CN101111511A

CN101111511A - Process for protein isolation

Info

Publication number: CN101111511A
Application number: CNA200580026695XA
Authority: CN
Inventors: A·利赫姆
Original assignee: AVT PLASMA Ltd
Current assignee: AVT PLASMA Ltd
Priority date: 2004-06-07
Filing date: 2005-06-06
Publication date: 2008-01-23
Also published as: ZA200700167B

Abstract

In one aspect, the present invention is directed to a process for isolating proteins from a solution comprising proteins, wherein the solution is selected from the group consisting of: crude bood plasma, blood serum, cryosupernatant derived from plasma, fractionated human plasma, cryoprecipitate derived from plasma and recombinant broths. The process involves providing a solid separation medium having the formula: M-S-L wherein M is a matrix backbone, S is an optional spacer arm, and L is a ligand which is mercaptonicotinic acid, contacting the solid separation medium with the solution comprising the proteins, such that at least one of the proteins becomes reversibly bound bound to said solid separation medium. At least one elution step is then performed to selectively elute at least one protein fraction from the solid separation medium. In another aspect, the present invention is directed to a process for isolating Factor VIII and/or Factor IX.

Description

The method of protein separation

Technical field

The present invention relates to from the biogenic method of separating protein.More specifically, the present invention relates to method of separating protein from blood.

Background technology

Blood plasma is one of starting material of nature most worthy, and the protein of purifying is that the life and health of the many individualities in the whole world is necessary from blood plasma.This most of protein is wherein produced by Cohn cold ethanol precipitation fractional separation method.This method by Edwin doctor Cohn of Harvard University in the early development forties of 20th century.Cohn finds can be according to the protein in the proteinic precipitation feature separated plasma under the different condition (pH, ionic strength, protein concn, temperature and alcohol concn).By changing these parameters, different proteins is precipitated out in the substep mode.The Cohn technology has been used for blood plasma industry many decades, but there are some limitation in this method aspect purity and the efficient.Although this method can be produced the product of certain quality, on the purity of the handiness of step and the product of producing, there is limitation.The commercial the most common protein that still uses this method to produce comprises albumin, immunoglobulin (Ig), Antithrombin III, zymoplasm and fibrinogen.

The one class technical progress of producing blood plasma product comprises that the protein molecule selective adsorption was to fixed solid surface (solid phase) when the use of chromatography, the protein in this chromatographic separation blood plasma were based on liquid (moving phase) and penetrate the sealing post that contains stationary phase.According to absorption or interactional efficient, the motion of different proteins is delayed to some extent, and this makes it realize separating and collecting at the bottom of post.

Chromatography is the means of the more gentle isolated protein of the more traditional cold Ethanol Method of Cohn in essence, and the time that the cold Ethanol Method of Cohn makes protein be exposed in the high concentration ethanol is longer.This will make protein denaturation and produce unwanted aggregation, and this transfers that the patient who accepts these products is had unfavorable treatment consequence.On the contrary, the chromatography fractional separation can effectively be removed impurity, and does not have influence on proteinic natural structure.With extensive repeatedly precipitated phase ratio related in the cold alcohol grading partition method of Cohn, chromatography is a kind of more direct separation method, and it can improve the proteinic amount that every liter of blood plasma is produced.

Adopted up to now chromatography makes the productive rate optimization keep simultaneously or improve aspect the necessary purity level still challenging.Still need effectively and the chromatographic technique of high performance-price ratio, this chromatographic technique with respect to existing method with higher purity and output selective elution plasma component.

The sixties in 20th century is early stage, it should be noted that when low temperature is hatched blood plasma, can be observed throw out and has formed.Confirmed that this throw out contains Factor IX, the Von Willebrand factor and many other plasma proteinss.Initial this cryoprecipitate is used to treat the haemophilia A patient, but needs to improve the tolerance of patient to this product, can adopt the Factor IX of the higher form of purity.The cryoprecipitate method remains the primary basis of the Factor IX production of industry use now.Unfortunately, this method efficient is not high, although and carried out multiple trial and used blood plasma but not the recovery of cryoprecipitate raising Factor IX by direct, this wherein seldom has trial in commercial achieving success.

Isolate factors IX is used for the treatment of the haemophilia B patient, and it is from supernatant liquor I---secondary flow branch of perfect Cohn blood plasma fractional separation method---.In basic all existing preparation methods, factors IX among the supernatant liquor I adopts the affinity chromatography step to use heparin sepharose (Heparin Sepharose) to be further purified usually, but because entire method is to be separated into the basis with the Cohn lease making, so on preparation efficiency, still exist significant limitations.

In this case, the more efficiently method that still needs from the mixture that contains Factor IX and/or factors IX separation factor VIII and/or factors IX.

Summary of the invention

According to first aspect, the invention provides a kind of described method of protein that from contain proteinic solution, separates, described solution is selected from rough blood plasma, serum, derives from the human plasma of the cold supernatant liquor of blood plasma, fractional separation, derives from the cryoprecipitate of blood plasma and the meat soup (recombinantbroth) of recombinating, and described method comprises:

(i) provide solid separating medium with following formula:

M-S-L

Wherein M is a matrix scaffold, and S is optional spacerarm, and L is a part sulfydryl nicotinic acid;

(ii) described solid separating medium is contacted with described solution, be attached to at least a so described protein reversible on the described solid separating medium;

(iii) carry out at least one-step elution with at least a protein flow point of selective elution from this solid separating medium.

Following feature relates to a first aspect of the present invention.

In one embodiment, protein be mammalian source or the people source.

Spacerarm S can derive from compound (as butanediol diglycidyl ether (butane dioldiglycidylether) or Epicholorohydrin) or other the suitable coupling agent that is used for covalently bound part known in the art that contains epoxy group(ing).

Matrix scaffold M can be resin, and this solid separating medium is the resin that works with part thus.In addition, this resin can be part can connected any resin.Further, this resin can be the high-density resin that is suitable for such as the non-packed bed absorption of fluidized-bed absorption and expanded bed adsorption, for example based on highly cross-linked microballoon sample agarose derivative, agarose-wolfram varbide condensation product, agarose stainless steel condensation product, agarose-quartzy condensation product, porous ceramics microballoon, the porous zirconia microballoon of 6% or 4% agarose, control microballoon that hole glass makes with and the hole in contain the porous inorganic material complex microsphere of organic polymer.

In one embodiment, this part can be 2-sulfydryl nicotinic acid (2-mercaptonicotinicacid).

Because protein is reversible with combining of part, this protein can separate under following suitable elution requirement with in the solid separating medium.

The solid separating medium can comprise high-density resin, this resin mean particle size is between about 10 microns to about 150 microns, microballoon density is between about 1.5g/ml to 15g/ml, perhaps, mean particle size is between about 10 microns to about 120 microns, microballoon density is between about 2.0g/ml to 15g/ml, perhaps, mean particle size is between about 15 microns to about 100 microns, microballoon density is between about 2.3g/ml to 15g/ml, perhaps, mean particle size is between about 15 microns to about 80 microns, and microballoon density is between about 3g/ml to 15g/ml.

Can be selected from for example IgG by the isolating protein of the method for first aspect, IgA, IgM, the immunoglobulin (Ig) of IgD or IgE, Transferrins,iron complexes (Tf), the fibrinogen or derivatives thereof, the plasma proteins enzyme inhibitors is antithrombin such as Antithrombin III for example, blood is urged solidifying egg white (bloodpro-coagulation protein), the solid albumen of blood anticoagulant, cytokine, somatomedin, the albumin or derivatives thereof, thrombolytic agent, angiogenesis inhibitor albumen, the Regular Insulin or derivatives thereof, α-1-proteinase inhibitor or derivatives thereof is α-1-antitrypsin for example, α-2-antiplasmin or derivatives thereof, C-1 esterase inhibitor, lipophorin, HDL, fibronectin or derivatives thereof, β-2-glycoprotein I, Profibrinolysin, plasmin, plasminogen activator, Profibrinolysin inhibitor, the urokinase or derivatives thereof, the streptokinase or derivatives thereof, inter-, α-2-macroglobulin, amyloid, Xue Qingleiniandanbai, ferritin, prealbumin, the GC-sphaeroprotein, blood clotting galactenzyme (haemopexin) and complement C3.

The protein flow point can contain a kind of protein.Perhaps, the protein flow point can contain multiple proteins.

This method can comprise 1 to 50,1 to 30,1 to 20,1 to 10,1 to 5 step wash-outs, 1 to 3 step or one-step elution.

In one embodiment, various elutriants can have the pH between about 4.0 to about 9.0.Perhaps, various elutriants can have the pH between about 4.0 to 8.5.Perhaps, various elutriants can have the pH between about 4.0 to about 8.0.In addition, various elutriants can have the pH between about 5.0 to about 8.0.In addition, various elutriants can have the pH between about 4.5 to 8.0.In addition, various elutriants can have the pH between about 5.5 to about 8.0.Further, various elutriants can have the pH between about 6.0 to 8.0.

Various elutriants can have between about 0.00005 Siemens/cm (S/cm) to the ionic strength between about 10.0S/cm.Perhaps, various elutriants can have between about 0.0005S/cm to the ionic strength between about 10.0S/cm.Perhaps, various elutriants can have between about 0.0001S/cm to the ionic strength between about 6.0S/cm.In addition, various elutriants can have between about 0.001S/cm to the ionic strength between about 5.5S/cm.Further, various elutriants can have between about 0.001S/cm to the ionic strength between about 5.0S/cm.Further again, various elutriants can have between about 0.005S/cm to the ionic strength between about 5.0S/cm.Further again, various elutriants can have between about 0.01S/cm to the ionic strength between about 4.0S/cm.

Various elutriants can have the interior pH of above-mentioned scope and the arbitrary combination of ionic strength value.

First kind of elutriant, second kind of elutriant or the third elutriant can have the pH between about 4.0 to about 9.0, and have between about 0.00005S/cm to the ionic strength between about 10.0S/cm.First kind of elutriant can have the pH between about 4.0 to about 8.0, and has between about 0.00005S/cm to the ionic strength between about 0.1S/cm.In addition, first kind of elutriant can have the pH between about 4.5 to about 6.5, and has between about 0.00005S/cm to the ionic strength between about 0.075S/cm.In addition, first kind of elutriant can have the pH between about 5.0 to about 6.0, and has between about 0.001S/cm to the ionic strength between about 0.05S/cm.

Second kind of elutriant can have the pH between about 5.0 to about 7.0, and has between about 0.0001S/cm to the ionic strength between about 0.1S/cm.Perhaps, second kind of elutriant can have the pH between about 5.5 to about 6.5, and has between about 0.0001S/cm to the ionic strength between about 0.075S/cm.In addition, second kind of elutriant can have the pH between about 5.5 to about 6.5, and has between about 0.001S/cm to the ionic strength between about 0.05S/cm.

