CN110462055A - The manufacturing method and darbepoetin of darbepoetin composition produce celliferous cultural method - Google Patents
The manufacturing method and darbepoetin of darbepoetin composition produce celliferous cultural method Download PDFInfo
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- CN110462055A CN110462055A CN201880015112.0A CN201880015112A CN110462055A CN 110462055 A CN110462055 A CN 110462055A CN 201880015112 A CN201880015112 A CN 201880015112A CN 110462055 A CN110462055 A CN 110462055A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/505—Erythropoietin [EPO]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Abstract
The method cultivated the purpose of the present invention is to provide a kind of pair of darbepoetin production cell and manufacture darbepoetin composition, in the manufacturing method of the darbepoetin composition, the total output of darbepoetin composition improves, and improves the sialic acid containing ratio in a molecule of the darbepoetin being secreted into culture solution by the cell.The present invention relates to a kind of manufacturing methods of darbepoetin composition comprising: darbepoetin production cell is cultivated in the culture medium of the protolysate added with plant origin and obtains the step 1 of culture solution；With the step 2 for recycling darbepoetin composition from the culture solution obtained by step 1.
Description
Technical field
The present invention relates to the manufacturing method of darbepoetin composition and with production darbepoetin ability zooblast
Cultural method etc..
Background technique
In recent years, it is actively being implemented the production using utilities such as the gene recombinant proteins of zooblast, in order to
Maximum generation yield is obtained, the ability to express of protein for increasing the zooblast etc. itself is not required nothing more than, also requires
Improve the proliferation of the zooblast.
On the other hand, even if gene recombination technology is in progress, still there is the gene recombinant protein for being difficult to produce, wherein it
One is glycoprotein.Known majority protein in the form of the glycoprotein for being attached with sugar chain to exist, and the difference of sugar chain structure is to egg
The function of white matter itself produces a very large impact.
For the biologics of application gene recombination technology, by the way that the channel genes of the protein of people are thin to animal
It is produced in born of the same parents.But using zooblast to produce glycoprotein, in the saliva generated by the production cell
Under the action of sugar chains catabolic enzymes such as liquid acid enzyme etc., sugar chain structure becomes unevenly, therefore can not show sufficient medicine sometimes
Effect.
The hematopoietin (EPO) for being known as Hemopoietic factor has 3 N- connection type sugar chains and 1 O- connecting-type
Sugar chain, half-life period changes with the sialic acid number of sugar-chain end in blood, when the sialic acid number of sugar-chain end is reduced, half-life period in blood
It reduces, hardly shows physiological activity (non-patent literature 1).Additionally, it was found that following darbepoetins, is in order to further
Extend half-life period in the blood of EPO and changes a part of the amino acid sequence of EPO and then increase sialic acid additional number and obtain
(non-patent literature 2, patent document 1) arrived.
Darbepoetin has 5 N- connection type sugar chains and 1 O- connection type sugar chain, increases with lasting red blood cell and makees
With can reduce administration frequency compared with existing EPO preparation, can reduce the burden of patient.
But for the reasons mentioned above, there is the equal of the more sugar chain containing sialic acid compared with EPO in order to efficiently produce
Even darbepoetin requires higher sugar chain control technique while improving yield.
Regulate and control method as sugar chain, it is known that following methods: in order to inhibit the sugar chain catabolic enzyme institute by being secreted into culture solution
The sialic acid of cause, will be for example as 2- deoxidation -2,3- dehydrogenation-N- acetyl nerve of sialidase inhibitor from the disengaging of glycoprotein
The low molecular compounds such as propylhomoserin (NeuAc2en) are added in culture medium and are cultivated.
On the other hand, in order to improve the proliferation of zooblast and the yield of the substance generated by the zooblast, existed in the past
Added in the culture medium used when cultivating the zooblast albumin, transferrins or insulin etc, serum or
Substance from serum, the protein from animal.Serum or the substance from serum, source are added into culture medium
When the protein of animal, there are cell culture and generation substance as obtained in it is by hepatitis, virus or ox statural spongiosus
The risk of pollutions such as sick (BSE), therefore, to without containing serum or the substance from serum and the protein from animal
Complete synthesis culture medium (chemically defined culture medium, chemically defined medium) is developed.
As the constituent of typical complete synthesis culture medium, amino acid, organic salt or inorganic salts, dimension life can be enumerated
Element, minerals, trace meter, sugar, lipid and nucleic acid etc., it is known that by adding in complete synthesis culture medium from soybean etc.
The protolysate of plant can equally improve the proliferation of zooblast and by the zooblast with serum-containing media
The yield (patent document 2,3) of obtained generation substance.
Existing technical literature
Patent document
Patent document 1: No. 2938572 specifications of Japanese Patent No.
Patent document 2: International Publication No. 2001/023527
Patent document 3: International Publication No. 2006/045438
Non-patent literature
Non-patent literature 1:Glycoconjugate J., 1993；10；263
Non-patent literature 2:Nephrol.Dial.Transplant., 2001；16；3-13
Summary of the invention
Problems to be solved by the invention
But it has no knowledge about the total output for improving darbepoetin composition and improves and culture solution is secreted by the cell
In darbepoetin a molecule in sialic acid containing ratio method.
Therefore, it is cultivated the purpose of the present invention is to provide a kind of pair of darbepoetin production cell and to manufacture sialic acid attached
The method of the high darbepoetin composition of addend, the manufacturing method of the darbepoetin composition, which is improved, is secreted into culture by the cell
The total output of darbepoetin composition in liquid, and improve a molecule of the darbepoetin being secreted into culture solution by the cell
In sialic acid containing ratio.
Means for solving the problems
The inventors discovered that being produced by using the culture medium of the protolysate containing plant origin to darbepoetin thin
Born of the same parents cultivate, and the total output that the darbepoetin composition in culture solution is secreted by the cell improves, also, by inhibiting to divide
The sialidase in culture solution is secreted, the sialic acid in a molecule of the darbepoetin being secreted into culture solution by the cell adds
Rate improves, and can produce the high darbepoetin composition of sialic acid additional number significantly, so as to complete the present invention.
That is, the present invention provides following (1)~(19).
