected from the alginate derivatives described in the above aspect [1-2-1] or the preferred alginate derivatives described in the above aspect [1-2-5]; the antibody-producing cells are selected from the cells described in the above aspects [1B-3-1] to [1B-3-9]; and the cationic polymer layer is selected from the cationic polymers described in the above aspect [1-15-1].

[0085] [1-17-2] The polymer-coated cross-linked alginate gel fiber of the above aspect [1] or [1A] is more preferably a polymer-coated cross-linked alginate gel fiber in which the alginate derivative represented by formula (I) used to form the cross-linked alginate gel is selected from the alginate derivatives described in the above aspect [1-1-2] or the more preferred alginate derivatives described in the above aspect [1-1-5], the alginate derivative represented by formula (II) is selected from the alginate derivatives described in the above aspect [1-2-2] or the more preferred alginate derivatives described in the above aspect [1-2-5]; the antibody-producing cells are selected from the cells described in the above aspects [1-3-2] to [1-3-3]; and the cationic polymer layer is selected from the cationic polymers described in the above aspects [1-15-2] to [1-15-4].

[0086] [1-17-2B] The polymer-coated cross-linked alginate gel fiber of the above aspect [1] or [1A] is more preferably a polymer-coated cross-linked alginate gel fiber in which the alginate derivative represented by formula (I) used to form the cross-linked alginate gel is selected from the alginate derivatives described in the above aspect [1-1-2] or the more preferred alginate derivatives described in the above aspect [1-1-5], the alginate derivative represented by formula (II) is selected from the alginate derivatives described in the above aspect [1-2-2] or the more preferred alginate derivatives described in the above aspect [1-2-5]; the antibody-producing cells are selected from the cells described in the above aspects [1B-3-2] to [1B-3-9]; and the cationic polymer layer is selected from the cationic polymers described in the above aspects [1-15-2] to [1-15-4].

[0087] [1-17-3] The polymer-coated cross-linked alginate gel fiber of the above aspect [1] or [1A] is more preferably a polymer-coated cross-linked alginate gel fiber in which the alginate derivative represented by formula (I) used to form the cross-linked alginate gel is selected from the alginate derivatives described in the above aspect [1-1-3], the alginate derivative represented by formula (II) is selected from the alginate derivatives described in the above aspect [1-2-3]; the antibody-producing cells are selected from the cells described in the above aspects [1-3-2] to [1-3-3]; and the cationic polymer layer is selected from the cationic polymers described in the above aspect [1-15-2] or [1-15-4].

[0088] [1-17-3B] The polymer-coated cross-linked alginate gel fiber of the above aspect [1] or [1A] is more preferably a polymer-coated cross-linked alginate gel fiber in which the alginate derivative represented by formula (I) used to form the cross-linked alginate gel is selected from the alginate derivatives described in the above aspect [1-1-3], the alginate derivative represented by formula (II) is selected from the alginate derivatives described in the above aspect [1-2-3]; the antibody-producing cells are selected from the cells described in the above aspect [1B-3-3] or [1B-3-9]; and the cationic polymer layer is selected from the cationic polymers described in the above aspect [1-15-2] or [1-15-4].

[0089] [1-17-4] The polymer-coated cross-linked alginate gel fiber of the above aspect [1] or [1A] is particularly preferably a polymer-coated cross-linked alginate gel fiber in which the alginate derivative represented by formula (I) used to form the cross-linked alginate gel is selected from the alginate derivatives described in the above aspect [1-1-4], and the alginate derivative represented by formula (II) is selected from the alginate derivatives described in the above aspect [1-2-4]; the antibody-producing cells are antibody-producing CHO cells; and the cationic polymer layer is selected from poly-L-ornithine, polyallylamine (PAA), polyethyleneimine, or polymethylene-co-guanidine (PMCG).

[0090] [1-17-5] In the above aspects [1-17-1] to [1-17-4], the cross-linked alginate gel of the polymer-coated cross-linked alginate gel fiber contains any of the addable components described in the above aspects [1-4] to [1-4-1].

[0091] In the polymer-coated cross-linked alginate gel fiber described in the above embodiment, by combining the elements of cells capable of producing antibodies, physiologically active substances, etc., the chemically modified alginic acid derivatives represented by formula (I) and formula (II) used to form the cross-linked alginate gel, and the cationic polymer (cationic polymer layer), it is possible to form any preferred embodiment of the method for producing polymer-coated cross-linked alginate gel fiber.

[0092] [1B] Aspect 1B is as follows: A multilayer polymer-coated crosslinked alginate gel fiber comprising a core layer containing cells capable of producing antibodies, physiologically active substances, etc. embedded in a crosslinked alginate gel obtained by crosslinking using chemically modified alginate derivatives represented by formulas (I) and (II), a cationic polymer layer (cationic polymer) coating the core layer, and an anionic polymer layer (anionic polymer) coating the cationic polymer layer. The chemically modified alginate derivatives represented by formulas (I) and (II) are the same as those defined in aspect [1] above.

[0093] [1C] Aspect 1C is as follows: A multilayer polymer-coated cross-linked alginate gel fiber obtained by coating a core layer containing cells capable of producing antibodies, physiologically active substances, etc., and a cross-linked alginate gel obtained by a cross-linking reaction using chemically modified alginate derivatives represented by formulas (I) and (II) with a cationic polymer and an anionic polymer. The chemically modified alginate derivatives represented by formulas (I) and (II) are the same as those defined in aspect [1] above.

[0094] [1D] Aspect 1D is as follows: A multilayer polymer-coated cross-linked alginate gel fiber comprising a core layer, a cationic polymer layer disposed on the outside of the core layer, and an anionic polymer layer disposed on the outside of the cationic polymer layer, wherein the core layer comprises cells capable of producing antibodies, physiologically active substances, etc., and cross-linked alginate gel formed by crosslinking using chemically modified alginate derivatives represented by formulas (I) and (II). The chemically modified alginate derivatives represented by formulas (I) and (II) are the same as those defined in aspect [1] above.

[0095] [1B-1-1] In the above aspects [1B] to [1D], the chemically modified alginic acid derivative Akn-L represented by formula (I) １ - is preferably the same as the definition described in the above-mentioned embodiment [1-1-1]; more preferably the same as the definition described in the above-mentioned embodiment [1-1-2]; even more preferably the same as the definition described in the above-mentioned embodiment [1-1-3]; particularly preferably the same as the definition described in the above-mentioned embodiment [1-1-4]; and most preferably the following partial structural formula (in each formula, the right side of the dashed line is not included): [1B-1-2] The above-mentioned aspect In the above-mentioned aspects [1B] to [1D], the Akn-L of the chemically modified alginic acid derivative represented by formula (I) is a group represented by the following formula: １ - is the same as defined in the above embodiment [1-1-5]; Preferred, more preferred, and even more preferred Akn-L １ - are each defined as in the above embodiment [1-1-5]; and particularly preferably, the following partial structural formulas (in each formula, the right side of the dashed line is not included): It is a group represented by the following formula:

[0096] [1B-2-1] The above-mentioned aspect In the above-mentioned aspects [1B] to [1D], -L of the chemically modified alginic acid derivative represented by formula (II) ２- is preferably the same as defined in the above-mentioned embodiment [1-2-1]; more preferably the same as defined in the above-mentioned embodiment [1-2-2]; even more preferably the same as defined in the above-mentioned embodiment [1-2-3]; particularly preferably the same as defined in the above-mentioned embodiment [1-2-4]; and most preferably the following partial structural formula (in each formula, the areas outside the dashed lines at both ends are not included): It is a group represented by the following formula:

[0097] [1B-2-2] The above-mentioned aspect In the above-mentioned aspects [1B] to [1D], -L of the chemically modified alginic acid derivative represented by formula (II) ２ - is the same as defined in the above embodiment [1-2-5]; preferred, more preferred, and even more preferred -L ２ - are each defined as in the above embodiment [1-2-5]; and particularly preferably, the following partial structural formulas (in each formula, the areas outside the dashed lines at both ends are not included): It is a group represented by the following formula:

[0098] [1B-2A] Akn, -L described in the above aspects [1], [1B-1-1] to [1B-2-2] １ -, -L ２ By using chemically modified alginic acid derivatives represented by formula (I) and formula (II) in which the definitions of - are appropriately combined, it is possible to arbitrarily form a preferred embodiment of the cross-linked alginic acid gel in the core layer of the multilayer polymer-coated cross-linked alginic acid gel fiber of the present invention.

[0099] [1B-3] In the above aspects [1], [1A], and [1B] to [1D], the cells capable of producing antibodies, physiologically active substances, etc., contained in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber include, for example, antibody-producing cells (various monoclonal antibodies such as human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, etc., or various modified antibodies such as their bispecific antibodies, minibodies, and glycosylated antibodies), physiologically active substance-producing cells (enzymes, cytokines, hormones, blood coagulation factors, vaccines, etc.), and cells capable of producing various useful substances useful as pharmaceutical raw materials, chemical raw materials, food raw materials, etc.; preferably, antibody-producing cells or physiologically active substance-producing cells.

[0100] [1B-3-1] In the above aspects [1], [1A], [1B], [1C] or [1D], the antibody-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber are hybridomas obtained from antibody-producing B cells (antibody-producing hybridomas) or cultured cells transformed with an antibody expression vector (antibody-producing genetically modified cells).

[0101] [1B-3-2] In the above aspects [1], [1A], and [1B] to [1D], the antibody-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber are preferably genetically modified animal cells that produce antibodies.

[0102] [1B-3-3] In the above aspect [1B-3-2], the animal cells used as the host include CHO cells, CHO cell sublines (CHO-K1 cells, CHO-DG44 cells, CHO-DXB11 cells, or CHO cells transformed to modify their sugar chains, etc.), COS cells, Sp2 / 0 cells, NS0 cells, SP2 cells, PERC6 cells, YB2 / 0 cells, YE2 / 0 cells, 1R The cell is selected from 983F cells, Namalwa cells, Wil-2 cells, Jurkat cells, Vero cells, Molt-4 cells, HEK293 cells, BHK cells, HT-1080 cells, KGH6 cells, P3X63Ag8.653 cells, C127 cells, JC cells, LA7 cells, ZR-45-30 cells, hTERT cells, NM2C5 cells, and UACC-812 cells.

[0103] [1B-3-4] In the above aspect [1B-3-2], the animal cells used as the host are preferably cells selected from CHO cells, CHO cell sublines, COS cells, Sp2 / 0 cells, NS0 cells, SP2 cells, PERC6 cells, HEK293 cells, BHK cells, HT-1080 cells, and C127 cells; more preferably cells selected from CHO cells, CHO cell sublines, Sp2 / 0 cells, NS0 cells, HEK293 cells, and BHK cells; and even more preferably CHO cells or CHO cell sublines.

[0104] [1B-3-5] In the above aspects [1], [1A], and [1B] to [1D], the host cells of the antibody-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber are preferably cells selected from CHO cells, CHO cell sublines, Sp2 / 0 cells, or NS0 cells; more preferably, CHO cells or CHO cell sublines.

[0105] [1B-3-6] In the above aspects [1], [1A], and [1B] to [1D], the antibody-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber are cells that produce antibodies used as biopharmaceuticals or biopharmaceutical raw materials.

[0106] [1B-3-7] In the above aspects [1], [1A], and [1B] to [1D], antibody-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber include muromonab-CD3, trastuzumab, rituximab, palivizumab, infliximab, basiliximab, tocilizumab, bevacizumab, adalimumab, cetuximab, omalizumab, eculizumab, panitumumab, ustekinumab, golimumab, and canadian serotypes. Kinumab, denosumab, ofatumumab, pertuzumab, natalizumab, nivolumab, alemtuzumab, secukinumab, ramucirumab, ipilimumab, evolocumab, mepolizumab, alirocumab, ixekizumab, brodalumab, elotuzumab, pembrolizumab, sarilumab, bezlotoxumab, belimumab, daratumumab, avelumab, dupilumab, atezolizumab, emicizumab, guselkumab, durvalumab, vedolizumab, romosozumab, risankizumab, necitumab Mab, ravulizumab, burosumab, isatuximab, tildrakizumab, satralizumab, galcanezumab, dinutuximab, fremanezumab, erenumab, casirivimab, imdevimab, anifrolumab, sotrovimab, ocrelizumab, naxitamab, aducanumab, tafasitamab, marjetuximab, gantenerumab, tiragolumab, clovalimab, nemolizumab, catumaxomab, pramotamab, faricimab, gemtuzumab, ibritumomab, brentuximab, i The cells are selected from cells that produce antibodies such as notuzumab, polatuzumab, enfortumab, sacituzumab, belantamab, loncustuximab, tisotumab, datopotamab, and patritumab; cells that produce antibodies with modified sugar chains such as mogamulizumab, benralizumab, obinutuzumab, and inebilizumab; and cells that produce low molecular weight antibodies consisting of antibody fragments such as ranibizumab, idarucizumab, blinatumomab, brolucizumab, abciximab, caplacizumab, and certolizumab.

[0107] [1B-3-8] In the above aspects [1], [1A], and [1B] to [1D], the antibody-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber are antibody-producing animal cells, preferably antibody-producing CHO cells, antibody-producing Sp2 / 0 cells, or antibody-producing NS0 cells, and more preferably antibody-producing CHO cells.

[0108] [1B-3-9] In the above aspects [1], [1A], and [1B] to [1D], the antibody-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber are preferably antibody-producing CHO cells whose host cells are CHO cells, such as muromonab-CD3-producing CHO cells, trastuzumab-producing CHO cells, rituximab-producing CHO cells, palivizumab-producing CHO cells, infliximab-producing CHO cells, basiliximab-producing CHO cells, tocilizumab-producing CHO cells, and gelatinocyte-producing CHO cells. Mutuzumab-producing CHO cells, bevacizumab-producing CHO cells, ibritumomab-producing CHO cells, adalimumab-producing CHO cells, cetuximab-producing CHO cells, ranibizumab-producing CHO cells, omalizumab-producing CHO cells, eculizumab-producing CHO cells, panitumumab-producing CHO cells, ustekinumab-producing CHO cells, golimumab-producing CHO cells, canakinumab-producing CHO cells, denosumab-producing CHO cells, mogamulizumab-producing CHO cells, certolizumab-producing CHO cells, ofatumumab-producing CHO cells, pertuzumab-producing CHO cells, brentuximab CHO cells producing ribozyme, CHO cells producing natalizumab, CHO cells producing nivolumab, CHO cells producing alemtuzumab, CHO cells producing secukinumab, CHO cells producing ramucirumab, CHO cells producing ipilimumab, CHO cells producing evolocumab, CHO cells producing mepolizumab, CHO cells producing alirocumab, CHO cells producing ixekizumab, CHO cells producing brodalumab, CHO cells producing idarucizumab, CHO cells producing elotuzumab, CHO cells producing pembrolizumab, CHO cells producing sarilumab, CHO cells producing bezlotoxumab, CHO cells producing belimumab HO cells, daratumumab-producing CHO cells, avelumab-producing CHO cells, dupilumab-producing CHO cells, atezolizumab-producing CHO cells, benralizumab-producing CHO cells, inotuzumab-producing CHO cells, emicizumab-producing CHO cells, guselkumab-producing CHO cells, durvalumab-producing CHO cells, obinutuzumab-producing CHO cells, vedolizumab-producing CHO cells, romosozumab-producing CHO cells, risankizumab-producing CHO cells, necitumumab-producing CHO cells, ravulizumab-producing CHO cells, burosumab-producing CHO cells, isatuximab-producing CHO cells,Tildrakizumab-producing CHO cells, satralizumab-producing CHO cells, galcanezumab-producing CHO cells, dinutuximab-producing CHO cells, fremanezumab-producing CHO cells, erenumab-producing CHO cells, casirivimab-producing CHO cells, imdevimab-producing CHO cells, anifrolumab-producing CHO cells, sotrovimab-producing CHO cells, ocrelizumab-producing CHO cells, naxitamab-producing CHO cells, aducanumab-producing CHO cells, tafasitamab-producing CHO cells, marjetuximab-producing CHO cells, polatuzumab-producing CHO cells, enfortumab-producing CHO cells, sacituzumab-producing CHO cells , belantamab-producing CHO cells, loncastuximab-producing CHO cells, tisotumab-producing CHO cells, inebilizumab-producing CHO cells, blinatumomab-producing CHO cells, brolucizumab-producing CHO cells, abciximab-producing CHO cells, caplacizumab-producing CHO cells, or anti-GPVI antibody-producing CHO cells; or trastuzumab-producing CHO cells, rituximab-producing CHO cells, infliximab-producing CHO cells, tocilizumab-producing CHO cells, adalimumab-producing CHO cells, nivolumab-producing CHO cells, or anti-GPVI antibody-producing CHO cells.

[0109] [1B-3-10] In the above aspects [1], [1A], [1B] to [1D], the physiologically active substance-producing cells contained in the core layer of the polymer-coated cross-linked alginate gel fiber or the multi-layered polymer-coated cross-linked alginate gel fiber are the same as the physiologically active substance-producing cells described in the above aspect [1-3-4].

[0110] [1B-3-11] In the above aspects [1], [1A], and [1B] to [1D], the physiologically active substance-producing cells contained in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber are cells selected from the group consisting of insulin-secreting cells, pancreatic islets, pancreatic islet cells, or MIN6 cells derived from pancreatic β cells.

[0111] [1B-3-12] In the above aspects [1], [1A], and [1B] to [1D], the physiologically active substance-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber are cultured cells transformed with a physiologically active substance expression vector (biologically active substance-producing genetically modified cells).

[0112] [1B-3-13] In the above aspects [1], [1A], and [1B] to [1D], the physiologically active substance-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or multilayer polymer-coated cross-linked alginate gel fiber are genetically modified animal cells that produce physiologically active substances.

[0113] [1B-3-14] In the above aspect [1B-3-13], the animal cell used as the host is a cell selected from CHO cells, CHO cell sublines, COS cells, Sp2 / 0 cells, NS0 cells, SP2 cells, PERC6 cells, HEK293 cells, BHK cells, HT-1080 cells, and C127 cells; preferably, a cell selected from CHO cells, CHO cell sublines, Sp2 / 0 cells, and NS0 cells, HEK293 cells, and BHK cells; more preferably, a CHO cell or a CHO cell subline.

[0114] [1B-3-15] In the above aspects [1], [1A], and [1B] to [1D], the host cells of the physiologically active substance-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber are preferably cells selected from CHO cells, CHO cell sublines, HEK293 cells, or BHK cells; more preferably, CHO cells or CHO cell sublines.

[0115] [1B-3-16] In the above aspects [1], [1A], and [1B] to [1D], the physiologically active substance-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or multilayer polymer-coated cross-linked alginate gel fiber are cells that produce physiologically active substances used as biopharmaceuticals or biopharmaceutical raw materials.

[0116] [1B-3-17] In the above aspects [1], [1A], [1B] to [1D], the physiologically active substance-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or the multi-layered polymer-coated cross-linked alginate gel fiber include alteplase, monteplase, imiglucerase, velaglucerase, agalsidase, laronidase, alglucosidase, avalglucosidase, idursulfase, galsulfase, elosulfase, rasbufase, and the like. cells that produce enzymes such as ribosomal enzymes, dornase, cerliponase, glucarpidase, hyaluronidase, and asfotase; cells that produce blood coagulation factors and blood-related proteins such as eptacog, octocog, rurioctocog, turoctocog, lonoctocog, damoctocog, simoctocog, nonacog, albutrepenonacog, catridecacog, efflaloctocog, eftrenonacog, thrombomodulin, antithrombin, vonicog, and albumin; insulin, insulin Cells that produce hormones such as lispro, insulin aspart, insulin glargine, insulin detemir, insulin glulisine, insulin degludec, somatropin, somapsitan, mecasermin, carperitide, vosoritide, glucagon, follitropin, choriogonadotropin, dulaglutide, liraglutide, semaglutide, teduglutide, teriparatide, and metreleptin; cells that produce interferons such as interferon alpha-2a, interferon alpha-2b, interferon beta-1a, interferon beta-1b, and interferon gamma-1a; cells that produce hematopoietic factors such as epoetin, darbepoetin, and romiplostim; filgrastim, lenograstim, teceleukin, trafermin, belfermin, etanercept, aflibercept, and denileukin The cells are selected from cells that produce cytokines such as diftitox and their receptors; and cells that produce cell surface antigens such as abatacept, cell surface receptors and their ligands.

[0117] [1B-3-18] In the above aspects [1], [1A], and [1B] to [1D], the physiologically active substance-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber are physiologically active substance-producing animal cells, preferably physiologically active substance-producing CHO cells, physiologically active substance-producing HEK293 cells, or physiologically active substance-producing BHK cells, and more preferably physiologically active substance-producing CHO cells.

[0118] [1B-3-19] In the above aspects [1], [1A], and [1B] to [1D], the physiologically active substance-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber are preferably physiologically active substance-producing CHO cells whose host cells are CHO cells, for example, cells selected from alteplase-producing CHO cells, alglucosidase-producing CHO cells, lurioctocog-producing CHO cells, dulaglutide-producing CHO cells, interferon beta-1a-producing CHO cells, darbepoetin-producing CHO cells, etanercept-producing CHO cells, aflibercept-producing CHO cells, and abatacept-producing CHO cells.

[0119] [1B-4] In the above aspects [1B] to [1D], the components that can be additionally contained in the core layer of the multilayer polymer-coated cross-linked alginate gel fiber are the same as the components described in the above aspect [1-4]; preferred components are the same as the components described in the above aspect [1-4-1].

[0120] [1B-5] In the above aspects [1B] to [1D], the weight-average molecular weight measured by gel filtration chromatography of the chemically modified alginic acid derivative represented by formula (I) used to form the cross-linked alginic acid gel contained in the core layer of the multilayer polymer-coated cross-linked alginic acid gel fiber is the same as the range of weight-average molecular weight described in the above aspect [1-5].

[0121] [1B-6] In the above aspects [1B] to [1D], the weight-average molecular weight measured by gel filtration chromatography of the chemically modified alginic acid derivative represented by formula (II) used to form the cross-linked alginic acid gel contained in the core layer of the multilayer polymer-coated cross-linked alginic acid gel fiber is the same as the range of weight-average molecular weight described in the above aspect [1-6].

[0122] [1B-7] In the above aspects [1B] to [1D], the reactive group (Akn-L) of the chemically modified alginic acid derivative represented by formula (I) used to form the crosslinked alginic acid gel contained in the core layer of the multilayer polymer-coated crosslinked alginic acid gel fiber is １ -NH ２ Base: Akn-L １ The introduction rate of (- is the same as defined in the above embodiment [1], [1B-1-1] or [1B-1-2]) is the same as the introduction rate range described in the above embodiment [1-7].

[0123] [1B-8] In the above aspects [1B] to [1D], the reactive group (N) of the chemically modified alginic acid derivative represented by formula (II) used to form the crosslinked alginic acid gel contained in the core layer of the multilayer polymer-coated crosslinked alginic acid gel fiber is ３ -L ２ -NH ２ Base:-L ２ The introduction rate of (- is the same as defined in the above embodiment [1], [1B-2-1] or [1B-2-2]) is the same as the introduction rate range described in the above embodiment [1-8].

[0124] [1B-9] In the above aspects [1B] to [1D], the weight-average molecular weight measured by gel permeation chromatography (GPC) of the alginic acid solution that can be additionally contained in the core layer of the multilayer polymer-coated cross-linked alginic acid gel fiber, or of the alginic acid (e.g., sodium alginate) used to prepare the alginic acid solution used to form the alginic acid gel, is the same as the range of weight-average molecular weight described in the above aspect [1-9-1].

[0125] [1B-10-1] In the above aspects [1B] to [1D], the concentration of the solution of the chemically modified alginic acid derivative represented by formula (I) used to form the cross-linked alginic acid gel contained in the core layer of the multilayer polymer-coated cross-linked alginic acid gel fiber is the same as the concentration range described in the above aspect [1-10-1].

[0126] [1B-10-2] In the above aspects [1B] to [1D], the concentration of the solution of the chemically modified alginic acid derivative represented by formula (II) used to form the cross-linked alginic acid gel contained in the core layer of the multilayer polymer-coated cross-linked alginic acid gel fiber is the same as the concentration range described in the above aspect [1-10-2].

[0127] [1B-10-3] In the above aspects [1B] to [1D], the concentration of the mixed solution of the chemically modified alginic acid derivative represented by formula (I) and the chemically modified alginic acid derivative represented by formula (II) used to form the cross-linked alginic acid gel contained in the core layer of the multilayer polymer-coated cross-linked alginic acid gel fiber is the same as the concentration range described in the above aspect [1-10-3].

[0128] [1B-10-4] In the above aspects [1B] to [1D], the concentration of the alginate solution that can be additionally contained in the core layer of the multilayer polymer-coated cross-linked alginate gel fiber, or the alginate solution used to form the alginate gel, is the same as the concentration range described in the above aspect [1-10-4].

[0129] [1B-11-1] In the above aspects [1B] to [1D], when the crosslinked alginate gel contained in the core layer of the multilayer polymer-coated crosslinked alginate gel fiber contains an alginate solution or an alginate gel formed from an alginate solution, the total concentration of the mixed solution containing the chemically modified alginate derivatives represented by formula (I) and formula (II) used to form the core layer and the alginate solution is preferably about 0.5 to about 2.0 wt %; more preferably, a concentration selected from about 1.0 wt %, about 1.5 wt %, and about 2.0 wt %; and even more preferably, about 1.5 wt %.

[0130] [1B-11-2] In the above aspects [1B] to [1D], when the cross-linked alginate gel contained in the core layer of the multilayer polymer-coated cross-linked alginate gel fiber includes an alginate solution or an alginate gel formed from an alginate solution, the combination of the concentration (C1 (wt %)) of the mixed solution containing the chemically modified alginic acid derivatives represented by formula (I) and formula (II) used to form the core layer and the concentration (C2 (wt %)) of the alginate solution is preferably a combination selected from the group consisting of (C1:C2) = (about 0.2:about 1.3), (about 0.5:about 1.0), (about 1.0:about 0.5), (about 1.5:0), (about 0.66:about 1.34), and (about 0.34:about 0.66).

[0131] [1B-11-3] In the above aspects [1B] to [1D], when the crosslinked alginate gel contained in the core layer of the multilayer polymer-coated crosslinked alginate gel fiber contains an alginate solution or an alginate gel formed from an alginate solution, the concentration (C1A (wt%)) of the solution of the chemically modified alginate derivative represented by formula (I) used to form the core layer, the concentration (C1A (wt%)) of the solution of the chemically modified alginate derivative represented by formula (II) The combination of the concentration (C1N (wt%)) and the concentration of the alginic acid solution (C2 (wt%)) is preferably a combination selected from the group consisting of (C1A:C1N:C2) = (about 0.1:about 0.1:about 1.3), (about 0.25:about 0.25:about 1.0), (about 0.5:about 0.5:about 0.5), (about 0.75:about 0.75:0), (about 0.33:about 0.33:about 1.34), and (about 0.17:about 0.17:about 0.66).

[0132] [1B-11-4] In the above aspects [1B] to [1D], in the mixed solution of the chemically modified alginic acid derivative solution represented by formula (I) and the chemically modified alginic acid derivative solution represented by formula (II) used to form the cross-linked alginic acid gel contained in the core layer of the multilayer polymer-coated cross-linked alginic acid gel fiber, the volume ratio (v1, v2) of the solutions of each derivative is, for example, v1 + v2 = 15, e.g., (v1:v2) = (7.5:7.5).

