HK1137185B

HK1137185B - Method for producing soluble thrombomodulin of high purity

Info

Publication number: HK1137185B
Application number: HK10104518.3A
Authority: HK
Inventors: 大东晋
Original assignee: 旭化成制药株式会社
Priority date: 2007-03-23
Filing date: 2008-03-21
Publication date: 2013-08-09

Description

Method for producing high-purity soluble thrombomodulin

Technical Field

The present invention relates to a method for producing a high-purity soluble thrombomodulin that does not substantially contain a denatured product of soluble thrombomodulin that can be produced under acidic conditions.

Technical Field

Thrombomodulin is known as a substance that blocks the blood coagulation activity of thrombin by specifically binding to thrombin and at the same time remarkably promotes the protein C activation energy of thrombin, and it is also known to have a strong blood coagulation blocking effect. It is known to prolong the coagulation time based on the thrombin action and to inhibit platelet aggregation based on the thrombin action. Protein C is a vitamin K-dependent protein that plays an important role in the blood coagulation-fibrinolysis system, and is activated by thrombin to become activated protein C. It is known that this activated protein C inactivates the active type factor V and the active type factor VIII of blood coagulation system factors in vivo, and is also known to be involved in the production of plasminogen activator having thrombolytic action (non-patent document 1). Therefore, it is considered that thrombomodulin promotes the activation of protein C by this thrombin and is useful as an anti-blood clotting agent or a thrombolytic agent, and further, there have been reports of animal experiments that have indicated that thrombomodulin is effective in the treatment and prevention of diseases accompanied by hypercoagulation (non-patent document 2).

Conventionally, thrombomodulin has been discovered as a glycoprotein expressed on vascular endothelial cells of various animal species including humans, and has been successfully cloned thereafter. That is, a gene of a precursor of human thrombomodulin containing a signal peptide is cloned from a human lung cDNA library by a genetic engineering method, and the entire gene sequence of thrombomodulin is analyzed to clarify an amino acid sequence of 575 residues containing the signal peptide (generally, 18 amino acid residues are exemplified) (patent document 1). The mature thrombomodulin in which the signal peptide is cleaved is composed of the following 5 regions: an N-terminal region (No. 1-226: indicating the position where the signal peptide is considered to be 18 amino acid residues, the same applies hereinafter) from the N-terminal side of the mature peptide, a region having 6 EGF-like structures (227-.

Full-length thrombomodulin is difficult to dissolve without the presence of a surfactant, and a surfactant must be added as a preparation, whereas there is soluble thrombomodulin that can be dissolved well without the presence of a surfactant. The soluble thrombomodulin may be prepared so as not to contain at least a part or all of the membrane-penetrating region, and for example, a soluble thrombomodulin composed of only 3 regions of the N-terminal region, the region having 6 EGF-like structures and the O-type sugar chain-increasing region (i.e., composed of the amino acid sequences at positions 19 to 516 of SEQ ID NO: 1) can be obtained by applying a recombinant technique, and the recombinant soluble thrombomodulin has the activity of a natural thrombomodulin (patent document 1). In addition, some reports have been made on soluble thrombomodulin (patent documents 2 to 9). Alternatively, a natural type may be exemplified by soluble thrombomodulin derived from human urine, and the like (patent documents 10 and 11).

Incidentally, in the gene, polymorphism mutation has been found in humans as confirmed in many cases by natural mutation or mutation at the time of acquisition, and it has been confirmed that the amino acid at the 473 rd position of the human thrombomodulin precursor composed of the amino acid sequence of 575 residues is Val and Ala. The nucleotide sequence encoding this amino acid corresponds to a T mutation and a C mutation at position 1418, respectively (non-patent document 4). However, the activity and the physical properties were completely the same, and it could be judged that both were substantially the same.

Thrombomodulin has been reported to have an effect on the treatment of DIC (non-patent document 5). In addition to the above, the use of thrombomodulin is expected to be used for, for example, Acute Coronary Syndrome (ACS), thrombosis, peripheral vascular occlusion, arteriosclerosis obliterans, vasculitis, functional disorder secondary to cardiac surgery, organ transplantation complications, angina pectoris, transient ischemic attack, gestational toxicosis, diabetes, Liver VOD (Liver Veno-occlusive disease; hepatic venous occlusion after acute hepatitis and bone marrow transplantation), deep venous thrombosis (DVT; deep venous thrombosis), and the like; and Adult Respiratory Distress Syndrome (ARDS), are disclosed.

As denatured products of thrombomodulin, aggregates formed in a freeze-drying step and aggregates formed during long-term storage in a freeze-dried state are known (patent documents 12 to 16).

As a method for producing soluble thrombomodulin at an industrial level in view of utilization in pharmaceuticals, for example, the following methods are known: a method for producing a high-purity soluble thrombomodulin that does not substantially contain a serum derivative and an antibody derivative, characterized in that, as a purification step, a method of affinity chromatography in which an antibody against thrombomodulin is supported is used, and that the soluble thrombomodulin obtained by affinity chromatography is brought into contact with a cation exchanger under conditions of a conductivity of 25 to 34ms/cm and a pH of 3 to 4, and in this step, the soluble thrombomodulin is obtained as a pass-through fraction (patent document 17); a method for purifying thrombomodulin, characterized in that a sample containing human urinary thrombomodulin is preliminarily purified by thrombin-binding affinity chromatography, and then purified by adsorption chromatography using hydroxyapatite as an adsorbent (patent document 18).

Patent document 1: japanese laid-open patent publication No. Sho 64-6219

Patent document 2: japanese laid-open patent publication No. 2-255699

Patent document 3: japanese laid-open patent publication No. 3-133380

Patent document 4: japanese laid-open patent publication No. 3-259084

Patent document 5: japanese laid-open patent publication No. 4-210700

Patent document 6: japanese patent laid-open publication No. 5-213998

Patent document 7: WO92/00325

Patent document 8: WO92/03149 publication

Patent document 9: WO93/15755 publication

Patent document 10: japanese laid-open patent publication No. 3-86900

Patent document 11: japanese laid-open patent publication No. 3-218399

Patent document 12: japanese laid-open patent publication No. 6-321085

Patent document 13: japanese patent No. 3007785

Patent document 14: japanese laid-open patent publication No. 11-171790

Patent document 15: w095/16460 publication

Patent document 16: japanese patent No. 3822383

Patent document 17: japanese laid-open patent publication No. 11-341990

Patent document 18: japanese patent No. 3745805

Non-patent document 1: suzuki hong Zhi, the medicine is located at あゆみ, Vol. 125, page 901 (1983)

Non-patent document 2: good mi et al, Blood 75.1396-1399(1990)

Non-patent document 3: M.Zushi et al, J.biol.chem., 246, 10351-10353(1989)

Non-patent document 4: D.Z.Wen et al, Biochemistry, 26, 4350-4357(1987)

Non-patent document 5: S.M.Bates et al, Br.J.Pharmacol, 144, 1017-

Disclosure of Invention

The present invention addresses the problem of providing a method for producing thrombomodulin substantially free of a denatured product of soluble thrombomodulin with high productivity.

The present inventors have found a novel problem in the process of producing soluble thrombomodulin on an industrial scale and also in the process of producing soluble thrombomodulin by a production method comprising a step of inactivating a virus under acidic conditions: when soluble thrombomodulin is left to stand under acidic conditions, a denatured product of the soluble thrombomodulin is produced and it is necessary to efficiently remove it. The present inventors have studied the use of Gel Filtration Chromatography (GFC), Size Exclusion Chromatography (SEC), and the like in order to remove a denatured product of the soluble thrombomodulin. However, it was found that Gel Filtration Chromatography (GFC) and Size Exclusion Chromatography (SEC) have a small processing capacity, usually 5% or less of the column volume, and therefore, it is necessary to increase the column volume or to perform multiple cycles. Further, as a method for improving efficiency, a method of increasing the linear velocity of chromatography may be considered, but since increasing the linear velocity results in a decrease in separation ability, this method is considered to be inappropriate. As another method, a method of increasing the concentration of the protein to be treated may be considered, but there are problems such as an increase in the concentration step for increasing the protein concentration and a decrease in the separation ability due to high viscosity caused by a high protein concentration.

Then, based on the above-described novel problems, the present inventors have considered that in order to produce soluble thrombomodulin on an industrial level, it is important to separate a denatured product of the soluble thrombomodulin by a method with high productivity that solves the above-described problems. The present inventors have conducted extensive studies on various conditions using various chromatographic supports in order to solve the above-mentioned novel problems, and as a result, have found a method for efficiently and stably separating a denatured product of the soluble thrombomodulin by using an anion exchanger or hydroxyapatite as a support and finding appropriate conditions for each support.

That is, the present invention can provide the following technical means.

[ A1 ] A method for producing a soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin that can be produced from the soluble thrombomodulin under acidic conditions, comprising the steps of:

(1) supplying an anion exchanger or hydroxyapatite with the soluble thrombomodulin-containing material containing or suspected of containing a denatured product of the soluble thrombomodulin; and

(2) under separation conditions under which the soluble thrombomodulin and a denatured product of the soluble thrombomodulin can be separated, a fraction containing the soluble thrombomodulin that does not substantially contain the denatured product of the soluble thrombomodulin is obtained.

[ A2 ] the method for producing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin that may be produced from the soluble thrombomodulin under acidic conditions, according to [ A1 ] above, comprising the steps of:

(0) placing the soluble thrombomodulin under an acidic condition at a pH of 5 or less;

(1) supplying the soluble thrombomodulin-containing material containing or suspected of containing the denatured product of the soluble thrombomodulin obtained in the above-mentioned step (0) to an anion exchanger or hydroxyapatite; and

[ A2-2 ] the method for producing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin that may be produced from the soluble thrombomodulin under acidic conditions, according to [ A1 ] above, comprising the steps of:

(xi) refining soluble thrombomodulin;

The production method according to any one of [ A2-3 ] above [ A1 ], [ A2 ] and [ A2-2 ], wherein the step of supplying the soluble thrombomodulin-containing material to an anion exchanger or hydroxyapatite is a step of supplying the soluble thrombomodulin-containing material to an anion exchanger.

The production method according to any one of [ A2-4 ] above [ A1 ], [ A2 ] and [ A2-2 ], wherein the step of supplying the soluble thrombomodulin-containing material to an anion exchanger or hydroxyapatite is a step of supplying the soluble thrombomodulin-containing material to hydroxyapatite.

The production method according to any one of [ A1 ] to [ A2-4 ] above, wherein the content of soluble thrombomodulin is 80% or more relative to the total proteins in the soluble thrombomodulin-containing material.

Note that, the term numbers cited as [ a 1] to [ a2-4 ] above are expressed as ranges, and when a term having a branch number such as [ a2-2 ] is disposed in the range, it means that a term having a branch number such as [ a2-2 ] is also cited. The same applies below.

The production method according to any one of [ A3-2 ] to [ A2-4 ] above, wherein the content of soluble thrombomodulin is 90% or more relative to the total proteins in the soluble thrombomodulin-containing material.

The production method according to any one of [ A3-3 ] to [ A2-4 ] above, wherein the content of soluble thrombomodulin is 95% or more relative to the total proteins in the soluble thrombomodulin-containing material.

The production method according to any one of [ A3-4 ] to [ A2-4 ] above, wherein the content of soluble thrombomodulin is 99% or more relative to total proteins in the soluble thrombomodulin-containing material.

The production method according to any one of [ A1 ] to [ A3-4 ] above, wherein the step (2) is a step of obtaining an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin by performing gradient elution that is linear or stepwise, or a combination of linear and stepwise.

Note that, the term numbers cited as [ a 1] to [ A3-4 ] above are expressed as ranges, and when a term having a branch number such as [ A3-2 ] is disposed in the range, it means that a term having a branch number such as [ A3-2 ] is also cited. The same applies below.

The production method according to any one of [ A1 ] to [ A3-4 ] above, wherein the step (2) is a step of obtaining a pass-through fraction, and a fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin is obtained in the step.

The production method according to any one of [ A1 ] to [ A3-4 ] above, wherein the step (2) is a step of obtaining an eluted fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin by performing isocratic elution.

