KR100302953B1 - A process for adsorbing proteins onto a copolymer using crosslinked poly(methacrylic anhydride) copolymer beads - Google Patents

Abstract

PURPOSE: Provided is a method for the adsorption of proteins by using crosslinked methacrylic anhydride copolymer beads useful for an adsorbent, a material for affinity chromatography, ion exchange resin and precursors thereof. CONSTITUTION: The method for the adsorption of proteins onto copolymers functionalized with functional groups having affinity to proteins comprises the step of contacting proteins with a poly(methacrylic anhydride)-based copolymer in the form of crosslinked spherical copolymer beads having a particle size of 10 micrometers to 2 mm. The protein is aqueous suspension and the beads are microporous. Preferably, the copolymer is functionalized with carboxylic acid functional groups.

Description

Protein adsorption using cross-linked methacrylic anhydride copolymers {A PROCESS FOR ADSORBING PROTEINS ONTO A COPOLYMER USING CROSSLINKED POLY (METHACRYLIC ANHYDRIDE) COPOLYMER BEADS}

The present invention relates to crosslinked, insoluble copolymers and, more particularly, to a method for adsorbing proteins using crosslinked copolymers of methacrylic anhydride suitable for use as adsorbents and ion exchange resin precursors.

Ion exchange resins prepared by copolymerizing acrylic and methacrylic acid with crosslinking monomers are well known and they are useful as weakly acidic cation exchange resins.

Because these acid monomers are hydrophilic and to some extent water soluble, it is more difficult to prepare these copolymers by suspension polymerization than resins made from acrylic and methacryl esters. The esters are relatively insoluble in water and hydrophobic and thus polymerize in oily droplets to form relatively clean beads. However, acids tend to partially polymerize in the aqueous suspending phase, and also consume monomers and form undesirable aqueous phase polymers that hinder the formation of clean copolymer beads. .

Special techniques must be used to minimize the tendency of acid monomers to dissolve in the aqueous phase. One such technique is to add an inorganic salt to the aqueous phase to 'salting out' the monomer, or to reduce its solubility in the resulting brine to the point where its polymerization is forced into the oil phase required. However, in these cases, not only must the salt be recovered for reuse or to dispose of the aqueous phase waste without causing a state problem, but this also leads to deformation of the oil phase droplets during the polymerization. This promotes the formation of non-spherical copolymer beads, which non-spherical copolymer beads have unexpected hydraulic properties and are difficult to fill in an ion exchange column.

Porous resin beads are particularly preferred for some applications, such as, for example, as ion exchangers or adsorbents with fast kinetics properties. Porosity is introduced into the beads in a number of different ways, including US Patent No. The use of a phase-separating agent to form macroreticular beads as disclosed in 4,221,871 is one common method of calculating this porosity. The choice of phase separator provides control over the total porosity, pore size and pore-size distribution in the final beads. Since methacrylic acid in the oil phase dissolves some of the water coming from the aqueous phase, water reduces the solubility of the total monomer solution in the oil phase, and phase separation occurs in the absence of the phase separator introduced. As a result, the preparation of the macrologous resins from methacrylic acid is simpler, but the adjustment of the pore parameters is limited.

Acid anhydrides are known to hydrolyze in the presence of strong acids or strong bases to form acids themselves. US Patent No. of Simon et al. 2,988,541 or US Pat. As disclosed in 3,871,964, anhydride polymers, such as allic anhydride polymers, introduce reactive sites into the copolymer that can be used for subsequent reactions such as ion exchange functionalization. Extensive data of esters, amides and nitriles of methacrylic acid have described methacrylic anhydride as a monomer for preparing copolymers which can be subsequently functionalized as weakly acidic cation exchangers (US Pat. No. 2,324,935 to kautter et al.). , Bayer, British Patent No. 894,391 suggested methacrylic anhydride as one of a wide range of olefinically unsaturated carboxylic acids, esters and anhydrides that can produce copolymers having a 'sponge structure', but disclosed a substantial process for preparing copolymers. There is no bar.

