CN1173771C - Polyamide Composite Affinity Membrane for Purification of Immunoglobulin in Human Plasma and Its Preparation - Google Patents

Abstract

A polyamide composite affinity membrane used for purification of immunoglobulin in human plasma is characterized in that: the membrane is a polyamide microstructure modified by hydroxyethyl cellulose bonded with a phenylalanine affinity ligand. Porous filter membrane composite membrane; the amount of hydroxyethyl cellulose is 150-225 mg per gram of membrane, and the amount of phenylalanine affinity ligand is 150-180 μmol per gram of membrane. The invention has good purification effect on gamma globulin in blood plasma, stable medium performance, convenient use and high recovery rate.

Description

Polyamide Composite Affinity Membrane for Purification of Immunoglobulin in Human Plasma and Its Preparation

The invention relates to separation membrane technology, and particularly provides a polyamide composite affinity membrane used for purifying immunoglobulin in human blood plasma and a preparation method thereof.

Most of the immunoglobulin purification methods reported in the literature are obtained by ethanol precipitation, but it can only precipitate various immunoglobulins together, and cannot separate different types of immunoglobulins, and the purity of the obtained products is not high. ideal. Some people use agarose, regenerated cellulose, and nylon membranes as raw materials to immobilize protein A or protein G to specifically adsorb immunoglobulins in plasma, and then use salt and organic solvents to absorb the immunoglobulins adsorbed on the above media. The protein is desorbed. However, this method has not been popularized and applied because the preparation of protein A and protein G is very troublesome, the price is also very expensive, and the immunoglobulin may be inactivated and denatured during the elution process. In addition, some people immobilized an exogenous lectin-Jacalin on agarose Sepharose, which can also purify immunoglobulins, but the purity and recovery rate of the obtained immunoglobulins are not very satisfactory.

The object of the present invention is to provide a polyamide composite affinity membrane and preparation method for the purification of immunoglobulin in human plasma, which has higher gamma globulin affinity capacity, and the obtained gamma globulin has higher purity .

The invention provides a polyamide composite affinity membrane for the purification of immunoglobulin in human plasma, which is characterized in that: the membrane is a hydroxyethyl cellulose modified membrane bonded with a phenylalanine affinity ligand. Polyamide microporous membrane composite membrane; the amount of hydroxyethyl cellulose is 150-225 mg per gram of membrane, and the amount of phenylalanine affinity ligand is 150-180 μmol per gram of membrane.

The present invention also provides the preparation method of the polyamide composite affinity membrane used for the purification of immunoglobulin in human plasma, characterized in that:

The polyamide microporous membrane (nylon 6 membrane) is hydrolyzed with concentrated hydrochloric acid;

Take 1g of the hydrolyzed polyamide film, put it into 50-150ml xylene system containing 30-60% dioxane, and then add 0.5-1g of trichlorotriazine to the system for activation. 30-60 minutes at room temperature;

The membrane activated by trichlorotriazine is then immersed in a solution containing 1-5% hydroxyethyl cellulose, and sodium carbonate is added to keep the pH value of the solution between 6 and 7, and react at 40-60°C for 2- 4 hours;

After drying the film compounded with hydroxyethyl cellulose under reduced pressure, immerse it again in the dioxane/xylene solution containing trichlorotriazine, add phenylalanine, and react at 40-50°C for 4-5 hours .

The difference between the present invention and prior art is:

1. For the first time, the microporous nylon 6 membrane was selected as the raw material, activated by the trichlorotriazine method, and hydroxyethyl cellulose was covalently bonded, which greatly increased the number of reactive groups on the membrane and reduced the resistance to removal. Non-specific adsorption of proteins other than immunoglobulins;

2. For the first time, the dioxane-xylene system was selected on the prepared nylon 6-hydroxyethyl cellulose composite membrane, and the phenylalanine affinity was bonded to the active chlorine atom on the trichlorotriazine. Ligands make the number of ligands on the membrane as high as 150-180 μmol/g, which greatly improves the affinity capacity of the medium;

3. Under the selected optimal affinity conditions, the affinity membrane for gamma globulin in human plasma can reach 15-25 mg/g membrane, which is about 50% higher than that of ordinary affinity media, and the obtained gamma globulin The purity is 80-85%.