[0133] [1B-11-5] In the above aspects [1B] to [1D], when the crosslinked alginate gel contained in the core layer of the multilayer polymer-coated crosslinked alginate gel fiber includes an alginate solution or an alginate gel formed from an alginate solution, the volume ratio of the volumes of each solution (v1, v2, v3) in the mixed solution of the solution of the chemically modified alginate derivative represented by formula (I), the solution of the chemically modified alginate derivative represented by formula (II), and the alginate solution used to form the core layer is, for example, v1 + v2 + v3 = 15, and combinations such as (v1:v2:v3) = (5:5:5), (2.5:2.5:10), and (1:1:13) are possible.

[0134] [1B-12] In the above aspects [1B] to [1D], the cross-linked alginate gel contained in the core layer of the multilayer polymer-coated cross-linked alginate gel fiber includes chemical cross-linking via a group represented by formula (III-L) (the definitions in the formula are the same as those in the above aspect [1-12]) described in the above aspect [1-12].

[0135] [1B-12-1] In the formula (III-L) of the above aspect [1B-12], preferred, more preferred, even more preferred, and particularly preferred -L １ - is each defined as in the above embodiment [1-12-1]; Most preferably, -X- is (CL-2) or (CL-2-r) and is represented by the following partial structural formula: (In each formula, the areas outside the dashed lines at both ends are not included.)

[0136] [1B-12-2] In the formula (III-L) of the above aspect [1B-12], preferred, more preferred, even more preferred, and particularly preferred -L ２ - are each defined as described in the above embodiment [1-12-2]; and most preferably, they are represented by the following partial structural formula: (In each formula, the areas outside the dashed lines at both ends are not included.)

[0137] [1B-12-3] In the above aspect [1B-12], -L in the group represented by formula (III-L) is preferred, more preferred, further preferred, and particularly preferred. ２ -XL１ Each of the combinations of -L has the same definition as that described in the above embodiment [1-12-3]; most preferably, -L ２ -XL １ - is represented by the following formula: (Each formula does not include the areas outside the dashed lines at both ends.)

[0138] [1B-12A] In the above aspects [1B] to [1D], the cross-linked alginate gel contained in the core layer of the multilayer polymer-coated cross-linked alginate gel fiber contains chemical cross-links via a group represented by formula (III-L) described in the above aspect [1-12A] [each definition in formula (III-L) is the same as the definition described in the above aspect [1-12A]].

[0139] [1B-12A-1] In the above aspect [1B-12A], preferred, more preferred, and even more preferred -L １ - is the same as the group described in the above embodiment [1-12A-1]; preferred, more preferred, and even more preferred -L ２ - is the same as the group described in the above embodiment [1-12A-1].

[0140] [1B-12A-2] In the above embodiment [1B-12A], -L of the group represented by formula (III-L) ２ -XL １ Preferred, more preferred, and even more preferred combinations of - are the same as those described in [1-12A-2]; particularly preferred combinations are those of the following formula: (Each formula does not include the areas outside the dashed lines at both ends.)

[0141] [1B-13] In the above aspects [1B] to [1D], the crosslinked alginate gel contained in the core layer of the multilayer polymer-coated crosslinked alginate gel fiber comprises chemical crosslinking via a group represented by formula (III-L) of the above aspect [1-12] [in formula (III-L), each definition is the same as that in the above aspect [1-12]] or formula (III-L) of the above aspect [1B-12A] [in formula (III-L), each definition is the same as that in the above aspect [1B-12A]] and ionic crosslinking via a divalent metal ion.

[0142] [1B-13A] In the above aspects [1B] to [1D], the divalent metal ions used to form ionic crosslinks in the crosslinked alginate gel contained in the core layer are the same as the divalent metal ions described in the above aspect [1-13A].

[0143] [1B-14] In the above aspects [1B] to [1D], the source of the divalent metal ions used to form ionic crosslinks in the crosslinked alginate gel contained in the core layer of the multilayer polymer-coated crosslinked alginate gel fiber is the same as the aqueous solution containing divalent metal ions described in the above aspect [1-14].

[0144] [1B-15-1] In the above embodiments [1B] to [1D], the cationic polymer in the cationic polymer layer of the multilayer polymer-coated cross-linked alginate gel fiber is the same as the cationic polymer described in the above embodiment [1-15-1].

[0145] [1B-15-2] In the above aspects [1B] to [1D], the preferred, more preferred, and even more preferred cationic polymers for the cationic polymer layer of the multilayer polymer-coated cross-linked alginate gel fiber are the same as the cationic polymers described in the above aspect [1-15-2].

[0146] [1B-15-3] In the above embodiments [1B] to [1D], the cationic polymer in the cationic polymer layer of the multi-polymer-coated cross-linked alginate gel fiber is chitosan.

[0147] [1B-15-4] In the above embodiments [1B] to [1D], the cationic polymer of the multilayer polymer-coated cross-linked alginate gel fiber is the same as the cationic polymer described in the above embodiment [1-15-4].

[0148] [1B-16] In the above aspects [1B] to [1D], the anionic polymer layer of the multilayer polymer-coated cross-linked alginate gel fiber is an anionic polymer selected from the group consisting of anionic polysaccharides, sulfated polysaccharides, synthetic polymers, anionic polyamino acids, chemically modified products thereof, cross-linked products thereof, mixtures thereof, etc.

[0149] [1B-16-1] In the above aspects [1B] to [1D], the anionic polymer layer of the multilayer polymer-coated cross-linked alginate gel fiber is an anionic polymer selected from the group consisting of anionic polysaccharides such as alginic acid, hyaluronic acid, caprygalacturonic acid, lagynan, succinoglucan, gum arabic, xanthan gum, alginic acid, pectin, pectic acid, carboxymethylcellulose, and agar, chemically modified products thereof, cross-linked products thereof, and mixtures thereof.

[0150] [1B-16-2] In the above embodiments [1B] to [1D], the anionic polymer layer of the multi-polymer-coated cross-linked alginate gel fiber is alginic acid.

[0151] [1B-16-3] In the above aspects [1B] to [1D], the anionic polymer layer of the multilayer polymer-coated cross-linked alginate gel fiber is an anionic polymer selected from the group consisting of the chemically modified alginic acid derivative represented by formula (I) of the above aspect [1] or [1-1-5], the chemically modified alginic acid derivative represented by formula (II) of the above aspect [1] or [1-2-5], a crosslinked product formed from the chemically modified alginic acid derivative represented by formula (I) of the above aspect [1] or [1-1-5] and the chemically modified alginic acid derivative represented by formula (II) of the above aspect [1] or [1-2-5], and a mixture thereof.

[0152] [1B-16-4] In the above aspects [1B] to [1D], the anionic polymer layer of the multilayer polymer-coated cross-linked alginate gel fiber is an anionic polymer selected from the group consisting of alginic acid, the chemically modified alginic acid derivative represented by formula (I) of the above aspect [1] or [1-1-5], the chemically modified alginic acid derivative represented by formula (II) of the above aspect [1] or [1-2-5], a crosslinked product formed from the chemically modified alginic acid derivative represented by formula (I) of the above aspect [1] or [1-1-5] and the chemically modified alginic acid derivative represented by formula (II) of the above aspect [1] or [1-2-5], and mixtures thereof.

[0153] [1B-16-4-1] In the above aspect [1B-16-3] or [1B-16-4], preferred, more preferred, even more preferred, and particularly preferred derivatives of the chemically modified alginic acid derivative represented by formula (I) of aspect [1] are the same as those described in the above aspects [1-1-1], [1-1-2], [1-1-3], and [1-1-4], respectively.

[0154] [1B-16-4-2] In the above aspect [1B-16-3] or [1B-16-4], preferred, more preferred, and even more preferred derivatives of the chemically modified alginic acid derivative represented by formula (I) in aspect [1-1-5] are the same as those described in the above aspect [1-1-5].

[0155] [1B-16-4-3] In the above aspect [1B-16-3] or [1B-16-4], preferred, more preferred, even more preferred, and particularly preferred derivatives of the chemically modified alginic acid derivative represented by formula (II) of aspect [1] are the same as those described in the above aspects [1-2-1], [1-2-2], [1-2-3], and [1-2-4], respectively.

[0156] [1B-16-4-4] In the above aspect [1B-16-3] or [1B-16-4], preferred, more preferred, and even more preferred derivatives of the chemically modified alginic acid derivative represented by formula (II) in aspect [1-2-5] are the same as those described in the above aspect [1-2-5].

[0157] [1B-16-5] In the above aspects [1B] to [1D], the anionic polymer layer of the multilayer polymer-coated cross-linked alginate gel fiber is an anionic polymer selected from the group consisting of sulfated polysaccharides such as chondroitin sulfate, dextran sulfate, heparan sulfate, dermatan sulfate, fucoidan, keratan sulfate, and heparin, chemically modified products thereof, cross-linked products thereof, and mixtures thereof.

[0158] [1B-16-6] In the above aspects [1B] to [1D], the anionic polymer layer of the multilayer polymer-coated cross-linked alginate gel fiber is an anionic polymer selected from the group consisting of synthetic polymers such as acrylic acid, methacrylic acid, ethylacrylic acid, polystyrene sulfonic acid, etc., chemically modified products thereof, cross-linked products thereof, mixtures thereof, etc.

[0159] [1B-16-7] In the above aspects [1B] to [1D], the anionic polymer layer of the multilayer polymer-coated cross-linked alginate gel fiber is an anionic polymer selected from the group consisting of anionic polyamino acids such as polyglutamic acid and polyaspartic acid, chemically modified products thereof, cross-linked products thereof, and mixtures thereof.

[0160] [1B-17] In the above embodiments [1B] to [1D], the outer diameter of the multi-polymer-coated cross-linked alginate gel fiber is, for example, in the range of about 0.1 to about 2000 μm.

[0161] Akn, -L as described in the above embodiment １ -, -L ２ By using chemically modified alginic acid derivatives represented by formula (I) and formula (II) in which the definitions of - and X are appropriately combined, it is possible to arbitrarily form a preferred embodiment of the cross-linked alginic acid gel in the core layer of the multilayer polymer-coated cross-linked alginic acid gel fiber of the above embodiment.

[0162] [1B-18-1] The multilayer polymer-coated cross-linked alginate gel fiber of Aspects [1B] to [1D] is preferably a multilayer polymer-coated cross-linked alginate gel fiber in which the alginate derivative represented by formula (I) used to form the cross-linked alginate gel is selected from the alginate derivatives described in Aspect [1-1-1] or the preferred alginate derivatives described in Aspect [1-1-5], the alginate derivative represented by formula (II) is selected from the alginate derivatives described in Aspect [1-2-1] or the preferred alginate derivatives described in Aspect [1-2-5]; the antibody-producing cells are selected from the cells described in Aspects [1B-3-1] to [1B-3-9]; the cationic polymer layer is selected from the cationic polymers described in Aspect [1-15-1]; and the anionic polymer layer is selected from the anionic polymers described in Aspects [1B-16] to [1B-16-7].

[0163] [1B-18-2] The multilayer polymer-coated cross-linked alginate gel fiber of any of Aspects [1B] to [1D] is more preferably a multilayer polymer-coated cross-linked alginate gel fiber in which the alginate derivative represented by formula (I) used to form the cross-linked alginate gel is selected from the alginate derivatives described in Aspect [1-1-2] or the more preferred alginate derivatives described in Aspect [1-1-5], the alginate derivative represented by formula (II) is selected from the alginate derivatives described in Aspect [1-2-2] or the more preferred alginate derivatives described in Aspect [1-2-5]; the antibody-producing cells are selected from the cells described in Aspects [1B-3-2] to [1B-3-9]; the cationic polymer layer is selected from the cationic polymers described in Aspects [1-15-2] to [1-15-4]; and the anionic polymer layer is selected from the anionic polymers described in Aspect [1B-16-1].

[0164] [1B-18-3] The multilayer polymer-coated cross-linked alginate gel fiber of any of the above aspects [1B] to [1D] is more preferably a multilayer polymer-coated cross-linked alginate gel fiber in which the alginate derivative represented by formula (I) used to form the cross-linked alginate gel is selected from the alginate derivatives described in the above aspect [1-1-3], and the alginate derivative represented by formula (II) is selected from the alginate derivatives described in the above aspect [1-2-3]; the antibody-producing cells are selected from the cells described in the above aspects [1B-3-3] to [1B-3-9]; the cationic polymer layer is selected from the cationic polymers described in the above aspects [1-15-2] or [1-15-4]; and the anionic polymer layer is selected from the anionic polymers described in the above aspects [1B-16-2] to [1B-16-4-4].

[0165] [1B-18-4] The multilayer polymer-coated cross-linked alginate gel fibers of the above aspects [1B] to [1D] are particularly preferably those in which the alginate derivative represented by formula (I) used to form the cross-linked alginate gel is selected from the alginate derivatives described in the above aspect [1-1-4], and the alginate derivative represented by formula (II) is selected from the alginate derivatives described in the above aspect [1-2-4]; the antibody-producing cells are selected from the cells described in the above aspects [1B-3-6] to [1B-3-9]; and the cationic polymer layer is selected from poly-L-ornithine, polyallylamine (PAA), polyethyleneimine, or polymethylene-co-guanidine (PMMA). and the anionic polymer layer is selected from alginic acid, an alginic acid derivative represented by formula (I) described in the above aspects [1-1-1] to [1-1-4] and [1-2-5], an alginic acid derivative represented by formula (II) described in the above aspects [1-2-1] to [1-2-4] and [1-2-5], and a crosslinked product formed from an alginic acid derivative represented by formula (I) described in the above aspects [1-1-1] to [1-1-4] and [1-1-5] and an alginic acid derivative represented by formula (II) described in the above aspects [1-2-1] to [1-2-4] and [1-2-5].

[0166] [1B-18-5] In the multilayer polymer-coated cross-linked alginate gel fiber of the above aspects [1B] to [1D], the alginate derivative represented by formula (I) used to form the cross-linked alginate gel is most preferably Akn-L １ - as the following partial structural formula (each formula does not include the right side of the dashed line): and the alginic acid derivative represented by formula (II) has a group represented by -L ２ - as the following partial structural formula (each formula does not include the areas outside the dashed lines at both ends): the antibody-producing cells are selected from the cells described in the above aspect [1B-3-5], and preferably the antibody-producing cells are antibody-producing CHO cells; the cationic polymer layer is selected from poly-L-ornithine, polyallylamine (PAA), polyethyleneimine, or polymethylene-co-guanidine (PMCG); the anionic polymer layer is selected from alginic acid, Akn-L １ - as the following partial structural formula (each formula does not include the right side of the dashed line): an alginic acid derivative represented by formula (I) having the formula -L ２ -, the following partial structural formula (each formula does not include the areas outside the dashed lines at both ends): and an alginic acid derivative represented by formula (II) having the formula: １ an alginic acid derivative represented by formula (I) having the partial structural formula (ALK-1a-3a) or (ALK-2-3) as -L ２ The multi-layer polymer-coated cross-linked alginate gel fiber is selected from cross-linked bodies formed from alginic acid derivatives represented by formula (II) having the partial structural formula (LN-1-3), (LN-3-3a) or (LN-5-3a) as the functional group.

[0167] [1B-18-6] In the above aspects [1B-18-1] to [1B-18-5], the cross-linked alginate gel of the multilayer polymer-coated cross-linked alginate gel fiber contains any of the addable components described in the above aspects [1-4] to [1-4-1].

[0168] In the multilayer polymer-coated cross-linked alginate gel fiber described in the above embodiment, by combining each element of the cells capable of producing antibodies, physiologically active substances, etc., the chemically modified alginic acid derivatives represented by formula (I) and formula (II) used to form the cross-linked alginate gel, the cationic polymer (cationic polymer layer), and the anionic polymer (multilayer anionic polymer), it is possible to form any preferred embodiment of the method for producing multilayer polymer-coated cross-linked alginate gel fiber.

[0169] [2] The second aspect is as follows. A method for producing a polymer-coated crosslinked alginate gel fiber is provided by coating a core layer containing a crosslinked alginate gel obtained by crosslinking cells capable of producing antibodies, physiologically active substances, etc., and the chemically modified alginate derivatives represented by formula (I) and formula (II) described in aspect [1] with a cationic polymer, the method comprising: step (1): injecting a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc., and the chemically modified alginate derivatives represented by formula (I) and formula (II) described in aspect [1] into a solution containing a divalent metal ion to obtain a crosslinked alginate gel fiber (CLA) containing cells capable of producing antibodies, physiologically active substances, etc., in its core layer; and step (2): contacting the crosslinked alginate gel fiber (CLA) containing cells capable of producing antibodies, physiologically active substances, etc., obtained in step (1) with a solution containing a cationic polymer to obtain a polymer-coated crosslinked alginate gel fiber (CFB) coated with a cationic polymer layer.

[0170] [2A] The polymer-coated cross-linked alginate gel fiber in the above aspect [2] is the polymer-coated cross-linked alginate gel fiber according to any one of the above aspects ([1] to [1-17-5]).

[0171] [2-1] In the above aspect [2], the cells capable of producing antibodies, physiologically active substances, etc. used in the production of polymer-coated cross-linked alginate gel fibers are the same as the cells capable of producing antibodies, physiologically active substances, etc. described in any one of the above aspects [1-3] to [1-3-5].

[0172] [2-1-1] In the above aspect [2], the cells capable of producing antibodies, physiologically active substances, etc. used in the production of polymer-coated cross-linked alginate gel fibers are the same as the cells capable of producing antibodies, physiologically active substances, etc. described in any one of the above aspects [1B-3] to [1B-3-19].

[0173] [2-2] In the above aspect [2], the weight-average molecular weight of the chemically modified alginic acid derivative represented by formula (I) used in the production of the polymer-coated cross-linked alginic acid gel fiber, as measured by gel filtration chromatography, is, for example, in the range of about 100,000 Da to about 3,000,000 Da; preferably in the range of about 300,000 Da to about 2,500,000 Da, and more preferably in the range of about 500,000 Da to about 2,000,000 Da.

[0174] [2-3] In the above aspect [2], the weight-average molecular weight of the chemically modified alginic acid derivative represented by formula (II) used in the production of the polymer-coated cross-linked alginic acid gel fiber, as measured by gel filtration chromatography, is, for example, in the range of about 100,000 Da to about 3,000,000 Da; preferably in the range of about 300,000 Da to about 2,500,000 Da, and more preferably in the range of about 500,000 Da to about 2,000,000 Da.

[0175] [2-4] In the above aspect [2], the reactive group of the chemically modified alginic acid derivative represented by formula (I) used for producing the polymer-coated crosslinked alginic acid gel fiber: Akn-L １ -NH ２ Group (Akn-L １ The introduction rate of the aryl group (- is the same as defined in the above embodiments [1] to [1-1-4]) is, for example, in the range of about 0.1 to about 30 mol %, preferably in the range of about 0.3 to about 20 mol %, and more preferably in the range of about 0.5 to about 10 mol %.

[0176] [2-5] In the above aspect [2], the reactive group of the chemically modified alginic acid derivative represented by formula (II) used for producing the polymer-coated crosslinked alginic acid gel fiber: N３ -L ２ -NH ２ Group (-L ２ - is the same as defined in the above embodiments [1], [1-2-1] to [1-2-4]) is, for example, in the range of about 0.1 to about 30 mol %, preferably in the range of about 0.3 to about 20 mol %, and more preferably in the range of about 0.5 to about 15 mol %.

[0177] [2-6] In the above aspect [2], components that can be added to the mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by formula (I) and formula (II) are, for example, components selected from the group consisting of alginic acid solution, culture medium, culture fluid, collagen solution, methylcellulose, sucrose solution, or mixtures thereof; preferably, components selected from the group consisting of alginic acid solution, culture medium, culture fluid, or mixtures thereof.

[0178] [2-7] In the above aspect [2], the weight-average molecular weight of the alginic acid (e.g., sodium alginate, etc.) used in preparing the alginic acid solution that can be added to the mixed solution containing cells capable of producing antibodies, physiologically active substances, etc., and the chemically modified alginic acid derivatives represented by formula (I) and formula (II), as measured by gel permeation chromatography (GPC), is, for example, in the range of about 150,000 Da to about 2,500,000 Da; preferably in the range of about 300,000 Da to about 2,000,000 Da, and more preferably in the range of about 700,000 Da to about 1,500,000 Da.

[0179] [2-7A] In the step (1) of producing the fiber of the above aspect [2], the weight-average molecular weight of alginic acid (e.g., sodium alginate) used in preparing the alginic acid solution that can be added to the mixed solution containing cells capable of producing an antibody, a physiologically active substance, etc., and the chemically modified alginic acid derivatives represented by formula (I) and formula (II) as measured by gel permeation chromatography (GPC) is, for example, about 15 preferably, in the range of about 300,000 Da to about 2,500,000 Da; and more preferably, in the range of about 700,000 Da to about 1,400,000 Da, about 800,000 Da to about 1,500,000 Da, about 1,400,000 Da to about 2,000,000 Da, or about 1,500,000 to about 2,500,000 Da.

[0180] [2-8] In the above aspect [2], the concentration of the solution of the chemically modified alginic acid derivative represented by formula (I) used in the production of polymer-coated cross-linked alginic acid gel fibers is, for example, in the range of about 0.01 to about 1.5 wt %, preferably about 0.05 to about 1.0 wt %, and more preferably about 0.08 to about 0.75 wt %.

[0181] In this specification, "% by weight" means w / v %.

[0182] [2-9] In the above aspect [2], the concentration of the solution of the chemically modified alginic acid derivative represented by formula (II) used in the production of the polymer-coated cross-linked alginic acid gel fiber is, for example, in the range of about 0.01 to about 1.5 wt %, preferably about 0.05 to about 1.0 wt %, and more preferably about 0.08 to about 0.75 wt %.

[0183] [2-10] In the above aspect [2], the concentration of the mixed solution of the chemically modified alginic acid derivative represented by formula (I) and the chemically modified alginic acid derivative represented by formula (II) used in the production of polymer-coated cross-linked alginic acid gel fiber is, for example, in the range of about 0.02 to about 2.0 wt %, preferably in the range of about 0.1 to about 2.0 wt %, and more preferably in the range of about 0.15 to about 1.5 wt %.

[0184] [2-11] In the above aspect [2], the concentration of the alginic acid solution that can be added to the mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by formula (I) and formula (II) is, for example, in the range of 0 to about 1.98 wt %, preferably 0 to about 1.8 wt %, and more preferably 0 to about 1.7 wt %.

[0185] [2-11-1] In the above aspect [2], when an alginic acid solution is added to a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and chemically modified alginic acid derivatives represented by formula (I) and formula (II), the total concentration of the mixed solution containing the chemically modified alginic acid derivatives represented by formula (I) and formula (II) and the alginic acid solution is preferably about 0.5 to about 2.0% by weight; more preferably, it is a concentration selected from about 1.0% by weight, about 1.5% by weight, and about 2.0% by weight.

[0186] [2-11-2] In the above aspect [2], when an alginic acid solution is added to a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and chemically modified alginic acid derivatives represented by formula (I) and formula (II), the combination of the concentration (C1 (wt %)) of the mixed solution containing the chemically modified alginic acid derivatives represented by formula (I) and formula (II) and the concentration (C2 (wt %)) of the alginic acid solution is preferably a combination selected from the group consisting of (C1:C2) = (about 0.2:about 1.3), (about 0.5:about 1.0), (about 1.0:about 0.5), (about 1.5:0), (about 0.66:about 1.34), and (about 0.34:about 0.66).

[0187] [2-11-3] In the above aspect [2], when an alginic acid solution is added to a mixed solution containing cells capable of producing an antibody, a physiologically active substance, etc. and the chemically modified alginic acid derivatives represented by formula (I) and formula (II), the concentration of the solution of the chemically modified alginic acid derivative represented by formula (I) (C1A (wt%)), the concentration of the solution of the chemically modified alginic acid derivative represented by formula (II) (C1N (wt%)) and The combination of the concentrations of the cellulose acetate solution and the alginic acid solution (C2 (wt%)) is preferably a combination selected from the group consisting of (C1A:C1N:C2) = (about 0.1:about 0.1:about 1.3), (about 0.25:about 0.25:about 1.0), (about 0.5:about 0.5:about 0.5), (about 0.75:about 0.75:0), (about 0.33:about 0.33:about 1.34), and (about 0.17:about 0.17:about 0.66).

[0188] [2-12-1] In the above aspect [2], in a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc., and chemically modified alginic acid derivatives represented by formula (I) and formula (II), the volume ratio (v1, v2) of the solution of the chemically modified alginic acid derivative represented by formula (I) to the solution of the chemically modified alginic acid derivative represented by formula (II) is, for example, a ratio of v1 + v2 = 15, e.g., (v1:v2) = (7.5:7.5).

[0189] [2-12-2] In the above aspect [2], when an alginate solution is added to a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc., and chemically modified alginate derivatives represented by formula (I) and formula (II) used to form the core layer of a polymer-coated cross-linked alginate gel fiber, the volume ratio of the volume (v1) of the chemically modified alginate derivative represented by formula (I), the volume (v2) of the solution of the chemically modified alginate derivative represented by formula (II), and the volume (v3) of the alginate solution in the mixed solution to which the alginate solution has been added is, for example, a ratio of v1 + v2 + v3 = 15, and combinations such as (v1:v2:v3) = (5:5:5), (2.5:2.5:10), and (1:1:13).

[0190] [2-13] In the above aspect [2], the divalent metal ion contained in the solution to be injected containing the mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by formula (I) and formula (II) is a divalent metal ion selected from the group consisting of calcium ions, magnesium ions, barium ions, strontium ions, zinc ions, etc.; preferably, calcium ions, barium ions, or strontium ions; more preferably, calcium ions or barium ions.

[0191] [2-14] In the above aspect [2], the solution to be injected containing the mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by formula (I) and formula (II) is an aqueous solution containing a divalent metal ion selected from the group consisting of an aqueous calcium chloride solution, an aqueous calcium carbonate solution, an aqueous calcium gluconate solution, an aqueous barium chloride solution, an aqueous strontium chloride solution, etc.; preferably, the aqueous calcium chloride solution or the aqueous barium chloride solution.

[0192] [2-15] In the above aspect [2] or [2-14], the concentration of the divalent metal ion is, for example, in the range of about 1 mM to about 1 M, or in the range of about 10 to about 500 mM; preferably, in the range of about 10 to about 100 mM.

[0193] [2-16-1] In the above aspect [2], a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by formula (I) and formula (II) can be prepared, for example, by using an apparatus XX having an inlet 1 and an outlet 2 as shown in Figure 3, by introducing the mixed solution into the inlet 1 of the apparatus XX and ejecting it from the outlet 2 of the apparatus XX.

[0194] [2-16-2] In the above aspect [2-16-1], a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by formula (I) and formula (II) can be extruded from the outlet 2 of the device XX using, for example, an extrusion tube YY as shown in Figure 3.

[0195] [2-17] In the above-mentioned aspect [2-16-2], the device XX and the extrusion tube YY may be combined into a syringe, for example. The syringe may be made of glass or plastic.

[0196] [2-18] In the above aspects [2], [2-16-1] and [2-16-2], the injection speed (flow rate) of the mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by formula (I) and formula (II) is, for example, in the range of about 100 to about 10,000 μL / min.

[0197] [2-19-1] In the above aspect [2], the solution containing a cationic polymer that is brought into contact with the cross-linked alginate gel fiber (CLA) containing cells capable of producing antibodies, physiologically active substances, etc. is a solution containing a cationic polymer selected from the group consisting of polyamino acids (polymers of basic amino acids), basic polysaccharides, basic polymers, salts thereof, etc.

