JPS61272654A - Carrier for hydrophobic chromatography - Google Patents

Abstract

PURPOSE:To obtain high mechanical strength and recovery rate as well as a large protein absorption capacity by constituting a chromatographic carrier of the crosslinking unit selected from the polyacrylate and polymethacrylate of polyhydric alcohol and prescribed hydrophobic group-contg. ester and prescribed glycerol ester unit. CONSTITUTION:The hydrophobic chromatographic carrier is constituted of the crosslinking unit selected from the polyacrylate and polymethacrylate of the polyhydric alcohol, the hydrophobic group-contg. ester unit expressed by formula I or II (where R<1> is a hydrogen atom or methyl group, n is 1-18, m is 0-2, X is a hydrogen atom or alkyl group) and the glycerol ester unit expressed by formula III (where R<2> is a hydrogen atom or methyl group). The crosslinking agent occupies 10wt% (more particularly 30-60%) in the resin and the hydrophobic group-contg. ester is incorporated with the hydrophobic group at 0.1-3.5meq/g. The glycerol ester unit is incorporated into the carrier at 15-98% of the total number of moles of the esters (II) and (III).

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a hydrophobic chromatography carrier, and more specifically, it has a high adsorption amount to biologically related substances such as proteins and fermentation products, and The present invention relates to a hydrophobic chromatography carrier that also has high mechanical strength.

[Conventional technology]

Hydrophobic chromatography is a chromatography that separates proteins based on differences in adsorption power (affinity) based on hydrophobic interactions between hydrophobic groups on a carrier and hydrophobic residues on proteins.

The advantage of hydrophobic chromatography is that it has a high adsorption power for proteins in solutions with high salt concentrations, where ion exchange chromatography is no longer effective, and that proteins adsorbed in this way can be easily absorbed by simply lowering the salt concentration. Chromatography is particularly effective in the early stages of purification, as it can be recovered in high yield.

Conventionally, cross-linked agarose with various argyl groups has been known as a hydrophobic chromatography carrier, but in general, it is often difficult to elute adsorbed proteins, and the mechanical strength is weak ( Because it was expensive, it was not necessarily industrially advantageous.

[Purpose of the invention]

In view of the above-mentioned circumstances, and as a result of various studies to develop an industrially advantageous hydrophobic chromatography carrier, the present invention has been developed by introducing a hydrophobic group into a hydrophilic crosslinked copolymer. We have completed the present invention by discovering that it is possible to obtain a carrier that has a high adsorption capacity (particularly for proteins) and a low recovery rate based on the hydrophobic interaction.

[Structure of the invention]

That is, the gist of the present invention is to use a crosslinking agent unit selected from polyacrylic acid and polymethacrylic ester of polyhydric alcohols, and general formula (1) or 'i, (ll) (wherein R1 is a hydrogen atom or Indicates a methyl group, n is /-
A hydrophobic group-containing ester unit represented by W/l, m represents O-1 and X represents a hydrogen atom or an alkyl group, and a general formula (R1) (wherein R1 represents a hydrogen atom or a methyl group). The hydrophobic chromatography carrier has a structure consisting essentially of the glycerin ester units shown.

The present invention will be explained in detail below.

The hydrophobic chromatography carrier according to the present invention comprises a polyacrylic acid ester or a polymethacrylic acid ester of a polyhydric alcohol that imparts a crosslinked structure to the carrier, and the above general formula (1) that expresses hydrophobic interaction, including (11). The hydrophobic group-containing ester shown by and the general formula that imparts hydrophilicity (
It is essentially made up of three structural units with different functions: glycerin ester shown by 厘.

Polyacrylic esters or polymethacrylic esters of polyhydric alcohols are usually ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, propylene glycol dimethacrylate, diethylene glycol dimethacrylate, Trinolopylene glycol dimethacrylate, trimethylolpropane trimethacrylate, pentaerythritol tetramethacrylate, 3-butylene glycol dimethacrylate and their corresponding acrylates can be used alone or in combination. Preferably, ethylene glyco-k (D diacrylate or dimethacrylate is used. These crosslinking agents are used for lθ(
weight)% or more. Preferably resin 1
It accounts for o-to (weight) %, especially 30-6θ (weight) %. If the proportion of the crosslinking agent in the resin is too small, the swelling property of the resin will increase, resulting in a decrease in mechanical strength, making it unsuitable as a carrier for chromatography. On the other hand, when the ratio of the crosslinking agent becomes too large, the protein adsorption capacity decreases due to a decrease in the hydrophobic group-combined ester moiety, or the hydrophilicity decreases due to a decrease in the glycerin ester moiety.

The hydrophobic group of the hydrophobic group-containing ester and 1 to -0-O
H,,-0-0,H,,-0-C,lH7,-0-1]
,H,,"6H111%-0-011'+7 groups≠lower general formula (IVI) or the following general formula (Vl) is used.