The production method according to any one of [ A1 ] to [ A3-4 ] above, wherein in the step (1), the soluble thrombomodulin-containing material is supplied to the anion exchanger using a buffer solution having a pH of 4 to 9; performing a gradient elution of linear or stepwise or a combination of linear and stepwise elution in step (2) using a buffer solution having a pH of 5 to 9 and a salt concentration of 0 to 1M, (a) confirming in advance the position of an elution fraction of soluble thrombomodulin to obtain an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin; or (b) obtaining an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin, while confirming the presence or absence of the soluble thrombomodulin in the elution fraction.

[ A7-2 ] the production method according to [ A7 ] above, wherein the step (a) or (b) is the step (a).

[ A7-3 ] the production method according to [ A7 ] above, wherein the step (a) or (b) is the step (b).

The production method according to any one of [ A1 ] to [ A3-4 ] above, wherein in the step (1), the soluble thrombomodulin-containing material is supplied to the anion exchanger using a buffer solution having a pH of 5 to 8 and a salt concentration of 0.1 to 0.2M; in the step (2), a flow-through fraction is obtained by using a buffer solution having a pH of 5 to 8 and a salt concentration of 0.1 to 0.2M, thereby obtaining a fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin.

The production method according to any one of [ A1 ] to [ A3-4 ] above, wherein in the step (1), a soluble thrombomodulin-containing material is supplied to hydroxyapatite using a buffer solution having a pH of 6 to 9 and a phosphate concentration of 8mM or less; performing a gradient elution of linear or stepwise, or a combination of linear and stepwise elution using a buffer solution having a pH of 6 to 9 and a phosphate concentration of 0 to 0.5M in the step (2), (a) confirming in advance the position of an elution fraction of soluble thrombomodulin to obtain an elution fraction of soluble thrombomodulin that contains substantially no denatured product of the soluble thrombomodulin; or (b) obtaining an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin, while confirming the presence or absence of the soluble thrombomodulin in the elution fraction.

[ A9-2 ] the production method according to [ A9 ] above, wherein the step (a) or (b) is the step (a).

[ A9-3 ] the production method according to [ A9 ] above, wherein the step (a) or (b) is the step (b).

The production method according to any one of [ A1 ] to [ A3-4 ] above, wherein in the step (1), the soluble thrombomodulin-containing material is supplied to hydroxyapatite using a buffer solution having a pH of 6 to 9 and a phosphate concentration of 5 to 20 mM; in the step (2), a pass-through fraction is obtained by using a buffer solution having a pH of 6 to 9 and a phosphate concentration of 5 to 20mM, thereby obtaining a fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin.

The production method according to any one of [ A1 ] to [ A10 ] above, which does not include a step of adjusting the pH of an eluted fraction to 4 or less after the fraction contains soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin.

The production method according to any one of [ A1 ] to [ A11 ] above, which comprises obtaining an eluted fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin, and then performing a concentration step and/or a desalting step without adjusting the pH of the fraction to 4 or less, and which is used for producing the soluble thrombomodulin as a pharmaceutical raw material.

The production method according to any one of [ A1 ] to [ A12 ] above, wherein the content of a denatured product of soluble thrombomodulin in the soluble thrombomodulin that does not substantially contain the denatured product of soluble thrombomodulin is 3% or less.

The production method according to any one of [ A1 ] to [ A13 ] above, wherein the soluble thrombomodulin is obtained from a transformed cell prepared by transfecting a host cell with a DNA encoding the amino acid sequence according to SEQ ID NO. 9 or SEQ ID NO. 11.

[ A15 ] A method for purifying soluble thrombomodulin such that the soluble thrombomodulin does not substantially contain a denatured product of the soluble thrombomodulin that may be produced from the soluble thrombomodulin under acidic conditions, the method comprising the steps of:

(1) supplying an anion exchanger or hydroxyapatite with the soluble thrombomodulin-containing material containing or suspected of containing a denatured product of the soluble thrombomodulin;

(2) obtaining an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin under separation conditions that allow the soluble thrombomodulin to be separated from the denatured product of the soluble thrombomodulin,

alternatively, in the purification method, in the step of supplying the soluble thrombomodulin-containing material to an anion exchanger or hydroxyapatite, a buffer solution of the soluble thrombomodulin-containing material is prepared, and soluble thrombomodulin substantially not containing a denatured product of the soluble thrombomodulin is obtained as a pass-through fraction.

The purification method according to [ A15-2 ] above [ A15 ], wherein the method has any one of the features described in [ A1 ] to [ A14 ] above.

The production method according to any one of [ A1 ] to [ A15 ] above, wherein the content of a denatured product of soluble thrombomodulin in the thrombomodulin that does not substantially contain the denatured product of soluble thrombomodulin is 3.0% or less.

[ B1 ] A method for producing thrombomodulin that does not substantially contain a denatured product of soluble thrombomodulin from a soluble thrombomodulin-containing material that contains or is suspected to contain the denatured product of soluble thrombomodulin that can be produced from the soluble thrombomodulin under acidic conditions, which comprises the steps of:

(1) supplying the thrombomodulin-containing material to an anion exchanger or hydroxyapatite;

(2) under separation conditions that allow the soluble thrombomodulin to be separated from a denatured product of the soluble thrombomodulin, an eluted fraction that contains the soluble thrombomodulin substantially free of the denatured product of the soluble thrombomodulin is obtained.

[ B2 ] A method for producing thrombomodulin that does not substantially contain a denatured product of soluble thrombomodulin from a soluble thrombomodulin-containing material that contains or is suspected to contain the denatured product of soluble thrombomodulin that can be produced from soluble thrombomodulin under acidic conditions, wherein in the step of supplying the thrombomodulin-containing material to an anion exchanger or hydroxyapatite, a buffer solution of the thrombomodulin-containing material is prepared, and soluble thrombomodulin that does not substantially contain the denatured product of soluble thrombomodulin is obtained in the form of a flow-through fraction.

The production method according to [ B3 ] or [ B1 ], wherein in the step (1), the anion exchanger is supplied with a soluble thrombomodulin-containing material, and the pH of the buffer solution used is 4 to 9; in the step (2), linear or stepwise gradient elution or a combination of linear and stepwise gradient elution is performed using a buffer solution having a pH of 5 to 9 and a salt concentration of 0 to 1M, and an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin is obtained by confirming in advance, or an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin is obtained by confirming the elution fraction.

[ B4 ] the production method according to [ B1 ], wherein in the step (1), the anion exchanger is supplied with a soluble thrombomodulin-containing material, and the pH of the buffer solution used is 4 to 5; in the step (2), linear or stepwise gradient elution or a combination of linear and stepwise gradient elution is performed using a buffer solution having a pH of 5 to 9 and a salt concentration of 0 to 1M, and an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin is obtained by confirming in advance, or an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin is obtained while confirming the elution fraction.

[ B5 ] the production method according to [ B2 ], wherein the pH of the buffer solution is 5 to 8, the salt concentration is 0.1 to 0.2M, and the soluble thrombomodulin-containing material is supplied to the anion exchanger to obtain a soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin in the form of a pass-through fraction.

[ B6 ] the production method according to [ B1 ], wherein in the step (1), the soluble thrombomodulin-containing material is supplied to hydroxyapatite, and a buffer solution having a pH of 6 to 9 and a phosphate concentration of 8mM or less is used; in the step (2), linear or stepwise gradient elution or a combination of linear and stepwise gradient elution is performed using a buffer solution having a pH of 6 to 9 and a phosphate concentration of 0 to 0.5M, and an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin is obtained by confirming in advance, or an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin is obtained by confirming the elution fraction.

[ B7 ] the production method according to [ B1 ], wherein in the step (1), the soluble thrombomodulin-containing material is supplied to hydroxyapatite, a buffer solution having a pH of 6 to 9 and a phosphate concentration of 1 to 4mM is used; in the step (2), a gradient elution is performed linearly or stepwise or in a combination of linear steps using a buffer solution having a pH of 6 to 9 and a phosphate concentration of 1 to 40mM, and an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin is obtained by confirming in advance, or an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin is obtained by confirming the elution fraction.

[ B8 ] the production method according to [ B2 ], wherein the pH of the buffer solution is 6 to 9, the phosphate concentration is 5 to 20mM, the soluble thrombomodulin-containing material is supplied to hydroxyapatite, and the soluble thrombomodulin substantially not containing a denatured product of the soluble thrombomodulin is obtained as a pass-through fraction.

The production method according to any one of [ B9 ] to [ B1 ] to [ B8 ], wherein after an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin is obtained, the pH of the elution fraction is not set to 4 or less.

The production method according to any one of [ B10 ] to [ B1 ] to [ B8 ], wherein an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin is obtained, and then the elution fraction is subjected to a concentration step or a desalting step so as not to adjust the pH of the elution fraction to 4 or less to prepare a raw material for a pharmaceutical product.

The production method according to any one of [ B11 ] to [ B10 ] of [ B1 ] or [ B10 ], wherein the content of a denatured product of soluble thrombomodulin in the soluble thrombomodulin that does not substantially contain the denatured product of soluble thrombomodulin is 3% or less.

The production method according to any one of [ B12 ] to [ B11 ] of [ B1 ] or [ B11 ], wherein the content of a denatured product of soluble thrombomodulin in the soluble thrombomodulin that does not substantially contain the denatured product of soluble thrombomodulin is 1% or less.

The production method according to any one of [ B1 ] to [ B12 ] of [ B13 ], wherein the thrombomodulin is a peptide obtained from a transformed cell prepared by transfecting a host cell with a DNA encoding the amino acid sequence according to SEQ ID NO. 9 or SEQ ID NO. 11.

By using the production method of the present invention, a concentration step for increasing the concentration of the treated protein is not required, the treatment volume is not limited, the denatured product of the soluble thrombomodulin can be separated with high productivity, and a high-purity soluble thrombomodulin substantially not containing the denatured product of the soluble thrombomodulin can be obtained.

Drawings

FIG. 1 shows a chromatogram for separating a denatured product of soluble thrombomodulin by anion exchanger linear gradient elution in example 4.

FIG. 2 shows a chromatogram for separating a denatured product of soluble thrombomodulin by anion exchanger linear gradient elution in example 5.

FIG. 3 shows a chromatogram for separating a denatured product of soluble thrombomodulin by anion exchanger linear gradient elution in example 6.

FIG. 4 shows the results of measuring the content of a denatured product of soluble thrombomodulin in the purified product obtained in example 3.

FIG. 5 shows a chromatogram of example 8 in which a denatured product of soluble thrombomodulin was removed by stepwise gradient elution using an anion exchanger.

FIG. 6 is a chromatogram showing the removal of a denatured product of soluble thrombomodulin by hydroxyapatite stepwise gradient elution in example 11.

FIG. 7 is a chromatogram showing the purification and removal of a denatured product of soluble thrombomodulin by a strong anion exchanger in example 14.

Detailed Description

It is known that the thrombomodulin of the present invention (1) binds selectively to thrombin and (2) has an action of promoting the activation of protein C by thrombin. Further, the following effects of thrombomodulin in the present invention are generally recognized: (3) the effect of prolonging the coagulation time based on the thrombin action and/or (4) the effect of inhibiting platelet aggregation based on the thrombin action, which is desirable. The action of these thrombomodulins is sometimes referred to as thrombomodulin activity.

The thrombomodulin activity preferably has the effects of (1) and (2) above, and more preferably all of the effects of (1) to (4) above.

The activity of promoting the activation of protein C by thrombin can be easily confirmed by, for example, a test method which is described in various publicly known documents including Japanese patent application laid-open No. Sho 64-6219, and the amount of activity of promoting the activation of protein C and the presence or absence of the activity can be easily confirmed. In addition, the effect of extending the coagulation time by thrombin action and/or the effect of inhibiting platelet aggregation by thrombin action can be confirmed similarly and easily.

Examples of the soluble thrombomodulin described in the present invention include soluble thrombomodulin soluble in water in the absence of a surfactant. Preferred examples of the solubility of the soluble thrombomodulin include solubility in water, for example, distilled water for injection (in the absence of a surfactant such as TritonX-100 or polidocanol, and in the vicinity of neutral pH in general), of 1mg/ml or more, or 10mg/ml or more, preferably 15mg/ml or more, or 17mg/ml or more, more preferably 20mg/ml or more, 25mg/ml or more, or 30mg/ml or more, particularly preferably 60mg/ml or more, and in some cases, 80mg/ml or more, or 100mg/ml or more, respectively. In judging whether or not soluble thrombomodulin can be dissolved, it can be understood as follows: after dissolution, the resultant was clear and transparent when observed with the naked eye at a position of brightness of about 1000lux immediately below a white light source, and it was directly indicated that the insoluble matter was not contained to such an extent that it could be clearly confirmed. Further, the presence or absence of the residue after filtration may be checked.