[0198] [2-19-2] In the above aspect [2], the solution containing a cationic polymer that is brought into contact with the cross-linked alginate gel fiber (CLA) containing cells capable of producing antibodies, physiologically active substances, etc. is preferably a polyamino acid, such as poly-L-ornithine (PLO), poly-D-ornithine (PDO), poly-DL-ornithine, poly-D-lysine (PDL), poly-L-lysine (PLL), poly-DL-lysine, poly-L-arginine (PLA), poly-D-arginine (PDA), poly-DL-arginine, or poly-L-homoarginine. The cationic polymer is a solution containing a cationic polymer selected from the group consisting of poly-L-ornithine, poly-L-lysine, and salts thereof; more preferably, a solution containing a cationic polymer selected from the group consisting of poly-L-ornithine, poly-L-lysine, and salts thereof; and even more preferably, a solution containing a cationic polymer selected from poly-L-ornithine or a salt thereof.

[0199] [2-19-3] In the above aspect [2], the solution containing a cationic polymer with which the cross-linked alginate gel fiber (CLA) containing cells capable of producing antibodies, physiologically active substances, etc. is brought into contact is, for example, a solution containing a cationic polymer selected from the group consisting of chitosan, which is a basic polysaccharide, or a salt thereof.

[0200] [2-19-4] In the above aspect [2], the solution containing a cationic polymer with which the crosslinked alginate gel fiber (CLA) containing cells capable of producing antibodies, physiologically active substances, etc. is brought into contact is, for example, a solution containing a cationic polymer selected from the group consisting of basic polymers such as polymethylene-co-guanidine (PMCG), polyallylamine (PAA), polyvinylamine (PVA), polyethyleneimine, allylamine-diallylamine copolymer, allylamine-maleic acid copolymer, and salts thereof.

[0201] [2-19-4-1] In the above aspect [2-19-4], the solution containing a cationic polymer is preferably a solution containing a cationic polymer selected from the group consisting of polyallylamine (PAA), polyethyleneimine, polymethylene-co-guanidine (PMCG), and salts thereof.

[0202] [2-20] In the above aspect [2], the solution containing a cationic polymer that is brought into contact with the cross-linked alginate gel fiber (CLA) containing cells capable of producing antibodies, physiologically active substances, etc. can contain components such as calcium chloride solution and buffer solution.

[0203] [2-21] In the above aspect [2], the temperature during production of the polymer-coated cross-linked alginate gel fiber is, for example, in the range of about 4 to about 37°C.

[0204] By combining the methods for producing polymer-coated cross-linked alginate gel fibers and the various elements described in the above embodiments, any preferred embodiment of the method for producing polymer-coated cross-linked alginate gel fibers can be formed.

[0205] [2B] Aspect 2B is as follows: A method for producing a multilayer polymer-coated crosslinked alginate gel fiber formed by sequentially coating a core layer containing a crosslinked alginate gel obtained by crosslinking cells capable of producing antibodies, physiologically active substances, etc., and the chemically modified alginate derivatives represented by formula (I) and formula (II) described in aspect [1] with a cationic polymer and an anionic polymer, the method comprising: Step (1): Injecting a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc., and the chemically modified alginate derivatives represented by formula (I) and formula (II) described in aspect [1] into a solution containing bivalent metal ions to obtain a crosslinked alginate gel fiber (CLA) containing cells capable of producing antibodies, physiologically active substances, etc., in the core layer; The method comprises the steps of: step (2): contacting the cross-linked alginate gel fiber (CLA) obtained in step (1), which contains cells capable of producing antibodies, physiologically active substances, etc. in its core layer, with a solution containing a cationic polymer to obtain a polymer-coated cross-linked alginate gel fiber (CFB) coated with a cationic polymer layer; and step (3): contacting the polymer-coated cross-linked alginate gel fiber (CFB) obtained in step (2) with a solution containing an anionic polymer to obtain a multi-layer polymer-coated cross-linked alginate gel fiber (ACFB) coated with an anionic polymer layer.

[0206] [2C] The multi-polymer-coated cross-linked alginate gel fiber in the above aspect [2B] is a multi-polymer-coated cross-linked alginate gel fiber according to any one of the above aspects ([1B] to [1B-18-6]).

[0207] [2B-1] In the above aspect [2B], the cells capable of producing the antibodies, physiologically active substances, etc. used in the production of the fiber are the same as the cells capable of producing the antibodies, physiologically active substances, etc. described in any one of the above aspects [1-3] to [1-3-5] and [1B-3] to [1B-3-19].

[0208] [2B-2] In the above aspect [2B], the weight-average molecular weight of the chemically modified alginic acid derivative represented by formula (I) used in step (1) or step (3) of fiber production, as measured by gel filtration chromatography, is within the same range of weight-average molecular weight as described in the above aspect [2-2].

[0209] [2B-3] In the above aspect [2B], the weight-average molecular weight of the chemically modified alginic acid derivative represented by formula (II) used in step (1) or step (3) of fiber production, as measured by gel filtration chromatography, is within the range of weight-average molecular weight described in the above aspect [2-3].

[0210] [2B-4] In the above aspect [2B], the reactive group of the chemically modified alginic acid derivative represented by formula (I) used in step (1) or step (3) of fiber production: Akn-L １ -NH ２ Group (Akn-L １ The introduction rate of (- is the same as defined in the above embodiment [1], [1B-1-1] or [1B-1-2]) is the same as the introduction rate range described in the above embodiment [2-4].

[0211] [2B-5] In the above aspect [2B], the reactive group of the chemically modified alginic acid derivative represented by formula (II) used in step (1) or step (3) of fiber production: N ３ -L ２ -NH ２ Group (-L ２ The introduction rate of (- is the same as defined in the above embodiment [1], [1B-2-1], or [1B-2-2]) is the same as the introduction rate range described in the above embodiment [2-5].

[0212] [2B-6] In the above aspect [2B], in step (1) of fiber production, the components that can be added to the mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by formula (I) and formula (II) are the same as the components described in the above aspect [2-6].

[0213] [2B-7] The weight-average molecular weight of the alginic acid (e.g., sodium alginate, etc.) used in preparing an alginic acid solution that can be added to a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and chemically modified alginic acid derivatives represented by formula (I) and formula (II) in step (1) of fiber production in aspect [2B], or the alginic acid (e.g., sodium alginate, etc.) used in preparing the solution when alginic acid is used as an anionic polymer in step (3) of fiber production in aspect [2B], as measured by gel permeation chromatography (GPC), is the same as the molecular weight described in aspect [2-7A].

[0214] [2B-8] In the above aspect [2B], the concentration of the solution of the chemically modified alginic acid derivative represented by formula (I) used in step (1) of fiber production is the same as the concentration range described in the above aspect [2-8].

[0215] [2B-9] In the above aspect [2B], the concentration of the solution of the chemically modified alginic acid derivative represented by formula (II) used in step (1) of fiber production is the same as the concentration range described in the above aspect [2-9].

[0216] [2B-10] In the above aspect [2B], the concentration of the mixed solution of the chemically modified alginic acid derivative represented by formula (I) and the chemically modified alginic acid derivative represented by formula (II) used in step (1) of fiber production is the same as the concentration range described in the above aspect [2-10].

[0217] [2B-11] In the above aspect [2B], the concentration of the alginic acid solution that can be added to the mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by formula (I) and formula (II) in step (1) of fiber production is the same as the concentration range described in the above aspect [2-11].

[0218] [2B-11-1] In the above aspect [2B], when an alginic acid solution is added to a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and chemically modified alginic acid derivatives represented by formulas (I) and (II) in step (1) of fiber production, the total concentration of the mixed solution containing the chemically modified alginic acid derivatives represented by formulas (I) and (II) and the alginic acid solution is the same as the concentration range described in the above aspect [2-11-1]; more preferably, it is about 1.5 wt%.

[0219] [2B-11-2] In the above aspect [2B], when an alginic acid solution is added to a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and chemically modified alginic acid derivatives represented by formulas (I) and (II) in step (1) of fiber production, the combination of the concentration (C1 (wt %)) of the mixed solution containing the chemically modified alginic acid derivatives represented by formulas (I) and (II) and the concentration (C2 (wt %)) of the alginic acid solution is the same as the concentration range described in the above aspect [2-11-2].

[0220] [2B-11-3] In the above aspect [2B], when an alginic acid solution is added to a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and chemically modified alginic acid derivatives represented by formula (I) and formula (II) in step (1) of fiber production, the combination of the concentration of the solution of the chemically modified alginic acid derivative represented by formula (I) (C1A (wt %)), the concentration of the solution of the chemically modified alginic acid derivative represented by formula (II) (C1N (wt %)), and the concentration of the alginic acid solution (C2 (wt %)) is the same as the concentration range described in the above aspect [2-11-3].

[0221] [2B-12-1] In the above aspect [2B], in the step (1) of fiber production, in the mixed solution containing cells capable of producing antibodies, physiologically active substances, etc., and chemically modified alginic acid derivatives represented by formula (I) and formula (II), the volume ratio (v1, v2) of the solution of the chemically modified alginic acid derivative represented by formula (I) to the solution of the chemically modified alginic acid derivative represented by formula (II) is, for example, a ratio of v1 + v2 = 15, e.g., (v1:v2) = (7.5:7.5).

[0222] [2B-12-2] In the above aspect [2B], when an alginic acid solution is added to a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and chemically modified alginic acid derivatives represented by formula (I) and formula (II) in the fiber production step (1), the volume ratio of the volume (v1) of the chemically modified alginic acid derivative represented by formula (I), the volume (v2) of the solution of the chemically modified alginic acid derivative represented by formula (II), and the volume (v3) of the alginic acid solution in the mixed solution to which the alginic acid solution has been added is the same as the combination described in the above aspect [2-12-2].

[0223] [2B-13] In the above aspect [2B], in the fiber production step (1), the divalent metal ion contained in the solution into which the mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by formula (I) and formula (II) is injected is the same as the divalent metal ion described in the above aspect [2-13].

[0224] [2B-14] In the above aspect [2B], in the fiber production step (1), the solution into which the mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by formula (I) and formula (II) is injected is the same as the aqueous solution containing divalent metal ions described in the above aspect [2-14].

[0225] [2B-15] In the above aspect [2B] or [2B-14], the concentration of the divalent metal ion used in step (1) of fiber production is, for example, in the range of about 1 mM to about 1 M, or in the range of about 10 to about 500 mM; preferably, about 10 to about 100 mM.

[0226] [2B-16-1] In the above aspect [2B], a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by formula (I) and formula (II) can be prepared, for example, by using an apparatus XX having an inlet 1 and an outlet 2 as shown in Figure 10, by introducing the mixed solution into the inlet 1 of the apparatus XX and ejecting it from the outlet 2 of the apparatus XX.

[0227] [2B-16-2] In the above aspect [2B-16-1], a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by formula (I) and formula (II) can be extruded from the outlet 2 of the device XX using, for example, an extrusion tube YY as shown in Figure 10.

[0228] [2B-17] In the above-mentioned aspect [2B-16-2], the device XX and the extrusion tube YY can be combined into a syringe, for example. The syringe can be made of glass or plastic.

[0229] [2B-18] In the above aspects [2B], [2B-16-1] and [2B-16-2], the injection speed (flow rate) of the mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by formula (I) and formula (II) is, for example, in the range of about 100 to about 10,000 μL / min.

[0230] [2B-19-1] In the above aspect [2B], the solution containing a cationic polymer that is brought into contact with the cross-linked alginate gel fiber (CLA) containing cells capable of producing antibodies, physiologically active substances, etc. in the fiber production step (2) is the same as the solution described in the above aspect [2-19-1].

[0231] [2B-19-2] In the above aspect [2B], the preferred, more preferred, and even more preferred solutions containing cationic polymers that are brought into contact with cross-linked alginate gel fiber (CLA) containing cells capable of producing antibodies, physiologically active substances, etc. in step (2) of fiber production are each the same as the solutions described in the above aspect [2-19-2].

[0232] [2B-19-3] In the above aspect [2B], the solution containing a cationic polymer that is brought into contact with the cross-linked alginate gel fiber (CLA) containing cells capable of producing antibodies, physiologically active substances, etc. in the fiber production step (2) is, for example, a solution containing a cationic polymer selected from the group consisting of chitosan, which is a basic polysaccharide, or a salt thereof.

[0233] [2B-19-4] In the above aspect [2B], the solution containing a cationic polymer that is brought into contact with the cross-linked alginate gel fiber (CLA) containing cells capable of producing antibodies, physiologically active substances, etc. in the fiber production step (2) is the same as the solution described in the above aspect [2-19-4].

[0234] [2B-20] In the above aspect [2B], the solution containing a cationic polymer that is brought into contact with the cross-linked alginate gel fiber (CLA) containing cells capable of producing antibodies, physiologically active substances, etc. in the fiber production step (2) can contain components such as calcium chloride solution and buffer solution.

[0235] [2B-21] In the above embodiment [2B], the temperature during production of the multi-polymer-coated cross-linked alginate gel fiber is, for example, in the range of about 4 to about 37°C.

[0236] [2B-22] In the above aspect [2B], the solution containing an anionic polymer that is brought into contact with the polymer-coated cross-linked alginate gel fiber (CFB) in step (3) of fiber production is a solution containing an anionic polymer selected from the group consisting of anionic polysaccharides, sulfated polysaccharides, synthetic polymers, anionic polyamino acids, chemically modified products thereof, cross-linked products thereof, salts thereof, and mixtures thereof.

[0237] [2B-23] In the above aspect [2B], the solution containing an anionic polymer with which the polymer-coated cross-linked alginate gel fiber (CFB) is brought into contact in the fiber production step (3) is a solution containing an anionic polymer selected from the group consisting of alginic acid, a chemically modified alginic acid derivative represented by formula (I) described in the above aspect [1], a chemically modified alginic acid derivative represented by formula (II) described in the above aspect [1], a cross-linked product formed from the chemically modified alginic acid derivative represented by formula (I) and / or the chemically modified alginic acid derivative represented by formula (II) described in the above aspect [1], a salt thereof, and a mixture thereof.

[0238] [2B-24] In the above aspect [2B], the solution containing an anionic polymer that is brought into contact with the polymer-coated cross-linked alginate gel fiber (CFB) in step (3) of fiber production is a solution containing an anionic polymer selected from the group consisting of anionic polysaccharides such as alginic acid, hyaluronic acid, caprygalacturonic acid, lageenan, succinoglucan, gum arabic, xanthan gum, alginic acid, pectin, pectic acid, carboxymethylcellulose, agar, etc., chemically modified products thereof, cross-linked products thereof, salts thereof, and mixtures thereof.

[0239] [2B-25] In the above aspect [2B], the solution containing an anionic polymer that is brought into contact with the polymer-coated cross-linked alginate gel fiber (CFB) in step (3) of fiber production is a solution containing a cationic polymer selected from the group consisting of sulfated polysaccharides such as chondroitin sulfate, dextran sulfate, heparan sulfate, dermatan sulfate, fucoidan, keratan sulfate, or heparin, chemically modified products thereof, cross-linked products thereof, salts thereof, and mixtures thereof.

[0240] [2B-26] In the above aspect [2B], the solution containing an anionic polymer that is brought into contact with the polymer-coated cross-linked alginate gel fiber (CFB) in step (3) of fiber production is a solution containing a cationic polymer selected from the group consisting of synthetic polymers such as acrylic acid, methacrylic acid, ethylacrylic acid, polystyrene sulfonic acid, etc., chemically modified products thereof, cross-linked products thereof, salts thereof, and mixtures thereof.

[0241] [2B-27] In the above aspect [2B], the solution containing an anionic polymer that is brought into contact with the polymer-coated cross-linked alginate gel fiber (CFB) in step (3) of fiber production is a solution containing a cationic polymer selected from the group consisting of anionic polyamino acids such as polyglutamic acid and polyaspartic acid, chemically modified products thereof, cross-linked products thereof, salts thereof, and mixtures thereof.

[0242] By combining the methods for producing multilayered polymer-coated cross-linked alginate gel fibers and the elements described in the above embodiments, any preferred embodiment of the method for producing multilayered polymer-coated cross-linked alginate gel fibers can be formed.

[0243] [3] A third aspect is as follows: A method for producing antibodies, physiologically active substances, etc., using a polymer-coated cross-linked alginate gel fiber formed by coating a core layer with a cationic polymer, the core layer including a cross-linked alginate gel obtained by carrying out a cross-linking reaction using cells capable of producing antibodies, physiologically active substances, etc., and the chemically modified alginate derivative represented by formula (I) and the chemically modified alginate derivative represented by formula (II) described in aspect [1]. In one aspect, the method for producing antibodies, physiologically active substances, etc., involves placing the polymer-coated cross-linked alginate gel fiber in a culture vessel, adding a medium to impregnate the polymer-coated cross-linked alginate gel fiber, and culturing the fiber.

[0244] [3A] The polymer-coated cross-linked alginate gel fiber in the above aspect [3] is the polymer-coated cross-linked alginate gel fiber according to any one of the above aspects ([1] to [1-17-5]).

[0245] [3-1] In the above aspect [3], the culture vessel is, for example, a vessel selected from the group consisting of tissue culture plates, Erlenmeyer flasks, T-flasks, spinner flasks, culture bags, animal cell culture vessels, etc.; Erlenmeyer flasks or animal cell culture vessels are preferred. For culture, any method such as static culture or shake culture may be selected, and any method such as batch culture, fed-batch culture, or continuous culture may be used, although fed-batch culture or continuous culture is preferred.

[0246] [3-2] In any one of the above aspects [3] to [3-1], the temperature during culture is, for example, in the range of about 28°C to about 39°C, for example, in the range of about 30°C to about 37°C.

[0247] [3-3] In any one of the above aspects [3] to [3-2], the stirring speed during culture is, for example, about 50 to about 250 rpm, for example, about 125 rpm.

[0248] [3-4] In any one of the above aspects [3] to [3-3], the culture conditions are, for example, a culture temperature in the range of about 28°C to about 39°C, 5% CO ２ The culture is carried out in an atmosphere in a culture device at an agitation speed of about 125 rpm.

[0249] [3-5] In any one of the above aspects [3] to [3-4], the culture period is, for example, 7 days, 14 days, 28 days, 42 days, 56 days, or 70 days.

[0250] In this specification, when the term "about" is used in relation to the culture temperature, it may include a value up to ±10% of the numerical value, and in one embodiment, a value up to ±20% of the numerical value.

[0251] [3-6] In the above aspect [3], the cells capable of producing antibodies, physiologically active substances, etc. contained in the core layer of the polymer-coated cross-linked alginate gel fiber are the same as the cells capable of producing antibodies, physiologically active substances, etc. described in any one of the above aspects [1-3] to [1-3-5].

[0252] [3-6-1] In the above aspect [3], the cells capable of producing antibodies, physiologically active substances, etc. contained in the core layer of the polymer-coated cross-linked alginate gel fiber are the same as the cells capable of producing antibodies, physiologically active substances, etc. described in any one of the above aspects [1B-3] to [1B-3-19].

[0253] [3-7] A method for producing an antibody, a physiologically active substance, or the like, in any one of the above aspects [3] to [3-5], comprising adding a cell growth inhibitor.

[0254] By combining the methods for producing antibodies, physiologically active substances, etc. using the polymer-coated cross-linked alginate gel fiber described in the above embodiments and each element, any preferred embodiment of the method for producing antibodies, physiologically active substances, etc. can be formed.

[0255] [3B] Aspect 3B is as follows: A method for producing antibodies, physiologically active substances, etc., using a multilayer polymer-coated crosslinked alginate gel fiber formed by sequentially coating a core layer containing a crosslinked alginate gel obtained by crosslinking cells capable of producing antibodies, physiologically active substances, etc., and the chemically modified alginate derivative represented by formula (I) and the chemically modified alginate derivative represented by formula (II) described in aspect [1] with a cationic polymer and an anionic polymer. In one aspect, the method for producing antibodies, physiologically active substances, etc., involves placing the multilayer polymer-coated crosslinked alginate gel fiber in a culture vessel, adding a medium to impregnate the multilayer polymer-coated crosslinked alginate gel fiber, and culturing the fiber.

[0256] [3C] The multi-polymer-coated cross-linked alginate gel fiber in the above aspect [3B] is a multi-polymer-coated cross-linked alginate gel fiber according to any one of the above aspects ([1B] to [1B-18-6]).

[0257] [3B-1] In the above-mentioned embodiment [3B], the culture vessel and culture are the same as those described in the above-mentioned embodiment [3-1].

[0258] [3B-2] In any one of the above aspects [3B] to [3B-1], the temperature during culture is, for example, in the range of about 28°C to about 39°C, for example, in the range of about 30°C to about 37°C.

[0259] [3B-3] In any one of the above aspects [3B] to [3B-2], the stirring speed during culture is, for example, about 50 to about 250 rpm, for example, about 125 rpm.

[0260] [3B-4] In any one of the above aspects [3] to [3B-3], the culture conditions are, for example, a culture temperature in the range of about 28°C to about 39°C, 5% CO ２ The culture is carried out in an atmosphere in a culture device at an agitation speed of about 125 rpm.

[0261] [3B-5] In any one of the above aspects [3B] to [3B-4], the culture period is, for example, 7 days, 14 days, 28 days, 42 days, 56 days, or 70 days.

[0262] [3B-6] In the above aspect [3B], the cells capable of producing antibodies, physiologically active substances, etc. contained in the core layer of the multilayer polymer-coated cross-linked alginate gel fiber are the same as the cells capable of producing antibodies, physiologically active substances, etc. described in any one of the above aspects [1-3] to [1-3-5] and [1B-3] to [1B-3-19].

[0263] [3B-7] A method for producing an antibody, a physiologically active substance, or the like, in any one of the above aspects [3B] to [3B-5], which comprises adding a cell growth inhibitor.

[0264] By combining the methods for producing antibodies, physiologically active substances, etc. using the multilayer polymer-coated cross-linked alginate gel fiber described in the above embodiments and each element, any preferred embodiment of the method for producing antibodies, physiologically active substances, etc. can be formed.

[0265] [4] In a fourth aspect, the antibody that penetrates the cationic polymer layer produced in the core layer of the polymer-coated cross-linked alginate gel fiber obtained by the antibody production method according to any one of the above aspects [3] to [3-7] is an antibody having an isotype selected from the group consisting of, for example, IgG, IgA, IgM, IgD, and IgE.

[0266] [4B] Aspect 4B is the method for producing an antibody according to any one of aspects [3B] to [3B-7], wherein the antibody produced in the core layer of the multilayer polymer-coated cross-linked alginate gel fiber and permeating the cationic polymer layer and the anionic polymer layer has an isotype selected from the group consisting of, for example, IgG, IgA, IgM, IgD, and IgE.

[0267] [5] In the fifth aspect, the molecular weight of the antibody that penetrates the cationic polymer layer produced in the core layer of the polymer-coated cross-linked alginate gel fiber obtained by the antibody production method according to any one of the above aspects [3] to [3-7] is in the range of, for example, about 45,000 to about 1,000,000 Da, about 3,000 to about 1,000,000 Da, about 20,000 to about 1,000,000 Da, about 20,000 to about 400,000 Da, about 45,000 to about 400,000 Da, about 20,000 to about 200,000 Da, or about 45,000 to about 200,000 Da.

[0268] [5B] Aspect 5B is an antibody production method according to any one of aspects [3B] to [3B-7], wherein the molecular weight of the antibody produced in the core layer of the multilayer polymer-coated cross-linked alginate gel fiber and permeating the cationic polymer layer and the anionic polymer layer is in the range of, for example, about 3,000 to about 1,000,000 Da, about 20,000 to about 1,000,000 Da, about 45,000 to about 1,000,000 Da, about 20,000 to about 400,000 Da, about 45,000 to about 400,000 Da, about 20,000 to about 200,000 Da, or about 45,000 to about 200,000 Da.

[0269] [6] The sixth aspect is insulin produced using MIN6 cells by the method according to any one of the above aspects [3] to [3-7], and the molecular weight of the insulin is, for example, in the range of about 5,000 to 10,000.

[0270] [6B] Aspect 6B is a physiologically active substance produced using physiologically active substance-producing cells by the production method described in aspects [3] to [3-7] and [3B] to [3B-7], and having a molecular weight in the range of, for example, about 3,000 to about 1,000,000 Da, about 20,000 to about 1,000,000 Da, about 45,000 to about 1,000,000 Da, about 20,000 to about 400,000 Da, about 45,000 to about 400,000 Da, about 20,000 to about 200,000 Da, or about 45,000 to about 200,000 Da.

[0271] [7] The seventh aspect is an antibody obtained by the method for producing an antibody according to any one of the aspects [3] to [3-7], for example, muromonab-CD3 produced by muromonab-CD3-producing CHO cells, trastuzumab produced by trastuzumab-producing CHO cells, rituximab produced by rituximab-producing CHO cells, palivizumab produced by palivizumab-producing CHO cells, infliximab produced by infliximab-producing CHO cells, basiliximab produced by basiliximab-producing CHO cells, tocilizumab produced by tocilizumab-producing CHO cells, gemtuzumab-producing CHO cells, or the like. Gemtuzumab is produced using CHO cells producing bevacizumab, bevacizumab is produced using CHO cells producing bevacizumab, ibritumomab is produced using CHO cells producing ibritumomab, adalimumab is produced using CHO cells producing adalimumab, cetuximab is produced using CHO cells producing cetuximab, ranibizumab is produced using CHO cells producing ranibizumab, omalizumab is produced using CHO cells producing omalizumab, eculizumab is produced using CHO cells producing eculizumab, panitumumab is produced using CHO cells producing panitumumab, ustekinumab is produced using CHO cells producing ustekinumab, and golimumab is produced using CHO cells producing golimumab. golimumab using canakinumab-producing CHO cells, canakinumab using denosumab-producing CHO cells, mogamulizumab using mogamulizumab-producing CHO cells, certolizumab using certolizumab-producing CHO cells, ofatumumab using ofatumumab-producing CHO cells, pertuzumab using pertuzumab-producing CHO cells, brentuximab using brentuximab-producing CHO cells, natalizumab using natalizumab-producing CHO cells, nivolumab using nivolumab-producing CHO cells, and alemtuzumab using alemtuzumab-producing CHO cells. Mutuzumab, secukinumab using secukinumab-producing CHO cells, ramucirumab using ramucirumab-producing CHO cells, ipilimumab using ipilimumab-producing CHO cells, evolocumab using evolocumab-producing CHO cells, mepolizumab using mepolizumab-producing CHO cells, alirocumab using alirocumab-producing CHO cells, ixekizumab using ixekizumab-producing CHO cells, brodalumab using brodalumab-producing CHO cells, idarucizumab using idarucizumab-producing CHO cells, elotuzumab using elotuzumab-producing CHO cells,Pembrolizumab is produced using pembrolizumab-producing CHO cells, sarilumab is produced using sarilumab-producing CHO cells, bezlotoxumab is produced using bezlotoxumab-producing CHO cells, belimumab is produced using belimumab-producing CHO cells, daratumumab is produced using daratumumab-producing CHO cells, avelumab is produced using avelumab-producing CHO cells, dupilumab is produced using dupilumab-producing CHO cells, and atezolizumab is produced using atezolizumab-producing CHO cells , benralizumab using benralizumab-producing CHO cells, inotuzumab using inotuzumab-producing CHO cells, emicizumab using emicizumab-producing CHO cells, guselkumab using guselkumab-producing CHO cells, durvalumab using durvalumab-producing CHO cells, obinutuzumab using obinutuzumab-producing CHO cells, vedolizumab-producing CHO cells, or an anti-GPVI antibody using anti-GPVI antibody-producing CHO cells.