The third elutriant can have the pH between about 5.0 to about 9.0, and has between about 0.0001S/cm to the ionic strength between about 4.0S/cm.Perhaps, the third elutriant can have the pH between about 6.0 to about 8.0, and has between about 0.01S/cm to the ionic strength between about 3.0S/cm.In addition, the third elutriant can have the pH between about 6.0 to about 8.0, and has between about 0.05S/cm to the ionic strength between about 2.0S/cm.

First kind of elutriant can be the elutriant that can not cause proteinic biological function forfeiture, the inorganic salt solution of demineralized water or one or more inorganic acids for example, described salt is hydrochloride, vitriol and nitrate for example, as sodium-chlor, Repone K, ammonium chloride, sodium sulfate, vitriolate of tartar and ammonium sulfate.Perhaps, first kind of elutriant can be the aqueous buffer solution that contains inorganic acid salt and/or organic acid salt, this inorganic acid salt and/or organic acid salt can not cause proteinic biological function forfeiture, for example contain Citrate trianion, acetate, succinate, lactic acid salt, tartrate, formate, propionic salt, phosphoric acid salt or boratory damping fluid.

Second kind of elutriant can be the aqueous buffer solution that contains inorganic acid salt and/or organic acid salt, this inorganic acid salt and/or organic acid salt can not cause proteinic biological function forfeiture, for example contain Citrate trianion, acetate, succinate, lactic acid salt, tartrate, formate, propionic salt, phosphoric acid salt or boratory damping fluid.In one embodiment, second elutriant contain have non-aromatic, the electronegative molecule (for example medium chain is to chain alkyl carboxylic acid and alkylsulphonic acid) and the electronegative washing composition (for example sodium lauryl sulphate and Sodium desoxycholate) of aromatic series or heteroaromatic hydrophobic part.For example, second kind of elutriant can comprise the salt that one or more are selected from following acid: caproic acid, enanthic acid, sad, n-nonanoic acid (perlagonic acid) and capric acid, undeeanoic acid, laurostearic acid, tridecylic acid, TETRADECONIC ACID and pentadecylic acid, with and unsaturated derivative and alkyl-substituted derivative.Perhaps, second kind of elutriant can comprise the salt that one or more are selected from following acid: own sulfonic acid, hot sulfonic acid, the last of the ten Heavenly stems sulfonic acid, ten disulfonic acid, hexane vitriol, octane vitriol, decane vitriol and dodecyl sulphate.

The third elutriant can be the aqueous buffer solution that contains inorganic acid salt and/or organic acid salt, this inorganic acid salt and/or organic acid salt can not cause proteinic biologic activity forfeiture, for example contain Citrate trianion, acetate, succinate, lactic acid salt, tartrate, formate, propionic salt, phosphoric acid salt or boratory damping fluid.Perhaps the third elutriant can contain lyophobicity (lyotrophobicity) higher relatively inorganic salt or organic salt, for example ammonium sulfate, sodium sulfate, vitriolate of tartar, ammonium phosphate, sodium phosphate, potassiumphosphate, ammonium citrate, Trisodium Citrate, Tripotassium Citrate.

The method of first aspect can comprise with the 4th kind of elutriant wash-out solid separating medium with selective elution the 4th protein flow point.The 4th kind of elutriant can have the pH between about 5.0 to about 9.0, and has between about 0.01S/cm to the ionic strength between about 2S/cm.Perhaps, the 4th kind of elutriant can have the pH between about 6.0 to about 8.0, and has between about 0.01S/cm to the ionic strength between about 1.0S/cm.In addition, the 4th kind of elutriant can have the pH between about 6.0 to about 8.0, and has between about 0.05S/cm to the ionic strength between about 1.0S/cm.The 4th kind of elutriant can be and contain the inorganic acid salt compatible with protein to be separated and/or the aqueous buffer solution of organic acid salt, for example contains Citrate trianion, acetate, succinate, lactic acid salt, tartrate, formate, propionic salt, phosphoric acid salt or boratory damping fluid.

The 4th kind of elutriant also can contain the particularly aqueous solution of the inorganic salt of inorganic acid of one or more mineral acids, this salt can not cause the forfeiture of proteinic biological function, for example hydrochloride, vitriol and nitrate are as sodium-chlor, Repone K, ammonium chloride, sodium sulfate, vitriolate of tartar, ammonium sulfate.

The first protein flow point can comprise one or more following albumin, Xue Qingleiniandanbai, prealbumin, α-1-proteinase inhibitor (protein of α-1-PI), Transferrins,iron complexes and fibrinogen that is selected from.

The second protein flow point can comprise one or more following for example protein of Antithrombin III, albumin, immunoglobulin (Ig), Transferrins,iron complexes and fibrinogen of antithrombin that is selected from.

The protein iii flow point can comprise one or more following for example protein of IgA, IgD, IgE, IgG and/or IgM, Transferrins,iron complexes and fibrinogen of immunoglobulin (Ig) that is selected from.

The 4th protein flow point can comprise one or more following Transferrins,iron complexes, the immunoglobulin (Ig) of α-2-macroglobulin, for example IgM and protein of fibrinogen of being selected from.

According to second aspect, the invention provides a kind of from the solution that contains Factor IX and/or factors IX the method for separation factor VIII and/or factors IX, described solution is selected from: the human plasma of rough blood plasma, serum, the cold supernatant liquor that derives from blood plasma, fractional separation, the cryoprecipitate that derives from blood plasma and reorganization meat soup, and described method comprises:

(i) provide solid separating medium with following formula:

M-S-L

Wherein M is that matrix scaffold, S are optional spacerarm, and L is the part benzene dimethylamine;

(ii) described solid separating medium is contacted with described solution, Factor IX and/or factors IX will reversibly be attached on the described solid separating medium so at least;

(iii) carry out the first step wash-out with wash-out unbinding protein from this solid separating medium;

(iv) carry out the second step wash-out with wash-out Factor IX and/or factors IX from this solid separating medium.

M can be the high-density resin that is suitable for such as the non-packed bed absorption of fluidized-bed absorption and expanded bed adsorption, for example based on highly cross-linked microballoon sample agarose derivative, agarose-wolfram varbide condensation product, agarose stainless steel condensation product, agarose-quartzy condensation product, porous ceramics microballoon, the porous zirconia microballoon of 6% or 4% agarose, control microballoon that hole glass makes with and the hole in contain the porous inorganic material complex microsphere of organic polymer.

This high-density resin has between about 10 microns to about 300 microns, or the mean particle size between about 15 microns to about 150 microns and between about 1.1g/ml to 15g/ml, or the microballoon density between about 1.5g/ml to 15g/ml, perhaps has mean particle size between about 10 microns to about 120 microns and the microballoon density between about 2.0g/ml to 15g/ml, perhaps have mean particle size between about 15 microns to about 100 microns and the microballoon density between about 2.3g/ml to 15g/ml, perhaps have median size between about 15 microns to about 80 microns and the microballoon density between about 3g/ml to 15g/ml.

Spacerarm S can derive from compound (as butanediol diglycidyl ether or Epicholorohydrin) or other the suitable coupling agent that is used for covalently bound part known in the art that contains epoxy group(ing).

In one embodiment, L can be m-xylene diamine.Perhaps L can be the p dimethylamine.L also can be adjacent dimethylamine.

Unbinding protein can be selected from but be not limited to: IgG, IgA, IgM, IgD or IgE, Transferrins,iron complexes (Tf), fibrinogen or derivatives thereof, the plasma proteins enzyme inhibitors is antithrombin such as Antithrombin III for example, and blood is urged solidifying egg white, the solid albumen of blood anticoagulant, cytokine, somatomedin, albumin or derivatives thereof, thrombolytic agent, angiogenesis inhibitor albumen, Regular Insulin or derivatives thereof, α-1-proteinase inhibitor or derivatives thereof is α-1-antitrypsin for example, α-2-antiplasmin or derivatives thereof, C-1 esterase inhibitor, lipophorin, HDL, fibronectin or derivatives thereof, β-2-glycoprotein I, Profibrinolysin, plasmin, plasminogen activator, Profibrinolysin inhibitor, the urokinase or derivatives thereof, the streptokinase or derivatives thereof, inter-, α-2-macroglobulin, amyloid, Xue Qingleiniandanbai, ferritin, prealbumin, the GC-sphaeroprotein, blood clotting galactenzyme and complement C3.

The elutriant that uses can have the pH between about 5.0 to about 9.0 in the elution step.This elutriant can have between about 0.0001S/cm to the conductivity between about 10.0S/cm.This elutriant can have the pH between about 6.0 to about 9.0, and has between about 0.03S/cm to the conductivity between about 0.2S/cm.

Elutriant can have approximately identical pH and different on ionic strength.For example, various elutriants can comprise different buffer systems, and/or randomly comprise other salt, and for example hydrochloride, vitriol and nitrate are as sodium-chlor, Repone K, ammonium chloride, sodium sulfate, vitriolate of tartar, ammonium sulfate.

Various elutriants can have the interior pH of above-mentioned scope and the arbitrary combination of ionic strength value.This elutriant can contain the aqueous buffer solution of inorganic acid salt or organic acid salt, this inorganic acid salt and/or organic acid salt can not cause the biological function forfeiture of Factor IX and/or factors IX, for example contain Citrate trianion, acetate, succinate, lactic acid salt, tartrate, formate, propionic salt, phosphoric acid salt or boratory damping fluid.This elutriant also can contain the aqueous solution of the inorganic salt of one or more inorganic acids, this salt can not cause that proteinic biological function loses, for example hydrochloride, vitriol and nitrate are as sodium-chlor, Repone K, ammonium chloride, sodium sulfate, vitriolate of tartar, ammonium sulfate.

First kind of elutriant and second kind of elutriant can have the pH between about 6.0 to about 8.0, and have between about 0.0001S/cm to the ionic strength between about 1.0S/cm.Perhaps, first kind of elutriant can have the pH between about 5.5 to about 6.0, and has between about 0.0001S/cm to the ionic strength between about 0.01S/cm.

In addition, first kind of elutriant can have the pH between about 5.5 to about 6.0, and has between about 0.0001S/cm to the ionic strength between about 0.05S/cm.In addition, first kind of elutriant can have the pH between about 6.0 to about 6.5, and has between about 0.001S/cm to the ionic strength between about 0.1S/cm.

Second kind of elutriant can have the pH between about 6.0 to about 8.0, and has between about 0.0001S/cm to the ionic strength between about 0.1S/cm.Perhaps, second kind of elutriant can have the pH between about 6.0 to about 8.0, and has between about 0.0001S/cm to the ionic strength between about 0.5S/cm.In addition, second kind of elutriant can have the pH between about 7.0 to about 9.0, and has between about 0.001S/cm to the ionic strength between about 1S/cm.