The soluble thrombomodulin of the present invention preferably contains the amino acid sequences at positions 19 to 132 of SEQ ID No. 1, which are known as the central site of thrombomodulin activity in human thrombomodulin, but is not particularly limited as long as it contains the amino acid sequences at positions 19 to 132 of SEQ ID No. 1. The amino acid sequences at positions 19 to 132 of SEQ ID No. 1 may be naturally or artificially modified, that is, one or more than 2 amino acids may be substituted, deleted or added in the amino acid sequences at positions 19 to 132 of SEQ ID No. 1, as long as they have thrombomodulin activity, which is an activity of promoting the activation of protein C by thrombin. The degree of the allowable variation is not particularly limited as long as it has thrombomodulin activity, and examples of the amino acid sequence include amino acid sequences having 50% or more identity, preferably 70% or more identity, more preferably 80% or more identity, still more preferably 90% or more identity, particularly preferably 95% or more identity, and most preferably 98% or more identity. Such amino acid sequences are referred to as identical variant sequences. These variants can be easily obtained by a general gene manipulation technique as described later.

The sequence of SEQ ID No. 3 is a sequence in which the 125 th amino acid Val of the sequence of SEQ ID No. 1 is mutated to Ala, but the soluble thrombomodulin of the present invention preferably comprises the 19 th to 132 th amino acid sequences of SEQ ID No. 3.

As described above, the soluble thrombomodulin of the present invention is not particularly limited as long as it contains a peptide sequence having at least thrombomodulin activity, including at least the 19 th to 132 th sequences of SEQ ID NO. 1 or SEQ ID NO. 3 or the same variant sequence thereof, and preferable examples thereof include a peptide consisting of the 19 th to 132 th sequences or the 17 th to 132 th sequences of SEQ ID NO. 1 or SEQ ID NO. 3, respectively, or a peptide consisting of the same variant sequence thereof and having at least thrombomodulin activity, and more preferable examples thereof include a peptide consisting of the 19 th to 132 th sequences of SEQ ID NO. 1 or SEQ ID NO. 3. There are other embodiments of a peptide having at least thrombomodulin activity, which is more preferably composed of the same variant sequence at positions 19 to 132 or 17 to 132 of SEQ ID NO. 1 or 3, respectively.

In another embodiment of the soluble thrombomodulin of the present invention, the soluble thrombomodulin preferably contains the amino acid sequences at positions 19 to 480 of SEQ ID NO. 5, and is not particularly limited as long as it contains the amino acid sequences at positions 19 to 480 of SEQ ID NO. 5. The amino acid sequence at positions 19 to 480 of SEQ ID No. 5 may be the same variant sequence as long as it has thrombomodulin activity by promoting the activation of protein C by thrombin.

The sequence No. 7 is a sequence in which the amino acid Val at the 473 rd position of the sequence No. 5 is mutated to Ala, but the soluble thrombomodulin of the present invention preferably also comprises the amino acid sequences at the 19 th to 480 th positions of the sequence No. 7.

As described above, the soluble thrombomodulin of the present invention is not particularly limited as long as it contains a peptide sequence having at least thrombomodulin activity, including at least the sequence from positions 19 to 480 of SEQ ID NO. 5 or SEQ ID NO. 7 or the same variant sequence thereof, and preferable examples thereof include a peptide consisting of the sequence from positions 19 to 480 or 17 to 480 of SEQ ID NO. 5 or SEQ ID NO. 7, respectively, or a peptide consisting of the same variant sequence thereof and having at least thrombomodulin activity, and more preferable examples thereof include a peptide consisting of the sequence from positions 19 to 480 of SEQ ID NO. 5 or SEQ ID NO. 7. There are also other embodiments of a peptide having at least thrombomodulin activity, which is more preferably composed of the same variant sequence at positions 19 to 480 or 17 to 480 of SEQ ID NO. 5 or SEQ ID NO. 7.

In another embodiment of the soluble thrombomodulin of the present invention, the soluble thrombomodulin preferably comprises the amino acid sequences from 19 th to 515 th of SEQ ID NO. 9, and is not particularly limited as long as it comprises the amino acid sequences from 19 th to 515 th of SEQ ID NO. 9. The amino acid sequence at positions 19 to 515 of SEQ ID No. 9 may be the same variant sequence as long as it has the thrombomodulin activity that promotes the activation of protein C by thrombin.

The sequence of SEQ ID NO. 11 is a sequence in which the amino acid Val at position 473 of the sequence of SEQ ID NO. 9 is modified to Ala, but the thrombomodulin of the present invention preferably also comprises the amino acid sequences of positions 19 to 515 of SEQ ID NO. 11.

As described above, the soluble thrombomodulin of the present invention is not particularly limited as long as it contains a peptide sequence having at least thrombomodulin activity, including at least the 19 th to 515 th sequences of SEQ ID NO. 9 or SEQ ID NO. 11, or the same variant sequence thereof, and more preferably includes a peptide comprising at least the 19 th to 516 th, 19 th to 515 th, 17 th to 516 th or 17 th to 515 th sequences of SEQ ID NO. 9 or SEQ ID NO. 11, or a peptide comprising at least thrombomodulin activity, and particularly preferably a peptide comprising the 19 th to 516 th, 19 th to 515 th, 17 th to 516 th or 17 th to 515 th sequences of SEQ ID NO. 9. As a preferred example, a mixture thereof may be mentioned. In addition, there are other embodiments in which a peptide consisting of the sequence at positions 19 to 516, 19 to 515, 17 to 516 or 17 to 515 of SEQ ID NO. 11 is particularly preferable. As a preferred example, a mixture thereof may be mentioned. In addition, a peptide having at least thrombomodulin activity, which is composed of the same variant sequence of these sequences, is also included as a preferable example.

As described above, the peptides having the same variant sequence also mean peptides in which one or more, that is, one or 2 or more, amino acids, and preferably several (for example, 1 to 20, preferably 1 to 10, more preferably 1 to 5, and particularly preferably 1 to 3) amino acids can be substituted, deleted, or added to the amino acid sequence of the subject peptide. The degree of the allowable variation is not particularly limited as long as it has thrombomodulin activity, and examples of the amino acid sequence include amino acid sequences having 50% or more identity, preferably 70% or more identity, more preferably 80% or more identity, still more preferably 90% or more identity, particularly preferably 95% or more identity, and most preferably 98% or more identity.

Further, as a preferable example of the soluble thrombomodulin of the present invention, there can be mentioned a peptide consisting of the sequence of SEQ ID NO. 14 (462 amino acid residues), a peptide consisting of the sequence of SEQ ID NO. 8 (272 amino acid residues), or a peptide consisting of the sequence of SEQ ID NO. 6 (236 amino acid residues) in Japanese patent laid-open No. Sho 64-6219.

The soluble thrombomodulin of the present invention is not particularly limited as long as it has at least the amino acid sequences at positions 19 to 132 of SEQ ID NO. 1 or SEQ ID NO. 3, and among them, a peptide having at least the amino acid sequences at positions 19 to 480 of SEQ ID NO. 5 or SEQ ID NO. 7 is preferable, and a peptide having at least the amino acid sequences at positions 19 to 515 of SEQ ID NO. 9 or SEQ ID NO. 11 is more preferable. More preferred examples of the peptide having at least the amino acid sequence from position 19 to 515 of SEQ ID NO. 9 or SEQ ID NO. 11 include peptides having a sequence from positions 19 to 516, 19 to 515, 17 to 516 or 17 to 515 of SEQ ID NO. 9 or SEQ ID NO. 11, respectively. Further, as a more preferable example, there is also a mixture of peptides consisting of the 19 th to 516 th, 19 th to 515 th, 17 th to 516 th or 17 th to 515 th positions of SEQ ID NO. 9 or 11, respectively, and SEQ ID NO. 9 or 11, respectively.

The mixture includes, for example, (30: 70) to (50: 50) as a mixing ratio of the peptide from the 17 th position to the peptide from the 19 th position in SEQ ID NO. 9 or 11, and preferable examples thereof include (35: 65) to (45: 55).

The mixing ratio of the peptide terminating at position 515 and the peptide terminating at position 516 in SEQ ID NO. 9 or 11 is, for example, (0: 100) to (90: 10), and in some cases, (70: 30) to (90: 10) and (0: 100) to (30: 70).

The mixing ratio of these peptides can be determined by a usual method.

The 19 th to 132 th sequences of SEQ ID No. 1 correspond to the 367 th to 480 th sequences of SEQ ID No. 9, and the 19 th to 480 th sequences of SEQ ID No. 5 correspond to the 19 th to 480 th sequences of SEQ ID No. 9.

The 19 th to 132 th bit sequences of SEQ ID No. 3 correspond to the 367 th to 480 th bit sequences of SEQ ID No. 11, and the 19 th to 480 th bit sequences of SEQ ID No. 7 correspond to the 19 th to 480 th bit sequences of SEQ ID No. 11.

The sequences at positions 1 to 18 in SEQ ID Nos. 1, 3, 5, 7, 9 and 11 are identical to each other.

As described later, these thrombomodulin of the present invention can be obtained from transformed cells prepared by transfecting host cells with a vector with DNA encoding the peptides of SEQ ID Nos. 1, 3, 5, 7, 9, 11 or the like (specifically, the nucleotide sequences of SEQ ID Nos. 2, 4, 6, 8, 10, 12 or the like, respectively). As the thrombomodulin, thrombomodulin obtained from transformed cells prepared by transfecting a host cell with a vector with DNA encoding the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 11 (specifically, the base sequence of SEQ ID NO. 10 or SEQ ID NO. 12, respectively) is preferable.

These peptides may have a sugar chain or no sugar chain as long as they have the above-mentioned amino acid sequence, and are not particularly limited in this regard. In addition, in gene manipulation, the kind of sugar chain, the position of addition, and the degree of addition vary depending on the kind of host cell used, and these peptides can be used arbitrarily. The fact described in Japanese patent application laid-open No. 11-341990 is known as to the binding position and type of sugar chain, and the thrombomodulin of the present invention may be added with the same sugar chain at the same position. As described later, it is not particularly limited to the case of obtaining the signal sequence by gene manipulation, but in the case of obtaining the signal sequence by gene manipulation, the nucleotide sequence encoding the amino acid sequences at positions 1 to 18 of SEQ ID NO. 9, the nucleotide sequence encoding the amino acid sequences at positions 1 to 16 of SEQ ID NO. 9, and other known signal sequences (for example, a signal sequence of human histiotype plasminogen activator) can be used as the signal sequence to be used for expression (International patent publication No. 88/9811).

When a DNA sequence encoding thrombomodulin is introduced into a host cell, a method of introducing a DNA sequence encoding thrombomodulin into a vector (particularly preferably an expression vector capable of expression in animal cells) is preferable. The expression vector is a DNA molecule comprising a promoter sequence, a sequence which imparts a ribosome binding site to mRNA, a DNA sequence encoding a protein to be expressed, a splicing signal, a terminator sequence for termination of transcription, a replication initiation sequence, etc., and examples of preferred animal cell expression vectors include pSV2-X reported by R.C. Mullgan et al [ Proc.Natl.Acad.Sci.U.S.A.78.2072(1981) ], and P.M.wlHoey et al [ method Emzymatology, 101, Academic Press (1983) ], etc. the expression vector also includes pBP69T (69-6).

Examples of host cells that can be used for producing these peptides include Chinese Hamster Ovary (CHO) cells, COS-1 cells, COS-7 cells, VERO (ATCC CCL-81) cells, BHK cells, canine kidney-derived MDCK cells, hamster AV-12-664 cells, and human-derived HeLa cells, WI38 cells, and human 293 cells. CHO cells are very general and preferred, and among CHO cells, DHFR-CHO cells are more preferred.

