CN102858978B - Soluble thrombomodulin of high purity and manufacture method thereof - Google Patents
Soluble thrombomodulin of high purity and manufacture method thereof Download PDFInfo
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- CN102858978B CN102858978B CN201180021497.XA CN201180021497A CN102858978B CN 102858978 B CN102858978 B CN 102858978B CN 201180021497 A CN201180021497 A CN 201180021497A CN 102858978 B CN102858978 B CN 102858978B
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- soluble thrombomodulin
- thrombomodulin
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Abstract
The present invention relates to soluble thrombomodulin of high purity, it is for by converting soluble thrombomodulin produced by cell, this conversion cell is to transfect the DNA of the base sequence comprising encoding soluble thrombomodulin to obtain to host cell, wherein, the protein content ratio of host cell resources is relative to 10, and 000U soluble thrombomodulin is less than 10ng.
Description
Technical field
The present invention relates to soluble thrombomodulin of high purity and manufacture method thereof.
Background technology
Thrombomodulin is as having by being combined the blood clotting activity blocking thrombin with Thrombin specificity
And remarkably promote the material of the effect of the protein C activation ability of thrombin and known, and known its has strength simultaneously
Blood coagulation blocking effect.Known its extends setting time based on thrombin action, suppresses platelet based on thrombin
Assemble.Protein C is the vitamin k-dependent protein matter played an important role in blood coagulation fibrinolytic system, at thrombin
Effect under be activated, become activated protein C.This activated protein C known makes the work of the blood clotting system factor in vivo
Property type accelerin and active form VIII factors inactive, and also with there is the plasminogen activator of thrombolytic effect
Generation relevant (non-patent literature 1).Result, it is believed that thrombomodulin utilize this thrombin to promote the activation of protein C,
Being useful as resisting blood coagulation agent or thrombolytic agent, the most also Report on Animal points out that thrombomodulin is companion
It is effectively (non-patent literature 2) with solidifying in the treatment of hyperfunction disease, prevention.
In the past, thrombomodulin was as the sugar expressed on the vascular endothelial cell in the various animal kinds headed by people
Protein and be found obtain, the most successfully carry out cloning.That is, gene engineering method is utilized, from people's lung cDNA library
The gene of the human thrombomodulin precursor containing signal peptide is cloned, and the complete genome sequence to thrombomodulin
Resolve, specify that the aminoacid sequence of 575 residues containing signal peptide (generally exemplifying 18 amino acid residues) (specially
Profit document 1).The ripe thrombomodulin that signal peptide is cut off is made up of following 5 regions: from the N-terminal side of its mature peptide
The N-terminal region risen (No. 1-226: represent to be position during 18 amino acid residues at handshaking signal peptide, lower with), there are 6
The region (No. 227-462) of EGF spline structure, O type sugar chain increase region (No. 463-498), the through region of film (No. 499-521),
And Cytoplasm inner region (No. 522-557), and as there is the part of identical activity with total length thrombomodulin (
Little activity unit), it is known that among the region with 6 EGF spline structures main by the 4th from N-terminal side, 5, No. 6
The part (non-patent literature 3) that EGF spline structure is constituted.
The thrombomodulin of total length is then difficult to dissolve without the existence of surfactant, must add as preparation
Surfactant, on the other hand, also can occur the solvable of dissolving well even if existing in the case of not having surfactant
Property thrombomodulin.As long as making soluble thrombomodulin at least not contain part or all of the through region of film to enter
Prepared by row, such as, the region only by N-terminal region, having 6 EGF spline structures and O type sugar chain increase these 3 districts, region
Territory constitutes the soluble thrombomodulin of (that is, by the 19th～516 amino acids Sequence composition of sequence numbering 1) can be by answering
Obtain with recombinant technique, and confirm this recombinant soluble thrombomodulin there is natural thrombomodulin
Activity (patent documentation 1).In addition the example as soluble thrombomodulin also has some to report (patent documentation 2～9).Separately
The outer soluble thrombomodulin also having enumerated Urina Hominis source as natural type etc. (patent documentation 10,11).
Incidentally, in gene, the variation by naturally variation or when obtaining, confirmed such as most cases that
Sample, have also discovered pleomorphism variation, the human thrombomodulin that the above-mentioned aminoacid sequence by 575 residues is constituted in people
473rd amino acids of precursor is by Val's with being confirmed now by Ala.In encoding this amino acid whose base sequence,
It is respectively equivalent to have T variation and C to make a variation (non-patent literature 4) on the 1418th.But, it is complete in terms of activity and physical property
Zero difference, can determine that both are substantially the same.
Thrombomodulin is effective (non-patent literature 5,6) in the treatment of DIC to have report to point out.Adjust as thrombosis
The purposes of joint albumen, in addition to the foregoing, is desirable to its treatment and prevention for the most following disease: acute coronary is combined
Simulator sickness (ACS), thrombosis, peripheral vessel obliterans, Arteriosclerosis obliterans, vasculitis, the Secondary cases function of operation on heart
Sexual disorders, the complication of organ transplantation, angina pectoris, temporal cerebral ischemia seizure, hypertensive state of pregnancy, diabetes, liver VOD (Liver
veno-occlusive disease；Occlusion of hepatic vein disease after acute hepatitis and bone marrow transplantation), deep vein thrombosis
(DVT；Deep venous thrombosis) etc.；And adult respiratory distress syndrome (ARDS；Adult respiratory
distress syndrome)。
Premised in view of the application in medicine, need in large quantities and manufacture solubility thrombosis with lower cost to adjust
Joint albumen, this is from it goes without saying that, but has document to point out, derive from manufacturing process foreign protein matter (such as host cell resources
The bovine serum albumin in protein, culture medium source, the mouse IgG etc. in antibody column source) there is immunogenicity, in terms of safety
Consider possible problematic (non-patent literature 7).
In view of the application in medicine, as the method manufacturing soluble thrombomodulin with industrial level, such as
The known affinity column chromatography having employing to be supported with the antibody for thrombomodulin is as the method for main refining step；Enter one
Walking the known manufacture method having following soluble thrombomodulin of high purity, it is for containing substantially no serum origin thing and resisting
The manufacture method of the soluble thrombomodulin of high purity of body source thing, this manufacture method is characterised by, will be by affine
This soluble thrombomodulin that chromatography obtains electrical conductivity be 25ms/cm～34ms/cm, pH be under conditions of 3～4 with
Cation exchange column contacts, and in this operation, obtains this solubility thrombomodulin with the form of the fraction that circulates (the logical り of element draws and divides)
In vain (patent documentation 12).It is known that combination strong anion exchange column color after the affinity column chromatography operation of main refining step
The process for purification (patent documentation 13) of spectrometry.
Prior art literature
Patent documentation
Patent documentation 1: Japanese Laid-Open Patent Publication 64-6219 publication
Patent documentation 2: Japanese Unexamined Patent Publication 2-255699 publication
Patent documentation 3: Japanese Unexamined Patent Publication 3-133380 publication
Patent documentation 4: Japanese Unexamined Patent Publication 3-259084 publication
Patent documentation 5: Japanese Unexamined Patent Publication 4-210700 publication
Patent documentation 6: Japanese Unexamined Patent Publication 5-213998 publication
Patent documentation 7:WO92/00325 publication
Patent documentation 8:WO92/03149 publication
Patent documentation 9:WO93/15755 publication
Patent documentation 10: Japanese Unexamined Patent Publication 3-86900 publication
Patent documentation 11: Japanese Unexamined Patent Publication 3-218399 publication
Patent documentation 12: Japanese Unexamined Patent Publication 11-341990 publication
Patent documentation 13:WO2008/117735 publication
Non-patent literature
Non-patent literature 1: Suzuki is grand to be controlled, medical science あ ゆ body (Advances in Medicine), volume 125, page 901 (nineteen eighty-three)
Non-patent literature 2:K.Gomi etc., Blood75.1396-1399 (1990)
Non-patent literature 3:M.Zushi etc., J.Biol.Chem., 264,10351-10353 (1989)
Non-patent literature 4:D.Z.Wen etc., Biochemistry, 26,4350-4357 (1987)
Non-patent literature 5:S.M.Bates etc., Br.J.Pharmacol., 144,1017-1028 (2005)
Non-patent literature 6:H.Saito etc., J.Thromb Haemost, 5 (1), 31 (2007)
Non-patent literature 7: early river husband, バ イ オ doctor's product と product safety protects (biologics
Exploitation and quality-safety guarantee), 273-274 (2007)
Summary of the invention
Invent problem to be solved
The problem of the present invention is that the protein concentration ratio providing host cell resources is relative to 10,000U solubility
Thrombomodulin is less than soluble thrombomodulin of high purity and the manufacture method thereof of 10ng.
Solve the means of problem
Patent documentation 13 is recorded through refined soluble thrombomodulin.Particularly in the embodiment of the document
The concentration of the protein (following, to be also referred to as " HCP " in this manual) disclosing Hosts in 14 is designated as " N.D. "
Although the soluble thrombomodulin of (be not expressly recited, but think it means that " detection ").See the ability of this record
Field technique personnel will be considered that the soluble thrombomodulin of high purity being minimized that is mixed into having adequately achieved HCP, also
Would not expect attempting to reduce the HCP in soluble thrombomodulin, i.e. attempt to reduce HCP in soluble thrombomodulin
The motivation being mixed into etc. the most non-existent.
But, the present inventor recognizes following significant problem strongly: using refined soluble thrombomodulin as
In the case of medicine uses, if being mixed into HCP, then it can cause the unforeseen situations such as anaphylactic shock, according to circumstances can
There is lethal risk.So, it is " N.D. " described in the embodiment 14 of above-mentioned patent documentation 13, thus people in the art
Member can be generally thought being mixed into of HCP and obtained abundant reduction, and the present inventor is to described in the embodiment 14 of above-mentioned patent documentation 13
The HCP concentration through refined soluble thrombomodulin be measured found that, although there is quantitation limit, but
The HCP concentration being mixed into is relative to soluble thrombomodulin 10, and 000U is (described in reference example 1 as be described hereinafter, as long as no spy
Not limiting, " U " means to represent the unit of the effect (being hereinafter also referred to as APC activity) promoting protein C activation.Lower same)
Showing 70ng～the ratio less than 80ng, the present inventor first confirms that the reduction about HCP may still have more than improvement
Ground.About this HCP concentration, the present inventor is alone it is considered that there is no disclosure by using in measuring at HCP in patent documentation 13
This new process of enrichment process, concentration accurately can be measured.
First the present inventor is found that the above-mentioned fact, it is contemplated that using safer for soluble thrombomodulin as medicine
Product utilize, it is thus achieved that the thrombosis through refining that the HCP content in soluble thrombomodulin is minimized further
Regulation albumen, thus it is found that the risk making anaphylactic shock is this novel problem of irreducible minimum.
The present inventor adjusts for the high-purity solubility thrombosis being minimized further that is mixed into manufacturing HCP with industrial level
Joint albumen, the importing to Novel pole chromatograph operation is studied.That is, attempted by it is believed that HCP removal effect is the highest
Affinity column chromatography in combine multiple column chromatography to reduce HCP, but add column chromatography operation and may create the problem that and not only increase
Manufacture labour and the time but also the yield of soluble thrombomodulin can be reduced.Further, in affinity column chromatography, i.e.
Make the multiple column chromatography of combination, also cannot obtain the high-purity solubility blood that HCP concentration is further reduced to more than existing level
Bolt regulation albumen.Vary slightly making HCP adjust with solubility thrombosis additionally, column chromatography also has only pH or ionic strength etc.
The problem that the separation of joint albumen changes, can not reproduce expected result, it is difficult to obtain the regulation of high-purity solubility thrombosis
Albumen.
Therefore, the present inventor in order to find out more easy and simple to handle than column chromatography, soluble thrombomodulin will not be reduced
Yield, effectively can remove with industrial level HCP, can obtain HCP be mixed into the high-purity solubility blood being minimized further
The method of bolt regulation albumen conducts in-depth research, it was found that by using nylon and/or polyether sulfone, especially nylon, can
It is less than the high-purity solubility thrombomodulin of 10ng relative to 10,000U soluble thrombomodulin with acquisition HCP concentration
In vain, above-mentioned problem can be solved, thus complete the present invention.
That is, the present invention can enumerate herein below.
(1) a kind of soluble thrombomodulin of high purity, it is for by converting solubility thrombosis regulation produced by cell
Albumen, this conversion cell is that the DNA of the base sequence by comprising encoding soluble thrombomodulin transfects to host cell
Obtaining, wherein, the protein content ratio of this host cell resources is relative to 10, and 000U soluble thrombomodulin is little
In 10ng.
(1-2) soluble thrombomodulin of high purity as described in above-mentioned (1), wherein, high-purity solubility thrombosis is adjusted
Joint albumen purity in gross protein is more than 99%.
Soluble thrombomodulin of high purity described in (1-3) above-mentioned (1) or (1-2), wherein, high-purity solubility blood
Bolt regulation albumen is the form of aqueous solution.
Soluble thrombomodulin of high purity described in (1-4) above-mentioned (1-3), wherein, high-purity solubility thrombosis is adjusted
The concentration that soluble thrombomodulin concentration is more than 8mg/mL in joint protein solution.
(2) soluble thrombomodulin of high purity as described in any one of above-mentioned (1)～(1-4), wherein, solubility
Thrombomodulin is by this conversion cell is carried out the thrombomodulin that serum-free culture produces.
It should be noted that the item numbering carrying out quoting as above-mentioned (1)～(1-4) represents with scope, at its model
In the case of enclosing the item that interior configuration has branch's numberings such as (1-2), it is meant that also refer to that there is branch's numberings such as (1-2)
?.Lower same.
(2-2) soluble thrombomodulin of high purity as described in any one of above-mentioned (1)～(2), is wherein measuring
During the protein content of this host cell resources, at least include that the method for following operation is measured by utilizing, confirm phase
Protein concentration for the 10,000U soluble thrombomodulin host cell resources less than 10ng；
A the DNA comprising the base sequence of encoding soluble thrombomodulin is transfected to host cell, to obtained by ()
Convert cell or its host cell one of arbitrarily carry out serum-free culture, obtained culture supernatant prepare host
The operation of the protein of cell derived；
B () makes rabbit sensitization with the protein of this host cell resources of above-mentioned (a), obtained antiserum refine anti-
The operation of the antibody of host cell resources protein；
Structure includes the operation of the mensuration system of following (c1)～(c6):
(c1) antibody making this anti-host cell resources protein of above-mentioned (b) is adsorbed in the operation of solid phase；
(c2-1) suspection is made to be mixed into the treating containing soluble thrombomodulin of protein of this host cell resources
Detection solution carry out with this solid phase of the antibody being adsorbed with this anti-host cell resources protein the operation that contacts and make containing
The solution of the protein of host cell resources known to concentration be adsorbed with this anti-host cell resources protein antibody should
Solid phase carries out the operation contacted；
(c3) in this solid phase, add the operation of the antibody of biotinylated anti-host cell resources protein；
(c4) in this solid phase, add the operation of avidinated Peroxidase Solution；
(c5) operation that enzyme substrate solution carries out developing the color is added；And
(c6) colour developing, the operation that its absorbance is measured are stopped；
(d) utilize said determination system to suspect be mixed into this host cell resources protein containing solubility thrombosis
The protein concentration of the host cell resources in the solution to be detected of regulation albumen is measured, it is judged that should be containing solubility thrombosis
Whether the protein concentration of the host cell resources in the solution to be detected of regulation albumen is included in determining of said determination system
Operation in weight range, this can quantification range be next by utilizing said determination system to use containing host cell known to concentration
The solution of the protein in source is measured and confirms in advance；
(e-1) in above-mentioned (d), adjusting containing solubility thrombosis of the protein being mixed into this host cell resources is being suspected
The protein concentration of host cell resources in the solution to be detected of joint albumen be included in can be in quantification range in the case of, should
Concentration is as the operation of the protein concentration of this host cell resources；
(e-2-1) in above-mentioned (d), suspect be mixed into this host cell resources protein containing solubility thrombosis
The protein concentration of host cell resources in the solution to be detected of regulation albumen be not included in can be in quantification range in the case of,
For become be included in said determination system can measured concentration in quantification range, as required to containing solubility blood
The solution to be detected of bolt regulation albumen concentrates or dilutes, the operation recording this enrichment factor or dilution rate；
(e-2-2) (c2-1) in the mensuration system that will represent with above-mentioned (c1)～(c6) replaces with (c2-as follows
2), utilize this mensuration system to carried out concentrating in above-mentioned (e-2-1) or dilution containing soluble thrombomodulin
The protein concentration of the host cell resources in solution to be detected is measured, it is considered to enrichment factor or dilution rate obtain this host
The operation of the protein concentration of cell derived；
(c2-2) make to have carried out as required concentrating or the solution to be detected containing soluble thrombomodulin of dilution
Carry out the operation that contacts with this solid phase of the antibody being adsorbed with this anti-host cell resources protein and make containing concentration known
The solution of protein of host cell resources carry out with this solid phase of the antibody being adsorbed with this anti-host cell resources protein
The operation of contact；And
F () additionally determines the thrombosis regulation in the solution to be detected that per unit volume contains soluble thrombomodulin
The APC activity of albumen, calculates the protein concentration of the host cell resources obtained in (e-1) or (e-2-2) relative to this APC
The operation of the ratio of activity.
(2-3) soluble thrombomodulin of high purity as described in above-mentioned (2-2), wherein, in above-mentioned (2-2) (a)
Host cell is Chinese hamster ovary cell DXB11 strain.
(3) soluble thrombomodulin of high purity as described in any one of above-mentioned (1)～(2-3), wherein, this host
Cell is Chinese hamster ovary cell.
(4) soluble thrombomodulin of high purity as described in any one of above-mentioned (1)～(3), wherein, this solubility
Thrombomodulin is the soluble thrombomodulin of the character with following (1)～(5)；
(1) with the effect of thrombin selective binding；
(2) effect of the activation of protein C based on thrombin is promoted；
(3) effect of setting time based on thrombin is extended；
(4) effect of platelet aggregation based on thrombin is suppressed；And
(5) antiinflammatory action.
(4-2) soluble thrombomodulin of high purity as described in any one of above-mentioned (1)～(3), wherein, this is solvable
Property thrombomodulin is the soluble thrombomodulin of the character with following (1)～(4)；
(1) with the effect of thrombin selective binding；
(2) effect of the activation of protein C based on thrombin is promoted；
(3) effect of setting time based on thrombin is extended；And
(4) effect of platelet aggregation based on thrombin is suppressed.
(5) soluble thrombomodulin of high purity as described in any one of above-mentioned (1)～(4-2), wherein, this is solvable
The molecular weight of property thrombomodulin is 50,000 to 80,000.
(6) soluble thrombomodulin of high purity as described in any one of above-mentioned (1)～(5), wherein, this high-purity
Soluble thrombomodulin is by including what the method for following operation manufactured:
A the DNA comprising the base sequence of encoding soluble thrombomodulin is transfected to host cell by (), it is thus achieved that convert
The operation of cell；
B () cultivates this conversion cell, it is thus achieved that the operation of the solution containing soluble thrombomodulin；And
C () makes this solution containing soluble thrombomodulin contact with nylon and/or polyether sulfone, obtaining high-purity can
The operation of dissolubility thrombomodulin, in this soluble thrombomodulin of high purity, the protein content of host cell resources
Ratio is less than 10ng relative to 10,000U soluble thrombomodulin.
(7) soluble thrombomodulin of high purity as described in any one of above-mentioned (1)～(6), wherein, this solubility
Thrombomodulin is following soluble thrombomodulin:
In its aminoacid sequence described in any one containing (i) sequence numbering 9 or sequence numbering 11 the 367th～
480 amino acids sequences and the peptide of the arbitrary amino acid sequence containing following (ii-1) or (ii-2), this peptide has following (1)
～the character of (5),
(ii-1) the 19th～244 in the aminoacid sequence described in any one of sequence numbering 9 or sequence numbering 11
Aminoacid sequence；Or
(ii-2) aminoacid sequence above-mentioned (ii-1) has the aminoacid of 1 or more than 2 replace, lack or
The aminoacid sequence added,
(1) with the effect of thrombin selective binding；
(2) effect of the activation of protein C based on thrombin is promoted；
(3) effect of setting time based on thrombin is extended；
(4) effect of platelet aggregation based on thrombin is suppressed；And
(5) antiinflammatory action.
(7-2) soluble thrombomodulin of high purity as described in any one of above-mentioned (1)～(6), wherein, this is solvable
Property thrombomodulin is following soluble thrombomodulin:
In its aminoacid sequence described in any one containing (i) sequence numbering 9 or sequence numbering 11 the 367th～
480 amino acids sequences and the peptide of the arbitrary amino acid sequence containing following (ii-1) or (ii-2), this peptide has following (1)
～the character of (4)；
(ii-1) the 19th～244 in the aminoacid sequence described in any one of sequence numbering 9 or sequence numbering 11
Aminoacid sequence；Or
(ii-2) aminoacid sequence above-mentioned (ii-1) has the aminoacid of 1 or more than 2 replace, lack or
The aminoacid sequence added,
(1) with the effect of thrombin selective binding；
(2) effect of the activation of protein C based on thrombin is promoted；
(3) effect of setting time based on thrombin is extended；And
(4) effect of platelet aggregation based on thrombin is suppressed.