By being controlled at the pH and the conductivity of the elutriant described in the top paragraph, this method can be used for separating Factor IX and the factors IX as mixture from the solution that contains Factor IX and factors IX and other material.Perhaps, when solution only contains Factor IX (for example solution be derive from the cryoprecipitate of blood plasma or wherein only Factor IX express reorganization meat soup the time), by controlling the pH and the conductivity of the elutriant described in the top paragraph, present method can be used for only separation factor VIII.When solution only contains factors IX (for example solution be derive from the cold supernatant liquor of blood plasma or wherein only factors IX express reorganization meat soup the time), by controlling the pH and the conductivity of the elutriant described in the top paragraph, present method can be used for only separation factor IX.

Description of drawings

Fig. 1 illustrates the method according to this invention, and being further purified of isolating protein flow point is shown.

Fig. 2 illustrates the product recovery rate of use according to the method for one embodiment of the invention.

The SDS-PAGE that Fig. 3 illustrates an embodiment according to a first aspect of the invention analyzes.

Fig. 4 illustrates from the unidirectional radioimmunodiffusion analysis of the flow point of an embodiment acquisition of a first aspect of the present invention.

Fig. 5 illustrates one embodiment of the invention, and wherein a first aspect of the present invention is used to separate some protein flow point continuously with second aspect.

The SDS-PAGE that Fig. 6 illustrates the gained flow point of Factor IX absorption analyzes.Swimming lane 1 illustrates rough blood plasma, and swimming lane 2 illustrates effluent (run-through) flow point, and swimming lane 3 illustrates the wash-out of Factor IX/factors IX.Factor IX/factors IX eluate is diluted with respect to elution volume so that directly compare with use blood plasma, can visual estimation productive rate.

Fig. 7 illustrates from the SDS-PAGE of the protein flow point of the method for protein isolation acquisition of first aspect and analyzes, and wherein the starting material that use (containing described proteinic solution) are the effluent flow point from the method acquisition of second aspect.Swimming lane 1 illustrates human plasma, and swimming lane 2 illustrates the effluent flow point, and (α-1-PI), swimming lane 3 illustrates wash-out 2 (albumin), and swimming lane 4 illustrates wash-out 3 (IgG), and swimming lane 5 illustrates wash-out 4 (fibrinogen).

Fig. 8 illustrates from the unidirectional radioimmunodiffusion analysis of the flow point of the method for protein isolation acquisition of first aspect, and wherein the starting material that use (containing described proteinic solution) are the effluent flow point of the method acquisition of second aspect.Flow point 1 to 4 is represented effluent flow point, wash-out 2, wash-out 3 and wash-out 4 respectively.Fig. 8 A illustrates albuminous quantitative, and Fig. 8 B illustrates the quantitative of IgG.Last two row of Fig. 8 A and 8B illustrate the typical curve (twice mensuration) of rough blood plasma 100-20%.Following two row of A and B illustrate following material: 1: the effluent flow point; 2: wash-out 2; 3: wash-out 3; 4: wash-out 4.

Definition

The below is some definition that help to understand description of the invention.

In the context of the present specification, term " comprises " and means " mainly comprise but needn't for only comprising ". In addition, the version that " comprises " of word for example " comprises " and " containing " has the corresponding implication that changes to some extent.

In the context of the present invention, term " elution step ", " wash-out " or " carrying out wash-out " are used interchangeably, be intended to refer to obtain to comprise one or more may be in conjunction with or not in conjunction with and the step of the protein flow point of the protein that separates with the solid separating medium subsequently.

In the context of the present specification, term " washing step " is intended to refer to use the step of liquid wash solid separating medium, and this step can not discharge any protein substantially from the solid separating medium.

In the context of the present specification, term " equilibrium step " is intended to refer to so step, wherein make sufficient flow of solution cross the solid separating medium, so about identical with inflow liquid (incoming solution) of gegenion concentration, conductibility and the pH of efflux (outgoing solution).

In the context of the present specification, term " restructuring meat soup " refers to the soluble protein by the cells in vitro expression of carrying out genetic manipulation. Protein by these cellular expressions of being handled in the restructuring meat soup comprises: solidify for example factor VII of pathway protein, Factor IX, factors IX or FXIII, immunoglobulin (Ig) is IgG for example, IgA, IgM, IgD or IgE, transferrins (Tf), the fibrinogen or derivatives thereof, the plasma protein enzyme inhibitor is antithrombase such as Antithrombin III for example, α l-protease inhibitors, blood is urged coagulated protein, the solid albumen of blood anticoagulant, cell factor, growth factor, the albumin or derivatives thereof, thrombolytic agent, anti-angiogenic generation albumen, the insulin or derivatives thereof, α-1-protease inhibitors or derivatives thereof is α-1-antitrypsin for example, α-2-antiplasmin or derivatives thereof, C-1 esterase inhibitor, apolipoprotein, HDL, fibronectin or derivatives thereof, β-2-glycoprotein I, plasminogen, fibrinolysin, activator of plasminogen, plasminogen inhibitor, the urokinase or derivatives thereof, the streptokinase or derivatives thereof, inter-α-trypsin inhibitor, α-2-macroglobulin, amyloid, acid seromucoid, ferritin, prealbumin, the GC-globulin, blood clotting galactenzyme and complement C3.

In the context of the present specification, term " blood plasma " refers to the liquid part of blood, is a kind of composite solution that contains 90% above water. The main solute of blood plasma is one group of heterogeneous protein. Other plasma fraction comprises fatty material (lipid), inorganic electrolyte, glucose, amino acid, vitamin, hormone and metabolic waste.

In the context of the present specification, term " serum " refers to remove the blood plasma of fibrinogen in the blood clotting process.

In the context of the present specification, term " cold supernatant " and " cryoprecipitate " should be understood according to following description: cold supernatant is the solution that is made by blood plasma after removing cryoprecipitate. Cryoprecipitate is by forming by plasma exposure temperature between 1 ℃ to 10 ℃ is precipitated the protein that obtains by blood plasma.

In the context of the present specification, term " human plasma that classification separates " is interpreted as referring to any component of blood plasma or the mixture of component, and they derive from carries out separating the blood plasma of processing such as precipitation, filtration, chromatography etc.

In the context of the present specification, term " effluent " is interpreted as referring to the protein flow point that obtains from a second aspect of the present invention, the eluate that obtains when comprising the plasma solutions upper prop, this eluate mix with the wash-out phase that obtains from first step wash-out.

Embodiment

Protein separation

A first aspect of the present invention relates to method of separating protein from biological solution.More specifically, the present invention relates to from human plasma, the cold supernatant liquor that derives from human plasma, fractional separation human plasma, derive from separation of human method of protein the cryoprecipitate of human plasma, human serum or reorganization meat soup.

Contain proteinic solution with the suitable liquid diluting of available another kind before described solid separating medium contacts.For example, blood plasma can be by the aqueous solution dilution of the inorganic salt of demineralized water or one or more inorganic acids compatible with protein to be separated, described salt is hydrochloride, vitriol and nitrate for example, as sodium-chlor, Repone K, ammonium chloride, sodium sulfate, vitriolate of tartar, ammonium sulfate.

Contain proteinic solution can with another kind of suitable liquid with from about 1000: 1 to about 1: 1000 dilution proportion.For example, Dilution ratio can be about 1000: 1, and about 750: 1, about 500: 1, about 250: 1, about 100: 1, about 50: 1, about 25: 1, about 10: 1, about 7.5: 1, about 5: 1, about 3: 1, about 2.5: 1, about 2: 1, about 1: 1, about 1: 2, about 1: 2.5, about 1: 3, about 1: 5, about 1: 7.5, about 1: 10, about 1: 25, about 1: 50, about 1: 100, about 1: 250, about 1: 500, about 1: 750, or about 1: 1000.

Contain proteinic solution pH can with liquid with regulate before the solid separating medium contacts.Perhaps, pH can proteinaceous solution with regulate after the solid separating medium contacts.PH or also can reduce pH can raise.Perhaps, pH is remained unchanged.PH can be adjusted to the pH between about 3.0 to about 6.0, perhaps, can be the pH between about 4.5 to about 6.0 with pH regulator.

For example, can be about pH 3.0,3.5,4.0,4.5,5.0,5.5,6.0 with pH regulator.PH can use suitable acid to regulate, and described acid is hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, succsinic acid, acetic acid etc. for example.

PH can use suitable alkali to regulate, and described alkali is carbonate, supercarbonate, ammonia, oxyhydroxide etc. for example.PH can regulate with suitable buffer system.Buffer system is known for those skilled in the art, comprise for example Citrate trianion, acetate, phosphoric acid salt, formate, succinate, MES, ADA, 1, two ((trishydroxymethyl) methylamino) propane (bis-tris propane) of 3-, PIPES, ACES, imidazoles, MOPS, TES, HEPES, HEPPS, TRICINE, hydrochloric acid G-NH2, TRIS, BICINE, glycylglycine, boric acid, CHES, CAPS.Many buffer systems are commercially available, can obtain from for example Sigma Chemical company.Those skilled in the art can determine suitable buffer system according to required pH.

Elutriant can be different on pH.Elution step can comprise with the elutriant that pH raises gradually or pH reduces gradually handles the solid separating medium.

Elutriant can have approximately identical pH, but can be different on ionic strength.For example, each elutriant can comprise different buffer systems, and/or optional other salt that comprises, and for example hydrochloride, vitriol and nitrate are as sodium-chlor, Repone K, ammonium chloride, sodium sulfate, vitriolate of tartar, ammonium sulfate.

Every kind of proteinic productive rate of institute's wash-out can be at least 50% of proteinic amount in the starting material, or at least 60%, or at least 70%, or at least 80%, or at least 90%.

Elution step can be carried out under the temperature between 0 ℃ to 40 ℃.For example, temperature can be about 5 ℃, and about 10 ℃, about 15 ℃, about 20 ℃, about 25 ℃, about 30 ℃, about 35 ℃ or about 40 ℃.

The method of first aspect can be chosen wantonly and comprise a step or multistep washing.Washing step can carry out in any stage of the method for first aspect, for example the solid separating medium with contain before proteinic solution contacts, the solid separating medium with contain after proteinic solution contacts, or between per step wash-out.Available any solution that can not cause the forfeiture of proteinic biological function for example water, salt solution or buffered soln for example citrate buffer carry out washing step.

Washing step can carry out after with second kind of elutriant wash-out solid separating medium.Lavation buffer solution can comprise higher relatively inorganic salt of lyophobicity or organic salt, for example ammonium sulfate, sodium sulfate, vitriolate of tartar, ammonium phosphate, sodium phosphate, potassiumphosphate, ammonium citrate, Trisodium Citrate, Tripotassium Citrate.The ionic strength of lavation buffer solution can be 0.001S/cm at least, or 0.01S/cm at least, or 0.1S/cm or further 1.0S/cm at least at least.