In addition, in the genetic engineering process and the peptide production process, microorganisms such as Escherichia coli are often used, and host-vector systems suitable for the respective microorganisms are preferably used, and among the above-mentioned host cells, suitable vector systems can be selected. The gene of thrombomodulin used in gene recombination technology has been cloned, and examples of production of gene recombination technology using thrombomodulin have been disclosed, and purification methods for obtaining purified products thereof are also known [ Japanese patent application laid-open No. Sho 64-6219, Japanese patent application laid-open No. Hei 2-255699, Japanese patent application laid-open No. Hei 5-213998, Japanese patent application laid-open No. Hei 5-310787, Japanese patent application laid-open No. Hei 7-155176, J.biol.chem., 264: 10351-10353(1989)]. Therefore, the soluble thrombomodulin used in the present invention can be produced by using the methods described in the above-mentioned reports or based on the methods described in these reports. For example, Japanese patent application laid-open No. Sho-64-6219 discloses Escherichia coli K-12 strain DH5(ATCC accession No. 67283) containing plasmid pSV2 thrombomodulin J2, the plasmid pSV2 thrombomodulin J2 containing DNA encoding the full length thrombomodulin. Further, a strain (E.coli DH5/pSV2TM J2) (FERM BP-5570) which was again deposited in the life research (International patent organism depositary, national institute of independent administrative sciences, institute of advanced Industrial science and technology, institute of Integrated technology and technology) (No. 1 Bu 3 (ZIP code 305), Tokyo, Takagaku, N.K.) at 19.6.1996 can also be used. The thrombomodulin of the present invention can be produced by a known gene manipulation technique using the DNA encoding the full-length thrombomodulin as a raw material.

The soluble thrombomodulin to be used in the present invention may be produced by a conventionally known method or by a method based on the method, and for example, the method described above in Yamamoto et al [ Japanese patent application laid-open No. Sho 64-6219 ] or Japanese patent application laid-open No. Hei 5-213998 can be referred to. That is, the human thrombomodulin-derived gene may be prepared, for example, as a DNA encoding the amino acid sequence of SEQ ID No. 9 by gene manipulation techniques, and may be further modified as necessary. For example, in order to prepare a DNA encoding the amino acid sequence of seq id No. 11 (specifically, consisting of the base sequence of seq id No. 12), the DNA is modified according to the following method in Enzymology, 100: 468 (1983), Academic Press ], wherein site-specific mutagenesis was performed on the codon of the 473 rd amino acid (particularly, the base at the 1418 th position) of the amino acid sequence encoding SEQ ID No. 9. For example, a DNA obtained by converting base T at position 1418 of SEQ ID NO. 10 into base C can be prepared by using a synthetic DNA for mutation having the base sequence shown in SEQ ID NO. 13.

The DNA thus prepared is incorporated into, for example, Chinese Hamster Ovary (CHO) cells to prepare transformed cells, and thrombomodulin purified by a known method can be produced from a culture solution obtained by culturing the cells by appropriate selection. As described above, the DNA encoding the amino acid sequence of SEQ ID No. 9 (SEQ ID No. 10) is preferably transfected into the above-mentioned host cell. The method for producing thrombomodulin to be used in the present invention is not limited to the above-mentioned methods, and for example, it may be extracted and purified from a tissue from which thrombomodulin is produced, a culture solution of the tissue, or the like, or may be subjected to a cleavage treatment with a protease as required.

When the thrombomodulin of the present invention is produced by the above cell culture method, the N-terminal amino acid may be found to be diversified by post-translational modification of the protein. For example, the amino acid at position 17, 18, 19 or 22 in SEQ ID NO. 9 may be the N-terminus. In addition, for example, the N-terminal amino acid may be modified so as to convert glutamic acid at position 22 to pyroglutamic acid. The amino acid at position 17 or 19 is preferably the N-terminus, and the amino acid at position 19 is more preferably the N-terminus. In addition, there are other modes in which the 17 th amino acid is preferably the N-terminus. The same modification, diversity, and the like as described above can be exemplified by SEQ ID NO 11.

In addition, in the case of using DNA having the base sequence of SEQ ID NO. 10 to produce thrombomodulin, the diversity of C-terminal amino acids was sometimes confirmed, and a peptide 1 amino acid residue shorter was sometimes produced. That is, the C-terminal amino acid may be modified so that the 515 th amino acid is C-terminal and the 515 th amino acid is amidated. Therefore, a peptide rich in diversity of N-terminal amino acid and C-terminal amino acid, or a mixture thereof may be produced. Preferably, the amino acid at position 515 is C-terminal. In addition, there are other modes in which the amino acid at position 516 is preferably the C-terminus. The same applies to the above modifications, variations, and the like, with respect to DNA having the base sequence of SEQ ID NO. 12.

The thrombomodulin obtained by the above method may be a mixture of peptides in which the diversity of the N-terminal and C-terminal is confirmed. Specifically, there can be mentioned a mixture of peptides consisting of the 19 th to 516 th, 19 th to 515 th, 17 th to 516 th or 17 th to 515 th sequences in SEQ ID NO. 9.

According to the present invention, there is provided a method for producing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin that can be produced from the soluble thrombomodulin under acidic conditions. The production method is not particularly limited as long as it includes the following two steps: (1) supplying an anion exchanger or hydroxyapatite with the soluble thrombomodulin-containing material containing or suspected of containing a denatured product of the soluble thrombomodulin; and (2) obtaining a fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin under separation conditions that allow the soluble thrombomodulin to be separated from the denatured product of the soluble thrombomodulin. However, it is preferable that the process (1) includes, before the process (0): the soluble thrombomodulin is placed under acidic conditions at a pH of 5 or less, and more preferably, the method comprises (0) a step of placing the soluble thrombomodulin under acidic conditions at a pH of 5 or less before the step (1) and a step of purifying the soluble thrombomodulin. In the production method of the present invention, the position of the step (, x) is not particularly limited, but the step (, x) is preferably performed before the step (1).

The acidic condition under which the soluble thrombomodulin of the present invention can form a denatured product of the soluble thrombomodulin under acidic conditions may include, for example, a pH of 5 or less, preferably a pH of 4 or less, more preferably a pH of 3 or less, and the lower limit of the pH may include, for example, a pH of 1 or more, preferably a pH of 2 or more, more preferably a pH of 3 or more. The time under acidic conditions may be, for example, 0.5 hours or more, preferably 1 hour or more, and more preferably 2 hours or more. The temperature under acidic conditions may be, for example, room temperature, preferably 15 ℃ or lower, more preferably 8 ℃ or lower, and the lower limit of the temperature may be usually 0 ℃ or higher, preferably 2 ℃ or higher, more preferably 5 ℃ or higher. Under the acidic conditions described above, the mixture becomes soluble thrombomodulin and a denatured product of the soluble thrombomodulin.

The upper limit of the content of the denatured product of soluble thrombomodulin in the "thrombomodulin substantially not containing a denatured product of soluble thrombomodulin" produced by the method of the present invention is preferably 3.0% or less, more preferably 2.0% or less, still more preferably 1.0% or less, particularly preferably 0.5% or less, and the lower limit thereof is 0.01% or more, but is preferably not more than the detection limit. The content of the denatured product of soluble thrombomodulin can be determined by chromatography, for example, by using TSKgelG3000SWXL (7.8mm I.D.. times.30 cm; TOSOH Co.) for analysis by size exclusion chromatography, supplying 150. mu.l of an analytical sample using a sodium sulfate-containing phosphate buffer as a mobile phase, and measuring the absorbance at 280nm at a flow rate of 0.9 ml/min.

The denatured product of the soluble thrombomodulin that can be formed from the soluble thrombomodulin of the present invention under acidic conditions is detected by a retention time of about 9 times the retention time of the soluble thrombomodulin (for example, the retention time of the denatured product of the soluble thrombomodulin is about 8 minutes when the retention time of the soluble thrombomodulin is about 9 minutes) when it is analyzed by size exclusion chromatography using TSKgelG3000SWXL (7.8 mmI.D.. times.30 cm; TOSOH Co.) and it is analyzed at a flow rate of 0.9 ml/minute by supplying 150. mu.l of an analysis sample using a phosphate buffer containing sodium sulfate. The denatured product of the soluble thrombomodulin that can be produced from the soluble thrombomodulin of the present invention under acidic conditions migrates in Native-PAGE (Native-PAGE) that is a non-denaturing product of electrophoresis, is found on the polymer side of the thrombomodulin, and is not detected in SDS-PAGE.

The soluble thrombomodulin-containing material (hereinafter, may be simply referred to as "denatured material-containing material") containing or suspected of containing a denatured product of soluble thrombomodulin that can be produced under acidic conditions, which is used in the present invention, is not particularly limited as long as it contains soluble thrombomodulin and a denatured product of soluble thrombomodulin, and examples thereof include: culturing cells capable of producing soluble thrombomodulin using a serum-component-containing medium or a serum-free medium, and obtaining a soluble thrombomodulin liquid from the prepared culture supernatant; it is preferable that: culturing cells capable of producing soluble thrombomodulin using a serum-containing medium or a serum-free medium, and purifying the obtained soluble thrombomodulin liquid or the soluble thrombomodulin liquid obtained by leaving the culture supernatant in an acidic condition; more preferably: culturing cells capable of producing soluble thrombomodulin using a serum-containing medium or a serum-free medium, and purifying the resulting culture supernatant to obtain a soluble thrombomodulin liquid. In addition, there are other ways of obtaining the soluble thrombomodulin liquid preferably by exposure to an acidic condition.

The purification step is not particularly limited as long as it is a step capable of removing at least proteins other than soluble thrombomodulin, and examples thereof include the following steps: eluting and recovering the soluble thrombomodulin under acidic conditions using affinity chromatography loaded with an antibody against thrombomodulin; or contacting the soluble thrombomodulin obtained by the affinity chromatography with a cation exchanger under conditions of a conductivity of 25 to 34ms/cm and a pH of 3 to 4, and recovering the soluble thrombomodulin as a pass-through fraction.

The soluble thrombomodulin-containing material (denatured product-containing material) is not particularly limited as long as it contains soluble thrombomodulin and a denatured product of soluble thrombomodulin, and may include a material that does not substantially contain proteins other than soluble thrombomodulin and a denatured product of soluble thrombomodulin. Specifically, the lower limit of the content of soluble thrombomodulin with respect to total proteins in the soluble thrombomodulin-containing material is 50% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, particularly preferably 90% or more, most preferably 95% or more, and most preferably 99% or more. In addition, it may be preferably 99.9% or more. The content of the soluble thrombomodulin may be the content of the soluble thrombomodulin itself, but may be the total content of the soluble thrombomodulin and a denatured product of the soluble thrombomodulin. When the content of the soluble thrombomodulin represents the total content of the soluble thrombomodulin and the denatured product of the soluble thrombomodulin, the lower limit of the content of the soluble thrombomodulin relative to the total protein in the soluble thrombomodulin-containing material is, for example, 50% or more, preferably 60% or more, more preferably 70% or more, further preferably 80% or more, particularly preferably 90% or more, most preferably 95% or more, and most preferably 99% or more. In addition, it may be preferably 99.9% or more.

The content of the soluble thrombomodulin can be determined by chromatography, for example, by subjecting a mobile phase of distilled water (solution A) containing 0.1% trifluoroacetic acid and acetonitrile (solution B) containing 0.1% trifluoroacetic acid to 150. mu.l of an analytical sample by reverse phase chromatography for analysis using AsahiPak C4P-50(4.6mm I.D.. times.15 cm; Showa Denko K.K.), and measuring the absorbance at 280nm of the eluate at a flow rate of 0.8 ml/min by linearly changing the mixing ratio of the solution A and the solution B from 96: 4 to 0: 100 after 48 minutes, whereby the content of the soluble thrombomodulin can be determined from the peak areas of the soluble thrombomodulin and other proteins. Alternatively, the content of soluble thrombomodulin may be determined by measuring proteins other than soluble thrombomodulin by the ELISA method described in Japanese patent application laid-open No. 11-341990.

In the present invention, it is preferable that the thrombomodulin-containing material containing or suspected of containing a denatured product of the soluble thrombomodulin is supplied to an anion exchanger or hydroxyapatite after the step of leaving the thrombomodulin in an acidic condition of pH5 or less. The pH under acidic conditions preferably has an upper limit of 5 or less, more preferably 4 or less, still more preferably 3 or less, and a lower limit of 1 or more, more preferably 2 or more, still more preferably 3 or more.

The time under acidic conditions may be, for example, 0.5 hours or more, preferably 1 hour or more, and more preferably 2 hours or more. The temperature under acidity is, for example, room temperature, preferably 15 ℃ or lower, more preferably 8 ℃ or lower, and the lower limit of the temperature under acidity is usually 0 ℃ or higher, preferably 2 ℃ or higher, more preferably 5 ℃ or higher.