(7-3) soluble thrombomodulin of high purity as described in any one of above-mentioned (1)～(6), wherein, this is solvable
Property thrombomodulin is following soluble thrombomodulin: it is the arbitrary amino acid sequence containing following (i-1) or (i-2)
Row and the peptide of the arbitrary amino acid sequence containing following (ii-1) or (ii-2), this peptide has following (1)～the character of (5),
(i-1) the 367th～480 in the aminoacid sequence described in any one of sequence numbering 9 or sequence numbering 11
Aminoacid sequence；Or
(i-2) aminoacid sequence above-mentioned (i-1) there is the aminoacid of 1 or more than 2 replace, lack or add
The aminoacid sequence added,
(ii-1) the 19th～244 in the aminoacid sequence described in any one of sequence numbering 9 or sequence numbering 11
Aminoacid sequence；Or
(ii-2) aminoacid sequence above-mentioned (ii-1) has the aminoacid of 1 or more than 2 replace, lack or
The aminoacid sequence added,
(1) with the effect of thrombin selective binding；
(2) effect of the activation of protein C based on thrombin is promoted；
(3) effect of setting time based on thrombin is extended；
(4) effect of platelet aggregation based on thrombin is suppressed；And
(5) antiinflammatory action.
(8) soluble thrombomodulin of high purity as described in any one of above-mentioned (1)～(6), wherein, this solubility
Thrombomodulin is following soluble thrombomodulin: it is the arbitrary amino acid sequence with following (i-1) or (i-2)
Peptide, this peptide has following (1)～the character of (5)；
(i-1) the 19th～516 ammonia in the aminoacid sequence described in any one of sequence numbering 9 or sequence numbering 11
Base acid sequence；Or
(i-2) aminoacid sequence above-mentioned (i-1) there is the aminoacid of 1 or more than 2 replace, lack or add
The aminoacid sequence added,
(1) with the effect of thrombin selective binding；
(2) effect of the activation of protein C based on thrombin is promoted；
(3) effect of setting time based on thrombin is extended；
(4) effect of platelet aggregation based on thrombin is suppressed；And
(5) antiinflammatory action.
(9) soluble thrombomodulin of high purity as described in any one of above-mentioned (1)～(6), wherein, comprises coding
The DNA of the base sequence of this soluble thrombomodulin is described in any one of coded sequence numbering 9 or sequence numbering 11
The DNA of aminoacid sequence.
(10) a kind of pharmaceutical composition, its high-purity solubility thrombosis described in any one containing above-mentioned (1)～(9) is adjusted
Joint albumen and pharmaceutically suitable carrier.
(11) a kind of manufacture method, it is the manufacture method of soluble thrombomodulin of high purity, and described high-purity can
In dissolubility thrombomodulin, the protein content ratio of host cell resources is relative to 10, and 000U solubility thrombosis regulates
Albumen is less than 10ng, and wherein, the method includes following operation: will comprise the base sequence of encoding soluble thrombomodulin
DNA transfects to host cell, obtains converting cell, makes the solution containing soluble thrombomodulin that this conversion cell produces
Contact with nylon and/or polyether sulfone.
(12) manufacture method as described in above-mentioned (11), wherein, soluble thrombomodulin is by thin to this conversion
Born of the same parents carry out serum-free culture and produce.
(13) manufacture method of the soluble thrombomodulin of high purity as described in above-mentioned (11) or (12), wherein, should
Soluble thrombomodulin is the soluble thrombomodulin of the character with following (1)～(5):
(1) with the effect of thrombin selective binding；
(2) effect of the activation of protein C based on thrombin is promoted；
(3) effect of setting time based on thrombin is extended；
(4) effect of platelet aggregation based on thrombin is suppressed；And
(5) antiinflammatory action.
(14) manufacture method as described in any one of above-mentioned (11)～(13), wherein, this host cell is Chinese hamster
Gonad cell.
(15) manufacture method as described in any one of above-mentioned (11)～(14), wherein, this soluble thrombomodulin
Molecular weight be 50,000 to 80,000.
(16) manufacture method as described in any one of above-mentioned (11)～(15), wherein, this soluble thrombomodulin
For following soluble thrombomodulin:
In its aminoacid sequence described in any one containing (i) sequence numbering 9 or sequence numbering 11 the 367th～
480 amino acids sequences and the peptide of the arbitrary amino acid sequence containing following (ii-1) or (ii-2), this peptide has following (1)
～the character of (5)；
(ii-1) the 19th～244 in the aminoacid sequence described in any one of sequence numbering 9 or sequence numbering 11
Aminoacid sequence；Or
(ii-2) aminoacid sequence above-mentioned (ii-1) has the aminoacid of 1 or more than 2 replace, lack or
The aminoacid sequence added,
(1) with the effect of thrombin selective binding；
(2) effect of the activation of protein C based on thrombin is promoted；
(3) effect of setting time based on thrombin is extended；
(4) effect of platelet aggregation based on thrombin is suppressed；And
(5) antiinflammatory action.
(17) manufacture method as described in any one of above-mentioned (11)～(15), wherein, this soluble thrombomodulin
For following soluble thrombomodulin: it is the peptide of the arbitrary amino acid sequence with following (i-1) or (i-2), this peptide has
There are following (1)～the character of (5),
(i-1) the 19th～516 ammonia in the aminoacid sequence described in any one of sequence numbering 9 or sequence numbering 11
Base acid sequence；
(i-2) aminoacid sequence above-mentioned (i-1) there is the aminoacid of 1 or more than 2 replace, lack or add
The aminoacid sequence added,
(1) with the effect of thrombin selective binding；
(2) effect of the activation of protein C based on thrombin is promoted；
(3) effect of setting time based on thrombin is extended；
(4) effect of platelet aggregation based on thrombin is suppressed；And
(5) antiinflammatory action.
(18) manufacture method as described in any one of above-mentioned (11)～(15), wherein, comprises this solubility thrombosis of coding
The DNA that DNA is the aminoacid sequence described in coded sequence numbering 9 or sequence numbering 11 of the base sequence of regulation albumen.
(19) manufacture method as described in any one of above-mentioned (11)～(18), wherein, nylon and/or the form of polyether sulfone
Form for filter membrane.
(20) manufacture method as described in above-mentioned (19), wherein, the membrane area of filter membrane is relative to 1mg host cell resources
Protein is 0.01m2To 0.5m2。
(21) manufacture method as described in any one of above-mentioned (11)～(20), wherein, nylon and/or polyether sulfone are Buddhist nun
Dragon.
(22) a kind of manufacture method, it is the system of the soluble thrombomodulin of high purity described in above-mentioned (11)～(20)
Making method, the method has (1-2)～(1～4), (2-2), (2-3), the feature described in any one of (7-2) or (7-3).
(23) manufacture method as described in any one of above-mentioned (11)～(22), it is high-purity solubility thrombomodulin
White manufacture method, in described soluble thrombomodulin of high purity, the protein content ratio of host cell resources is phase
For 10,000U soluble thrombomodulin is less than 10ng, and the method includes following operation:
A the DNA of the aminoacid sequence described in coded sequence numbering 9 or sequence numbering 11 is transfected to host cell by (),
Obtain the operation converting cell；
B () cultivates this conversion cell, it is thus achieved that the operation of the solution containing soluble thrombomodulin；
C thrombomodulin purity that this solution containing soluble thrombomodulin is refined in gross protein by ()
It it is the operation of more than 99%；And
D () makes this solution containing soluble thrombomodulin contact with nylon, regulated by high-purity solubility thrombosis
The operation of Protein Separation, in this soluble thrombomodulin of high purity, the protein content ratio of host cell resources is phase
For 10,000U soluble thrombomodulin is less than 10ng,
Described host cell is Chinese hamster ovary cell.
(24) a kind of soluble thrombomodulin of high purity, it is to be manufactured by the method described in above-mentioned (23)
's.
(25) a kind of method, it is the side of the protein removing the host cell resources in soluble thrombomodulin
Method, the method includes following operation: transfect thin to host by the DNA comprising the base sequence of encoding soluble thrombomodulin
Born of the same parents, obtain converting cell, make the solution containing soluble thrombomodulin and nylon and/or polyethers that this conversion cell produces
Sulfone contacts.
(26) method as described in above-mentioned (25), it is to remove the host cell resources in soluble thrombomodulin
Method of protein, the method includes following operation:
A the DNA of the aminoacid sequence described in coded sequence numbering 9 or sequence numbering 11 is transfected to host cell by (),
Obtain the operation converting cell；
B () cultivates this conversion cell, it is thus achieved that the operation of the solution containing soluble thrombomodulin；
C thrombomodulin purity that this solution containing soluble thrombomodulin is refined in gross protein by ()
It it is the operation of more than 99%；And
D () makes the operation that this solution containing soluble thrombomodulin contacts with nylon,
Described host cell is Chinese hamster ovary cell.
(27) a kind of method, it is the host cell resources removed in soluble thrombomodulin described in above-mentioned (25)
Method of protein, the method has the feature described in any one of above-mentioned (1)～(9).
The effect of invention
By use the present invention manufacture method, it is possible to obtain host cell resources protein content ratio be relative to
10,000U soluble thrombomodulin less than 10ng, host cell resources protein be mixed into obtained reduce height
Purity soluble thrombomodulin.Thereby, it is possible to reduce soluble thrombomodulin as allergy during medicine utilization
Property shock risk.
Detailed description of the invention
Below some optimal ways of the present invention (are used for implementing the optimal way of the present invention: following, in this manual
Also referred to as " embodiment ") it is specifically described, but the scope of the present invention is not limited to the ad hoc fashion of the description below.
The soluble thrombomodulin of high purity of present embodiment can use as medicine material.Also can be by this
The soluble thrombomodulin of high purity of embodiment uses as medicine with the combination of other pharmaceutically suitable carrier.
It addition, the soluble thrombomodulin of high purity of present embodiment can be to contain substantially no solubility thrombosis
Material, pharmaceutical composition containing highly purified soluble thrombomodulin form beyond regulation albumen makes
With.The medicine that can also combine with other pharmaceutically suitable carrier with the soluble thrombomodulin of high purity of present embodiment
The form of compositions uses.
Hereinafter, sometimes include that the medicine material of soluble thrombomodulin of high purity is referred to as the regulation of high-purity thrombosis
Albumen.It addition, sometimes include containing substantially no the material beyond soluble thrombomodulin containing high-purity solubility
Thrombomodulin pharmaceutical composition is referred to as soluble thrombomodulin of high purity.
For the thrombomodulin in present embodiment, it is known that it has: (1) and thrombin selective binding, (2)
Promote the effect of the activation of protein C based on thrombin.And the most generally approve that it has (3) and extends based on thrombin
The effect of setting time, (4) suppress effect and/or (5) antiinflammatory action of platelet aggregation based on thrombin.By these blood
The effect that bolt regulation albumen is had is referred to as thrombomodulin activity.
As thrombomodulin activity, preferably have above-mentioned (1) and the effect of (2), further have above-mentioned (1)～
(4) effect.Further, as thrombomodulin activity, more preferably possess whole effects of (1)～(5).
Thrombomodulin such as can utilize with the combination of thrombin with Thrombosis and
Haemostasis1993 70 (3): 418-422 be representative various known document in described test method carry out really
Recognize.The effect promoting the activation of protein C based on thrombin such as can utilize and with Japanese Laid-Open Patent Publication 64-6219 publication be
The test method being expressly recited in the various known document represented easily live vol to the effect promoting protein C activation
Or it is with or without confirming.Alternatively, it is also possible to similarly easy to extending the effect of setting time based on thrombin and/or pressing down
The effect making platelet aggregation based on thrombin confirms.Further, for antiinflammatory action, it is also possible to utilize such as with
Blood 2008112:3361-3670, The Journal of Clinical Investigation 20051155:1267-
In the various known document that 1274 is representative, described test method confirms.
As soluble thrombomodulin, can enumerate do not have in the presence of surfactant water-soluble solvable
Property thrombomodulin is as example.As the deliquescent preferred exemplary of soluble thrombomodulin, can exemplify water,
Such as distilled water for injection is (under conditions of there is not the surfactant such as TritonX-100 or polidocanol, generally in neutrality
Near) in be more than 1mg/ml or for more than 10mg/ml, preferably more than 15mg/ml or be more than 17mg/ml, enter
One step is preferably more than 20mg/ml, more than 25mg/ml or more than 30mg/ml, can preferably enumerate especially into 60mg/ml with
On, according to circumstances, more than 80mg/ml or more than 100mg/ml can be enumerated respectively.To soluble thrombomodulin it is being
When no solubilized judges it can be understood as, after dissolution, such as in the underface of white light source, bright at about 1000lux
The position of degree, for clarification in the case of utilizing naked eyes to observe, not contain the insoluble substance work that can substantially confirm degree
Situation for direct indicator.Additionally, it is possible to carry out filtering the presence or absence confirming residue.
For soluble thrombomodulin, as long as there is thrombomodulin activity as mentioned above, for solubility, then
Its molecular weight indefinite, as the upper limit of molecular weight, preferably 100, less than 000, more preferably 90, less than 000, further
Be preferably 80, less than 000, particularly preferably 70, less than 000, as the lower limit of molecular weight, more preferably 50,000 with
Above, particularly preferably more than 60,000.The molecular weight of soluble thrombomodulin may utilize and measures the logical of protein molecular weight
Chang Fangfa is easily measured, but is preferably measured by mass analysis, more preferably MALDI-TOF-MS method.
In order to obtain the soluble thrombomodulin of purpose Range molecular weight, as described later, by encoding soluble thrombosis
The DNA of regulation albumen utilizes carrier transfection in host cell, and by cultivating prepared conversion cell, obtain can
Dissolubility thrombomodulin, utilizes column chromatography etc. that obtained soluble thrombomodulin is carried out classification, it is possible to
Obtain the soluble thrombomodulin of purpose Range molecular weight.
As soluble thrombomodulin, preferably comprising following aminoacid sequence, this aminoacid sequence is human-like thrombosis
In regulation albumen the 19th～132 amino acids sequences of sequence numbering 1 and sequence numbering 9 or the 19th of sequence numbering 11 the～
In 244 amino acids sequences or this aminoacid sequence one or the aminoacid of more than 2 can replace, lacks, add
Aminoacid sequence.19th～132 amino acids sequences of this sequence numbering 1 are and the promotion base in thrombomodulin activity
In the sequence that the effect of the activation of the protein C of thrombin is relevant.On the other hand, sequence numbering 9 or the 19th of sequence numbering 11 the
～244 amino acids sequence be the sequence relevant to the antiinflammatory action in thrombomodulin activity.Solubility thrombosis is adjusted
For joint albumen, as long as there is thrombomodulin activity, then this sequence numbering 1 as soluble thrombomodulin entirety
19th～132 amino acids sequences can also nature or artificial variation, i.e. the 19th～132 amino acids sequences of sequence numbering 1
In the aminoacid of one or more than 2 can replace, lack, add.As long as having thrombomodulin activity, the most right
The degree of variation allowed is not particularly limited, such as, can exemplify the homology, preferably of more than 50% as aminoacid sequence
The homology of more than 70%, the homology of more preferably more than 80%, the homology of further preferred more than 90%, particularly preferred 95% with
On homology, the homology of most preferably more than 98%.By in such aminoacid sequence or the aminoacid of more than 2
The aminoacid sequence replace, lack, added is referred to as identical series of variation.These are made a variation, as described later, as long as using
Common gene manipulation techniques can be readily available.For soluble thrombomodulin, as long as having above-mentioned sequence, conduct
Soluble thrombomodulin entirety at least has and thrombin selective binding, the work of promotion protein C based on thrombin
The effect changed, is just not particularly limited, has antiinflammatory action the most simultaneously.
The sequence of sequence numbering 3 is the sequence that the 125th amino acids Val variation is Ala of the sequence of sequence numbering 1, makees
For the soluble thrombomodulin in present embodiment, the most also comprise the 19th～132 amino acids sequences of sequence numbering 3
Row.
As such soluble thrombomodulin, if at least have sequence numbering 1 or the 19th of sequence numbering 3 the～
132 bit sequences or their identical series of variation and sequence numbering 9 or the 19th～244 bit sequences of sequence numbering 11 or they
Identical series of variation, as soluble thrombomodulin entirety at least have with thrombin selective binding and promote based on
The effect of the protein C activation of thrombin, is just not particularly limited, and can enumerate by sequence numbering 1 or sequence numbering 3
Peptide that 19～132 or the 17th～132 bit sequences are constituted or by the identical series of variation of above-mentioned sequence and sequence numbering 9
Or the 19th～244 bit sequences of sequence numbering 11 or their identical series of variation are constituted and as soluble thrombomodulin
Overall at least have the peptide of thrombomodulin activity as preferred example, more preferably by sequence numbering 1 or sequence numbering 3
The 19th～132 bit sequences and sequence numbering 9 or the 19th～244 bit sequences of sequence numbering 11 or their identical variation sequence
The peptide that row are constituted.It addition, there is also more preferably by the 19th～132 in sequence numbering 1 or sequence numbering 3 or the 17th～
The identical series of variation of 132 and sequence numbering 9 or the 19th～244 bit sequences of sequence numbering 11 or their identical variation
Sequence composition and the alternate manner as the overall peptide at least with thrombomodulin activity of soluble thrombomodulin.
Soluble thrombomodulin has antiinflammatory action preferably as soluble thrombomodulin entirety simultaneously.
It addition, as the alternate manner of soluble thrombomodulin, preferably comprise the 19th～480 of sequence numbering 5
Aminoacid sequence, as long as the 19th～480 amino acids sequences comprising sequence numbering 5 are just not particularly limited.This sequence numbering 5
The 19th～480 amino acids sequences as long as there is thrombomodulin activity can also be for its identical series of variation.
The sequence of sequence numbering 7 is the sequence that the 473rd amino acids Val variation is Ala of the sequence of sequence numbering 5, makees
For the soluble thrombomodulin in present embodiment, it is also preferred that comprise the 19th～480 amino acids sequences of sequence numbering 7
Row.
So, as soluble thrombomodulin, as long as comprise, at least there is sequence numbering 5 or sequence numbering 7
The 19th～480 bit sequences or their identical series of variation, at least have thrombomodulin activity peptide sequence, just do not have
It is particularly limited to, can enumerate and be made up of sequence numbering 5 or sequence numbering 7 respective 19th～480 or the 17th～480 bit sequences
Peptide or be made up of the identical series of variation of above-mentioned sequence and at least have thrombomodulin activity peptide as preferably
Example, the peptide being more preferably made up of the 19th～480 bit sequences of sequence numbering 5 or sequence numbering 7.It addition, there is also more excellent
Choosing is made up of the identical series of variation of respective 19th～480 or the 17th～480 of sequence numbering 5 or sequence numbering 7 and extremely
There is the alternate manner of the peptide of thrombomodulin activity less.
Soluble thrombomodulin has antiinflammatory action the most simultaneously.
It addition, as the particularly preferred alternate manner of soluble thrombomodulin, preferably comprise the 19th of sequence numbering 9 the
～515 amino acids sequences, as long as the 19th～515 amino acids sequences comprising sequence numbering 9 are just not particularly limited.This sequence
As long as the 19th～515 amino acids sequences of column number 9 have thrombomodulin activity, it is also possible to be its identical variation sequence
Row.
The sequence of sequence numbering 11 is the sequence that the 473rd amino acids Val variation is Ala of the sequence of sequence numbering 9, makees
For the thrombomodulin in present embodiment, the most also comprise the 19th～515 amino acids sequences of sequence numbering 11.
As such soluble thrombomodulin, as long as comprise, at least there is sequence numbering 9 or sequence numbering
19th～515 bit sequences of 11 or their identical series of variation, at least there is the peptide sequence of thrombomodulin activity, just do not have
Be particularly limited to, can enumerate by respective 19th～516 of sequence numbering 9 or sequence numbering 11, the 19th～515, the 19th
～the peptide of the Sequence composition of 514, the 17th～516, the 17th～515 or the 17th～514 or by above-mentioned sequence
Identical series of variation constitute and at least have thrombomodulin activity peptide as preferred example, particularly preferably by sequence
In column number 9 the 19th～516, the 19th～515, the 19th～514, the 17th～516, the 17th～515 or the 17th
～the peptide of the Sequence composition of 514.These mixture can also be enumerated as preferred example.It addition, also have particularly preferably
By respective 19th～516 of sequence numbering 11, the 19th～515, the 19th～514, the 17th～516, the 17th～515
The alternate manner of the peptide that position or the 17th～514 bit sequences are constituted.These mixture can also be enumerated as preferred example.
The peptide conduct being made up of and at least having thrombomodulin activity their identical series of variation can also be enumerated further
Preferably example.
Soluble thrombomodulin has antiinflammatory action the most simultaneously.