[0272] [7-1] In the method for producing an antibody according to any one of the above aspects [3] to [3-7], examples of antibodies that can be produced include trastuzumab using trastuzumab-producing CHO cells, rituximab using rituximab-producing CHO cells, infliximab using infliximab-producing CHO cells, tocilizumab using tocilizumab-producing CHO cells, adalimumab using adalimumab-producing CHO cells, nivolumab using nivolumab-producing CHO cells, and an anti-GPVI antibody using anti-GPVI antibody-producing CHO cells; for example, tocilizumab using tocilizumab-producing CHO cells, or an anti-GPVI antibody using anti-GPVI antibody-producing CHO cells.

[0273] [7B] Aspect 7B is directed to an antibody obtained by the method for producing an antibody according to any one of aspects [3] to [3-7] and [3B] to [3B-7], wherein the antibody is selected from the group consisting of muromonab-CD3, trastuzumab, rituximab, palivizumab, infliximab, basiliximab, tocilizumab, bevacizumab, adalimumab, cetuximab, omalizumab, eculizumab, panitumumab, ustekinumab, golimumab, canakinumab, denosumab, ofatumumab, and pertuzumab. , natalizumab, nivolumab, alemtuzumab, secukinumab, ramucirumab, ipilimumab, evolocumab, mepolizumab, alirocumab, ixekizumab, brodalumab, elotuzumab, pembrolizumab, sarilumab, bezlotoxumab, belimumab, daratumumab, avelumab, dupilumab, atezolizumab, emicizumab, guselkumab, durvalumab, vedolizumab, romosozumab, risankizumab, necitumumab, ravli Zumab, burosumab, isatuximab, tildrakizumab, satralizumab, galcanezumab, dinutuximab, fremanezumab, erenumab, casirivimab, imdevimab, anifrolumab, sotroximab, ocrelizumab, naxitamab, aducanumab, tafasitamab, marjetuximab, gantenerumab, tiragolumab, clovalimab, nemolizumab, catumaxomab, pramotamab, faricimab, gemtuzumab, ibritumab antibodies such as mab, brentuximab, inotuzumab, polatuzumab, enfortumab, sacituzumab, belantamab, loncustuximab, tisotumab, datopotamab, and patritumab; antibodies with modified sugar chains such as mogamulizumab, benralizumab, obinutuzumab, and inebilizumab; and low molecular weight antibodies consisting of antibody fragments such as ranibizumab, idarucizumab, blinatumomab, brolucizumab, abciximab, caplacizumab, and certolizumab.

[0274] [7C] Aspect 7C is directed to the antibody production method according to any one of aspects [3] to [3-7] and [3B] to [3B-7], wherein the antibody obtained is selected from muromonab-CD3-producing CHO cells, trastuzumab-producing CHO cells, rituximab-producing CHO cells, palivizumab-producing NS0 cells, palivizumab-producing CHO cells, infliximab-producing Sp2 / 0 cells, infliximab-producing CHO cells, basiliximab-producing Sp2 / 0 cells, basiliximab-producing CHO cells, tocilizumab-producing CHO cells, bevacizumab-producing CHO cells, adalimumab-producing CHO cells, cetuximab-producing CHO cells, Sp2 / 0 cells, cetuximab-producing CHO cells, omalizumab-producing CHO cells, eculizumab-producing NS0 cells, eculizumab-producing CHO cells, panitumumab-producing CHO cells, ustekinumab-producing Sp2 / 0 cells, ustekinumab-producing CHO cells, golimumab-producing Sp2 / 0 cells, golimumab-producing CHO cells, canakinumab-producing Sp2 / 0 cells, canakinumab-producing CHO cells, denosumab-producing CHO cells, ofatumumab-producing NS0 cells, ofatumumab-producing CHO cells, pertuzumab-producing CHO cells, natalizumab-producing NS0 cells, natalizumab-producing CHO cells, nivolumab-producing CHO cells, alemtuzumab-producing CHO cells, secukinumab-producing CHO cells, ramucirumab-producing NS0 cells, ramucirumab-producing CHO cells, ipilimumab-producing CHO cells, evolocumab-producing CHO cells, mepolizumab-producing CHO cells, alirocumab-producing CHO cells, ixekizumab-producing CHO cells, brodalumab-producing CHO cells, elotuzumab-producing NS0 cells, elotuzumab-producing CHO cells, pembrolizumab-producing CHO cells, sarilumab-producing CHO cells, bezlotoxumab-producing CHO cells, belimumab-producing NS0 cells, belimumab-producing CHO cells Viable CHO cells, daratumumab-producing CHO cells, avelumab-producing CHO cells, dupilumab-producing CHO cells, atezolizumab-producing CHO cells, emicizumab-producing CHO cells, guselkumab-producing CHO cells, durvalumab-producing CHO cells, vedolizumab-producing CHO cells, romosozumab-producing CHO cells, risankizumab-producing CHO cells, necitumumab-producing NS0 cells, necitumumab-producing CHO cells, ravulizumab-producing CHO cells, burosumab-producing CHO cells, isatuximab-producing CHO cells, tildrakizumab-producing CHO cells, satralizumab-producing CHO cells,Galcanezumab-producing CHO cells, dinutuximab-producing Sp2 / 0 cells, dinutuximab-producing CHO cells, fremanezumab-producing CHO cells, erenumab-producing CHO cells, casirivimab-producing CHO cells, imdevimab-producing CHO cells, anifrolumab-producing NS0 cells, anifrolumab-producing CHO cells, sotrovimab-producing CHO cells, ocrelizumab-producing CHO cells, naxitamab-producing CHO cells, aducanumab-producing CHO cells, tafasitamab-producing CHO cells, marjetuximab-producing CHO cells, gemtuzumab-producing NS0 cells, gemtuzumab-producing CHO cells, ibritumomab-producing CHO cells, brentuximab-producing CHO cells, inotuzumab-producing CHO cells, pola Antibodies produced from CHO cells such as tuzumab-producing CHO cells, enfortumab-producing CHO cells, sacituzumab-producing Sp2 / 0 cells, sacituzumab-producing CHO cells, belantamab-producing CHO cells, loncustuximab-producing CHO cells, tisotumab-producing CHO cells, mogamulizumab-producing CHO cells, benralizumab-producing CHO cells, obinutuzumab-producing CHO cells, inebilizumab-producing CHO cells, ranibizumab-producing CHO cells, idarucizumab-producing CHO cells, blinatumomab-producing CHO cells, brolucizumab-producing CHO cells, abciximab-producing CHO cells, caplacizumab-producing CHO cells, certolizumab-producing CHO cells, and anti-GPVI antibody-producing CHO cells.

[0275] [7C-1] The antibody obtained by the method for producing an antibody according to the above aspects [3] to [3-7] and [3B] to [3B-7] is selected from the group consisting of muromonab-CD3-producing CHO cells, trastuzumab-producing CHO cells, rituximab-producing CHO cells, palivizumab-producing CHO cells, infliximab-producing CHO cells, basiliximab-producing CHO cells, tocilizumab-producing CHO cells, gemtuzumab-producing CHO cells, bevacizumab-producing CHO cells, ibritumomab-producing CHO cells, adalimumab-producing CHO cells, cetuximab-producing CHO cells, ranibizumab-producing CHO cells, and omali Ipilimumab-producing CHO cells, eculizumab-producing CHO cells, panitumumab-producing CHO cells, ustekinumab-producing CHO cells, golimumab-producing CHO cells, canakinumab-producing CHO cells, denosumab-producing CHO cells, mogamulizumab-producing CHO cells, certolizumab-producing CHO cells, ofatumumab-producing CHO cells, pertuzumab-producing CHO cells, brentuximab-producing CHO cells, natalizumab-producing CHO cells, nivolumab-producing CHO cells, alemtuzumab-producing CHO cells, secukinumab-producing CHO cells, ramucirumab-producing CHO cells, ipilimumab-producing CHO cells CHO cells, evolocumab-producing CHO cells, mepolizumab-producing CHO cells, alirocumab-producing CHO cells, ixekizumab-producing CHO cells, brodalumab-producing CHO cells, idarucizumab-producing CHO cells, elotuzumab-producing CHO cells, pembrolizumab-producing CHO cells, sarilumab-producing CHO cells, bezlotoxumab-producing CHO cells, belimumab-producing CHO cells, daratumumab-producing CHO cells, avelumab-producing CHO cells, dupilumab-producing CHO cells, atezolizumab-producing CHO cells, benralizumab-producing CHO cells, inotuzumab-producing CHO cells cells, emicizumab-producing CHO cells, guselkumab-producing CHO cells, durvalumab-producing CHO cells, obinutuzumab-producing CHO cells, vedolizumab-producing CHO cells, romosozumab-producing CHO cells, risankizumab-producing CHO cells, necitumumab-producing CHO cells, ravulizumab-producing CHO cells, burosumab-producing CHO cells, isatuximab-producing CHO cells, tildrakizumab-producing CHO cells, satralizumab-producing CHO cells, galcanezumab-producing CHO cells, dinutuximab-producing CHO cells, fremanezumab-producing CHO cells, erenumab-producing CHO cells,Antibodies produced from CHO cells such as casirivimab-producing CHO cells, imdevimab-producing CHO cells, anifrolumab-producing CHO cells, sotrovimab-producing CHO cells, ocrelizumab-producing CHO cells, naxitamab-producing CHO cells, aducanumab-producing CHO cells, tafasitamab-producing CHO cells, marjetuximab-producing CHO cells, polatuzumab-producing CHO cells, enfortumab-producing CHO cells, sacituzumab-producing CHO cells, belantamab-producing CHO cells, loncustuximab-producing CHO cells, tisotumab-producing CHO cells, inebilizumab-producing CHO cells, blinatumomab-producing CHO cells, brolucizumab-producing CHO cells, abciximab-producing CHO cells, caplacizumab-producing CHO cells, and anti-GPVI antibody-producing CHO cells.

[0276] [7C-2] The antibody obtained by the method for producing an antibody according to any one of the above aspects [3] to [3-7] and [3B] to [3B-7] is an antibody produced from CHO cells such as trastuzumab-producing CHO cells, rituximab-producing CHO cells, infliximab-producing CHO cells, tocilizumab-producing CHO cells, adalimumab-producing CHO cells, nivolumab-producing CHO cells, and anti-GPVI antibody-producing CHO cells; or an antibody produced from tocilizumab-producing CHO cells or anti-GPVI antibody-producing CHO cells; or an antibody produced from tocilizumab-producing CHO cells.

[0277] Each aspect will be described in more detail below.

[0278] 1. Alginic Acid In this specification, the alginic acid used as a raw material for synthesizing the chemically modified alginic acid derivatives represented by Formula (I) and Formula (II), the alginic acid used as a raw material for the alginic acid solution or alginic acid gel that can be contained in the core layer, and the alginic acid used to form the anionic polymer layer are described below. In this specification, the term "alginic acid" refers to at least one alginic acid (sometimes referred to as "alginic acids") selected from the group consisting of alginic acid, alginic acid esters, and salts thereof (e.g., sodium alginate). The alginic acid used may be naturally occurring or synthetic, but naturally occurring alginic acid is preferred. The preferred alginic acids are bioabsorbable polysaccharides extracted from brown algae such as Lessonia, Macrocystis, Laminaria, Ascophyllum, Durvillea, Ecklonia cava, Eisenia bicolor, and Laminaria kelp. These alginic acids are linear polymers of two uronic acids, D-mannuronic acid (M) and L-guluronic acid (G). More specifically, it is a block copolymer in which a homopolymer fraction of D-mannuronic acid (MM fraction), a homopolymer fraction of L-guluronic acid (GG fraction), and a fraction in which D-mannuronic acid and L-guluronic acid are randomly arranged (M / G fraction) are arbitrarily bonded.

[0279] Alginic acid is a natural polysaccharide extracted and purified from brown seaweed. It is a polymer formed by the polymerization of D-mannuronic acid (M) and L-guluronic acid (G). The composition ratio (M / G ratio) of alginic acid to L-guluronic acid, i.e., gel strength, varies primarily depending on the type of organism from which the seaweed is derived and is also influenced by the habitat and season of the organism. It ranges widely from a high G type (M / G ratio of approximately 0.2) to a high M type (M / G ratio of approximately 5). The physicochemical properties of alginic acid, as well as the preferred uses, vary depending on the M / G ratio and the arrangement of M and G moieties. The gelling ability of alginic acids and the properties of the resulting gel are affected by the M / G ratio; it is generally known that a higher G ratio results in higher gel strength. The M / G ratio also affects the hardness, brittleness, water absorption, flexibility, and other properties of the gel. Therefore, the alginic acid used in the present invention should have an appropriate M / G ratio and viscosity depending on the final use.

[0280] Industrial methods for producing alginic acid include the acid method and the calcium method, and alginic acid produced by either method can be used in the present invention. After purification, alginic acid whose quantitative value by HPLC falls within the range of 80 to 120% by mass is preferred, more preferably 90 to 110% by mass, and even more preferably 95 to 105% by mass. In the present invention, alginic acid whose quantitative value by HPLC falls within the above range is referred to as high-purity alginic acid. The alginic acid or its salt used in the present invention is preferably high-purity alginic acid. Commercially available products include, for example, those sold by Kimica Co., Ltd. under the Kimica Algin series, preferably high-purity food and pharmaceutical grade products. Commercially available products can also be further purified as appropriate. For example, low-endotoxin treatment is preferred. Purification and low-endotoxin treatment methods, such as those described in JP 2007-75425 A, can be used. In this specification, "mass %" refers to w / w % or w / v %.

[0281] The salt of alginic acid in the "alginic acid" used in the present invention is a "monovalent metal salt of alginic acid," which is a salt prepared by ion-exchanging the hydrogen ion of the carboxylic acid of D-mannuronic acid or L-guluronic acid of alginic acid with a monovalent metal ion such as Na or K. Specific examples of the monovalent metal salt of alginic acid include sodium alginate and potassium alginate, with sodium alginate being particularly preferred.

[0282] In this specification, alginic acid may be expressed as (ALG)-COOH, where alginic acid is (ALG) and any one of the carboxyl groups of alginic acid is -COOH.

[0283] The alginic acid used in the present invention has a weight-average molecular weight appropriate for its final use. The weight-average molecular weight (GPC) of the alginic acid used in the present invention is, for example, 10,000 to 10,000,000, preferably 100,000 to 5,000,000, and more preferably 150,000 to 3,000,000.

[0284] In some embodiments, the alginic acid is sodium alginate. Commercially available sodium alginate can be used as the sodium alginate. Here, the sodium alginate used in the examples described below is selected from sodium alginates A-1, A-2, A-3, B-1, B-2, and B-3 (sold by Mochida Pharmaceutical Co., Ltd.) shown in the table below. The viscosity, weight-average molecular weight, and M / G ratio of a 1 w / w% aqueous solution of each sodium alginate are shown in the table below.

[0285]

[0286] The physical properties of sodium alginate A-1, A-2, A-3, B-1, B-2, and B-3 were measured by the following methods. The measurement methods are not limited to these, but the physical properties may differ from those described above depending on the measurement method.

[0287] [Viscosity measurement of sodium alginate] Measurement was performed using a rotational viscometer (cone-plate rotational viscometer) according to the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). Specific measurement conditions are as follows: The sample solution was prepared using MilliQ water. The measurement equipment used was a cone-plate rotational viscometer (viscosity and viscoelasticity measuring device Rheostress RS600 (Thermo Haake GmbH) sensor: 35 / 1). The rotation speed was 1 rpm when measuring 1 w / w% sodium alginate solution. The reading time was 2 minutes, and the average value from 1 minute to 2 minutes was used. The average value of three measurements was used as the measured value. The measurement temperature was 20°C.

[0288] [Measurement of weight-average molecular weight of sodium alginate] Measurement was carried out using two methods: (1) gel permeation chromatography (GPC) and (2) GPC-MALS. The measurement conditions were as follows:

[0289] [Pretreatment Method] The sample was dissolved in an eluent, and then filtered through a 0.45 μm membrane filter to prepare a measurement solution.

[0290] (1) Gel Permeation Chromatography (GPC) Measurement [Measurement Conditions (Relative Molecular Weight Distribution Measurement)] Column: TSKgel GMPW-XL x 2 + G2500PW-XL (7.8 mm ID x 300 mm x 3) Eluent: 200 mM sodium nitrate aqueous solution Flow rate: 1.0 mL / min Concentration: 0.05% Detector: RI detector Column temperature: 40°C Injection volume: 200 μL Molecular weight standards: standard pullulan, glucose

[0291] (2) GPC-MALS Measurement [Refractive Index Increment (dn / dc) Measurement (Measurement Conditions)] Differential Refractometer: Optilab T-rEX Measurement Wavelength: 658 nm Measurement Temperature: 40°C Solvent: 200 mM aqueous sodium nitrate solution Sample Concentration: 0.5 to 2.5 mg / mL (5 concentrations)

[0292] [Measurement conditions (absolute molecular weight distribution measurement)] Column: TSKgel GMPW-XL x 2 + G2500PW-XL (7.8 mm ID x 300 mm x 3) Eluent: 200 mM sodium nitrate aqueous solution Flow rate: 1.0 mL / min Concentration: 0.05% Detector: RI detector, light scattering detector (MALS) Column temperature: 40°C Injection volume: 200 μL

[0293] In this specification, the molecular weight of alginic acid, alginic acid derivatives, crosslinked alginic acid, and crosslinked alginic acid may be expressed in units of Da (Dalton).

[0294] The composition ratio (M / G ratio) of D-mannuronic acid to L-guluronic acid in alginates varies mainly depending on the type of organism from which seaweed is derived, and is also influenced by the location and season of the organism. It ranges widely, from a high M / G ratio of approximately 0.2 (high G type) to a high M / G ratio of approximately 5 (high M type). The gelling ability of alginates and the properties of the resulting gel are affected by the M / G ratio, and it is generally known that a higher G ratio results in higher gel strength. The M / G ratio also affects the hardness, brittleness, water absorption, flexibility, and other properties of the gel. The M / G ratio of the alginates and / or their salts used is typically 0.1 to 4.0, in some embodiments 0.1 to 3.0, in some embodiments 0.1 to 2.0, in some embodiments 0.5 to 1.8, and in some embodiments 0.8 to 1.2. In other embodiments, it is 0.1 to 0.5.

[0295] The alginic acid used in the present invention should have an appropriate viscosity and M / G ratio depending on the final use.

[0296] In this specification, a numerical range indicated using "to" indicates a range that includes the numerical values ​​before and after "to" as the minimum and maximum values, respectively.

[0297] The terms "alginate ester" and "alginate salt" used herein are not particularly limited, but must not have a functional group that inhibits the crosslinking reaction in order to react with a crosslinking agent. Preferred examples of alginate esters include propylene glycol alginate.

[0298] Alginic acid can be in the form of, for example, a monovalent salt of alginic acid or a divalent salt of alginic acid. Examples of monovalent salts of alginic acid include sodium alginate, potassium alginate, and ammonium alginate, with sodium alginate or potassium alginate being preferred, and sodium alginate being more preferred. Examples of divalent salts of alginic acid include calcium alginate, magnesium alginate, barium alginate, and strontium alginate.

[0299] Alginic acid is a polymeric polysaccharide, and although it is difficult to accurately determine its molecular weight, its weight-average molecular weight is generally 1,000 to 10,000,000, preferably 10,000 to 8,000,000, and more preferably 20,000 to 3,000,000. It is known that when measuring the molecular weight of naturally occurring polymeric substances, the values ​​can vary depending on the measurement method.

[0300] In this specification, when specifying the molecular weight of the alginic acid derivative or alginic acid or its salt of the present invention, unless otherwise specified, it is the weight-average molecular weight calculated by size exclusion chromatography (SEC). It is desirable to use alginic acid or its salt used in the present invention with an appropriate molecular weight distribution depending on its final use.

[0301] For example, under the measurement conditions of gel permeation chromatography (GPC) or gel filtration chromatography (collectively referred to as size exclusion chromatography (SEC)) described in the Examples below, the molecular weight is preferably 100,000 to 5,000,000, more preferably 150,000 to 3,000,000. In one embodiment, the molecular weight is 500,000 to 3,000,000, more preferably 1,000,000 to 2,500,000, and even more preferably 1,000,000 to 2,000,000.

[0302] Furthermore, for example, the absolute weight-average molecular weight can be measured by GPC-MALS (SEC-MALS). The weight-average molecular weight (absolute molecular weight) measured by GPC-MALS is preferably 10,000 or more, more preferably 50,000 or more, and even more preferably 60,000 or more, and is preferably 1,000,000 or less, more preferably 800,000 or less, even more preferably 700,000 or less, and particularly preferably 500,000 or less. The preferred range is 10,000 to 1,000,000, more preferably 500,000 to 800,000, and even more preferably 60,000 to 500,000.

[0303] Typically, when the molecular weight of a high molecular weight polysaccharide is calculated using the above-mentioned SEC or SEC-MALS method, a measurement error of about 10% to about 30% can occur. For example, if the molecular weight is 500,000, the value can vary within a range of about 350,000 to 650,000, and if the molecular weight is 1,000,000, the value can vary within a range of about 700,000 to 1,300,000. In this specification, when the term "about" is used in describing molecular weight measurements, it can include values ​​up to ±10% of the numerical value, and in some embodiments, up to ±20% of the numerical value.

[0304] Generally, naturally occurring polymeric substances do not have a single molecular weight but are aggregates of molecules with various molecular weights, and are therefore measured as a molecular weight distribution with a certain range. A typical measurement method is gel filtration chromatography. Typical information on molecular weight distribution obtained by gel filtration chromatography includes the weight average molecular weight (Mw), number average molecular weight (Mn), and polydispersity ratio (Mw / Mn).

[0305] The weight average molecular weight is determined by taking into account the contribution of large molecular weight polymers to the average molecular weight, and is expressed by the following formula.

[0306] Mw = Σ(WiMi) / W = Σ(HiMi) / Σ(Hi) The number average molecular weight is calculated by dividing the total weight of polymers by the total number of polymers.

[0307] Mn = W / ΣNi = Σ(MiNi) / ΣNi = Σ(Hi) / Σ(Hi / Mi) where W is the total weight of polymers, Wi is the weight of the i-th polymer, Mi is the molecular weight at the i-th elution time, Ni is the number of molecular weights Mi, and Hi is the height at the i-th elution time.

[0308] It is known that when measuring the molecular weight of naturally occurring polymeric substances, the values ​​can vary depending on the measurement method (for example, hyaluronic acid: Chikako YOMOTA et. al. Bull. Natl. Health Sci., Vol. 117, pp. 135-139 (1999), Chikako YOMOTA et. al. Bull. Natl. Inst. Health Sci., Vol. 121, pp. 30-33 (2003)). Regarding the measurement of the molecular weight of alginic acid, there are documents describing a method of calculation from intrinsic viscosity and a method of calculation by SEC-MALLS (Size Exclusion Chromatography with Multiple Angle Laser Light Scattering Detection) (ASTM F2064-00 (2006), published by ASTM International). In the present invention, the weight average molecular weight can be a value calculated by measuring the molecular weight by a conventional method as shown in the above document, for example, by size exclusion chromatography (SEC), and using a calibration curve using pullulan as a standard substance. In addition, in the present invention, the weight average molecular weight can be an absolute molecular weight measured by a conventional method as shown in the above document, for example, by size exclusion chromatography (SEC)-MALS.

[0309] The molecular weight of alginic acids can be measured by a conventional method.

[0310] In this specification, when the molecular weight of alginic acid or a salt thereof is specified, unless otherwise specified, it refers to the weight-average molecular weight calculated by gel filtration chromatography. Typical conditions for using gel filtration chromatography to measure molecular weight include, for example, the conditions in the Examples described below. The column can be, for example, a Superose 6 Increase 10 / 300 GL column (GE Healthcare Sciences). The developing solvent can be, for example, 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl. The molecular weight standards can be, for example, blue dextran, thyroglobulin, ferritin, aldolase, conalbumin, ovalbumin, ribonuclease A, and aprotinin.

[0311] The viscosity of the alginic acid used in this specification is not particularly limited, but when measured as a 1 wt% aqueous solution of alginic acids, it is preferably 10 mPa·s to 1000 mPa·s, more preferably 50 mPa·s to 800 mPa·s.

[0312] The viscosity of an aqueous solution of alginic acid can be measured according to a conventional method. For example, the viscosity can be measured using a rotational viscometer such as a coaxial double-cylinder rotational viscometer, a single-cylinder rotational viscometer (Brookfield viscometer), or a cone-and-plate rotational viscometer (cone-and-plate viscometer). Preferably, the viscosity is measured according to the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). More preferably, a cone-and-plate viscometer is used.

[0313] When alginic acids are first extracted from brown algae, they have a large molecular weight and a high viscosity, but the molecular weight decreases and the viscosity decreases during processes such as thermal drying and purification. Alginic acids with different molecular weights can be produced by controlling conditions such as temperature during the production process, selecting the brown algae used as raw materials, and molecular weight fractionation during the production process. Furthermore, alginic acids with the desired molecular weight can be produced by mixing them with other lots of alginic acids with different molecular weights or viscosities.

[0314] In some embodiments, the alginate used herein is alginate that has not been treated with low endotoxins, or in other embodiments, alginate that has been treated with low endotoxins. Low endotoxins refers to an endotoxin level that is low enough to not substantially induce inflammation or fever. Low endotoxin alginates are more preferred.

[0315] The endotoxin reduction treatment can be carried out by a known method or a method similar thereto. For example, the purification of sodium hyaluronate can be carried out by the method of Suga et al. (see, for example, Japanese Patent Application Laid-Open No. 9-324001), the purification of β1,3-glucan by Yoshida et al. (see, for example, Japanese Patent Application Laid-Open No. 8-269102), the purification of biopolymer salts such as alginate and gellan gum by Williams et al. (see, for example, Japanese Translation of PCT International Publication No. 2002-530440), the purification of polysaccharides by James et al. (see, for example, the pamphlet of International Publication No. 93 / 13136), the method of Lewis et al. (see, for example, the specification of U.S. Patent No. 5,589,591), the purification of alginate by Herman Frank et al. (see, for example, Appl Microbiol Biotechnol (1994) 40:638-643), or methods similar thereto. Low-endotoxin treatment can be carried out by known methods such as, but not limited to, washing, filtration through a filter (endotoxin removal filter, charged filter, etc.), ultrafiltration, purification using a column (endotoxin-adsorbing affinity column, gel filtration column, ion exchange resin column, etc.), adsorption to hydrophobic substances, resins or activated carbon, organic solvent treatment (extraction with organic solvent, precipitation / sedimentation by adding organic solvent, etc.), surfactant treatment (see, for example, JP 2005-036036 A), or an appropriate combination of these. These treatment steps may also be appropriately combined with known methods such as centrifugation. It is desirable to select an appropriate method depending on the type of alginate.

[0316] The endotoxin level can be confirmed by known methods, and can be measured, for example, by a method using Limulus Limulus Aqueous Solution (LAL) or a method using Endospecy (registered trademark) ES-24S Set (Seikagaku Corporation).

[0317] The method of endotoxin treatment used is not particularly limited, but as a result, the endotoxin content of alginic acids is preferably 500 endotoxin units (EU) / g or less, more preferably 100 EU / g or less, particularly preferably 50 EU / g or less, and particularly preferably 30 EU / g or less, when endotoxin is measured using Limulus reagent (LAL). In the present invention, "substantially endotoxin-free" means that the endotoxin value measured by the Japanese Pharmacopoeia endotoxin test is within the above-mentioned numerical range. Low-endotoxin treated sodium alginate is available, for example, from Sea Matrix (registered trademark) (Mochida Pharmaceutical Co., Ltd.), PRONOVA ＴＭ It is available commercially as UP LVG (FMC BioPolymer).

