JP2023043798A - Filter for liquid chromatography column and manufacturing method thereof - Google Patents

Abstract

To provide a filter for a liquid chromatography column having mechanical strength to withstand high pressure, and further resistant to non-specific adsorption of proteins and nucleic acids, which are samples to be analyzed, and a manufacturing method thereof.SOLUTION: A surface of a porous metal filter for a liquid chromatography column is treated with a polyfunctional silane coupling agent having an epoxy group and an amino acid.SELECTED DRAWING: Figure 1

Description

TECHNICAL FIELD The present invention relates to a liquid chromatography column filter used as analytical means in fields such as biochemistry and medical chemistry, and a method for producing the same.

In the fields of biochemistry, medical chemistry, and the like, liquid chromatography is used as an analysis method using proteins, nucleic acids, and the like as samples to be analyzed. Liquid chromatography includes various techniques such as ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, and affinity chromatography. Conduct analysis.

Columns used for liquid chromatography are those in which both ends of a column housing filled with a packing material are sealed with column ends provided with filters. Metal (stainless steel sintered body, titanium), glass, polyetheretherketone (PEEK), tetrafluoropolyethylene (Teflon (registered trademark)), polyethylene, etc. known (see, for example, Patent Documents 1 to 8).

Further, Patent Document 9 discloses porous glass filters modified with various silane compounds, and Patent Documents 10 and 11 disclose techniques for introducing a silicone coating into the filter. In addition, Non-Patent Documents 4 and 5 disclose a method of introducing 3-Glycidoxypropyltrimethoxysilane, which is a silane treatment agent, into the metal surface and introducing various functional groups into the introduced epoxy groups, in order to prevent protein adsorption to stainless steel. there is

On the other hand, as a method for preventing non-specific adsorption of proteins, etc. to metals, resins, etc., Non-Patent Document 1 describes a method of preconditioning using proteins such as albumin and casein, and polysaccharides such as dextran. Non-Patent Document 2 describes a method using a water-soluble synthetic polymer. Furthermore, in recent years, a technique having a betaine structure described in Non-Patent Document 3 has been used.

A filter for a liquid chromatography column seals the packing material inside the column, that is, prevents the packing material from flowing out, but does not easily cause clogging, allowing solids, etc. in the sample and eluent to flow into the column. It is a porous body used for the purpose of protecting the filler by preventing In recent years, a packing material with a particle size of 2 microns or less has been put into practical use, and the pressure when packing the packing material into a column and the operating pressure (pressure for feeding the liquid to be analyzed) during analysis is 100 MPa. has reached close to For this reason, column filters are required to have high pressure resistance, and the filter materials used are also limited in terms of pressure resistance, such as those made of glass, tetrafluoroethylene, and polyethylene. does not have a pressure resistance that can withstand 100 MPa. A filter made of polyetheretherketone, which can withstand 100 MPa, has been put into practical use, but it has the drawback of being highly hydrophobic.

A column filter using sintered stainless steel or porous titanium has high pressure resistance because it is a filter in which metals are fused. However, since these filters are made of metal, they are known to adsorb basic proteins and metal-coordinating proteins, and high-molecular-weight proteins such as antibody aggregates adsorb to the column at the initial stage of injection. The phenomenon (hereinafter referred to as "initial adsorption") is known. Although initial adsorption can be improved by injecting a large amount of sample into the column (this technique is hereinafter referred to as "conditioning"), initial adsorption may occur again when the column is stored. Therefore, it is necessary to confirm the presence or absence of initial adsorption before analysis.

In order to meet these requirements, filters for liquid chromatography columns are required to have mechanical strength to withstand high pressure, and also have properties that make it difficult for proteins and nucleic acids, which are the samples to be analyzed, to adsorb. was wanted.

As described above, 1) high pressure resistance and 2) low nonspecific adsorption are required for filters used in liquid chromatography columns used for analysis of biopolymers such as proteins and nucleic acids.