”nH2nx GV
I (In the formula, n represents 7 to 1 g.) Ge'mH*m (V) (In the formula, m represents O-1, and X represents a water atom or an alkyl group.) The hydrophobic group-containing ester is The amount of hydrophobic groups introduced into the carrier is 0./
~, y, smeq/11 is present in the carrier. If the introduced amount is smaller than θ, /mθq/I, it will be impractical as a carrier for hydrophobic chromatography, and if the introduced amount is smaller than 3. If it becomes thicker than smeq/I, the hydrophilicity of the carrier decreases, resulting in moderate practical problems such as a decrease in protein recovery.

A more preferable amount of hydrophobic groups introduced into the carrier according to the present invention is θ,
j~3mθq/&. Further, the glycerin ester unit is preferably present in the carrier in an amount such that the number of moles of the glycerin ester unit is from 9r% to the total number of moles of (glycerin ester unit + hydrophobic group-containing ester unit). When the number of glycerin esters is small, the hydrophilicity of the carrier decreases.

The carrier according to the present invention is obtained by copolymerizing the above-mentioned crosslinking agent unit, hydrophobic group-containing ester unit, and glycerin ester by copolymerizing the crosslinking agent and glycidyl ester of acrylic acid or methacrylic acid. Then, the epoxy ring of the glycidyl ester is reacted with an alcohol or phenol to convert a part of the glycidyl ester into an ester containing a hydrophobic group, and then the remaining epoxy ring of the glycidyl ester is reacted with water to open it. It can be easily manufactured by ringing.

The polymerization reaction may be carried out by any known method, but it is usually preferable to carry out suspension polymerization in an aqueous medium in the presence of a suitable dispersion stabilizer to obtain a particulate copolymer. As the dispersion stabilizer, gelatin, sodium polyacrylate, carboxymethyl cellulose, polyvinyl alcohol, etc. are usually used. Further, it is preferable to dissolve salts in the aqueous N-form to prevent dissolution of the first reaction raw material in the aqueous medium. As the salt, sodium chloride, calcium chloride, sodium sulfate, sodium carbonate, etc. are used. The polymerization reaction is generally carried out using a suitable polymerization initiator.

Polymerization initiators generally include benzoyl peroxide, 1-butyl hydroperoxide, azobisisobutyronitrile, dimethylazobis(methylvalate), azobis(+I, α-dimethylvaleronitrile), co-, 2'-
Organic peroxides such as azobis-,2,+-dimethylvaleronitrile and organic azobis compounds are used. These polymerization initiators are usually used in an amount such that θ, 0/−, t (weight)% of the reaction raw materials.

The polymerization reaction is usually carried out at 60-90°C under stirring for 6 to 90°C. Photon in 20 hours. Further, in this one-coupling reaction, by employing the following method, the entire porous crosslinked one-coupling can be obtained. In other words, methods for producing porous crosslinked copolymers are well known, and usually include (1) adding a solvent to the reaction raw material that is homogeneously mixed therein but has low affinity for the crosslinked copolymer to be produced; (2) A method in which a linear polymer such as polystyrene is polymerized in the presence of a reaction raw material, and the linear polymer is extracted and removed from the resulting crosslinked copolymer; (3) Polymerization is carried out in the presence of a linear polymer such as polystyrene and a solvent that has an affinity for the resulting crosslinked copolymer, such as toluene, ethyl acetate, or dimethylformamide, in the reaction raw materials, and the resulting crosslinked copolymer is then polymerized. A method for extracting and removing linear polymers, (4) adding (1) to the reaction raw materials;
Any solvent that can be used is one that can be uniformly mixed with the solvent and reaction raw materials prepared above, and can be polymerized in the coexistence of a solvent that has an affinity for the crosslinked copolymer to be produced.

The reaction between the crosslinked copolymer and an alcohol (b) is a phenol (IVI formula or (V1 formula), such as methanol, ethanol, /-glopanol, -monoglobanol, /-butanol, copetanol, isobutyl alcohol, / - The cross-linked copolymer is suspended in alcohols and phenols such as pentanol, l-hexanol, cetyl alcohol, stearyl alcohol, benzyl alcohol, no-phenyl ethanol, -mono-phenyl ethanol, phenol, and cresol, and then refluxed. The reaction can be carried out at 90-110°C for a time of S to 0. At this time, it is preferable to use concentrated sulfuric acid, or in some cases boron trifluoride, as a catalyst.The degree of introduction of a hydrophobic group into the epoxy ring is as follows:
By selecting the reaction conditions, it is possible to perform the following steps.

The ring-opening reaction of the remaining epoxy ring can be carried out by suspending the crosslinked copolymer into which all of the hydrophobic groups have been introduced as described above in water containing sulfuric or phosphoric acid, and holding the suspension at ao-ioo°C for S to -0 hours.