As another example, US Patent No. 4,070,384, US Pat. 3,764,477 and Barnes, US Patent No. 2,308,581 discloses the preparation of copolymers of methacrylic anhydride by bulk or precipitation polymerization or reversible suspension polymerization in which the continuous, suspended phase is organic rather than an aqueous solution. Riemann et al. Mentioned only the possibility of suspension polymerization in the above patents, but did not exemplify the actual preparation method.

It is therefore an object of the present invention to provide a method for adsorbing proteins using crosslinked methacrylic anhydride copolymer beads useful as adsorbents, affinity-chromatography materials, ion exchange resins and their precursors.

The beads used in the present invention are polymerized from a mixture comprising at least 50 wt% methacrylic anhydride and about 0.1-50 wt% polyethylene-based unsaturated crosslinking monomers. The inventors have found a way to allow such copolymer beads to be produced with controlled surface area and particle size and to control the porosity when porous beads are produced. The method comprises the following steps.

a. Methacrylic acid in an amount of at least 50 wt% based on the weight of monomers in the aqueous suspension medium.

Anhydrides, cross-linking monomers and monomer-soluble free-radical polymerization initiators

Suspending droplets of monomer mixture;

b. The droplets until the monomer mixture polymerized to form copolymer beads,

Heating above the decomposition temperature of the polymerization initiator; And

c. Separating copolymer beads from the suspension medium.

According to the present invention, there is provided a protein comprising contacting a protein with a poly (methylcrylic anhydride) -based functional copolymer in the form of a crosslinked spherical copolymer bead having a particle size ranging from 10 μm to 2 mm prepared by the method. A method of adsorbing a protein onto a copolymer functionalized with a functional group having an affinity for is provided.

Although other monomers are copolymerized with methacrylic acid to form the copolymer beads of the present invention, the copolymer is primarily poly (methacrylic anhydride). The term 'predominantly' as used herein means that the polymer contains 50 wt% or more of polymerized units of the polymer (in this case, poly (methacrylic anhydride)).

Monomers copolymerized with methacrylic anhydride to form copolymer beads of the present invention include at least one polyethylene-based unsaturated cross-linking monomer present at levels of about 0.1-50 wt% of the total monomers. Cross-linking monomers or monomers are for example ethylene glycol dimethacrylate, ethylene glycol diacrylate, trimethylolpropane di- and tri-acrylate, trimethylolpropane di- and tri-methacrylate, divinyl ketone, allyl Acrylate, diallyl malate, diallyl fumarate, diallyl succinate, diallyl carbonate, diallyl malonate, diallyl oxalate, diallyl adipate, diallyl sebacate, divinyl sebacate, N, N Polyvinyl or polyallyl ethers of '-methylenediacrylamide, N, N'-methylenedimethacrylamide, and glycols, polyvinyl or polyallyl ethers of glycerol, polyvinyl or polyallyl ethers of pentaerythritol , Acrylic crosslinkers such as polyvinyl or polyallyl ethers of mono- or di-thio-derivatives of glycols or resorcinol and the like It may be aliphatic, as (acrylic crosslinkers); Or aromatic, such as, for example, styrenic crosslinkers such as divinylbenzene, trivinylbenzene, divinyltoluenes, divinylnaphthylenes, diallyl phthalate, divinylxylene, divinylethylbenzene, and the like. May be; Or heterocyclic crosslinkers such as divinylpyridine. Such cross-linking monomers are well known to those skilled in the art.