The method of first aspect also can comprise a step or a multistep balance.Equilibrium step can contain the solid separating medium proteinic solution and carrying out before contacting with described.Equilibrium step can comprise water or suitable buffered soln is handled described solid separating medium to regulate the pH and the ionic strength of solid separating medium.The solution that is used for equilibrium step can be the aqueous solution of the inorganic salt of demineralized water or one or more inorganic acids compatible with protein to be separated, described salt is hydrochloride, vitriol and nitrate for example, as sodium-chlor, Repone K, ammonium chloride, sodium sulfate, vitriolate of tartar, ammonium sulfate.Perhaps, level pad can be and contains the inorganic acid salt compatible with protein to be separated and/or the aqueous buffer solution of organic acid salt, for example contains Citrate trianion, acetate, succinate, lactic acid salt, tartrate, formate, propionic salt, phosphoric acid salt or boratory damping fluid.

Equilibrium step can carry out under the temperature between about 10 ℃ to 40 ℃.For example, temperature can be about 10 ℃, and about 15 ℃, about 20 ℃, about 25 ℃, about 30 ℃ or about 40 ℃.

Contain proteinic solution pH can be used for described solid separating medium with contain proteinic described solution contact before the pH of buffered soln of balance solid separating medium identical.Contain proteinic solution pH can be used for described solid separating medium with contain proteinic described solution contact before the pH of buffered soln of balance solid separating medium different.

The method of first aspect also can comprise a step or multistep regeneration.Regeneration step comprises with the suitable agent that residuals can be removed from the solid separating medium handles the solid separating medium.Regeneration step can be carried out after the first step wash-out is finished any time.Suitable regeneration reagent comprises alkali for example hydroxide solution such as sodium hydroxide or potassium hydroxide, peracid solutions or superoxol, the solution that contains reactive chlorine such as hypochlorite solutions, denaturing agent example hydrochloric acid guanidine, organic solvent such as ethanol.

According to the method for first aspect, in the chromatography column device that can pack into the solid separating medium suitable.This method can be carried out with the packed bed pattern in filling column.Perhaps, this method can be carried out with the expanded bed pattern in expanded bed adsorption (EBA) post.In the context of first aspect, in fact all human plasma protein fraction matter all can be adsorbed on the solid separating medium in the single post.The described continuous elution step of the application can be used for the elute protein flow point of selective elution enrichment specified protein then.

The EBA post is known in the art, and suitable column device and equipment, comprise the method that liquid is imported the expansion column, can buy from the GE Healthcare of Sweden, perhaps described in WO99/65586, WO 01/85329 and WO92/00799, their full content is included the application in by cross reference.

An embodiment of first aspect comprises to be selected the EBA post and the solid separating medium of appropriate amount is packed in the post.The amount of employed solid separating medium depends on the amount that contains proteinic solution that will use and the protein concn in this protein soln.When protein soln was human plasma or CPP, general 1 liter of solid separating medium was used for per 0.5 to 1.5 liter of blood plasma.If when protein soln was the reorganization fermenting broth, one liter of solid separating medium can be used for 1 liter of fermenting broth, is up to 1000 L fermenting broths.Can prove conclusively circulation (flow through) at the bottom of post is fluidized up to the solid separating medium.Suitable post linear rate of flow comprise scope 0.5 to 20cm/min or about 5cm/min to 15cm/min between flow velocity.The solid separating medium can use appropriate solution (for example water, aqueous electrolyte liquid or buffered soln) balance then, after this for example blood plasma, serum, cold supernatant liquor, dissolved cryoprecipitate or reorganization meat soup solution can be introduced at the bottom of the post.After solution is loaded on the solid separating medium, can choose wantonly and carry out other washing step.Can carry out the protein flow point of elution step then with selective elution enrichment specified protein.

Being eluted in separately of per step is fit to carry out under the condition that selective elution comprises one or more proteinic protein flow points.For example, be used for the pH and/or the ionic strength of the elutriant of each elution step by change, but the wash-out different proteins.Can use suitable damping fluid that elutriant is adjusted to and have suitable pH and ionic strength.Ionic strength also can use salt to regulate.Elution step can comprise the elutriant processing solid separating medium that raises gradually with pH continuously.Perhaps elution step can comprise the elutriant processing solid separating medium that reduces gradually with pH continuously.

Another embodiment according to first aspect relates to the EBA post that foundation contains the agarose-wolfram varbide cohesion microballoon that works with 2-sulfydryl nicotinic acid.Agarose-tungsten carbide microspheres size distribution is between the 40-120 micron, and mean diameter is 70 microns.The density of microballoon is 2.9g/ml.Equilibrium step uses pH to be about 4.5 citrate buffer to carry out, following closely be to be about 5.0 the further equilibrated step of citrate buffer with pH.Then will be to transfer pH be 5.0 human plasma is applied to this post with the ratio of 1.5 liters of diluting plasmas of every liter of solid separating medium by 2 parts of water dilutions and with HCl.Use following this post of buffered soln wash-out then:

Wash-out 1. 10mM Trisodium Citrates, pH 5.0

Wash-out 2. 5g/ rise Sodium octoate and HCl, and pH 6.0

Wash-out 3. 1M Trisodium Citrates, pH 8.0

Wash-out 4. 20mM Trisodium Citrates and 0.1 M sodium-chlor, pH 8.0

The solid separating medium is regenerated with 1 M NaOH then.

The flow velocity of all operations is 7.5cm/ minute, and the amount of employed each solution provides at table 2.

The volume of the elutriant that uses generally depends on many correlative factors, for example:

(i) employed flow velocity in application of sample, washing, wash-out, regeneration and the equilibrium process;

The (ii) number of institute's wash-out product flow point;

The selection of (iii) per step elutriant that uses is because the selection of elutriant influences the output and the purity of each flow point;

The (iv) optimal spacing between each flow point, it is also influential to the output and the purity of acquisition product;

(the v) height of bed of solid separating medium and since the height of bed of solid separating medium when reducing the common wash volumes that consumes and elution volume reduce.

Table 2 illustrates the best liquor capacity in per step in the above-mentioned embodiment.

With reference to Fig. 3, from the visible protein of top embodiment: α-1 proteinase inhibitor, albumin, IgG and fibrinogen can effectively be separated.

Table 2

Step	Liquor capacity *
Step	Liquor capacity *	Balance	5.0CV
Elutriant		Balance	5.0CV
Elutriant	1	4.2CV
Elutriant	1	4.2CV
Elutriant	2	2.9CV
Elutriant	2	2.9CV
Elutriant	3	4.4CV
Elutriant	3	4.4CV
Elutriant	4	2.1CV
Regeneration	4	2.1CV	1.0CV
Regeneration	Amount to	19.6CV	1.0CV

^*CV is a column volume

Following table 3 illustrates by SRI and measures the protein yields with respect to the rough blood plasma of use that obtains.

Table 3

	Wash-out 1	Wash-out 2	Wash-out 3	Wash-out 4
	Wash-out 1	Wash-out 2	Wash-out 3	Wash-out 4	Albumin	＜5％	95+％
Immunoglobulin (Ig)			95+％	＜5％	Albumin	＜5％	95+％
Immunoglobulin (Ig)			95+％	＜5％	α-1 proteinase inhibitor	95+％
Fibrinogen			＜5％	95+％	α-1 proteinase inhibitor	95+％

For unidirectional radioimmunodiffusion (SRI) is carried out in the fractional yield of measuring range protein in the elutriant flow point 1 to 4.The SRI that Fig. 4 illustrates albumin, IgG, α-1-proteinase inhibitor and factor I analyzes.

Aforesaid method can be steadily, can reappear and uncomplicated mode is carried out.Find that all final eluates are clarified liq, any protein does not all have remarkable sex change/sedimentary sign.The sign that destruction, gathering or the immunoreactivity of wash-out product change is not seen in test behind the post of SDS-PAGE and above-mentioned SRI.

Those skilled in the art should know that the method for first aspect and second aspect can be by measuring the ultraviolet absorptivity monitoring of the liquid of discharging from post.The existence of protein and other UV absorbing material can detect in the process of carrying out method of the present invention and quantitatively, and can correctly collect the different proteins flow point.Equally, the continuous monitoring to pH, conductivity and specific refractory power can be used for record and controls method of the present invention.

The separation of Factor IX and/or factors IX

The solution that contains described Factor IX and/or factors IX is generally rough undiluted blood plasma, it directly can be contacted with the solid separating medium.Perhaps, rough blood plasma can with before described solid separating medium contacts with another kind of suitable liquid diluting.For example, blood plasma can be with the aqueous solution dilution of the inorganic salt of demineralized water or one or more inorganic acids compatible with Factor IX and/or factors IX, described salt is hydrochloride, vitriol and nitrate for example, for example sodium-chlor, Repone K, ammonium chloride, sodium sulfate, vitriolate of tartar, ammonium sulfate.

The solution that contains Factor IX and/or factors IX can be with another kind of suitable liquid with from about 1000: 1 to 1: 1000 dilution proportion.For example, Dilution ratio can be about 1000: 1, and about 750: 1, about 500: 1, about 250: 1, about 100: 1, about 50: 1, about 25: 1, about 10: 1, about 7.5: 1, about 5: 1, about 3: 1, about 2.5: 1, about 2: 1, about 1: 1, about 1: 2, about 1: 2.5, about 1: 3, about 1: 5, about 1: 7.5, about 1: 10, about 1: 25, about 1: 50, about 1: 100, about 1: 250, about 1: 500, about 1: 750 or about 1: 1000.

Contain Factor IX and/or factors IX solution pH can with regulate before the solid separating medium contacts.Perhaps, pH can the solution that contains Factor IX and/or factors IX with regulate after the solid separating medium contacts.PH or also can reduce pH can raise.Perhaps, pH is remained unchanged.PH can be transferred between about 5.0 to about 9.0.For example, pH can be adjusted to about pH of 5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5 or 9.0.Can use suitable acid for adjusting pH, described acid is hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, succsinic acid, acetic acid etc. for example.

PH can use suitable alkali to regulate described alkali such as carbonate, supercarbonate, ammonia, oxyhydroxide etc.PH can regulate with suitable buffer system.Buffer system is known for those skilled in the art, comprise for example Citrate trianion, acetate, phosphoric acid salt, formate, succinate, MES, ADA, 1, two ((trishydroxymethyl) methylamino) propane of 3-, PIPES, ACES, imidazoles, MOPS, TES, HEPES, HEPPS, TRICINE, hydrochloric acid G-NH2, TRIS, BICINE, glycylglycine, boric acid, CHES, CAPS.Many buffer systems are commercially available, can obtain from for example Sigma Chemical company.Those skilled in the art can determine suitable buffer system according to required pH.