(1) a kind of manufacturing method of darbepoetin composition comprising following step 1 and step 2,
Step 1: darbepoetin production cell being trained in the culture medium of the protolysate added with plant origin
Support the step of obtaining culture solution；
Step 2: the step of darbepoetin composition is recycled from culture solution obtained in step 1.
(2) a kind of manufacturing method for the darbepoetin composition for inhibiting sialic acid to be detached from from darbepoetin molecule, packet
Following step 1 and step 2 are included,
Step 1: darbepoetin production cell being trained in the culture medium of the protolysate added with plant origin
Support the step of obtaining culture solution；
Step 2: the step of darbepoetin composition is recycled from culture solution obtained in step 1.
(3) manufacturing method as described in (1) or (2), wherein each molecule in culture solution obtained in step 1 reaches shellfish
The above are each molecule darbepoetins to contain 18~22 for the 67% of the darbepoetin composition that Bo Ting contains 12~22 sialic acids
The darbepoetin composition of a sialic acid.
(4) manufacturing method as described in any one of (1)~(3), wherein each in culture solution obtained in step 1
The above are each molecule darbepoetins to contain for the 55% of the darbepoetin composition that molecule darbepoetin contains 12~22 sialic acids
There is the darbepoetin composition of 19~22 sialic acids.
(5) manufacturing method as described in any one of (1)~(4), wherein each in culture solution obtained in step 1
The above are each molecule darbepoetins to contain for the 44% of the darbepoetin composition that molecule darbepoetin contains 12~22 sialic acids
There is the darbepoetin composition of 20~22 sialic acids.
(6) manufacturing method as described in any one of (1)~(5), wherein each in culture solution obtained in step 1
The above are each molecule darbepoetins to contain for the 18% of the darbepoetin composition that molecule darbepoetin contains 12~22 sialic acids
There is the darbepoetin composition of 22 sialic acids.
(7) manufacturing method as described in any one of (1)~(6), wherein the Da Bei in culture solution obtained in step 1
It is high when mooring the total output of spit of fland composition than cultivating in the culture medium of the protolysate without containing plant origin.
(8) manufacturing method as described in any one of (1)~(7), wherein it is in host cell that darbepoetin, which produces cell,
Transformed cells obtained from the middle carrier for importing the gene containing coding darbepoetin.
(9) manufacturing method as described in (8), wherein host cell is zooblast.
(10) manufacturing method as described in (9), wherein zooblast is Chinese hamster ovary (CHO) cell, mouse black
Plain tumor (NS0) cell or mouse myeloma (SP2/0) cell or the cell from these cells.
(11) manufacturing method as described in any one of (1)~(10), wherein add the protolysate of plant origin
Culture medium is complete synthesis culture medium.
(12) manufacturing method as described in any one of (1)~(11), wherein the plant origin added into culture medium
The concentration of protolysate is 1~5g/l.
(13) manufacturing method as described in any one of (1)~(12), wherein the protolysate of plant origin is source
In the protolysate of soybean.
(14) manufacturing method as described in any one of (1)~(13), wherein further comprise to darbepoetin composition
The purification step purified.
(15) manufacturing method as described in (14), wherein purification step be selected from anion-exchange chromatography, reverse-phase chromatography or
At least one of gel filtration.
(16) manufacturing method as described in any one of (1)~(15), wherein obtained darbepoetin composition is every
One molecule contains the darbepoetin of average 18 or more sialic acids.
(17) a kind of cultural method of darbepoetin production cell comprising: in the proteolysis for being added with plant origin
Darbepoetin production cell is cultivated in the culture medium of object, is secreted into darbepoetin composition in culture solution.
(18) a kind of cultural method of darbepoetin production cell comprising: in the proteolysis for being added with plant origin
Darbepoetin production cell is cultivated in the culture medium of object, sialic acid in culture is inhibited to be detached from from darbepoetin molecule.
It is (19) a kind of to inhibit the method that sialic acid is detached from from darbepoetin molecule in culture solution preservation comprising: it is adding
Have to cultivate darbepoetin production cell in the culture medium of the protolysate of plant origin and obtains culture solution.
Invention effect
In the manufacturing method of the present invention, by added with plant origin protolysate culture medium in reach Bei Bo
Spit of fland production cell is cultivated, and can be improved the total output for the darbepoetin composition being secreted into culture solution by the cell, and
And it can be improved the sialic acid containing ratio in a molecule of the darbepoetin being secreted into culture solution by the cell.
Detailed description of the invention
Fig. 1 is the figure for showing the structure of darbepoetin.
Fig. 2 be show darbepoetin N- connection type sugar chain connected with O- type sugar chain structure figure.In Fig. 2, NeuAc table
Show that sialic acid is 1 N-acetyl-neuraminate.
Fig. 3 is to show the culture supernatant cultivated in soy hydrolyzate addition culture medium and soy hydrolyzate removing liquid
In darbepoetin sialic acid isotype distribution ongoing change figure.Swimming lane 1 and swimming lane 14 indicate that sialic acid additional number is
18~22 darbepoetins.Swimming lane 2 indicate by soy hydrolyzate removing liquid saved at 8 DEG C 0 it is small in the case of saliva
The distribution of sour isotype, swimming lane 3 indicate to save soy hydrolyzate removing liquid at 8 DEG C 24 it is small in the case of sialic acid it is of the same race
Type distribution, swimming lane 4 indicate by soy hydrolyzate removing liquid is saved at 8 DEG C 48 it is small in the case of sialic acid isotype divide
Cloth, swimming lane 5 indicate by soy hydrolyzate removing liquid is saved at 8 DEG C 72 it is small in the case of sialic acid isotype distribution.Swimming
Road 6 indicate by the culture supernatant cultivated in the culture medium containing soy hydrolyzate saved at 8 DEG C 0 it is small in the case of
The distribution of sialic acid isotype, swimming lane 7 indicate will the culture supernatant that be cultivated in the culture medium containing soy hydrolyzate 8
Sialic acid isotype distribution in the case of preservation 24 is small at DEG C, the expression of swimming lane 8 will be in the culture medium containing soy hydrolyzate
It is middle cultivate obtained culture supernatant is saved at 8 DEG C 48 it is small in the case of the distribution of sialic acid isotype, the expression of swimming lane 9 will be
The culture supernatant cultivated in culture medium containing soy hydrolyzate saved at 8 DEG C 72 it is small in the case of sialic acid it is same
Kind type distribution.Swimming lane 10, which indicates to remove in soy hydrolyzate, adds the inhibitor NeuAc2en of sialidase and at 8 DEG C in liquid
Save 0 it is small in the case of sialic acid isotype distribution, swimming lane 11 indicate soy hydrolyzate remove liquid in adds sialic acid
The inhibitor NeuAc2en of enzyme and at 8 DEG C save 24 it is small in the case of sialic acid isotype distribution, swimming lane 12 indicate exist
Soy hydrolyzate removes the inhibitor NeuAc2en that sialidase is added in liquid and saliva in the case of preservation 48 is small at 8 DEG C
Liquid acid isotype distribution, swimming lane 13 indicate soy hydrolyzate remove liquid in add sialidase inhibitor NeuAc2en and
Sialic acid isotype distribution in the case of preservation 72 is small at 8 DEG C.
