JPH0489500A - Method for purifying substance by affinity chromatography and apparatus for purification - Google Patents

Abstract

PURPOSE:To efficiently and economically separate protein-related substances by using a separation membrane unit prepared by linking a ligand to a membranous support obtained by forming cellular glass into a tubular shape as an affinity adsorbent. CONSTITUTION:Cellular glass is initially formed into a tubular or platy shape and a ligand having a moderate affinity for the objective substance is then linked to the resultant membranous support. The obtained support is subsequently used as a separation membrane unit and a sample liquid for separation is then passed therethrough to carry out purification by affinity chromatography. A support having 0.3-2mm thickness for the tubular shape and 0.3-1mm thickness for the platy shape and 0.3-0.6cc/g pore volume, 100-400m<2>/g specific surface area and 0.1-10/mum pore diameter is preferred as the aforementioned support. Furthermore, e.g. protein A for IgG is cited as the ligand.

Description

[Detailed description of the invention] [Background of the invention] The present invention relates to the purification of substances that can be separated by chromatography, and more specifically to a method and apparatus for purifying protein-related substances based on affinity chromatography with high separation and purification efficiency.

<Conventional technology> Affinity chromatography is a separation method based on specific interactions between molecules of substances such as proteins. It is widely used for the separation and purification of various physiologically active substances.

In the separation and purification of proteins and the like by affinity chromatography, a ligand is usually bound to a gel-like adsorbent, which is packed into a column, and a series of separation and purification operations are performed by column chromatography. That is, by first supplying a solution containing the target substance to a column filled with a carrier immobilized with a highly specific ligand, the target substance selectively and reversibly binds to the ligand while flowing through the column. Most of the impurities flow out of the column. Subsequently, impurities remaining in the column are washed out with a buffer solution. Thereafter, by flowing a suitable eluent through the column, the target substance bound to the ligand from which impurities have been removed is dissociated and eluted, and recovered.

Gel materials used here include cellulose, dextran, agarose, polyacrylamide, porous glass, as well as vinyl polymers, nylon and polystyrene, amino acid copolymers, and ultra gels.
el) etc. are used.

[Summary of the invention]

<Problem to be solved by the invention> However, with the conventional separation and purification method of proteins, etc. using column chromatography as described above, it is difficult to scale up due to the following points, and it is not economical to use it on an industrial scale. It is disadvantageous.

- In agarose-based materials, compaction is severe, resulting in increased pressure loss and decreased separation efficiency.

- Silica-based systems reduce compaction, but in order to improve separation efficiency, it is necessary to reduce the particle size, which requires operation under high pressure, which increases equipment costs.

・Since mass transfer is rate-limiting, the amount of ligand used increases, making it uneconomical.

In order to overcome these drawbacks, a membrane affinity method having the following characteristics has recently been reported.

・The filling height of the adsorbent is the minimum, the operating pressure is low, and the processing capacity is maximized.

- Increasing the membrane area increases the amount of ligand introduced and the adsorption capacity of the target substance.

- Mass transfer is large, which increases the efficiency of ligand utilization.

However, since the membrane material used here is an organic membrane such as cellulose or nylon, it has the following drawbacks.

-Poor chemical resistance, which limits the activation method of the desorbent.

-Poor heat resistance, which makes steam sterilization impossible and poses a sanitary problem.

・The strength of the membrane is low and the life of the membrane is short.

The present invention has been made with the object of solving the above-mentioned problems and providing an economical industrial purification method and purification apparatus for protein-related substances etc. with excellent separation efficiency.

<Technical means for solving the problem> As a result of extensive research in order to solve the above problems, the present inventors have discovered that porous glass formed into a membrane can be used as an affinity adsorbent. It was discovered that protein-related substances can be efficiently and economically separated by this method, and the present invention was completed based on this knowledge.

That is, the substance purification method according to the present invention is a substance purification method using affinity chromatography. The method is characterized in that a ligand having the above-mentioned properties is bound to form a separation membrane, and a sample liquid for separation is passed through the separation membrane.

