CN1068887C - Synthesis of affinity film medium using cellulose as substrate - Google Patents

Abstract

A kind of synthetic method of the affinity membrane medium that takes cellulose as matrix is to adopt glycidyl methacrylate or glycidyl acrylate to carry out self-polymerization reaction to obtain polymer, carry out grafting reaction with cellulose again, carry out finally Ring-opening reaction to obtain an affinity medium in which the active group is an aldehyde group or an amino group. The self-polymerization and branching reaction is carried out in a neutral medium, and the reaction temperature is 50-100 ° C. The ring-opening reaction temperature of the amino group is 40-80 ℃. The affinity medium prepared by this method improves the binding capacity of cellulose, and will have broad application prospects in the large-scale separation and purification of biological products and human immunotherapy.

Description

Synthetic method of affinity membrane medium based on cellulose

The invention relates to a method for synthesizing an affinity membrane medium with cellulose as a matrix. Specifically, a composite medium is synthesized by copolymerization and grafting of cellulose as a substrate and glycidyl methacrylate, and on this basis, ring-opening oxidation or ring-opening is connected to spacer arms of different lengths such as amino, ethylenediamine, hydrazine, etc. Synthesis of affinity media that can attach bioligands with reactive groups.

In recent years, with the development of bioengineering technology, people's understanding of biological products has continued to deepen, and the demand has continued to increase. Subsequently, various analytical separation methods for the purification of biological products have been greatly developed. Among them, affinity chromatography has become an important means of analyzing biological macromolecules such as proteins and nucleic acids due to its good specificity and high purification multiples. At present, the components that can be used as affinity media include agarose, dextran, cellulose, silicon limbs, polyacrylamides, porous glass, and various polymers. Among them, agarose is the earliest and most widely used normal-pressure chromatographic medium. It has a uniform structure and extremely low non-specific adsorption. It is a very good chromatographic medium material. In the next few years, agarose will be the dominant carrier of affinity chromatography It is difficult to be replaced by other substrates. However, agarose is a gel-like substance, and even if it is cross-linked, the pressure it can withstand is extremely limited. The same is true for colloidal carriers such as dextran, which is difficult to meet the requirements of large-scale production. Jaosoo J.C〖Agr.Foodchem.19(1971)581〗studied the relationship between the high flow rate and the pressure drop of Sephadex G150 gel column. When the flow rate increases, the pressure increases, but when the flow rate decreases to a certain limit, the pressure drop decreases. This shows that the gel has irreversible deformation, increasing the column height, and the inflection point of the curve is the allowable flow rate to decrease. It can be seen that the gel matrix is difficult to scale up and cannot meet the requirements of large-scale preparation. Another common chromatographic medium, silica gel, is especially common in high-efficiency affinity chromatography because of its good mechanical properties and high pressure resistance. However, the silanol on the surface of silica gel has some ionic properties, which makes the surface of silica gel strong non-specific adsorption, and silica gel The narrow pH range limits the application of silica gel in affinity chromatography.

In recent years, although commercial agarose, silica gel and other separation media have been widely used in the literature, the study of media has left the traditional media such as simple agarose and silica gel, and the search for composite media has become a direction of affinity media research. The use of affinity media in large-scale production has the following requirements: (1) low cost, (2) withstand high pressure, (3) high affinity capacity, (4) low degree of expansion, (5) no deformation under compression. As an affinity medium, cellulose has good mechanical properties and high chemical stability, and is the cheapest and easy-to-obtain raw material among all chromatographic media. Cellulose is the preferred affinity medium for large-scale production and amplification. However, the cellulose medium itself has great defects, which limit its application in affinity chromatography. Mainly caused by the structure of cellulose. Cellulose is composed of many β-D-glucose connected by 1-4 glycosidic bonds into straight chains, the straight chains are parallel to each other, and the hydroxyl groups of glucose between chains can form hydrogen bonds. This very ordered structure is the crystalline region of cellulose. Glycosidic chains leaving this crystalline region form the non-crystalline region. The crystalline and non-crystalline regions are particularly sensitive to the physical and chemical conditions of affinity chromatography. The derivatization reaction of cellulose generally occurs in the low-degree crystallization or non-crystalline regions, which makes the linked ligands quite heterogeneous microscopically and can be activated. The group content of cellulose is much lower than that of agarose, which makes it difficult to achieve the desired affinity effect with pure cellulose media.