Fig. 4 is to be illustrated to the area value of the darbepoetin composition of each sialic acid additional number with column relative to sialic acid
The figure of the result for the table 1 that the ratio (%) of the gross area value for the darbepoetin composition that additional number is 12~22 is measured.
The longitudinal axis indicates the ratio (%) of the area value of the darbepoetin composition of each sialic acid additional number.The sialic acid additional number of table 1 is
The ratio of 22 area values in Fig. 4 indicate sialic acid additional number be 22 darbepoetin composition 22,22-1S and
The total of the ratio (%) of the area value of these isotypes of 22-2S indicates.
Specific embodiment
The manufacturing method of darbepoetin composition of the invention includes the following steps 1 and step 2,
Step 1: darbepoetin production cell being trained in the culture medium of the protolysate added with plant origin
Support the step of obtaining culture solution；
Step 2: the step of darbepoetin composition is recycled from culture solution obtained in step 1.
Hereinafter, being illustrated to each step.
[step 1]
Step 1 is in the culture medium of the protolysate added with plant origin to the ability with production darbepoetin
Cell the step of being cultivated, darbepoetin composition is made to be secreted into culture medium and obtain culture solution.
In the present invention, darbepoetin is expressed as [Asn30Thr32Val87Asn88Thr90] EPO people's promoting erythrocyte generate
Plain analog, be 30 in the amino acid sequence of the human forcing erythrogenin in International Publication No. 95/05465,32,87,
88 and 90 amino acid residues are replaced into made of Asn, Thr, Val, Asn and Thr respectively, have structure shown in FIG. 1.
24,30,38, the 83 of darbepoetin be combined with N- on 88 Asn residues and connect type sugar chain, at 126
It is combined with O- connection type sugar chain on Ser residue, connects the primary structure of type sugar chain with O- as N- connection type sugar chain, can enumerate
Structure shown in Fig. 2.
4 sialic acids are contained up on the N- connection type sugar chain in conjunction with darbepoetin molecule, in O- connection type sugar chain
End contain up to 2 sialic acids, 22 sialic acids are contained up in a molecule darbepoetin.
In the present invention, as darbepoetin, also including, for example, Aranesp or the Aranesp of genetic recombination
Or their biological Counterfeit Item.In addition, novel RBC acceptor garland rate disclosed in International Publication No. 2003/020299 stimulates egg
White (novel erythropoiesis stimulating protein) and NESP (trade mark) or Aranesp (trade mark) or
Their biological Counterfeit Item is also contained in darbepoetin of the invention.
The host cell of darbepoetin used in the present invention production cell, belong to mammality, birds, reptile class,
The zooblast etc. of any type in amphibian animal, fish and insects etc. can be used, but it is preferable to use belong to mammality
Zooblast, more preferably using from the zooblast of primates such as people or monkey or deriving from mouse, rat or hamster
The zooblast of equal rodents.
As mammiferous cell is belonged to, can enumerate such as myeloma cell or from myeloma cell cell,
Gonad cell, nephrocyte, haemocyte, uterine cell, phoirocyte, mammary glandular cell or embryonic retinoblast etc..It is special
It is not preferably selected from myeloma cell or the cell in the cell and gonad cell of myeloma cell, specifically, can be with
Enumerate such as Chinese hamster ovary (CHO) cell, murine melanoma (NS0) cell or mouse myeloma (SP2/0) cell or
Person derives from the cell of these cells.
As mammiferous cell is belonged to, can enumerate for example as the HL-60 of human cell line (ATCC CCL-240),
HT-1080(ATCC CCL-121)、HeLa(ATCC CCL-2)、293(ECACC 85120602)、Namalwa(ATCC CRL-
1432), Namalwa KJM-1 (Cytotechnology, 1,151 (1988)), NM-F9 (DSM ACC2605, International Publication No.
No. 05/17130) or PER.C6 (No. 6855544 ECACC No.96022940, U.S. Patent No. specifications)；As monkey cells strain
VERO (ATCC CCL-1651) or COS-7 (ATCC CRL-1651)；C127I (ATCC CRL- as mouse cell strain
1616),Sp2/0-Ag14(ATCC CRL-1581),NIH3T3(ATCCCRL-1658),NS0(ATCC CRL-1827)；As
Y3 Ag1.2.3. (ATCC CRL 1631), YO (ECACCNo:85110501) and YB2/0 (the ATCC CRL- of rat cell strain
1662)；CHO-K1 (ATCC CCL-61), CHO/dhfr- (ATCC CRL-9096), CHO/DG44 as hamster cell strain
[Proc.Natl.Acad.Sci.USA, 77,4216 (1980)] or BHK21 (ATCC CRL-10)；Or as dog cell
MDCK (ATCC CCL-34) etc..
It as the cell for belonging to birds, can enumerate such as chicken cell strain SL-29 (ATCC CRL-29), as belonging to
The cell of fish can be enumerated such as zebra fish cell strain ZF4 (ATCC CRL-2050), as the cell for belonging to insects,
Such as moth (Spodopterafrugiperda, Spodoptera frugiperda) cell strain Sf9 (ATCC CRL-1711) etc. can be enumerated.Separately
Outside, it can enumerate for example as the primary MK cells of primary cultured cell, Primary rabbit kidney cell, primary chick embryo cell or original
For Quail embryo cell etc..
