CN1355064A - Composite affinity polyamide film for purifying immone globulin in human blood plasma and preparing process thereof - Google Patents

Abstract

A composite microporous filtering polyamide membrane with affinity for puritying immunoglobulin used in human blood plasma contains phenylalnine as affinity ligand and hydroxyethylcellulose as modifier. Its advantages include high effect, high stability and high recovery rate.

Description

Be used for the composite affinity polyamide film and the preparation of human plasma immunoglobulin purification

The present invention relates to the diffusion barrier technology, a kind of composite affinity polyamide film and preparation method who is used for the human plasma immunoglobulin purification is provided especially.

The present immunoglobulin purification method of bibliographical information, great majority obtain with ethanol precipitation, but it can only get off various immunoglobulin (Ig) coprecipitations, different types of immunoglobulin (Ig) can not be separated, and products obtained therefrom purity is also undesirable.Someone adopts with agarose, regenerated cellulose, nylon membrane is raw material, immobilized A albumen or the G albumen gone up, and the immunoglobulin (Ig) in the adsorbed plasma gets off the immunoglobulin (Ig) desorption that adsorbs on the above-mentioned medium with salt and organic solvent more specifically.Yet this method is very trouble because A albumen and G protein Preparation are got up, and price is also very expensive, also might cause the inactivation and the sex change of immunoglobulin (Ig) in the elution process, also fails the application that puts it over.Somebody a kind of ectogenous agglutinine-Jacalin immobilized on the agarose Sepharose in addition, but also purifying immunoglobulin (Ig), but the purity of gained immunoglobulin (Ig) and the rate of recovery are all not really desirable.

The object of the present invention is to provide a kind of composite affinity polyamide film and preparation method who is used for the human plasma immunoglobulin purification, it has the higher affine capacity of gamma globulin matter, and the gained gamma globulin has higher purity.

The invention provides a kind of composite affinity polyamide film that is used for the human plasma immunoglobulin purification, it is characterized in that: this film be bonding have the phenylalanine affinity ligand by hydroxyethyl cellulose modified polyamide miillpore filter composite membrane; The amount of hydroxyethylcellulose is every gram film 15～225mg, and the amount of phenylalanine affinity ligand is every gram film 150～180 μ mol.

The present invention also provides the above-mentioned preparation method who is used for the composite affinity polyamide film of human plasma immunoglobulin purification, it is characterized in that:

Polyamide miillpore filter (nylon 6 films) is hydrolyzed with concentrated hydrochloric acid;

Get the PA membrane after the 1g hydrolysis, put into the dimethylbenzene system that 50～150ml contains 30～60% dioxane, the three chlorotriazine methods that add 0.5～1g again in this system respectively activate, and reaction was at room temperature carried out 30～60 minutes;

Film after the activation of three chlorotriazines immerses and contains in 1～5% hydroxy ethyl fiber cellulose solution, and adds sodium carbonate, and the pH value of solution value is remained between 6～7, reacts 2～4 hours down in 40～60 ℃;

With compound behind the film drying under reduced pressure of hydroxyethylcellulose, immerse once more in the above-mentioned dioxane/xylene solution that contains three chlorotriazines, add phenylalanine, 40～60 C reaction 4～5 hours.

Difference with the prior art of the present invention is:

1. selecting micropore nylon 6 films first for use is raw material, activate with three chlorotriazine methods, on the covalent bonding hydroxyethylcellulose, but increased the number of reactive group on the film greatly, and reduced non-specific adsorption to protein except that immunoglobulin (Ig);

2. on prepared nylon 6-hydroxyethylcellulose composite membrane, select dioxane-dimethylbenzene system for use first, by with three chlorotriazines on the reaction key of the active chlorine atom phenylalanine affinity ligand that closed, make that the aglucon number has improved the affine capacity of medium greatly up to 150～180 μ mol/g on the film;

3. under selected best affine condition, this affinity membrane can reach 15～25mg/g film to the affine capacity of gamma globulin matter in the human plasma, and is higher by about 50% than common affinity media, and the purity of gained gamma globulin is 80～85%.

In a word, the affinity membrane that the present invention developed has aglucon bonded amount height, purification effect to gamma globulin in the blood plasma is good, dielectric behavior is stable, easy to use, characteristics such as rate of recovery height are effective tool and the means of producing high purity immune globulin, have very strong practicality and application value.