What is called is had to the peptide of identical series of variation, as it has been described above, it also implies that the aminoacid of the peptide as object
Sequence has more than 1, i.e. 1 or the aminoacid of more than 2, the most multiple (such as 1 to 20, preferably 1 to 10
Individual, more preferably 1 to 5, particularly preferred 1 to 3) the aminoacid peptide that can replace, lack, add.As long as having thrombosis
Regulation protein active, the degree for allowable variation is just not particularly limited, such as aminoacid sequence can exemplify 50% with
On homology, the homology of preferably more than 70%, the homology of more preferably more than 80%, the homology of further preferred more than 90%
Property, the homology of particularly preferred more than 95%, the homology of most preferably more than 98%.
Further, as soluble thrombomodulin, it is also possible to enumerate in Japanese Laid-Open Patent Publication 64-6219 publication
The peptide that is made up of the sequence (462 amino acid residues) of sequence numbering 14, by the sequence of sequence numbering 8, (272 aminoacid are residual
Base) peptide that constitutes or the peptide that is made up of the sequence (236 amino acid residues) of sequence numbering 6 be as preferred example.
As soluble thrombomodulin, as long as at least have sequence numbering 1 or the 19th～132 of sequence numbering 3 the
Amino acids sequence and sequence numbering 9 or the 19th～244 bit sequences of sequence numbering 11 or their identical series of variation,
At least there is as soluble thrombomodulin entirety the peptide of thrombomodulin activity, be just not particularly limited, Qi Zhongyou
Elect the peptide of the 19th～480 amino acids sequences at least with sequence numbering 5 or sequence numbering 7 as, more preferably at least have
The peptide of the 19th～515 amino acids sequences of sequence numbering 9 or sequence numbering 11.As at least having sequence numbering 9 or sequence
The peptide of the 19th～515 amino acids sequences of numbering 11, can enumerate by sequence numbering 9 or sequence numbering 11 the respective 19th～
516, the 19th～515, the 19th～514, the 17th～516, the 17th～515 or the Sequence composition of the 17th～514
Peptide as preferred example.Alternatively, it is also possible to enumerate by respective 19th～516 of sequence numbering 9 or sequence numbering 11,
The peptide that 19th～515, the 19th～514, the 17th～516, the 17th～515 or the 17th～514 bit sequences are constituted
, sequence numbering 9 or the respective mixture of sequence numbering 11 be as preferred example.
In the case of said mixture, as from respective 17th peptide started of sequence numbering 9 or sequence numbering 11
With from the 19th blending ratio of peptide started, (30:70)～(50:50) can be exemplified, can enumerate (35:65)～(45:
55) as preferred example.
It addition, as in respective 514th of sequence numbering 9 or sequence numbering 11, the 515th and the 516th termination
The blending ratio of peptide, can exemplify (0:0:100)～(0:90:10), according to circumstances can exemplify (0:70:30)～(10:
90:0), (10:0:90)～(20:10:70).
The blending ratio of these peptides can be obtained by usual way.
It should be noted that the sequence of the 19th～132 of sequence numbering 1 the is equivalent to the 367th～480 of sequence numbering 9
The sequence of position, the sequence of the 19th～480 of sequence numbering 5 is equivalent to the sequence of the 19th～480 of sequence numbering 9.
Additionally, the sequence of the 19th～132 of sequence numbering 3 the is equivalent to the sequence of the 367th～480 of sequence numbering 11
Row, the sequence of the 19th～480 of sequence numbering 7 is equivalent to the sequence of the 19th～480 of sequence numbering 11.
Further, sequence numbering 1,3,5,7,9 and 11 sequence of respective 1st～18 is identical sequence
Row.
For these soluble thrombomodulins, as described later, such as, can be obtained by following conversion cell, should
Converting cell is to utilize carrier will to encode the DNA of these peptides (specifically, for the alkali of the 1st～732 with sequence numbering 10
The base sequence of the base sequence of the 55th～396 of basic sequence and sequence numbering 2；There is the 1st～732 of sequence numbering 10
Base sequence and the base sequence of the base sequence of the 55th～396 of sequence numbering 4；Sequence numbering 6, sequence numbering 8, sequence
The base sequence of column number 10 or sequence numbering 12 etc.) transfect and prepare to host cell.
As long as it addition, these peptides have above-mentioned aminoacid sequence, it can have sugar chain or also can not have sugar
Chain, is not particularly limited in this.It addition, in genetic manipulation, according to the difference of the host cell species used, sugar
The kind of chain, Adding sites and addition degree are different, and they all can use.For binding site and the kind of sugar chain,
Know the fact that have described in Japanese Unexamined Patent Publication 11-341990 publication, for the thrombomodulin in present embodiment, also deposit
There iing the situation at the same sugar chain of same position addition.In the soluble thrombomodulin of present embodiment, in conjunction with
There are two days line styles (Off U シ Le バ イ ア Application テ Na リ type) of fucosido and fucosido triantennary type (Off U シ Le ト リ ア
Application テ Na リ type) both N conjunction type sugar chains, its ratio can example be (100:0)～(60:40), be preferably (95:5)～
(60:40) (90:10)～(70:30) can, be enumerated as preferred example.The ratio of these sugar chains can pass through biochemistry
Laboratory method 23 glycoprotein sugar chain organon, association's publishing centre (biochemical process 23 glycoprotein sugar organon,
セ Application タ can be published) 2 dimension sugar chain collection of illustrative plates described in (nineteen ninety) etc. are measured.Further, if to present embodiment
Soluble thrombomodulin sugar composition study, then detect neutral sugar, amino sugar and sialic acid, relative to albumen
Matter content, can enumerate independently of one another with the ratio that mass ratio range is 1～30%, preferably 2～20%, more preferably 5～10%.These
Sugar content can by neonatology experiment lecture 3 saccharic I glycoprotein (on), Tokyo chemistry same people (neonatology seat 3
The method described in (nineteen ninety) of sugar I sugar タ Application パ Network (on), the capital same people of chemistry) (neutral sugar: phend-sulphuric acid,
Amino sugar: Ai Erxun-Mo Genfa, sialic acid: periodic acid-Resorcinol Method) it is measured.
In the case of obtaining soluble thrombomodulin by genetic manipulation, as can use when expressing
Signal sequence, it is possible to use the base sequence of the 1st～18 amino acids sequences of above-mentioned coded sequence numbering 9, coded sequence
The base sequence of the 1st～16 amino acids sequences of numbering 9, signal sequence, such as human histiotype plasminogen known to other
The signal sequence (No. 88/9811 publication of International Publication) of activator.
In the case of the DNA sequence of encoding soluble thrombomodulin is directed in host cell, it may be preferred to lift
Go out to recombinate the DNA sequence of encoding soluble thrombomodulin the carrier (table particularly preferably can expressed in zooblast
Reach carrier) in carry out the method that imports.So-called expression vector is by promoter sequence, gives ribosome binding site to mRNA
Sequence, the coding DNA sequence of albumen to be expressed, splicing signal, the terminator sequence of tanscription termination, replication initiation sequence etc.
The DNA molecular constituted, as the example of preferred animal cell expression vectors, can enumerate R.C.Mulligan etc.
[the Method in such as pSV2-X, P.M.Howley that [Proc.Natl.Acad.Sci.U.S.A.78.2072 (1981)] report
Emzymology, 101,387, Academic Press (1983)] pBP69T (69-6) etc. that reports.It addition, also there is restructuring to arrive
Other optimal way in the expression vector can expressed in microorganism.
As the host cell that can use when manufacturing these peptides, zooblast can be enumerated.
As zooblast, Chinese hamster ovary (CHO) cell, COS-1 cell, COS-7 cell, VERO can be enumerated
The kidney derived mdck cell of (ATCC CCL-81) cell, bhk cell, dog, hamster AV-12-664 cell, NS0 cell etc., as
Human archeocyte can enumerate HeLa cell, WI 38 cell, people 293 cell, PER.C6 cell.Chinese hamster ovary celI is extremely widespread and preferred,
In Chinese hamster ovary celI, further preferred dihydrofolate reductase (DHFR) defect Chinese hamster ovary celI.
Additionally, in the process of genetic manipulation and the manufacture process of peptide, more than use the microorganisms such as escherichia coli, preferably make
With being suitable to the host-vector system of each microorganism, in above-mentioned host cell, it is also possible to select suitable carrier system.With
In gene being cloned of the thrombomodulin of gene recombination technology, and the base that use thrombomodulin is had been disclosed
Because of the manufacture example of recombinant technique, additionally become known for obtaining process for purification [the Japanese Laid-Open Patent Publication 64-6219 public affairs of its highly finished product
Report, Japanese Unexamined Patent Publication 2-255699 publication, Japanese Unexamined Patent Publication 5-213998 publication, Japanese Unexamined Patent Publication 5-310787 public affairs
Report, Japanese Unexamined Patent Publication 7-155176 publication, J.Biol.Chem., 264:10351-10353 (1989)].Therefore, this embodiment party
Soluble thrombomodulin used in formula can be by using the method described in above-mentioned report or described with these reports
Method on the basis of manufacture.Such as, in Japanese Laid-Open Patent Publication 64-6219 publication, disclose and comprise plasmid pSV2 blood
The e. coli k-12 bacterial strain DH5 (ATCC deposit number 67283) of bolt regulation albumen J2, this plasmid pSV2 thrombomodulin
J2 comprises the DNA of encoding full leng thrombomodulin.Grind (existing only furthermore it is also possible to this bacterial strain is preserved in life by use again
Vertical independent administrative institution's industrial technology comprehensive study institute's Patent Organism preservation center) bacterial strain (bacillus coli DH 5/pSV2TM J2)
(FERMBP-5570).Can be using the DNA of this encoding full leng thrombomodulin as raw material, by known genetic manipulation skill
Art prepares the thrombomodulin of present embodiment.
Soluble thrombomodulin can use existing known method or be prepared, such as based on these methods
It is referred to method [Japanese Laid-Open Patent Publication 64-6219 publication] or the Japanese Unexamined Patent Publication 5-213998 of above-mentioned Yamamoto et al.
Publication.That is, by gene manipulation techniques, people source soluble thrombomodulin gene is made the ammonia of such as coded sequence numbering 9
The DNA of base acid sequence, and then the most also can be changed.As this change, such as, in order to make coded sequence numbering 11
The DNA (specifically, be made up of the base sequence of sequence numbering 12) of aminoacid sequence, for the ammonia of coded sequence numbering 9
The codon (base of particularly the 1418th) of the 473rd amino acids of base acid sequence, according to Methods in
Enzymology, 100:468 (nineteen eighty-three), the method described in Academic Press carries out mutation site-specific.Such as,
The variation synthetic DNA with base sequence shown in sequence numbering 13 can be used to make the 1418th of sequence numbering 10 the
Base T be converted into the DNA of base C.
Thus prepared DNA can be recombinated in such as Chinese hamster ovary (CHO) cell, make conversion cell, enter
Row is suitable to be selected, and produce in obtained culture fluid can through refined by cultivating this cell to utilize known method
Dissolubility thrombomodulin.As described above, it is preferred to the DNA (sequence numbering 10) of the aminoacid sequence by coded sequence numbering 9 turns
Contaminate in above-mentioned host cell.
When above-mentioned conversion cell is cultivated, it is possible to use be generally used for the culture medium that cell is cultivated, preferably pass through
Utilize various culture medium that this conversion cell is cultivated in advance and select optimal culture medium.It is, for example possible to use MEM is trained
Support the known culture medium such as base, DMEM culture medium, 199 culture medium as minimal medium and to carry out further improveing or adding respectively
The additive of kind of culture medium and the culture medium that obtains.As cultural method, can enumerate and utilize the culture medium being added with serum
Carry out the serum free culture system cultivated or utilize the culture medium without serum to carry out the serum-free culture cultivated.Cultural method is also
It is not particularly limited, but preferably serum-free culture.
In serum free culture system, in the case of adding serum in culture medium, preferably Ox blood serum.Ox blood serum has tire Sanguis Bovis seu Bubali
Clearly, newborn calf serum, calf serum, ABS etc., cultivate as long as being suitable to cell, it is possible to use any.The opposing party
Face, in serum-free culture, the serum-free medium used can use commercially available culture medium.Be suitable to the depletion of blood of various cell
The clear commercially available product of culture medium, such as, for Chinese hamster ovary celI, have CD-CHO, CHO-S-SFMII, CHO-that Invitrogen society is on sale
III-PFM, has IS CHO, ISCHO-CD culture medium etc. that Irvine Scientific society is on sale.These culture medium can be direct
Use, carry out improveing or add additive and use.It addition, as serum-free medium, can exemplify respectively by 5mg/L
Add insulin, siderophillin and the DMEM culture medium of Monohydrated selenium dioxide.So, as long as being the thrombosis that can produce present embodiment
The culture medium of regulation albumen, is just not particularly limited.Cultural method is not particularly limited, and can be batch culture, the most in batches
The culture methods such as cultivation, fed-batch culture, perfusion cultivation.
Utilize above-mentioned cell culture processes in the case of manufacturing soluble thrombomodulin, sometimes can pass through albumen
The post translational modification of matter confirms the amino acid whose multiformity of N-terminal.Such as, the 17th in sequence numbering 9,18,19 or
The aminoacid that person is 22 becomes N-terminal sometimes.It addition, the most also the glutamic acid rotating of the 22nd is changed to pyroglutamic acid
Carry out the amino acid whose modification of N-terminal.The aminoacid of preferably the 17th or 19 is N-terminal, and the aminoacid of more preferably the 19th is N
End.It addition, there is also the alternate manner that aminoacid is N-terminal of preferably the 17th.About above modification and multiformity etc.,
Same example can also be enumerated for sequence numbering 11.
Further, using the DNA of the base sequence with sequence numbering 10 to manufacture soluble thrombomodulin
In the case of, confirm the amino acid whose multiformity of C-terminal sometimes, produce the peptide of short 1 amino acid residue sometimes.That is,
There are the aminoacid of the 515th be C-terminal so that the 515th be amidated such adorned feelings of C-terminal aminoacid
Condition.It addition, also there is the situation of the peptide that can produce short 2 amino acid residues.That is, the aminoacid that there are the 514th is C end
The situation of end.Thus, produce N-terminal aminoacid and C-terminal aminoacid sometimes and be imbued with multifarious peptide or theirs is mixed
Compound.The aminoacid of the aminoacid of preferably the 515th or the 516th be C-terminal, the aminoacid of more preferably the 516th be C end
End.And there is also the alternate manner that aminoacid is C-terminal of preferably the 514th.About above modification and multiformity etc., right
Also it is same in the DNA of the base sequence with sequence numbering 12.
The thrombomodulin utilizing said method to obtain is the multifarious peptide having confirmed N-terminal and C-terminal sometimes
Mixture.Specifically, can enumerate by the 19th～516 in sequence numbering 9, the 19th～515, the 19th～514,
The mixture of the peptide of the Sequence composition of the 17th～516, the 17th～515 or the 17th～514.
In accordance with the invention it is possible to provide HCP's to be mixed into the soluble thrombomodulin of high purity being reduced.
As the soluble thrombomodulin of high purity of present embodiment, can enumerate and contain substantially no solubility blood
The highly purified soluble thrombomodulin of the protein beyond bolt regulation albumen.Specifically, such as essence can be enumerated
On do not contain the soluble thrombomodulin of HCP.Preferably can enumerate and contain substantially no HCP, mouse IgG and Ox blood serum
The soluble thrombomodulin of protein.
The soluble thrombomodulin of high purity of present embodiment does not contains the protein in people source.
For the content of HCP, as long as being that the state containing substantially no HCP is just not particularly limited, preferably with respect to can
Dissolubility thrombomodulin 10,000U is the ratio of the ratio that HCP is less than 10ng, more preferably less than 8ng, is further preferably no larger than
The ratio of the ratio of the ratio of 7ng, particularly preferably less than 6ng, particularly preferably smaller than 5ng.The high-purity of present embodiment can
Dissolubility thrombomodulin is to utilize to convert cell generation, and this conversion cell will be by comprising encoding soluble thrombomodulin
The DNA transfection of white base sequence obtains to host cell, according to the consideration, refines in any case, the most also has trace journey
Degree HCP is mixed into.The content of HCP is the lowest more preferred, such as, as the lower limit of HCP content, can exemplify relative to solubility blood
Bolt regulation protein 10,000U, HCP are the ratio of 0.001ng.
For the content of mouse IgG, as long as being that the state containing substantially no mouse IgG is just not particularly limited, preferably
Relative to soluble thrombomodulin 10,000U, mouse IgG is the ratio of the ratio less than 10ng, more preferably less than 2ng
The ratio of rate, further preferably less than 0.6ng.
For the content of bovine serum protein, as long as being to contain substantially no the state of bovine serum protein the most especially
Limiting, preferably with respect to soluble thrombomodulin 10,000U is the ratio of the ratio less than 10ng, more preferably less than 2ng
The ratio of rate, further preferably less than 0.6ng.These protein concentrations are preferably measured by ELISA method, Ke Yican
Examine biochemistry laboratory method 11 enzyme immunoassay Tokyo chemistry same people (biochemical method 11 エ Application ザ イ system イ system ノ ア Star
The セ イ capital same people of chemistry) (1992) etc. be measured.
It addition, in the soluble thrombomodulin of high purity of present embodiment, as the solubility in gross protein
Thrombomodulin purity, is preferably more than 99%, more preferably more than 99.5%, more preferably 99.7% in HPLC method
Above, the purity of particularly preferably more than 99.9%.As long as can be to soluble thrombomodulin for the chromatography in HPLC method
Purity be measured the most not limiting, gel filtration liquid chromatography, ion-exchange selectivity and anti-phase can be exemplified
Liquid chromatography etc., preferably gel filtration liquid chromatography.In the case of using gel filtration liquid chromatography, for being made
Post, as long as the molecular weight combining soluble thrombomodulin carries out selecting, such as can enumerate use TOSOH
TSKgel G3000SWXL (Cao Japanese, eastern), utilizes the method that the phosphate buffer of pH7.3 carries out launching.Can be according to day
Test implemented by the liquid chromatography<2.01>of side of this officina.
Further, in the soluble thrombomodulin of high purity of present embodiment, the DNA for Hosts becomes
Point, preferably with respect to soluble thrombomodulin 10,000U is the ratio of the ratio less than 2ng, more preferably less than 0.2ng
The ratio of rate, further preferably less than 0.02ng.For the mensuration of amount of DNA, as long as use thresholding system (U.S.,
Molecular Devices) can easily be measured.
For the soluble thrombomodulin of high purity of present embodiment, as long as the content of HCP is relative to this solubility
Thrombomodulin 10,000U is the ratio less than 10ng, then its form is just not particularly limited, can be with solution or powder
Form exists.Preferably exist with the form of solution.Further, also with the presence of preferably with the alternate manner of powder morphology.As solution
Concentration under state status, is preferably below 100mg/mL, more preferably below 60mg/mL, more preferably in terms of the upper limit
Below 30mg/mL, particularly preferably below 15mg/mL, be preferably in terms of lower limit more than 2mg/mL, more preferably 4mg/mL with
Upper, more preferably more than 6mg/mL, particularly preferably more than 8mg/mL, most preferably more than 10mg/mL.It addition, with
In the presence of powder morphology, cryodesiccated form can be enumerated as preferred example.Lyophilization body is referred to
Method described in WO03/061687 obtains.
The soluble thrombomodulin of high purity of present embodiment can be to contain substantially no endotoxic form
Obtain.As endotoxin content, preferably with respect to soluble thrombomodulin 10,000U is less than 1 endotoxin unit (EU),
More preferably less than 0.2EU, further preferably less than 0.04EU.Endotoxin amount can be according to Pharmacopeia of Japan ordinary test method
Endotoxin test method<4.01>be measured.Further, since the soluble thrombomodulin of high purity of present embodiment is not
Containing TFA etc. to the harmful material of organism, can obtain close to aseptic state, thus can enter as the raw material of medicine
Exercise and use.
The soluble thrombomodulin of high purity that being mixed into of the HCP of present embodiment is reduced can by make containing
The solution of soluble thrombomodulin contacts with nylon and/or polyether sulfone and obtains, and nylon is preferably used.It addition, also have excellent
Choosing uses the alternate manner of polyether sulfone.
As the nylon contacted with the solution containing soluble thrombomodulin of present embodiment, can enumerate containing
The polyamide of aliphatic skeleton, can exemplify such as nylon 6, nylon66 fiber, Stanyl (DSM). or nylon MXD 6.If it is adsorbable
HCP, its kind indefinite, preferably nylon 6.For nylon, such as can be with the Minisart NY purchased from Sartorius society
Form obtain.For the form of nylon, if be the form that can contact with solution of membranaceous, non-woven fabrics, pearl etc. the most especially
Do not limit, be preferably moulded as membranaceous, use with the form of filter membrane.In this case, as long as the aperture of filter membrane is
The size that HCP can pass through the most does not limits, and can exemplify 0.01 μm～10 μm, preferably 0.1 μm～1 μm, more preferably 0.01 μ
M～0.06 μm.The amount of the solution containing soluble thrombomodulin for contacting with nylon, can make a part of molten in advance
Liquid contacts with a small amount of nylon, is evaluated the removal ability of HCP, be thus readily determined contact with nylon containing solvable
The amount of the solution of property thrombomodulin.