This method can be chosen wantonly and comprise a step or multistep washing.Washing step can the solid separating medium with carry out before the solution that contains Factor IX and/or factors IX contacts, perhaps the solid separating medium with carry out after the solution that contains Factor IX and/or factors IX contacts.Any solution of the available biological function forfeiture that can not cause Factor IX and/or factors IX for example water, salt solution or buffered soln for example citrate buffer carry out washing step.

Perhaps, lavation buffer solution can comprise higher relatively inorganic salt of lyophobicity or organic salt, for example ammonium sulfate, sodium sulfate, vitriolate of tartar, ammonium phosphate, sodium phosphate, potassiumphosphate, ammonium citrate, Trisodium Citrate, Tripotassium Citrate.Lavation buffer solution can have the pH between about 5.0 to about 9.0.The pH of lavation buffer solution can be 5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,7.4,7.6,7.8,8.0,8.2,8.4,8.6,8.8 or 9.0.Lavation buffer solution can have between about 0.1mS/cm to the conductivity between about 100mS/cm.Perhaps, lavation buffer solution can have between about 0.1mS/cm to the conductivity between about 40mS/cm.

Lavation buffer solution also can comprise the aqueous solution of the inorganic salt of one or more inorganic acids that can not cause the biological functions of protein forfeiture, described salt is hydrochloride, vitriol and nitrate for example, for example sodium-chlor, Repone K, ammonium chloride, sodium sulfate, vitriolate of tartar, ammonium sulfate.

This method also can comprise a step or a multistep balance.Equilibrium step can with the solid separating medium with carry out before the solution that contains Factor IX and/or factors IX contacts.Equilibrium step can comprise water or suitable buffered soln is handled described solid separating medium to regulate the pH and the ionic strength of solid separating medium.The solution that is used for equilibrium step can be the aqueous solution of the inorganic salt of demineralized water or one or more inorganic acids compatible with Factor IX and/or factors IX, described salt is hydrochloride, vitriol and nitrate for example, for example sodium-chlor, Repone K, ammonium chloride, sodium sulfate, vitriolate of tartar, ammonium sulfate.Perhaps, level pad can be and contains the inorganic acid salt compatible with Factor IX and/or factors IX and/or the aqueous buffer solution of organic acid salt, for example contains Citrate trianion, acetate, succinate, lactic acid salt, tartrate, formate, propionic salt, phosphoric acid salt or boratory damping fluid.

Carry out under the temperature between reducible 2 ℃ to about 28 ℃ of the equilibrium step.For example, temperature can be about 2,4,6,8,10,12,14,16,18,20,22,24,26 or 28 ℃.

Contain Factor IX and/or factors IX solution pH be used in that described solid separating medium is identical with the pH of the buffered soln of balance solid separating medium before the described solution that contains Factor IX and/or factors IX contacts.Contain Factor IX and/or factors IX solution pH can be used in that described solid separating medium is different with the pH of the buffered soln of balance solid separating medium before the described solution that contains Factor IX and/or factors IX contacts.

Present method also can comprise a step or multistep regeneration.Regeneration step comprises with the suitable agent that residuals can be removed from the solid separating medium handles the solid separating medium.Regeneration step can be in that described solid separating medium carries out after with wash-out Factor IX and/or factors IX with the elutriant wash-out.Suitable regeneration reagent comprises alkali, for example hydroxide solution such as sodium hydroxide or potassium hydroxide, peracid solutions or superoxol, the solution that contains reactive chlorine such as hypochlorite solutions, denaturing agent example hydrochloric acid guanidine, organic solvent such as ethanol.

This method can be carried out with the packed bed pattern in filling column.Perhaps, this method can be carried out with the expanded bed pattern in the EBA post.

As mentioned above, in one embodiment, this method can be used for separating Factor IX and the factors IX as mixture from the solution that contains Factor IX and factors IX and other material.In this embodiment, select the EBA post for use, the solid separating medium of the capacity of wherein having packed into.The amount of the solid separating medium that uses will depend on the amount of the solution that contains Factor IX and factors IX that will use, and the concentration of Factor IX and factors IX in this solution.When protein soln was human plasma or CPP, general 1 liter of solid separating medium was used for per 5 to 30 liters blood plasma.If protein soln is the reorganization fermenting broth, one liter of solid separating medium can be used for 1 liter of fermenting broth, is up to 1000 L fermenting broths.Can prove conclusively circulation at the bottom of post is fluidized up to the solid separating medium.Suitable post linear rate of flow comprise scope at 0.5cm/min to the flow velocity between 40cm/min or the about 3cm/min to 15cm/min.Equilibrium step can use appropriate solution (for example water, aqueous electrolyte liquid or buffered soln) to carry out then, after this for example rough blood plasma of the solution that contains Factor IX and factors IX can be introduced at the bottom of the post, the solution that contains Factor IX and factors IX contacts with the solid separating medium thus.Carry out the first step wash-out then with unbinding protein wash-out from the post.Washing step can be chosen wantonly after the first step wash-out and carry out.Carry out the second step wash-out then with Factor IX and factors IX wash-out from the solid separating medium.

A first aspect of the present invention and second aspect can two-step approach be used continuously, wherein the first step relates to the second aspect of using invention, be used for from containing the mixture and/or the factors IX of the listed any proteinic solution separation factor VIII of specification sheets page 4 or itself and the von Willebrand factor, its coefficient of concentration is at least 10, second step related to the first aspect of using invention, not conjugated protein being separated into that is used for obtaining from the first step of second aspect contained for example α-1-proteinase inhibitor, albumin, IgG, the proteinic flow point of Antithrombin III and fibrinogen, its all proteins output is all very high.

The method of second aspect can be used continuously with other method for protein isolation.For example, the method of second aspect can be used for separation factor VIII and/or factors IX from plasma solutions, and the residual protein that will exist from the flow point that effluent obtains is as using various other fractionation methods to separate other proteinic starting material, described other protein is albumin, fibrinogen, immunoglobulin (Ig), α-1-proteinase inhibitor for example, described other fractionation method separate for example precipitate, filtration, ion exchange chromatography etc.

Can be with the protein isolate of the first aspect of aspect of the present invention and second aspect with suitable form as forming preparation with powder, solution, liquid form etc.The example of some product formulation provides in table 4.

Table 4

Product	Preparation
Product	Preparation	Factor IX	Dry powder: 250 IU, 500 IU, 1000IU
α-1-PI	Dry powder, 1g	Factor IX	Dry powder: 250 IU, 500 IU, 1000IU
α-1-PI	Dry powder, 1g	Albumin
	5%, 25% solution	Albumin
	5%, 25% solution	Antithrombin III	Dry powder	1000 IU
IVIG	10% liquid, pH 4.25	Antithrombin III	Dry powder	1000 IU
IVIG	10% liquid, pH 4.25	Fibrinogen	Dry powder 1.5g

Other purification process of isolated protein flow point also can as required or properly use in method of the present invention.

So a kind of method can relate to and comprise a series of anion-exchange chromatographies or hydrophobic interaction chromatography step.Suchlike method plays a part to concentrate and be further purified the protein flow point that obtains from method of the present invention.By selecting the optimal fixation medium and the elution requirement of a step or multistep series chromatography, all proteins that obtains from method of the present invention can be attached on the post.Conjugated protein can be gone up selective elution from second post (secondary column) under one group of different electrochemical conditions then, and this electrochemical conditions can cause that protein significantly concentrates and might be further purified.Other chromatographic technique for example affinity chromatography, metal chelate chromatography and gel-filtration also can be used singly or in combination.

Method of the present invention can be chosen wantonly and comprise the inactivation of virus step.For example, the virally inactivated/removing step of every kind of product is shown in the table 5, and wherein every kind of product can separate by method of the present invention.

Table 5

Product	Step	1	Step 2
Product	Step	1	Step 2	Factor IX	Solvent detergent	Xeothermic, 80 ℃, 72h
α-1 proteinase inhibitor	Solvent detergent	Virus filtration		Factor IX	Solvent detergent	Xeothermic, 80 ℃, 72h
α-1 proteinase inhibitor	Solvent detergent	Virus filtration	Antithrombin III	Pasteurization, 60 ℃, 1h	Virus filtration
Albumin	The S-200 chromatography	Pasteurization, 60 ℃, 1h	Antithrombin III	Pasteurization, 60 ℃, 1h	Virus filtration
Albumin	The S-200 chromatography	Pasteurization, 60 ℃, 1h	IVIG	The solvent stain remover	Virus filtration
Fibrinogen	The solvent stain remover	Damp and hot, 62 ℃, 10h	IVIG	The solvent stain remover	Virus filtration

The present invention now will only describe in detail by way of example with regard to the following example.Embodiment is intended to be used to explain the present invention, does not describe disclosed ubiquity and should not be interpreted as limiting in the whole text in the specification sheets.

Embodiment

Embodiment 1 α-1-proteinase inhibitor, Antithrombin III, fibrinogen, immune ball Albumen separates with albuminous

Step 1.Solid separating medium: the agarose-tungsten carbide microspheres that works with 2-sulfydryl nicotinic acid.Agarose-tungsten carbide microspheres size distribution is between the 40-120 micron, and mean diameter is 70 microns.The density of microballoon be 2.9g/ml (FastLine UFC NNSDW cat.No.:CS48, UpFront Chromatography A/S, Copenhagen, Denmark.).With microballoon pack into the EBA post (FastLine 100, UpFront Chromatography A/S, Copenhagen, Denmark) (diameter is 10cm; The height of bed is made as 50cm; Set bed volume=3.926 L) in.When temperature is 25 ℃, with 2.5 column volumes (9.8L) 40mM Trisodium Citrate (pH 4.5), use another 2.5 column volume (9.8L) 40mM Trisodium Citrate (pH 5.0) then, carry out balance with the 7.5cm/min linear rate of flow.

Step 2.2 L thaw human plasma by 4 L water with 1: 2 dilution proportion, will contain on 6 liters of diluting plasma solution of the human plasma that thaws in this post.Plasma solutions its pH before being used for post of dilution is adjusted to pH 5.0 with 1 MHCl, and goes up sample than being 1.5 liters of diluting plasma solution of every liter of resin.

Step 3.Behind sample on the diluting plasma solution, this post elution buffer 1 wash-out, this elution buffer contains the buffered soln that 4.2 column volumes (16.49 L) contain 10mM Trisodium Citrate (pH 5.0).Remove the unbinding protein that comprises 95% above α-1-proteinase inhibitor, lipid and other material that the diluting plasma solution that is used for post exists thus.