In addition, in the substance purification device using affinity stomatography according to the present invention, a separation membrane body configured by bonding a ligand having an appropriate affinity for a target substance to a carrier is used to supply a sample liquid for separation. This apparatus is characterized in that the carrier is formed by molding porous glass into a tube or plate shape.

Effect The present invention has the following effects.

(1) Adoption of a tubular or plate-shaped membrane adsorbent allows operation at low pressure and eliminates the problem of compaction.

(2) Since a porous glass membrane with controlled pore size is used, it is possible to prevent the decline in mass transfer, which was a problem with conventional gel methods, allowing for effective use of ligands and economical use of materials. separation and purification becomes possible.

(3) By employing an inorganic membrane, the adsorbent has excellent chemical resistance, and therefore the method of activating the membrane adsorbent for ligand binding is not limited, and various methods can be used for this purpose.

(4) Since it has excellent heat resistance, it can be sterilized in an autoclave, improving sanitary properties. Furthermore, it has excellent membrane strength and has a long lifespan, making it economical.

[Detailed description of the invention]

Purification method> The method of purifying a substance according to the present invention is a method of purifying a substance using Affinity chromatography, in which a membrane carrier made of porous glass is formed into a tube or a plate, and a target substance is added to the membrane carrier. As mentioned above, the method is characterized in that a ligand having a suitable affinity is bound to form a separation membrane, and a sample liquid for separation is passed through the separation membrane. The basic principle is to separate target substances using a glass membrane as a carrier for affinity chromatography.

In the present invention, the target substance to be separated can be separated by affinity chromatography, that is, chromatography that utilizes affinity, which is a specific interaction between substances, and such substances include Examples include various protein-related substances (for example, various hormones, enzymes, antibodies, albumin, etc.) such as pharmaceuticals produced by biotechnology, as well as polynucleotides, glycolipids, and polysaccharides.

In addition, sample solutions for separation containing these target substances include:
Examples include fermentation liquid, extracts of crushed bacterial cells, and purified liquids thereof.

As mentioned above, the carrier is a membrane formed from porous glass into a tubular or plate shape, and its thickness is preferably 0.
．． About 3 to 4.0 wm, more preferably 0.3 to 2.0 wm in the case of a tubular shape. 0wm, 0.3 in case of plate shape
~1.01, preferably with a pore volume of 0.3~1.
About 0 cc/g, more preferably 0.3 to 0.6 cc/
g, the specific surface area is preferably about 1 to 500 i/g, more preferably 100 to 400 rrf/g, and the pore diameter is preferably about 0.05 to 50 μm, more preferably 0.1 to 10 μm. be.

Porous glass as a carrier is SiO, Al2O3,
It is fired from one or more components selected from general inorganic glass components such as B2O3, and the method for firing this porous glass is described in general literature, such as "Fine Ceramics Handbook" by Ken Hamano. You can refer to ``Bioceramics'' (Gihodo Publishing), edited by Yaya (Asakura Shoten), etc.

The target substance can be bound to a carrier or adsorbed by Gantt, or the ligand can be prepared from general literature such as "Experiment and Application Affinity Chromatography".
(Kodansha), etc., specific receptors for various hormone proteins, substrates for various enzymes, antibodies (or antigens) for various antigens (or antibodies), complementary proteins for protein A1 polynucleotide for IgG, etc. Polynucleotide, NAD (
P) Related enzymes, ATP-related enzymes, synthetic dyes for serum albumin, etc., lectins for glycoproteins, glycolipids, polysaccharides, etc., heparin for blood coagulation factors, etc. are generally mentioned.