Other monomers that may be copolymerized with methacrylic anhydride include other esters, such as acrylic acid and methacrylic acid, and their esters such as their C ₁ -C ₁₈ alkyl, cycloalkyl, aryl, aralkyl and alkaryl alcohols. Acrylic monomers. Other monomers that can be copolymerized with methacrylic anhydride include acrylonitrile and methacrylonitrile, vinyl alkyl ethers such as vinyl chloride, vinyl formate, methylvinyl ether, acrylamide, methacrylamide, and ethylvinyl. Aromatic monomers such as benzene, styrene, α-methylstyrene, vinyltoluene, vinylnaphthalene and the like. The other monomers may be present alone or as a mixture of one or more other monomers. Methacrylic anhydride is present at a level of at least 50 wt% in all monomer mixtures useful for the preparation of the copolymer beads of the invention.

The macroporous copolymer beads of the present invention can be prepared by suspension polymerizing a monomer mixture comprising at least 50 wt% methacrylic anhydride.

In suspension polymerization, the monomer mixture is suspended as droplets in an aqueous suspension medium optionally containing stabilizers and other additives to promote stable and uniformly sized droplets. The reaction is initiated by the production of free radicals in the monomer mixture, which may be derived from the thermal decomposition of free radical-producing initiators such as peroxides, peracid salts, azo initiators in the presence of monomers, and also t May be derived from redox initiators such as -butyl hydrogen peroxide / sodium formaldehyde hydrosulfite, or from ultraviolet or other ionizing radiation. Preferred in the initiation of the polymerization is the inclusion of a monomer-stable, heat-labile initiator in the monomer mixture and heating the suspension medium in which the monomer droplets are suspended to a temperature above the thermal decomposition temperature of the initiator.

However, monomer-stable, thermally unstable initiators include, for example, benzoyl peroxide, t-butyl hydrogen peroxide, cumene peroxide, α-cumyl peroxy neodecanoate, tetralin peroxide, peroxide Peroxides such as acetyl, caproyl peroxide, t-butyl perbenzoate, t-butyl diperphthalate, methyl ethyl ketone and the like and hydrogen peroxide compounds and related initiators are included. Other useful ones include azodiisobutyronitrile, azodiisobutyrylamide, azobis (α, α-dimethylvaleronitrile), azobis (α-methylbutyronitrile) and dimethyl, diethyl or dibutyl azobis ( Azoinitiators such as methyl valerate). Peroxide initiators are preferably used at a level of about 0.01-3 wt% based on the total weight of monomers, and azo initiators are preferably used at a level of about 0.01-2 wt% based on the total weight of monomers.

Preferred initiators are preferred, such as α-cumyl peroxy neodecanoate and di (4-t-butylcyclohexyl) peroxydicarbonate, which have relatively low decomposition temperatures, i. This is because polymerization is possible at relatively low temperatures that are less hydrolyzed or less reactive with other components present in the reactants.

Preferred copolymer beads of the present invention are macroporous. That is, they include pores having a diameter in the range of about 5-10,000 nm, which impart an internal pore volume (5% pore volume) to at least about 5% of the volume of the beads themselves. As a result of the porosity, the surface area of the macroporous beads is significantly larger than the surface area of gel polymer beads (those without macroporosity).

One method of introducing such porosity is described in U.S. Pat. 4,221,871, which comprises the introduction of porogens, also known as precipitants, phase extenders and phase separators, into the monomer mixture. Porogens are good solvents for monomers and poor solvents for polymers, and preferred porogens do not react with monomers or polymers. Particularly preferred porogens useful for preparing the copolymer beads of the present invention are iso-octane, methyl isobutyl ketone, methylisobutyl carbinol, xylene, toluene, di-n-butyl ether and the like. Particularly preferred are more hydrophobic porogens such as iso-octane and xylene because they promote less hydrolysis of anhydride groups during polymerization. The pore size and surface area of macroporous polymer beads can be controlled by techniques known to those skilled in the art, such techniques as porogen type and concentration, crosslinker type and concentration, initiator type and concentration, polymerization temperature. And the like. Other methods of introducing macroporosity into copolymer beads, well known to those skilled in the art, may also be used.