As myeloma cell or from the cell of myeloma cell, such as Sp2/0-Ag14, NS0, Y3 can be enumerated
Ag1.2.3., YO or YB2/0 etc..As gonad cell or from the cell of gonad cell, can enumerate such as CHO-K1,
CHO/dhfr-Or CHO/DG44 etc..
In addition, can be enumerated such as 293, VERO, COS-7, BHK21 or MDCK as nephrocyte, as haemocyte,
HL-60, Namalwa, Namalwa KJM-1 or NM-F9 etc. can be enumerated, as uterine cell, can be enumerated such as HeLa,
It as phoirocyte, can enumerate such as HT-1080 or NIH3T3, as mammary glandular cell, can enumerate for example
C1271I etc. can be enumerated such as PER.C6 as embryonic retinoblast.
Cell is produced as darbepoetin, can enumerate and be imported for example in above-mentioned host cell containing coding darbepoetin
Gene carrier obtained from transformed cells, implement mutation handle so that its generate darbepoetin cell or as production
The hybridoma etc. of the cell fusion with myeloma cells cell of raw darbepoetin.Further implementing to above-mentioned cell makes Da Bei
The cell etc. of the raised mutation processing of the expression quantity of Bo Ting is also contained in darbepoetin production cell of the invention.
The transformed cells of the carrier of the gene containing coding darbepoetin have been imported by that will include the life with darbepoetin
The recombinant vector etc. for producing relevant DNA and promoter etc. imported into host cell used in aforementioned present invention and obtains.
As DNA relevant to the production of darbepoetin, it can be used and for example encode the DNA of darbepoetin, encode and reach
Any one in DNA of the relevant enzyme of the biosynthesis of Bei Boting or protein etc..
As the carrier used to prepare the recombinant vector, such as pcDNAI, pcDM8 can be enumerated
(manufacture of Funakoshi company), pAGE107 [Japanese Unexamined Patent Publication 3-22979 bulletin；Cytotechnology,3,133
(1990)], pAS3-3 (Japanese Unexamined Patent Publication 2-227075 bulletin), pcDM8 [Nature, 329,840 (1987)], pcDNAI/
Amp (manufacture of Invitrogen company), pREP4 (manufacture of Invitrogen company), pAGE103 [J.Biochem., 101,1307
Or pAGE210 etc. (1987)].
It, can be with as long as functioning in the zooblast that can be used in the present invention can be used as promoter
Enumerate IE (early stage at once, immediateearly) promoter of gene of such as cytomegalovirus (CMV), the early stage of SV40 opens
Mover, the promoter of retrovirus, metallothionein promoter, heat-shock promoters or SR α promoter etc..In addition, can also
The enhancer etc. of the IE gene of people CMV to be used together with promoter.
As introduction method of the recombinant vector into host cell, as long as example DNA is imported into the cell
Method can be used, and can enumerate such as electroporation [Cytotechnology, 3,133 (1990)], calcium phosphate method (Japan
Japanese Laid-Open Patent Publication 2-227075 bulletin) or lipofection [Proc.Natl.Acad.Sci.USA, 84,7413 (1987) or
Virology, 52,456 (1973)] etc..
As the protolysate of plant origin, as long as from the proteolysis of the plants such as wheat, rice or soybean
Any protolysate can be used in object, particularly preferably derives from the protolysate of soybean.
The protolysate of plant origin is using ammonia obtained from the breaks down proteins that will be obtained as the plant as raw material
Mixture of the short-chain peptides such as base acid and dipeptides or tripeptides as main component, in addition to this also containing trace meter etc..In plant
In the manufacture of the protolysate in source, the acid hydrolyzation using the acid such as hydrochloric acid or the enzymatic hydrolysis using enzymes such as protease can be enumerated
Method carries out film process as needed.
The average molecular weight of the protolysate of plant origin is preferably 800Da hereinafter, following be more preferably step by step
600Da or less, further preferably 450Da or less, particularly preferably 300Da or less.
The molecular weight distribution of protolysate as plant origin, less than 500Da be preferably 45~80%, 500~
It is preferably 5~15%, 2000~5000Da is preferably 2~10% that 1000Da, which is preferably 15~30%, 1000~2000Da,.Or
Person is more preferably 90~98% less than 2000Da, is more preferably 95~98% less than 2000Da.
As derive from soybean protolysate, can enumerate such as Amysoy, Hy-Soy, N-Z-Soy, HyPep (with
Upper is Quest International company), Soy peptone (Gibco company), Bac-Soyton (Difco company),
SE50MK (DMV International Nutritions company), Peptone Hy-Soy T or Soy Hydrolysate UF
(the above are the manufactures of Sigma-Aldrich company) etc. can enumerate as the protolysate from wheat or rice
HyPep (Quest International company) etc..
It may be culture when can start in the period of adding the protolysate of plant origin in culture solution for culture
In the process, it is not particularly limited, but is preferably added in culture medium when culture starts to use.With fed-batch cultivation method or filling
In the case that the form of supplemented medium in stream cultivation etc. is added during the cultivation process, the proteolysis of plant origin
Object can be individually added in culture medium in the form of supplemented medium, can also to be mixed in advance with other medium components and
At the form of supplemented medium be added in culture medium.
As during culture, be not particularly limited, as final main culture during, preferably 8 days or more.As stream
During adding the main culture in cultivation, preferably 8 days~1 month, more preferably 10 days~21 days, particularly preferably 10 days~
14 days.
Be added to concentration cell according to used in culture of the protolysate of the plant origin in culture medium type,
Addition period of the protolysate of plant origin etc. suitably selects, preferably 0.1~100g/L, further preferably 1
~10g/L, particularly preferably 1~5g/L.
Culture medium used in the present invention can make as long as can be used in the culture of darbepoetin production cell
With, but it is preferable to use the complete synthesis culture mediums without containing serum or the substance from serum or the protein from animal
(chemically defined culture medium, chemically defined medium).