The bonding of the ligand to the carrier is usually through a covalent bond, and the ligand (often a protein or peptide) is attached to -NH2, -COOH or -○H by bonding a cyano group, epoxy group, amino group, etc. to the carrier. The ligand can be immobilized on the carrier by covalently bonding the like using a conventional method. Furthermore, in order to immobilize the ligand on the carrier by covalent bonding, it is necessary to activate the porous glass as the carrier. General literature such as "Experiments and Applied Affinity Chromatography" (Kodansha) can be referred to for the method of activating such a carrier and the method of immobilizing a ligand by covalent bonding, but the An overview of the example is explained below.

First, porous glass is treated with a silane derivative, such as γ-aminopropyltriethoxysilane, to prepare alkylamino glass. This alkylamino glass can be prepared in the form of aromatic amino derivatives, isocyanate derivatives, carboxylic acid derivatives, acid chloride derivatives, etc., depending on the type of ligand and the purpose of separation and purification.

Furthermore, it is also possible to immobilize amino groups on alkylamino glass using a ligand having a carboxyl group (proteins or polypeptides are typical examples thereof) or a crosslinking reagent such as glutaraldehyde.

Furthermore, a spacer of appropriate length (
For example -(CH2). - etc.), affinity chromatography can be performed more advantageously. There are two methods for introducing a spacer: one is to immobilize a ligand on a carrier to which a spacer has been bound in advance, and the other is to form a combination of a ligand and spacer in advance and immobilize it on a carrier. Both methods are used. It is possible. For information on how to introduce a spacer, you can refer to general literature such as "Experiments and Applications of Affinity Chromatography" (Kodansha).

In the present invention, among the various activation methods for porous glass described above, the most efficient method can be appropriately selected depending on the target substance.

Preferred specific examples of the method for binding the ligand to the carrier are shown in detail in the experimental examples below.

By binding a ligand to a porous glass carrier as described above, a separation membrane body for adsorbing and separating a target substance is constructed. Regarding this separation membrane body, a tubular one is shown in the purification apparatus shown in FIG. 2, which will be described later, and a plate-shaped one is shown in the purification apparatus shown in FIG. 3, respectively.

In order to separate and purify the target substance using this separation membrane, the method is basically the same as that of conventional membrane affinity chromatography. A separation sample solution containing the target substance is supplied and passed through the membrane while applying an appropriate pressure, and the target substance is adsorbed onto the separation membrane. Next, impurities remaining in the separation membrane body are washed out with an appropriate buffer, and then the target substance bound to the ligand is dissociated and eluted with an appropriate eluent and recovered. In order to actually use the separation membrane body, a purification device incorporating the tubular separation membrane body shown in Fig. 2 or a purification device incorporating the plate-shaped separation membrane body shown in Fig. 3, as described later, is required. A device or the like may be used. Further, the above buffer solution and eluate may be appropriately selected according to the target substance as in the past.

Methods for separating and purifying target substances using separation membranes are described in general literature, such as S. Brandt et al.
t at,, Bio/technology, v
ol 6°P779 (1988), etc., and specific examples of the present invention are shown in detail in Experimental Examples below.

<Purification Apparatus> The substance purification apparatus according to the present invention includes a separation membrane body constituted by bonding a ligand having an appropriate affinity for the target substance to a carrier in a casing for supplying a sample liquid for separation. As described above, there is provided an apparatus for purifying a substance by holding a substance, in which the carrier is formed by molding porous glass into a tube shape or a plate shape. The basic principle is the purification method. The purification apparatus according to the present invention may employ any known structure used for this type of purpose, except for the separation membrane body.

Fig. 2 shows an example of the basic configuration of a purification device that incorporates a separation membrane body 1 formed into a tubular shape, and Fig. 3 shows an example of the basic configuration of a purification device incorporating a separation membrane body 1' formed into a plate shape. Examples of basic configurations of purification devices are shown. These separation membrane bodies have already been explained in detail in the section on purification methods.