As the salt used in the present invention, a suitable salt is a chloride, and a most suitable salt is sodium chloride. In addition, for hydroxyapatite, suitable phosphates are sodium phosphate, potassium phosphate.

Examples of the separation conditions capable of separating soluble thrombomodulin from a denatured product of soluble thrombomodulin include gradient elution conditions of linear or stepwise or a combination of linear and stepwise, conditions under which soluble thrombomodulin does not elute in the pass-through fraction, or isocratic elution conditions, preferably gradient elution conditions of linear or stepwise or a combination of linear and stepwise, or conditions under which soluble thrombomodulin does not elute in the pass-through fraction, more preferably gradient elution conditions of linear or stepwise or a combination of linear and stepwise. In addition, there are other modes in which the conditions under which the soluble thrombomodulin is not eluted in the flow-through fraction are preferable. In addition, isocratic elution conditions may also be preferred.

The pass-through fraction includes a fraction in which soluble thrombomodulin is not adsorbed to an anion exchanger or hydroxyapatite after the soluble thrombomodulin-containing material is supplied to the anion exchanger or hydroxyapatite. The flow-through fraction is not particularly limited as long as it contains substantially no denatured product of thrombomodulin, and examples thereof include 1 to 20 column volumes, 1 to 50 column volumes, and 1 to 100 column volumes.

The gradient elution conditions may be appropriately set according to the column used. Specific conditions include those described later. The buffer used as the conditions under which the soluble thrombomodulin does not elute in the flow-through fraction may be appropriately set depending on the column used. Specific conditions include those described later. The isocratic elution conditions may be appropriately set according to the column used.

The anion exchanger is preferably a strong anion exchanger, that is, an anion exchanger having a quaternary ammonium group (for example, SOURCEQ (GE Healthcare Bio-Sciences corporation)), and the anion exchanger may be equilibrated with several column volumes of a buffer solution having a pH of 4 to 9 (the type of the buffer solution is not limited, and for example, 0.1M acetic acid, pH4, or 0.02M phosphoric acid, pH7.3, or 0.02M Tris, pH8) before the denatured product-containing material is supplied. After the equilibration, the denatured product-containing material is supplied, and before the elution of the soluble thrombomodulin, the material may be washed with a buffer solution having a pH of 4 to 9 (for example, 0.1M acetic acid, pH4, or 0.02M phosphoric acid, pH7.3, or 0.02M Tris, pH8, without limitation). The soluble thrombomodulin and the denatured product of the soluble thrombomodulin are separated and eluted by gradient elution of the adsorbed denatured product-containing material at a salt concentration of 0 to 1M using a buffer solution of pH5 to 9, preferably pH7 to 8 (the type of buffer solution is not limited, and may be, for example, 0.02M phosphoric acid, pH7.3, or 0.02M Tris, pH 8). The separation conditions are not particularly limited as long as they are separation conditions capable of separating soluble thrombomodulin from a denatured product of soluble thrombomodulin, and examples of the gradient elution conditions include linear or stepwise conditions, or a combination of linear and stepwise conditions. Preferably, the salt concentration of the gradient elution is 0 to 0.3M, and more preferably, the salt concentration is 0.03 to 0.26M. The amount of the elution buffer is 2 to 40 column volumes, preferably 5 to 20 column volumes.

When gradient elution conditions are used, examples of the step of obtaining an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin include: (a) confirming the position of the soluble thrombomodulin-eluting fraction in advance to obtain an eluting fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin; or (b) obtaining an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin, while confirming the presence or absence of the soluble thrombomodulin in the elution fraction.

Specifically, the step (a) includes the following steps: the position of an elution fraction containing substantially no denatured product of soluble thrombomodulin is confirmed in advance by an analytical method (for example, size exclusion chromatography described in example 7 below) in which the presence of soluble thrombomodulin and a denatured product of soluble thrombomodulin can be confirmed, and the elution fraction at the position is collected. Further, the step (b) is specifically described, and the following steps may be mentioned: an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin is obtained while confirming that the elution fraction contains the soluble thrombomodulin and that the elution fraction does not substantially contain the denatured product of the soluble thrombomodulin by an analytical method (for example, size exclusion chromatography described in example 7 below) in which the presence of soluble thrombomodulin and the denatured product of soluble thrombomodulin can be confirmed. The step (b) is preferred. In some cases, the step (a) may be preferred.

In addition, soluble thrombomodulin substantially free of denatured product of thrombomodulin may also be obtained as a flow-through fraction by replacing the denatured product-containing material with an appropriate buffer solution before supplying the denatured product-containing material. The pass-through fraction includes a fraction in which soluble thrombomodulin is not adsorbed to an anion exchanger after the soluble thrombomodulin-containing material is supplied to the anion exchanger. In the case of utilizing the difference in the adsorption force to the column between the soluble thrombomodulin and the denatured product of the soluble thrombomodulin, a strong anion exchanger, that is, an anion exchanger having a quaternary ammonium group, such as SOURCEQ, QSepharoseFF (GE Healthcare Bio-Sciences Co., Ltd.), Sartobind (Sartorius Co., Ltd.), Mustang (PALL Co., Ltd.) can be preferably used as the anion exchanger. A suitable buffer for substitution is preferably a buffer having a pH of 5-8 and a salt concentration of 0.1-0.2M, and the type of buffer is not limited, and for example, 0.02M phosphoric acid, 0.18M sodium chloride, and pH7.3 can be used. As a method for obtaining a pass-through fraction, it is sufficient if the fraction is appropriately obtained while confirming that the fraction contains the soluble thrombomodulin and does not substantially contain a denatured product of the soluble thrombomodulin by an analysis method such as size exclusion chromatography described in example 7 described later.

In addition, isocratic elution conditions may also be used. In this case, the gradient elution condition and the condition under which soluble thrombomodulin is not eluted in the pass-through fraction may be referred to set appropriate conditions.

Hydroxyapatite can be equilibrated with, for example, Macro-Prep (R) Ceramic HydroxyPatiteTYPE 1 (BIO-RAD)) using a buffer having a phosphate concentration of 8mM or less, preferably 5mM or less, more preferably 2mM or less (the type of buffer is not limited, and examples thereof include 5mM phosphate, 0.2M sodium chloride, pH7, 1mM phosphate, 25mM TRIS, 0.2M sodium chloride, pH7.7, 2mM phosphate, 20mM HEPES, 0.17M sodium chloride, and pH7) in a volume of several columns before the supply of the denatured product-containing material. The buffer containing the denatured product is supplied using a buffer having a phosphate concentration of 8mM or less, preferably 5mM or less, more preferably 2mM or less at pH 6-9, and after equilibration, the denatured product-containing product is supplied, and before elution of the soluble thrombomodulin, the solution can be washed with a buffer having a phosphate concentration of 8mM or less, preferably 5mM or less, more preferably 2mM or less at pH 6-9 (the type of the buffer is not limited, and examples thereof include 5mM phosphate, 0.2M sodium chloride, pH7, 1mM phosphate, 25mM TRIS, 0.2M sodium chloride, pH7.7, or 2mM phosphate, 20mM HEPES, 0.17M sodium chloride, pH 7). Gradient elution of the adsorbed denatured product-containing material is carried out using a buffer solution having a pH of 6 to 9, preferably 7 to 8, at a phosphate concentration of 0 to 0.5M, thereby separating and eluting the soluble thrombomodulin and the denatured product of the soluble thrombomodulin. The separation conditions are not particularly limited as long as they are separation conditions capable of separating soluble thrombomodulin from a denatured product of soluble thrombomodulin, and examples of the gradient elution conditions include linear or stepwise. The phosphate concentration of the gradient is preferably 1 to 40mM, more preferably, the gradient is performed in stages, and an elution fraction containing soluble thrombomodulin substantially free of a denatured product of the soluble thrombomodulin can be obtained at a phosphate concentration of 8 to 10 mM. By further increasing the concentration of phosphate, a denatured product of soluble thrombomodulin is eluted.

When gradient elution conditions are used, the method for obtaining an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin may be the same as the method for separating soluble thrombomodulin and a denatured product of the soluble thrombomodulin by an anion exchanger.

In addition, soluble thrombomodulin substantially free of denatured product of thrombomodulin may also be obtained as a flow-through fraction by replacing the denatured product-containing material with an appropriate buffer solution before supplying the denatured product-containing material. The pass-through fraction includes a fraction in which soluble thrombomodulin is not adsorbed to hydroxyapatite after the soluble thrombomodulin-containing material is supplied to hydroxyapatite. The difference in adsorption force to the column between the soluble thrombomodulin and a denatured product of the soluble thrombomodulin is utilized. In this case, for example, Macro-Prep (R) Ceramic Hydroxycalcium TYPE1 (BIO-RAD) can be used as the HydroxyaPatite. A suitable buffer for substitution is a buffer having a pH of 6 to 9 and a phosphate concentration of 5 to 20mM (preferably a pH of 6 to 7 and a phosphate concentration of 5 to 10mM), and the type of buffer is not limited, and for example, a buffer having a pH of 10mM phosphoric acid, 10mM sodium chloride, or 7 can be used. As a method for obtaining a pass-through fraction, it is sufficient if the fraction can be appropriately obtained while confirming that the fraction contains the soluble thrombomodulin and does not substantially contain a denatured product of the soluble thrombomodulin by an analysis method such as size exclusion chromatography described in example 7 described later.

In the production method of the present invention, it is preferable that the method does not include a step of adjusting the pH of an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of soluble thrombomodulin to 4 or less after the elution fraction is obtained by supplying the soluble thrombomodulin-containing material to an anion exchanger or hydroxyapatite. It is also preferable that the pH is not adjusted to 4 or less in the subsequent concentration step and/or desalting step for preparing the soluble thrombomodulin into a pharmaceutical material.

As a preferred method for purifying soluble thrombomodulin from the prepared culture supernatant by culturing cells capable of producing soluble thrombomodulin using a serum component-containing medium or a serum-free medium, there can be mentioned a purification method using, as an index, the activity of thrombomodulin, for example, the activity of promoting the activation of protein C by thrombin, and examples thereof include the following purification methods: the culture supernatant was roughly purified by Q-Sepharose FF of an ion exchange column, the fraction having thrombomodulin activity was recovered, then, the fraction having strong thrombomodulin activity was purified by an anti-mouse thrombomodulin monoclonal antibody of an affinity column, the recovered fraction was purified by SP-Sepharose FF of a cation exchange column, the flow-through fraction was concentrated, and the fraction having thrombomodulin activity was obtained by gel filtration. After purification by the SP-Sepharose FF, a high-purity thrombomodulin substantially free of a denatured product of soluble thrombomodulin can be easily obtained by using an anion exchanger of the present invention, preferably a strong anion exchanger, that is, an anion exchanger having a quaternary ammonium group (for example, SOURCEQ (GE healthcare Bio-Sciences Co., Ltd.) or hydroxyapatite of the present invention (for example, Macro-Prep (R)) under optimum conditions.

Further, by purifying with an anti-mouse thrombomodulin monoclonal antibody and then using an anion exchanger of the present invention, preferably an anion exchanger having a quaternary ammonium group (for example, SOURCEQ (GE Healthcare Bio-Sciences)) under optimum conditions, a denatured product of soluble thrombomodulin, a mouse antibody derivative, and a serum derivative in the case of using a serum-containing medium can be simultaneously removed, and a high-purity thrombomodulin substantially not containing a denatured product of soluble thrombomodulin can be easily obtained.

Further, a highly purified product in which the buffer solution is exchanged can be obtained by a concentration step and/or a desalting step without adjusting the pH of the highly purified thrombomodulin obtained by the above-mentioned methods to 4 or less. The soluble thrombomodulin obtained by the above-mentioned method can be used as a raw material for pharmaceuticals.

Further, the present invention provides a method for purifying soluble thrombomodulin, which comprises subjecting the purified soluble thrombomodulin to a purification treatment in which a denatured product of the soluble thrombomodulin that can be produced from the soluble thrombomodulin under an acidic condition is substantially not contained. The purification method is not particularly limited as long as it comprises the following steps: (1) supplying an anion exchanger or hydroxyapatite with the soluble thrombomodulin-containing material containing or suspected of containing a denatured product of the soluble thrombomodulin; and (2) obtaining a fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin under separation conditions that allow the soluble thrombomodulin to be separated from the denatured product of the soluble thrombomodulin. The purification method may be a purification method having the characteristics of the method for producing soluble thrombomodulin of the present invention.