On the other hand, other known methods for preparing copolymer beads can be used, as gel or macroporous beads, using methacrylic anhydride monomers or mixtures of the foregoing monomers to produce the copolymer beads of the present invention. The monomer is known as seed beads and can be introduced into an aqueous suspension of pre-formed polymer beads that can swell in the monomer, then the monomer taken in the beads to swell the seed beads is polymerized. . In this process the polymer beads may be crosslinked, in which case the methacrylic anhydride may be the sole monomer or the monomer mixture may not have any additional crosslinking monomers. The polymer beads may not be crosslinked, in which case the monomer mixture preferably contains crosslinking monomers. Preferably, the monomer mixture preferably contains a polymerization initiator. Since the growth of seed particles can be controlled in this process, this seed process makes it possible to effectively control the final particle size. In addition, if the seed beads have a uniform diameter as a result of particle size classification or as a result of a process of producing particles of essentially uniform size, such as emulsion polymerization, the resulting copolymer beads will be uniform in size and seed It will be larger than the beads.

Another alternative process for producing beads of uniform diameter is to introduce the monomer mixture into the suspension medium by spraying the monomer mixture into the moving stream of the suspension medium at a controlled rate through one or more uniform diameter orifices. . The monomer droplets thus formed have a uniform diameter and when they are polymerized by heating to temperatures above the initiator decomposition temperature in the suspension medium, the resulting copolymer beads maintain particle-size uniformity. These beads may be used as formed or as seed beads in the above-described process of growing the seed beads to a larger and controlled size.

In all of the above-described optional processes and in other processes that are apparent to those skilled in the art as useful for preparing the copolymer beads of the present invention, it is important to introduce macroporosity into the beads, as described above. .

The size of the beads produced by any of the processes described above may be adjusted in the range of about 10 μm-2 mm, more preferably about 50 μm-1 mm. Particle size control in suspension polymerization is well known to those of ordinary skill in the art and includes varying the interfacial tension at the oil-water interface, changing the viscosity of the phases, and the stirring speed. There are techniques that seem to change.

A particular advantage of the present invention which is evident in the process of preparing the copolymer beads of the present invention by suspension polymerization is that the aqueous phase is used to reduce monomer solubility in water, for example to reduce the formation of aqueous polymers. There is no need for any inorganic salts or other water soluble ingredients, such as inhibitors, to be included in the aqueous suspension medium. Since methacrylic anhydride is relatively insoluble in water, there is no need for such salts and other water soluble components, but the presence of such is not necessarily excluded from the present invention.

Once formed, the copolymer beads can be reacted to introduce ion exchange groups or chelate functional groups, certain affinity ligands, or a wide variety of hydrophilic or hydrophobic fractions or combinations thereof into the beads. Anhydride functional groups from methacrylic anhydride present in the copolymer are particularly reactive and provide convenient reaction sites for the attachment of other functional groups. One route is the hydrolysis of the anhydride to weak acid carboxyl groups or the formation of cation-exchange functional groups on the copolymer. It is to be noted that at least a portion of the anhydride groups are hydrolyzed during the polymerization to form carboxyl groups, which are about 20% or less, mainly about 15% or less of the total anhydride groups that may theoretically be present in the copolymer. Further hydrolysis with acids to form weak acid carboxyl groups in hydrogen form, or further hydrolysis with alkali metal hydroxides to form weak acid carboxyl groups in alkali metal form may be used to increase weak acid functional groups.