The purpose of the present invention is to provide an affinity membrane medium and synthesis method with cellulose as the matrix. The affinity media prepared in this way greatly enhanced the binding capacity of cellulose. The cellulose composite medium can be easily formed into a membrane. The membrane medium has the advantages of small expansion, good permeability, high pressure resistance, and not easily deformed. Membrane media can be easily packed into axial columns and radial columns. Axial columns are suitable for small-scale separation and purification. Radial columns are suitable for large-scale separation and purification due to their linear amplification and fast flow rate. The method will have broad application prospects in the large-scale separation and purification of biological products and human immunotherapy.

In order to achieve the above object, the present invention uses cellulose and glycidyl methacrylate copolymerization grafting, so that the few derivatizable hydroxyl groups in the cellulose are bonded to a polymer chain, and each monomer in this polymer chain Containing a reactive active functional group can greatly increase the ligand capacity of cellulose, and the content of the active functional group can be controlled by changing the polymerization conditions and bonding conditions. The active group content is 1.0-2.5mmol/g (medium), which solves the problem of low derivatization capacity of cellulose itself. At the same time, due to the accumulation of high molecular substances on the surface of cellulose, the degree of swelling of cellulose is reduced, and because the cellulose composite medium is easy to form a membrane, the membrane medium produced has good permeability and fast flow rate, and is suitable for large biological macromolecules. scale separation. The above-mentioned method for synthesizing an affinity medium of the present invention is characterized in that it adopts glycidyl methacrylate or glycidyl acrylate to carry out self-polymerization to obtain a polymer, then carries out grafting reaction with cellulose, and finally ring-opening reaction, The affinity medium whose active group is aldehyde group or amino group is obtained, self-polymerization and grafting reaction are carried out in neutral medium, the reaction temperature is 50-100 DEG C, and the ring-opening reaction temperature is 40-80 DEG C. At the same time, an initiator, such as azobisisobutyronitrile, n-butyllithium or ammonium persulfate and sodium thiosulfate, should be added in the self-polymerization and grafting reactions, and the amount of the initiator added is 1 to 5% of the weight of the reaction monomer. . In addition, the ring-opening reaction should be controlled at a certain pH value, and the pH value is 8-12 when the ring-opening reactant is hexamethylenediamine. Specifically, the present invention takes cellulose as the preparation of the matrix-based affinity medium by the following three steps:

1. Glycidyl methacrylate or glycidyl acrylate self-polymerizes into polymers of a certain size;

2. Grafting reaction between cellulose and polymer;

3. Preparation of affinity media with different spacer arm lengths.

In above-mentioned 1, 2 reaction, reaction medium is neutral aqueous solution, wherein the content of cellulose is 1.5～2.5%, the content of glycidyl methacrylate or glycidyl acrylate is 3～10%, control reaction temperature The temperature is 50-100°C, and the self-polymerization grafting reaction time is preferably 0.5-3 hours. In the reaction process, an initiator should be added to induce the reaction. The initiator of the anionic polymerization reaction used is azobisisobutyronitrile, butyllithium or ammonium persulfate and sodium thiosulfate, and the amount of the initiator added is the amount of the reaction monomer. 1-5% of the total. The ring-opening reaction of the amino group is carried out at 40-80°C and at a certain pH value, and the specific operation process can be carried out according to conventional techniques. Below by example the technology of the present invention is given further illustration:

Example 1 Preparation of Composite Medium

Add 100ml of water to a 250ml flask with a self-installed mixer, thermometer and condenser, add 2 grams of cellulose when the temperature rises to 80°C, stir well, add 5ml of glycidyl methacrylate, stir for 5 minutes, add Contain a mixed solution (about 10ml) of 0.5 grams each of ammonium persulfate and sodium thiosulfate, react at a constant temperature for 2 hours, stop the reaction, wash with a large amount of water after cooling down to room temperature, and then wash off the residual organic matter on the surface of the cellulose with acetone or ethanol , and dried in a vacuum oven to synthesize a composite medium with active epoxy groups. The active epoxy group content in the composite medium is 1.0-2.5mmol/g (dry medium).