[0318] In other embodiments, the sodium alginate used herein as a raw material for synthesizing the chemically modified alginic acid derivatives represented by formula (I) and formula (II), and the sodium alginate used as a raw material for the alginic acid solution or alginic acid gel that can be contained in the core layer, are not particularly limited, and can be selected from, for example, sodium alginates A-1, A-2, A-3, B-1, B-2, or B-3 listed in Table 8.

[0319] In some other embodiments, sodium alginate, which is a raw material for the alginic acid solution that can be used in the anionic polymer layer, is not particularly limited, and can be selected from, for example, sodium alginate A-1, A-2, A-3, B-1, B-2, or B-3 listed in Table 8.

[0320] In this specification, the concentration of the alginic acid solution (also referred to as sodium alginate solution) prepared using the sodium alginate ranges, for example, from about 0.1 to about 3.3% by weight.

[0321] In this specification, sodium alginate, which is a raw material for synthesizing the chemically modified alginic acid derivatives represented by formula (I) and formula (II), is preferably A-2, A-3, B-2, or B-3, more preferably A-2 or A-3, as shown in Table 8. The concentration of the sodium alginate solution used in the synthesis of the chemically modified alginic acid derivatives represented by formula (I) and formula (II) is preferably in the range of 1.5 to 2.0% by weight.

[0322] In this specification, the sodium alginate used to prepare the alginate solution that can be contained in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber, or the alginate solution used to form the alginate gel, is preferably A-2, A-3, B-2, or B-3, more preferably A-2 or A-3, listed in Table 8. The concentration of the alginate solution prepared using the sodium alginate is preferably in the range of about 0.3 to about 1.5% by weight.

[0323] In this specification, the sodium alginate used in preparing an alginic acid solution that can be used to form an anionic polymer layer of a multilayer polymer-coated cross-linked alginate gel fiber is preferably A-2 or B-2 listed in Table 8. The concentration of the alginic acid solution prepared using the sodium alginate is, for example, in the range of about 0.01 to about 5.0% by weight, about 0.05 to about 1.0% by weight, about 0.1 to about 0.5% by weight, or about 0.15 to about 0.4% by weight.

[0324] In this specification, an alginate solution refers to a solution in which alginate is dissolved in a solvent. The solvent is not particularly limited, but examples thereof include culture medium, cell culture medium, culture solution, isotonic buffer solution, water, phosphate-buffered saline (PBS), and physiological saline. When sodium alginate is dissolved in a solvent, it is called a sodium alginate solution.

[0325] In some embodiments, the solution of the chemically modified alginic acid derivative represented by formula (I) or formula (II) and the alginic acid solution used to form the core layer of the polymer-coated cross-linked alginic acid gel fiber and the multilayer polymer-coated cross-linked alginic acid gel fiber are not particularly limited, and may be mixed with a collagen solution, a culture medium, a culture solution, etc. The solvents used to prepare the solution of the chemically modified alginic acid derivative represented by formula (I) or formula (II) and the alginic acid solution are as described below.

[0326] 2. Chemically Modified Alginic Acid Derivative In some embodiments, the chemically modified alginic acid derivative in the present specification is one in which a reactive group in the Huisgen reaction described below, or a reactive group complementary to the reactive group, is introduced to any one or more carboxyl groups of alginic acid via an amide bond and a bivalent linker. More specifically, it is represented by the following formula (I): [In formula (I), (ALG), Akn-L １ The definitions of - and -NH-CO- are the same as those in the first aspect above], and an alginic acid derivative represented by the following formula (II): [In formula (II), (ALG), -L ２ The definitions of -, and -NH-CO- are the same as those in the first aspect above.

[0327] Divalent linker (-L １ -or-L ２ Specifically, the linker (-) can be selected from the divalent linkers described in the above aspects.

[0328] In some embodiments, the bivalent linker (-L １ -or-L ２ Any linker can be used for the cyclic alkyne group (Akn-) as long as it does not inhibit the reaction with the azide group (Huisgen reaction). Specifically, a linear alkylene group (-(CH ２ ) ｎ -, n = 1 to 30) [-CH ２- may be replaced by a plurality of (for example, 1 to 10, or 1 to 5) groups such as -C(═O)-, -CONH-, -O-, -NH-, -S-, a cycloalkyl ring having 3 to 8 carbon atoms, a benzene ring, or a heterocycle (a 5- or 6-membered aromatic heterocycle or a 5- or 6-membered non-aromatic heterocycle such as a pyridine ring, a piperidine ring, or a piperazine ring); ２ -) hydrogen atom is an oxo group (=O), C １－６ and the like, but are not limited to these.

[0329] In the -NH-CO- group of the chemically modified alginic acid derivative represented by the formula (I) or (II), the hydrogen atom of the imino group (-NH-) can be substituted with a methyl group to form a -N(Me)-CO- group. １ -, -L ２ The bond between the alginic acid and the hydroxyl group is an —NH—CO— bond or an —N(Me)—CO— bond, preferably an —NH—CO— bond. The —CO— in the —NH—CO— bond or the —N(Me)—CO— bond is derived from the carboxyl group of alginic acid.

[0330] In the present specification, the chemically modified alginic acid derivatives represented by formula (I) or formula (II) can be produced, for example, by the synthesis method of chemically modified alginic acid derivatives described below.

[0331] The weight-average molecular weight of the chemically modified alginic acid derivative represented by formula (I) herein, as measured by gel filtration chromatography, is in the range of about 100,000 Da to about 3,000,000 Da, preferably about 300,000 Da to about 2,500,000 Da, and more preferably about 500,000 Da to about 2,000,000 Da. The weight-average molecular weight of the chemically modified alginic acid derivative represented by formula (II), as measured by gel filtration chromatography, is in the range of about 100,000 Da to about 3,000,000 Da, preferably about 300,000 Da to about 2,500,000 Da, and more preferably about 500,000 Da to about 2,000,000 Da.

[0332] As used herein, Akn-L of formula (I) １ The —NH— group does not necessarily have to be bonded to all the carboxyl groups of the alginic acid building block. ３ -L ２ It is not necessary for the --NH-- group to be bonded to all of the carboxyl groups of the alginic acid building block.

[0333] As used herein, Akn-L of formula (I) １ When the —NH— group is referred to as a reactive group, N in formula (II) ３ -L ２ The —NH— group serves as a complementary reactive group. ３ -L ２ When the —NH— group is referred to as a reactive group, Akn-L in formula (I) １ The —NH— group serves as a complementary reactive group.

[0334] In the present specification, the introduction rate of reactive groups into the chemically modified alginic acid derivative represented by formula (I) is, for example, in the range of about 0.1 to about 30 mol%, preferably about 0.3 to about 20 mol%, and more preferably about 0.5 to about 10 mol%. Also, the introduction rate of reactive groups into the chemically modified alginic acid derivative represented by formula (II) is, for example, in the range of about 0.1 to about 30 mol%, preferably about 0.3 to about 20 mol%, and more preferably about 0.5 to about 15 mol%.

[0335] The introduction rate of the reactive group or the complementary reactive group is the number of uronic acid monosaccharide units into which each reactive group has been introduced, expressed as a percentage, among the uronic acid monosaccharide units, which are the repeating units of alginic acid. The introduction rate of each reactive group or the complementary reactive group can be determined by the method described in the Examples below.

[0336] In the present specification, the cyclic alkyne group (Akn) in formula (I) and the azide group in formula (II) form a triazole ring by Huisgen reaction, thereby forming a bridge.

[0337] In this specification, the chemically modified alginic acid derivatives represented by formula (I) or formula (II) also include those in which any carboxyl group in the molecule forms a monovalent salt (e.g., sodium salt, etc.).

[0338] In this specification, the chemically modified alginic acid derivatives represented by formula (I) and formula (II) can be used to form the crosslinked alginic acid gel contained in the core layer of a polymer-coated crosslinked alginic acid gel fiber and a multi-polymer-coated crosslinked alginic acid gel fiber, and can also be used to form the anionic polymer layer of a multi-polymer-coated crosslinked alginic acid gel fiber.

[0339] 3. Huisgen Reaction The Huisgen reaction (1,3-dipolar cycloaddition reaction) is a condensation reaction between compounds having a terminal azide group and a terminal alkyne group, as shown in the following formula. As a result of the reaction, a disubstituted 1,2,3-triazole ring is obtained in good yield, and is characterized by the absence of unnecessary by-products. This reaction is thought to produce a 1,4- or 1,5-disubstituted triazole ring, but the use of a copper catalyst makes it possible to obtain the triazole ring regioselectively.

[0340]

[0341] In addition, Wittig and Krebs have reported a Huisgen reaction that does not use a copper catalyst. That is, this reaction is a reaction in which a cycloadduct can be obtained by simply mixing cyclooctyne and phenyl azide (in the following formula, R ３(=phenyl). Because the triple bond of cyclooctyne is highly distorted, the reaction with phenyl azide relieves this distortion, driving the reaction, which proceeds spontaneously, eliminating the need for a catalyst.

[0342]

[0343] As described above, the Huisgen reaction can be used with azide compounds having substituted primary azides, secondary azides, tertiary azides, aromatic azides, etc., and compounds having terminal or cyclic alkyne groups, which are reactive groups complementary to the azide group. Furthermore, since the Huisgen reaction reacts almost exclusively with azide groups and alkyne groups, it is possible to substitute various functional groups (e.g., ester groups, carboxyl groups, alkenyl groups, hydroxyl groups, amino groups, etc.) in the reaction substrate.

[0344] In some embodiments, the alkyne group in the Huisgen reaction is, for example, a cyclic alkyne group (cyclooctyl group) as described in embodiment [1] above, to form crosslinks between alginic acid molecules via 1,2,3-triazole rings in a short time, easily, and efficiently without using a copper catalyst in order to avoid the production of undesired by-products and to avoid the cytotoxicity of copper catalysts.

[0345] In a preferred embodiment of the crosslinking method for chemically modified alginic acid derivatives, the reaction (Huisgen reaction) does not cause undesirable side reactions and hardly produces any by-products, making it possible to incorporate cells capable of producing antibodies, physiologically active substances, etc. into the core layer of the polymer-coated crosslinked alginate gel fiber of the present invention.

[0346] 4. Method for Producing Chemically Modified Alginic Acid Derivatives In the present specification, the chemically modified alginic acid derivatives represented by formula (I) or formula (II) can be produced by reacting a compound represented by formula (AM-1) (Akn-L) with a compound represented by formula (AM-2) (Akn-L) as shown in the following reaction formula: １ -NH ２ : Akn-L １ - is the same as defined in the above embodiment [1]), or an amine represented by formula (AM-2) (N ３ -L ２ -NH ２ :-L ２The amine represented by the formula (where "-" is the same as defined in the embodiment [1]) can be produced by a condensation reaction using any carboxyl group of alginic acid and any condensing agent. The detailed conditions for each reaction are in accordance with the conditions described in WO 2019 / 240219.

[0347]

[0348] In the method for producing a chemically modified alginic acid derivative represented by formula (I) or formula (II), the introduction rate of the amine represented by formula (AM-1) or formula (AM-2) (which has the same meaning as the introduction rate of the reactive group of the chemically modified alginic acid derivative represented by formula (I) or formula (II) in the above embodiment) can be adjusted by appropriately selecting and combining reaction conditions such as (i) to (v) below, taking into consideration the properties of the amine. (i) increasing or decreasing the equivalent amount of condensing agent, (ii) increasing or decreasing the reaction temperature, (iii) extending or shortening the reaction time, (iv) adjusting the concentration of alginic acid as a reaction substrate, (v) adding a water-miscible organic solvent to increase the solubility of the amine of formula (AM-1) or formula (AM-2), etc.

[0349] The following describes a method for producing a more specific amine, among the amines represented by formula (AM-1) or formula (AM-2), used in the present specification.

[0350] In the following production methods, the definitions of x1a, x1b, y1b, x2, y2, z2, x3a, y3a, z3a, x3b, y3b, z3b, x4, y4, x5a, y5a, z5a, x5b, y5b, z5b, x6, y6, z6, x7a, y7a, z7a, v7a, x7b, y7b, z7b, v7b, a1, b1, a2, b2, a3, b3, a4, b4, a5, and a6 are the same as those in the above embodiment [1]; R Ａ = C, such as methyl group, ethyl group, etc. １～６ is an alkyl group; １ represents a —C(O)O-tertBu group, a —C(O)O-Bn group, a —C(O)CH ３ group, -C(O)CF ３ group, —SO ２ Ph, -SO ２ PhMe group, -SO２ Ph(NO ２ E is a leaving group such as a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), an -OTs group, an -OMs group, etc., and X is a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.). In addition, in each of the following manufacturing methods, formula (SM-A1), formula (SM-A2), formula (SM-B), formula (SM-C1), formula (SM-C2), formula (SM-D), formula (SM-E1), formula (SM-E2), formula (SM-F), formula (SM-G1), formula ( SM-G2), Formula (SM-H), Formula (SM-J), Formula (SM-K), Formula (SM-L), Formula (RG-A1), Formula (RG-A2), Formula (RG-B1), Formula (RG-C1), Formula (RG-C2), Formula (RG-D1), Formula (RG-E1), Formula ( The compounds represented by formula (RG-E2), formula (RG-F1), formula (RG-F2), formula (RG-F3), formula (RG-G1-1), formula (RG-G1-2), formula (RG-G1-3), formula (RG-G2-1), formula (RG-G2-2), formula (RG-G2-3), formula (RG-H1), formula (RG-H2), formula (RG-I1), formula (RG-J1), formula (RG-K), formula (RG-L1) and formula (RG-M) are commercially available compounds or compounds that can be produced from commercially available compounds by production methods known in the literature.

[0351] In addition, in each of the following production methods, the protecting group P １ The protection and deprotection of the above can be carried out in accordance with a method known in the literature, for example, the deprotection method described in "Protective Groups in Organic Synthesis 4th Edition, 4th Edition, 2007, John Wiley & Sons, Greene et al."

[0352] In the following production methods, when a condensation reaction is mentioned, it means the same reaction as the above-mentioned condensation reaction.

[0353] [Production Method A] Production method for amines represented by formula (AM-1-1A) and formula (AM-1-1B):

[0354] <Step 1> A condensation reaction is carried out using the compound of formula (SM-A) and the compound of formula (RG-A1) to obtain a condensation product. Subsequently, bromine is added, and then a debromination reaction is carried out using a base such as tert-BuOK to form an alkyne group. Subsequently, a protecting group P １ By deprotecting the compound, an amine represented by formula (AM-1-1A) or a salt thereof can be produced.

[0355] <Step 2> A condensation reaction is carried out using the compound of formula (SM-A2) and the compound of formula (RG-A2) obtained by the same method as in <Step 1> of the above [Production Method A], and then a protecting group P １ By deprotecting the group, an amine represented by formula (AM-1-1B) or a salt thereof can be produced.

[0356] [Production Method B] Production method for amine represented by formula (AM-1-2):

[0357] A condensation product is obtained by carrying out a condensation reaction using the compound of formula (SM-B) and the compound of formula (RG-B1). １ By deprotecting the group, an amine represented by formula (AM-1-2) or a salt thereof can be produced.

[0358] [Production Method C] Production method for amines represented by formula (AM-1-3A) and formula (AM-1-3B):

[0359] A condensation reaction is carried out using the compound of formula (SM-C1) and the compound of formula (RG-C1) to obtain a condensate. １ By deprotecting the compound, an amine represented by formula (AM-1-3A) or a salt thereof can be produced.

[0360] A condensation reaction is carried out using the compound of formula (SM-C2) and the compound of formula (RG-C2) to obtain a condensate. １ By deprotecting the compound, an amine represented by formula (AM-1-3B) or a salt thereof can be produced.

[0361] [Production Method D] Production method for amine represented by formula (AM-1-D):

[0362] A condensation reaction is carried out using the compound of formula (SM-D) and the compound of formula (RG-D1) to obtain a condensate. １ An amine represented by formula (AM-1-D) or a salt thereof can be prepared by deprotecting the group.

[0363] [Production Method E] Production method for amines represented by formula (AM-1-E1) and formula (AM-1-E2):

[0364] A condensation reaction is carried out using the compound of formula (SM-E1) and the compound of formula (RG-E1) to obtain a condensate. １ By deprotecting the group, an amine represented by formula (AM-1-E1) or a salt thereof can be produced.

[0365] A condensation reaction is carried out using the compound of formula (SM-E2) and the compound of formula (RG-E2) to obtain a condensate. １ By deprotecting the group, an amine represented by formula (AM-1-E2) or a salt thereof can be produced.

[0366] [Production Method F] Production method for amine represented by formula (AM-1-F):

[0367] <Step 1> A condensation reaction is carried out using the compound of formula (SM-F) and the compound of formula (RG-F1) to obtain a condensate. １By deprotecting the amine represented by formula (AM-1-F), or a salt thereof, can be produced. <Step 2> A condensation reaction is carried out using the compound of formula (SM-F) and the compound of formula (RG-F2) to obtain a condensate. Subsequently, in the presence of a base such as sodium hydroxide, the ester group is hydrolyzed in a reaction-inert solvent such as methanol, ethanol, tetrahydrofuran, water, or a mixed solvent thereof to produce a carboxylic acid represented by formula (IM-F1), or a salt thereof. <Step 3> [Production Method F] A condensation reaction is carried out using the compound of formula (IM-F1) obtained in <Step 2> and the compound of formula (RG-F3), to obtain a condensate. Subsequently, a protecting group P １ An amine represented by formula (AM-1-F) or a salt thereof can be produced by deprotecting the group.

[0368] [Production Method G] Production method for amines represented by formula (AM-1-G1) and formula (AM-1-G2):

[0369] <Step 1> A condensation reaction is carried out using the compound of formula (SM-G1) and the compound of formula (RG-G1-1) to obtain a condensate. １ By deprotecting the compound represented by formula (AM-1-G1), an amine represented by formula (AM-1-G1) or a salt thereof can be produced. <Step 2> A condensation reaction is carried out using the compound represented by formula (SM-G1) and the compound represented by formula (RG-G1-2) to obtain a condensate. Subsequently, by hydrolyzing the ester group, a carboxylic acid represented by formula (IM-G1) or a salt thereof can be produced.

[0370] <Step 3> [Production Method G] The compound of formula (IM-G1) obtained in <Step 2> and the compound of formula (RG-G1-3) are subjected to a condensation reaction to obtain a condensate. １ The amine represented by formula (AM-1-G1) or a salt thereof can be produced by deprotecting the protecting group P. <Step 4> A condensation reaction is carried out using the compound of formula (SM-G2) and the compound of formula (RG-G2-1) to obtain a condensate. １By deprotecting the group, an amine represented by formula (AM-1-G2) or a salt thereof can be produced.

[0371] <Step 5> A condensation reaction is carried out using the compound of formula (SM-G2) and the compound of formula (RG-G2-2) to obtain a condensate. Subsequently, the ester group is hydrolyzed to produce a carboxylic acid represented by formula (IM-G2) or a salt thereof (for example, a lithium salt, a sodium salt, a potassium salt, etc.). <Step 6> [Production Method G] A condensation reaction is carried out using the compound of formula (IM-G2) obtained in <Step 5> and the compound of formula (RG-G2-3) to obtain a condensate. Subsequently, a protecting group P １ By deprotecting the group, an amine represented by formula (AM-1-G2) or a salt thereof can be produced.

[0372] [Production Method H] Production method for amine represented by formula (AM-2-H):

[0373] <Step 1> Using the compound of formula (SM-H) and the compound of formula (RG-H1), (i) PPh ３ , and N ２ (CO ２ CHMe ２ ) ２ In the presence of the reagent (I), a Mitsunobu reaction is carried out in a reaction-inert solvent such as tetrahydrofuran, followed by hydrolysis in the same manner as in Step 2 of [Production Method F], thereby producing a compound represented by Formula (IM-H1) or a salt thereof (for example, a lithium salt, a sodium salt, a potassium salt, etc.).

[0374] <Step 2> [Production Method H] The compound of formula (IM-H1) obtained in <Step 1> and the compound of formula (RG-H2) are subjected to a condensation reaction to obtain a condensate. １ By deprotecting the group, an amine represented by formula (AM-2-H) or a salt thereof can be produced.

[0375] [Production Method I] Production method for an amine represented by formula (AM-2-I):

[0376] <Step 1A> [Production Method H] The compound of formula (IM-H1) obtained in <Step 1> and the compound of formula (RG-I1) are subjected to a condensation reaction to obtain a condensate. １ An amine represented by formula (AM-2-I) or a salt thereof can be prepared by deprotecting the compound.

[0377] [Production Method J] Production method for amine represented by formula (AM-2-J):

[0378] <Step 1> Compound of formula (IM-J1) can be produced by condensation reaction using compound of formula (SM-J) and compound of formula (RG-J1). <Step 2> [Production Method J] Compound of formula (IM-J1) obtained in <Step 1> is condensed with NaN in a reaction-inert solvent such as dimethyl sulfoxide in accordance with a method known in the literature, for example, the method described in "Organometallics, 29(23), pp. 6619-6622; 2010". ３ After the azide group is introduced by the reaction of １ An amine represented by formula (AM-2-J) or a salt thereof can be produced by deprotecting the group.

[0379] [Production Method K] Production method for an amine represented by formula (AM-2-K):

[0380] <Step 1> Compound of formula (IM-K) can be produced by carrying out a condensation reaction using compound of formula (SM-K) and compound of formula (RG-K). <Step 2> [Production method K] Compound of formula (IM-K) obtained in <Step 1> is subjected to the same reaction as in <Step 2> in [Production method J], and the protecting group P １ An amine represented by formula (AM-2-K) or a salt thereof can be produced by deprotecting the above-mentioned compound.

[0381] [Production Method L] Production method for amine represented by formula (AM-2-L):

[0382] A condensation reaction is carried out using the compound of formula (SM-L) and the compound of formula (RG-L1) to obtain a condensate. １ By deprotecting the group, an amine represented by formula (AM-2-L) or a salt thereof can be produced.

[0383] [Production Method M] Production method for an amine represented by formula (AM-2-M):

[0384] A condensation reaction is carried out using the compound of formula (SM-L) and the compound of formula (RG-M) to obtain a condensate. １ An amine represented by formula (AM-2-M) or a salt thereof can be prepared by deprotecting the group.

[0385] Amines other than the amines represented by formula (AM-1) or formula (AM-2) that can be produced by the above [Production Method A] to [Production Method M], for example, linker-L of formula (AM-1) １ - or a linker -L of formula (AM-2) ２ - is a straight chain alkylene group (-(CH ２ ) ｎ -, n = 1 to 30) [-CH ２ - may be replaced by a plurality of (for example, 1 to 10, or 1 to 5) groups such as -C(═O)-, -CONH-, -O-, -NH-, -S-, a cycloalkyl ring having 3 to 8 carbon atoms, a benzene ring, or a heterocycle (a 5- or 6-membered aromatic heterocycle or a 5- or 6-membered non-aromatic heterocycle such as a pyridine ring, a piperidine ring, or a piperazine ring); ２ -) hydrogen atom is an oxo group (=O), C １－６and optionally substituted with a plurality (e.g., 1 to 10 or 1 to 5) of groups selected from alkyl groups (e.g., methyl, ethyl, n-propyl, isopropyl, etc.), halogen atoms (e.g., fluorine, chlorine, bromine, iodine), hydroxyl groups (—OH), and the like), the desired amine can be produced by appropriately combining known methods in the literature, for example, synthetic methods described in “Experimental Chemistry Lectures, 5th Edition, each edition, 2007, Maruzen,” “Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 3rd Edition (Edited by Richard C. Larock), 2018,” “Strategic Applications of Named Reactions in Organic Synthesis, (Edited by Laszlo Kurti, Barbara Czako), Academic Press, 2005,” and the like.

[0386] In the present specification, the amines represented by formula (AM-1) or formula (AM-2) (including subformulas of each formula) may form pharmaceutically acceptable salts (e.g., acid addition salts; for example, hydrochloride, hydrobromide, sulfate, acetate, trifluoroacetate, p-toluenesulfonate, etc.).

[0387] The compounds herein can form salts, which can be obtained by a conventional method, for example, by mixing a solution containing an appropriate amount of acid or base to form the desired salt, followed by fractional filtration or by distilling off the mixed solvent. A general review of salts is available in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use," Stahl & Wermuth (Wiley-VCH, 2002), which provides a detailed description.

[0388] In the present specification, the amine represented by formula (AM-1) or formula (AM-2) (including subformulas of each formula) or a salt thereof can form a solvate with a solvent such as water, ethanol, or glycerol.

[0389] Unless otherwise specified, in this specification, when a variable substituent is substituted on a cyclic group, the variable substituent is not bonded to a specific carbon atom of the cyclic group. For example, the variable substituent Rs in the following formula A means that it can be substituted on any of carbon atoms i, ii, iii, iv, or v in the formula A.

[0390]

[0391] 5-1. Crosslinked Alginate Gel In this specification, the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber and the multilayer polymer-coated crosslinked alginate gel fiber is formed using the chemically modified alginate derivative described in Section 2. Chemically Modified Alginate Derivatives. The crosslinked alginate gel (which can also be referred to as crosslinked alginate or chemically crosslinked alginate) is formed by (i) crosslinking via divalent metal ion bonds, (ii) crosslinking via chemical bonds, or (iii) crosslinking via both divalent metal ion bonds and chemical bonds. The crosslinked alginate gel contained in the core layer of each fiber is a crosslinked alginate gel that includes both chemical crosslinking via triazole rings formed by a Huisgen reaction (crosslinking reaction) as crosslinking, and ionic crosslinking formed by the coexistence of divalent metal ions (e.g., calcium ions). The crosslinked alginate gel contained in the core layer of each fiber is a crosslinked alginate gel that includes chemical crosslinking via triazole rings formed by a Huisgen reaction (crosslinking reaction).

[0392] In this specification, the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber and the multilayer polymer-coated crosslinked alginate gel fiber can be obtained by performing a Huisgen reaction (crosslinking reaction) using chemically modified alginate derivatives represented by formulas (I) and (II) to form chemical crosslinks between the derivatives. Alternatively, the crosslinked alginate gel can be obtained by forming ionic crosslinks between the chemically modified alginate derivatives represented by formulas (I) and (II) in the presence of divalent metal ions. Alternatively, the crosslinked alginate gel can be obtained by performing a Huisgen reaction (crosslinking reaction) using chemically modified alginate derivatives represented by formulas (I) and (II) to form chemical crosslinks between the derivatives, and then further forming ionic crosslinks between the derivatives in the presence of divalent metal ions.

[0393] In this specification, "crosslinking is formed," "crosslinking is formed," or "crosslinking reaction is performed" means that by performing a Huisgen reaction using a chemically modified alginic acid derivative represented by the formula (I) and the formula (II), a chemical crosslink (chemical bond) is formed between the chemically modified alginic acid derivative represented by the formula (I) and the formula (II), or by causing a divalent metal ion to coexist with the chemically modified alginic acid derivative represented by the formula (I) and the chemically modified alginic acid derivative represented by the formula (II), an ionic crosslink (ionic bond) is formed between each derivative, or both chemical crosslinking by the Huisgen reaction and ionic crosslinking by divalent metal ions is formed. It also means that ionic crosslinking is formed in alginic acid by causing a divalent metal ion to coexist with alginic acid (sodium alginate, etc.).