Other examples of reactions that can be used to make functional groups on beads include reaction with ammonia to form ammonium salts of carboxylic acid groups, and amines to form amine salts (primary, secondary, tertiary amines and polyethyleneamines). And the like). Other suitable derivatives of anhydride polymers are alkyl or other esters and amides, preferably C ₁ -C ₁₈ alkyl esters and amides prepared by the reaction of carboxyl groups on the polymer with suitable amines or alkyl or phenylalkylalcohols. Alkylamides, dialkylamides, arylamides, alkarylamides and allylalkylamides with amine substituents, as well as aminoesters, amino amides, hydroxy amides and hydroxy esters, Wherein the functional groups are separated by alkylene, phenyl, alkyleneamine, alkylene oxide, phenylalkyl, phenylalkylphenyl or alkylphenylalkyl or other aryl groups. Substituents having amine salts, including amine or quaternary amine salt groups, may be used to separate the carboxyl groups on the polymer such that amide chains are formed with the polymer or in certain cases where higher temperatures are used, amide chains are formed with adjacent carboxyl groups. It is conveniently formed by reacting with the same multifunctional amines, or by reacting anhydride groups on the polymer with the same multifunctional amines so that ester chains with the polymer are formed. Likewise, sulfur-containing derivatives can be prepared by reacting anhydride groups with thiols and thioalcohols. How such functionalization of the copolymer beads can be performed will be apparent to those skilled in the art, as will additional functional groups that may be introduced into the copolymer beads.

The pores of the macroporous copolymer beads or functionalized resin beads of the present invention are filled with a monomer, which may be the same as or different from the monomer used to prepare the macroporous beads, and the monomer is polymerized, followed by a double functional group. Hybrid resins can be produced by functionalizing a newly formed polymer in the pores with a functional group different from that on the beads so that beads with dual functionality are produced. Optionally, the surface of macroporous copolymer beads or functionalized resin beads prepared from the prior art is applied with methacrylic anhydride monomers of the present invention or their pores are filled with methacrylic anhydride monomers of the present invention and then double functional The polymer may be polymerized to form a macrolog-type beads having a hybrid resin having a polymer or a polymer of methacrylic anhydride of the present invention. Advantages provided by the latter structure include control of swelling and diffusion kinetics by selection of the base macroporous polymer, improved chemical stability of the applied beads, and reduced bead contamination potential.

Hereinafter, the present invention will be described through examples. Unless stated otherwise, all percentages and ratios are by weight and all reagents used are of good commercial quality.

Example 1

In this embodiment, the macroporous resin beads of the present invention are produced from methacrylic anhydride.

462.45 g of deionized water, 27.5 g of 2% aqueous hydroxyethylcellulose solution, 9.0 g of 10% aqueous, 98% in a 1-litre four-neck round bottom plastic with stirrer, liquid-cooled condenser and thermocouple probe -Hydrolyzed polyvinyl alcohol having a molecular weight of 11,000-31,000, and 0.15 g sodium nitrite were added. The input was stirred at 300 rpm for 1 hour. Of 12.5 g of 0-xylene, 4.7 g of 80% divinylbenzene (mainly ethylvinylbenzene), 32.8 g of methacrylic anhydride (99.5% purity), and 0.5 g of α-cumyl peroxyneodecanoate A separate organic mixture was prepared by mixing a 75% mineral alcohol solution and 0.375 g of di (4-t-butylcyclohexyl) peroxydicarbonate and then stirring the mixture until the initiators were completely dissolved in the solution. . At the end of the 1-hour stirring period, stirring of the flask input was continued while slowly adding the organic mixture to the flask. When the addition of the organic mixture was complete, the flask contents were heated to 35 ° C. at 1 ° C./min with stirring and then to 75 ° C. at 0.2 ° C./min. The flask contents were kept at 75 ° C. for 1 hour with stirring and then cooled to room temperature. The resulting beads were drained, washed three times with the same amount of acetone as the bead volume and then vacuum dried in a buchner funnel. After drying, copolymer beads A 25 g sample was treated with 120 g of 50% (w / w) aqueous sodium hydroxide solution and 300 mL of deionized water for about 3 hours at a temperature of 80-90 ° C. and cooled overnight with stirring to dry the anhydride groups in the copolymer in sodium form. Switched to weak acid groups. Following the reaction, the beads were washed with deionized water, dehydrated and dried. The properties of these beads and the beads of Examples 2-6 are shown in Table 1 below.