Example 2 Synthesis of an affinity medium with 4 atom spacers

The epoxy-type composite medium prepared in Example 1 is placed in a hydrochloric acid solution of pH=0.5, and stirred evenly, so that the epoxy is fully ring-opened to become an ortho hydroxyl group. Generally, it takes 3 days at room temperature, and the epoxy in the medium cannot be detected. Stop the reaction, filter, wash fully with water until neutral, then add to newly prepared 1.5% sodium iodate (NalO ₄ ) for 30 minutes, filter, wash with a large amount of water, and dry the membrane. The membrane medium was packed in a small column for the ligand experiment. Equilibrate the small column with 0.1mol/l pH=7.6 phosphate buffer, take a certain amount of protein and dissolve it in the equilibrium buffer, load the sample at room temperature or 4°C for 4 hours or longer, and wash with the equilibrium buffer until A280 is free. Absorption, balance with boric acid buffer solution of 0.1mol/lpH=8.2, passivate with glycine ethyl ester hydrochloride solution for 1 hour, then add a certain amount of NaBH ₄ or NaCHBH ₃ to reduce carbon-nitrogen bond, and reduce for 12 hours or more , and then passed through 0.1mol/lpH=8.2 boric acid buffer, 0.2mol/lpH=2.3 glycine solution, 0.1mol/lpH=7.6 phosphate buffer containing 1mol/lNaCl, and finally 0.1mol/lpH=7.6 phosphoric acid Buffer equilibration, synthesis of affinity media with arm lengths of 4 atoms.

Example 3: Synthesis of an affinity medium with an arm length of 11 atoms

Use 3 grams of the epoxy-type composite medium prepared in Example 1, add 150ml of water, stir vigorously until the dispersion is even, after heating up to 80°C, add 10-30ml of ammonia water, control the pH value to 8-12, reflux for 1-2 hours, pump Filter until the filtrate is neutral. Make a membrane column, equilibrate with 0.1mol/l boric acid buffer solution with pH=8.2, circulate with a certain amount of 0.25% glutaraldehyde solution for 2 hours, pass through 2mol/l acetic acid solution, wash with pure water until A280 has no absorption, and use 0.1mol/l l Phosphate buffer with pH=7.6 is equilibrated, and the procedure for binding ligands is the same as in Example 1. Affinity media with arms length 11 atoms were synthesized.

Example 4: Synthesis of an affinity medium with an arm length of 18 atoms

Add 3 grams of the epoxy-type composite medium prepared in Example 1 into 150ml of water, stir vigorously until the dispersion is uniform, and after heating up to 80°C, add ethylenediamine solids equivalent to 1 to 5 times the epoxy content, and control the pH to 8 to 12, react for 1 to 2 hours, take out and wash to neutrality, make a membrane, dry and pack into a column, activate with glutaraldehyde and attach a ligand. The steps are the same as in Example 1, and the synthetic arm length is 18 atoms.

Comparative example 1

Comparison of the QHprotein A adsorption column prepared by the technology of the present invention and the ZETAFINITY-10 protein A adsorption column produced by the U.S. CUNO company

(1) Experimental materials and methods

The bovine serum albumin used in the experiment was electrophoretic pure, and the IgG was immunologically pure, both purchased from Huamei Biochemical Reagent Company. The pump used is Cole Parmer model 7518-10, the detector is GILSON model 112 ultraviolet detector, the detection wavelength is 254 (in)/280 (out), Sensitivity (AUFS) 1.0, KNUER recorder, paper speed 1.0mm/min. 752C UV-Vis spectrophotometer (produced by Shanghai No. 2 Analytical Instrument Factory). Determination of protein concentration: A280 ultraviolet detection method, determined by bovine serum albumin standard concentration curve.

(2) Brief introduction of QH protein A column and ZETA-10 protein A column

QH protein A column: The membrane medium is synthesized according to the technical method of the present invention, the column size is 8×20mmI.D., the medium weight is 1.04g, and when the flow rate is 4ml/min, the pre-column pressure is 10psi. The column shell is plexiglass, ZETA-10protein A column: the medium is an affinity medium synthesized with cellulose as the matrix, the size is 38×10mmI.D., and the medium can hold 0.9～1.0g. The column shell is made of plastic products. When the pressure is 3ml/min, the column pressure increases and the column is easily broken.

(3) Comparison of the maximum adsorption capacity of human IgG

Human IgG was dissolved in 0.01mol/pH=7.5 phosphate buffer, the concentration was 10.6mg/ml, 4ml was put on the column, circulated for 10 minutes, the baseline of the recorder was washed with equilibrium buffer, and 0.2mol/l pH-2.3 glycine buffer was used Elute to the baseline, collect the eluate, and measure the concentration and volume. The sample loading and processing methods of the two columns are exactly the same. The result is that when the QH protein A column and the ZETA-10 protein A column are used, the maximum adsorption capacity for human IgG is 32.0mg/g dry medium and 12.0mg/g dry medium respectively .