[0394] By contacting the chemically modified alginic acid derivatives represented by formula (I) and formula (II) with divalent metal ions, ionic crosslinks are formed, and the time required for the formation of an ionically crosslinked alginic acid gel is, for example, instantaneous (e.g., 1 to 5 seconds) to several hours (e.g., 1 to 3 hours). Furthermore, the time required for the Huisugen reaction between the chemically modified alginic acid derivatives represented by formula (I) and formula (II) to form chemical crosslinks and form a chemically crosslinked alginic acid gel is, for example, several seconds to 24 hours, several seconds to 12 hours, or several seconds to 30 minutes.

[0395] The divalent metal ions used to obtain the cross-linked alginate gel are not particularly limited, but examples thereof include calcium ions, magnesium ions, barium ions, strontium ions, zinc ions, etc., and are preferably calcium ions, barium ions, or strontium ions, and more preferably calcium ions or barium ions.

[0396] The solution containing a divalent metal ion is not particularly limited, but examples thereof include a solution containing calcium ions (e.g., an aqueous solution of calcium chloride, an aqueous solution of calcium carbonate, an aqueous solution of calcium gluconate, etc.), a solution containing barium ions (e.g., an aqueous solution of barium chloride, etc.), and a solution containing strontium ions (e.g., an aqueous solution of strontium chloride, etc.), of which a solution containing calcium ions or a solution containing barium ions is preferred, and a calcium chloride aqueous solution or a barium chloride aqueous solution is more preferred.

[0397] The divalent metal ion concentration (e.g., calcium ion or barium ion concentration) of the divalent metal ion-containing solution is not particularly limited, but is, for example, in the range of about 1 mM to about 1 M, or in the range of about 10 to about 500 mM; preferably, about 10 to about 100 mM.

[0398] The solvent used in preparing a solution containing a divalent metal ion or the like is not particularly limited, and examples thereof include tap water, pure water (e.g., distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water (MilliQ water), culture medium, cell culture medium, culture solution, phosphate buffered saline (PBS), and physiological saline; physiological saline or ultrapure water is preferred.

[0399] When the crosslinked alginate gel contained in the core layer of each fiber contains both ionic and chemical crosslinks, the ionic crosslinking reaction is instantaneous and reversible, whereas the chemical crosslinking reaction proceeds slowly under relatively mild conditions and is irreversible. By utilizing this property and appropriately combining chemical and ionic crosslinking, the crosslinked alginate gel of the present invention can be efficiently produced. For example, using the apparatus XX described below in "11. Method for producing polymer-coated crosslinked alginate gel fiber," a mixed solution of chemically modified alginate derivatives represented by formulas (I) and (II) is injected into a solution containing divalent metal ions, forming ionic crosslinks and instantly producing a crosslinked alginate gel in the form of a fiber (fiber). At the same time, a Huisgen reaction (crosslinking reaction) occurs between the chemically modified alginate derivatives represented by formulas (I) and (II), forming chemical crosslinks, resulting in a crosslinked alginate gel in the form of a fiber (fiber) containing both ionic and chemical crosslinks. The physical properties of the cross-linked alginate gel can be adjusted, for example, by changing the concentration of the aqueous solution containing the divalent metal ions used (e.g., calcium chloride aqueous solution) or by changing the introduction rate of reactive groups introduced into the chemically modified alginate derivative.

[0400] By utilizing the crosslinking reaction described above and using the chemically modified alginic acid derivatives represented by formulas (I) and (II), the crosslinked alginic acid gel contained in the core layer of the polymer-coated crosslinked alginic acid gel fiber and the multilayer polymer-coated crosslinked alginic acid gel fiber of the present invention can be produced in the form of fiber (fiber) (also referred to as "crosslinked alginic acid gel fiber"). The crosslinked alginic acid gel fiber can be produced by adding an alginic acid (e.g., sodium alginate) solution to a mixed solution of the chemically modified alginic acid derivatives represented by formulas (I) and (II).

[0401] In the present invention, chemical crosslinking (Huisugen reaction) is used as one method for strengthening the fiber structure (for example, to achieve long-term stability, etc.). When crosslinked alginate gel fibers having both ionic and chemical crosslinks prepared as described above are cultured in a culture medium, the divalent metal ions forming the ionic crosslinks are gradually and reversibly released, leaving crosslinked alginate gel fibers with only chemical crosslinks remaining, but the gel structure is maintained by the irreversible chemical crosslinks, allowing for stable, continuous culture.

[0402] The crosslinked alginate gel formed by a crosslinking reaction using the chemically modified alginate derivatives represented by formula (I) and formula (II) of the present invention may contain other components, such as, but not limited to, a collagen solution, a collagen gel, a culture medium, a cell culture medium, a culture solution, methylcellulose, a sucrose solution, an alginate solution, an alginate gel, etc.

[0403] In this specification, when the term "alginate gel" is simply used, it means an alginate gel in which ionic crosslinks are formed by allowing divalent metal ions to coexist in alginic acid (e.g., sodium alginate) or a solution thereof.

[0404] 5-2. Chemical Crosslinking in Crosslinked Alginate Gel In the present specification, the crosslinked alginate gel can be obtained by mixing the chemically modified alginic acid derivatives represented by the formula (I) and the formula (II) and carrying out the Huisgen reaction.

[0405] In this specification, the crosslinked alginate gel forms a three-dimensional network structure through chemical crosslinking (crosslinking by triazole rings formed from alkyne groups and azide groups). A preferred chemically modified alginate derivative improves the stability of the crosslinked alginate gel after crosslinking. The physical properties of the crosslinked alginate gel can be adjusted, for example, by adjusting the introduction rate of each reactive group in the raw material chemically modified alginate represented by formula (I) or formula (II).

[0406] In some embodiments, the cross-linked alginate gel has the following formula (III-L): [In formula (III-L), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; -L １ -, -L ２ - and -X- are the same as defined in the above embodiment [1-12]].

[0407] In some embodiments, when preparing a crosslinked alginate gel, the mixing ratio of the chemically modified alginic acid derivative represented by formula (I) to the chemically modified alginic acid derivative represented by formula (II) is, for example, 1:1.0-4.0, or 1:1.0-3.0, or 1:1.0-2.0, or 1:1.0-1.5, or 1:1, in terms of the weight ratio of the chemically modified alginic acid derivative represented by formula (I) to the chemically modified alginic acid derivative represented by formula (II); preferably 1:1.0-3.0.

[0408] In some embodiments, when preparing a crosslinked alginate gel, the mixing ratio of the chemically modified alginic acid derivative represented by formula (II) to the chemically modified alginic acid derivative represented by formula (I) is, for example, 1:1.0 to 4.0, or 1:1.0 to 3.0, or 1:1.0 to 2.0, or 1:1.0 to 1.5, or 1:1 by weight.

[0409] In some embodiments, when preparing a crosslinked alginate gel, the mixing ratio of the chemically modified alginic acid derivative represented by formula (I) to the chemically modified alginic acid derivative represented by formula (II) is, more preferably, the ratio of the introduction rate (mol%) of the reactive group of the chemically modified alginic acid derivative represented by formula (I) to the chemically modified alginic acid derivative represented by formula (II), for example, 1:1.0 to 4.0, or 1:1.0 to 3.0, or 1:1.0 to 2.0, or 1:1.0 to 1.5, or 1:1; preferably 1:1.0 to 3.0.

[0410] In some embodiments, when preparing a crosslinked alginate gel, the mixing ratio of the chemically modified alginic acid derivative represented by formula (II) to the chemically modified alginic acid derivative represented by formula (I) is more preferably, for example, 1:1.0 to 4.0, or 1:1.0 to 3.0, or 1:1.0 to 2.0, or 1:1.0 to 1.5, or 1:1, in terms of the ratio of the introduction rate (mol%) of the reactive group of the chemically modified alginic acid derivative represented by formula (II) to the chemically modified alginic acid derivative represented by formula (I).

[0411] In a crosslinked alginate gel, it is not necessary for all carboxyl groups in the alginic acid constituent units to have crosslinks of the formula (III-L) above. The introduction rate of crosslinks of the formula (III-L) above (also referred to as the crosslinking rate) in the crosslinked alginate gel is, for example, in the range of about 0.1 to about 80%, about 0.3 to about 60%, about 0.5 to about 30%, or about 1.0 to about 10%.

[0412] The concentration of the solution of the alginic acid derivative represented by formula (I) or formula (II) in the Huisgen reaction to obtain the crosslinked alginic acid gel contained in the core layer of the polymer-coated crosslinked alginic acid gel fiber and multilayer polymer-coated crosslinked alginic acid gel fiber of the present invention is, for example, in the range of about 0.01 to about 1.5 wt %; preferably, in the range of about 0.05 to about 1.0 wt %; and more preferably, in the range of about 0.08 to about 0.75 wt %.

[0413] In the Huisgen reaction using the alginic acid derivatives represented by formulas (I) and (II) to obtain the crosslinked alginic acid gel contained in the core layer of the polymer-coated crosslinked alginic acid gel fiber and multilayer polymer-coated crosslinked alginic acid gel fiber of the present invention, when an alginic acid solution is added to a mixed solution of the alginic acid derivatives represented by formulas (I) and (II), the concentration of the alginic acid solution is, for example, in the range of 0% to about 1.98% by weight; preferably, in the range of 0% to about 1.8% by weight; and more preferably, in the range of 0 to about 1.7% by weight.

[0414] The reaction temperature for the Huisgen reaction (the temperature at which the cross-linked alginate gel and cross-linked alginate gel fiber are produced) is usually about 4 to about 60°C, preferably about 15 to about 37°C.

[0415] 6. Polymer-Coated Cross-Linked Alginate Gel Fiber In this specification, the term "polymer-coated cross-linked alginate gel fiber" refers to a fibrous (fibrous) structure obtained by coating a core layer containing cells capable of producing antibodies, physiologically active substances, etc., and a cross-linked alginate gel obtained by a cross-linking reaction using the chemically modified alginate derivatives represented by formula (I) and formula (II) with a cationic polymer (cationic polymer layer) (the method for producing polymer-coated cross-linked alginate gel fiber will be described later).

[0416] The polymer-coated cross-linked alginate gel fiber is a fibrous structure comprising a core layer and a cationic polymer layer disposed on the outer surface of the core layer. The core layer comprises cells capable of producing antibodies, physiologically active substances, etc., and a cross-linked alginate gel formed by cross-linking using the chemically modified alginate derivatives represented by formulas (I) and (II), and the cationic polymer layer is a cationic polymer.

[0417] The polymer-coated cross-linked alginate gel fiber is a fibrous structure comprising a core layer and a cationic polymer layer disposed on the outer surface of the core layer. The core layer comprises cells capable of producing antibodies, physiologically active substances, etc., and a cross-linked alginate gel. The cross-linked alginate gel comprises cross-links obtained by a cross-linking reaction using the chemically modified alginate derivatives represented by formulas (I) and (II). The cationic polymer layer is a cationic polymer.

[0418] 1 shows a cross-sectional view of an example of a polymer-coated cross-linked alginate gel fiber formed by coating a cross-linked alginate gel fiber with a cationic polymer. This polymer-coated cross-linked alginate gel fiber has an outer diameter c and comprises a core layer 5 with a diameter a and a cationic polymer layer 4 with a thickness b. The core layer 5 contains a cross-linked alginate gel containing cells 6 that produce antibodies, physiologically active substances, etc. The cross-linked alginate gel of the core layer 5 is a cross-linked alginate gel formed by a cross-linking reaction using chemically modified alginate derivatives represented by formulas (I) and (II).

[0419] The core layer of the polymer-coated cross-linked alginate gel fiber in some embodiments may contain, in addition to the cross-linked alginate gel formed by a cross-linking reaction using the chemically modified alginate derivatives represented by formula (I) and formula (II) described in embodiment [1], other components such as collagen solution, collagen gel, culture medium, cell culture medium, culture fluid, methylcellulose, sucrose solution, alginate solution, alginate gel, etc., as long as they are not cytotoxic; preferably, they may contain a component selected from the group consisting of alginate solution, alginate gel, culture medium, and culture fluid.

[0420] The "polymer-coated cross-linked alginate gel fiber" is a fibrous structure having an outer diameter (c in Figure 2) of, for example, about 0.1 to about 2000 μm, and is therefore sometimes called a "polymer-coated cross-linked alginate microfiber."

[0421] The cross-sectional shape perpendicular to the central axis of the polymer-coated cross-linked alginate gel fiber is not limited to circular, but may also be an asymmetric structure or a deformed shape. For example, the cross-sectional shape may be a variety of shapes such as circular, elliptical, or polygonal (e.g., triangular, rectangular, pentagonal, etc.), and preferably is a circular cross-sectional shape as shown in Figure 2.

[0422] The outer diameter of the polymer-coated cross-linked alginate gel fiber (in the case of a non-circular fiber, the major axis or maximum diameter is considered to be the outer diameter) is, for example, in the range of about 0.1 to about 2000 μm, about 0.2 μm to about 2000 μm, about 1 to about 1000 μm, about 2 to about 500 μm, or about 2 to about 200 μm.

[0423] The diameter of the core layer of the polymer-coated cross-linked alginate gel fiber ranges, for example, from about 0.1 to about 2000 μm, from about 0.2 μm to about 2000 μm, from about 1 to about 1000 μm, from about 2 to about 500 μm, from about 2 to about 200 μm, etc. The cross-sectional diameter of the core layer is preferably at least 50% less than the cross-sectional diameter of the fiber.

[0424] The thickness of the polymer layer (b) of the polymer-coated cross-linked alginate gel fiber can be calculated by (outer diameter of the polymer-coated cross-linked alginate gel fiber - diameter of the core layer) / 2 (b = (c - a) / 2 in Figure 2). The thickness of the polymer layer is, for example, about 0.1 to about 200 µm, about 1 to about 200 µm, or about 5 µm to about 200 µm.

[0425] The diameter and outer diameter of the core layer and the inner diameter of the polymer layer of the polymer-coated cross-linked alginate gel fiber can be measured, for example, by preparing a fiber using a cationic polymer that emits fluorescence in the polymer layer and measuring the diameter and outer diameter of the core layer and the inner diameter of the polymer layer using a phase-contrast optical microscope. The diameter and outer diameter of the core layer and the inner diameter of the polymer layer can be measured, for example, by preparing a fiber using a cationic polymer that emits fluorescence in the polymer layer and measuring the diameter and inner diameter of the polymer-coated cross-linked alginate gel fiber. The diameter and inner diameter of the polymer-coated cross-linked alginate gel fiber are expressed as the average values ​​measured at several points on the polymer-coated cross-linked alginate gel fiber. The core layer and polymer layer of the polymer-coated cross-linked alginate gel fiber typically have a substantially uniform thickness, and preferably, each layer has a thickness uniformity within a range of ±10%.

[0426] The length of the polymer-coated cross-linked alginate gel fiber is not particularly limited, but may be, for example, about 0.01 to about 100 m, or about 0.1 to about 75 m, or about 0.3 to about 50 m.

[0427] In some embodiments of the present invention, the core layer of the polymer-coated cross-linked alginate gel fiber can be formed using a mixed solution of chemically modified alginic acid derivatives represented by formula (I) and formula (II) containing cells capable of producing antibodies, physiologically active substances, etc. In this case, the concentration of the solution of the chemically modified alginic acid derivative represented by formula (I) or formula (II) is, for example, in the range of about 0.01 to about 1.5 wt %, preferably about 0.05 to about 1.0 wt %, and more preferably about 0.08 to about 0.75 wt %. The concentration of the mixed solution of the chemically modified alginic acid derivatives represented by formula (I) and formula (II) is, for example, in the range of about 0.02 to about 2.0 wt %, preferably about 0.1 to about 2.0 wt %, and more preferably about 0.15 to about 1.5 wt %. Furthermore, when an alginic acid solution is added to the mixed solution of the chemically modified alginic acid derivatives represented by formula (I) and formula (II), the concentration of the alginic acid is, for example, in the range of 0 to about 1.98 wt %; preferably, in the range of 0 to about 1.8 wt %; and more preferably, in the range of 0 to about 1.7 wt %.

[0428] In the present specification, when an alginic acid solution is added to a mixed solution of chemically modified alginic acid derivatives represented by formula (I) and formula (II) containing cells capable of producing antibodies, physiologically active substances, etc., used to form the core layer of a polymer-coated cross-linked alginate gel fiber, the combination of the concentration (C1 (wt %)) of the mixed solution containing the chemically modified alginic acid derivatives represented by formula (I) and formula (II) and the concentration (C2 (wt %)) of the alginic acid solution is not particularly limited, and examples include combinations of (C1:C2) = (about 0.2:about 1.3), (about 0.5:about 1.0), (about 1.0:about 0.5), (about 1.5:0), (about 0.66:about 1.34), (about 0.34:about 0.66), (about 0.16:about 0.34), etc. In addition to these concentrations, the concentrations of C1 and C2 can also be appropriately combined to prepare the fiber.

[0429] In the present specification, when an alginic acid solution is added to a mixed solution of chemically modified alginic acid derivatives represented by formula (I) and formula (II) containing cells capable of producing antibodies, physiologically active substances, etc., which is used to form a core layer of a polymer-coated cross-linked alginic acid gel fiber, the concentration of the solution of the chemically modified alginic acid derivative represented by formula (I) (C1A (wt %)) and the concentration of the solution of the chemically modified alginic acid derivative represented by formula (II) (C1N (wt %)) The combination of the concentrations of the alginic acid solution (C2 (wt%)) is not particularly limited, but examples include (C1A:C1N:C2) = (about 0.1:about 0.1:about 1.3), (about 0.25:about 0.25:about 1.0), (about 0.5:about 0.5:about 0.5), (about 0.75:about 0.75:0), (about 0.33:about 0.33:about 1.34), (about 0.17:about 0.17:about 0.66), (about 0.08:about 0.08:about 0.34), etc. In addition to these concentrations, the concentrations of C1A, C1N, and C2 can be appropriately combined to prepare the solution.

[0430] In this specification, in a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc., and chemically modified alginic acid derivatives represented by formula (I) and formula (II), which is used to form the core layer of a polymer-coated cross-linked alginic acid gel fiber, the volume ratio (v1, v2) of the solution of the chemically modified alginic acid derivative represented by formula (I) to the solution of the chemically modified alginic acid derivative represented by formula (II) is, for example, a ratio of v1 + v2 = 15, e.g., (v1:v2) = (7.5:7.5).

[0431] In this specification, when an alginate solution is added to a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc., and chemically modified alginate derivatives represented by formula (I) and formula (II) used to form the core layer of a polymer-coated cross-linked alginate gel fiber, the volume ratio of the volume (v1) of the chemically modified alginate derivative represented by formula (I), the volume (v2) of the solution of the chemically modified alginate derivative represented by formula (II), and the volume (v3) of the alginate solution in the mixed solution to which the alginate solution has been added is, for example, v1 + v2 + v3 = 15, and combinations such as (v1:v2:v3) = (5:5:5), (2.5:2.5:10), and (1:1:13) are possible.

[0432] In the present specification, when an alginic acid solution is added to a mixed solution of chemically modified alginic acid derivatives represented by formula (I) and formula (II) containing cells capable of producing antibodies, physiologically active substances, etc., used to form the core layer of a polymer-coated cross-linked alginate gel fiber, the molecular weight of the alginic acid (e.g., sodium alginate, etc.) used to prepare the alginic acid solution is not particularly limited, and the weight-average molecular weight measured by gel permeation chromatography (GPC) is, for example, in the range of about 150,000 Da to about 2,500,000 Da, about 300,000 Da to about 2,000,000 Da, about 700,000 Da to about 2,000,000 Da, etc.

[0433] In the present specification, when an alginic acid solution is added to a mixed solution of chemically modified alginic acid derivatives represented by formula (I) and formula (II) containing cells capable of producing antibodies, physiologically active substances, etc., which is used to form the core layer of a polymer-coated cross-linked alginic acid gel fiber, the molecular weight of alginic acid (e.g., sodium alginate, etc.) used to prepare the alginic acid solution is not particularly limited, but may be determined by gel filtration chromatography. The weight average molecular weight measured by the Fee method (GPC method) is, for example, in the range of about 150,000 Da to about 2,500,000 Da, about 300,000 Da to about 2,500,000 Da, about 700,000 Da to about 1,400,000 Da, about 800,000 Da to about 1,500,000 Da, about 1,400,000 Da to about 2,000,000 Da, or about 1,500,000 to about 2,500,000 Da.

[0434] The solvent used to prepare the solution of the chemically modified alginic acid derivative represented by formula (I) or formula (II), the alginic acid solution, etc., used in producing the core layer of the polymer-coated cross-linked alginate gel fiber is not particularly limited, but examples thereof include culture medium, cell culture medium, culture solution, isotonic buffer solution, phosphate-buffered saline (PBS), and saline; culture medium, cell culture medium, culture solution, saline, or isotonic buffer solution is preferred.

[0435] 7. Cationic Polymer A polycation refers to a compound having two or more cationic groups in one molecule, and a cationic group refers to a cationic group or a group that can be derived to a cationic group. Examples of cationic groups include amino groups; monoalkylamino groups such as methylamino groups and ethylamino groups; dialkylamino groups such as dimethylamino groups and diethylamino groups; imino groups; and guanidino groups. An amino group is a -NH ３ ＋ It may also be a group.

[0436] In this specification, the term "cationic polymer" refers to a polymer having two or more cationic groups in one molecule. Examples of cationic polymers include those obtained by polymerizing a monomer having a cationic group. Furthermore, the cationic polymer is preferably one that is hydrophilic enough to be soluble in water and has the property of becoming positively charged when the cationic group dissociates in water. As the cationic polymer, a polymer having two or more amino groups in one molecule is particularly preferred.

[0437] In this specification, the cationic polymer is preferably a substance that can increase the strength of a crosslinked alginate gel fiber by coating the surface of the crosslinked alginate gel fiber with the cationic polymer (see Figure 2) due to electrostatic interaction between the carboxyl groups of the crosslinked alginate gel fiber and the cationic groups of the cationic polymer on the surface of the crosslinked alginate gel fiber formed by crosslinking using chemically modified alginate derivatives represented by formulas (I) and (II) containing cells capable of producing antibodies, physiologically active substances, etc. Furthermore, the cationic polymer is preferably a substance that allows antibodies, physiologically active substances, etc. produced by cells producing antibodies, physiologically active substances, etc. contained in the core layer to permeate the cationic polymer (polymer layer) coating the core layer and be released to the outside of the polymer-coated crosslinked alginate gel fiber.

[0438] In the present specification, examples of the cationic polymer include cationic polymers of polyamino acids (polymers of basic amino acids), basic polysaccharides (e.g., chitosan, etc.), and basic polymers (e.g., polymethylene-co-guanidine (PMCG), polyallylamine (PAA), polyvinylamine (PVA), polyethyleneimine, allylamine-diallylamine copolymer, and allylamine-maleic acid copolymer), and preferably, polyamino acids such as poly-L-ornithine (PLO), poly-D-ornithine (PDO), poly-DL-ornithine, and poly-D-lysine (PD The cationic polymer is a cationic polymer selected from the group consisting of poly-L-lysine (PLL), poly-DL-lysine, poly-L-arginine (PLA), poly-D-arginine (PDA), poly-DL-arginine, poly-L-homoarginine (PLHA), poly-D-homoarginine (PDHA), poly-DL-homoarginine, poly-L-histidine (PLH), poly-D-histidine (PDH), and poly-DL-histidine; more preferably, it is poly-L-ornithine, poly-L-lysine, or polyallylamine; and even more preferably, it is poly-L-ornithine.

[0439] In this specification, examples of the cationic polymer used in preparing a solution containing a cationic polymer include the polyamino acids, basic polysaccharides, basic polymers, and salts thereof (hydrochlorides, hydrobromides, etc.). The cationic polymers that can be used are commercially available products or those prepared from commercially available products.

[0440] In this specification, the degree of polymerization of the cationic polymer is not particularly limited, and examples thereof include a degree of polymerization of 50 to 6,000, a degree of polymerization of 50 to 2,000, and a degree of polymerization of 100 to 1,500. In the case of poly-L-ornithine, the degree of polymerization is, for example, 130 to 1,300, in the case of polyallylamine, the degree of polymerization is, for example, 50 to 1,800, and in the case of chitosan, the degree of polymerization is, for example, 60 to 6,000.

[0441] In this specification, the weight average molecular weight (Mw) of the cationic polymer is not particularly limited, and examples thereof include a range of 500 to 1,000,000, a range of 1,000 to 500,000, a range of 3,000 to 300,000, a range of 5,000 to 100,000, a range of 10,000 to 50,000, etc. The weight average molecular weight (Mw) of the cationic polymer can be measured by gel permeation chromatography (GPC).

[0442] For example, in the case of poly-L-ornithine, commercially available poly-L-ornithine hydrobromide [e.g., molecular weight: 70,000 to 150,000 (manufactured by Fujifilm-Wako Pure Chemical Industries), molecular weight: 15,000 to 30,000, 30,000 to 70,000, 5,000 to 15,000 (manufactured by Sigma-Aldrich)] can be used; for example, in the case of polyallylamine, commercially available polyallylamine [e.g., molecular weight: 1,600, 3,000, 5,000, 8,000, 15,000, 25,000 (manufactured by Nitto Boseki), 15,000, 65,000, 75,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 200,000, 250,000, 300,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 150,000, 160,000, 170,000, 200,000, 250,000, 300,000, 300,000, 400,000, 500,000, 500,000 00 (manufactured by Sigma-Aldrich), etc.], commercially available polyallylamine hydrochloride [for example, molecular weight: 1,600, 3,000, 5,000, 15,000, 100,000 (manufactured by Nitto Boseki), up to 17,500, 50,000 (manufactured by Sigma-Aldrich), etc.] can be used; for example, in the case of chitosan, commercially available chitosan [for example, molecular weight: up to 15,000 (manufactured by Fujifilm-Wako Pure Chemical Industries), 5,000, 50,000, 100,000, 160,000, 180,000 (manufactured by Sigma-Aldrich), etc.] can be used.

[0443] Chitosan, which is one of the cationic polymers, is a deacetylated product of chitin, and from the viewpoint of its water solubility, chitosan having a degree of deacetylation within the range of, for example, 40 to 100%, 45 to 90%, or 50 to 80% can be used.

[0444] The concentration of the solution containing the cationic polymer is not particularly limited, but may be any concentration that allows the surface of the alginate gel fiber to be uniformly coated. For example, concentrations of about 0.01 to about 10.0% by weight, about 0.01 to about 5.0% by weight, or about 0.02 to about 1.0% by weight are included, and a concentration of about 0.02 to about 5.0% by weight is preferred, and a concentration of about 0.05 to about 1.0% by weight is more preferred.

[0445] The viscosity of the solution containing the cationic polymer is not particularly limited, but is, for example, in the range of 10.0 to 500.0 mPa·s, in the range of 20.0 to 300.0 mPa·s, or in the range of 50.0 to 200.0 mPa·s.

[0446] In the solution containing the cationic polymer, two or more kinds of cationic polymers can be used in combination.

[0447] The solvent for the solution containing the cationic polymer is not particularly limited as long as it can dissolve the cationic polymer. Examples of the solvent include water (tap water, pure water (e.g., distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water (MilliQ water)), aqueous solutions of inorganic salts (phosphate buffered saline (PBS), saline, etc.), and the like. Ultrapure water, water, or saline is preferred, as it can increase the amount of charge on the cationic polymer.