If a biopolymer such as protein is used to prevent non-specific adsorption of a filter for a liquid chromatography column, there is a concern that bacterial cells may be generated depending on the storage conditions of the column. In addition, there is a concern that biopolymers such as proteins used for filter adsorption prevention may be mixed into the fractionated sample, and there is concern about using the fractionated sample for further investigation. When synthetic polymers are used, there are no concerns as with biopolymers, but since synthetic polymers often have a hydrophobic part in the main chain of the polymer, there is concern about interaction with the sample to be measured. occur. Furthermore, if the synthetic polymer is eluted into the fractionated sample, there is concern about its toxic effects.

The method of coating the filter with a silicone film is not suitable because the base polymer is hydrophobic and proteins and the like are hydrophobically adsorbed. In addition, in the method of modifying the metal surface with a silane treatment agent shown in Non-Patent Documents 4 and 5, it is necessary to treat the metal surface in advance, and the introduction of the silane treatment agent is performed in a water-insoluble organic solvent such as toluene. It requires a complicated procedure because it is necessary, and it is necessary to further introduce a functional group to the introduced epoxy group.

JP-A-2000-081424 JP-A-2000-131302 Japanese Patent Application Laid-Open No. 2001-249120 Japanese Patent Publication No. 2005-508398 JP 2006-138724 A JP 2006-227022 A JP-A-06-242094 JP-A-07-260763 JP-A-1998-282078 Japanese Patent No. 3990503 Japanese Patent No. 3857439

Immunodiagnostics Oxford University Press, Oxford, UK 1999 Surface Modification of Polymeric Biomaterials, Plenum, New York, N.W. Y. (1997) Biointerface, bioconjugation, and biomatrix based on bioinspired phospholipid polymers, Handbook of Nanostructured Biomaterials Kang CK1, Lee YS. , J Mater Sci Mater Med. 2007 Jul;18(7):1389-98 The surface modification of stainless steel and the correlation between the surface properties and protein adsorption Hairen Wang, Anti-Corrosion Methods and Materials Vol. 61 Iss: 5, pp. 307-313 2014, Corrosion protection of stainless steel by a self-assembled organosilane bilayer

An object of the present invention is to provide a filter for a liquid chromatography column, which has mechanical strength to withstand high pressure and which is resistant to non-specific adsorption of proteins and nucleic acids, which are samples to be analyzed, and a method for producing the same. It is in.

The present inventors have conducted intensive research to solve the problems of conventionally used 1) liquid chromatography columns using porous metal filters having high pressure resistance and 2) non-specific adsorption. The present invention has been completed. That is, the present invention provides a filter for a liquid chromatography column, which is a filter obtained by coating the surface of a porous metal filter with a reaction condensate of a polyfunctional silane coupling agent having an epoxy group and an amino acid, and which contains proteins, nucleic acids, and the like. Adsorption of biopolymers can be reduced. When the filter of the present invention is used in a column for liquid chromatography, the adsorption of basic proteins and metal-coordinating proteins, which has been a drawback of porous metal filters, is reduced, and the initial adsorption of high-molecular-weight proteins is also reduced. The present invention will be described in detail below.

The present invention relates to a method for reducing the initial adsorption of basic proteins, metal-coordinating proteins and, in particular, macromolecular proteins, in a liquid chromatography column using a porous metal filter. A liquid chromatography column using this improves the elution peak shape of basic proteins and metal-coordinating proteins, and reduces the initial adsorption of proteins. Since it can be used immediately, it is particularly suitable for analyzing biopolymers such as proteins and nucleic acids.

In the porous metal filter of the present invention, the surface of the filter is coated with a condensate of γ-GOP, which is a silane coupling agent. The thickness of the coating layer is not particularly limited as long as it does not block the through-holes of the filter, and is preferably 0.5 nm to 100 nm.

The porous metal filter of the present invention can be produced by treating a porous metal filter with a polyfunctional silane coupling agent having an epoxy group and an amino acid in a neutral phosphate buffer solution, followed by drying and heating. Polyfunctional silane coupling agents having epoxy groups include (3-glycidoxypropyl)trimethoxysilane or (3-glycidoxypropyl)triethoxysilane, (3-glycidoxypropyl)methyldimethoxysilane or ( Examples include 3-glycidoxypropyl)methyldiethoxysilane, 5,6-epoxyhexyltrimethoxysilane, and the like. In particular, (3-glycidoxypropyl)trimethoxysilane and (3-glycidoxypropyl)triethoxysilane are preferably used because adsorption can be effectively suppressed.