Examples

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.

Example 1 crude purification Using a Strong anion exchanger column

According to Japanese patent application laid-open No. 11-341990, a DNA encoding the amino acid sequence of SEQ ID NO. 9 is prepared by gene manipulation techniques, incorporated into Chinese hamster ovary cells by a transgene, and the incorporated Chinese hamster ovary cells are cultured to obtain a culture supernatant. The obtained 411 culture supernatant was filtered through a 0.2 μm membrane filter (Millipore, Millipak 20). The filtered culture supernatant was supplied to a column (diameter 44cm, height 26.3cm) of Q-Sepharose (GE Healthcare Bio-Sciences) equilibrated with 20mM Tris salt buffer (pH7.4) containing 150mM sodium chloride. Then, the column was washed with 6 column volumes of 20mM acetic acid buffer containing 180mM sodium chloride, and further washed with 8 column volumes of 20mM Tris salt buffer (pH7.4) containing 180mM sodium chloride, and elution was started with 20mM Tris salt buffer (pH7.4) containing 300mM sodium chloride, whereby an eluate of 0.5 column volume capacity rising from the peak of absorbance at 280nm of the eluate was obtained as a roughly purified product. The flow rate was 760 mL/min.

Example 2 purification Using monoclonal antibody

According to Japanese patent application laid-open No. 11-341990, an anti-thrombomodulin monoclonal antibody using human lung-derived thrombomodulin as an antigen was prepared, and subjected to a contact reaction with CNBr-activated Sepharose (cyanogen bromide activated Sepharose) 4FastFlow (GE Healthcare Bio-Sciences Co.) to couple an anti-thrombomodulin monoclonal, thereby preparing an anti-thrombomodulin monoclonal-bound Sepharose 4 FastFlow. 16.51 of the eluted fractions obtained in example 1 were supplied to a monoclonal antibody column (diameter 44cm, height 10.5cm) equilibrated with 20mM phosphate buffer (pH7.3) containing 0.3M sodium chloride. The column was washed with 20mM phosphate buffer (pH7.3) containing 1.0M sodium chloride flowing through 6 column volumes, and further washed with 0.1M acetate buffer (pH5.0) flowing through 3 column volumes, and elution was started with 0.1M glycine hydrochloride buffer (pH3.0) containing 0.3M sodium chloride to obtain an eluate in which the peak of the absorbance at 280nm of the eluate increased to decreased, and 0.5M phosphate buffer (pH7.3) containing 1/10 in the volume of the eluate was added to the eluate to obtain a purified solution. The flow rate was 760 mL/min.

Example 3 purification Using Strong cation exchanger column

193ml of the purified solution obtained in example 2 was adjusted to pH3.5 with 1.0M glycine hydrochloride buffer (pH2.0), and the adjusted solution was supplied to a column (diameter 16mm, height 12cm) of SP-Sepharose FF (GE healthcare Bio-Sciences) equilibrated with 0.1M glycine hydrochloride buffer (pH3.5) containing 0.3M NaCl. Washing was started with 100mM glycine hydrochloride buffer (pH3.5) containing 300mM NaCl to obtain a flow-through fraction in which the peak of absorbance at 280nm decreased from the rise, and the flow-through fraction was immediately neutralized to pH7 with 0.5M phosphate buffer (pH7.3) to obtain a highly purified product. The flow rate was 3.3 mL/min. The content of soluble thrombomodulin in the purified product of high purity was found to be 99% or more by chromatography using AsahiPak C4P-50 or ELISA described in Japanese patent application laid-open No. 11-341990.

Example 4 separation of denatured product of soluble thrombomodulin by anion exchanger Linear gradient elution (1)

10ml of the purified product of high purity obtained in example 3 was diluted with 30ml of purified water. The diluted 40ml solution was supplied to a sourreQ 30(GE Healthcare Bio-Sciences) column (diameter 0.5cm, height 9.8cm) equilibrated with 20mM phosphate buffer (pH6) containing 0.1M sodium chloride. The column was washed with 3 column volumes of 20mM phosphate buffer (pH6), elution was started under a linear gradient of 0.1M to 0.3M sodium chloride in 20mM phosphate buffer (pH6) and with an elution volume of 20 column volumes, and separation was carried out in units of 1 column volume from the increase in the peak absorbance at 280nm of the eluate. The flow rate for sample addition and washing was set to 2.2 mL/min, and the flow rate for elution was set to 0.3 mL/min. The chromatogram is shown in FIG. 1.

Example 5 separation of denatured product of soluble thrombomodulin by anion exchanger Linear gradient elution (2)

10ml of the purified product of high purity obtained in example 3 was diluted with 30ml of purified water. The diluted 40ml solution was supplied to a sourreQ 30(GE Healthcare Bio-Sciences) column (diameter 0.5cm, height 9.8cm) equilibrated with 20mM phosphate buffer (pH7) containing 0.1M sodium chloride. The column was washed with 3 column volumes of 20mM phosphate buffer (pH7), elution was started under a linear gradient of 0.1M to 0.3M sodium chloride in 20mM phosphate buffer (pH7) and with an elution volume of 20 column volumes, and separation was carried out in units of 1 column volume from the increase in the peak absorbance at 280nm of the eluate. The flow rate for sample addition and washing was set to 2.2 mL/min, and the flow rate for elution was set to 0.3 mL/min. The chromatogram is shown in FIG. 2.

Example 6 separation of denatured product of soluble thrombomodulin by anion exchanger Linear gradient elution (3)

10ml of the purified product of high purity obtained in example 3 was diluted with 30ml of purified water. The diluted 40ml solution was supplied to a sourreQ 30(GE Healthcare Bio-Sciences) column (diameter 0.5cm, height 9.8cm) equilibrated with 20mM Tris buffer (pH8) containing 0.1M sodium chloride. The column was washed with 3 column volumes of 20mM Tris buffer (pH8), elution was started with a linear gradient of 0.1M to 0.3M sodium chloride in 20mM Tris buffer (pH8) and an elution volume of 20 column volumes, and separation was performed in units of 1 column volume from the increase in the peak absorbance at 280nm of the eluate. The flow rate for sample addition and washing was set to 2.2 mL/min, and the flow rate for elution was set to 0.3 mL/min. The chromatogram is shown in FIG. 3.

Example 7 evaluation of denatured product content of soluble thrombomodulin in examples 4 to 6

Each fraction was evaluated for the content of a denatured product of soluble thrombomodulin by size exclusion chromatography using TSKgelG3000SWXL (7.8 mmI.D.. times.30 cm; TOSOH). The column was equilibrated with a mobile phase, and the mobile phase was analyzed by using 50mM phosphate buffer (pH7.3) containing 0.1M sodium sulfate at a flow rate of 0.9 ml/min and supplying 150. mu.l of each fraction. The recovery of each fraction was calculated by measuring the absorbance at 280 nm. The results of examples 4 to 6 are shown in Table 1 below. The content of a denatured product of soluble thrombomodulin in the purified product of high purity obtained in example 3 was also measured by the above-mentioned method, and the measurement result was 7.0% (FIG. 4). Under any of the conditions of examples 4 to 6, a high-purity soluble thrombomodulin substantially not containing a denatured product of the soluble thrombomodulin can be obtained at a recovery rate of 76% to 81%.

[ Table 1]

Example 8 separation of denatured product of soluble thrombomodulin by anion exchanger stepwise gradient elution

70ml of the purified product of high purity obtained in example 3 was diluted with 210ml of purified water. The diluted solution was supplied to a sourceQ30(GE Healthcare Bio-Sciences) column (diameter 0.5cm, height 20.5cm) equilibrated with 20mM phosphate buffer (pH7.3) containing 30mM sodium chloride. The column was washed with 3 column volumes of 20mM phosphate buffer (pH7.3) containing 30mM sodium chloride, and elution was started with 20mM phosphate buffer (pH7.3) containing 0.18M sodium chloride, to obtain an eluate in which the peak of absorbance at 280nm of the eluate increased to 6 column volume volumes. The content of the denatured product of soluble thrombomodulin in the eluate was evaluated by size exclusion chromatography using TSKgelG3000SWXL (TOSOH Co.) analysis under the conditions of example 7, and as a result, the content ratio of the denatured product of soluble thrombomodulin was 0.0%, and a high-purity soluble thrombomodulin substantially not containing the denatured product of soluble thrombomodulin was obtained. The recovery of the eluent was 89%. The flow rate at the time of sample addition and washing was 0.8 mL/min, and the flow rate at the time of elution was 0.4 mL/min. The chromatogram is shown in FIG. 5.

Example 9 preparation and transgenesis of human-derived thrombomodulin Gene by Gene manipulation technique and serum-free culture of transfer-incorporated Chinese hamster ovary cells to obtain a culture supernatant containing soluble thrombomodulin

Two types of culture media, a seed culture medium and a perfusion culture medium, were prepared for a serum-free medium containing no animal components, which was used for obtaining a culture supernatant containing soluble thrombomodulin.

The seed medium was prepared by sterile mixing of 1L of IS CHO-CD (liquid Medium, catalog No. 91119-1L, manufacturer Irvine Scientific) with 5mL of HT supplement (liquid, catalog No. 11067-030, manufacturer Invitrogen) and 40mL of L-Glutamine 200mM (liquid, catalog No. 25030-081, manufacturer Invitrogen). The seed culture medium is stored in a refrigerated state (2-8 ℃), and is heated in a constant-temperature water bath at 36 ℃ immediately before use.

The culture medium for perfusion culture was prepared by adding and dissolving 20.8g of ISCHO-CD-A3 (powder culture medium, catalog No. 98688, manufacturer Irvine Scientific), 2.6g of common salt (grade: special grade, manufacturer: Wako Junyaku) and 4.4g of sodium bicarbonate common salt (grade: special grade, manufacturer: Wako Junyaku) in 1L of deionized ultrafiltration water, and aseptically filtering the mixture through a 0.2 μm filter (PVDF, manufacturer: Millipore). The perfusion culture medium is refrigerated for storage (2-8 degrees) during storage and use.

At the start of seed culture, cryopreserved cells capable of producing soluble thrombomodulin (Japanese patent application laid-open No. H11-341990) were rapidly thawed in a 37 ℃ constant temperature water bath, suspended in a seed medium, centrifuged (2000rpm, 2 minutes, domestic chemistry) to remove the centrifugal supernatant, and then the cell pellet was suspended in 100mL of seed medium and dispensed into 225cm²In a T-flask culture vessel (manufacturer: BD Biosciences), seed culture of paragraph 1 was started. The seed culture of the 1 st stage was performed by static culture in a 36 ℃ 5% carbon dioxide incubator. The density of the living planktonic cells in the 1 st seed culture reaches about 8 x 10⁵cells/mL, subculture in the 2 nd stage seed culture.

The seed culture of the 2 nd stage is followed by agitation culture. The amount of subculture solution was successively increased in the scale of seed culture in the agitation culture and in the agitation culture vessel in such a manner that the 2 nd stage seed culture was 400mL (glass spinner flask, manufacturer: Kai-tian-science), the 3 rd stage seed culture was 1.6L (glass spinner flask, manufacturer: Kai-science), and the 4 th stage seed culture was 6L (glass sphere spinner flask, manufacturer: Kai-science). The density of the seed cultured floating living cells reaches about 8 x 10⁵cells/mL, subculture was performed in the next seed culture. In the subculture, the same amount of medium for seed culture as the value obtained by subtracting the culture scale before subculture from the culture scale after subculture was added to perform expansion. The agitation culture was carried out in a 36-degree, 5% carbon dioxide incubator using a magnetic stirrer (manufacturer: Takeda scientific) at an agitation speed of 60 to 100 rpm.

The density of the living planktonic cells in the 4 th seed culture reaches about 8 x 10⁵At cells/mL, the seed culture solution was transferred to 3 perfusion culture tanks (2L perfusion culture apparatus, manufacturer: Mitsubishi organism) and 2L of each was transferred to start perfusion culture.

The quantitative transfer of the perfusion culture medium supplied to the perfusion culture tank in perfusion culture was performed at a flow rate of 2L/day using a peristaltic pump (manufacturer: Masterflex).