[0448] 8. Multilayer Polymer-Coated Cross-Linked Alginate Gel Fiber In this specification, the term "multilayer polymer-coated cross-linked alginate gel fiber" refers to a fibrous (fibrous) structure obtained by coating the outside of the cationic polymer layer of the polymer-coated cross-linked alginate gel fiber described above in "6. Polymer-Coated Cross-Linked Alginate Gel Fiber" with an anionic polymer (anionic polymer layer). That is, the fibrous structure is obtained by coating the outside of the cationic polymer layer of the polymer-coated cross-linked alginate gel fiber obtained by coating, with a cationic polymer, a core layer containing cells capable of producing antibodies, physiologically active substances, etc., and a cross-linked alginate gel obtained by cross-linking using the chemically modified alginate derivatives represented by formulas (I) and (II) (the method for producing the multilayer polymer-coated cross-linked alginate gel fiber will be described later).

[0449] The multilayer polymer-coated cross-linked alginate gel fiber is a fibrous structure comprising a core layer, a cationic polymer layer disposed on the outer surface of the core layer, and an anionic polymer layer disposed on the outer surface of the cationic polymer layer. The core layer comprises cells capable of producing antibodies, physiologically active substances, etc., and a cross-linked alginate gel formed by cross-linking using the chemically modified alginate derivatives represented by formulas (I) and (II). The cationic polymer layer is a cationic polymer, and the anionic polymer layer is an anionic polymer.

[0450] The multilayer polymer-coated cross-linked alginate gel fiber is a fibrous (fibrous) structure comprising a core layer, a cationic polymer layer disposed on the outer surface of the core layer, and an anionic polymer layer disposed on the outer surface of the cationic polymer layer. The core layer comprises cells capable of producing antibodies, physiologically active substances, etc., and a cross-linked alginate gel. The cross-linked alginate gel comprises cross-links obtained by a cross-linking reaction using the chemically modified alginate derivatives represented by formulas (I) and (II). The cationic polymer layer is a cationic polymer, and the anionic polymer layer is an anionic polymer.

[0451] 8 shows a cross-sectional view of an example of a multilayer polymer-coated cross-linked alginate gel fiber. This multilayer polymer-coated cross-linked alginate gel fiber has an outer diameter e and comprises a core layer 5 with a diameter a, a cationic polymer layer 4 with a thickness b, and an anionic polymer layer 7 with a thickness d. The core layer 5 contains a cross-linked alginate gel containing cells 6 that produce antibodies, physiologically active substances, etc. The cross-linked alginate gel of the core layer 5 is a cross-linked alginate gel formed by a cross-linking reaction using chemically modified alginate derivatives represented by formulas (I) and (II).

[0452] The core layer of the multilayer polymer-coated cross-linked alginate gel fiber in some embodiments contains, in addition to the cross-linked alginate gel formed by a cross-linking reaction using the chemically modified alginate derivatives represented by formula (I) and formula (II) described in embodiment [1], other components such as collagen solution, collagen gel, culture medium, cell culture medium, culture fluid, methylcellulose, sucrose solution, alginate solution, alginate gel, etc., as long as they are not cytotoxic; preferably, they contain a component selected from the group consisting of alginate solution, alginate gel, culture medium, and culture fluid.

[0453] The multi-layer polymer-coated cross-linked alginate gel fiber is a fibrous structure having an outer diameter (e in Figure 8) of, for example, about 0.1 to about 2000 μm, and is therefore sometimes called a multi-layer polymer-coated cross-linked alginate microfiber.

[0454] The cross-sectional shape perpendicular to the central axis of the multilayer polymer-coated cross-linked alginate gel fiber is not limited to circular, but may also be an asymmetric structure or a deformed shape.For example, the cross-sectional shape may be a variety of shapes such as circular, elliptical, or polygonal (e.g., triangular, rectangular, pentagonal, etc.), and preferably is a circular cross-sectional shape as shown in Figure 8.

[0455] The outer diameter of the multilayer polymer-coated cross-linked alginate gel fiber (in the case of a non-circular fiber, the major axis or maximum axis is considered to be the outer diameter) is, for example, in the range of about 0.1 to about 2000 μm, about 0.2 μm to about 2000 μm, about 1 to about 1000 μm, about 2 to about 500 μm, or about 2 to about 200 μm.

[0456] The diameter of the core layer of the multi-polymer-coated cross-linked alginate gel fiber ranges, for example, from about 0.1 to about 2000 μm, from about 0.2 μm to about 2000 μm, from about 1 to about 1000 μm, from about 2 to about 500 μm, from about 2 to about 200 μm, etc. The diameter of the core layer is preferably at least 50% less than the diameter of the fiber.

[0457] The thickness of the cationic polymer layer (b) of the multilayer polymer-coated cross-linked alginate gel fiber can be determined by the method described above in "6. Polymer-coated cross-linked alginate gel fiber." The thickness of the anionic polymer layer (e) of the multilayer polymer-coated cross-linked alginate gel fiber can be determined by the formula "(outer diameter of the multilayer polymer-coated cross-linked alginate gel fiber - outer diameter of the polymer-coated cross-linked alginate gel fiber) / 2 (e = (d - a) / 2 in Figure 8)." The thickness of the anionic polymer layer is, for example, about 0.1 to about 200 μm, about 1 to about 200 μm, or about 5 μm to about 200 μm.

[0458] The diameter and outer diameter of the core layer, the inner diameter of the cationic polymer layer, and the inner diameter of the anionic polymer layer of the above-mentioned multi-polymer-coated cross-linked alginate gel fiber can be measured, for example, by preparing a fiber using a fluorescent polymer in each polymer layer and measuring it from images taken with a phase-contrast optical microscope. The values ​​are expressed as the average of measurements taken at several locations on the multi-polymer-coated cross-linked alginate gel fiber. The core layer and polymer layers of the above-mentioned multi-polymer-coated cross-linked alginate gel fiber usually have a substantially uniform thickness, and preferably, each layer has a thickness uniformity within a range of ±10%.

[0459] The length of the multi-layered polymer-coated cross-linked alginate gel fiber is not particularly limited, but may be, for example, about 0.01 to about 100 m, or about 0.1 to about 75 m, or about 0.3 to about 50 m.

[0460] In the present specification, the core layer of some embodiments of the multilayer polymer-coated cross-linked alginate gel fiber can be formed using a mixed solution of chemically modified alginate derivatives represented by formula (I) and formula (II) containing cells capable of producing antibodies, physiologically active substances, etc. In this case, the concentration range of the solution of the chemically modified alginate derivative represented by formula (I) or formula (II) is the same as that described in the above-mentioned 6. Polymer-coated cross-linked alginate gel fiber.

[0461] In this specification, when an alginate solution is added to a mixed solution of chemically modified alginate derivatives represented by formula (I) and formula (II) containing cells capable of producing antibodies, physiologically active substances, etc., used to form the core layer of a multilayer polymer-coated cross-linked alginate gel fiber, the concentrations of the mixed solution containing the chemically modified alginate derivatives represented by formula (I) and formula (II) and the alginate solution, the combination of concentrations, and the volume ratio of each solution are the same as those described above in "6. Polymer-coated cross-linked alginate gel fiber."

[0462] The solvents used to prepare the solution of the chemically modified alginic acid derivative represented by formula (I) or formula (II), the alginic acid solution, etc. used to produce the core layer of the multilayer polymer-coated cross-linked alginic acid gel fiber are the same as those described in the above section "6. Polymer-coated cross-linked alginic acid gel fiber."

[0463] 9. Anionic Polymers A polyanion is a compound having two or more anionic groups per molecule, and the anionic group is an anionic group or a group that can be derived to an anionic group. Examples of anionic groups include acidic functional groups such as carboxyl groups and sulfate groups.

[0464] In this specification, the anionic polymer layer refers to a layer that coats the outside of the cationic polymer layer through electrostatic interaction with the cationic polymer. The anionic polymer that forms the anionic polymer layer is not particularly limited as long as it can form a membrane through electrostatic interaction with the cationic polymer, and examples thereof include polymers having acidic functional groups such as carboxyl groups or sulfate groups. Specific examples include polysaccharides such as alginic acid, polygalacturonic acid, pectin, pectic acid, carboxymethylcellulose, carrageenan, hyaluronic acid, and chondroitin sulfate, synthetic polymers such as polyacrylic acid and polystyrene sulfonic acid, and chemically modified versions thereof, including chemically crosslinked versions thereof. Furthermore, the anionic polymer may be a polymer that exhibits neutrality overall, as long as it has the property of being able to form a membrane through electrostatic interaction with the cationic polymer.

[0465] In this specification, the anionic polymer is preferably a substance that can coat the cationic polymer layer of the polymer-coated cross-linked alginate gel fiber as an anionic polymer layer, thereby forming a multi-layer polymer-coated cross-linked alginate gel fiber (see Figure 9).Furthermore, the anionic polymer is preferably a substance that can allow antibodies, physiologically active substances, etc. produced in the core layer of the multi-layer polymer-coated cross-linked alginate gel fiber to permeate the cationic polymer layer, and then permeate the anionic polymer layer coating the cationic polymer layer and be released to the outside of the fiber.

[0466] In this specification, the anionic polymer layer refers to an anionic polymer selected from the group consisting of, for example, anionic polysaccharides, sulfated polysaccharides, synthetic polymers, anionic polyamino acids, chemically modified products thereof, crosslinked products thereof, mixtures thereof, etc., and specifically, anionic polymers selected from the group consisting of, for example, anionic polysaccharides such as alginic acid, hyaluronic acid, polygalacturonic acid, carrageenan, succinoglucan, gum arabic, xanthan gum, pectin, pectic acid, carboxymethylcellulose, agar, etc., chemically modified products thereof, crosslinked products thereof, mixtures thereof, etc.

[0467] In the present specification, the anionic polymer layer is, for example, an anionic polymer selected from the group consisting of alginic acid, the chemically modified alginic acid derivative represented by the above formula (I), the chemically modified alginic acid derivative represented by formula (II), a crosslinked product formed from the chemically modified alginic acid derivative represented by formula (I) and / or the chemically modified alginic acid derivative represented by formula (II), and a mixture thereof; for example, a sulfated polysaccharide such as chondroitin sulfate, dextran sulfate, heparan sulfate, dermatan sulfate, fucoidan, keratan sulfate, or heparin. , chemically modified products thereof, crosslinked products thereof, mixtures thereof, etc.; for example, anionic polymers selected from the group consisting of synthetic polymers such as acrylic acid, methacrylic acid, ethylacrylic acid, polystyrene sulfonic acid, etc., chemically modified products thereof, crosslinked products thereof, mixtures thereof, etc.; for example, anionic polymers selected from the group consisting of anionic polyamino acids such as polyglutamic acid, polyaspartic acid, etc., chemically modified products thereof, crosslinked products thereof, mixtures thereof, etc.

[0468] In this specification, the anionic polymer layer is preferably an anionic polymer selected from the group consisting of alginic acid, a chemically modified alginic acid derivative represented by the aforementioned formula (I), a chemically modified alginic acid derivative represented by formula (II), a crosslinked product formed from the chemically modified alginic acid derivative represented by formula (I) and / or the chemically modified alginic acid derivative represented by formula (II), and a mixture thereof.

[0469] The solvent used to prepare a solution containing an anionic polymer is not particularly limited as long as it can dissolve the anionic polymer, and examples thereof include water (tap water, pure water (e.g., distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water (MilliQ water)), aqueous solutions of inorganic salts (phosphate buffered saline (PBS), saline, etc.), and preferably water or saline. However, when alginic acid or a chemically modified alginic acid derivative represented by formula (I) or formula (II) is used as the anionic polymer, solutions containing polyvalent (divalent, trivalent) metal ions that cause gelation are excluded.

[0470] In this specification, the weight average molecular weight (Mw) of the anionic polymer is not particularly limited, but is, for example, in the range of about 500 to about 5,000,000, about 300,000 to about 2,000,000, about 150,000 to about 2,500,000, or about 100,000 to about 3,000,000. The weight average molecular weight (Mw) of the anionic polymer can be measured by gel permeation chromatography (GPC).

[0471] When alginic acid is used as the anionic polymer herein, the weight average molecular weight (Mw) thereof is, for example, in the range of about 150,000 Da to about 2,500,000 Da, about 150,000 Da to about 800,000 Da, about 300,000 Da to about 700,000 Da, about 700,000 Da to about 1,400,000 Da, about 800,000 Da to about 1,500,000 Da, about 1,400,000 Da to about 2,000,000 Da, or about 1,500,000 to about 2,500,000 Da.

[0472] When the chemically modified alginic acid derivative represented by formula (I) or (II) is used as the anionic polymer herein, the weight average molecular weight (Mw) thereof is, for example, in the range of about 100,000 to about 3,000,000, in the range of about 300,000 Da to about 2,500,000, in the range of about 500,000 to about 2,000,000, etc.

[0473] The concentration of the solution containing the anionic polymer is not particularly limited, but may be any concentration that allows the surface of the cationic polymer layer of the polymer-coated cross-linked alginate gel fiber to be uniformly coated, and may be, for example, about 0.01 to about 5.0% by weight, about 0.05 to about 1.0% by weight, about 0.1 to about 0.5% by weight, or about 0.15 to about 0.4% by weight, with 0.15% by weight being preferred.

[0474] In the solution containing an anionic polymer, two or more kinds of anionic polymers can be used in combination.

[0475] When a solution containing the chemically modified alginic acid derivative represented by formula (I) is used to form the anionic polymer layer of the multilayer polymer-coated cross-linked alginic acid gel fiber herein, the concentration of the solution is, for example, in the range of about 0.01 to about 5.0 wt %, about 0.05 to about 1.0 wt %, about 0.1 to 0.5 wt %, about 0.15 to about 0.4 wt %, or about 0.05 to about 0.15 wt %, about 0.075 to about 0.2 wt %, or about 0.15 to about 0.4 wt %.

[0476] When a solution containing the chemically modified alginic acid derivative represented by formula (II) is used to form the anionic polymer layer of the multilayer polymer-coated cross-linked alginic acid gel fiber herein, the concentration of the solution is, for example, in the range of about 0.01 to about 5.0 wt %, about 0.05 to about 1.0 wt %, about 0.1 to about 0.5 wt %, about 0.15 to about 0.4 wt %, about 0.05 to about 0.15 wt %, about 0.075 to about 0.2 wt %, or about 0.15 to about 0.4 wt %.

[0477] When an alginic acid solution is used to form the anionic polymer layer of the multilayer polymer-coated cross-linked alginic acid gel fiber herein, the concentration of the solution is, for example, in the range of about 0.01 to about 5.0% by weight, about 0.05 to about 1.0% by weight, about 0.1 to about 0.5% by weight, about 0.15 to about 0.4% by weight, about 0.05 to about 0.15% by weight, about 0.075 to about 0.2% by weight, or about 0.15 to about 0.4% by weight.

[0478] In the present specification, when a mixed solution containing the chemically modified alginic acid derivatives represented by formula (I) and formula (II) is used to form the anionic polymer layer of the multilayer polymer-coated cross-linked alginate gel fiber, the concentration of the mixed solution is, for example, in the range of about 0.01 to about 5.0 wt %, about 0.05 to about 1.0 wt %, about 0.1 to about 0.5 wt %, or about 0.15 to about 0.4 wt %, preferably 0.15 wt %.

[0479] In the present specification, when a mixed solution containing alginic acid and the chemically modified alginic acid derivatives represented by formula (I) and formula (II) is used to form the anionic polymer layer of the multilayer polymer-coated cross-linked alginate gel fiber, the concentration of the mixed solution is, for example, in the range of about 0.01 to about 5.0 wt %, about 0.05 to about 1.0 wt %, about 0.1 to about 0.5 wt %, or about 0.15 to about 0.4 wt %, preferably 0.15 wt %.

[0480] In this specification, when a mixed solution containing chemically modified alginic acid derivatives represented by formula (I) and formula (II) is used to form the anionic polymer layer of a multilayer polymer-coated cross-linked alginic acid gel fiber, the volume ratio (v1, v2) of the solution of the chemically modified alginic acid derivative represented by formula (I) to the solution of the chemically modified alginic acid derivative represented by formula (II) in the mixed solution is, for example, a ratio of v1 + v2 = 15, e.g., (v1:v2) = (7.5:7.5).

[0481] In this specification, when a mixed solution containing alginic acid and chemically modified alginic acid derivatives represented by formula (I) and formula (II) is used to form the anionic polymer layer of a multilayer polymer-coated cross-linked alginic acid gel fiber, the volume ratio (v1, v2, v3) of the solution of the chemically modified alginic acid derivative represented by formula (I), the solution of the chemically modified alginic acid derivative represented by formula (II), and the alginic acid solution in the mixed solution is, for example, v1 + v2 + 3v3 = 15, and examples of combinations include (v1:v2:v3) = (5:5:5), (2.5:2.5:10), and (1:1:13).

[0482] In the present specification, when the chemically modified alginic acid derivatives represented by formula (I) and formula (II) are used to form the anionic polymer layer of the multilayer polymer-coated cross-linked alginic acid gel fiber, the chemically modified alginic acid derivatives represented by formula (I) and formula (II) may form chemical cross-links in the anionic polymer layer after coating. The chemical cross-links are represented by the following formula (III-LA): [In formula (III-LA), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; １Ａ -, -L ２Ａ - and -X Ａ - represents the corresponding -L in the above embodiment [1-12]. １ -, -L ２ - and -X- are as defined above.

[0483] 10. Cells contained in the core layer In this specification, the cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber are not particularly limited, and examples thereof include antibody-producing cells (various monoclonal antibodies such as human antibodies, humanized antibodies, chimeric antibodies, and mouse antibodies, or various modified antibodies such as their bispecific antibodies, minibodies, and glycosylated antibodies), physiologically active substance-producing cells (enzymes, cytokines, hormones, blood coagulation factors, vaccines, etc.), and cells capable of producing various useful substances useful as pharmaceutical raw materials, chemical raw materials, food raw materials, etc. Antibody-producing cells or physiologically active substance-producing cells are preferred.

[0484] In this specification, examples of antibody-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber include hybridomas obtained from antibody-producing B cells (antibody-producing hybridomas), or cultured cells transformed with an antibody expression vector (antibody-producing genetically modified cells).

[0485] In this specification, examples of physiologically active substance-producing cells that can be encapsulated in the core layer of polymer-coated cross-linked alginate gel fibers and multilayer polymer-coated cross-linked alginate gel fibers include cultured cells transformed with a physiologically active substance expression vector (biologically active substance-producing genetically modified cells).

[0486] The cultured cells used as hosts for these genetic recombinations are not particularly limited, but examples include microorganisms such as bacteria or yeast, plant cells, insect cells, and animal cells.

[0487] Examples of microorganisms used as hosts include Escherichia coli, budding yeast, fission yeast, and Pichia yeast, and examples of insect cells used as hosts include Sf9 cells, Sf21 cells, and High five cells.

[0488] Examples of animal cells used as hosts include CHO cells, CHO cell sublines (CHO-K1 cells, CHO-DG44 cells, CHO-DXB11 cells, or CHO cells transformed to modify their sugar chains, etc.), COS cells, Sp2 / 0 cells, NS0 cells, SP2 cells, PERC6 cells, YB2 / 0 cells, YE2 / 0 cells, 1R983F cells, Namalwa cells, Wil-2 cells, Jurkat cells, Vero cells, Molt-4 cells, HEK293 cells, BHK cells, HT-1080 cells, KGH6 cells, P3X63Ag8.653 cells, C127 cells, JC cells, LA7 cells, ZR-45-30 cells, hTERT cells, NM2C5 cells, and UACC-812 cells (these cells are commercially available from American (Some of these are listed in the ATCC cell line catalog available from the American Type Culture Collection.) In this specification, unless otherwise specified, the term "CHO ​​cells" refers to cells including "CHO ​​cell sublines," and also refers to other cells including their respective cell sublines.

[0489] In this specification, antibody-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber are preferably animal cells transformed with an antibody expression vector, i.e., genetically modified animal cells that produce antibodies. Also, physiologically active substance-producing cells that can be encapsulated in the core layer are preferably animal cells transformed with a physiologically active substance expression vector, i.e., genetically modified animal cells that produce physiologically active substances.

[0490] Specific examples of animal cells used as hosts for these include CHO cells, CHO cell sublines, COS cells, Sp2 / 0 cells, NS0 cells, SP2 cells, PERC6 cells, HEK293 cells, BHK cells, HT-1080 cells, and C127 cells; more preferably, cells selected from the group consisting of CHO cells, CHO cell sublines, Sp2 / 0 cells, and NS0 cells, HEK293 cells, and BHK cells; even more preferably, CHO cells or CHO cell sublines. Furthermore, in one aspect, host cells for antibody-producing cells are preferably CHO cells, CHO cell sublines, Sp2 / 0 cells, or NS0 cells; more preferably, CHO cells or CHO cell sublines. Furthermore, host cells for physiologically active substance-producing cells are preferably CHO cells, CHO cell sublines, HEK293 cells, or BHK cells; more preferably, CHO cells or CHO cell sublines.

[0491] In this specification, antibody-producing cells that can be contained in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber include, but are not limited to, cells that produce antibodies used as biopharmaceuticals or biopharmaceutical raw materials. Furthermore, physiologically active substance-producing cells include, but are not limited to, cells that produce physiologically active substances used as biopharmaceuticals or biopharmaceutical raw materials.

[0492] Examples of biopharmaceuticals include pharmaceuticals for a variety of diseases such as various cancers, autoimmune diseases / inflammatory diseases, eye diseases, blood diseases, neurological diseases, rare genetic diseases, endocrine and metabolic diseases, cardiovascular diseases, respiratory diseases, digestive diseases, skin diseases, muscle and bone diseases, and infectious diseases.

[0493] Among biopharmaceuticals, specific targets of antibody drugs are not particularly limited, but include C5 (complement), CD3, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD52, CD79, IL-1β, IL-4R, IL-5, IL-6, IL-6R, IL-12, IL-17, IL-17R, IL-23, IFNAR, PCSK9, CGRP, CGRPR, GD2 (ganglioside), HER2, HER3, TROP2, BCMA, PD-1, Examples of target proteins include PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, KIR, SLAMF7, RANKL, TNF-α, BLyS, EGFR, VEGF, VEGFR, FGF, nectin, integrin, EpCAM, CCR4, TfR, TF, FIXa, FX, GPVI, sclerostin, amyloid β, IgE, and various viruses (including their subtypes, subunits, and fragments), and cells that produce antibodies against these targets can be contained in the core layer.

[0494] In the present specification, antibody-producing cells that can be contained in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber are not particularly limited, but specific examples include muromonab-CD3, trastuzumab, rituximab, palivizumab, infliximab, basiliximab, tocilizumab, bevacizumab, adalimumab, cetuximab, omalizumab, eculizumab, panitumumab, ustekinumab, golimumab, and cannabinoids. Kinumab, denosumab, ofatumumab, pertuzumab, natalizumab, nivolumab, alemtuzumab, secukinumab, ramucirumab, ipilimumab, evolocumab, mepolizumab, alirocumab, ixekizumab, brodalumab, elotuzumab, pembrolizumab, sarilumab, bezlotoxumab, belimumab, daratumumab, avelumab, dupilumab, atezolizumab, emicizumab, guselkumab, durvalumab, vedolizumab, romosozumab, risankizumab, neci Tumumab, ravulizumab, burosumab, isatuximab, tildrakizumab, satralizumab, galcanezumab, dinutuximab, fremanezumab, erenumab, casirivimab, imdevimab, anifrolumab, sotroximab, ocrelizumab, naxitamab, aducanumab, tafasitamab, marjetuximab, gantenerumab, tiragolumab, clovalimab, nemolizumab, catumaxomab, pramotamab, faricimab, gemtuzumab, ibritumomab, brentuximab Examples of such antibodies include cells that produce antibodies such as tuzumab, inotuzumab, polatuzumab, enfortumab, sacituzumab, belantamab, loncustuximab, tisotumab, datopotamab, and patritumab; cells that produce antibodies with modified sugar chains such as mogamulizumab, benralizumab, obinutuzumab, and inebilizumab; and cells that produce low molecular weight antibodies consisting of antibody fragments such as ranibizumab, idarucizumab, blinatumomab, brolucizumab, abciximab, caplacizumab, and certolizumab.