In addition, there is no particular limit to the size of the porous glass of the separation membrane when it is actually used as a carrier, as long as the membrane surface is supported by a suitable support such as a mesh.
When a support is not used, from the viewpoint of strength, a tubular carrier preferably has an inner diameter of about 2 to 15 mm and a length of about 50 to 500 mm, and a plate-shaped carrier has an area of 3 to 100 mm.
- is preferable.

In the purification device shown in FIG. 2, a plurality of tubular separation membrane bodies 1 are arranged in parallel in the axial direction and fixed in a cylindrical sample liquid supply casing 2 with openings a maintained at both ends thereof. This is what I did. Casing 2 is made of polypropylene,
Commonly used synthetic resins such as polycarbonate and Teflon, or commonly used metals such as stainless steel can be used.

To separate and purify the target substance using this purification device,
The sample liquid is supplied into the tube of the separation membrane body 1 at one end without applying appropriate pressure, and unnecessary liquid is removed from the tube (inside the cylinder of the casing 2).
Either the sample solution is allowed to flow out from the outside of the tube of the separation membrane body 2 and discharged from the outflow port 3, or the sample solution is supplied from the outflow port 3 to flow from outside the tube of the separation membrane body 2 into the tube, and then, in the same manner as the sample solution, a buffer solution for washing is added. and eluate are supplied, and the target substance is collected from the outlet 3 side or the other end side. In this case, a cylindrical portion of an appropriate length having a supply port (or collection port) for supplying a sample or recovering a target substance may be provided in the casing 2 at the end on the side of the opening a.

The purification apparatus shown in FIG. 3 has a plate-shaped separation membrane 1' sandwiched between a pair of casings 2', 2'. In the case of such a purification device, as shown in the figure, a plurality of separation membrane bodies 1' are arranged in a plurality of membranes having concentrically and radially channel grooves 5, 5' and a communication hole 6 in the center. It is more preferable that the distributor 4 and the mesh-like spacer 7 be arranged symmetrically and stacked in the casings 2', 2'. The casing 2', the distributor 4, and the spacer 7 are
It can be molded from commonly used synthetic resins such as polypropylene, polycarbonate, and Teflon. To separate and purify a target substance using this purification device, a sample liquid is supplied from the supply port 8 of one casing 2' while applying an appropriate pressure, and unnecessary liquid is discharged from the other outlet 8'. Thereafter, a washing buffer and an eluate are supplied in the same manner as the sample solution, and the target substance is recovered from the outlet 8'.

〔Example〕

The following is intended to be illustrative of the present invention and is not intended to limit the invention thereto.

Examples The following examples use porous glass membranes made by Asahi Glass Co., Ltd.
This example shows the use of a tubular membrane manufactured by Inner diameter 2mm
, inner thickness 0. Three porous glass membrane tubes each having a diameter of 3 mm and a length of 100 mm were used for the separation and purification experiment. The pore diameter of the porous glass membrane is 1.0 μm, and the membrane area is 2 based on the outer diameter.
1.7 c4, membrane volume 0, 304 cIll was used. Activation of the membrane was performed by the following procedure. First, a porous glass membrane was prepared using a toluene solution of γ-aminopropyltriethoxysilane (10% v/v).
The mixture was refluxed for 3 hours, and then excess silane was washed away using methanol to obtain alkylamino glass. Here, this alkylamino glass was further subjected to the following reaction to obtain two types of derivatives, namely, an aromatic amino derivative (1) and a carboxylic acid derivative (2).

(1) Aromatic amino derivative (2) Carboxylic acid derivative carrier HH ↓ As a case of separation and purification, in this example, human IgG was separated and purified by group-specific affinity chromatography using protein A as a ligand. ,In addition,
As protein A, one produced by recombinant E. coli into which a protein production gene derived from Staphylococcus aureus Cowan 1 strain was incorporated was used.

The flow of a series of separation and purification tests is as follows.

Equilibration of deadsorbent ↓ Immobilization of protein A ↓ Blocking of deadsorbent ↓ Washing ↓ Equilibration ↓ Adsorption of IgG fraction ↓ Washing ↓ Elution of IgG Note that this series of operations can be performed as shown in Figure 1. It can be implemented by a process flow diagram.