After confirming to make the solution containing soluble thrombomodulin containing HCP contact with nylon, the HCP in solution contains
Amount is less than obtaining the high-purity of present embodiment while the ratio of 10ng relative to soluble thrombomodulin 10,000U
Degree soluble thrombomodulin, at HCP content relative to soluble thrombomodulin 10, stops when 000U is more than 10ng
Obtain, or can be transformed to not make articles for use by the nylon in using, re-start obtaining of soluble thrombomodulin of high purity
?.If exemplifying the relation of HCP amount and nylon amount, the most such as in the case of nylon is filter membrane, relative to the film of 1mg HCP
Area can exemplify 50m as the upper limit2Hereinafter, preferably 5m2Below, it is more preferably 0.5m2Below, more preferably 0.1m2
Hereinafter, it is preferably 0.01m as lower limit2Above, it is more preferably 0.02m2Above, more preferably 0.03m2Above.Important
It is to set membrane area according to HCP amount to be reduced.
The polyether sulfone contacted with the solution containing soluble thrombomodulin of present embodiment such as can be purchased from
The form of the Minisart High-Flow of Sartorius society obtains.About the form of polyether sulfone, as long as being membranaceous, nonwoven
Cloth, pearl etc. and can contacting with solution does not just limit especially.It is preferably moulded as the membranaceous form with filter membrane to use.This feelings
As long as the size that the aperture of the filter membrane under condition can be passed through for HCP does not the most limit, 0.01 μm～10 μm can be exemplified, preferably
0.1 μm～1 μm, more preferably 0.01 μm～0.06 μm.For molten containing soluble thrombomodulin contacted with polyether sulfone
The amount of liquid, can make a part of solution contact with a small amount of polyether sulfone in advance, be evaluated the removal ability of HCP, thus hold
Change places and determine.
In confirming the solution after making the solution containing soluble thrombomodulin containing HCP contact with polyether sulfone
HCP content is less than obtaining present embodiment while the ratio of 10ng relative to soluble thrombomodulin 10,000U
Soluble thrombomodulin of high purity, at HCP content relative to soluble thrombomodulin 10,000U is more than 10ng
Time stop obtain, or can by use in polyether sulfone be transformed to not make articles for use, re-start high-purity solubility thrombosis regulation
The acquisition of albumen.If exemplifying the relation of HCP amount and polyether sulfone amount, the most such as in the case of polyether sulfone is filter membrane, relatively
Membrane area in 1mg HCP can exemplify 50m as the upper limit2Hereinafter, preferably 5m2Below, it is more preferably 0.5m2Hereinafter, as
Lower limit is preferably 0.02m2Above, it is more preferably 0.03m2Above, more preferably 0.05m2Above.It is important that according to wanting
The HCP amount reduced sets membrane area.
Present embodiment is made to the manufacture work being mixed into the soluble thrombomodulin of high purity being reduced of HCP
For sequence, as long as comprising operation that the solution made containing soluble thrombomodulin contacts with nylon and/or polyether sulfone, making
HCP content is relative to this soluble thrombomodulin 10, and 000U is that the ratio less than 10ng is just not particularly limited, and can show
Example goes out following manufacturing process.
The manufacturing process being made up of (a)～(g), it includes making the solution containing soluble thrombomodulin and nylon
And/or the operation of polyether sulfone contact.
A () is cultivated and is converted cell and reclaim the operation of culture fluid (production liquid)；
B () is filtered and is produced the operation producing liquid after liquid is filtered；
C () is supplied in producing liquid after filtration in Anion exchange column chromatography, obtain the operation of thick refined liquid；
D thick refined liquid is supplied in the affinity column chromatography being loaded with antithrombotic regulation protein monoclonal antibody by (), obtain
The operation of refined liquid 1；
E refined liquid 1 is supplied in cation exchange column chromatography operation by (), obtain the operation of refined liquid 2；
Refined liquid 2 f () will concentrate after is supplied in gel filtration column chromatography, concentrates its dissolution fluid, obtains essence
The operation of liquid 3 processed；And
G () utilizes the operation that refined liquid 3 is filtered by virus removal film and aseptic filter membrane.
In above-mentioned manufacturing process, the solution containing soluble thrombomodulin is made to contact with nylon and/or polyether sulfone
Operation may be embodied in the operation of the arbitrary of (b)～(g) or more than 2, preferably be contained in the arbitrary of (d)～(g) or
In the operation that person is more than 2.In order to effectively remove HCP, particularly preferably the finishing operation of manufacturing process, i.e. at (g) after increase
Add the operation contacted with nylon and/or polyether sulfone.
It addition, be 0 to make being mixed into of bovine serum protein, the more preferably cultivation converting cell of (a) is serum-free training
Support.
Further, make HCP's to be mixed into the high-purity solubility thrombomodulin being reduced as present embodiment
White manufacturing process, can enumerate manufacturing process shown below.
The manufacturing process being made up of (a)～(g), it includes making the solution containing soluble thrombomodulin and nylon
And/or the operation of polyether sulfone contact.
A () is cultivated and is converted cell and reclaim the operation of culture fluid (production liquid)；
B () is filtered and is produced the operation producing liquid after liquid is filtered；
C () is supplied in producing liquid after filtration in Anion exchange column chromatography, obtain the operation of thick refined liquid 1；
D thick refined liquid 1 is supplied in drainage column chromatograph by (), obtain the operation of thick refined liquid 2；
E thick refined liquid 2 is supplied in the affinity column chromatography being loaded with antithrombotic regulation protein monoclonal antibody by (), obtain
The operation of refined liquid 1；
Refined liquid 1 f () will concentrate after is supplied in gel filtration column chromatography, obtains the operation of refined liquid 2；And
G () utilizes the operation that refined liquid 2 is filtered by aseptic filter membrane.
In above-mentioned manufacturing process, the solution containing soluble thrombomodulin is made to contact with nylon and/or polyether sulfone
Operation may be embodied in the operation of the arbitrary of (b)～(g) or more than 2, preferably be contained in the arbitrary of (e)～(g) or
In the operation that person is more than 2.In order to effectively remove HCP, sometimes it is also preferred that increase after the finishing operation of manufacturing process, i.e. (g)
Add the operation contacted with nylon and/or polyether sulfone.
It addition, be 0 to make being mixed into of bovine serum protein, the more preferably cultivation converting cell of (a) is serum-free training
Support.
After confirming to make the solution containing soluble thrombomodulin containing HCP contact with nylon and/or polyether sulfone
HCP content in solution is less than obtaining this while the ratio of 10ng relative to soluble thrombomodulin 10,000U
The soluble thrombomodulin of high purity of embodiment, at HCP content relative to soluble thrombomodulin 10,000U be
Stop during more than 10ng obtaining, or can be transformed to not make articles for use by the nylon in using and/or polyether sulfone, re-start high-purity
The acquisition of degree soluble thrombomodulin.It addition, being mixed into of HCP that make as described previously for present embodiment is reduced
Soluble thrombomodulin of high purity manufacturing process for, as long as include making containing soluble thrombomodulin is molten
Operation that liquid contacts with nylon and/or polyether sulfone, the HCP content is made relative to this soluble thrombomodulin 10,000U to be
Ratio less than 10ng is just not particularly limited.I.e., it is possible to exist refined after the operation contacted with nylon and/or polyether sulfone
Operation, as result, as long as the content ratio that can obtain HCP is that 000U soluble thrombomodulin is less than relative to 10
The soluble thrombomodulin of high purity of 10ng.
As the refining step that can exist after the operation contacted with nylon and/or polyether sulfone, can enumerate cloudy from
Sub-exchange column chromatography, affinity column chromatography, cation exchange column chromatography method, gel filtration column chromatography or drainage column color
The column chromatography operations such as spectrometry；Membrance concentration operation；The membrane filtration operation of virus removal, aseptic filtration etc.；Or they two or more
Combination, preferred cationic exchange column chromatography, gel filtration column chromatography, membrance concentration, virus removal, aseptic filtration or
They combinations of more than two kinds.The most preferably after the operation contacted with nylon and/or polyether sulfone, there is cation exchange column color
Spectrometry, gel filtration column chromatography, membrance concentration, virus removal and aseptic filtration.It addition, connecing with nylon and/or polyether sulfone
After the operation touched, cation exchange column chromatography method, gel filtration column chromatography, membrance concentration, virus removal and aseptic filtration
Refining step can enumerate and go with cation exchange column chromatography method, membrance concentration, gel filtration column chromatography, membrance concentration, virus
Remove, situation that the order of aseptic filtration exists is as preferred example.It addition, as cation exchange column chromatography method, can show
Example goes out SP Sepharose FastFlow, DEAE Sepharose Fast Flow, Capto S, Capto DEAE (GE
Healthcare society)；SHyperCel (Poul society)；TOYOPEARL GigaCap S-650 (Dong Caoshe), preferably SP
Sepharose FastFlow.As concentrating film, MICROZA UF (chemistry society of Asahi Chemical Industry), Kvick Flow can be exemplified
10KD (GEHealthcare society), Pellicon 2 (Millipore society), preferably MICROZA UF.As solvent resistant column color
Spectrum, can exemplify Sephacryl S-300HR, Superose 12pg (GE Healthcare society), TOYOPEARL HW (east
Cao), preferably Sephacryl S-300HR.As virus removal film, can exemplify PLANOVA 15N (Asahi Kasei medical treatment),
Biresolve NFP (Millipore society), Ultipor VF (Poul society), preferably PLANOVA 15N.As aseptic filter membrane,
MilliPak, Millidisk (Millipore society), Supore EVA (Poul society), Sartopore2 can be exemplified
(Sartorius Stedium society), preferably MilliPak.
So, by making the solution containing soluble thrombomodulin and nylon and/or the polyether sulfone of present embodiment
The soluble thrombomodulin of high purity that obtains of contact is that HCP contains ratio relative to soluble thrombomodulin 10,
The 000U material less than 10ng.
The 1U of the soluble thrombomodulin of present embodiment is defined as activating as index with protein C
APC produced the amount of 0.1 μm ol paranitroanilinum in 1 minute in analyzing, it is according to Biologicals (2002) 30,69-76 institute
The method recorded utilizes and includes that the method for following operation is measured.
A () adds human thrombin in the solution to be detected containing soluble thrombomodulin and carries out the work heated
Sequence；
B () adds human protein C and carries out the operation heated；
C () adds heparin-Antithrombin III and carries out the operation heated；
D () adds synthesis substrate S-2366 (pyroGlu-Pro-Arg-pNA HCl) and carries out the operation heated；
E () adds acetic acid, terminate substrate and cut off the operation of reaction；
F () measures the operation of the absorbance of 405nm；And
G () measures the operation of the activity of the solution to be detected containing soluble thrombomodulin according to following formula.
[several 1]
Asample: the absorbance of sample solution
Ablank: the absorbance of comparison (water)
The molar absorption coefficient 9.6 × 10 of M: paranitroanilinum-3[1/μM]
V1: measure the liquid measure (L) during absorbance
V2: the liquid measure (mL) of sample solution
T: substrate cuts off the response time (minute)
K: the activation of protein C generated by the thrombomodulin 1U molal quantity 0.1 (μ being dissociated the paranitroanilinum
Mol/ minute/U)
For the soluble thrombomodulin of high purity of present embodiment, every 1mg protein can be exemplified and have
The activity of 3,000U, preferably 4,000U～9,000U, more preferably 5,000U～8,000U, more preferably 6,000U～
7,000U.Protein concentration can be measured with bovine serum albumin as standard substance according to known determination of protein concentration method.
Such as, Lowry method, Bradford method, BCA method etc. can be exemplified.
The HCP content of the soluble thrombomodulin of high purity of present embodiment utilizes the side at least including following operation
Method is measured.
A the DNA comprising the base sequence of encoding soluble thrombomodulin is transfected to host cell, to obtained by ()
Convert cell or its host cell one of arbitrarily carry out serum-free culture, obtained culture supernatant prepare host
The operation of the protein of cell derived；
B () makes rabbit sensitization with the protein of this host cell resources of above-mentioned (a), obtained antiserum refine anti-
The operation of the antibody of host cell resources protein；
Structure includes the operation of the mensuration system of following (c1)～(c6):
(c1) antibody making this anti-host cell resources protein of above-mentioned (b) is adsorbed in the operation of solid phase；
(c2-1) suspection is made to be mixed into the treating containing soluble thrombomodulin of protein of this host cell resources
Detection solution carries out the operation contacted with this solid phase of the antibody being adsorbed with this anti-host cell resources protein；
(c3) in this solid phase, add the operation of the antibody of biotinylated anti-host cell resources protein；
(c4) in this solid phase, add the operation of avidinated Peroxidase Solution；
(c5) operation that enzyme substrate solution carries out developing the color is added；And
(c6) colour developing, the operation that its absorbance is measured are stopped；
(d) utilize said determination system to suspect be mixed into this host cell resources protein containing solubility thrombosis
The protein concentration of the host cell resources in the solution to be detected of regulation albumen is measured, it is judged that should be containing solubility thrombosis
Whether the protein concentration of the host cell resources in the solution to be detected of regulation albumen is included in determining of said determination system
Operation in weight range, this can quantification range be next by utilizing said determination system to use containing host cell known to concentration
The solution of the protein in source is measured and confirms in advance；
(e-1) in above-mentioned (d), adjusting containing solubility thrombosis of the protein being mixed into this host cell resources is being suspected
The protein concentration of host cell resources in the solution to be detected of joint albumen be included in can be in quantification range in the case of, should
Concentration is as the operation of the protein concentration of this host cell resources；
(e-2-1) in above-mentioned (d), suspect be mixed into this host cell resources protein containing solubility thrombosis
The protein concentration of host cell resources in the solution to be detected of regulation albumen be not included in can be in quantification range in the case of,
For become be included in said determination system can measured concentration in quantification range, as required to containing solubility blood
The solution to be detected of bolt regulation albumen concentrates or dilutes, the operation recording this enrichment factor or dilution rate；
(e-2-2) (c2-1) in the mensuration system that will represent with above-mentioned (c1)～(c6) replaces with (c2-as follows
2), utilize this mensuration system to carried out concentrating in above-mentioned (e-2-1) or dilution containing soluble thrombomodulin
The protein concentration of the host cell resources in solution to be detected is measured, it is considered to enrichment factor or dilution rate obtain this host
The operation of the protein concentration of cell derived；
(c2-2) make to have carried out as required concentrating or the solution to be detected containing soluble thrombomodulin of dilution
Carry out the operation that contacts with this solid phase of the antibody being adsorbed with this anti-host cell resources protein and make containing concentration known
The solution of protein of host cell resources carry out with this solid phase of the antibody being adsorbed with this anti-host cell resources protein
The operation of contact；And
F () additionally determines the thrombosis regulation in the solution to be detected that per unit volume contains soluble thrombomodulin
The APC activity of albumen, calculates the protein concentration of the host cell resources obtained in (e-1) or (e-2-2) relative to this
The operation of the ratio of APC activity.
In this specification, HCP is that making is used when the gene-recombinated cell producing soluble thrombomodulin
Host cell resources protein, it does not contains soluble thrombomodulin.HCP can be by following culture supernatant system
Standby, this culture supernatant is for used for of the same race with the transfection of the DNA of the base sequence comprising coding thrombomodulin
Host cell carries out cultivating and obtaining.For the host cell that what is called is of the same race, such as in the case of Chinese hamster ovary celI, it represents
Comprise be categorized as CHO-K1 strain (ATCC numbering CCL-61), CHO-S strain (U.S., Invitrogen catalog number 11619-012),
The concept of the bacterial strain of the Chinese hamster ovary celIs such as CHO-DXB11, CHO-DG44 strain (U.S., Invitrogen catalog number 12610-010),
As long as be categorized as the bacterial strain of Chinese hamster ovary celI, it is possible to use arbitrary bacterial strain is prepared.If mentioning Chinese hamster ovary celI, then work is preferably used
DXB11 strain or CHO-K1 strain, more preferably use DXB11 strain for host cell of the same race.It addition, CHO-K1 strain is also preferably used
Alternate manner.
The host cell resources that HCP is used when being the gene-recombinated cell making and producing soluble thrombomodulin
Protein, its method being defined as utilizing the operation at least containing above-mentioned (a)～(f) and the material that determines, as HCP's
Constitutive requirements, can enumerate the histone H2B (Biochimie, 61 (1), 61-69 (1979)) shown in test example 6.
It addition, in the host cell that the DNA transfection of the base sequence by comprising coding thrombomodulin is the most of the same race, right
In the case of obtained conversion cell carries out cultivating and being prepared by obtained culture supernatant, as long as by this culture supernatant
It is supplied in and uses the antibody specific binding with thrombomodulin as in the antibody column of ligand, non-adsorbed fraction is carried out
Reclaim.After confirming this non-adsorbed fraction and can not detect the APC activity of thrombomodulin, can carry out as HCP
Use.HCP utilizes ultrafilter membrane to concentrate the most as required.It should be noted that for cultivating host cell or conversion
The culture medium used during cell, in order to avoid being mixed into of other oroteins, preferably serum-free medium, more preferably serum-free
Culture medium is protein-free culture.Make rabbit sensitization with HCP, obtained anti-HCP antiserum permissible when refining anti-HCP antibody
Utilize column chromatography, the combination of ammonium sulfate salting-out process and column chromatography can be exemplified further.Post color during anti-HCP antibody is refined
Spectrum is preferably used a-protein post.When preparing HCP, turn using the DNA by the base sequence comprising coding thrombomodulin
In the case of the conversion cell that dye obtains to host cell, when refined anti-HCP antibody, also have and preferably utilizing a-protein
Use thrombomodulin post after post is refined, obtain this non-adsorbed fraction to make the alternate manner of anti-HCP antibody.
Construct HCP concentration measure system time, need clear and definite its can quantification range, as long as can be to relative to every 10,000U
Thrombomodulin HCP content is measured less than the situation of 10ng, can the most not limit by quantification range, but more can measure to
Low concentration is the most preferred.For can quantification range, more than 100ng/mL, preferably more than 50ng/mL, more can be exemplified as lower limit
It is preferably more than 25ng/mL, 500ng/mL can be exemplified as the upper limit.
In the case of detected solution is concentrated, as long as carrying out according to common protein compression method, do not have
It is particularly limited to, but concentrates preferably by ultrafilter membrane.It addition, also have preferably by utilize after lyophilization a small amount of water or
Buffer carries out dissolving the alternate manner of the method concentrated.It addition, by concentrating, the composition beyond HCP is also concentrated, and it can
HCP can be measured system and bring impact, thus tested solution needs to bring HCP mensuration system in the range of impact
Concentrate.Such as, to be detected molten containing soluble thrombomodulin of impact is brought as HCP will not being measured system
The upper limit of liquid concentration range, can enumerate 5mg/mL.
HCP content in every 10,000U thrombomodulin calculates according to following formula.
a/b×10,000
A: the HCP content (ng/mL) in every 1mL sample
B: APC activity (U/mL) of the thrombomodulin in every 1mL sample
The soluble thrombomodulin of high purity of present embodiment promotes the activation of protein C based on this thrombin,
A large amount of generations demonstrate the active form protein C of resisting blood coagulation effect and thrombolytic effect.Thus, the height of present embodiment
Purity soluble thrombomodulin goes far towards the resisting blood coagulation in organism and thromboembolism.Due to present embodiment
Soluble thrombomodulin of high purity there is resisting blood coagulation effect and inhibition on platelet aggregation and thromboembolism
Effect, thus can be as that regulate blood coagulation or use for regulating the pharmaceutical composition of platelet aggregation, tool
Say body, can be used for such as myocardial infarction, thrombosis, gland disease, peripheral vessel obliterans, Arteriosclerosis obliterans, blood vessel
The treatment and prevention of the diseases such as interior blood coagulation syndrome (DIC), angina pectoris, temporal cerebral ischemia seizure, hypertensive state of pregnancy.
When preparing the pharmaceutical composition of present embodiment, as long as the high-purity solubility thrombosis of present embodiment is regulated
Albumen mixes with pharmaceutically suitable carrier.I.e., it is possible to by treatment or prevent in above-mentioned disease as the present embodiment of effective dose
Soluble thrombomodulin of high purity mix with the carrier of appropriate amount, prepare the medicine being suitable to effectively be applied to patient
Compositions.As the pharmaceutical composition of present embodiment, it is preferably made cryodesiccated preparation.It addition, the medicine of present embodiment
Compositions is preferably used as intravascular injection preparation.Further preferably make drop intravenous preparation.It addition, manufacturing freezing
During drying agent, the method described in WO03/061687 that is referred to is carried out.
In the case of using as injection, above-mentioned carrier can be administered with medicine type, and preferably solubilized
Carrier in normal saline solution or glucose injection, as this carrier, can exemplify the free sucrose of choosing, refined gelatin, in vain
In the group of albumen, mannitol, glucose and sodium chloride composition more than a kind, and the various inorganic salts of interpolation can also be enumerated
The methods such as pH adjusting agent are as preferred example, in this case, regulate at the high-purity solubility thrombosis with present embodiment
In the combination of albumen, as pharmaceutical composition generally solubility, and can thoroughly carry out lyophilization, therefore be preferred.
It addition, in the present embodiment, above-mentioned carrier is additionally also preferably glycerol.Above-mentioned carrier preferably adds when preparing preparation,
But adding when the used time dissolves also is to allow.