Example 2

This example describes the preparation of macroporous resin beads of the present invention similar to Example 1 using the same organic mixture and different suspending agents and using a more rapid temperature profile to a lower final polymerization temperature.

The initial charge into the flask was 449.15 g of deionized water, 0.75 g of carboxymethyl-methylcellulose and 0.05 g of sodium lauryl sulfate, the reaction mixture heated to 60 ° C. at 1 ° C./min and then to room temperature. The procedure of Example 1 was repeated except that it was kept at that temperature for 5 hours before cooling.

Example 3

This example prepared macroporous polymer beads of the present invention similar to Example 2 using trimethylolpropane trimethacrylate instead of divinylbenzene as the crosslinking agent and methyl isobutyl ketone instead of 0-xylene as the organic solvent. Explain what to do.

23.0 g of methyl isobutyl ketone, 10.0 g of trimethylolpropane trimethacrylate, 27.5 g of methacryl as separately prepared organic mixtures using the procedure of Example 1 and the flask initial input and heating schedule of Example 2 Acid anhydride (99.5% purity), 0.5 g of α-cumyl peroxyneodecanoate 75 g mineral spirits solution and 0.375 g of di (4-t-butylcyclohexyl) peroxydicarbonate Using, polymer beads were prepared.

Example 4

This example describes another example of preparing the macroporous resin beads of the present invention using different conditions and reagents.

Using the procedure of Example 1, an aqueous mixture of 750 g deionized water, 1.26 g carboxymethylmethylcellulose, and 0.09 g pure powdered sodium lauryl sulfate was prepared in a reaction flask, where 50.0 g Methyl isobutyl ketone, 30.0 g trimethylolpropane trimethacrylate, 82.5 g methacrylic anhydride (92% purity), 1.0 g α-cumyl peroxyneodecanoate 75% mineral spirit solution and 0.76 An organic mixture of g of di (4-t-butylcyclohexyl) peroxydicarbonate was added. The reaction mixture was heated to 50 ° C. at 1 ° C./min, from 50 ° C. to 58 ° C. at 0.5 ° C./min and held at 58 ° C. for 1.5 hours, then cooled to room temperature, the resin beads of the present invention were recovered, dried and run Post-treatment as described in Example 1.

Some of these beads were functionalized with the polyamine according to the following procedure to form an anion exchange resin. A 0.677 g sample of dry copolymer beads was transferred to a 1 L round bottom flask and suspended in 250 g of deionized water with stirring. 3.0 g of triethylenetetraamine (TETA) was suspended in 250 ml of deionized water in a 500-ml beaker and the suspended amine was added dropwise to the flask containing the copolymer beads. After the amine addition, the flask contents were heated to 50 ml and kept at that temperature for 5 hours, then they were cooled, washed with deionized water and dried. Particle size measurement results for these TETA-functionalized beads are shown in Table 1 below.

Example 5

This embodiment describes another example of preparing the macroporous resin beads of the present invention using another organic mixture.

50.0 g of methylisobutyl ketone, 15.0 g of trimethylolpropane trimethacrylate, 97.5 g of methacrylic anhydride, using the procedure of Example 1 and the aqueous mixture of Example 4 and the temperature profile, as an organic mixture 92% purity), 1.0 g of a 75% mineral spirit solution of α-cumyl peroxyneodecanoate and 0.76 g of di (4-t-butylcyclohexyl) peroxydicarbonate were used to prepare the resin beads. The copolymer beads were recovered, dried and worked up according to the procedure of Example 1.

After drying, a sample of the copolymer was converted to TETA form as described in Example 4, and the particle size measurement results for these TETA-functionalized beads are shown in Table 1 below.

Example 6

This example describes the preparation of the macroporous resin beads of the present invention using low levels of the divinylbenzene crosslinkers of Examples 1 and 2 and the methyl isobutyl ketone porogen of Examples 3-5.