(4) Comparison of maximum non-specific adsorption capacity

Dissolve bovine serum albumin in 0.01mol/l pH=7.5 phosphate buffer with a concentration of 1mg/ml, take 4ml of sample and cycle for 10 minutes, wash the baseline of the recorder with equilibrium buffer, and wash with pH=7.5mol/l NaCl Remove to the baseline, collect the eluate, measure the concentration and volume, the loading and processing methods of the two columns are exactly the same, the result is that the QH protein A column and ZETA-10 protein A column are used, and the maximum non-specific adsorption capacity is 1.30 mg/g dry medium and 2.11 mg/g dry medium.

Comparative example 2

Comparison of protein A membrane adsorption columns and protein A agarose adsorption columns for immunosorbent therapy.

Immunoadsorption therapy is to remove endogenous and exogenous pathogenic factors in human blood through antigen-antibody immune reaction or physical and chemical action, and purify the blood, so as to achieve the purpose of treating some suspected diseases. In recent years, immunoadsorption therapy has gradually become an important branch of blood purification technology, and has increasingly attracted the attention of the medical community. It is protein A that has been used in clinical treatment with better effect. At present, what has been commercially sold is the protein A immunoadsorption column with agarose as the matrix produced by Gambro Company of Sweden. This column contains 62.5ml of agar, the binding capacity of protein A is 20mgIgG/ml agar, the shell is made of acrylic ester into a cylindrical shape of 50×40mm, and the plasma perfusion rate is 15-35ml/min. Immunoadsorption column has not been reported yet. A protein A immunoadsorption column with a spacer arm length of 18 atoms was synthesized by the technical method of the present invention. There are two types of columns, one is the axial column, the size is 14×60mm, I.D, the medium is 12g, the plasma perfusion rate is 20-80ml/min, the protein A binding capacity is 32mg lgG/g dry medium, the other is The column is in the form of a radial column, which adopts a spiral-wound membrane module structure. The liquid flows from the periphery of the column to the column core. The size is 20×60mmI.D., and the inner medium is 13g. The ability to bind remains unchanged. Normal human plasma passes through the above protein A membrane adsorption column, and the adsorbed IgG is eluted with glycine=HCL buffer solution with pH=2.3, and the co-purity is checked by mass spectrometry. The results show that the protein A membrane adsorption column prepared by the technology of the present invention Good specificity. The proteinA membrane adsorption column has been sterilized and depyrogenated, and the clinical animal experiment effect is good.

As can be seen from the above comparison, the adsorption capacity of the protein A immunosorbent column prepared by the technology of the present invention is nearly 2 times higher than that of the ZETA-10protein A column of the U.S. CUNO company, and the adsorption capacity per unit volume is equivalent to the agarose-based column. One-half of the protein A adsorption column, but its flow rate is significantly higher than that of the agarose column.

The results of the above examples and comparative examples show that the synthetic affinity membrane medium of the technical method of the present invention avoids the weakness of soft substrates such as agarose, and has strong pressure resistance. It can still exist stably at a high flow rate, and the separation operation can be completed in a short time. The macromolecule flows in the membrane gap in a convective manner, and can quickly combine with the ligand, which improves the mass transfer in the column. Theoretically speaking , radial membrane chromatography has the characteristics of linearity and large size, and is suitable for large-scale separation and purification. The method will have broad application prospects in the large-scale separation and purification of biological products and human immunotherapy.

Claims (2)

1. one kind is the synthetic method of the affinity film medium of matrix with the Mierocrystalline cellulose, it is characterized in that it being to adopt to carry out self-polymeric reaction with glytidyl methacrylate or acrylic acid epoxy propyl ester and obtain polymkeric substance, carry out graft reaction with Mierocrystalline cellulose again, carry out ring-opening reaction at last, obtain active group and be aldehyde radical or be amino affinity media, autohemagglutination and graft reaction carry out in neutral medium, temperature of reaction is 50～100 ℃, amino ring-opening reaction temperature is 40～80 ℃, to add the initiator Diisopropyl azodicarboxylate in autohemagglutination and the graft reaction, n-Butyl Lithium or ammonium persulphate and Sulfothiorine, initiator add-on be reaction monomers heavy 1～5%.

2. according to the described synthetic method of claim 1, the pH value is 8～12 when it is characterized in that the ring-opening reaction thing is quadrol.