The soluble thrombomodulin of high purity of present embodiment for adult's dosage of every 1 time according to age, property
, body weight, the difference of symptom etc. and different, typically about 0.1mg～200mg, can exemplify and be administered once a day or according to need
To be administered for several times, such as by intravascular injection, the method that is preferably administered by drop intravenous.For present embodiment
Pharmaceutical composition, can convert using the soluble thrombomodulin as effective ingredient and be administered one by 0.1mg～200mg every day
Secondary or be administered as required for several times, by such as intravascular injection, be preferably administered by drop intravenous.
Embodiment
The present invention is more specifically described by below example, but the scope of the present invention is not by their any limit
Fixed.
The assay method of the APC activity of (reference example 1) thrombomodulin
According to Biologicals (2002) 30,69-76, for activating the thrombomodulin as index with protein C
APC activity be measured.
Add in 20mM calcium chloride solution 75 μ L and utilize the Tris-imidazole buffer containing 0.05% polysorbate20
The sample solution 25 μ L that is diluted also cools down with ice, adds human thrombin (U.S., the Sigma) solution of 40U/mL afterwards
25 μ L are stirred, and heat at 37 DEG C.The human protein C of 12U/mL is added after 10 minutes from adding human thrombin solution
(U.S., Enzyme Research) solution 25 μ L is stirred, heats at 37 DEG C.From adding human protein C solution 10
After minute, add heparin-Antithrombin III solution 100 μ L and be stirred, heat at 37 DEG C.From adding heparin-anticoagulation
After enzyme III solution plays 10 minutes, add synthesis substrate S-2366 (Sweden, the Chromogenix) solution being previously heated to 37 DEG C
250 μ L are stirred, and heat at 37 DEG C.From adding substrate solution after 10 minutes, add 50% acetic acid 1.5mL and stir
Mix, with water for comparison, measure the absorbance of 405nm.
The APC activity of thrombomodulin calculates according to following formula.It addition, thrombomodulin 1U is defined as 1 minute
Generate the amount of 0.1 μm ol paranitroanilinum.
[several 2]
Asample: the absorbance of sample solution
Ablank: the absorbance of comparison (water)
The molar absorption coefficient 9.6 × 10 of M: paranitroanilinum-3[1/μM]
V1: measure the liquid measure 2.0 × 10 during absorbance-3(L)
V2: the liquid measure 0.025 (mL) of sample solution
T: substrate cuts off the response time 10 (minute)
K: the activation of protein C generated by the thrombomodulin 1U molal quantity 0.1 (μ being dissociated the paranitroanilinum
Mol/ minute/U)
It addition, reagent is as described below.
<Tris-imidazole buffer>
Adding A liquid in B liquid 100mL, adjust to pH8.4, profit is diluted with water to 10 times.
A liquid: TRIS 3.03g and imidazoles 1.70g is dissolved in 1M hydrochloric acid 50mL,
Add water and become 100mL, add sodium chloride 11.7g and dissolve.
B liquid: 2-amino-2-hydroxymethyl-1,3-propylene glycol 4.04g, imidazoles 2.27g and sodium chloride 1.95g are dissolved in
In water so that it is for 100mL, add sodium chloride 11.7g and dissolve.
<20mM calcium chloride solution>
Tris-imidazole buffer 2mL is added in 60mM calcium chloride solution 1mL.
<heparin-Antithrombin III solution>
Add Antithrombin III (Japan, Mitsubishi Pharma) solution 7.5 μ L, Tris-imidazole buffer of 2U/mL
The heparin of liquid 42.5 μ L and 30U/mL (Japan, hold field pharmacy) solution 50 μ L carries out vibration mixing.Prepared by the used time, until to use
Before be cooled with ice.
The assay method of (reference example 2) HCP concentration
Serum-free culture is carried out for being imported with the gene recombinaton Chinese hamster ovary celI of thrombomodulin gene.By culture supernatant
It is supplied in antithrombotic regulation protein antibodies post, obtains non-adsorbed fraction.For this non-adsorbed fraction, survey according to reference example 1
Determine the APC activity of thrombomodulin, after confirming inactive detection, utilize ultrafilter membrane to concentrate, prepare HCP.With HCP it is
Antigen carries out sensitization to rabbit, after utilizing ammonium sulfate precipitation and a-protein post to carry out obtained anti-HCP antiserum refining, supplies
Award in the affinity column with thrombomodulin as ligand, obtain non-adsorbed fraction.So obtain identifying that thrombosis regulates
The rabbit anti-HCP antibody of albumen.
The PBS containing 0.05% polysorbate80 is utilized to be diluted, so that the HCP concentration predicted is 0～500ng/
ML, makes sample solution.It addition, in the case of the HCP concentration of sample solution is low, ultrafilter membrane etc. is used to be concentrated into suitable dense
Degree.Additionally add containing the PBS of 0.05% polysorbate80 in HCP, prepare in its 1mL containing 500,400,300,200,
100,8 kinds of solution of 50,25 and 0ng, make standard solution.
The rabbit anti-HCP antibody through 25 μ g/mL of sodium carbonate buffer dilution is added molten in the microplate of polystyrene system 96 hole
Liquid, in every 1 hole, each 100 μ L that add, stand about 2 hours at 25 DEG C afterwards.It follows that utilize 250 μ L containing 0.05% poly-Pyrusussuriensis
After each hole is carried out 5 times cleaning by the PBS of alcohol ester 80, each addition 200 μ L contain the PBS of 1% gelatin, stand about 1 hour at 25 DEG C.
After each hole is carried out 5 times cleaning by the PBS containing 0.05% polysorbate80 utilizing 250 μ L, in each hole, add 100 μ L's
Sample solution and standard solution, stand about 16 hours at 25 DEG C.It follows that utilize 250 μ L containing 0.05% polysorbate80
PBS carry out each hole 5 times cleaning after, each add the biotinylated rabbit of 100 μ L anti-HCP antibody-solutions, stand about 2 at 25 DEG C
Hour.After each hole is carried out 5 times cleaning by the PBS containing 0.05% polysorbate80 utilizing 250 μ L, each 100 μ L of addition resist
Biotin protein-Peroxidase Solution, stands about 2 hours at 25 DEG C.Utilize 250 μ L containing 0.05% polysorbate80
PBS carry out each hole 5 times cleaning after, each add 100 μ L enzyme substrate solutions, under room temperature, dark place stands.After appropriateness colour developing, respectively add
25% sulphuric acid entering 50 μ L makes reaction stop, and utilizes 96 hole microplate absorbance meters (Japan, Tecan Japan) to measure 492nm's
Absorbance.The HCP content in 1mL sample is obtained by the calibration trace obtained based on standard solution.It should be noted that this mensuration
One example of the quantitation limit of method is 25ng/mL.As required, every 10 are calculated according to the following formula, in 000U thrombomodulin
HCP content.
HCP content (ng/10,000U) in every 10,000U thrombomodulin
=a/b×10,000
A: the HCP content (ng/mL) in every 1mL sample
B: APC activity (U/mL) of the thrombomodulin in every 1mL sample
It addition, reagent is as described below.
<sodium carbonate buffer>
In natrium carbonicum calcinatum 0.16g and sodium bicarbonate 0.29g, add water dissolve, be made for 100mL.
<Avidin-Peroxidase solution>
Utilize the PBS containing 0.05% polysorbate80 by former for the horseradish peroxidase being combined with avidin D
Liquid (U.S., Vector Laboratories) is diluted to about 30,000 times.
<enzyme substrate solution>
O-phenylenediamine dihydrochloride 10mg is joined citrate-phosphate salt buffer (by citric acid monohydrate compound 2.56g
It is dissolved in water with disodium hydrogen phosphate dodecahydrate 9.12g, is made for 500mL) 20mL dissolves, adding before using
Enter hydrogen peroxide 10 μ L.
The assay method of (reference example 3) mouse IgG concentration
With mouse IgG as antigen, rabbit is carried out sensitization, utilize ammonium sulfate precipitation and a-protein post to obtained anti-little
Mus IgG antiserum refines, and obtains rabbit anti-mouse IgG antibody.
Utilize and be diluted containing the PBS of 0.05% polysorbate80 so that the mouse IgG concentration predicted be 0～
25ng/mL, makes sample solution.It addition, in the case of the IgG concentration of sample solution is low, use ultrafilter membrane etc. to be concentrated into suitable
When concentration.Additionally add containing the PBS of 0.05% polysorbate80 in mouse IgG, prepare in its 1mL containing 25,20,
15,9 kinds of solution of 10,5,2.5,1.25,0.63 and 0ng, make standard solution.
In the microplate of polystyrene system 96 hole, add rabbit anti-mouse IgG through 1.5 μ g/mL of sodium carbonate buffer dilution resist
Liquid solution, in every 1 hole, each 100 μ L that add, stand about 2 hours at 25 DEG C afterwards.It follows that utilize gathering containing 0.05% of 250 μ L
The PBS of PS80 carries out 5 times and cleans each hole, and each addition 200 μ L contain the PBS of 1% gelatin, stands about 1 at 25 DEG C little
Time.After each hole is carried out 5 times cleaning by the PBS containing 0.05% polysorbate80 utilizing 250 μ L, each hole adds 100 μ L
Sample solution and standard solution, 25 DEG C stand about 16 hours.It follows that utilize 250 μ L containing 0.05% polysorbate
After each hole is carried out 5 times cleaning by the PBS of 80, each 100 μ L biotinylated rabbit anti-mouse IgG antibody solution that adds, quiet at 25 DEG C
Put about 2 hours.After each hole is carried out 5 times cleaning by the PBS containing 0.05% polysorbate80 utilizing 250 μ L, each addition 100 μ
L Avidin-Peroxidase solution, stands about 2 hours at 25 DEG C.Utilize 250 μ L containing 0.05% polysorbate
After each hole is carried out 5 times cleaning by the PBS of 80, each enzyme substrate solution adding 100 μ L, under room temperature, dark place stands.Appropriateness colour developing
After, each 25% sulphuric acid adding 50 μ L makes reaction stop, and utilizes 96 hole microplate absorbance meters (Japan, Tecan Japan) to measure
The absorbance of 492nm.The mouse IgG content in 1mL sample is obtained by the calibration trace obtained based on standard solution.This algoscopy
An example of quantitation limit be 0.63ng/mL.As required, every 10 are calculated according to the following formula, in 000U thrombomodulin
Mouse IgG content.
Mouse IgG content (ng/10,000U) in every 10,000U thrombomodulin
=a/b×10,000
A: the mouse IgG content (ng/mL) in every 1mL sample
B: APC activity (U/mL) of the thrombomodulin in every 1mL sample
It addition, reagent is as described below.
<sodium carbonate buffer>
In natrium carbonicum calcinatum 0.16g and sodium bicarbonate 0.29g, add water dissolve, be made for 100mL.
<Avidin-Peroxidase solution>
Utilize the PBS containing 0.05% polysorbate80 by former for the horseradish peroxidase being combined with avidin D
Liquid (U.S., Vector Laboratories) is diluted to about 30,000 times.
<enzyme substrate solution>
O-phenylenediamine dihydrochloride 10mg is joined citrate-phosphate salt buffer (by citric acid monohydrate compound 2.56g
It is dissolved in water with disodium hydrogen phosphate dodecahydrate 9.12g, is made for 500mL) 20mL dissolves, adding before using
Enter hydrogen peroxide 10 μ L.
The assay method of (reference example 4) bovine serum protein concentration
With Ox blood serum as antigen, rabbit is carried out sensitization, utilize ammonium sulfate precipitation and a-protein post to obtained anti-Sanguis Bovis seu Bubali
Albumin matter antiserum refines, and obtains rabbit anti-bovine serum protein antibody.
The PBS containing 0.05% polysorbate80 is utilized to be diluted, so that the bovine serum protein concentration predicted is
0～25ng/mL, make sample solution.It addition, in the case of the bovine serum protein concentration of sample solution is low, use ultrafiltration
Films etc. are concentrated into suitable concentration.In Ox blood serum, additionally add the PBS containing 0.05% polysorbate80, prepare in its 1mL
Containing 8 kinds of solution of 25,20,15,10,5,2.5,1.25 and 0ng, make standard solution.
The anti-bovine serum albumin of rabbit of the 10 μ g/mL diluted through sodium carbonate buffer is added in the microplate of polystyrene system 96 hole
Matter antibody-solutions, in every 1 hole, each 100 μ L that add, stand about 2 hours at 25 DEG C afterwards.It follows that utilize containing of 250 μ L
The PBS of 0.05% polysorbate80 carries out 5 times and cleans each hole, and each addition 200 μ L contain the PBS of 1% gelatin, 25 DEG C of standings
About 1 hour.After each hole is carried out 5 times cleaning by the PBS containing 0.05% polysorbate80 utilizing 250 μ L, add in each hole
The sample solution of 100 μ L and standard solution, stand about 16 hours at 25 DEG C.It follows that utilize 250 μ L containing 0.05% poly-mountain
After each hole is carried out 5 times cleaning by the PBS of pears alcohol ester 80, each addition biotinylated rabbit of 100 μ L anti-bovine serum protein antibody is molten
Liquid, stands about 2 hours at 25 DEG C.The PBS containing 0.05% polysorbate80 utilizing 250 μ L carries out 5 times and cleans each hole
After, each Avidin-Peroxidase solution adding 100 μ L, stand about 2 hours at 25 DEG C.Utilize containing of 250 μ L
After each hole is carried out 5 times cleaning by the PBS having 0.05% polysorbate80, each addition 100 μ L enzyme substrate solutions, dark place under room temperature
Stand.After appropriateness colour developing, each 25% sulphuric acid adding 50 μ L makes reaction stop, utilizing 96 hole microplate absorbance meters (Japanese,
Tecan Japan) measure 492nm absorbance.The Sanguis Bovis seu Bubali in 1mL sample is obtained by the calibration trace obtained based on standard solution
Albumin matter content.One example of the quantitation limit of this algoscopy is 1.25ng/mL.As required, calculate every 10 according to the following formula,
Bovine serum protein content in 000U thrombomodulin.
Bovine serum protein content (ng/10,000U) in every 10,000U thrombomodulin
=a/b×10,000
A: the bovine serum protein content (ng/mL) in every 1mL sample
B: APC activity (U/mL) of the thrombomodulin in every 1mL sample
It addition, reagent is as described below.
<sodium carbonate buffer>
In natrium carbonicum calcinatum 0.16g and sodium bicarbonate 0.29g, add water dissolve, be made for 100mL.
<Avidin-Peroxidase solution>
Utilize the PBS containing 0.05% polysorbate80 by former for the horseradish peroxidase being combined with avidin D
Liquid (U.S., Vector Laboratories) is diluted to about 30,000 times.
<enzyme substrate solution>
O-phenylenediamine dihydrochloride 10mg is joined citrate-phosphate salt buffer (by citric acid monohydrate compound 2.56g
It is dissolved in water with disodium hydrogen phosphate dodecahydrate 9.12g, is made for 500mL) 20mL dissolves, adding before using
Enter hydrogen peroxide 10 μ L.
The manufacture 1 of (comparative example 1) soluble thrombomodulin
According to the embodiment 1 of Japanese Unexamined Patent Publication 11-341990 publication, by the aminoacid described in coded sequence numbering 9
The DNA of sequence carries out restructuring by gene manipulation techniques and makes gene recombinaton Chinese hamster ovary celI, sows afterwards containing 150mg/L
L-PROLINE (Japan, aginomoto), the Kanamycin Sulfate (Japan, Meiji Seika Kaisba) of 60mg/L, 1mg/L tartaric acid safe
Happy rhzomorph (Japan, Mercian) and the DMEM culture medium (U.S., Invitrogen) of 10% Ox blood serum (U.S., HyClone)
In, at CO2Cultivate in 37 DEG C in incubator.It is centrifuged culture fluid separating, obtained cell suspension is being contained
The L-PROLINE of 150mg/L (Japan, aginomoto), the Kanamycin Sulfate (Japan, Meiji Seika Kaisba) of 60mg/L, 1mg/L
Tylosin tartrate (Japan, Mercian), 10% dimethyl sulfoxide (Germany, Merck) and 10% Ox blood serum (U.S.,
HyClone), in DMEM culture medium (U.S., Invitrogen), (4 × 10 it are dispensed in bottle afterwards7Cells/ props up), freezing
It is saved in liquid nitrogen.
The NaHCO of the culture medium shown in component list 2 by Japanese Unexamined Patent Publication 11-341990 publication3Concentration becomes 5,
700mg/L, NaCl concentration is become 2,410mg/L, gained culture medium is carried out cell cultivation as minimal medium.For increasing
Grow culture medium, in minimal medium, add the Kanamycin Sulfate (U.S., Invitrogen) of 60mg/L, the wine of 1mg/L
Stone acid tylosin (U.S., Sigma-Aldrich) and 8% Ox blood serum (U.S., HyClone) use.It addition, for
Production medium, the serum-concentration making proliferated culture medium is 3%.
1 cell bottle is melted, by this cell sowing to the proliferated culture medium of 100mL, uses suspension culture bottle to exist
CO25 days stir culture are carried out in 37 DEG C in incubator.It is 7.0 × 10 at viable cell density5The moment of more than cells/mL, will training
Nutrient solution total amount is passaged in the proliferated culture medium of 0.9L, uses suspension culture bottle at CO2Stirring in 5 days is carried out in 37 DEG C in incubator
Cultivate.It is 7.0 × 10 at viable cell density5In the moment of more than cells/mL, culture fluid total amount is passaged to the enrichment culture of 9L
In base, use culture tank, 37 DEG C, pH7.2, carry out 5 days stir culture under conditions of dissolved oxygen 50%.At viable cell density it is
7.0×105In the moment of more than cells/mL, culture fluid total amount is passaged in the proliferated culture medium of 120L, uses perfusion to cultivate
Groove, 37 DEG C, pH7.2, carry out 7 days stir culture under conditions of dissolved oxygen 50%.It is 7.0 × 10 at viable cell density5cells/
In the moment of more than mL, start perfusion and cultivate, be carried out continuously the interpolation of production medium and the recovery of culture supernatant.Condition of culture
For: 37 DEG C, pH7.2, dissolved oxygen 50%, culture medium exchange 130～200L/ days, above pressurization 0～0.2MPa.At viable cell density
Reach 7.5 × 106Proceed after cells/mL 36 days to cultivate, culture supernatant is reclaimed as producing liquid.Used
The production liquid reclaimed is carried out fining by filter SUPRAdisc II (U.S., Pall) and Supor EBV (U.S., Pall),
Liquid is produced 2～10 DEG C of preservations after making filtration.
It is supplied in the utilization 20mM Tris salt buffer containing 150mM sodium chloride by producing liquid after the filtration of about 700L
(pH7.7) Q-Sepharose Fast Flow (U.S., GE Healthcare) post (diameter 63cm, the height of equilibrating have been carried out
Degree 25cm) in.It follows that utilize the 20mM acetate buffer (pH5.5) containing 180mM sodium chloride of 6 column volumes (CV) to carry out
Clean, be carried out further with 20mM Tris salt buffer (pH7.7) containing 180mM sodium chloride until the suction of 280nm
Till receipts are returned to baseline, 20mM Tris salt buffer (pH7.7) containing 300mM sodium chloride is utilized to start eluting.Washed
The eluent of the 0.5 column volume capacity that the absorbance of de-liquid has begun to ramp up at the peak of 280nm is as thick refined liquid.Repeatedly implement
6 same operations, it is thus achieved that the thick refined liquid of 6 batches.It addition, be 2 DEG C～10 DEG C, column flow rate (Network ロ マ ト flow velocity) in temperature
Implemented under conditions of 109L/ hour.
According to the embodiment 10 of Japanese Unexamined Patent Publication 11-341990 publication, it is anti-with the thrombomodulin in people's lung source
Originally make antithrombotic regulation protein monoclonal antibody, with CNBr-activated Sepharose (cyanogen bromide-activated agarose
Gel) 4Fast Flow (U.S., GE Healthcare) carries out haptoreaction, carries out antithrombotic regulation protein monoclonal antibody
Coupling, make antithrombotic regulation protein monoclonal antibody and combine Sepharose 4Fast Flow, fill to post, make list
Clonal antibody post.The thick refined liquid of about 40L is supplied in and balances through the 20mM phosphate buffer (pH7.3) containing 0.3M sodium chloride
In the monoclonal anti scapus (diameter 44cm, highly 13cm) changed.The 20mM phosphate buffer containing 1.0M sodium chloride of circulation 6CV
(pH7.3), further the 0.1M acetate buffer (pH5.0) of circulation 3CV be carried out, utilize the 0.1M containing 0.3M sodium chloride
Glycine HCI buffer (pH3.0) starts eluting.Obtain the absorbance of eluent at the peak of 280nm from beginning to ramp up to opening
Begin the eluent till declining, and adds the 0.5M phosphate buffer (pH7.3) shape with refined liquid 1 of 1/10 capacity in eluent
Formula obtains.Repeatedly implement 6 same operations, it is thus achieved that the refined liquid 1 of 6 batches.It should be noted that be 2 DEG C～10 in temperature
DEG C, column flow rate be to implement under conditions of 46L/ hour.