Cell separation in perfusion culture is performed by using a spin filter provided in a perfusion culture tank. Different rotary filters were used for each of the 3 perfusion culture tanks. The first rotary filter was made of a stainless steel mesh (FP-10, pore size 10 μm, manufacturer: Fuji Filter industry), the second rotary filter was made of a stainless steel mesh (FP-30, pore size 30 μm, manufacturer: Fuji Filter industry), and the third rotary filter was made of a polyester PETP mesh (pore size 10 μm, catalog No. BB-8808571, manufacturer: Sartorius).

When the amount of culture medium is 2L or more, culture supernatant separated from cells by a rotary filter provided in the perfusion culture tank is intermittently discharged by the action of a liquid level sensor that displays the liquid level of 2L in the perfusion culture tank. The culture supernatant discharged intermittently is transferred to a container for cold storage and is stored at a temperature of 2 to 8 ℃.

For perfusion culture conditions, the temperature in the tank is controlled to be 35-37 ℃, the dissolved oxygen concentration is 10-90%, and the pH is 6.8-7.6.

The perfusion culture was carried out for 30 days, during which the average viable cell density was 17X 10 of that of the first stage⁶cells/mL, second stage 10X 10⁶cells/mL, third stage 18X 10⁶cells/mL。

Culture supernatants obtained from the day three to the day 30 of perfusion culture were mixed in 3 perfusion culture vessels, filtered through a 0.2 μm filter (manufactured by PVDF, manufacturer: Millipore), and the filtered culture supernatants were stored in a refrigerated storage (2 to 8 degrees).

Example 10 purification Using Strong anion exchanger column

850ml of the culture supernatant obtained in example 9 was supplied to a Q-Sepharose FF (GEHealthcare Bio-Sciences) column (diameter: 1.6cm, height: 29cm) equilibrated with 20mM sodium acetate containing 30mM sodium chloride and 120mM acetic acid buffer. Subsequently, the column was washed with 12 column volumes of 20mM sodium acetate containing 30mM sodium chloride and 120mM acetic acid buffer (pH3.8), elution was started with 140mM sodium chloride and 20mM sodium acetate containing 40mM acetic acid buffer (pH4.2), and 1/5 column volume of 1M HEPES buffer (pH8) containing 10mM potassium phosphate was added to the eluate of 2 column volume rising from the main peak of the absorbance at 280nm of the eluate to obtain a purified product. The flow rate was 2 mL/min.

Example 11 separation of denatured product of soluble thrombomodulin by hydroxyapatite stepwise gradient elution

20ml of the purified product obtained in example 10 was supplied to a Macro-Prep (R) Ceramic HydroxyPatite TYPE1 (BIO-RAD) column (diameter: 0.5cm, height: 9.7cm) equilibrated with 20mM HEPES buffer (pH7) containing 0.17M sodium chloride and 2mM sodium phosphate. The column was washed with a 20mM HEPES buffer (pH7) containing 0.17M sodium chloride and 2mM sodium phosphate in a volume of 6 columns, and eluted with a phosphate buffer (pH7) containing 10mM sodium chloride in a volume of 4 columns to give an eluate in which the peak of absorbance at 280nm of the eluate increased to decreased. The flow rate was 0.4 mL/min. The eluate was evaluated for the denatured product content of soluble thrombomodulin by size exclusion chromatography using TSKgelG3000SWXL (TOSOH Co.) analysis under the conditions of example 7, and as a result, the denatured product content ratio of soluble thrombomodulin was 0.8% or less. The column was regenerated with 0.5M phosphate buffer (pH 7). The chromatogram is shown in FIG. 6.

Example 12 Rough purification Using Strong anion exchanger column (2)

5.2l of the culture supernatant obtained in example 9 was supplied to a CaptoQ (GE Healthcare Bio-Sciences) column (diameter 1.6cm, height 26cm) equilibrated with 20mM Tris salt buffer (pH7.7) containing 150mM sodium chloride. Next, washing was carried out with 15 column volumes of 20mM Tris salt buffer (pH7.7) containing 0.18M sodium chloride, and elution was started with 20mM Tris salt buffer (pH7.7) containing 0.3M sodium chloride, to obtain an eluate of 2 column volume rising from the peak of absorbance at 280nm of the eluate as a roughly purified product. The flow rate was set at 10 mL/min.

Example 13 purification with monoclonal antibody (2)

Monoclonal antibodies were prepared according to patent document 2. 72mL of the eluted fraction obtained in example 12 was supplied to a monoclonal antibody column (diameter 5cm, height 11cm) for equilibration with 20mM phosphate buffer (pH7.3) containing 0.3M sodium chloride. A purified solution was obtained by washing with a 20mM phosphate buffer (pH7.3) containing 1.0M sodium chloride flowing through 6 column volumes and a 0.1M acetate buffer (pH5.0) flowing through 3 column volumes, starting elution with a 0.1M glycine hydrochloride buffer (pH3.0) containing 20mM sodium chloride, to obtain an eluate in which the peak of the absorbance at 280nm of the eluate increased to decreased, and adding a 0.3M phosphate buffer (pH7.3) having a volume capacity of 15% of the eluate to the eluate. The flow rate was set to 9.8 mL/min. The purified solution was found to have a soluble thrombomodulin content of 99% or more by chromatography using AsahiPak C4P-50, ELISA described in Japanese patent application laid-open No. 11-341990, or the like.

Example 14 purification of a protein Using a Strong anion exchanger and removal of denatured product of soluble thrombomodulin

75mL of the purified solution obtained in example 13 was supplied to a sourceQ30(GE Healthcare Bio-Sciences) column (diameter: 0.5cm, height: 20.5cm) equilibrated with a 0.1M acetic acid buffer solution (pH4) containing 40mM sodium chloride. The eluate was passed through 2 column volumes of a 0.1M acetate buffer (pH4) containing 40mM sodium chloride, further passed through 4 column volumes of a 0.1M acetate buffer (pH4) containing 50mM sodium chloride, and further washed with 3 column volumes of a 20mM phosphate buffer (pH7.3) containing 30mM sodium chloride, and the elution was started with a 20mM phosphate buffer (pH7.3) containing 0.18M sodium chloride, to obtain 6 column volumes of eluate from the rise of the peak of the absorbance at 280nm of the eluate. The flow rate at the time of sample addition was 1 mL/min, and the flow rate at the time of washing and elution was 0.3 mL/min. The content of the denatured product of soluble thrombomodulin in the eluate was evaluated by size exclusion chromatography using TSKgelG3000SWXL (TOSOH Co.) analysis under the conditions of example 7, and as a result, the content ratio of the denatured product of soluble thrombomodulin was 0.1%, and a high-purity soluble thrombomodulin substantially not containing the denatured product of soluble thrombomodulin was obtained. The recovery of the eluent was 71%. The chromatogram is shown in FIG. 7.

Example 15 evaluation of the amount of Mixed heterologous proteins (mouse antibody derivative and cell derivative of Chinese hamster ovary cell)

The elution fractions of examples 13 to 14 were evaluated by the ELISA method described in Japanese patent application laid-open No. 11-341990. The absorbance at 280nm of each elution fraction was measured, and the mixing amount was evaluated with the absorbance of 1, and the evaluation results are shown in Table 2 below. The mixing amounts of the respective components can be reduced at the same time.

[ Table 2]

Sequence listing

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Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn

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Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu

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Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val

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Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg

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Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr

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Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro

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Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys

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Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro

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Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys

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Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala

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Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys

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Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly

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Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln

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His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys

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Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr

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Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro

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Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr

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Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp

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atgcttgggg tcctggtcct tggcgcgctg gccctggccg gcctggggtt ccccgcaccc 60

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gaccagccgg gctcctactc gtgcatgtgc gagaccggct accggctggc ggccgaccaa 960