Below by embodiment in detail the present invention is described in detail.

Embodiment 1

At first polyamide miillpore filter (nylon 6 films) is hydrolyzed with concentrated hydrochloric acid, makes the amide groups on the film change primary amino radical as much as possible into, number of amino groups is carried out quantitative assay with improved ninhydrin method on the film of hydrolysis front and back.

PA membrane after the hydrolysis soaks with NaOH solution, and after fully neutralizing remaining HCl, uses D-H ₂O fully washs, suction filtration, and carries out vacuum drying, uses three chlorotriazine method (CyCl then ₃) activate, add three chlorotriazines of 0.8g by the 1g PA membrane, to carry out in (V/V) dioxane/dimethylbenzene system that is reflected at 1: 1, reaction was at room temperature carried out 60 minutes, after reaction finishes, used dioxane earlier, used a large amount of D-H again ₂O fully washs, and the reduced vacuum drying.

Film after the activation of three chlorotriazines immerses and contains in the 3% hydroxy ethyl fiber cellulose solution, and adds an amount of sodium carbonate, and the pH value of solution value is remained between 6～7.Reacted 4 hours down in 60 ℃, wash 3 times with 0.1mol NaOH solution then, use massive laundering again, and decompressing and extracting; Adopt phenol-sulfuric acid method to measure the amount of hydroxyethylcellulose compound on the nylon membrane, by glucose unit on the cellulosic molecule.The film of compound hydroxyethylcellulose increases about 17 times than the group number that can react on the original nylon membrane, and composite membrane has also significantly reduced the non-specific adsorption to large biological molecule except that immunoglobulin (Ig).

With compound behind the film drying under reduced pressure of hydroxyethylcellulose, immerse once more in the above-mentioned dioxane/xylene solution that contains three chlorotriazines, add phenylalanine, reaction is 5 hours under 40～50 ℃ of stirrings, thoroughly wash with dioxane, aqueous isopropanol and deionized water respectively then, and drying under reduced pressure, so just made the nylon 6-hydroxyethylcellulose composite affinity membrane that contains the phenylalanine affinity ligand, the content of phenylalanine can reach 165 μ mol on every gram film.

With 20 diameters of preparation as stated above is the affinity membrane of 47mm band phenylalanine dentate, is assemblied in the dish formula affinity membrane separator.Use 0.05mol/L NaH ₂PO ₄Solution dilution 4ml human plasma pumps into affinity membrane separator with plasma extender with the flow velocity of 1ml/min, makes that phenylalanine dentate and gamma globulin produce affine interaction on the film.Earlier under the 2ml/min flow velocity, wash away foreign protein residual on the film with 1mol/L sodium chloride solution 50ml, used 1mol/L sodium chloride/ethylene glycol again instead (1: 1, V/V), the immunoglobulin (Ig) of affine absorption on wash-out film under the same flow (γ-Globulin), collect eluent, after the bag filter dialysis, sample is done electrophoretic analysis, prove that gained gamma immune globular protein purity is 83.5%.The rate of recovery of gamma globulin can reach 78% in the blood plasma, than the rate of recovery height of other purification process.

Embodiment 2

PA membrane after embodiment 1 hydrolysis is soaked with NaOH solution, and after fully neutralizing remaining HCl, use D-H ₂O fully washs, suction filtration, and carries out vacuum drying, uses three chlorotriazine method (CyCl then ₃) activate, add three chlorotriazines of 1g by the 1g PA membrane, carry out in (V/V) dioxane/dimethylbenzene system that is reflected at 1: 1.

Film after the activation of three chlorotriazines immerses and contains in the 5% hydroxy ethyl fiber cellulose solution, and adds an amount of sodium carbonate, and the pH value of solution value is remained between 6～7.The film of compound hydroxyethylcellulose increases about 20 times than the group number that can react on the original nylon membrane.

With compound behind the film drying under reduced pressure of hydroxyethylcellulose, immerse once more in the above-mentioned dioxane/xylene solution that contains three chlorotriazines, add phenylalanine, reaction is 5 hours under 40～50 ℃ of stirrings, thoroughly wash with dioxane, aqueous isopropanol and deionized water respectively then, and drying under reduced pressure, so just made the nylon 6-hydroxyethylcellulose composite affinity membrane that contains the phenylalanine affinity ligand, the content of phenylalanine can reach 175 μ mol on every gram film.