The porous metal filter to be used is not particularly limited as long as it has through holes suitable for a liquid chromatography column, and the surface of the porous metal filter does not need to be electropolished in advance.

A porous metal filter is treated by heating in a phosphate buffer solution in which a polyfunctional silane coupling agent having an epoxy group and an amino acid are dissolved. The concentration of the polyfunctional silane coupling agent having an epoxy group may be in the range of 1 to 30% by weight, and the concentration of the amino acid may be in the range of 1 to 20% by weight. Since the coupling agent or condensates of amino acids may block the through-pores of the filter, the amount used may be adjusted according to the pore characteristics of the filter member used. Although the amino acid is not particularly limited, it is preferable to use a basic amino acid, particularly lysine and/or arginine, in terms of the effect of reducing adsorption to the filter.

Although the concentration of the phosphate buffer is not particularly limited, it is 1 to 500 mmol/L, preferably 1 to 100 mmol/L. Also, the pH is preferably 4-8. The heating temperature is preferably 60-100°C.

A filter treated with a polyfunctional silane coupling agent having an epoxy group and an amino acid in a phosphate buffer is removed from the solution, washed with an aqueous ethanol solution, washed with water, dried and heated. Dry heating is not necessarily required, but dry heating is desirable since strongly basic proteins may be adsorbed. The drying heating temperature is preferably 50 to 100° C. If the temperature is high, initial adsorption of high-molecular-weight proteins may occur. The porous metal filter thus obtained can be used as it is, or after washing with water, it can be attached to a column for liquid chromatography.

The filter for a liquid chromatography column of the present invention is obtained by: 1) treating a porous metal filter having high pressure resistance with a polyfunctional silane coupling agent having an epoxy group and an amino acid; 2) It is a filter that reduces non-specific adsorption of samples such as proteins and nucleic acids.

FIG. 10 is a diagram showing the ratio of aggregates to the total peak area when mouse antibodies containing aggregates were continuously injected.

The filter for liquid chromatography column will be described in more detail below, but the present invention is not limited thereto.

Example 1
The filter was removed from a 4.6 mm ID x 15 cm length liquid chromatography column using a commercially available stainless steel sintered filter. Put 10 mmol/L phosphate buffer (pH 6.0) in a glass container, (3-glycidoxypropyl) trimethoxysilane (Sila Ace S510 JNC) and L (+) -Lysine Hydrochloride (manufactured by Fujifilm Wako Pure Chemical) : 125-01465) were added to each 0.15 mol/L and dissolved by stirring in an ultrasonic bath. The filter removed from the column was placed in this solution, degassed and washed in an ultrasonic bath. The washed filter and solution were placed in a separable flask, immersed in an oil bath set at 90° C., and heated and stirred for 16 hours. After cooling, the filter was removed from the solution and excess solution was removed with a paper filter. A 20% ethanol aqueous solution and a filter were placed in a glass container, washed in an ultrasonic bath, then the filter was washed again with ultrapure water in the same procedure, and dried by heating in an oven set at 60 ° C. for 5 hours ( These techniques are hereinafter referred to as "filter processing"). The resulting filter was attached to the original column. As a packing material, the packing material was extracted from TSKgel UP-SW3000 manufactured by Tosoh Corporation and packed in the prepared column. A sample was continuously injected into the column under the following conditions, and the peak shape was measured to observe the degree of adsorption.
<Measurement condition>
Eluent: 100 mmol/L sodium sulfate in 100 mmol/L phosphate buffer (pH 6.7)
Flow rate: 0.35mL/min
Temperature: 25°C
Sample: 1.5g/L mouse monoclonal IgG1 5uL
FIG. 1 shows the ratio of aggregates to the total peak area when a mouse antibody containing about 20% aggregates was continuously injected. Initial adsorption of aggregate components was greatly improved by performing filter treatment.