Step 4.After the step 3, this post contains elution buffer 2 (pH 6.0) wash-out of 5g/L Sodium octoate/HCl with 2.9 column volumes (11.39 L).Step 4 is albumin wash-out from the post, and its output is higher than 95% of albuminous amount that the diluting plasma solution that is used for post exists.This step also wash-out be used for 60% Antithrombin III that the diluting plasma solution of post exists.

Step 5.After post wash-out as described in the top step 4, this post contains elution buffer 3 wash-outs of 1 M Trisodium Citrate (pH 8.0) with 4.4 column volumes (17.27L).This step wash-out be used for the 95% above immunoglobulin (Ig) that the diluting plasma solution of post exists.

Step 6.After post wash-out as described in the top step 5, this post is with elution buffer 4 (pH8.0) wash-out, and this elution buffer 4 contains the 20mM Trisodium Citrate that 2.1 column volumes (8.24 L) contain 0.1 M sodium-chlor.

Step 7.After post wash-out as described in the step 6, this post is with the regeneration of 1 column volume (3.926 L), 1 M sodium hydroxide, and with 2.5 column volumes (9.8 L) 40mM Trisodium Citrates (pH 4.5), 2.5 column volumes (9.8 L) 40mM Trisodium Citrates (pH 5.0) balance once more again.

Embodiment 2 α-1-proteinase inhibitor, albumin, immunoglobulin (Ig) and fibrinogen Separation, wherein also comprise washing step

The solid separating medium that is used for present embodiment is identical with the solid separating medium that is used for embodiment 1.

Step 1.Under 21 ℃ of temperature, with 2.5 column volumes (196.3mL) 40mM Trisodium Citrate (pH 4.5), (diameter is 2cm with the linear rate of flow balance EBA post (FastLine 20, UpFront Chromatography A/S, Copenhagen Denmark) of 5.0cm/min; The height of bed is 25cm; The setting bed volume=78.5mL).

Step 2.39.3mL thaw human plasma by 78.5mL water with 1: 2 dilution proportion, on the 117.8mL diluting plasma solution of the human plasma that will thaw in this post.Diluting plasma solution its pH before being used for post is adjusted to pH 5.0 with 1 M HCl, and goes up sample than being 1.5 liters of plasma solutions of every liter of resin.

Step 3.Behind sample on the diluting plasma solution, this post contains elution buffer 1 wash-out of 10mM Trisodium Citrate (pH 5.0) with 3.3 column volumes (259.2 mL).Remove the unbinding protein that comprises α-1-proteinase inhibitor, lipid and other material that the diluting plasma solution that is used for post exists thus.

Step 4. is above-mentionedAfter the step 3, this post contains elution buffer 2 (pH 6.0) wash-out of 5g/L Sodium octoate/HCl with 2.6 column volumes (204.2mL).Step 4 wash-out be used for the albumin that the diluting plasma solution of post exists.

Step 4a.After post wash-out as described in the top step 4, this post washs with 1.0 column volumes (78.5mL), 1 M Trisodium Citrate (pH 8.0).

Step 5.After post washing as described in the top step 4a, this post contains elution buffer 3 wash-outs of 0.3 M Trisodium Citrate (pH 8.0) with 4.9 column volumes (384.8mL).This step wash-out be used for the immunoglobulin (Ig) that the diluting plasma solution of post exists.

Step 6.After post wash-out as described in the top step 5, this post is with elution buffer 4 (pH8.0) wash-out, and this elution buffer 4 contains the 20mM Trisodium Citrate that 2.6 column volumes (204.2mL) contain 0.1 M sodium-chlor.This step wash-out be used for the fibrinogen that the diluting plasma solution of post exists.

Step 7.After post wash-out as described in the step 6, this post is with the regeneration of 1 column volume (78.5mL), 1 M sodium hydroxide, and with 2.0 column volumes (157mL) 40mM Trisodium Citrate (pH 4.5) balance once more.

Embodiment 3 α-1-proteinase inhibitor, albumin, Transferrins,iron complexes, immunoglobulin (Ig) and The separation of fibrinogen

Step 1.Under 21 ℃ of temperature, with 2.5 column volumes (196.3mL) 40mM Trisodium Citrate (pH 5.0), (diameter is 2cm with the linear rate of flow balance EBA post (FastLine 20, UpFront Chromatography A/S, Copenhagen Denmark) of 15.0cm/min; The height of bed is 25cm; The setting bed volume=78.5mL).

Step 2.Human plasma, will be contained on the 117.8mL diluting plasma solution of the human plasma that thaws in this post with 1: 2 dilution proportion by 78.5mL water 39.3mL thaw.Diluting plasma solution its pH before being used for post is adjusted to pH 5.0 with 1 M HCl, and goes up sample than being 1.5 liters of plasma solutions of every liter of resin.

Step 3.Behind sample on the diluting plasma solution, this post elution buffer 1 wash-out that contains 9.4 column volumes (738.3mL) demineralized water.Remove the unbinding protein that comprises 100% α-1-proteinase inhibitor, 10% albumin, 5% Transferrins,iron complexes and 10% factor I, lipid and other material that the diluting plasma solution that is used for post exists thus.

Above the step 4.After the step 3, this post contains elution buffer 2 (pH 6.0) wash-out of 5g/L Sodium octoate/HCl with 8.9 column volumes (699mL).Step 4 from the post wash-out albumin, its output is to be used for 90% of albuminous amount that the diluting plasma solution of post exists.This step also wash-out be used for 5% immunoglobulin (Ig) that the diluting plasma solution of post exists.

Step 5.After post wash-out as described in the top step 4, this post contains elution buffer 3 wash-outs of 0.3 M Trisodium Citrate (pH 8.0) with 9.0 column volumes (706.8mL).This step wash-out be used for the 85% above immunoglobulin (Ig) that the diluting plasma solution of post exists.This step also wash-out be used for 95% Transferrins,iron complexes and 30% fibrinogen that the diluting plasma solution of post exists.

Step 6.After post wash-out as described in the top step 5, this post is with elution buffer 4 (pH8.0) wash-out, and this elution buffer 4 contains the 20 mM Trisodium Citrates that 5.0 column volumes (392.7mL) contain 0.1 M sodium-chlor.This step wash-out be used for 60% fibrinogen and 10% immunoglobulin (Ig) that the diluting plasma solution of post exists.

Step 7.After post wash-out as described in the step 6, this post is with the regeneration of 1 column volume (78.5mL), 1 M sodium hydroxide, and with 2.0 column volumes (157mL) 40mM Trisodium Citrate (pH 5.0) balance once more.

Embodiment 4 α-1-proteinase inhibitor, albumin, immunoglobulin (Ig), Transferrins,iron complexes and The separation of fibrinogen

Step 1.Under 21 ℃ of temperature, with 2.5 column volumes (196.3mL) 40mM Trisodium Citrate (pH 5.0), (diameter is 2cm with the linear rate of flow balance EBA post (FastLine 20, UpFront Chromatography A/S, Copenhagen Denmark) of 5.0cm/min; The height of bed is 25cm; The setting bed volume=78.5mL).

Step 3.Behind sample on the diluting plasma solution, this post elution buffer 1 wash-out that contains 6.8 column volumes (533.8mL) demineralized water.Remove the unbinding protein that comprises 100% α-1-proteinase inhibitor, 10% albumin, 5% Transferrins,iron complexes and 10% factor I, lipid and other material that the diluting plasma solution that is used for post exists thus.

Step 4.After the described post washing of top step 3, this post contains elution buffer 2 (pH 6.0) wash-out of 5g/L Sodium octoate/HCl with 5.5 column volumes (431.75mL).Step 4 from the post wash-out albumin, its output is to be used for 90% of albuminous amount that the diluting plasma solution of post exists.This step also wash-out be used for 5% immunoglobulin (Ig) that the diluting plasma solution of post exists.

Step 5.After the top step 4, this post contains elution buffer 3 wash-outs of 0.3 M Trisodium Citrate (pH 8.0) with 5.0 column volumes (392.5mL).This step wash-out be used for the 85% above immunoglobulin (Ig) that the diluting plasma solution of post exists.This step also wash-out be used for 95% Transferrins,iron complexes and 30% fibrinogen that the diluting plasma solution of post exists.

Step 6.After post wash-out as described in the top step 5, this post is with elution buffer 4 (pH8.0) wash-out, and this elution buffer 4 contains the 20mM Trisodium Citrate that 3.1 column volumes (243.35mL) contain 0.1 M sodium-chlor.This step wash-out be used for 60% fibrinogen and 10% immunoglobulin (Ig) that the diluting plasma solution of post exists.

Embodiment 5 α-1-proteinase inhibitor, albumin, immunoglobulin (Ig), fibrinogen With separating of Transferrins,iron complexes

Step 1.Under 21 ℃ of temperature, with 2.5 column volumes (196.3mL) 40mM Trisodium Citrate (pH 5.0), (diameter is 2cm with the linear rate of flow balance EBA post (FastLine 20, UpFront Chromatography A/S, Copenhagen Denmark) of 10.0cm/min; The height of bed is 25cm; The setting bed volume=78.5mL).

Step 3.Behind sample on the diluting plasma solution, this post elution buffer 1 wash-out that contains 9.5 column volumes (745.75mL) demineralized water.Remove the unbinding protein that comprises 100% α-1-proteinase inhibitor, 10% albumin, 5% Transferrins,iron complexes and 10% factor I, lipid and other material that the diluting plasma solution that is used for post exists thus.

Step 4.After the top step 3, this post contains elution buffer 2 (pH 6.0) wash-out of 5 g/L Sodium octoate/HCl with 7.1 column volumes (557.35mL).Step 4 from the post wash-out albumin, its output is to be used for 90% of albuminous amount that the diluting plasma solution of post exists.This step also wash-out be used for 5% immunoglobulin (Ig) that the diluting plasma solution of post exists.

Step 5.After the top step 4, this post contains elution buffer 3 wash-outs of 0.3 M Trisodium Citrate (pH 8.0) with 5.9 column volumes (463.15mL).This step wash-out be used for the 85% above immunoglobulin (Ig) that the diluting plasma solution of post exists.This step also wash-out be used for 95% Transferrins,iron complexes and 30% fibrinogen that the diluting plasma solution of post exists.

Embodiment 6 α-1-proteinase inhibitor, albumin, immunoglobulin (Ig), Transferrins,iron complexes and The separation of fibrinogen

Step 1.Under 21 ℃ of temperature, with 2.5 column volumes (196.3mL) 40mM Trisodium Citrate (pH 5.0), (diameter is 2cm with the linear rate of flow balance EBA post (FastLine 20, UpFront Chromatography A/S, Copenhagen Denmark) of 20.0cm/min; The height of bed is 25cm; The setting bed volume=78.5mL).