〔Example〕

The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to the following Examples unless it exceeds the gist thereof.

Examples Glycidyl methacrylate 5ioy, ethylene glycol dimethacrylate 9θ11 Toluene Jooy and co,co'-azobis-,2-dimethylvaleronitrile 3
A mixture of g was added to a solution in which demineralized water 2/QOdK polyvinyl alcohol co/y and sodium chloride I was dissolved, and suspension polymerization was carried out at 70° C. for t hours with stirring. After cooling the reaction product, the produced copolymer particles were taken from door to door and washed with water. Next, this copolymer was stirred for 3 hours at room temperature with methanol added thereto, which was repeated 3 times, and then filtered and dried at 0.degree. C. for 16 hours.

Porous copolymer particles having an average diameter of 1 da Oμ were obtained, and the yield was 96%.

This copolymer particle qo11 was reacted for 3 hours with various alcohols λOO or phenol shown in Table 7. The reactants were separated and thoroughly washed with methanol and water.

Next, this was placed in a 70% sulfuric acid aqueous solution -〇〇- and maintained at 50°C for S hours without stirring.

After cooling, the reactants were separated and thoroughly washed with water to obtain hydrophobic chromatography carriers 4 (a) to (f). Experimental conditions e
The results are summarized in Table 7.

In addition, the amount of hydrophobic group introduced is based on the application example using solid state 0-NMR.
/ θ, - Jusha%. 7.7.3, /Sk
0,/M) containing i% ammonium sulfate) 1) Sulfuric acid buffer #
It was dissolved in (p)i'/-jO).

Support obtained according to Example 77′f, %(di (n
-Butyl group introduced carrier) Each S, Ornlf O, / V
) After sufficient equilibration with lithium-hydrochloric acid (pH, sO), centrifugation was performed to remove the adhering solution, and the 4 different concentrations of ammonium sulfate were added to 100 d of the serum r-globulin solution of the seeds.
The mixture was shaken and stirred at io° C. for one hour. After prefecture, UV lfi luminous intensity of supernatant liquid (8℃ and Onm)'T
The adsorbed amount of r-globulin was determined by determining h 1ffl+.

Then, the entire adsorption carrier mass was separated by 0.1M Tris.
Field acid gauze solution (pH 9,!0)! Washed well with rO-. Next, after centrifuging and removing the adhering solution, 0,/
M) Squirrel acid tolerant buffer solution (TIHq, so) i
o.

ml and shaken and stirred at 10° C. for 1 hour.

The amount of r-globulin eluted was determined by measuring the UV intensity of the supernatant at 1 nm.

Table 1 shows the results of adsorption and elution of r-globulin.
Shown all in one place.

Clothing -- Comparative Example 1 The same operation as in Example was performed up to the middle to obtain porous copolymer particles of ethylene glycol dimethacrylate and glycidyl methacrylate.

This copolymer particle θIi was mixed with a 7θ% sulfuric acid aqueous solution and 20θ
- It was kept at 50°C for a long time without stirring. After cooling, the reactants were separated and thoroughly washed with water. The obtained phase does not contain any hydrophobic groups, but contains many hydroxyl groups and is highly hydrophilic.

Regarding these eight bodies, the operation was exactly the same as in Application Example 7. The results are shown in Table-9.

Tables 3 and 3 clearly show the effect of introducing a hydrophobic group.

Application example - Bovine serum and monoglobulin were added to a concentration of 7. It was dissolved in θ,/M Tris/HCl buffer solution (pH 7,50) containing n% S weight ammonium sulfate. The carrier obtained in the example, /kA (at, (t)l,
(C), (θ), (f) Each S, O resistance is θ, / M)
1) After sufficient equilibration with acid-resistant buffer solution (pH 7, JO), centrifugation and removal of adhesion solution, the above-mentioned serum and one globulin solution were added to each /θθd, and incubated at 70°C. The mixture was shaken and stirred for 7 hours.

UV absorbance of supernatant (at, 2gOnm) ft1
The amount of r-globulin adsorbed on each carrier was determined from the determination of K. The results are shown in Table y.

Table - It can be seen that the amount of adsorption of r-globulin is large for the carrier phase in which a daphenyl group is introduced and the carrier phase in which a long-chain alkyl group is introduced.

〔Effect of the invention〕

Claims (1)

[Claims] (1) A crosslinking agent unit selected from polyacrylic esters and polymethacrylic esters of polyhydric alcohols, and the following general formula (I) or (II) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) ▲Mathematical formula , chemical formulas, tables, etc.▼(II) (In the formula, R^1 represents a hydrogen atom or a methyl group, and n is 1
~18, m represents 0 to 2, X represents a hydrogen atom or an alkyl group), and the general formula (III) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(III) (Formula A hydrophobic chromatography carrier having a structure consisting essentially of a glycerin ester unit (wherein R^2 represents a hydrogen atom or a methyl group).