Example 2
As in Example 1, the filter was removed from a 4.6 mm inner diameter×15 cm long liquid chromatography column using a commercially available stainless steel sintered filter. Put 10 mmol/L phosphate buffer (pH 6.0) in a glass container, (3-glycidoxypropyl)trimethoxysilane (Sila Ace S510 JNC) 0.3 mol/L and L(+)-Lysine (Fuji Film Wako Pure Chemical Industries: 124-06212) was added to 0.15 mol/L and dissolved by stirring in an ultrasonic bath. Filter treatment was performed in the same manner as in Example 1, and the obtained filter was attached to the original column. As a packing material, a packing material was extracted from TSKgel UP-SW3000 manufactured by Tosoh Corporation, packed in a prepared column, and the degree of adsorption was evaluated in the same manner as in Example 1. The results are shown in FIG. The initial adsorption of aggregate components was greatly improved.

Example 3
The filter was removed from a 4.6 mm ID x 15 cm length liquid chromatography column using a commercially available stainless steel sintered filter. Put 10 mmol/L phosphate buffer (pH 6.0) in a glass container, add 0.17 mol/L (3-glycidoxypropyl)trimethoxysilane (Sila Ace S510, manufactured by JNC) and L(+)-Arginine Hydrochloride (Fuji Film (manufactured by Wako Pure Chemical Industries: 018-04625) was added at a concentration of 0.25 mol/L and dissolved by stirring in an ultrasonic bath. Filter treatment was performed in the same manner as in Example 1, and the obtained filter was attached to the original column. As a packing material, a packing material was extracted from TSKgel UP-SW3000 manufactured by Tosoh Corporation, packed in a prepared column, and the degree of adsorption was evaluated in the same manner as in Example 1. The results are shown in FIG. The initial adsorption of aggregate components was greatly improved.

Comparative example 1
In the same manner as in Examples 1, 2 and 3, a liquid chromatography column of 4.6 mm inner diameter and 15 cm length was prepared using a commercially available sintered stainless steel filter. The peak shape of the mouse antibody was measured without filtering, and the degree of adsorption was observed. The results are shown in FIG. Although antibody aggregates were eluted, the peak areas were smaller than those of Examples 1, 2 and 3, indicating initial adsorption.

Claims (15)

A filter for a liquid chromatography column coated with a silane coupling agent, wherein the porous metal filter contains a condensate of an amino acid and a polyfunctional silane coupling agent having an epoxy group. 2. The filter for a liquid chromatography column according to claim 1, wherein said porous metal filter is made of sintered stainless steel or titanium. 3. The liquid chromatography column according to claim 1, wherein the silane coupling agent is (3-glycidoxypropyl)trimethoxysilane and/or (3-glycidoxypropyl)triethoxysilane. filter for. 4. The liquid chromatography column filter according to any one of claims 1 to 3, wherein said amino acid is arginine and/or lysine. The filter for a liquid chromatography column according to any one of claims 1 to 4, wherein the coating layer coated with the silane coupling agent has a thickness of 0.5 nm to 100 nm. A liquid chromatography column characterized by heat-treating a porous metal filter in an aqueous solvent in which a polyfunctional silane coupling agent having an epoxy group and an amino acid are dissolved, followed by washing and heating in a dry state. filter manufacturing method. 7. The method for producing a filter for a liquid chromatography column according to claim 6, wherein the concentration of said polyfunctional silane coupling agent having an epoxy group is 1 to 30% by weight. 8. The method for producing a filter for a liquid chromatography column according to claim 6, wherein said amino acid concentration is 1 to 20% by weight. The method for producing a filter for a liquid chromatography column according to any one of claims 6 to 8, wherein the aqueous solvent has a pH of 4 to 8. The method for producing a filter for a liquid chromatography column according to any one of claims 6 to 9, wherein the aqueous solvent is a phosphate buffer having a concentration of 1 to 500 mmol/L. The method for producing a filter for a liquid chromatography column according to any one of claims 6 to 10, wherein the heat treatment is performed at 60 to 100°C. The method for manufacturing a filter for a liquid chromatography column according to any one of claims 6 to 11, wherein the washing is an ethanol aqueous solution and/or water washing. The method for producing a filter for a liquid chromatography column according to any one of claims 6 to 12, wherein the heating temperature in the dry state is 50 to 100°C. A liquid chromatography column comprising the filter for a liquid chromatography column according to any one of claims 1 to 5. A method of using the liquid chromatography column of claim 14 for size exclusion chromatography.

Images