In a word, the affinity membrane developed by the present invention has the characteristics of high ligand binding capacity, good purification effect on gamma globulin in plasma, stable medium performance, convenient use, high recovery rate, etc., and is the best choice for producing high-purity immunoglobulin. Effective tools and means have strong practicability and promotion and application value.

The present invention is described in detail below by way of examples.

Example 1

First, the polyamide microporous filter membrane (nylon 6 membrane) is hydrolyzed with concentrated hydrochloric acid, so that the amide groups on the membrane are converted into primary amino groups as much as possible, and the number of amino groups on the membrane before and after hydrolysis is quantitatively determined by the improved ninhydrin method .

The hydrolyzed polyamide membrane is soaked in NaOH solution, and after fully neutralizing the remaining HCl, it is fully washed with DH ₂ O, suction filtered, and vacuum-dried, and then activated by the trichlorotriazine method (CyCl ₃ ) , add 0.8g trichlorotriazine meter according to 1g polyamide membrane, react in 1: 1 (V/V) dioxane/xylene system, carry out reaction at room temperature for 60 minutes, after reaction finishes, first Wash thoroughly with dioxane and a large amount of DH ₂ O, and dry under reduced pressure in vacuo.

The membrane activated by trichlorotriazine is then immersed in a solution containing 3% hydroxyethyl cellulose, and an appropriate amount of sodium carbonate is added to keep the pH value of the solution between 6 and 7. React at 60°C for 4 hours, then wash 3 times with 0.1mol NaOH solution, wash with a large amount of water, and dry under reduced pressure; use the phenol-sulfuric acid method to determine the amount of hydroxyethyl cellulose composited on the nylon membrane, according to the fiber Glucose unit on the element molecule. The membrane compounded with hydroxyethyl cellulose has about 17 times more reactive groups than the original nylon membrane, and the composite membrane also greatly reduces the non-specific adsorption of biological macromolecules other than immunoglobulin.

After drying the film compounded with hydroxyethyl cellulose under reduced pressure, immerse it again in the dioxane/xylene solution containing trichlorotriazine, add phenylalanine, and react for 5 hours under stirring at 40-50°C , and then washed thoroughly with dioxane, isopropanol solution and deionized water, and dried under reduced pressure, so that the nylon 6-hydroxyethyl cellulose composite affinity ligand containing phenylalanine was made And membrane, the content of phenylalanine per gram of membrane can reach 165μmol.

The 20 affinity membranes with a diameter of 47 mm and phenylalanine ligands prepared by the above method were assembled in a disc-type affinity membrane separator. Dilute 4ml of human plasma with 0.05mol/L NaH ₂ PO ₄ solution, and pump the diluted plasma solution into the affinity membrane separator at a flow rate of 1ml/min to make the phenylalanine ligand on the membrane and gamma globulin generate affinity interaction. First use 50ml of 1mol/L sodium chloride solution at a flow rate of 2ml/min to wash off the remaining impurity proteins on the membrane, then use 1mol/L sodium chloride/ethylene glycol (1:1, V/V) instead, in the same The affinity-adsorbed immunoglobulin (γ-Globulin) on the membrane was eluted under the flow rate, and the eluate was collected. After dialysis with a dialysis bag, the sample was analyzed by electrophoresis, which proved that the purity of the obtained gamma immunoglobulin was 83.5%. The recovery rate of gamma globulin in plasma can reach 78%, which is higher than that of other purification methods.

Example 2

Soak the hydrolyzed polyamide membrane in Example 1 with NaOH solution, and fully neutralize the remaining HCl, then fully wash with DH ₂ O, suction filter, and vacuum-dry, and then use the trichlorotriazine method (CyCl ₃ ) To activate, add 1 g of trichlorotriazine to 1 g of polyamide membrane, and the reaction is carried out in a 1:1 (V/V) dioxane/xylene system.

The membrane activated by trichlorotriazine is then immersed in a solution containing 5% hydroxyethyl cellulose, and an appropriate amount of sodium carbonate is added to keep the pH value of the solution between 6 and 7. The membrane compounded with hydroxyethyl cellulose has about 20 times more reactive groups than the original nylon membrane.