First, two types of desorbents are activated. In the case of adsorbent (1), 0.2.5% of glutaraldehyde was used.
An activated membrane was prepared by circulating 1M borate buffer (pH 8,2) (for 2 hours). After washing excess glutaraldehyde with the same buffer, protein A (5 mg
/ml) dissolved in the same buffer was circulated for 5 hours to immobilize protein A. It was then reduced with NaB H4 and excess active groups were blocked with 1% glycine ethyl ester hydrochloride (pi(8,1)). This operation continued for 1.0 h.

Furthermore, 0.05M phosphate buffer (pH 7, 6, 0, 2
5M NaCl (containing), washed with 0.2M glycine-HC1 buffer (pH 12, 3), and finally equilibrated with 0.05M phosphate buffer (pH 7, 6, 0, containing 25M NaCl) for adsorption and elution of IgG. Tested.

In the case of adsorbent (2), separation was carried out as follows. Protein A was dissolved in distilled water to a concentration of 5 a+g/ml, and the pH was adjusted to 4.5 with 0.1M HCl.

On the other hand, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was dissolved in distilled water to a concentration of 10 mg/ml, and adjusted to pH 4.5. Protein A is immobilized by mixing and circulating these two types of solutions. Furthermore, blocking washing and equilibration operations were performed in the same manner as for adsorbent (1).

The IgG fraction obtained by partially purifying human serum is 10 m
IgG was adsorbed by circulating at a flow rate of 1/min. In addition, for elution of IgG, glycine-HCl buffer (pH 2,
3) was carried out. In addition, the protein amount is Lory (Lo
vry) method (OD during circulation) was measured using a 280n box. In addition, the purity of eluted IgG was tested using 5D
This was done by S-PAGE.

Adsorption f of protein A obtained with adsorbents (1) and (2)
The results of the recovery rates of fi (mg ProtcinA/cJ-membrane) and IgG are shown in Tables 1 and 2. In addition,
By 5DS-PAGE, 98 proteins were eluted.
% or more was confirmed to be IgG.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram showing an outline of an affinity chromatography system using a separation membrane. FIG. 2 is a partially cutaway view showing an example of the basic configuration of a purification apparatus according to the present invention using a separation membrane body formed into a tubular shape. Figure 3 shows a method using a separation membrane body formed into a plate membrane shape.
FIG. 1 is a configuration diagram showing an example of the basic configuration of a purification apparatus according to the present invention.

Claims (1)

[Claims] 1. In a method for purifying a substance using affinity chromatography, a ligand having an appropriate affinity for the target substance is placed on a membrane carrier made of porous glass in the shape of a tube or plate. 1. A method for purifying a substance, the method comprising: combining the two to form a separation membrane body, and passing a sample liquid for separation through the separation membrane body. 2. The pore volume of the carrier is 0.3 to 1.0 cc/g, the specific surface area is 1 to 500 m^2/g, and the pore diameter is 0.05 to 50 μ
2. The purification method according to claim 1, wherein the membrane has a thickness of 0.3 to 4.0 mm. 3. The purification method according to claim 1 or 2, wherein the target substance is a protein. 4. A substance purification device comprising a separation membrane body, which is composed of a carrier bound to a ligand having an appropriate affinity for the target substance, held in a casing for supplying a sample liquid for separation. . An apparatus for purifying a substance by affinity chromatography, characterized in that the carrier is formed by molding porous glass into a tube shape or a plate shape. 5. The pore volume of the carrier is 0.3 to 1.0 cc/g, the specific surface area is 1 to 500 m^2/g, and the pore diameter is 0.05 to 50 p.
5. The purification apparatus according to claim 4, wherein the membrane thickness is 0.3 to 4.0 mm. 6. The purification device according to claim 4 or 5, wherein the target substance is a protein.