caccggtgcg aggacgtgga tgactgcata ctggagccca gtccgtgtcc gcagcgctgt 1020

gtcaacacac agggtggctt cgagtgccac tgctacccta actacgacct ggtggacggc 1080

gagtgtgtgg agcccgtgga cccgtgcttc agagccaact gcgagtacca gtgccagccc 1140

ctgaaccaaa ctagctacct ctgcgtctgc gccgagggct tcgcgcccat tccccacgag 1200

ccgcacaggt gccagatgtt ttgcaaccag actgcctgtc cagccgactg cgaccccaac 1260

acccaggcta gctgtgagtg ccctgaaggc tacatcctgg acgacggttt catctgcacg 1320

gacatcgacg agtgcgaaaa cggcggcttc tgctccgggg tgtgccacaa cctccccggt 1380

accttcgagt gcatctgcgg gcccgactcg gcccttgtcc gccacattgg caccgactgt 1440

<210>7

<211>480

<212>PRT

<213>human

<400>7

Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly Leu Gly

1 5 10 15

Phe Pro Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu

20 25 30

His Asp Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala

35 40 45

Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser

50 55 60

Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly

65 70 75 80

Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys

85 90 95

Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr

100 105 110

Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn

115 120 125

Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu

130 135 140

Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val

145 150 155 160

Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg

165 170 175

Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr

180 185 190

Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro

195 200 205

Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys

210 215 220

Thr Ala Pro Pro Gly Ala ValGln Gly His Trp Ala Arg Glu Ala Pro

225 230 235 240

Gly Ala Trp Asp Cys Ser ValGlu Asn Gly Gly Cys Glu His Ala Cys

245 250 255

Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala

260 265 270

Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys

275 280 285

Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly

290 295 300

Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln

305 310 315 320

His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys

325 330 335

Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr

340 345 350

Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro

355 360 365

Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr

370 375 380

Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu

385 390 395 400

Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp

405 410 415

Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile

420 425 430

Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly

435 440 445

Gly Phe Cys Ser Gly ValCys His Asn Leu Pro Gly Thr Phe Glu Cys

450 455 460

Ile Cys Gly Pro Asp Ser Ala Leu Ala Arg His Ile Gly Thr Asp Cys

465 470 475 480

<210>8

<211>1440

<212>DNA

<213>human

<400>8

atgcttgggg tcctggtcct tggcgcgctg gccctggccg gcctggggtt ccccgcaccc 60

gcagagccgc agccgggtgg cagccagtgc gtcgagcacg actgcttcgc gctctacccg 120

ggccccgcga ccttcctcaa tgccagtcag atctgcgacg gactgcgggg ccacctaatg 180

acagtgcgct cctcggtggc tgccgatgtc atttccttgc tactgaacgg cgacggcggc 240

gttggccgcc ggcgcctctg gatcggcctg cagctgccac ccggctgcgg cgaccccaag 300

cgcctcgggc ccctgcgcgg cttccagtgg gttacgggag acaacaacac cagctatagc 360

aggtgggcac ggctcgacct caatggggct cccctctgcg gcccgttgtg cgtcgctgtc 420

tccgctgctg aggccactgt gcccagcgag ccgatctggg aggagcagca gtgcgaagtg 480

aaggccgatg gcttcctctg cgagttccac ttcccagcca cctgcaggcc actggctgtg 540

gagcccggcg ccgcggctgc cgccgtctcg atcacctacg gcaccccgtt cgcggcccgc 600

ggagcggact tccaggcgct gccggtgggc agctccgccg cggtggctcc cctcggctta 660

cagctaatgt gcaccgcgcc gcccggagcg gtccaggggc actgggccag ggaggcgccg 720

ggcgcttggg actgcagcgt ggagaacggc ggctgcgagc acgcgtgcaa tgcgatccct 780

ggggctcccc gctgccagtg cccagccggc gccgccctgc aggcagacgg gcgctcctgc 840

accgcatccg cgacgcagtc ctgcaacgac ctctgcgagc acttctgcgt tcccaacccc 900

gaccagccgg gctcctactc gtgcatgtgc gagaccggct accggctggc ggccgaccaa 960

caccggtgcg aggacgtgga tgactgcata ctggagccca gtccgtgtcc gcagcgctgt 1020

gtcaacacac agggtggctt cgagtgccac tgctacccta actacgacct ggtggacggc 1080

gagtgtgtgg agcccgtgga cccgtgcttc agagccaact gcgagtacca gtgccagccc 1140

ctgaaccaaa ctagctacct ctgcgtctgc gccgagggct tcgcgcccat tccccacgag 1200

ccgcacaggt gccagatgtt ttgcaaccag actgcctgtc cagccgactg cgaccccaac 1260

acccaggcta gctgtgagtg ccctgaaggc tacatcctgg acgacggttt catctgcacg 1320

gacatcgacg agtgcgaaaa cggcggcttc tgctccgggg tgtgccacaa cctccccggt 1380

accttcgagt gcatctgcgg gcccgactcg gcccttgccc gccacattgg caccgactgt 1440

<210>9

<211>516

<212>PRT

<213>human

<400>9

Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly Leu Gly

1 5 10 15

Phe Pro Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu

20 25 30

His Asp Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala

35 40 45

Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser

50 55 60

Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly

65 70 75 80

Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys

85 90 95

Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr

100 105 110

Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn

115 120 125

Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu

130 135 140

Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val

145 150 155 160

Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg

165 170 175

Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr

180 185 190

Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro

195 200 205

Val Gly Ser Ser Ala Ala ValAla Pro Leu Gly Leu Gln Leu Met Cys

210 215 220

Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro

225 230 235 240

Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys

245 250 255

Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala

260 265 270

Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys

275 280 285

Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly

290 295 300

Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln

305 310 315 320

His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys

325 330 335

Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr

340 345 350

Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro

355 360 365

Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr

370 375 380

Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu

385 390 395 400

Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp

405 410 415

Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile

420 425 430

Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly

435 440 445

Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys

450 455 460

Ile Cys Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys

465 470 475 480

Asp Ser Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro

485 490 495

Pro Ser Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala ValGly Leu

500 505 510

Val His Ser Gly

515

<210>10

<211>1548

<212>DNA

<213>human

<400>10

atgcttgggg tcctggtcct tggcgcgctg gccctggccg gcctggggtt ccccgcaccc 60

gcagagccgc agccgggtgg cagccagtgc gtcgagcacg actgcttcgc gctctacccg 120

ggccccgcga ccttcctcaa tgccagtcag atctgcgacg gactgcgggg ccacctaatg 180

acagtgcgct cctcggtggc tgccgatgtc atttccttgc tactgaacgg cgacggcggc 240

gttggccgcc ggcgcctctg gatcggcctg cagctgccac ccggctgcgg cgaccccaag 300

cgcctcgggc ccctgcgcgg cttccagtgg gttacgggag acaacaacac cagctatagc 360

aggtgggcac ggctcgacct caatggggct cccctctgcg gcccgttgtg cgtcgctgtc 420

tccgctgctg aggccactgt gcccagcgag ccgatctggg aggagcagca gtgcgaagtg 480

aaggccgatg gcttcctctg cgagttccac ttcccagcca cctgcaggcc actggctgtg 540

gagcccggcg ccgcggctgc cgccgtctcg atcacctacg gcaccccgtt cgcggcccgc 600

ggagcggact tccaggcgct gccggtgggc agctccgccg cggtggctcc cctcggctta 660

cagctaatgt gcaccgcgcc gcccggagcg gtccaggggc actgggccag ggaggcgccg 720

ggcgcttggg actgcagcgt ggagaacggc ggctgcgagc acgcgtgcaa tgcgatccct 780

ggggctcccc gctgccagtg cccagccggc gccgccctgc aggcagacgg gcgctcctgc 840

accgcatccg cgacgcagtc ctgcaacgac ctctgcgagc acttctgcgt tcccaacccc 900

gaccagccgg gctcctactc gtgcatgtgc gagaccggct accggctggc ggccgaccaa 960

caccggtgcg aggacgtgga tgactgcata ctggagccca gtccgtgtcc gcagcgctgt 1020

gtcaacacac agggtggctt cgagtgccac tgctacccta actacgacct ggtggacggc 1080

gagtgtgtgg agcccgtgga cccgtgcttc agagccaact gcgagtacca gtgccagccc 1140

ctgaaccaaa ctagctacct ctgcgtctgc gccgagggct tcgcgcccat tccccacgag 1200

ccgcacaggt gccagatgtt ttgcaaccag actgcctgtc cagccgactg cgaccccaac 1260

acccaggcta gctgtgagtg ccctgaaggc tacatcctgg acgacggttt catctgcacg 1320

gacatcgacg agtgcgaaaa cggcggcttc tgctccgggg tgtgccacaa cctccccggt 1380

accttcgagt gcatctgcgg gcccgactcg gcccttgtcc gccacattgg caccgactgt 1440

gactccggca aggtggacgg tggcgacagc ggctctggcg agcccccgcc cagcccgacg 1500

cccggctcca ccttgactcc tccggccgtg gggctcgtgc attcgggc 1548

<210>11

<211>516

<212>PRT

<213>human

<400>11

Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly Leu Gly

1 5 10 15

Phe Pro Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu

20 25 30

His Asp Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala

35 40 45

Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser

50 55 60

Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly

65 70 75 80

Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys

85 90 95

Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr

100 105 110

Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn

115 120 125

Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu

130 135 140

Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val

145 150 155 160

Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg

165 170 175

Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr

180 185 190

Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro

195 200 205

Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys

210 215 220

Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro

225 230 235 240

Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys

245 250 255

Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala

260 265 270

Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys

275 280 285

Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly

290 295 300

Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln

305 310 315 320

His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys

325 330 335

Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr

340 345 350

Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro

355 360 365

Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr

370 375 380

Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu

385 390 395 400

Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp

405 410 415

Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile

420 425 430

Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly

435 440 445

Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys

450 455 460

Ile Cys Gly Pro Asp Ser Ala Leu Ala Arg His Ile Gly Thr Asp Cys

465 470 475 480

Asp Ser Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro

485 490 495

Pro Ser Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu

500 505 510

Val His Ser Gly

515

<210>12

<211>1548

<212>DNA

<213>human

<400>12

atgcttgggg tcctggtcct tggcgcgctg gccctggccg gcctggggtt ccccgcaccc 60

gcagagccgc agccgggtgg cagccagtgc gtcgagcacg actgcttcgc gctctacccg 120

ggccccgcga ccttcctcaa tgccagtcag atctgcgacg gactgcgggg ccacctaatg 180

acagtgcgct cctcggtggc tgccgatgtc atttccttgc tactgaacgg cgacggcggc 240

gttggccgcc ggcgcctctg gatcggcctg cagctgccac ccggctgcgg cgaccccaag 300

cgcctcgggc ccctgcgcgg cttccagtgg gttacgggag acaacaacac cagctatagc 360

aggtgggcac ggctcgacct caatggggct cccctctgcg gcccgttgtg cgtcgctgtc 420

tccgctgctg aggccactgt gcccagcgag ccgatctggg aggagcagca gtgcgaagtg 480

aaggccgatg gcttcctctg cgagttccac ttcccagcca cctgcaggcc actggctgtg 540

gagcccggcg ccgcggctgc cgccgtctcg atcacctacg gcaccccgtt cgcggcccgc 600

ggagcggact tccaggcgct gccggtgggc agctccgccg cggtggctcc cctcggctta 660

cagctaatgt gcaccgcgcc gcccggagcg gtccaggggc actgggccag ggaggcgccg 720

ggcgcttggg actgcagcgt ggagaacggc ggctgcgagc acgcgtgcaa tgcgatccct 780

ggggctcccc gctgccagtg cccagccggc gccgccctgc aggcagacgg gcgctcctgc 840

accgcatccg cgacgcagtc ctgcaacgac ctctgcgagc acttctgcgt tcccaacccc 900

gaccagccgg gctcctactc gtgcatgtgc gagaccggct accggctggc ggccgaccaa 960

caccggtgcg aggacgtgga tgactgcata ctggagccca gtccgtgtcc gcagcgctgt 1020

gtcaacacac agggtggctt cgagtgccac tgctacccta actacgacct ggtggacggc 1080

gagtgtgtgg agcccgtgga cccgtgcttc agagccaact gcgagtacca gtgccagccc 1140

ctgaaccaaa ctagctacct ctgcgtctgc gccgagggct tcgcgcccat tccccacgag 1200

ccgcacaggt gccagatgtt ttgcaaccag actgcctgtc cagccgactg cgaccccaac 1260

acccaggcta gctgtgagtg ccctgaaggc tacatcctgg acgacggttt catctgcacg 1320

gacatcgacg agtgcgaaaa cggcggcttc tgctccgggg tgtgccacaa cctccccggt 1380

accttcgagt gcatctgcgg gcccgactcg gcccttgccc gccacattgg caccgactgt 1440

gactccggca aggtggacgg tggcgacagc ggctctggcg agcccccgcc cagcccgacg 1500

cccggctcca ccttgactcc tccggccgtg gggctcgtgc attcgggc 1548

<210>13

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthesis of DNA

<400>13

aatgtggcgg gcaagggccg a 21

Claims

1. A method for producing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin that may be produced from the soluble thrombomodulin under acidic conditions, the method comprising the steps of:

(2) obtaining a fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin under separation conditions that allow the soluble thrombomodulin to be separated from the denatured product of the soluble thrombomodulin,

the term "substantially free" means that the content of a denatured product of soluble thrombomodulin is 3.0% or less.

2. The production method according to claim 1, wherein the content of soluble thrombomodulin is 80% or more relative to the total protein in the soluble thrombomodulin-containing material.

3. The production method according to claim 1, wherein the step (2) is a step of obtaining an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin by performing gradient elution that is linear or stepwise, or a combination of linear and stepwise.

4. The production method according to claim 1, wherein the step (2) is a step of obtaining a pass-through fraction, in which a fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin is obtained,

the pass-through fraction is a fraction in which soluble thrombomodulin is not adsorbed to an anion exchanger or hydroxyapatite after the soluble thrombomodulin-containing material is supplied to the anion exchanger or hydroxyapatite.

5. The production method according to claim 1, wherein the step (2) is a step of obtaining an eluted fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin by performing isocratic elution.

6. The production method according to claim 1, wherein in the step (1), the soluble thrombomodulin-containing material is supplied to the anion exchanger using a buffer solution having a pH of 4 to 9; performing a gradient elution of linear or stepwise, or a combination of linear and stepwise elution using a buffer solution having a pH of 5 to 9 and a salt concentration of 0M to 1M in the step (2), (a) confirming in advance the position of an elution fraction of soluble thrombomodulin to obtain an elution fraction of soluble thrombomodulin that contains substantially no denatured product of the soluble thrombomodulin; or (b) obtaining an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin, while confirming the presence or absence of the soluble thrombomodulin in the elution fraction.

7. The production method according to claim 1, wherein in the step (1), the soluble thrombomodulin-containing material is supplied to the anion exchanger using a buffer solution having a pH of 5 to 8 and a salt concentration of 0.1M to 0.2M; in the step (2), a flow-through fraction is obtained by using a buffer solution having a pH of 5 to 8 and a salt concentration of 0.1M to 0.2M, thereby obtaining a fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin,

8. The production method according to claim 1, wherein in the step (1), a soluble thrombomodulin-containing material is supplied to hydroxyapatite using a buffer solution having a pH of 6 to 9 and a phosphate concentration of 8mM or less; performing a gradient elution of linear or stepwise or a combination of linear and stepwise elution in step (2) using a buffer solution having a pH of 6 to 9 and a phosphate concentration of 0M to 0.5M, (a) confirming in advance the position of an elution fraction of soluble thrombomodulin to obtain an elution fraction of soluble thrombomodulin that contains substantially no denatured product of the soluble thrombomodulin; or (b) obtaining an elution fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin, while confirming the presence or absence of the soluble thrombomodulin in the elution fraction.

9. The production method according to claim 1, wherein in the step (1), the soluble thrombomodulin-containing material is supplied to hydroxyapatite using a buffer solution having a pH of 6 to 9 and a phosphate concentration of 5mM to 20 mM; in the step (2), a flow-through fraction is obtained by using a buffer solution having a pH of 6 to 9 and a phosphate concentration of 5mM to 20mM, thereby obtaining a fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin,

10. The production method according to any one of claims 1 to 9, which does not include a step of adjusting the pH of an eluted fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin to 4 or less.

11. The production method according to any one of claims 1 to 9, which comprises a concentration step and/or a desalting step for not lowering the pH of an eluted fraction containing soluble thrombomodulin that does not substantially contain a denatured product of the soluble thrombomodulin to 4 or less, and which is used for preparing the soluble thrombomodulin into a pharmaceutical material.

12. The production method according to any one of claims 1 to 9, wherein the content of a denatured product of soluble thrombomodulin in soluble thrombomodulin that does not substantially contain the denatured product of soluble thrombomodulin is 3% or less.

13. The method according to any one of claims 1 to 9, wherein the soluble thrombomodulin is obtained from a transformed cell prepared by transfecting a host cell with a DNA encoding the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 11.