After drying the film compounded with hydroxyethyl cellulose under reduced pressure, immerse it again in the dioxane/xylene solution containing trichlorotriazine, add phenylalanine, and react for 5 hours under stirring at 40-50°C , and then washed thoroughly with dioxane, isopropanol solution and deionized water, and dried under reduced pressure, so that the nylon 6-hydroxyethyl cellulose composite affinity ligand containing phenylalanine was made And the film, the content of phenylalanine per gram of film can reach 175μmol.

Example 3

Immerse 1g of polyamide membrane hydrolyzed by hydrochloric acid in 20ml of reaction system containing 25mmol/L Na ₂ CO ₃ (pH8.0) and 1,4-butanediol diglycidyl ether (BDE), at 40°C-50°C React while stirring for 8-10h. After the reaction, wash the membrane with 1:1 isopropanol/deionized water (DH ₂ O) and deionized water in large quantities to completely wash away unreacted BDE. Then immerse the membrane in an aqueous solution containing 2% HEC (HEC is hydroxyethyl cellulose), adjust the pH value to 11 with 2mol/L KOH solution, react at 25°C-35°C for 2-3h, and use 0.5 mol/L NaHCO ₃ solution and deionized water to wash thoroughly until the washing liquid is neutral. Put the polyamide membrane combined with HEC into the reaction solution consisting of 20ml deionized water, 4ml BDE and 80mg KBH ₄ to react at 25°C-35°C for 2-4h, and then wash the membrane with a large amount of deionized water. Then put the washed membrane into 20ml of a solution consisting of 0.5mol/L NaHCO ₃ and 100mg of phenylalanine (Phe) to react at 60-70°C for 2-4h, and leave it overnight at room temperature. The reacted membrane was then thoroughly washed with deionized water, immersed in a 0.2mol/L β-mercaptoethanol solution (pH 7.0), and left at room temperature for 4 hours, during which the membrane was often stirred to seal the unreacted activity on the membrane. groups, washed thoroughly with deionized water, and dried in vacuum. The content of the bound affinity ligand (Phe) on the membrane was determined to be 205 μmol/g, slightly higher than that of the affinity membrane prepared by the trichlorotriazine bonding method.

Put 20 prepared affinity membranes into a disc-type affinity membrane separator with a diameter of 47 mm, and pump 5 ml of human plasma samples containing 0.05 mol/L NaH ₂ PO ₄ solution into the separator at a flow rate of 1 ml/min. In the device, the ligands on the membrane have affinity interactions with the gamma globulin in the plasma. First use 25ml1mol/L NaCl/ethylene glycol (1:1, V/V) to elute the gamma globulin with affinity on the membrane at the same flow rate, collect the eluate, and after dialysis, use electrophoresis for purity analysis to prove The gamma globulin has a purity of 86.4% and a recovery rate of 82.0%, slightly higher than the trichlorotriazine method.

comparative example

Foreign literature reports adopt the agarose gel Sepharose 4B produced by Pharmacia Company as the matrix material, and use the cyanogen bromide activation method to bond the phenylalanine ligand, and the ligand bonding amount is 78 μ mol/ml, which is lower than that of the present invention. Amount of bonding. A Sepharose 4B-Phe affinity column with a volume of about 20ml packed with this affinity medium can once purify 5ml plasma samples, and the purity of the resulting immunoglobulin is 80-82%, which is slightly lower than the present invention. The recovery rate of gamma globulin in plasma was 75%. close to the results 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; Described bonding process is: polyamide miillpore filter composite membrane is hydrolyzed with concentrated hydrochloric acid, the activation of three chlorotriazines, behind the compound upward hydroxyethylcellulose, immersion contains in the dioxane/xylene solution of three chlorotriazines again, add phenylalanine, covalent bond phenylalanine aglucon.

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, putting into 50～150ml, to contain the dioxane percent by volume be 30～60% dimethylbenzene system, adds three chlorotriazines of 0.5～1g again in this system, activates, 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, under 40～50 ℃ of stirrings, reacted 4～5 hours.