Embodiment 3

1g contains 25mmol/L Na through the PA membrane immersion 20ml of hydrochloric acid hydrolysis ₂CO ₃(pH8.0) and 1, in the reaction system of 4-butanediol diglycidyl ether (BDE), under 40 ℃-50 ℃, react 8-10h while stirring.Reaction is used isopropyl alcohol/deionized water (D-H of 1: 1 respectively after finishing ₂O) and deionized water take out in a large number and wash film, thoroughly to wash unreacted BDE off.And then film immersed contain in the aqueous solution of 2%HEC, with 2mol/L KOH solution pH value is transferred to 11, react 2-3h in 25 ℃-35 ℃ times, use 0.5mol/L NaHCO respectively ₃Solution and deionized water are thoroughly taken out and are washed till cleaning solution and are neutral.The PA membrane that combines HEC is inserted the deionized water by 20ml, 4ml BDE and 80mg KBH ₄Under 25 ℃-35 ℃, react 2-4h in the reaction solution of forming, use a large amount of deionized water rinsing films then.Again the film of cleaning is inserted 20ml by 0.5mol/L NaHCO ₃In the solution of forming with 100mg phenylalanine (Phe) in 60-70 ℃ of reaction 2-4h down, kept at room temperature overnight.With deionized water reacted film is thoroughly cleaned again, and immersed in the 0.2mol/L beta-mercaptoethanol solution (pH7.0), place 4h under the room temperature, frequent during this time agitated-film, with unreacted active group on the closing membrane, thoroughly clean vacuum drying again with deionized water.Affinity ligand (Phe) content of bonding is 205 μ mol/g on this film after measured, a little more than the affinity membrane with the preparation of three chlorotriazine bonding methods.

With 20 prepared affinity membranes diameter of packing into is in the dish formula affinity membrane separator of 47mm, allows 5ml contain 0.05mol/L NaH ₂PO ₄The human plasma sample of solution pumps in this separator with the flow velocity of 1ml/min, makes aglucon and the gamma globulin in the blood plasma on the film produce affine interaction.Elder generation's 25ml 1mol/L NaCl/ ethylene glycol (1: 1, V/V) affine gamma globulin on wash-out film under the same flow is collected eluent, after dialysis, do purity analysis with electrophoresis, the purity that proves this gamma globulin is 86.4%, the rate of recovery is 82.0%, a little more than three chlorotriazine methods.

Comparative example

The Ago-Gel Sepharose 4B that the foreign literature report adopts Pharmacia company to produce is a host material, and with phenylalanine dentate on the cyanogen bromide activation bonding, the aglucon bonded amount is 78 μ mol/ml, is lower than bonded amount of the present invention.A volume with this affinity media filling is about 20mlSepharose 4B-Phe affinity column, a purifying 5ml plasma sample, and the purity of gained immunoglobulin (Ig) is 80～82%, a little less than the present invention.The rate of recovery of gamma globulin is 75% in the blood plasma.Approach result of the present invention.

Claims (2)

1, a kind of composite affinity polyamide film that is used for the human plasma immunoglobulin purification is characterized in that: this film be bonding have the phenylalanine affinity ligand by hydroxyethyl cellulose modified polyamide miillpore filter composite membrane; The amount of hydroxyethylcellulose is every gram film 150～225mg, and the amount of phenylalanine affinity ligand is every gram film 150～180 μ mol.

2, the described preparation method who is used for the composite affinity polyamide film of human plasma immunoglobulin purification of a kind of claim 1 is characterized in that:

The polyamide miillpore filter is hydrolyzed with concentrated hydrochloric acid;

Get the PA membrane after the 1g hydrolysis, put into the dimethylbenzene system that 50～150ml contains 30～60% dioxane, add three chlorotriazines of 0.5～1g more respectively in this system, activate, reaction was at room temperature carried out 30～60 minutes;

Film after the activation of three chlorotriazines immerses and contains in 1～5% hydroxy ethyl fiber cellulose solution, and adds sodium carbonate, and the pH value of solution value is remained between 6～7, reacts 2～4 hours down in 40～60 ℃;

With compound behind the film drying under reduced pressure of hydroxyethylcellulose, immerse once more in the above-mentioned dioxane/xylene solution that contains three chlorotriazines, add phenylalanine, under 40～50 ℃ of stirrings, reacted 4～5 hours.