Step 3.Behind sample on the diluting plasma solution, this post elution buffer 1 wash-out that contains 12.6 column volumes (989.1mL) demineralized water.Remove the unbinding protein that comprises 100% α-1-proteinase inhibitor, lipid and other material that the diluting plasma solution that is used for post exists thus.It should be noted that the flow velocity that the albumin of significant quantity also can 20cm/min in this step is able to wash-out.

Step 4.After the top step 3, this post contains elution buffer 2 (pH 6.0) wash-out of 5g/L Sodium octoate/HCl with 10.6 column volumes (832.1mL).Step 4 from the post wash-out albumin, its output is to be used for 90% of albuminous amount that the diluting plasma solution of post exists.This step also wash-out be used for 5% immunoglobulin (Ig) that the diluting plasma solution of post exists.

Step 5.After the top step 4, this post contains elution buffer 3 wash-outs of 0.3 M Trisodium Citrate (pH 8.0) with 6.9 column volumes (541.65mL).This step wash-out be used for the 85% above immunoglobulin (Ig) that the diluting plasma solution of post exists.This step also wash-out be used for 95% Transferrins,iron complexes and 30% fibrinogen that the diluting plasma solution of post exists.

Step 6.After post wash-out as described in the top step 5, this post is with elution buffer 4 (pH8.0) wash-out, and this elution buffer 4 contains the 20mM Trisodium Citrate that 6.8 column volumes (533.8mL) contain 0.1 M sodium-chlor.This step wash-out be used for 60% fibrinogen and 10% immunoglobulin (Ig) that the diluting plasma solution of post exists.

Embodiment 7 α-1-proteinase inhibitor, albumin and IgG separate

Step 3.Behind sample on the diluting plasma solution, this post contains elution buffer 1 wash-out of 10mM Trisodium Citrate (pH 5.0) with 3.3 column volumes (259.2mL).Remove the unbinding protein that comprises α-1-proteinase inhibitor, lipid and other material that the diluting plasma solution that is used for post exists thus.

Step 4.After the described post washing of top step 3, this post contains elution buffer 2 (pH 6.0) wash-out of 5g/L Sodium octoate/HCl with 2.7 column volumes (211.95mL).Step 4 wash-out be used for the albumin that the diluting plasma solution of post exists.

Step 5.Delete.

Step 6.After post wash-out as described in the top step 4, this post is with elution buffer 4 (pH8.0) wash-out, and this elution buffer 4 contains the 20mM Trisodium Citrate that 3.8 column volumes (298.3mL) contain 0.1 M sodium-chlor.This step wash-out be used for the IgG that the diluting plasma solution of post exists.

Embodiment 8 α-1-proteinase inhibitor, albumin, immunoglobulin (Ig) and fibrinogen Separation, wherein also comprise washing step

Step 1.Under 21 ℃ of temperature, with 2.5 column volumes (392.5mL) 40mM Trisodium Citrate (pH 4.5), (diameter is 2cm with the linear rate of flow balance EBA post (FastLine 20, UpFront Chromatography A/S, Copenhagen Denmark) of 5.0cm/min; The height of bed is 25cm; The setting bed volume=78.5mL).

Step 3.Behind sample on the diluting plasma solution, this post contains elution buffer 1 wash-out of 10mM Trisodium Citrate (pH 5.0) with 2.9 column volumes (455.3mL).Remove the unbinding protein that comprises α-1-proteinase inhibitor, lipid and other material that the diluting plasma solution that is used for post exists thus.

Step 4.After the top step 3, this post contains elution buffer 2 (pH 6.0) wash-out of 5g/L Sodium octoate/HCl with 2.8 column volumes (439.6mL).Step 4 wash-out be used for the albumin that the diluting plasma solution of post exists.

Step 4a.After post wash-out as described in the top step 4, this post washs with 1.0 column volumes (157mL), 1 M Trisodium Citrate (pH 8.0).

Step 5.After post washing as described in the top step 4a, this post contains elution buffer 3 wash-outs of 0.3 M Trisodium Citrate (pH 8.0) with 4.5 column volumes (706.5mL).This step wash-out be used for the immunoglobulin (Ig) that the diluting plasma solution of post exists.

Step 6.After post wash-out as described in the top step 5, this post is with elution buffer 4 (pH8.0) wash-out, and this elution buffer 4 contains the 20mM Trisodium Citrate that 1.9 column volumes (298.3mL) contain 0.1 M sodium-chlor.This step wash-out be used for the fibrinogen that the diluting plasma solution of post exists.

Step 7.After post wash-out as described in the step 6, this post is with the regeneration of 1 column volume (157mL), 1 M sodium hydroxide, and with 1.9 column volumes (298.3mL) 40mM Trisodium Citrate (pH 4.5) balance once more.

Embodiment 9 α-1-proteinase inhibitor, albumin, immunoglobulin (Ig) and fibrinogen Separation, wherein also comprise washing step

Step 1.Under 21 ℃ of temperature, with 2.5 column volumes (392.5mL) 40mM Trisodium Citrate (pH 4.5), (diameter is 2cm with the linear rate of flow balance EBA post (FastLine 20, UpFront Chromatography A/S, Copenhagen Denmark) of 10.0cm/min; The height of bed is 25cm; The setting bed volume=78.5mL).

Step 2.Human plasma, will be contained on the 117.8mL diluting plasma solution of the human plasma that thaws in this post with 1: 2 dilution proportion by 78.5mL water 39.3mL thaw.Diluting plasma solution its pH before being used for post is adjusted to pH 5.0 with 1MHCl, and goes up sample than being 1.5 liters of plasma solutions of every liter of resin.

Step 3.Behind sample on the diluting plasma solution, this post contains elution buffer 1 wash-out of 10mM Trisodium Citrate (pH 5.0) with 3.7 column volumes (580.9mL).Remove the unbinding protein that comprises α-1-proteinase inhibitor, lipid and other material that the diluting plasma solution that is used for post exists thus.

Step 4.After the top step 3, this post contains elution buffer 2 (pH 6.0) wash-out of 5g/L Sodium octoate/HCl with 3.6 column volumes (565.2mL).Step 4 wash-out be used for the albumin that the diluting plasma solution of post exists.

Step 5.After post washing as described in the top step 4a, this post contains elution buffer 3 wash-outs of 0.3 M Trisodium Citrate (pH 8.0) with 5.4 column volumes (847.8mL).This step wash-out be used for the immunoglobulin (Ig) that the diluting plasma solution of post exists.

Step 6.After post wash-out as described in the top step 5, this post is with elution buffer 4 (pH8.0) wash-out, and this elution buffer 4 contains the 20mM Trisodium Citrate that 1.7 column volumes (266.9mL) contain 0.1 M sodium-chlor.This step wash-out be used for the fibrinogen that the diluting plasma solution of post exists.

Step 7.After post wash-out as described in the step 6, this post is with the regeneration of 1 column volume (157mL), 1 M sodium hydroxide, and with 2.6 column volumes (408.2mL) 40mM Trisodium Citrate (pH 4.5) balance once more.

Assay determination:

All output of the foregoing description are determined (AgneteIngild in:Handbook of Immunoprecipitation-in-Gel Techniques by competitive list to radioimmunodiffusion, ed.NilsH.Axelsen, Scandinavian Journal of Immunology, Suppl.No.10, Vol17, pp.41-57,1983).

Embodiment 10 is separation factor VIII and factors IX from rough blood plasma

Step 1.The solid separating medium: matrix scaffold is agarose-wolfram varbide resin, and spacer derives from epoxy group(ing), and part is p dimethylamine or m-xylene diamine.With microballoon pack into the EBA post (FastLine 10, UpFront Chromatography A/S, Copenhagen, Denmark) (diameter is 1cm; The height of bed is made as 25cm; Set in the bed volume=20mL).When temperature is 25 ℃, with 20mM Trisodium Citrate (pH 6.0), carry out balance with the linear rate of flow of 5.0cm/min.

Step 2 (application of sample step).With on the undiluted rough blood plasma of 300mL in this post.Blood plasma its pH before being used for post is adjusted to pH 6.0 with 1 M HCl, and goes up sample than being 15 liters of blood plasma of every liter of resin.

Step 3 (wash-out 1).Behind sample on the blood plasma, this post contains elution buffer 1 (pH 6.0) wash-out of 20mM Trisodium Citrate with 9 column volumes (180mL).Remove unbinding protein thus.

Step 4.This post washs with 6.4 column volumes (128mL) lavation buffer solution then, and this lavation buffer solution contains 20mM Trisodium Citrate+0.2M sodium-chlor, and pH is 6.0.

Step 5 (wash-out 2).Above step 4 after, this post is with 6.0 column volumes (120mL) elution buffer, 2 wash-outs, this elution buffer contains 20mM Trisodium Citrate+1.0 M sodium-chlor, pH is 8.0.This step wash-out be used for the Factor IX activity more than 45% that the blood plasma of post exists.This step also wash-out be used for the factors IX activity more than 85% that the blood plasma of post exists.

Step 6.After the described post wash-out of step 5, this post is with 1 column volume (20mL) 1M sodium hydroxide regeneration, and with 2.0 column volumes (40mL) 20mM Trisodium Citrate (pH 6.0) balance once more.

The active mensuration of Factor IX:

Use is measured relative Factor IX activity available from the Factor IX active agent box of Coamatic (catalog number 822585-63) in rough blood plasma and EBA protein flow point.Use undiluted (100% activity) and dilution rough blood plasma preparation standard curve (not providing) as 80%, 60%, 40%, 20% and 0% (blank) of initial activity.Flow point from effluent, washes and the eluate (wash-out 2) of post has been carried out analyzing and having determined the activity yied in the eluate (referring to table 7) by the reference standard curve.As seen from Table 7, under employed condition combination of solid separating medium and wash-out 45% Factor IX activity.

Table 7: Factor IX, flow point volume and activity yied

	Volume	Relative Factor IX activity	Activity yied
	Volume	Relative Factor IX activity	Activity yied	Rough blood plasma effluent washes takes off thing (wash-out 2) earlier	300ml 480ml 128ml 120ml	100％ 13％ 0％ 113％	100％ 21％ 0％ 45％

The antigenic mensuration of factors IX:

Factors IX sandwich ELISA by commodity in use antibody is measured relative factors IX antigen concentration in rough blood plasma and EBA protein flow point.Use undiluted (100%) with the rough blood plasma preparation standard curve (do not provide) of dilution as 80%, 60%, 40%, 20% and 0% (blank) of starting point concentration.Flow point from effluent, washes and the eluate (wash-out 2) of post has been carried out analyzing and determined antigen yield (referring to table 8) in the eluate (wash-out 2) by the reference standard curve.As seen from Table 8, under employed condition combination of solid separating medium and wash-out 85% factors IX antigen.