In the case where the host cell of darbepoetin production cell is zooblast, as basic culture medium, using
Basal medium used in the culture of common zooblast.As basis used in the culture in common zooblast
Culture medium can enumerate such as RPMI1640 culture medium [The Journal of the American Medical
Association, 199,519 (1967)], the MEM culture medium [Science, 122,501 (1952)] of Iger (Eagle), Du
Family name improves MEM (DMEM) culture medium [Virology, 8,396 (1959)], 199 culture mediums [Proceeding of the
Society for the Biological Medicine, 73,1 (1950)], F12 culture medium (manufacture of LTI company)
[Proc.Natl.Acad.Sci.USA, 53,288 (1965)], Yi Sikefu improve Du Shi culture medium (IMDM culture medium)
[J.Experimental Medicine, 147,923 (1978)] or EX-CELL325PF culture medium (manufacture of JRH company) or
The improved culture medium of these culture mediums or mixed culture medium etc. are, it is preferable to use RPMI1640 culture medium, DMEM culture medium, F12 training
Support base, IMDM culture medium or EX-CELL 325PF culture medium etc..
Culture medium used in the present invention, needed for the growth for adding zooblast in basal medium as needed
Trophic factors or physiological activator for wanting etc..These additives are preferably before culture in advance containing in the medium.
As trophic factors, the sugar such as glucose, amino acid or vitamin etc. can be enumerated.
As amino acid, such as l-Alanine, L-arginine, altheine, L-Aspartic acid, L- Guang ammonia can be enumerated
Acid, Pidolidone, L-Glutamine, glycine, L-Histidine, l-Isoleucine, L-Leu, L-lysine, L- first sulphur ammonia
Acid, L-phenylalanine, L-PROLINE, Serine, L-threonine, L-Trp, l-tyrosine or Valine etc., use one
Kind is applied in combination two or more.
As vitamin, such as D-biotin, D-VB5, choline, folic acid, myo- inositol, niacinamide, pyrrole can be enumerated and trembled
Aldehyde, riboflavin, thiamine, cyanocobalamin or DL- alpha-tocopherol etc. using one kind or are applied in combination two or more.
In addition, as the object for replacing animal origin object in the complete synthesis culture medium without animal origin object and adding
Matter, can enumerate for example using the physiological activator of method of gene recombination manufacture, hydrolysate or lipid without animal origin object,
Minerals, trace meter or nucleic acid etc..As the physiological activator using method of gene recombination manufacture, such as gene can be enumerated
Recombulin, genetic recombination transferrins, genetic recombination albumin or genetic recombination growth factor etc..
As the lipid for being free of animal origin object, can enumerate such as cholesterol, linoleic acid or linolenic acid.
As complete synthesis culture medium, such as ADPF culture medium (animal origin-free protein culture medium, Animal can be enumerated
derived protein free medium；The manufacture of HyClone company), (Invitrogen is public for CD-Hybridoma culture medium
Department's manufacture), CD-CHO culture medium (manufacture of Invitrogen company), IS CD-CHO culture medium or KINSM-10 culture medium
(manufacture of Irvine Scientific company) etc..
In the case where being cultivated with long-time or high density, it is preferable to use containing amino acids and Wei Sheng in high concentration
The culture medium of plain class is for example mixed RPMI1640 culture medium, DMEM culture medium and F12 culture medium with the ratio of 1:1:1
Culture medium, the culture medium, the hybridoma SFM medium that mix DMEM culture medium and F12 culture medium with the ratio of 1:1
(manufacture of Invitrogen company) etc..
In the present invention, the total output raising of the darbepoetin composition in culture solution obtained in step 1 refers to, is containing
Da Bei in the unit Culture liquid measure of culture finish time when being cultivated in the culture medium of the protolysate of above-mentioned plant origin
The concentration for mooring spit of fland composition improves compared with when cultivating in the culture medium in the hydrolysate without containing above-mentioned plant origin.
The concentration of darbepoetin composition in unit Culture liquid measure can use egg well known to ELISA method or HPLC method etc.
Any one method in white matter method for measurement of concentration is found out, and can be enumerated for example using Biacore (GE Healthcare
Company manufacture) determination of protein concentration method.
As the darbepoetin composition in culture solution obtained in step 1, there is each molecule darbepoetin to be attached with
Preferably 18~22, the structure for being more preferably 19~22, being particularly preferably 20~22, most preferably 22 sialic acids.
As the darbepoetin composition in culture solution obtained in step 1, preferably each molecule darbepoetin contains 12
The above are each molecule darbepoetins to contain 18~22 sialic acids for the 67% of the darbepoetin composition of~22 sialic acids
Darbepoetin composition.Additionally, it is preferred that each molecule darbepoetin contains the darbepoetin composition of 12~22 sialic acids
55% the above are the darbepoetin compositions that each molecule darbepoetin contains 19~22 sialic acids.Additionally, it is preferred that each point
The above are each molecule darbepoetins to contain for the 44% of the darbepoetin composition that sub- darbepoetin contains 12~22 sialic acids
The darbepoetin composition of 20~22 sialic acids.Additionally, it is preferred that each molecule darbepoetin contains 12~22 sialic acids
The above are the darbepoetin compositions that each molecule darbepoetin contains 22 sialic acids for the 18% of darbepoetin composition.
As the darbepoetin composition in culture solution obtained in step 1, specifically, for example following reach can be enumerated
Bei Boting composition: preferably the 10th~14 day each molecule darbepoetin contains reaching for 12~22 sialic acids after culture starts
The above are the darbepoetin compositions that each molecule darbepoetin contains 18~22 sialic acids for the 67% of Bei Boting composition.Separately
Outside, specifically, for example following darbepoetin compositions: the 10th~14 day each molecule preferably after culture starts can be enumerated
The above are each molecule darbepoetins to contain 19 for the 55% of the darbepoetin composition that darbepoetin contains 12~22 sialic acids
The darbepoetin composition of~22 sialic acids.In addition, specifically, for example following darbepoetin compositions can be enumerated: excellent
Culture is selected in start rear 10th~14 day each molecule darbepoetin and contain the darbepoetin composition of 12~22 sialic acids
44% the above are the darbepoetin compositions that each molecule darbepoetin contains 20~22 sialic acids.In addition, specifically, can
To enumerate for example following darbepoetin compositions: the 10th~14 day each molecule darbepoetin contains 12 preferably after culture starts
The above are the Da Bei that each molecule darbepoetin contains 22 sialic acids for the 18% of the darbepoetin composition of~22 sialic acids
Moor spit of fland composition.