The procedure of Example 1, the aqueous mixture of Examples 4 and 5, and 30,645 g of methyl isobutyl ketone, 2.563 g of divinylbenzene (80% purity), 47.95 g of methacrylic anhydride (92% purity), 0.665 Resin beads were prepared using a 75% mineral spirit solution of g of α-cumyl peroxyneodecanoate and an organic mixture of 0.5 g of di (4-t-butylcyclohexyl) peroxydicarbonate. The reaction mixture was heated to 50 ° C. at 1 ° C./min, held at 50 ° C. for 4 hours and cooled to room temperature. The copolymer beads were recovered, dried and worked up according to the procedure of Example 1.

Cytochrome-c Capacity and Recovery

Each of the previously prepared resins was tested for their ability to adsorb and elute protein cytochrome-c. To determine the cytochrome-c capacity, ie the amount of cytochrome-c that the resin can adsorb from a solution per unit volume of resin, a 20 ml wet sample of each resin was prepared with a 2.5-cm inner diameter and a minimum of 20 Pretreatment was carried out by transfer to a glass chromatography column with a cm length. The free liquid was withdrawn from the column by applying 2 bar of air pressure at the top of the column. The column was then filled with HPLC grade methanol, similarly discharged through the column at a pressure of 2 bar, and this process was repeated until 200 mL of methanol was discharged through the column.

After methanol was discharged from the column, 200 mL of pure deionized water was similarly discharged through the column. Water was drained from the column and 200 mL of 0.05M acetate buffer (pH was adjusted to 5.4 using 3.0 g glacial acetic acid, 50% sodium hydroxide solution in 1000 mL of water) was similarly drained through the column.

Accurate volumes of approximately 2-ml samples of pretreated resins were measured as described above, and the samples were diluted with acetate buffer so that the total volume of resin + buffer was 80 ml. Each sample was transferred to a 250-ml container and 20 ml of a 20 mg / ml solution of Cytochrome-c (Sigma C-2506; Sigma Chemical Company, St. Louis, Missouri) in acetate buffer was quickly added.

The vessel was sealed and shaken for 30 minutes and the resin allowed to settle for 5 minutes.

Ultraviolet absorption of the supernatant liquid was measured at 550 nanometers (nm) and the free cytochrome-c concentration was determined compared to that of the standard cytochrome-c solution. The cytochrome-c capacity of the resin was calculated by the following equation (1).

Capacity = (400-(100 x C _s )) / Vr ... (1)

In Equation (1), C _s is the cytochrome-c concentration (mg / ml) in the supernatant and Vr is the volume (ml) of the resin sample.

Cytochrome-c recovery, ie, the amount of cytochrome-c eluted and adsorbed onto the resin, was measured as follows. All of the 2-ml samples in the above mentioned volume measurements were transferred to a glass chromatography column with a 1 cm inner diameter and rinsed with 4-5 ml of acetate buffer to remove as much residual cytochrome-c solution as possible. Exactly 100.0 ml of 0.5 M sodium chloride / acetate buffer was passed through each resin sample at a rate of 1 ml / min, and then the ultraviolet absorption of the collected sodium chloride solution was measured at 550 nm.

The free cytochrome-c concentration in the sodium chloride solution was determined by comparing it with the standard solutions as described above, and the recovery rate for the resin was calculated by the following equation (2).

Recovery (%) = (100 x Cr) / (Vr x capacity) .... (2)

In formula (2), Cr is the concentration of cytochrome-c in sodium chloride solution (mg / ml), the dose is the cytochrome-c dose calculated by formula (1), and Vr is the volume of the resin sample.