Utilize 1.0M glycine HCI buffer (pH2.0) that the refined liquid 1 about 170L of 6 batches is adjusted to pH3.5, supply
In the SP-SepharoseFF (U.S. GE utilizing 0.1M glycine HCI buffer (pH3.5) equilibrating containing 0.3M NaCl
Healthcare Bioscience) in post (diameter 45cm, highly 10cm).Utilize the 0.1M glycinate containing 0.3M NaCl
Acid buffer (pH3.5) proceeds by cleaning, obtains the absorbance peak at 280nm from beginning to ramp up to beginning to decline
Circulation (the logical り of element) fraction, is neutralized to pH7 with 0.5M phosphate buffer (pH7.3) immediately, obtains with the form of refined liquid 2.Need
Illustrate, temperature be 2 DEG C～10 DEG C, column flow rate be to implement under conditions of 160L/ hour.
The refined liquid 2 of about 200L utilize ultrafilter membrane Microza UF assembly SIP-2013 (Japan, Asahi Chemical Industry's chemistry) dense
After being reduced to about 10L, it is supplied in the Sephacryl utilizing 20mM phosphate buffer (pH7.3) equilibrating containing 50mM sodium chloride
In S-300HR (U.S. GE Healthcare Bioscience) post (diameter 63cm, highly 94cm).By the absorption of 280nm
After maximum eluting peak separates, ultrafilter membrane MicrozaUF assembly SIP-1013 (Japan, Asahi Chemical Industry's chemistry) is utilized to be concentrated into about
12L, obtains with the form of refined liquid 3.It should be noted that temperature be 2 DEG C～10 DEG C, column flow rate be the bar of 6.2L/ hour
Implement under part.
At room temperature, make refined liquid 3 by utilizing the 20mM phosphorus containing 50mM sodium chloride under 0.1MPa pressure below
Virus removal film PLANOVA 15N (Japan, Asahi Kasei medical treatment, the membrane area 1m of acid buffer (pH7.3) equilibrating2), afterwards
Further by the PVDF filter membrane (U.S., Millipore) of 0.22 μm, recovery total.Adjust as solubility thrombosis
Joint extract of protein goods.
By same operation, it is thus achieved that the highly finished product of 3 batches (A1, A2, A3).
The APC activity of the thrombomodulin of A1, A2, A3 is respectively 69000U/mL, 68000U/mL, 72000U/mL.
Soluble thrombomodulin concentration in the solution of A1, A2, A3 be respectively 10.5mg/mL, 10.2mg/mL,
10.3mg/mL。
The manufacture 2 of (comparative example 2) soluble thrombomodulin
Use the culture medium shown in component list 2 of Japanese Unexamined Patent Publication 11-341990 publication as minimal medium.For
Proliferated culture medium, adds the Kanamycin Sulfate (U.S., Invitrogen) of 60mg/L, 1mg/L in minimal medium
Tylosin tartrate (Japan, the pharmacy of salt open country) and 8% Ox blood serum (U.S., HyClone) use.It addition, for life
Producing culture medium, the serum-concentration making proliferated culture medium is 4%.
1 the cell bottle made in comparative example 1 is melted, by this cell sowing to the proliferated culture medium of 100mL, makes
With suspension culture bottle at CO23 days stir culture are carried out in 37 DEG C in incubator.It is 5.0 × 10 at viable cell density5cells/mL
In the above moment, culture fluid total amount is passaged in the proliferated culture medium of 400mL, uses suspension culture bottle at CO2In incubator
3 days stir culture are carried out in 37 DEG C.It is 5.0 × 10 at viable cell density5In the moment of more than cells/mL, culture fluid total amount is passed
For to the proliferated culture medium of 2L, use aryballos at CO23 days stir culture are carried out in 37 DEG C in incubator.Close at living cells
Degree is 5.0 × 105In the moment of more than cells/mL, culture fluid total amount is passaged in the proliferated culture medium of 7.5L, uses spherical
Bottle is at CO24 days stir culture are carried out in 37 DEG C in incubator.It is 5.0 × 10 at viable cell density5The moment of more than cells/mL,
Start perfusion to cultivate, be carried out continuously the interpolation of production medium and the recovery of culture supernatant.Condition of culture is: 37 DEG C,
PH7.2, dissolved oxygen 50%, culture medium exchange 10L/ days, pressurization 0～0.2MPa above.Viable cell density reach 7.5 ×
106Proceed after cells/mL 40 days to cultivate, culture supernatant is reclaimed as producing liquid.
The filter (U.S., Pall) that liquid use aperture is 0.7 μm and 0.22 μm that produces reclaimed carries out fining, makes
Liquid is produced 2 DEG C～10 DEG C preservations after filtration.
It is supplied in the utilization 20mM Tris salt buffer containing 150mM sodium chloride by producing liquid after the filtration of about 400L
(pH7.4) Q-Sepharose Fast Flow (U.S., the GE Healthcare) post (diameter 44cm, highly 26cm) of equilibrating
In.It follows that utilize the 20mM acetate buffer (pH5.5) containing 180mM sodium chloride of 6CV to be carried out, further with
20mM Tris salt buffer (pH7.4) containing 180mM sodium chloride is carried out, until the absorption of 280nm is returned to baseline is
Only, utilizing 20mM Tris salt buffer (pH7.4) containing 300mM sodium chloride to start eluting, the absorbance obtaining eluent exists
The eluent of the 0.5 column volume capacity that the peak of 280nm has begun to ramp up is as thick refined liquid.It addition, be 2 DEG C～10 in temperature
DEG C, column flow rate be to implement under conditions of 45L/ hour.
The thick refined liquid of about 20L is supplied in 20mM phosphate buffer (pH7.3) balance utilized containing 0.3M sodium chloride
In the monoclonal anti scapus (diameter 44cm, highly 12cm) changed.The 20mM phosphate buffer containing 1.0M sodium chloride of circulation 6CV
(pH7.3), further the 0.1M acetate buffer (pH5.0) of circulation 3CV be carried out, utilize the 0.1M containing 0.3M sodium chloride
Glycine HCI buffer (pH3.0) starts eluting.Obtain the absorbance of eluent at the peak of 280nm from beginning to ramp up to opening
Begin the eluent till declining, and adds the 0.5M phosphate buffer (pH7.3) of 1/10 capacity, with refined liquid 1 in eluent
Form obtains.It addition, temperature be 2 DEG C～10 DEG C, column flow rate be to implement under conditions of 45L/ hour.
Utilize 1.0M glycine HCI buffer (pH2.0) that the refined liquid 1 of about 12L is adjusted to pH3.5, be supplied in utilization
SP-SepharoseFF (the U.S. GE of 0.1M glycine HCI buffer (pH3.5) equilibrating containing 0.3M NaCl
Healthcare Bioscience) in post (diameter 14cm, highly 13cm).Utilize the 0.1M glycinate containing 0.3M NaCl
Acid buffer (pH3.5) starts to clean, and obtains the circulation from beginning to ramp up to beginning to decline of the absorbance peak at 280nm
Fraction, is neutralized to pH7 with 0.5M phosphate buffer (pH7.3) immediately, obtains with the form of refined liquid 2.It addition, be 2 in temperature
DEG C～10 DEG C, column flow rate be to implement under conditions of 15L/ hour.
The refined liquid 2 of about 16L utilize ultrafilter membrane Microza UF assembly SIP-1013 (Japan, Asahi Chemical Industry's chemistry) concentrate
To about 1.2L, it is supplied in the Sephacryl utilizing 20mM phosphate buffer (pH7.3) equilibrating containing 50mM sodium chloride
In S-300HR (U.S. GE Healthcare Bioscience) post (diameter 25cm, highly 85cm).By the absorption of 280nm
After maximum eluting peak separates, ultrafilter membrane Microza UF assembly SIP-1013 (Japan, Asahi Chemical Industry's chemistry) is utilized to be concentrated into
About 0.8L, obtains with the form of refined liquid 3.It addition, temperature be 2 DEG C～10 DEG C, column flow rate be to carry out under conditions of 1L/ hour
Implement.
At room temperature, make refined liquid 3 by utilizing the 20mM phosphorus containing 50mM sodium chloride under 0.1MPa pressure below
The virus removal film PLANOVA 15N (Japan, Asahi Kasei medical treatment, membrane area 0.3m2) of acid buffer (pH7.3) equilibrating, it
After further by the PVDF filter membrane (U.S., Millipore) of 0.22 μm, recovery total.As solubility thrombosis
Regulation extract of protein goods (batch: B1).
The APC activity of the thrombomodulin of B1 is 79000U/mL.
Soluble thrombomodulin concentration in the solution of B1 is 12.6mg/mL.
The manufacture 3 of (comparative example 3) soluble thrombomodulin
Use DMEM culture medium (U.S., Invitrogen) as minimal medium.For proliferated culture medium, to basic training
Support and base adds the L-PROLINE (Japan, aginomoto) of 150mg/L, Kanamycin Sulfate (Japan, Mingzhi's system of 60mg/L
Really), tylosin tartrate (Japan, Mercian) and 10% Ox blood serum (U.S., HyClone) of 1mg/L uses.
It addition, for production medium, the serum-concentration making proliferated culture medium is 1%～3%.
1 the cell bottle made in comparative example 1 is melted, by this cell sowing to the proliferated culture medium of 100mL, makes
With suspension culture bottle at CO25 days stir culture are carried out in 37 DEG C in incubator.Culture fluid total amount is passaged to the propagation of 400mL
In culture medium, use suspension culture bottle at CO25 days stir culture are carried out in 37 DEG C in incubator.Culture fluid total amount is passaged to
In the proliferated culture medium of 1.6L, it is blown into air and CO2, use aryballos to carry out 5 days stir culture in 37 DEG C.Will training
Nutrient solution total amount is passaged in the proliferated culture medium of 6L, is blown into air, CO2And oxygen, use aryballos to carry out 5 in 37 DEG C
It stir culture.Culture fluid total amount is passaged in the proliferated culture medium of 56L, is blown into air, CO2And oxygen, use
Aryballos carry out 4 days stir culture in 37 DEG C.Carry out after carrying out total amount culture medium exchange 3 days cultivating, carry out total amount training further
After supporting base exchange, reach 1 × 10 at viable cell density6Production medium is replaced with after cells/mL.Use CC-100 continuous
Centrifugal separator (Sweden, Alfa Laval), reclaims the production liquid in sky, is exchanged for fresh culture.Productive culture
Implement 100 days.The filter (U.S., Pall) that liquid use aperture is 0.7 μm and 0.22 μm that produces reclaimed carries out fining, system
Become and produce liquid 2 DEG C～10 DEG C preservations after filtering.
It is supplied in the utilization 20mM Tris salt buffer containing 150mM sodium chloride by producing liquid after the filtration of about 2400L
(pH7.4) Q-Sepharose Fast Flow (U.S., the GE Healthcare) post (diameter 44cm, highly 25cm) of equilibrating
In.It follows that utilize the 20mM acetate buffer (pH5.5) containing 180mM sodium chloride of 6CV to be carried out, further with
20mM Tris salt buffer (pH7.4) containing 180mM sodium chloride of 2CV is carried out, and utilizes containing 300mM sodium chloride
20mM Tris salt buffer (pH7.4) starts eluting, obtains the pact that the absorbance of eluent has begun to ramp up at the peak of 280nm
The eluent of 15L is as thick refined liquid 1.It addition, temperature be 2 DEG C～10 DEG C, column flow rate be to carry out under conditions of 45L/ hour
Implement.
Above-mentioned thick refined liquid 1 is supplied in and utilizes 20mM phosphate buffer (pH7.0) equilibrating containing 0.3M NaCl
In Butyl-Sepharose FF (U.S. GE Healthcare Bioscience) post (diameter 25cm, highly 10cm).Utilize
20mM phosphate buffer (pH7.0) containing 0.3M NaCl starts to clean, and obtains absorbance at the peak of 280nm from beginning to ramp up
Play the circulation fraction to beginning to decline as thick refined liquid 2.It addition, temperature be 2 DEG C～10 DEG C, column flow rate be that 13L/ is little
Implement time under conditions of.
The thick refined liquid of about 20L is supplied in 20mM phosphate buffer (pH7.3) balance utilized containing 0.3M sodium chloride
In the monoclonal anti scapus (diameter 44cm, highly 18cm) changed.The 20mM phosphate buffer containing 1.0M sodium chloride of circulation 6CV
(pH7.3), further the 0.1M acetate buffer (pH5.0) of circulation 3CV be carried out, utilize the 0.1M containing 0.3M sodium chloride
Glycine HCI buffer (pH3.0) starts eluting.Obtain the absorbance of eluent at the peak of 280nm from beginning to ramp up to
Eluent till beginning to decline, add in eluent 1M Glycine-NaOH buffer (pH9.0) of 1/10 capacity with
The 0.5M phosphate buffer (pH7.3) of 1/25 capacity obtains with the form of refined liquid 1.It addition, be 2 DEG C～10 DEG C, post in temperature
Flow velocity is to implement under conditions of 50L/ hour.
The refined liquid 1 of about 15L utilize ultrafilter membrane Microza UF assembly SIP-1013 (Japan, Asahi Chemical Industry's chemistry) concentrate
To about 1L, it is supplied in the Sephacryl S-utilizing 20mM phosphate buffer (pH7.3) equilibrating containing 150mM sodium chloride
In 300HR (U.S. GE Healthcare Bioscience) post (diameter 25cm, highly 80cm).It addition, temperature be 2 DEG C～
10 DEG C, column flow rate be to implement under conditions of 1L/ hour.After being separated by the eluting peak absorbing maximum of 280nm, make liquid
The body PVDF filter membrane (U.S., Millipore) by 0.22 μm, reclaims about 3L.As solubility thrombomodulin
White highly finished product (batch: B2).
The APC activity of the thrombomodulin of B2 is 28000U/mL.
Soluble thrombomodulin concentration in the solution of B2 is 3.8mg/mL.
The manufacture 4 of (comparative example 4) soluble thrombomodulin
The cell bottle of 1 freezen protective in comparative example 1 is melted, sow containing 8mM L-glutaminate (U.S.,
Invitrogen), 50 μMs of hypoxanthine (U.S., Invitrogen) and 8 μMs of thymidines (U.S.,
Invitrogen) in serum-free medium IS CHO-CD (U.S., Irvine Scientific), at CO2In incubator in
Cultivate for 37 DEG C.It is centrifuged culture fluid separating, by obtained cell suspension (beautiful containing 8mM L-glutaminate
State, Invitrogen), 50 μMs of hypoxanthine (U.S., Invitrogen), 8 μMs of thymidines (U.S.,
Invitrogen) and 10% dimethyl sulfoxide (U.S., Sigma-Aldrich) serum-free medium ISCHO-CD (U.S.,
Irvine Scientific) in, it is dispensed in bottle (2 × 10 afterwards7Cells/ props up), freezen protective is in liquid nitrogen.
Proliferated culture medium be dissolve in 1L water 20.78g IS CHO-CD-A3 (U.S., Irvine Scientific),
The sodium chloride (field Japanese, rich pharmacy) of 4.06g and the sodium bicarbonate (Japan and Wako Pure Chemical Industries) of 2.20g make.Separately
Outward, production medium is to dissolve IS CHO-CD-A3 (U.S., IrvineScientific), the 2.63g of 20.78g in 1L water
Sodium chloride (field Japanese, rich pharmacy) and the sodium bicarbonate (Japan and Wako Pure Chemical Industries) of 4.40g make.
1 cell bottle is melted, by this cell sowing to the proliferated culture medium of 100mL, uses T-flask at CO2Training
5 days quiescent culture are carried out in 36 DEG C in supporting case.It is 7.0 × 10 at viable cell density5The moment of more than cells/mL, by culture fluid
40mL is passaged in the proliferated culture medium of 360mL, uses suspension culture bottle at CO2Stirring training in 7 days is carried out in 36 DEG C in incubator
Support.It is 7.0 × 10 at viable cell density5In the moment of more than cells/mL, culture fluid 80mL is passaged to the enrichment culture of 720mL
In base, use suspension culture bottle at CO26 days stir culture are carried out in 36 DEG C in incubator.Viable cell density be 7.0 ×
105In the moment of more than cells/mL, culture fluid total amount is passaged in the proliferated culture medium of 9.2L, uses perfusion culture tank,
36 DEG C, pH7.1, carry out 8 days stir culture under conditions of dissolved oxygen 50%.It is 7.0 × 10 at viable cell density5Cells/mL with
On moment, start perfusion cultivate, be carried out continuously the interpolation of production medium and the recovery of culture supernatant.Condition of culture is:
36 DEG C, pH7.1, dissolved oxygen 50%, culture medium exchange 10L/ days, pressurization 0～0.2MPa above.Viable cell density reach 7.5 ×
106Proceed after cells/mL 26 days to cultivate, culture supernatant is reclaimed as producing liquid.
Use filter SupraCap (U.S., Pall) and Supor EBV (U.S., Pall) that the production liquid reclaimed is entered
Row is fining, produces liquid 2 DEG C～10 DEG C preservations after making filtration.
It is supplied in the utilization 20mM Tris salt buffer containing 150mM sodium chloride by producing liquid after the filtration of about 250L
(pH7.7) Q-Sepharose Fast Flow (U.S., the GE Healthcare) post (diameter 25cm, highly 25cm) of equilibrating
In.Next the 20mM acetate buffer (pH5.6) containing 180mM sodium chloride utilizing 6CV is carried out, further with
20mM Tris salt buffer (pH7.7) containing 180mM sodium chloride of 4CV is carried out, and utilizes containing 290mM sodium chloride
20mM Tris salt buffer (pH7.7) starts eluting, obtain that the absorbance of eluent has begun to ramp up at the peak of 280nm 0.5
The eluent of column volume capacity is as thick refined liquid.It addition, temperature be 2 DEG C～10 DEG C, column flow rate be the condition of 18L/ hour
Under implement.
The above-mentioned thick refined liquid of about 6L is supplied in the 20mM phosphate buffer (pH7.3) utilized containing 0.3M sodium chloride put down
In the monoclonal anti scapus (diameter 44cm, highly 8cm) of weighing apparatusization.The 20mM phosphoric acid buffer containing 1.0M sodium chloride of circulation 6CV
Liquid (pH7.3), further the 0.1M acetate buffer (pH5.0) of circulation 3CV are carried out, and utilize containing 0.3M sodium chloride
0.1M glycine HCI buffer (pH3.0) starts eluting.Obtain the absorbance of eluent at the peak of 280nm from beginning to ramp up
Eluent to beginning to decline, adds the 0.5M phosphate buffer (pH7.3) of 1/10 capacity in eluent, as refined
Liquid 1 obtains.It addition, temperature be 2 DEG C～10 DEG C, column flow rate be to implement under conditions of 46L/ hour.
Utilize 1.0M glycine HCI buffer (pH2.0) that the refined liquid 1 of about 14L is adjusted to pH3.5, be supplied in utilization
SP-SepharoseFF (the U.S. GE of 0.1M glycine HCI buffer (pH3.5) equilibrating containing 0.3M NaCl
Healthcare Bioscience) in post (diameter 14cm, highly 13cm).Utilize the 0.1M glycinate containing 0.3M NaCl
Acid buffer (pH3.5) starts to clean, and obtains the circulation from beginning to ramp up to beginning to decline of the absorbance peak at 280nm
Fraction, is neutralized to pH7 with 0.5M phosphate buffer (pH7.3) immediately, obtains as refined liquid 2.It addition, be 2 DEG C in temperature
～10 DEG C, column flow rate be to implement under conditions of 15L/ hour.
The refined liquid 2 of about 20L utilize ultrafilter membrane Microza UF assembly SIP-1013 (Japan, Asahi Chemical Industry's chemistry) concentrate
To about 1L, it is supplied in the Sephacryl S-utilizing 20mM phosphate buffer (pH7.3) equilibrating containing 50mM sodium chloride
In 300HR (U.S. GE Healthcare Bioscience) post (diameter 25cm, highly 79cm).By the absorption of 280nm
After big eluting peak separates, ultrafilter membrane Microza UF assembly SIP-1013 (Japan, Asahi Chemical Industry's chemistry) is utilized to be concentrated into about
0.7L, obtains as refined liquid 3.It addition, temperature be 2 DEG C～10 DEG C, column flow rate be to carry out reality under conditions of 1L/ hour
Execute.
At room temperature, make refined liquid 3 total amount by utilizing containing 50mM sodium chloride under 0.1MPa pressure below
Virus removal film PLANOVA 15N (Japan, Asahi Kasei medical treatment, the membrane area of 20mM phosphate buffer (pH7.3) equilibrating
0.12m2), the most further by the PVDF filter membrane (U.S., Millipore) of 0.22 μm, recovery total.As
Soluble thrombomodulin highly finished product (batch: B3).
The manufacture 1 of (embodiment 1) soluble thrombomodulin of high purity
NaHCO by the culture medium shown in the component list 2 of Japanese Unexamined Patent Publication 11-341990 publication3Concentration becomes 5,
700mg/L, NaCl concentration is become 2,410mg/L, gained culture medium is carried out cell cultivation as minimal medium.For increasing
Grow culture medium, in minimal medium, add the Kanamycin Sulfate (U.S., Invitrogen) of 60mg/L, the wine of 1mg/L
Stone acid tylosin (U.S., Sigma-Aldrich) and 8% Ox blood serum (U.S., HyClone) use.It addition, for
Production medium, the serum-concentration making proliferated culture medium is 3%.