Sialic acid number in darbepoetin composition can be by using capillary electrophoresis (Beckman Coulter
Company's manufacture) etc. isoelectric focussing etc. the sialic acid isotype distribution of darbepoetin is measured and is found out.
[step 2]
Step 2 is the step of recycling darbepoetin composition from culture solution obtained in step 1.By in step 1
The darbepoetin composition cultivated and be secreted into culture solution can be purified according to well known method, manufacturer of the invention
Method may further include the purification step purified to darbepoetin composition.
For example, can use the methods of centrifuge separation obtains culture supernatant from culture solution obtained in step 1, pass through list
Solely or be applied in combination common gene recombinant protein isolates and purifies method, i.e. solvent extraction method, saltouing using ammonium sulfate etc.
Method, desalination process, using organic solvent the precipitation method, use Q- agarose or DIAION HPA-75 (Mitsubishi Chemical Ind's manufacture)
The anion-exchange chromatography of equal resins is exchanged using the cation of the resins such as S- agarose FF (manufacture of Pharmacia company)
Chromatography, the hydrophobic chromatography method using resins such as butyl-agarose or phenyl sepharoses, the gel filtration using molecular sieve,
The electrophoresis such as RP chromatography, chromatofocusing method, isoelectric focusing, using MF, UF/DF, virus except the films such as striping filtration method or
Concentration or exchange of solvent method etc., obtain darbepoetin composition from the culture supernatant.
It is that preferred each molecule contains 18 or more average, more preferable each molecule and contains as darbepoetin composition
The darbepoetin of average 18.5 or more sialic acids.As darbepoetin composition, specifically, can enumerate for example preferably
Each molecule obtained in rear 10th~14 day culture solution contains 18 or more average, more preferable each molecule since culture
Darbepoetin containing average 18.5 or more sialic acids.As described above, the sialic acid number in darbepoetin composition
It can use using the isoelectric focussing of capillary electrophoresis (manufacture of Beckman Coulter company) etc. etc. to darbepoetin
Sialic acid isotype distribution be measured and find out.
The present invention relates to a kind of cultural methods of darbepoetin production cell comprising: in the egg for being added with plant origin
Darbepoetin production cell is cultivated in the culture medium of white hydrolysate, is secreted into darbepoetin composition in culture solution.
It is identical with above-mentioned steps 1 about the culture of the darbepoetin production cell in the cultural method.
Moreover, it relates to a kind of cultural method of darbepoetin production cell comprising: it is being added with plant origin
Protolysate culture medium in darbepoetin production cell is cultivated, inhibit in culture sialic acid from darbepoetin point
Son is detached from.It is identical with above-mentioned steps 1 about the culture of the darbepoetin production cell in the cultural method.
Moreover, it relates to a kind of inhibit the method that sialic acid is detached from from darbepoetin molecule in culture solution preservation,
It include: to be cultivated in the culture medium of the protolysate added with plant origin darbepoetin production cell and trained
Nutrient solution.According to the method for the present invention, by the effect of the protolysate of the plant origin contained in culture solution, be able to suppress by
Sialic acid caused by sialidase from the disengaging of darbepoetin molecule, culture solution before starting after culture to purifying
The sialic acid that can be improved on darbepoetin molecule in preservation adds rate.The preservation condition of culture solution is not particularly limited, preferably
It can be saved 72 hours at 8 DEG C.
Using following embodiments, more specifically the present invention will be described, but embodiment only illustration of the invention,
The scope of the present invention is not defined.
Embodiment
What [embodiment 1] produced in the culture medium containing soy hydrolyzate and the culture medium without containing soy hydrolyzate reaches
The sialic acid of Bei Boting composition adds the comparison of rate
Using the Chinese hamster ovary celI of production darbepoetin, fed-batch cultivation is carried out in 1L conical flask, is containing soy hydrolyzate
Culture medium and culture medium without containing soy hydrolyzate in rate added to the sialic acid of darbepoetin be compared.Use EX-
Darbepoetin is generated Chinese hamster ovary celI and burnt from 125mL capacity triangle by CELL325PF culture medium (manufacture of Sigma-Aldrich company)
After bottle is expanded culture into 500mL capacity conical flask, further in 1L capacity conical flask (manufacture of Corning company)
In expand culture, obtain seed culture fluid required for main culture.
It is cultivated about expanding, about 10~30% culture base unit weight using each flask capacity is according to reaching 2 × 105Cell/
The mode inoculating cell of mL carries out the culture of expansion in 3 days or 4 days at 37 DEG C.With KINSM-10 culture medium (Irvine
Scientific company manufacture) based on culture medium in add 3g/L soy hydrolyzate (Soy Hydrolysate UF,
The manufacture of Sigma-Aldrich company；Molecular weight distribution: less than 500Da is 66%, 500~1000Da be 22%, 1000~
2000Da is 9%, 2000~5000Da is 3%) (to have Soy) or do not add (no Soy) and production medium is prepared, right
(1000rpm, 25 DEG C, 5 minutes) are centrifuged in seed culture fluid, by the cell after removal supernatant to reach 2.0 × 105Carefully
Born of the same parents/mL mode is inoculated into respectively in the 1L conical flask supplemented with above-mentioned production medium 200mL.Cell just after inoculation is close
Degree is 2.4 × 10 in the culture medium containing soy hydrolyzate5Cell/mL, without containing soy hydrolyzate culture medium in be
1.8×105Cell/mL.
Then, with 37 DEG C, 100rpm, be blown into 5%CO2Condition, start main culture.In the culture containing soy hydrolyzate
In base, 2% feed-batch culture of Culture liquid measure is added within the 4th, 5,6,7,8,9,10,11,12 and 13 day respectively after culture starts
Base [is added with the soy hydrolyzate (Soy of 2g/L in FMDF-7 culture medium (manufacture of Irvine Scientific company)
Hydrolysate UF, Sigma-Aldrich company manufacture) culture medium].
On the other hand, without containing soy hydrolyzate culture medium in, the 4th after culture starts, 5,6,7,8,9,10,
11,2% supplemented medium of Culture liquid measure is added within 12 and 13 days respectively [in FMDF-7 culture medium (Irvine Scientific
Company's manufacture) in the potassium chloride (and Wako Pure Chemical Industries company manufacture) added with 1.2g/L culture medium].