[0495] In the present specification, antibody-producing cells that can be contained in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber are not particularly limited, but specific examples include antibody-producing animal cells, preferably antibody-producing CHO cells, antibody-producing Sp2 / 0 cells, or antibody-producing NS0 cells, more preferably antibody-producing CHO cells. More specifically, the antibody-producing animal cells are not particularly limited, but examples include muromonab-CD3-producing CHO cells, trastuzumab-producing CHO cells, rituximab-producing CHO cells, palivizumab-producing NS0 cells, palivizumab-producing CHO cells, infliximab-producing Sp2 / 0 cells, infliximab-producing CHO cells, basiliximab-producing Sp2 / 0 cells, basiliximab-producing CHO cells, tocilizumab-producing CHO cells, bevacizumab-producing CHO cells, Viable CHO cells, adalimumab-producing CHO cells, cetuximab-producing Sp2 / 0 cells, cetuximab-producing CHO cells, omalizumab-producing CHO cells, eculizumab-producing NS0 cells, eculizumab-producing CHO cells, panitumumab-producing CHO cells, ustekinumab-producing Sp2 / 0 cells, ustekinumab-producing CHO cells, golimumab-producing Sp2 / 0 cells, golimumab-producing CHO cells, canakinumab-producing Sp2 / 0 cells, canakinumab-producing CHO cells, denosumab CHO cells producing nivolumab, CHO cells producing alemtuzumab, CHO cells producing secukinumab, CHO cells producing ramucirumab, CHO cells producing ipilimumab, CHO cells producing evolocumab, CHO cells producing mepolizumab, CHO cells producing alirocumab O cells, ixekizumab-producing CHO cells, brodalumab-producing CHO cells, elotuzumab-producing NS0 cells, elotuzumab-producing CHO cells, pembrolizumab-producing CHO cells, sarilumab-producing CHO cells, bezlotoxumab-producing CHO cells, belimumab-producing NS0 cells, belimumab-producing CHO cells, daratumumab-producing CHO cells, avelumab-producing CHO cells, dupilumab-producing CHO cells, atezolizumab-producing CHO cells, emicizumab-producing CHO cells,Guselkumab-producing CHO cells, durvalumab-producing CHO cells, vedolizumab-producing CHO cells, romosozumab-producing CHO cells, risankizumab-producing CHO cells, necitumumab-producing NS0 cells, necitumumab-producing CHO cells, ravulizumab-producing CHO cells, burosumab-producing CHO cells, isatuximab-producing CHO cells, tildrakizumab-producing CHO cells, satralizumab-producing CHO cells, galcanezumab-producing CHO cells, di Nutuximab-producing Sp2 / 0 cells, dinutuximab-producing CHO cells, fremanezumab-producing CHO cells, erenumab-producing CHO cells, casirivimab-producing CHO cells, imdevimab-producing CHO cells, anifrolumab-producing NS0 cells, anifrolumab-producing CHO cells, sotrovimab-producing CHO cells, ocrelizumab-producing CHO cells, naxitamab-producing CHO cells, aducanumab-producing CHO cells, and tafasitamab-producing CHO cells , margetuximab-producing CHO cells, gemtuzumab-producing NS0 cells, gemtuzumab-producing CHO cells, ibritumomab-producing CHO cells, brentuximab-producing CHO cells, inotuzumab-producing CHO cells, polatuzumab-producing CHO cells, enfortumab-producing CHO cells, sacituzumab-producing Sp2 / 0 cells, sacituzumab-producing CHO cells, belantamab-producing CHO cells, loncustuximab-producing CHO cells, tisotumab-producing CHO cells HO cells, mogamulizumab-producing CHO cells, benralizumab-producing CHO cells, obinutuzumab-producing CHO cells, inebilizumab-producing CHO cells, ranibizumab-producing CHO cells, idarucizumab-producing CHO cells, blinatumomab-producing CHO cells, brolucizumab-producing CHO cells, abciximab-producing CHO cells, caplacizumab-producing CHO cells, certolizumab-producing CHO cells, anti-GPVI antibody-producing CHO cells, and the like;

[0496] For example, the antibody-producing CHO cells include muromonab-CD3-producing CHO cells, trastuzumab-producing CHO cells, rituximab-producing CHO cells, palivizumab-producing CHO cells, infliximab-producing CHO cells, basiliximab-producing CHO cells, tocilizumab-producing CHO cells, gemtuzumab-producing CHO cells, bevacizumab-producing CHO cells, ibritumomab-producing CHO cells, adalimumab-producing CHO cells, cetuximab-producing CHO cells, ranibizumab-producing CHO cells, omalizumab-producing CHO cells, eculizumab-producing CHO cells, and panitumumab-producing CHO cells. Cells, ustekinumab-producing CHO cells, golimumab-producing CHO cells, canakinumab-producing CHO cells, denosumab-producing CHO cells, mogamulizumab-producing CHO cells, certolizumab-producing CHO cells, ofatumumab-producing CHO cells, pertuzumab-producing CHO cells, brentuximab-producing CHO cells, natalizumab-producing CHO cells, nivolumab-producing CHO cells, alemtuzumab-producing CHO cells, secukinumab-producing CHO cells, ramucirumab-producing CHO cells, ipilimumab-producing CHO cells, evolocumab-producing CHO cells, mepolizumab-producing CHO cells, alilokuma CHO cells producing ribozyme, CHO cells producing ixekizumab, CHO cells producing brodalumab, CHO cells producing idarucizumab, CHO cells producing elotuzumab, CHO cells producing pembrolizumab, CHO cells producing sarilumab, CHO cells producing bezlotoxumab, CHO cells producing belimumab, CHO cells producing daratumumab, CHO cells producing avelumab, CHO cells producing dupilumab, CHO cells producing atezolizumab, CHO cells producing benralizumab, CHO cells producing inotuzumab, CHO cells producing emicizumab, CHO cells producing guselkumab, CHO cells producing durvalumab O cells, obinutuzumab-producing CHO cells, vedolizumab-producing CHO cells, romosozumab-producing CHO cells, risankizumab-producing CHO cells, necitumumab-producing CHO cells, ravulizumab-producing CHO cells, burosumab-producing CHO cells, isatuximab-producing CHO cells, tildrakizumab-producing CHO cells, satralizumab-producing CHO cells, galcanezumab-producing CHO cells, dinutuximab-producing CHO cells, fremanezumab-producing CHO cells, erenumab-producing CHO cells, casirivimab-producing CHO cells, imdevimab-producing CHO cells, anifrolumab-producing CHO cells,sotrovimab-producing CHO cells, ocrelizumab-producing CHO cells, naxitamab-producing CHO cells, aducanumab-producing CHO cells, tafasitamab-producing CHO cells, marjetuximab-producing CHO cells, polatuzumab-producing CHO cells, enfortumab-producing CHO cells, sacituzumab-producing CHO cells, belantamab-producing CHO cells, loncustuximab-producing CHO cells, tisotumab-producing CHO cells, inebilizumab-producing CHO cells, blinatumomab-producing CHO cells, brolucizumab-producing CHO cells, abciximab-producing CHO cells, caplacizumab-producing CHO cells, anti-GPVI antibody-producing CHO cells, and the like are included;

[0497] For example, the CHO cells are selected from the group consisting of trastuzumab-producing CHO cells, rituximab-producing CHO cells, infliximab-producing CHO cells, tocilizumab-producing CHO cells, adalimumab-producing CHO cells, nivolumab-producing CHO cells, and anti-GPVI antibody-producing CHO cells; for example, tocilizumab-producing CHO cells.

[0498] The antibodies produced in this manner can also be modified or altered after production, specifically by PEGylation, drug binding modification, radiolabeling, etc. That is, in the production of modified antibodies such as PEGylated antibodies and antibody-drug conjugates, cells used to produce the starting antibody (starting antibody-producing cells) can be exemplified as cells that can be encapsulated in the core layer. The starting antibody-producing cells are not particularly limited, and examples of starting antibody-producing cells for PEGylated antibodies include cells that produce the starting antibody fragment of certolizumab pegol, specifically certolizumab-producing CHO cells; and examples of starting antibody-producing cells for antibody-drug conjugates include gemtuzumab ozogamicin, ibritumomab tiuxetan, trastuzumab emtansine, trastuzumab deruxtecan, brentuximab vedotin, inotuzumab ozogamicin, cetuximab sarotarocan sodium, polatuzumab vedotin, enfortumab vedotin, sacituzumab govitecan, belantamab mafodotin, loncastuximab tesirin, tisotumab vedotin, datopotamab deruxtecan, and patritumab. Examples of such cells include cells that produce raw material antibodies such as deruxtecan, and specific examples include gemtuzumab-producing NS0 cells, ibritumomab-producing CHO cells, trastuzumab-producing CHO cells, brentuximab-producing CHO cells, inotuzumab-producing CHO cells, cetuximab-producing Sp2 / 0 cells, polatuzumab-producing CHO cells, enfortumab-producing CHO cells, sacituzumab-producing Sp2 / 0 cells, belantamab-producing CHO cells, loncastuximab-producing CHO cells, and tisotumab-producing CHO cells.

[0499] The core layer may also contain cells that produce a fusion protein of an antibody or antibody fragment with another protein or peptide, such as Pavinafusp alpha-producing CHO cells, Vintrafusp alpha-producing CHO cells, etc. "Antibodies" will be described in detail in "14. Classification of antibodies" and "15. Methods for producing and purifying antibodies and physiologically active substances."

[0500] In this specification, the term "biologically active substance" refers to a substance or a group of compounds that exert a physiological or pharmacological action on an organism. Examples of substances and groups of compounds that exert a physiological or pharmacological action on an organism include enzymes, insulin, alkaloids, cytokines (interferons, interleukins, chemokines, tumor necrosis factors, etc.), plant hormones, neurotransmitters, pheromones, hormones (animal hormones), growth factors, growth regulators, growth inhibitors, activators, hematopoietic factors, blood coagulation factors, vaccines (attenuated vaccines, inactivated vaccines, protein vaccines, etc.), etc.

[0501] Furthermore, receptors for these substances, cell surface antigens, cell surface receptors, and their ligands are also substances that exhibit physiological or pharmacological effects and are included in the category of physiologically active substances. Furthermore, in addition to physiologically active substances inherent in living organisms, substances obtained by modifying or altering physiologically active substances, substances that activate or inhibit physiological activity, and fusion proteins that combine multiple physiologically active substances or their partial regions or fragments are also included in the category of physiologically active substances, so long as they exhibit physiological or pharmacological effects, and these are all referred to as physiologically active substances in this specification. In this specification, the physiologically active substance is preferably a proteinaceous physiologically active substance, i.e., a physiologically active substance consisting of a protein or peptide.

[0502] In this specification, the physiologically active substance-producing cells that can be contained in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber are not particularly limited, but as mentioned above, include cells that produce physiologically active substances used as biopharmaceuticals or biopharmaceutical raw materials.

[0503] Physiologically active substances used as biopharmaceuticals are not particularly limited, and examples thereof include enzymes such as t-PA, glucocerebrosidase, galactosidase, hyaluronidase, iduronidase, glucosidase, sulfatase, urate oxidase, DNase, adenosine deaminase, tripeptidyl peptidase, hyaluronidase, phenylalanine ammonia lyase, and alkaline phosphatase; blood coagulation factors and blood-related proteins such as FVIIa, FVIII, FIX, FXIII, thrombomodulin, antithrombin, and albumin; hormones such as insulin, growth hormone, diuretic peptides, gonadotropins, GLP-1, GLP-2, parathyroid hormone, and leptin; hematopoietic factors such as erythropoietin and thrombopoietin; cytokines and receptors thereof such as G-CSF, IL-2, IL-10, IL-2R, IL-4R, IL-5R, IL-6R, IL-17R, TNFR, EGF, EGFR, FGF, VEGF, VEGFR, PDGF, PDGFR, and TGF-β; cell surface antigens such as CTLA-4, cell surface receptors and their ligands; and proteins and peptides for vaccines such as hepatitis B virus-derived antigens, papillomavirus-derived antigens, varicella-zoster virus-derived antigens, and SARS-CoV-2-derived antigens, as well as subtypes, subunits, and active fragments thereof. Cells that produce these physiologically active substances can be contained in the core layer.

[0504] As used herein, the term "biologically active substance" includes structurally modified substances, such as substances whose amino acid sequence has been modified to alter the activity of the substance, specifically insulin analogs, GLP-1 analogs, erythropoietin analogs, etc. Furthermore, the term "biologically active substance" includes substances consisting of the amino acid sequence of a partial region or fragment of the original substance, and may also include substances that combine multiple sequences of the amino acid sequences of these partial regions or fragments; specifically, insulin analogs, FVIII analogs, parathyroid hormone analogs, etc. Furthermore, the term "biologically active substance" includes fusion proteins that combine two or more substances or partial regions or fragments thereof, such as fusion proteins of an enzyme and an antibody, fusion proteins of a cytokine receptor and an antibody Fc portion, fusion proteins of a cell surface antigen extracellular domain and an antibody Fc portion, fusion proteins of a blood coagulation factor and an antibody Fc portion, and fusion proteins of a blood coagulation factor and a plasma protein. Cells that produce these structurally modified bioactive substances can be contained in the core layer.

[0505] The physiologically active substances produced in this manner can also be modified or altered after production, and specific examples include PEGylation, glycosylation, drug binding modification, radiolabeling, etc. That is, they can be used to produce proteins and peptides that serve as raw materials in the production of modified proteins and peptides such as PEGylated proteins and fatty acid-added peptides, and specific examples include cells that produce raw material proteins and peptides such as PEGylated FVIII, PEGylated erythropoietin, and fatty acid-added insulin analogs, and the core layer can contain cells that produce these raw material physiologically active substances (raw material physiologically active substance-producing cells).

[0506] In the present specification, the physiologically active substance-producing cells that can be contained in the core layer of the polymer-coated cross-linked alginate gel fiber and the multi-layer polymer-coated cross-linked alginate gel fiber are not particularly limited, but specifically include alteplase, monteplase, imiglucerase, velaglucerase, agalsidase, laronidase, alglucosidase, avalglucosidase, idursulfase, galsulfase, elosulfase, rasburica, cells that produce enzymes such as hydroxylase, dornase, cerliponase, glucarpidase, hyaluronidase, and asfotase; cells that produce blood coagulation factors and blood-related proteins such as eptacog, octocog, rurioctocog, turoctocog, lonoctocog, damoctocog, simoctocog, nonacog, albutrepenonacog, catridecacog, efflaloctocog, eftrenonacog, thrombomodulin, antithrombin, vonicog, and albumin; insulin, insulin Cells that produce hormones such as lispro, insulin aspart, insulin glargine, insulin detemir, insulin glulisine, insulin degludec, somatropin, somapsitan, mecasermin, carperitide, vosoritide, glucagon, follitropin, choriogonadotropin, dulaglutide, liraglutide, semaglutide, teduglutide, teriparatide, and metreleptin; cells that produce interferons such as interferon alpha-2a, interferon alpha-2b, interferon beta-1a, interferon beta-1b, and interferon gamma-1a; cells that produce hematopoietic factors such as epoetin, darbepoetin, and romiplostim; filgrastim, lenograstim, teceleukin, trafermin, belfermin, etanercept, aflibercept, and denileukin Examples include cells that produce cytokines such as diftitox and their receptors; cells that produce cell surface antigens such as abatacept, cell surface receptors and their ligands; and cells that produce subtypes, subunits, and active fragments thereof.

[0507] In this specification, the physiologically active substance-producing cells that can be contained in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber are not particularly limited, but specific examples include physiologically active substance-producing animal cells, preferably physiologically active substance-producing CHO cells, physiologically active substance-producing HEK293 cells, or physiologically active substance-producing BHK cells, and more preferably physiologically active substance-producing CHO cells.More specifically, the physiologically active substance-producing animal cells are not particularly limited, but examples thereof include alteplase-producing CHO cells, imiglucerase-producing CHO cells, agalsidase-producing CHO cells, laronidase-producing CHO cells, alglucosidase-producing CHO cells, avalglucosidase-producing CHO cells, idursulfase-producing CHO cells, galsulfase-producing CHO cells, elosulfase-producing CHO cells, dornase-producing CHO cells, cells, cerliponase-producing CHO cells, hyaluronidase-producing CHO cells, asfotase-producing CHO cells, lurioctocog-producing CHO cells, turoctocog-producing CHO cells, lonoctocog-producing CHO cells, nonacog-producing CHO cells, albutrepenonacog-producing CHO cells, thrombomodulin-producing CHO cells, antithrombin-producing CHO cells, vonicog-producing CHO cells, follitropin-producing CHO cells, choriogonadotropin-producing CHO cells, du CHO cells producing physiologically active substances such as raglutide-producing CHO cells, interferon beta-1a-producing CHO cells, epoetin-producing CHO cells, darbepoetin-producing CHO cells, lenograstim-producing CHO cells, etanercept-producing CHO cells, aflibercept-producing CHO cells, and abatacept-producing CHO cells; and HEK293 cells producing physiologically active substances such as simoctocog-producing HEK293 cells, efflaloctocog-producing HEK293 cells, and eftrenonacog-producing HEK293 cells. physiologically active substance-producing BHK cells such as monteplase-producing BHK cells, eptacog-producing BHK cells, octocog-producing BHK cells, and damoctocog-producing BHK cells; physiologically active substance-producing HT-1080 cells such as velaglucerase-producing HT-1080 cells, agalsidase-producing HT-1080 cells, and idursulfase-producing HT-1080 cells; physiologically active substance-producing PERC6 cells such as follitropin-producing PERC6 cells; and the like.

[0508] For example, examples of the physiologically active substance-producing CHO cells include alteplase-producing CHO cells, alglucosidase-producing CHO cells, rurioctocog-producing CHO cells, dulaglutide-producing CHO cells, interferon beta-1a-producing CHO cells, darbepoetin-producing CHO cells, etanercept-producing CHO cells, aflibercept-producing CHO cells, and abatacept-producing CHO cells.

[0509] Although the physiologically active substances listed here may be modified or altered after production, as described above, the core layer may contain cells that produce the physiologically active substances that serve as raw materials. For example, in the case of PEGylated physiologically active substances such as elapeguademase, pegvalase, rurioctocog alfa pegol, turoctocog alfa pegol, damoctocog alfa pegol, nonacog beta pegol, pegvisomant, peginterferon alfa-2a, peginterferon alfa-2b, epoetin beta pegol, pegfilgrastim, and pegbelfermin, the core layer may contain cells that produce the physiologically active substances that serve as raw materials.

[0510] Cells capable of producing a physiologically active substance include, in addition to the aforementioned physiologically active substance-producing genetically modified cells, natural cells, and cells that have been artificially modified, as well as cell masses consisting of a plurality of cells, such as insulin-secreting cells, pancreatic islets, pancreatic islet cells, dopamine-secreting cells, pituitary cells, growth hormone-secreting cells, parathyroid cells, nerve growth factor-secreting cells, blood coagulation factor-secreting cells, hepatocytes, parathyroid cells, erythropoietin-secreting cells, norepinephrine-secreting cells, etc. In this specification, in one embodiment, the cells that produce a physiologically active substance are insulin-secreting cells, pancreatic islets or pancreatic islet cells, or MIN6 cells derived from pancreatic β cells.

[0511] "Insulin-secreting cells" refer to cells with the ability to secrete insulin, such as beta cells that secrete insulin among cells constituting pancreatic islets. Furthermore, "insulin-secreting cells" may also refer to cells that have acquired insulin secretion function through differentiation, maturation, modification, or the like. For example, "insulin-secreting cells" may include cells with insulin secretion function obtained by differentiating stem cells such as iPS cells, ES cells, or somatic stem cells (e.g., mesenchymal stem cells), cells with insulin secretion function obtained by maturing immature cells or progenitor cells, and cells conferred insulin secretion function through genetic recombination. Here, differentiating or maturing the cells includes culturing the cells, i.e., cells obtained by differentiation or maturation may include cells obtained by culture. "Pancreatic islets" are also known as islets of Langerhans and are cell clusters composed of an average of approximately 2,000 pancreatic islet cells. Pancreatic islets are composed of five types of cells: α cells that secrete glucagon, β cells that secrete insulin, δ cells that secrete somatostatin, ε cells that secrete ghrelin, and PP (pancreatic polypeptide) cells that secrete pancreatic polypeptide.

[0512] As used herein, the term "pancreatic islet cells" refers to cells that contain at least one of the five types of cells that constitute the pancreatic islets, and preferably includes at least β cells. In some embodiments, the pancreatic islet cells may be a mixture containing all of α cells, β cells, δ cells, ε cells, and PP cells, or may be cells contained in pancreatic islets.

[0513] Furthermore, "pancreatic islet cells" may be cells that have become pancreatic islet cells through differentiation, maturation, modification, etc. In this case, "pancreatic islet cells" may include, for example, pancreatic islet cells obtained by differentiating stem cells such as iPS cells, ES cells, and somatic stem cells (e.g., mesenchymal stem cells), as well as pancreatic islet cells obtained by maturing immature cells or progenitor cells.

[0514] When used for transplantation, "insulin-secreting cells" or "pancreatic islets (including pancreatic islet cells)" preferably have sufficient viability and function to recover a pathological condition of a patient when transplanted into the patient. The function of insulin-secreting cells, pancreatic islets, or pancreatic islet cells is, for example, to secrete insulin, and it is preferred that glucose responsiveness is maintained even after transplantation.

[0515] The donor of "insulin-secreting cells," "pancreatic islets," or "pancreatic islet cells" is an animal, preferably a vertebrate, more preferably a mammal, and specific examples include humans, pigs, monkeys, rats, or mice, and even more preferably humans or pigs. In some embodiments, the donor of "insulin-secreting cells," "pancreatic islets," or "pancreatic islet cells" is a pig, in order to alleviate the shortage of donors. "Insulin-secreting cells," "pancreatic islets," or "pancreatic islet cells" may be either pancreatic islets or pancreatic islet cells obtained from a donor animal, or insulin-secreting cells or pancreatic islet cells obtained from donor-derived cells, and may be, for example, insulin-secreting cells or pancreatic islet cells differentiated from human-derived ES cells or iPS cells.

[0516] When the "insulin-secreting cells," "pancreatic islets," or "pancreatic islet cells" are derived from pigs, examples include adult porcine pancreatic islets, or fetal, neonatal, or perinatal porcine pancreatic islets, or insulin-secreting cells or pancreatic islet cells obtained from such pancreatic islets. The pancreatic islets may be appropriately cultured before use, or islets obtained by maturing fetal, neonatal, or perinatal porcine pancreatic islets may also be used.

[0517] Examples of blood coagulation factor-secreting cells include factor VIII-secreting cells and factor IX-secreting cells.

[0518] 11. Method for Producing Polymer-Coated Cross-Linked Alginate Gel Fiber Herein, a method for producing a polymer-coated cross-linked alginate gel fiber is provided, in which a cross-linked alginate gel (core layer) containing cells capable of producing antibodies, physiologically active substances, etc. is formed by a cross-linking reaction using chemically modified alginate derivatives represented by formula (I) and formula (II) and the cross-linked alginate gel (core layer) is coated with a cationic polymer (cationic polymer layer). For example, a method for producing the fiber is provided, which includes using an apparatus XX shown in Figure 3.

[0519] The method for producing the polymer-coated cross-linked alginate gel fiber will be described below.

[0520] The method for producing the polymer-coated cross-linked alginate gel fiber is not particularly limited, but may be carried out, for example, using the apparatus XX shown in Fig. 3. The apparatus XX here is an apparatus preferably used for producing polymer-coated cross-linked alginate gel fiber.

[0521] The device XX is a device capable of creating a micro flow path having one inlet and one outlet, as shown in Figure 3, for example. By introducing a solution through the inlet and flowing it at an appropriate speed, the solution becomes fibrous and is discharged from the outlet.

[0522] The device XX can use, for example, an extrusion tube YY as shown in Figure 3 to extrude a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc., and chemically modified alginic acid derivatives represented by formula (I) and formula (II), which is introduced into the inlet of the device XX, and eject the mixed solution from the outlet of the device XX.

[0523] As an example of an apparatus including the device XX and the extrusion tube YY, a syringe can be used. In the case of a syringe, the device XX serves as an outer tube, and the extrusion tube YY serves as an inner tube for extruding the solution introduced into the device XX from an outlet. When a syringe is used, a syringe made of glass or plastic can be used.

[0524] As shown in Figure 3, a container DD such as a beaker containing a solution containing divalent metal ions is used as a container for receiving the fibrous material discharged from the outlet 2 of the device XX, and a container EE such as a beaker containing a solution containing a cationic polymer is used as a container for coating the surface of the cross-linked alginate gel fiber CLA with a cationic polymer.

[0525] 3 is a schematic diagram illustrating one embodiment of the process for producing polymer-coated cross-linked alginate gel fibers. As an example, a production method using a mixed solution of chemically modified alginate derivatives represented by formulas (I) and (II) containing cells (cells capable of producing antibodies, physiologically active substances, etc.) is described.

[0526] Polymer-coated cross-linked alginate gel fibers can be produced, for example, by a method including the following steps (S) to (2): step (S): introducing a mixed solution containing cells capable of producing antibodies, physiologically active substances, etc., and the chemically modified alginate derivatives represented by formulae (I) and (II) through inlet 1 of device XX; step (1): injecting the mixed solution containing cells capable of producing antibodies, physiologically active substances, etc., and the chemically modified alginate derivatives represented by formulae (I) and (II) through outlet 2 of device XX into a solution containing divalent metal ions to contact the mixed solution with the divalent metal ions, thereby obtaining cross-linked alginate gel fibers (CLA) containing cells capable of producing antibodies, physiologically active substances, etc.; step (2): contacting the cross-linked alginate ge...

Claims

1. A multi-layer polymer-coated cross-linked alginate gel fiber comprising a core layer containing antibody-producing cells or physiologically active substance-producing cells embedded in a cross-linked alginate gel obtained by carrying out a cross-linking reaction using a chemically modified alginate derivative represented by formula (I) or (II), a cationic polymer layer coating the core layer, and an anionic polymer layer coating the cationic polymer layer. The chemically modified alginate derivative represented by formula (I) is represented by the following formula (I): (each formula does not include the areas outside the dashed lines at both ends) represents a linker selected from the group consisting of the partial structural formulas shown in 2. The multilayer polymer-coated cross-linked alginate gel fiber according to claim 1, wherein the antibody-producing cells contained in the core layer are genetically modified animal cells that produce antibodies, and the host cells are cells selected from the group consisting of CHO cells, CHO cell sublines, COS cells, Sp2 / 0 cells, NS0 cells, SP2 cells, PERC6 cells, YB2 / 0 cells, YE2 / 0 cells, 1R983F cells, Namalwa cells, Wil-2 cells, Jurkat cells, Vero cells, Molt-4 cells, HEK293 cells, BHK cells, HT-1080 cells, KGH6 cells, P3X63Ag8.653 cells, C127 cells, JC cells, LA7 cells, ZR-45-30 cells, hTERT cells, NM2C5 cells, and UACC-812 cells.

3. The multilayer polymer-coated cross-linked alginate gel fiber of claim 1, wherein the physiologically active substance-producing cells contained in the core layer are cells selected from the group consisting of insulin-secreting cells, pancreatic islets, pancreatic islet cells, dopamine-secreting cells, pituitary cells, growth hormone-secreting cells, parathyroid cells, nerve growth factor-secreting cells, blood coagulation factor-secreting cells, hepatic cells, parathyroid cells, erythropoietin-secreting cells, norepinephrine-secreting cells and physiologically active substance expression vectors (genetically modified cells).

4. A multilayer polymer-coated cross-linked alginate gel fiber as described in any one of claims 1 to 3, wherein the component additionally contained in the core layer is a component selected from the group consisting of alginate solution, alginate gel, culture medium, culture fluid, collagen solution, methylcellulose and sucrose solution.

5. A multi-layer polymer-coated cross-linked alginate gel fiber according to any one of claims 1 to 4, wherein the cationic polymer layer is a cationic polymer selected from the group consisting of polyamino acids, basic polysaccharides, and basic polymers.

6. The cationic polymer layer is selected from the group consisting of poly-L-ornithine (PLO), poly-D-ornithine (PDO), poly-DL-ornithine, poly-D-lysine (PDL), poly-L-lysine (PLL), poly-DL-lysine, poly-L-arginine (PLA), poly-D-arginine (PDA), poly-DL-arginine, poly-L-homoarginine (PLHA), poly-D-homoarginine (PDHA), poly-DL-homoarginine, poly-L-histidine ( 6. The multilayer polymer coated crosslinked alginate gel fiber according to claim 1, wherein the cationic polymer is selected from the group consisting of poly(aminopropyl ether), poly(D-histidine) (PLH), poly-D-histidine (PDH), poly-DL-histidine, polymethylene-co-guanidine (PMCG), polyallylamine (PAA), polyvinylamine (PVA), polyethyleneimine, allylamine-diallylamine copolymer, and allylamine-maleic acid copolymer.

7. A multi-layer polymer-coated cross-linked alginate gel fiber according to any one of claims 1 to 6, wherein the anionic polymer layer is an anionic polymer selected from the group consisting of anionic polysaccharides, sulfated polysaccharides, synthetic polymers, anionic polyamino acids, chemically modified forms thereof, cross-linked forms thereof, and mixtures thereof.

8. A multi-layer polymer-coated cross-linked alginate gel fiber according to any one of claims 1 to 7, wherein the anionic polymer layer is an anionic polymer selected from the group consisting of alginic acid, a chemically modified alginic acid derivative represented by formula (I) according to claim 1, a chemically modified alginic acid derivative represented by formula (II) according to claim 1, a cross-linked body formed from the chemically modified alginic acid derivative represented by formula (I) and formula (II) according to claim 1, and mixtures thereof.

9. A method for producing a multi-layered polymer-coated cross-linked alginate gel fiber according to any one of claims 1 to 8, comprising the steps of: (1) injecting a mixed solution containing antibody-producing cells or bioactive substance-producing cells and the chemically modified alginate derivatives represented by formula (I) and formula (II) according to claim 1 into a solution containing a divalent metal ion to obtain a cross-linked alginate gel fiber containing cells capable of producing antibodies or bioactive substances; (2) contacting the cross-linked alginate gel fiber containing the antibody-producing cells or bioactive substance-producing cells obtained in (1) with a solution containing a cationic polymer to obtain a polymer-coated cross-linked alginate gel fiber coated with a cationic polymer layer; and (3) contacting the polymer-coated cross-linked alginate gel fiber obtained in (2) with a solution containing an anionic polymer to obtain a multi-layered polymer-coated cross-linked alginate gel fiber coated with an anionic polymer layer.

10. A method for producing an antibody or a physiologically active substance, which uses the multilayer polymer-coated cross-linked alginate gel fiber according to any one of claims 1 to 8.

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