1 the cell bottle made in comparative example 1 is melted, by this cell sowing to the proliferated culture medium of 100mL, makes
With suspension culture bottle at CO25 days stir culture are carried out in 37 DEG C in incubator.It is 7.0 × 10 at viable cell density5cells/mL
In the above moment, culture fluid total amount is passaged in the proliferated culture medium of 0.9L, uses suspension culture bottle at CO2In incubator in
37 DEG C carry out 5 days stir culture.It is 7.0 × 10 at viable cell density5In the moment of more than cells/mL, culture fluid total amount is passed on
To the proliferated culture medium of 9L, use culture tank 37 DEG C, pH7.2, carry out 5 days stir culture under conditions of dissolved oxygen 50%.?
Viable cell density is 7.0 × 105In the moment of more than cells/mL, culture fluid total amount is passaged in the proliferated culture medium of 120L,
Use perfusion culture tank, 37 DEG C, pH7.2, carry out 7 days stir culture under conditions of dissolved oxygen 50%.At viable cell density it is
7.0×105In the moment of more than cells/mL, start perfusion and cultivate, be carried out continuously interpolation and the culture supernatant of production medium
Recovery.Condition of culture is: 37 DEG C, pH7.2, dissolved oxygen 50%, culture medium exchange 130～200L/ days, above pressurization 0～
0.2MPa.7.5 × 10 are reached at viable cell density6Proceed after cells/mL 40 days to cultivate, using culture supernatant as life
Production fluid reclaims.Use filter SUPRAdisc II (U.S., Pall) and Supor EBV (U.S., Pall) to recovery
Production liquid carry out fining, make and produce liquid after filtration 2 DEG C～10 DEG C preservations.
It is supplied in the utilization 20mM Tris salt buffer containing 150mM sodium chloride by producing liquid after the filtration of about 700L
(pH7.7) Q-Sepharose Fast Flow (U.S., the GE Healthcare) post (diameter 63cm, highly 25cm) of equilibrating
In.Next the 20mM acetate buffer (pH5.5) containing 180mM sodium chloride utilizing 6CV is carried out, further with containing
20mM Tris salt buffer (pH7.7) having 180mM sodium chloride is carried out till the absorption of 280nm returns back to baseline,
Utilizing 20mM Tris salt buffer (pH7.7) containing 300mM sodium chloride to start eluting, the absorbance obtaining eluent exists
The eluent of the 0.5 column volume capacity that the peak of 280nm has begun to ramp up is as thick refined liquid.Repeatedly implement 3 same operations,
Obtain the thick refined liquid of 3 batches.It addition, temperature be 2 DEG C～10 DEG C, column flow rate be to implement under conditions of 109L/ hour.
The thick refined liquid of about 20L is supplied in 20mM phosphate buffer (pH7.3) balance utilized containing 0.3M sodium chloride
In the monoclonal anti scapus (diameter 44cm, highly 13cm) changed.The 20mM phosphate buffer containing 1.0M sodium chloride of circulation 6CV
(pH7.3), further the 0.1M acetate buffer (pH5.0) of circulation 3CV be carried out, sweet with the 0.1M containing 0.3M sodium chloride
Propylhomoserin hydrochloride buffer (pH3.0) starts eluting.Obtain the absorbance of eluent at the peak of 280nm from beginning to ramp up to beginning
Eluent till decline, adds the 0.5M phosphate buffer (pH7.3) of 1/10 capacity, as refined liquid 1 in eluent
Obtain.Repeatedly implement 6 same operations, it is thus achieved that the refined liquid 1 of 6 batches.It addition, temperature be 2 DEG C～10 DEG C, column flow rate be
Implement under conditions of 46L/ hour.
Make the refined liquid 1 about 130L of 6 batches by nylon filter membrane (Germany, Sartorius with the flow velocity of 5L/ minute
Society, SARTOLON Maxi Caps 5101307H3, aperture 0.4 μm+0.2 μm, membrane area 1.8m2) afterwards (relative to 1mg HCP
Use about 0.07m2Membrane area), utilize 1.0M glycine HCI buffer (pH2.0) to be adjusted to pH3.5, be supplied in utilization and contain
SP-SepharoseFF (the U.S. GE of 0.1M glycine HCI buffer (pH3.5) equilibrating of 0.3M NaCl
Healthcare Bioscience) in post (diameter 45cm, highly 10cm).Utilize the 0.1M glycinate containing 0.3M NaCl
Acid buffer (pH3.5) starts to clean, and obtains the circulation from beginning to ramp up to beginning to decline of the absorbance peak at 280nm
Fraction, is neutralized to pH7 with 0.5M phosphate buffer (pH7.3) immediately, obtains as refined liquid 2.It addition, be 2 DEG C in temperature
～10 DEG C, column flow rate be to implement under conditions of 160L/ hour.
The refined liquid 2 of about 160L utilize ultrafilter membrane Microza UF assembly SIP-2013 (Japan, Asahi Chemical Industry's chemistry) dense
After being reduced to about 10L, it is supplied in the Sephacryl utilizing 20mM phosphate buffer (pH7.3) equilibrating containing 50mM sodium chloride
In S-300HR (U.S. GE Healthcare Bioscience) post (diameter 63cm, highly 94cm).By the absorption of 280nm
After maximum eluting peak separates, ultrafilter membrane MicrozaUF assembly SIP-1013 (Japan, Asahi Chemical Industry's chemistry) is utilized to be concentrated into about
6L, obtains as refined liquid 3.It addition, temperature be 2 DEG C～10 DEG C, column flow rate be to carry out reality under conditions of 6.2L/ hour
Execute.
At room temperature, make refined liquid 3 by utilizing the 20mM phosphorus containing 50mM sodium chloride under 0.1MPa pressure below
Virus removal film PLANOVA 15N (Japan, Asahi Kasei medical treatment, the membrane area 1m of acid buffer (pH7.3) equilibrating2), afterwards
Further by the PVDF filter membrane (U.S., Millipore) of 0.22 μm, recovery total.As high-purity solubility
Thrombomodulin highly finished product (batch: A4).
The APC activity of the thrombomodulin of A4 is 60000U/mL.
Soluble thrombomodulin concentration in the solution of A4 is 9.3mg/mL.
The manufacture 2 of (embodiment 2) soluble thrombomodulin of high purity
It is supplied in utilization containing 150mM sodium chloride by producing liquid after the filtration of the about 2,000L obtained in embodiment 1
Q-Sepharose Fast Flow (U.S., the GE Healthcare) post of 20mM Tris salt buffer (pH7.7) equilibrating
In (diameter 63cm, highly 25cm).Next the 20mM acetate buffer (pH5.45) containing 170mM sodium chloride of 6CV is utilized
Being carried out, 20mM Tris salt buffer (pH7.7) containing 170mM sodium chloride further with 4CV is carried out, profit
Start eluting with 20mM Tris salt buffer (pH7.7) containing 300mM sodium chloride, obtain the absorbance of eluent at 280nm
The eluent of 0.5 column volume capacity that begun to ramp up of peak as thick refined liquid.Repeatedly implement 2 same operations, it is thus achieved that 2
The thick refined liquid of batch.It addition, temperature be 2 DEG C～10 DEG C, column flow rate be to implement under conditions of 109L/ hour.
The thick refined liquid of about 10L is supplied in 20mM phosphate buffer (pH7.3) balance utilized containing 0.3M sodium chloride
In the monoclonal anti scapus (diameter 44cm, highly 13cm) changed.The 20mM phosphate buffer containing 1.0M sodium chloride of circulation 6CV
(pH7.3), further the 0.1M acetate buffer (pH5.0) of circulation 3CV be carried out, utilize the 0.1M containing 0.3M sodium chloride
Glycine HCI buffer (pH3.0) starts eluting.Obtain the absorbance of eluent at the peak of 280nm from beginning to ramp up to opening
Begin the eluent till declining, and adds the 0.5M phosphate buffer (pH7.3) of 1/10 capacity, as refined liquid 1 in eluent
Obtain.Repeatedly implement 8 same operations, it is thus achieved that the refined liquid 1 of 8 batches.It addition, be 2 DEG C～10 DEG C, column flow rate in temperature
Implemented under conditions of 46L/ hour.
Make the refined liquid 1 about 180L of 6 batches by nylon filter membrane (Germany, Sartorius with the flow velocity of 5L/ minute
Society, SARTOLON Maxi Caps 5101307H3, aperture 0.4 μm+0.2 μm, membrane area 1.8m2) afterwards (relative to 1mg HCP
Use about 0.05m2Membrane area), utilize 1.0M glycine HCI buffer (pH2.0) to be adjusted to pH3.5, be supplied in utilization and contain
There is the SP-SepharoseFF (U.S. GE of 0.1M glycine HCI buffer (pH3.5) equilibrating of 0.3M NaCl
Healthcare Bioscience) in post (diameter 45cm, highly 10cm).Utilize the 0.1M glycinate containing 0.3M NaCl
Acid buffer (pH3.5) starts to clean, and obtains the circulation from beginning to ramp up to beginning to decline of the absorbance peak at 280nm
Fraction, is neutralized to pH7 with 0.5M phosphate buffer (pH7.3) immediately, obtains as refined liquid 2.It addition, be 2 DEG C in temperature
～10 DEG C, column flow rate be to implement under conditions of 160L/ hour.
The refined liquid 2 of about 220L utilize ultrafilter membrane Microza UF assembly SIP-2013 (Japan, Asahi Chemical Industry's chemistry) dense
After being reduced to about 5L, it is supplied in the Sephacryl utilizing 20mM phosphate buffer (pH7.3) equilibrating containing 50mM sodium chloride
In S-300HR (U.S. GE Healthcare Bioscience) post (diameter 63cm, highly 94cm).By the absorption of 280nm
After maximum eluting peak separates, ultrafilter membrane Microza UF assembly SIP-1013 (Japan, Asahi Chemical Industry's chemistry) is utilized to be concentrated into
About 10L, obtains as refined liquid 3.It addition, temperature be 2 DEG C～10 DEG C, column flow rate be to carry out under conditions of 6.2L/ hour
Implement.
At room temperature, make refined liquid 3 by utilizing the 20mM phosphorus containing 50mM sodium chloride under 0.1MPa pressure below
Virus removal film PLANOVA 15N (Japan, Asahi Kasei medical treatment, the membrane area 1m of acid buffer (pH7.3) equilibrating2After), enter
One step PVDF filter membrane (U.S., Millipore) by 0.22 μm, recovery total.As high-purity solubility blood
Bolt regulation extract of protein goods (batch: A5).
The APC activity of the thrombomodulin of A5 is 69000U/mL.
Soluble thrombomodulin concentration in the solution of A5 is 10.9mg/mL.
The manufacture 3 of (embodiment 3) soluble thrombomodulin of high purity
The NaHCO of the culture medium shown in component list 2 by Japanese Unexamined Patent Publication 11-341990 publication3Concentration becomes 5,
700mg/L, NaCl concentration is become 2,410mg/L, gained culture medium is carried out cell cultivation as minimal medium.For increasing
Grow culture medium, in minimal medium, add the Kanamycin Sulfate (U.S., Invitrogen) of 60mg/L, the wine of 1mg/L
Stone acid tylosin (U.S., Sigma-Aldrich) and 8% Ox blood serum (U.S., HyClone) use.It addition, for
Production medium, the serum-concentration making proliferated culture medium is 3%.
1 the cell bottle made in comparative example 1 is melted, by this cell sowing to the proliferated culture medium of 100mL, makes
With suspension culture bottle at CO25 days stir culture are carried out in 37 DEG C in incubator.It is 7.0 × 10 at viable cell density5cells/mL
In the above moment, culture fluid total amount is passaged in the proliferated culture medium of 0.9L, uses suspension culture bottle at CO2In incubator in
37 DEG C carry out 5 days stir culture.It is 7.0 × 10 at viable cell density5In the moment of more than cells/mL, culture fluid total amount is passed on
To the proliferated culture medium of 9L, use culture tank 37 DEG C, pH7.2, carry out 5 days stir culture under conditions of dissolved oxygen 50%.
It is 7.0 × 10 at viable cell density5In the moment of more than cells/mL, culture fluid total amount is passaged to the proliferated culture medium of 120L
In, use perfusion culture tank, 37 DEG C, pH7.2, carry out 7 days stir culture under conditions of dissolved oxygen 50%.At viable cell density
It is 7.0 × 105In the moment of more than cells/mL, start perfusion and cultivate, be carried out continuously interpolation and the culture supernatant of production medium
The recovery of liquid.Condition of culture is: 37 DEG C, pH7.2, dissolved oxygen 50%, culture medium exchange 130～200L/ days, above pressurization 0～
0.2MPa.7.5 × 10 are reached at viable cell density6Proceed after cells/mL 36 days to cultivate, using culture supernatant as life
Production fluid reclaims.Use filter SUPRAdisc II (U.S., Pall) and Supor EBV (U.S., Pall) for returning
The production liquid received carries out fining, produces liquid 2 DEG C～10 DEG C preservations after making filtration.
It is supplied in the utilization 20mM Tris salt buffer containing 150mM sodium chloride by producing liquid after the filtration of about 700L
(pH7.7) Q-Sepharose Fast Flow (U.S., the GE Healthcare) post (diameter 63cm, highly 25cm) of equilibrating
In.Next the 20mM acetate buffer (pH5.5) containing 180mM sodium chloride utilizing 6CV is carried out, further with containing
20mM Tris salt buffer (pH7.7) having 180mM sodium chloride is carried out till the absorption of 280nm returns back to baseline,
Utilizing 20mM Tris salt buffer (pH7.7) containing 300mM sodium chloride to start eluting, the absorbance obtaining eluent exists
The eluent of the 0.5 column volume capacity that the peak of 280nm has begun to ramp up is as thick refined liquid.Repeatedly implement 6 same operations,
Obtain the thick refined liquid of 6 batches.It addition, temperature be 2 DEG C～10 DEG C, column flow rate be to implement under conditions of 109L/ hour.
The thick refined liquid of about 20L is supplied in 20mM phosphate buffer (pH7.3) balance utilized containing 0.3M sodium chloride
In the monoclonal anti scapus (diameter 44cm, highly 13cm) changed.The 20mM phosphate buffer containing 1.0M sodium chloride of circulation 6CV
(pH7.3), further the 0.1M acetate buffer (pH5.0) of circulation 3CV be carried out, utilize the 0.1M containing 0.3M sodium chloride
Glycine HCI buffer (pH3.0) starts eluting.Obtain the absorbance of eluent at the peak of 280nm from beginning to ramp up to opening
Begin the eluent till declining, and adds the 0.5M phosphate buffer (pH7.3) of 1/10 capacity, as refined liquid 1 in eluent
Obtain.Repeatedly implement 12 same operations, it is thus achieved that the refined liquid 1 of 12 batches.It addition, be 2 DEG C～10 DEG C, post stream in temperature
Speed was implemented under conditions of 46L/ hour.
Make the refined liquid 1 about 270L of 12 batches under the flow velocity of 5L/ minute by nylon filter membrane (German,
Sartorius society, SARTOLON Maxi Caps 5101307H3, aperture 0.4 μm+0.2 μm, membrane area 1.8m2) afterwards (relatively
About 0.05m is used in 1mg HCP2Membrane area), utilize 1.0M glycine HCI buffer (pH2.0) to be adjusted to pH3.5, supply
In the SP-SepharoseFF (U.S. GE utilizing 0.1M glycine HCI buffer (pH3.5) equilibrating containing 0.3M NaCl
Healthcare Bioscience) in post (diameter 45cm, highly 10cm).Utilize the 0.1M glycine containing 0.3M NaCl
Hydrochloride buffer (pH3.5) starts to clean, and obtains the absorbance peak at 280nm stream from beginning to ramp up to beginning to decline
Logical fraction, is neutralized to pH7 with 0.5M phosphate buffer (pH7.3) immediately, obtains as refined liquid 2.It addition, be 2 in temperature
DEG C～10 DEG C, column flow rate be to implement under conditions of 160L/ hour.
The refined liquid 2 of about 300L utilize ultrafilter membrane Microza UF assembly SIP-2013 (Japan, Asahi Chemical Industry's chemistry) dense
After being reduced to about 11L, it is supplied in the Sephacryl utilizing 20mM phosphate buffer (pH7.3) equilibrating containing 50mM sodium chloride
In S-300HR (U.S. GE Healthcare Bioscience) post (diameter 63cm, highly 94cm).By the absorption of 280nm
After maximum eluting peak separates, ultrafilter membrane Microza UF assembly SIP-1013 (Japan, Asahi Chemical Industry's chemistry) is utilized to be concentrated into
About 13L, obtains as refined liquid 3.It addition, temperature be 2 DEG C～10 DEG C, column flow rate be to carry out under conditions of 6.2L/ hour
Implement.
At room temperature, make refined liquid 3 by utilizing the 20mM phosphorus containing 50mM sodium chloride under 0.1MPa pressure below
Virus removal film PLANOVA 15N (Japan, Asahi Kasei medical treatment, the membrane area 1m of acid buffer (pH7.3) equilibrating2), afterwards
Further by the PVDF filter membrane (U.S., Millipore) of 0.22 μm, recovery total.As high-purity solubility
Thrombomodulin highly finished product (batch: A6).
The APC activity of the thrombomodulin of A6 is 81000U/mL.
Soluble thrombomodulin concentration in the solution of A6 is 11.9mg/mL.
The manufacture 4 of (embodiment 4) soluble thrombomodulin of high purity
For proliferated culture medium, by the IS CHO-CD-A3 (U.S., Irvine Scientific) of 20.78g, 4.06g
The sodium bicarbonate (Japan and Wako Pure Chemical Industries) of sodium chloride (field Japanese, rich pharmacy) and 2.20g is dissolved in 1L water and makes
Make.It addition, for production medium, dissolve in 1L water 20.78g IS CHO-CD-A3 (U.S.,
IrvineScientific), (Japan and light are pure for the sodium bicarbonate of the sodium chloride (field Japanese, rich pharmacy) of 2.63g and 4.40g
Medicine industry) make.
The cell bottle made in 1 comparative example 4 is melted, by this cell sowing to the proliferated culture medium of 100mL, makes
With T-flask at CO25 days quiescent culture are carried out in 36 DEG C in incubator.It is 7.0 × 10 at viable cell density5More than cells/mL
Moment, culture fluid total amount is passaged in the proliferated culture medium of 0.9L, use suspension culture bottle at CO2In 36 DEG C in incubator
Carry out 5 days stir culture.It is 7.0 × 10 at viable cell density5In the moment of more than cells/mL, culture fluid total amount is passaged to 9L
Proliferated culture medium in, use culture tank, 36 DEG C, pH7.1, carry out 5 days stir culture under conditions of dissolved oxygen 50%.Living
Cell density is 7.0 × 105In the moment of more than cells/mL, culture fluid total amount is passaged in the proliferated culture medium of 120L, makes
Use perfusion culture tank, 36 DEG C, pH7.1, carry out 7 days stir culture under conditions of dissolved oxygen 50%.It is 7.0 at viable cell density
×105In the moment of more than cells/mL, start perfusion and cultivate, be carried out continuously the interpolation of production medium and returning of culture supernatant
Receive.Condition of culture is: 36 DEG C, pH7.1, dissolved oxygen 50%, culture medium exchange 130L/ days, pressurization 0～0.2MPa above.Thin living
Born of the same parents' density reaches 7.5 × 106Proceed after cells/mL 20 days to cultivate, culture supernatant is reclaimed as producing liquid.
Use filter SUPRAdisc II (U.S., Pall) and Supor EBV (U.S., Pall) for the life reclaimed
Production fluid carries out fining, produces liquid 2 DEG C～10 DEG C preservations after making filtration.
It is supplied in the utilization 20mM Tris salt buffer containing 150mM sodium chloride by producing liquid after the filtration of about 1,400L
(pH7.7) Q-Sepharose Fast Flow (U.S., the GE Healthcare) post (diameter 63cm, highly 25cm) of equilibrating
In.Next the 20mM acetate buffer (pH5.6) containing 180mM sodium chloride utilizing 6CV is carried out, further with
20mM Tris salt buffer (pH7.7) containing 180mM sodium chloride of 4CV is carried out, and utilizes containing 290mM sodium chloride
20mM Tris salt buffer (pH7.7) starts eluting, obtain that the absorbance of eluent has begun to ramp up at the peak of 280nm 0.5
The eluent of column volume capacity is as thick refined liquid.It is also carried out same operation, it is thus achieved that 2 for producing liquid after the filtration of about 900L
The thick refined liquid of batch.It addition, temperature be 2 DEG C～10 DEG C, column flow rate be to implement under conditions of 109L/ hour.