The 7th day, the 10th day and the 14th day acquisition culture solution, uses Biacore (GE Healthcare after culture starts
Company manufacture) measurement culture solution in darbepoetin composition total output.Further, sample is acquired for these, uses tool
There is the separator [AKTA explorer (manufacture of GE Healthcare company)] of anti-darbepoetin monoclonal antibody column, point
From, obtain each about 600 μ g of darbepoetin composition in acquisition sample.
Using capillary electrophoresis (manufacture of Beckman Coulter company), to obtained darbepoetin composition
Sialic acid isotype distribution be measured.About sialic acid isotype measure of spread, set cartridge (Cartridge) temperature as
25 DEG C, voltage value 6kV, using by 10mmol/L tri- (methylol) methylglycine, 10mmol/L NaCl, 10mmol/L second
The buffer that sour sodium, 7mmol/L urea, 2.5mmol/L putrescine, pH4.7 are constituted is implemented.Each sialic acid additional number will be reached
The gross area value for the darbepoetin composition that the area value of Bei Boting composition is 12~22 relative to sialic acid additional number
The result that ratio (%) is measured is shown in table 1 and Fig. 4.
Table 1
As shown in table 1 and Fig. 4, compared with the culture medium (no Soy) without containing soy hydrolyzate, containing soy hydrolyzate
Culture medium (having Soy) in, sialic acid additional number be 18~22 darbepoetin ratio increase.It is cultivating the 14th day,
The ratio is as follows: sialic acid additional number is the darbepoetin that 18 darbepoetins are 12.3%, sialic acid additional number is 19
It is for the 11.2%, darbepoetin that the darbepoetin that sialic acid additional number is 20 is 12.4%, sialic acid additional number is 21
13.6%, the darbepoetin that sialic acid additional number is 22 is 18.6%, especially sialic acid additional number be 22 reach Bei Bo
The ratio in spit of fland dramatically increases.
As a result, as shown in table 1, in the culture medium containing soy hydrolyzate, the average sialic acid of each molecule
Additional number is 18.7.In addition, compared with the culture medium without containing soy hydrolyzate, in the culture medium containing soy hydrolyzate
In, the total output for the darbepoetin composition that sialic acid additional number is 12~22 increases to about 1.7 times~about 2.2 times.
It is confirmed by result above, soy hydrolyzate is as manufacturing the darbepoetin composition more than sialic acid additional number
Culture media composition be effective.
The inhibition that soy hydrolyzate in [embodiment 2] culture medium is detached from sialic acid from darbepoetin molecule
The training that will be cultivated in the culture medium containing soy hydrolyzate using method preparation same as Example 1
It supports supernatant to save 0~72 hour at 8 DEG C, carries out stability test.In addition, use concentration film (manufacture of Millipore company,
Amicon Ultra-4 30000MWCO) in the training containing soy hydrolyzate prepared using method same as Example 1
It after the culture supernatant cultivated in feeding base is concentrated, is replaced with MilliQ water, prepares soy hydrolyzate and remove liquid, together
Saved 0~72 hour at 8 DEG C to sample.Further, addition in liquid is removed in prepared soy hydrolyzate be used as well known saliva
2- deoxidation -2,3- dehydrogenation of liquid acid enzyme inhibitor-N-acetyl-neuraminate (NeuAc2en) 0.5mM, similarly saves at 8 DEG C
0~72 hour.
Each sample after preservation is analyzed using isoelectric focussing, as a result, removed in liquid in soy hydrolyzate, confirmation
(Fig. 3, swimming lane 2~5) timely is detached from from the high darbepoetin molecule of sialic acid additional number to sialic acid.On the other hand, containing
There is the culture supernatant (Fig. 3, swimming lane 6~9) cultivated in the culture medium of soy hydrolyzate and is removed in liquid in soy hydrolyzate
Addition as sialidase inhibitor NeuAc2en and in the case where being saved, do not confirm sialic acid and added from sialic acid
The high darbepoetin molecule of number is detached from (Fig. 3, swimming lane 10~13).
It is enlightened by result above, the soy hydrolyzate in culture medium inhibits the sialic acid caused by sialidase from sialic acid
The disengaging of the high darbepoetin molecule of additional number.
Using ad hoc fashion, the present invention is described in detail, but be apparent to those skilled in the art
It is that can make various changes and deform in the case where not departing from the intent and scope of the present invention.It should be noted that this Shen
Please based on the Japanese patent application (Japanese Patent Application 2017-040747) that on March 3rd, 2017 submits, by reference to its entirety into
Row is quoted.
Claims (19)
1. a kind of manufacturing method of darbepoetin composition comprising following step 1 and step 2,
Step 1: added with plant origin protolysate culture medium in darbepoetin production cell cultivated and
The step of obtaining culture solution；
Step 2: the step of darbepoetin composition is recycled from culture solution obtained in step 1.
2. a kind of manufacturing method for the darbepoetin composition for inhibiting sialic acid to be detached from from darbepoetin molecule comprising following
Step 1 and step 2,
Step 1: added with plant origin protolysate culture medium in darbepoetin production cell cultivated and
The step of obtaining culture solution；
Step 2: the step of darbepoetin composition is recycled from culture solution obtained in step 1.
3. manufacturing method as claimed in claim 1 or 2, wherein each molecule in culture solution obtained in step 1 reaches Bei Bo
The above are each molecule darbepoetins to contain 18~22 for the 67% of the darbepoetin composition that 12~22 sialic acids are contained in spit of fland
The darbepoetin composition of sialic acid.
4. manufacturing method according to any one of claims 1 to 3, wherein each point in culture solution obtained in step 1
The above are each molecule darbepoetins to contain for the 55% of the darbepoetin composition that sub- darbepoetin contains 12~22 sialic acids
The darbepoetin composition of 19~22 sialic acids.
5. manufacturing method as described in any one of claims 1 to 4, wherein each point in culture solution obtained in step 1
The above are each molecule darbepoetins to contain for the 44% of the darbepoetin composition that sub- darbepoetin contains 12~22 sialic acids
The darbepoetin composition of 20~22 sialic acids.
6. such as manufacturing method according to any one of claims 1 to 5, wherein each point in culture solution obtained in step 1
The above are each molecule darbepoetins to contain for the 18% of the darbepoetin composition that sub- darbepoetin contains 12~22 sialic acids
The darbepoetin composition of 22 sialic acids.