Table 8: factors IX, flow point volume and antigen yield

	Volume	Relative factors IX antigen concentration	Activity yied
	Volume	Relative factors IX antigen concentration	Activity yied	Rough blood plasma effluent washes eluate (wash-out 2)	300ml 480ml 128ml 120ml	100％ 0％ 0％ 21 3％	100％ 0％ 0％ 85％

Selectivity

Fig. 8 illustrates eluate (wash-out 2) by SDS-PAGE and comprises protein that minor proportion is used.When for rough blood plasma directly relatively and during dilution, only visible IgG, albumin and other proteic atomic weak band, this show with respect to the FVIII/FIX flow point these proteinic lose not remarkable.Equally, for rough blood plasma directly relatively with effluent flow point dilution, do not observe and proteinicly significantly lose.RID is illustrated in that α-1-PI and fibrinogen are also reclaimed (not providing) fully in the effluent flow point.

The consistency of the method for embodiment 11 second aspects and the method for first aspect

In order to determine to remove Factor IX earlier and whether factors IX can have a negative impact to the separation method of first aspect, be used as the starting material of the method for first aspect from the effluent flow point of embodiment 10.

Step 1.Solid separating medium: the agarose-tungsten carbide microspheres that works with 2-sulfydryl nicotinic acid.Agarose-tungsten carbide microspheres size distribution is between the 40-120 micron, and mean diameter is 70 microns.The density of microballoon be 2.9g/ml (FastLine UFC NNSDW cat.No.:CS48, UpFront Chromatography A/S, Copenhagen, Denmark.).With microballoon pack into the EBA post (FastLine 20, UpFront Chromatography A/S, Copenhagen, Denmark) (diameter is 2cm; The height of bed is made as 50cm; The setting bed volume=157mL).Under 25 ℃ of temperature, with 2.5 column volumes (392.5mL) 40mM Trisodium Citrates (pH4.5), use another 2.5 column volume (392.5mL) 40mM Trisodium Citrate (pH 5.0) then, be that 7.5cm/min carries out balance with the linear rate of flow.

Step 2.Effluent from embodiment 10 described posts is adjusted to 1 part of rough blood plasma+2 part water, with on this effluent in this post.Diluting plasma solution its pH before being used for post is adjusted to pH 5.0 with 1 M HCl, and goes up sample than being 1.5 liters of blood plasma of every liter of resin.

Step 3.Behind sample on the diluting plasma solution, this post contains elution buffer 1 wash-out of 10mM Trisodium Citrate (pH 5.0) with 3.3 column volumes (518mL).Remove the unbinding protein that comprises α-1-proteinase inhibitor, lipid and other material that the diluting plasma solution that is used for post exists thus.

Step 4.After the top step 3, this post contains elution buffer 2 (pH 6.0) wash-out of 5g/L Sodium octoate/HCl with 2.6 column volumes (408mL).Step 4 wash-out be used for the albumin that the diluting plasma solution of post exists.

Step 5.After the described post washing of top step 4a, this post contains elution buffer 3 wash-outs of 0.3 M Trisodium Citrate (pH7.4) with 4.9 column volumes (769mL).This step wash-out be used for the immunoglobulin (Ig) that the diluting plasma solution of post exists.

Step 6.After post wash-out as described in the top step 5, this post is with elution buffer 4 (pH7.4) wash-out, and this elution buffer 4 contains the 20mM Trisodium Citrate that 2.6 column volumes (408mL) contain 0.1 M sodium-chlor.This step wash-out be used for the fibrinogen that the diluting plasma solution of post exists.

Step 7.After the described post wash-out of step 6, this post is with 1 column volume (157mL), 1 M sodium hydroxide regeneration, and with 2.0 column volumes (314mL) 40mM Trisodium Citrate (pH 4.5) balance once more.

As shown in Figure 7, the qualitative strips that is obtained by the blood plasma that lacks Factor IX/factors IX is identical with the strips of acquisition among the embodiment 4.The processing volume that each flow point obtains is also described identical with embodiment 4.

Fig. 8 A-B illustrates the quantitative analysis of albumin and IgG in the different proteins flow point, and just as observed, in fact all albumin reclaim in wash-out 2 and obtain, and in fact all IgG reclaim in wash-out 3 and obtain.Albumin and IgG in other flow point, almost detect less than.

Find that also α-1-proteinase inhibitor is identical with the described behavior of the method for first aspect with fibrinogen.

These results show that p dimethylamine or m-xylene diamine can be with effective means never selective extraction Factor IX and factors IX in the diluting plasma, and can significantly not reduce the level of other plasma proteins, other plasma proteins can be separated in the method for subsequently (one or more) method such as first aspect.The separation method of the albumin of the separation method of Factor IX/factors IX and first aspect/IgG/ fibrinogen/α-1-proteinase inhibitor is compatible.

Claims

1. one kind is separated described method of protein from contain proteinic solution, described solution is selected from: the human plasma of rough blood plasma, serum, the cold supernatant liquor that derives from blood plasma, fractional separation, the cryoprecipitate that derives from blood plasma and reorganization meat soup, and described method comprises:

(i) provide solid separating medium with following formula:

M-S-L

2. according to the process of claim 1 wherein that this solution is rough blood plasma.

3. according to the method for claim 1 or claim 2, wherein M is a high-density resin.

4. according to each method of claim 1 to 3, wherein L is a 2-sulfydryl nicotinic acid.

5. according to each method of claim 1 to 4, wherein S is the compound that comprises epoxy group(ing).

6. according to each method of claim 1 to 5, wherein this at least a protein stream branch comprises and is selected from following at least a protein: immunoglobulin (Ig) is IgG for example, IgA, IgM, IgD or IgE, Transferrins,iron complexes, the fibrinogen or derivatives thereof, the plasma proteins enzyme inhibitors is antithrombin such as Antithrombin III for example, blood is urged solidifying egg white, the solid albumen of blood anticoagulant, cytokine, somatomedin, albumin or derivatives thereof, thrombolytic agent, angiogenesis inhibitor albumen, the Regular Insulin or derivatives thereof, α-1-proteinase inhibitor or derivatives thereof is α-1-antitrypsin for example, α-2-antiplasmin or derivatives thereof, the C-1 esterase inhibitor, lipophorin, HDL, the fibronectin or derivatives thereof, β-2-glycoprotein I, Profibrinolysin, plasmin, plasminogen activator, the Profibrinolysin inhibitor, urokinase or derivatives thereof, streptokinase or derivatives thereof, inter-, α-2-macroglobulin, amyloid, Xue Qingleiniandanbai, ferritin, prealbumin, GC-sphaeroprotein, blood clotting galactenzyme and complement C3.

7. according to the method for claim 6, wherein this at least a protein stream branch comprises the following protein of at least a selection: immunoglobulin (Ig), Transferrins,iron complexes, fibrinogen or derivatives thereof, α-1-proteinase inhibitor and albumin, or derivatives thereof.

8. according to each method of claim 1 to 7, wherein contain described proteinic this solution and have pH between about 3.0 to about 6.0.

9. method according to Claim 8 wherein contains described proteinic this solution and has pH between about 4.5 to about 6.0.

10. according to each method of claim 1 to 9, wherein the first step wash-out comprise with pH between between about 4 to about 8, ionic strength between about 0.00005S/cm extremely this solid separating medium of eluant solution between about 0.1S/cm with the wash-out first protein flow point.

11. according to the method for claim 10, wherein this first protein stream branch comprises the protein of at least a α of being selected from-1-proteinase inhibitor, albumin, Xue Qingleiniandanbai and prealbumin.

12. according to the method for claim 10 or claim 11, wherein the second step wash-out comprise with pH between between about 5 to about 7, ionic strength between about 0.0001S/cm extremely this solid separating medium of eluant solution between about 0.1S/cm with the wash-out second protein flow point.

13. according to the method for claim 12, wherein this solution also comprises aromatic series, non-aromatic or heteroaromatic hydrophobic compound.

14. according to the method for claim 13, wherein this non-aromatic hydrophobic compound is alkyl carboxylate, sulfonate or electronegative washing composition.

15. according to each method of claim 12 to 14, wherein this second protein stream branch comprises at least a protein that is selected from albumin and immunoglobulin (Ig).

16. according to each method of claim 12 to 15, wherein the 3rd step wash-out comprise with pH between between about 5 to about 9, ionic strength between about 0.001S/cm extremely this solid separating medium of eluant solution between about 4S/cm with wash-out protein iii flow point.

17. according to the method for claim 16, wherein this protein iii flow point comprises at least a protein that is selected from immunoglobulin (Ig), Transferrins,iron complexes and fibrinogen.

18. according to the method for claim 16 or claim 17, wherein the 4th step wash-out comprise with pH between between about 5 to about 9, ionic strength between about 0.01S/cm extremely this solid separating medium of eluant solution between about 2S/cm with wash-out the 4th protein flow point.

19. according to the method for claim 18, wherein this solution also comprises inorganic acid salt.

20. according to the method for claim 18 or claim 19, wherein the 4th protein stream branch comprises at least a protein that is selected from Transferrins,iron complexes, immunoglobulin (Ig), fibrinogen and α-2-macroglobulin.

21. each method of claim 1 to 20, wherein said method is carried out with the expanded bed pattern in the expanded bed adsorption post.

22. each method of claim 1 to 20, wherein said method is carried out with the packed bed pattern in filling column.

23. the method for separation factor VIII and/or factors IX from a solution that contains Factor IX and/or factors IX, described solution is selected from: the human plasma of rough blood plasma, serum, the cold supernatant liquor that derives from blood plasma, fractional separation, the cryoprecipitate that derives from blood plasma and reorganization meat soup, and described method comprises:

(i) provide solid separating medium with following formula:

M-S-L

(ii) described solid separating medium is contacted with described solution, Factor IX and/or factors IX reversibly are attached on the described solid separating medium so at least;

24. according to the method for claim 23, the solution that wherein contains Factor IX and factors IX is rough blood plasma.

25. according to the method for claim 23 or claim 24, wherein M is a high-density resin.

26. according to each method of claim 23 to 25, wherein S is the compound that contains epoxy group(ing).

27. according to each method of claim 23 to 26, wherein the first step wash-out comprise with pH between between about 5.5 to about 6.5, ionic strength between about 0.001S/cm extremely the eluant solution solid separating medium between about 0.02S/cm with the wash-out first protein flow point.

28. according to the method for claim 27, wherein the second step wash-out comprise with pH between between about 7 to about 9, ionic strength between about 0.03S/cm extremely the eluant solution solid separating medium between about 0.2S/cm with the wash-out second protein flow point.

29. according to the method for claim 27 or claim 28, wherein this solution also comprises inorganic acid salt.