The thick refined liquid of about 13L is supplied in 20mM phosphate buffer (pH7.3) balance utilized containing 0.3M sodium chloride
In the monoclonal anti scapus (diameter 44cm, highly 13cm) changed.The 20mM phosphate buffer containing 1.0M sodium chloride of circulation 6CV
(pH7.3), further the 0.1M acetate buffer (pH5.0) of circulation 3CV be carried out, utilize the 0.1M containing 0.3M sodium chloride
Glycine HCI buffer (pH3.0) starts eluting.Obtain the absorbance of eluent at the peak of 280nm from beginning to ramp up to opening
Begin the eluent till declining, and adds the 0.5M phosphate buffer (pH7.3) of 1/10 capacity, as refined liquid 1 in eluent
Obtain.Repeatedly implement 5 same operations, it is thus achieved that the refined liquid 1 of 5 batches.It addition, be 2 DEG C～10 DEG C, column flow rate in temperature
Implemented under conditions of 46L/ hour.
The refined liquid 1 about 110L making 5 batches passes through nylon filter membrane (Germany, Sartorius with the flow velocity of 5L/ minute
Society, SARTOLON Maxi Caps 5101307H3, aperture 0.4 μm+0.2 μm, membrane area 1.8m2) (make relative to 1mgHCP afterwards
With about 0.03m2Membrane area), utilize 1.0M glycine HCI buffer (pH2.0) to be adjusted to pH3.5, be supplied in utilization and contain
SP-SepharoseFF (the U.S. GE of 0.1M glycine HCI buffer (pH3.5) equilibrating of 0.3M NaCl
Healthcare Bioscience) in post (diameter 45cm, highly 10cm).Utilize the 0.1M glycinate containing 0.3M NaCl
Acid buffer (pH3.5) starts to clean, and obtains the circulation from beginning to ramp up to beginning to decline of the absorbance peak at 280nm
Fraction, is neutralized to pH7 with 0.5M phosphate buffer (pH7.3) immediately, obtains as refined liquid 2.It addition, be 2 DEG C in temperature
～10 DEG C, column flow rate be to implement under conditions of 160L/ hour.
The refined liquid 2 of about 120L utilize ultrafilter membrane Microza UF assembly SIP-2013 (Japan, Asahi Chemical Industry's chemistry) dense
After being reduced to about 5L, it is supplied in the Sephacryl utilizing 20mM phosphate buffer (pH7.3) equilibrating containing 50mM sodium chloride
In S-300HR (U.S. GE Healthcare Bioscience) post (diameter 63cm, highly 94cm).By the absorption of 280nm
After maximum eluting peak separates, ultrafilter membrane Microza UF assembly SIP-1013 (Japan, Asahi Chemical Industry's chemistry) is utilized to be concentrated into
About 5L, obtains as refined liquid 3.It addition, temperature be 2 DEG C～10 DEG C, column flow rate be to carry out reality under conditions of 6.2L/ hour
Execute.
At room temperature, make refined liquid 3 total amount by utilizing containing 50mM sodium chloride under 0.1MPa pressure below
Virus removal film PLANOVA 15N (Japan, Asahi Kasei medical treatment, the membrane area of 20mM phosphate buffer (pH7.3) equilibrating
1m2), the most further by the PVDF filter membrane (U.S., Millipore) of 0.22 μm, recovery total.As high-purity
Degree soluble thrombomodulin highly finished product (batch: A7).
The APC activity of the thrombomodulin of A7 is 69000U/mL.
Soluble thrombomodulin concentration in the solution of A7 is 10.4mg/mL.
(test example 1) HCP based on various filter membranes removes evaluation
After making the refined liquid 1 (HCP concentration: 462ng/ml) obtained in comparative example 1 by the filter membrane of unlike material, right
HCP concentration compares.That is, the refined liquid 1 making 5ml is made by (1) PVDF (polyvinylidene fluoride) with the flow velocity of 1ml/ minute
(U.S., Millipore, Millex GV), (2) CA (cellulose acetate) make (Germany, Sartorius, Minisart), (3)
PES (polyether sulfone) makes (Germany, Sartorius, Minisart High-Flow), (4) nylon system (U.S., Thermo Fisher
Scientific, NALGENE Syringe Filter), (5) CA+GF (cellulose acetate+glass fibre) system (Sartorius,
Minisart Plus) each filter membrane, with the form recovery total of filtrate.
Liquid before and after filtering is carried out to the mensuration of protein concentration and HCP concentration.Protein concentration is by the suction of 280nm
Luminosity is tried to achieve, and HCP concentration is measured according to the method shown in reference example 2.Result understands, for all of filter membrane, almost
The most unconfirmed to the change of protein concentration before and after filtering, but nylon filter membrane and PES filter membrane have confirmed higher
HCP removal effect, HCP concentration is reduced to 28% and 36% (table 1) respectively.Although it addition, aperture is identical, but the fall of HCP concentration
The low difference according to film material and have relatively big difference, thus it is believed that it not removes thawless HCP for carrying out assembling, but
HCP Adsorption based on film.It addition, the film footpath of each filter membrane being evaluated is 25mm or 26mm, according to each manufacture quotient
According to the effective film area of the filter membrane that chart is recorded, the membrane area relative to 1mg HCP is 0.17m2Or 0.23m2。
[table 1]
(test example 2) HCP based on nylon system and PES filter membrane removal ability compares
In test example 1, it is judged that nylon filter membrane and PES filter membrane have higher HCP removal ability, thus
For these filter membranes, carry out the evaluation of HCP removal ability change based on logical liquid measure.It addition, in filter membrane, for each material
Matter prepares the commodity of Liang Zhong manufacturer, and for carrying out the refined liquid 1 of logical liquid, it is different for using from the batch used in test example 2
Batch (HCP concentration: 303ng/ml).In filter membrane, use PVDF system (U.S., Millipore, Millex as comparison
GV: aperture 0.22 μm, film footpath 25mm, effective film area 3.9cm2), use (1) U.S., Pall society as nylon system
Acrodisc AP-4436T: aperture 0.2 μm, film footpath 25mm, effective film area 3.9cm2, and (2) Germany, Sartorius society
Minisart NY25: aperture 0.2 μm, film footpath 25mm, effective film area 4.8cm2；(3) U.S. Poul is used as PES system
Acrodisc PN4612: aperture 0.2 μm of society, film footpath 25mm, effective film area 2.8cm2, and (4) Sartorius society
Minisart High-Flow: aperture 0.2 μm, film footpath 26mm, effective film area 5.3cm2.Refined liquid 1 in each filter membrane
Logical liquid measure is 100ml under the flow velocity of 10ml/ minute, often leads to liquid 20ml, samples filtrate, shown in reference example 2
Method measure HCP concentration, by the absorption measurement protein concentration of 280nm.
Its result, the change to protein concentration the most unconfirmed in all samples, but at nylon system and PES filter membrane
In all confirmed HCP removal effect (table 2).HCP removal effect is the highest at nylon filter membrane, and HCP concentration maximum reduces
To 25%.Understand, the logical liquid measure in filter membrane less, i.e. filter membrane area relative to 1mg HCP the biggest, HCP removal effect is more
High.
[table 2]
The HCP removal ability of the lower nylon filter membrane of (test example 3) different liquids composition is evaluated
The HCP of nylon filter membrane is gone decapacitation by the difference for buffer composition and soluble thrombomodulin concentration
The impact that power is brought is evaluated.After the refined liquid 1 obtained in comparative example 4, refined liquid 2, refined liquid 2 are concentrated, refine
Liquid 3 and highly finished product, make each 5mL pass through film footpath 25mm, effective film area 4.8cm with the flow velocity of 1mL/ minute2, aperture 0.2 μm
Nylon filter membrane (Germany, Sartorius society, MinisartNY25), with the form recovery total of filtrate.Relative to
The filter membrane area of HCP contained in 1mg each sample is: refined liquid 1:0.77m2, refined liquid 2:1.7m2, refined liquid 2 concentrates
Rear: 0.43m2, refined liquid 3:0.72m2, highly finished product: 0.81m2.For obtained filtrate, according to the side shown in reference example 2
Method measures HCP concentration, the absorption of 280nm obtain protein concentration.Its results verification has to nylon masking for whole samples
Having higher HCP removal effect, the protein concentration response rate has obtained the high value (table 3) of more than 90%.
[table 3]
(test example 4) is by removing HCP's in the soluble thrombomodulin highly finished product utilizing various manufacture method to obtain
Evaluate
The soluble thrombomodulin obtained by different manufacture methods is made to be ground by nylon filter membrane further
Study carefully whether HCP is removed.For in comparative example 1～3 obtain soluble thrombomodulin highly finished product (respectively A1, B1 and
B2), each 5mL is made to pass through film footpath 25mm, effective film area 4.8cm with the flow velocity of 1mL/ minute2, the nylon mistake of aperture 0.2 μm
Filter membrane (Germany, Sartorius society, Minisart NY25), with the form recovery total of filtrate.Relative to each solubility of 1mg
HCP contained in thrombomodulin highly finished product, filter membrane area is: A1:0.34m2, B1:0.46m2, B2:1.7m2.For
Solution before and after filtration, carries out HCP content, mouse IgG content and bovine serum protein according to the method shown in reference example 2～4
The mensuration of content.In whole highly finished product, confirm nylon filter membrane and only HCP is had stronger removal effect (table 4).
Thus it can be said that nylon filter membrane not has a non-specific adsorption effect to protein, but have HCP special
Property adsorption.
[table 4]
(test example 5) comparison based on the thrombomodulin highly finished product purity carried out with or without use nylon filter membrane
For by not using the industrial level manufacture (comparative example 1) of nylon filter membrane and by employing nylon mistake
The industrial level manufacture (embodiment 1～4) of filter membrane and the thrombomodulin highly finished product that obtain, shown in reference example 2～4
Method carries out HCP content, mouse IgG content and the mensuration of bovine serum protein content.
Not by 3 batches (A1, A2 and A3) of the comparative example 1 of nylon filter membrane, have 10ng/10,000U with
On high HCP content, and in having passed through 4 batches (A4, A5, A6 and A7) of embodiment 1～4 of nylon filter membrane, HCP contains
Amount is less than quantitation limit.For as the mouse IgG content of other impurity and bovine serum protein content, unconfirmed to or without making
By the difference (table 5) of its content that nylon filter membrane is brought.Thus, in the manufacture of industrial level, nylon filter membrane
Also there is the specificity removal ability to HCP, it is possible to manufacture HCP content less than 10ng/10,000U such high-purity solubility
Thrombomodulin.
Use gel filtration liquid chromatography and ion-exchange selectivity high-purity for what embodiment 1～4 obtained
Purity in the gross protein of degree soluble thrombomodulin is measured.In gel filtration liquid chromatography, use
TOSOH TSKgel G3000SWXL (Cao Japanese, eastern), the 50mM phosphate buffer (pH7.3) containing 0.1M sodium sulfate,
It is measured under the conditions of temperature 40 DEG C, flow velocity 0.9mL/ minute are such, the whole highly finished product of result (A4, A5, A6 and A7) pure
Degree is more than 99%.It addition, in ion-exchange selectivity, use TOSOH DEAE 5PW (Cao Japanese, eastern), at 30 points
Clock linear gradient eluting is (from 20mM piperazine salt acid buffer (pH5.6) containing 50mM sodium chloride to containing 350mM sodium chloride
20mM piperazine salt acid buffer (pH5.6)), temperature 40 DEG C, flow velocity 0.9mL/ minute such under the conditions of be measured, result is complete
The purity of portion's highly finished product (A4, A5, A6 and A7) is more than 99%.
It addition, utilizing SDS-PAGE for manufacturing based on comparative example 1 and confirming that molecular weight is through MALDI-TOF-MS method
The soluble thrombomodulin of high purity obtained in the soluble thrombomodulin highly finished product of 64,000 and embodiment 1～4
When the molecular weight of highly finished product compares, detect band in same position.Thus it is believed that high-purity solubility thrombosis regulates
The molecular weight of albumen is 64,000.
Further, implement according to the gelling technique of the Endotoxin test method<4.01>of Pharmacopeia of Japan ordinary test method
Test, result endotoxin content is 0.004～0.03EU/10,000U, for extremely low level (table 6).
[table 5]
[table 6]
(test example 6) utilizes the parsing of the HCP that nylon filter membrane removes
After utilizing method same as in Example 3 to manufacture soluble thrombomodulin of high purity, used in manufacturing
Nylon filter membrane (Germany, Sartorius, SARTOLON Maxi Caps 5101307H3, aperture 0.4 μm+0.2 μm, film
Area 1.8m2) take out from shell, film is cut into the fragment of about 3g.5 fragments are utilized the 20mM containing 50mM sodium chloride
After phosphate buffer (pH7.3) fully cleans, every 1 fragment is joined be incorporated with 40mL containing 0.5%CHAPS, 200mM
In the developmental tube of 50mM Tris hydrochloride buffer (pH8.0) of sodium chloride.At room temperature vibrate one, thus will film adsorb
Protein Extraction in buffer.Use ultrafilter membrane Vivaspin20 (Germany, Sartorius) and Amicon Ultra-
Extracting solution total amount is concentrated into 15 μ L by 0.5mL (U.S., Millipore).About 1/5 amount in this concentrated solution is supplied in SDS-
PAGE (Japan, Atto, e-PAGEL5/20), carries out CBB dyeing (Japan and Wako Pure Chemical Industries, Quick-CBB).Cut out molecule
Amount confirms 10, the band near 000, after this gel pieces utilizes dithiothreitol dithio reduction, utilizes iodoacetamide to carry out
Urea methylates, and carries out enzymic digestion based on insulin all night.Enzymic digestion thing is supplied in LC/MS/MS, uses the data base of NCBI
Carried out Mascot retrieval by obtained mass spectrometric data, the aminoacid sequence of enzymic digestion thing is resolved.
The condition determination of LC/MS/MS
LC/MS/MS (determinator):
The automatic spraying system of DiNa-2A multidimensional (Japan, KYA technologies)
MS measurement range:
MS1 (m/z400-1500) MS2 (m/z50-1500) × 3 (data dependent scan pattern)
Ionization mode: nanoESI+
Post:
PicoFrit Column BataBasic C18 (U.S., New Objective)
Mobile phase:
Mobile phase A:0.1% formic acid/2% acetonitrile
Mobile phase B:0.1% formic acid/80% acetonitrile
Gradient: 0 → 30 minute mobile phase B 5 → 40%
30 → 40 minutes mobile phases B 40 → 100%
40 → 60 minutes mobile phases B 100%
Flow: 300nL/ minute
Shown in the aminoacid sequence following (1) of each segment predicted by mass spectrometric data～(7), with group egg as follows
The partial sequence of white H2B (Biochimie, 61 (1), 61-69 (1979)) is consistent.It follows that remove through nylon filter
One of HCP is histone H2B.
(1)KESYSVYVYK
(2)VLKQVHPDTGISSK
(3)STITSREIQTAVR
(4)EIQTAVR
(5)EIQTAVRLLLPGELAK
(6)LLLPGELAK
(7)LLLPGELAKHAVSEGTK
[table 7]
Histone 2B aminoacid sequence
PEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSR
(sequence impaled by shows in histone H2B aminoacid sequence above-mentioned with predicted by mass spectrometric data
～the amino acid sequence range of consensus amino acid sequence of (7) (1).)
Claims (15)
1. a soluble thrombomodulin of high purity, it is for by converting soluble thrombomodulin produced by cell,
This conversion cell is that the DNA transfection of the base sequence by encoding soluble thrombomodulin obtains to host cell, its
In, the protein content ratio of host cell resources is relative to 10, and 000U soluble thrombomodulin is less than 10ng and is
More than 0.001ng, this host cell is Chinese hamster ovary cell.
2. soluble thrombomodulin of high purity as claimed in claim 1, wherein, soluble thrombomodulin is for passing through
The soluble thrombomodulin this conversion cell being carried out serum-free culture and produce.
3. soluble thrombomodulin of high purity as claimed in claim 1 or 2, wherein, this soluble thrombomodulin
For having the soluble thrombomodulin of the character of following (1)～(5):
(1) with the effect of thrombin selective binding；
(2) effect of the activation of protein C based on thrombin is promoted；
(3) effect of setting time based on thrombin is extended；
(4) effect of platelet aggregation based on thrombin is suppressed；And
(5) antiinflammatory action.
4. soluble thrombomodulin of high purity as claimed in claim 1 or 2, wherein, this soluble thrombomodulin
The scope that molecular weight is 50,000 to 80,000.
5. soluble thrombomodulin of high purity as claimed in claim 1 or 2, wherein, this high-purity solubility thrombosis is adjusted
Joint albumen is by including what the method for following operation manufactured:
A the DNA of the base sequence of encoding soluble thrombomodulin is transfected to host cell by (), it is thus achieved that convert the work of cell
Sequence；
B () cultivates this conversion cell, it is thus achieved that the operation of the solution containing soluble thrombomodulin；And
C () makes this solution containing soluble thrombomodulin contact with nylon and/or polyether sulfone, obtain high-purity solubility
The operation of thrombomodulin, in this soluble thrombomodulin of high purity, the protein content ratio of host cell resources
For being less than 10ng relative to 10,000U soluble thrombomodulin.
6. soluble thrombomodulin of high purity as claimed in claim 1 or 2, wherein, this soluble thrombomodulin
For following soluble thrombomodulin: its aminoacid sequence described in any one of sequence numbering 9 or sequence numbering 11
In the peptide of the 19th～516 amino acids sequences.
7. soluble thrombomodulin of high purity as claimed in claim 1 or 2, wherein, encodes the regulation of this solubility thrombosis
The DNA of the base sequence of albumen is the aminoacid sequence described in any one of coded sequence numbering 9 or sequence numbering 11
DNA。
8. a pharmaceutical composition, it contains the soluble thrombomodulin of high purity described in any one of claim 1～7
And pharmaceutically suitable carrier.
9. a manufacture method, it is the manufacture method of soluble thrombomodulin of high purity, described high-purity solubility blood
In bolt regulation albumen, the protein content ratio of host cell resources is relative to 10, and 000U soluble thrombomodulin is little
In 10ng, wherein, the method includes following operation:
A the DNA of the base sequence of encoding soluble thrombomodulin is transfected to host cell by (), obtain converting the work of cell
Sequence；
B () cultivates this conversion cell, it is thus achieved that the operation of the solution containing soluble thrombomodulin；
C soluble thrombomodulin that this solution containing soluble thrombomodulin is refined in gross protein by () is pure
Degree is the operation of more than 99%；And
D () makes this solution containing soluble thrombomodulin contact with nylon and/or polyether sulfone, by high-purity solubility blood
The operation of bolt regulation Protein Separation, in this soluble thrombomodulin of high purity, the protein content ratio of host cell resources
Rate is relative to 10, and 000U soluble thrombomodulin is less than 10ng,
Described host cell is Chinese hamster ovary cell,
Further, the aminoacid sequence of this soluble thrombomodulin is remembered by any one of sequence numbering 9 or sequence numbering 11
The 19th～516 amino acids sequences in the aminoacid sequence carried.
10. manufacture method as claimed in claim 9, wherein, soluble thrombomodulin is by entering this conversion cell
Row serum-free culture and produce.
11. manufacture methods as described in claim 9 or 10, wherein, the molecular weight of this soluble thrombomodulin is 50,
000 to 80,000.
12. manufacture methods as described in claim 9 or 10, wherein, the form of nylon and/or polyether sulfone is the shape of filter membrane
State.
13. manufacture methods as claimed in claim 12, wherein, the membrane area of filter membrane is relative to 1mg host cell resources
Protein is 0.01m2To 0.5m2。
14. manufacture methods as described in claim 9 or 10, wherein, in operation (d), make this containing solubility thrombomodulin
White solution contacts with nylon.
15. manufacture methods as claimed in claim 9, wherein, encode the DNA of the base sequence of this soluble thrombomodulin
The DNA of the aminoacid sequence described in coded sequence numbering 9 or sequence numbering 11.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010105421 | 2010-04-30 | ||
| JP2010-105421 | 2010-04-30 | ||
| PCT/JP2011/060348 WO2011136313A1 (en) | 2010-04-30 | 2011-04-28 | High-purity soluble thrombomodulin and method for producing same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102858978A CN102858978A (en) | 2013-01-02 |
| CN102858978B true CN102858978B (en) | 2016-12-14 |
Family
ID=
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0689843A1 (en) * | 1993-12-17 | 1996-01-03 | Mochida Pharmaceutical Co., Ltd. | Composition containing soluble thrombomodulins |
| CN1151406A (en) * | 1995-10-24 | 1997-06-11 | 日本化学研究股份有限公司 | Method for purifying thrombomodulin |
| CN101522206A (en) * | 2006-10-06 | 2009-09-02 | 旭化成制药株式会社 | Therapeutic and/or ameliorating agent for disseminated intravascular coagulation |
| CN101641368A (en) * | 2007-03-23 | 2010-02-03 | 旭化成制药株式会社 | Method for producing soluble thrombomodulin of high purity |
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0689843A1 (en) * | 1993-12-17 | 1996-01-03 | Mochida Pharmaceutical Co., Ltd. | Composition containing soluble thrombomodulins |
| CN1151406A (en) * | 1995-10-24 | 1997-06-11 | 日本化学研究股份有限公司 | Method for purifying thrombomodulin |
| CN101522206A (en) * | 2006-10-06 | 2009-09-02 | 旭化成制药株式会社 | Therapeutic and/or ameliorating agent for disseminated intravascular coagulation |
| CN101641368A (en) * | 2007-03-23 | 2010-02-03 | 旭化成制药株式会社 | Method for producing soluble thrombomodulin of high purity |
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