The results of the above measurements on the resins of Examples 1-6 are shown in Table 1 below. Table 1 below also shows the results of streptomycin-dose measurements for the copolymers of Examples 4, 5 and 6, which are given in streptomycin active units per ml of wet resin. 1 mg of streptomycin sulfate corresponds to about 767 streptomycin activity units. Table 1 below also shows comparative results for two different milled and classified, weak acid, macroporous methacrylic acid-divinylbenzene resins of the prior art, one of which (Comparative Example A) ranges from 150-300 μm. And the other (Comparative Example B) has a particle size distribution of 75-150㎛.

Chart 1

Particle Size CEC ⁽³⁾ Cyto-c ⁽⁴⁾ Cyto-c Strep ⁽⁵⁾

Example (Μm) (meq / ml) Dose (mg / ml) % Recovery Recovery Rate (Strep Units)

1--72 91-

2 277 ⁽¹⁾ 6.86 144 82-

3 202 ⁽²⁾ 5.48 157 98-

4 160⁽²⁾6.95-7.72 19 85 305

5 160 ⁽²⁾ 8.73-9.17 44 81 251

6 500 6.9--101

A 150-300 11.7 82 100-

Comparison B 75-150 10.3 11 87-

(1) Particle size was measured in the form of sodium.

(2) Particle size was measured in the form of TETA.

(3) Cation Exchange Capacity

(4) cytochrome-c

(5) as Streptomycin activity units

Streptomycin dose

As can be seen from the results shown in Examples 1-6 and Table 1, the cation exchange capacity of the resins of the present invention is satisfactory for many purposes, and the capacity and recovery for proteins such as cytochrome-c and streptomycin It is also good to excellent, and the resins themselves can be prepared without the need for arduous processes, including salting to rule out monomer solubility problems.

Example 7

This example illustrates the preparation of the gel resin of the present invention using the suspension aids of Example 2 without the use of porogen.

Aqueous phase was prepared by mixing 958.47 g of deionized water, 1.44 g of carboxymethylmethyl reulose and 0.0864 g of sodium lauryl sulphate until 2-L, solid materials were dissolved in a round bottom flask. It was. 88.87 g of industrial divinylbenzene (55% purity), 163.97 g of methacrylic anhydride (94% purity), 4.8 g of di (4-t-butylcyclohexyl) peroxydicarbonate initiator and 3.2 g of α-cumyl Peroxyneodecanoate initiators were mixed until the initiators dissolved to form a separate organic phase. The organic phase was stirred and added to form a suspension of organic droplets in the aqueous phase. The mixture was heated from 25 ° C. to 60 ° C. at 0.5 ° C./min and held at 60 ° C. for 4 hours, due to exotherm, the temperature rose to 65 ° C. for 20 minutes. At the end of the reaction period the flask contents were cooled, the solids were separated from the liquid in a Buchner funnel and the solids were washed with acetone. This solid material was in the form of nonporous or gelled, copolymer beads.

As described above, the method of the present invention is good in terms of poor exchange rate and recovery rate for proteins while excluding the conventional problems.

Claims (8)

Functional groups having affinity for the protein, including contacting the protein with a poly (methacrylic anhydride) based functional copolymer in the form of a crosslinked spherical copolymer bead having a particle size ranging from 10 μm to 2 mm. A method of adsorbing a protein on a functionalized copolymer. The method of claim 1, wherein the protein is an aqueous suspension. The method of claim 1 wherein the beads are macroporous. The method of claim 1 wherein the copolymer is functionalized with carboxylic acid functional groups. The method of claim 2, wherein the protein adsorbed on the copolymer is subsequently transferred from the copolymer into a liquid different from the aqueous suspension. The method of claim 5, wherein at least 80 wt% of the protein adsorbed on the copolymer is subsequently transferred from the copolymer into a liquid different from the aqueous suspension. The method of claim 5, wherein at least 90 wt% of the protein adsorbed on the copolymer is subsequently transferred from the copolymer into a liquid different from the aqueous suspension. The method of claim 5, wherein at least 95 wt% of the protein adsorbed on the copolymer is subsequently transferred from the copolymer into a liquid different from the aqueous suspension.