7. such as manufacturing method according to any one of claims 1 to 6, wherein reach Bei Bo in culture solution obtained in step 1
It is high when the total output of spit of fland composition in the culture medium of the protolysate without containing plant origin than cultivating.
8. such as manufacturing method according to any one of claims 1 to 7, wherein it is in host cell that darbepoetin, which produces cell,
Transformed cells obtained from the middle carrier for importing the gene containing coding darbepoetin.
9. manufacturing method as claimed in claim 8, wherein host cell is zooblast.
10. manufacturing method as claimed in claim 9, wherein zooblast is Chinese hamster ovary (CH0) cell, mouse is black
Melanoma (NS0) cell or mouse myeloma (SP2/0) cell or the cell from these cells.
11. such as manufacturing method according to any one of claims 1 to 10, wherein add the protolysate of plant origin
Culture medium is complete synthesis culture medium.
12. the manufacturing method as described in any one of claim 1~11, wherein the plant origin added into culture medium
The concentration of protolysate is 1~5g/l.
13. the manufacturing method as described in any one of claim 1~12, wherein the protolysate of plant origin is source
In the protolysate of soybean.
14. the manufacturing method as described in any one of claim 1~13, wherein further comprise to darbepoetin composition
The purification step purified.
15. manufacturing method as claimed in claim 14, wherein purification step is selected from anion-exchange chromatography, reverse-phase chromatography
Or at least one of gel filtration.
16. the manufacturing method as described in any one of claim 1~15, wherein obtained darbepoetin composition is every
One molecule contains the darbepoetin of average 18 or more sialic acids.
17. a kind of cultural method of darbepoetin production cell comprising: in the training of the protolysate added with plant origin
It supports in base and darbepoetin production cell is cultivated, be secreted into darbepoetin composition in culture solution.
18. a kind of cultural method of darbepoetin production cell comprising: in the training of the protolysate added with plant origin
It supports in base and darbepoetin production cell is cultivated, sialic acid in culture is inhibited to be detached from from darbepoetin molecule.
19. a kind of inhibit the method that sialic acid is detached from from darbepoetin molecule in culture solution preservation comprising: it is being added with plant
Darbepoetin production cell is cultivated in the culture medium of the protolysate in source and obtains culture solution.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2017-040747 | 2017-03-03 | ||
| JP2017040747A JP6258536B1 (en) | 2017-03-03 | 2017-03-03 | Method for producing darbepoetin composition and method for culturing darbepoetin-producing cells |
| PCT/JP2018/008156 WO2018159842A1 (en) | 2017-03-03 | 2018-03-02 | Production method for darbepoetin composition, and culture method for darbepoetin-producing cells |
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| CN110462055A true CN110462055A (en) | 2019-11-15 |
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| CN201880015112.0A Pending CN110462055A (en) | 2017-03-03 | 2018-03-02 | The manufacturing method and darbepoetin of darbepoetin composition produce celliferous cultural method |
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| JP (1) | JP6258536B1 (en) |
| KR (1) | KR102653666B1 (en) |
| CN (1) | CN110462055A (en) |
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| WO (1) | WO2018159842A1 (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999061650A1 (en) * | 1998-05-29 | 1999-12-02 | Genentech, Inc. | Cell culture process for producing glycoproteins |
| KR20070001569A (en) * | 2005-06-29 | 2007-01-04 | 주식회사 셀트리온 | A method of producing erythropoietin having the enhanced sialic acid content using a lower molecular weight fraction of soy hydrolysates and erythropoietin having the enhanced sialic acid content produced by the same |
| CN101065480A (en) * | 2004-10-29 | 2007-10-31 | 巴克斯特国际公司 | Animal protein-free media for cultivation of cells |
| CN102076705A (en) * | 2008-06-24 | 2011-05-25 | 雷迪博士实验室有限公司 | Purification of modified cytokines |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1057534C (en) * | 1993-08-17 | 2000-10-18 | 柯瑞英-艾格公司 | Erythropoietin analogs |
| AT409379B (en) | 1999-06-02 | 2002-07-25 | Baxter Ag | MEDIUM FOR PROTEIN- AND SERUM-FREE CELL CULTURE |
| KR101443257B1 (en) * | 2011-10-18 | 2014-09-19 | 주식회사 종근당 | Methods for Purifying Erythropoietin Analogs Having Lower Isoelectric Point |
| AU2013309506A1 (en) * | 2012-09-02 | 2015-03-12 | Abbvie Inc. | Methods to control protein heterogeneity |
| KR101414897B1 (en) * | 2013-11-29 | 2014-07-04 | 씨제이헬스케어 주식회사 | A method for purifying darbepoetin alfa |
-
2017
- 2017-03-03 JP JP2017040747A patent/JP6258536B1/en active Active
-
2018
- 2018-03-02 TW TW107107037A patent/TW201837050A/en unknown
- 2018-03-02 KR KR1020197025072A patent/KR102653666B1/en active IP Right Grant
- 2018-03-02 WO PCT/JP2018/008156 patent/WO2018159842A1/en active Application Filing
- 2018-03-02 CN CN201880015112.0A patent/CN110462055A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999061650A1 (en) * | 1998-05-29 | 1999-12-02 | Genentech, Inc. | Cell culture process for producing glycoproteins |
| CN101065480A (en) * | 2004-10-29 | 2007-10-31 | 巴克斯特国际公司 | Animal protein-free media for cultivation of cells |
| KR20070001569A (en) * | 2005-06-29 | 2007-01-04 | 주식회사 셀트리온 | A method of producing erythropoietin having the enhanced sialic acid content using a lower molecular weight fraction of soy hydrolysates and erythropoietin having the enhanced sialic acid content produced by the same |
| CN102076705A (en) * | 2008-06-24 | 2011-05-25 | 雷迪博士实验室有限公司 | Purification of modified cytokines |
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| JP6258536B1 (en) | 2018-01-10 |
| TW201837050A (en) | 2018-10-16 |
| KR102653666B1 (en) | 2024-04-03 |
| WO2018159842A1 (en) | 2018-09-07 |
| JP2018143157A (en) | 2018-09-20 |
| KR20190125314A (en) | 2019-11-06 |
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