JP2017227461A - Method for preventing adsorption of liquid chromatography column - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for manufacturing a filter for a liquid chromatography column which has a mechanical strength large enough to withstand high pressure and to which protein or nucleic acid as an analysis object sample are not easily adsorbed nonspecifically.SOLUTION: Conditioning is performed by injecting polylysine with a molecular weight of 100,000 or larger into a liquid chromatography column using a porous metal filter.SELECTED DRAWING: None

Description

  The present invention relates to a method for preventing adsorption of a liquid chromatography column used as an analysis means in the fields of biochemistry, medical chemistry and the like.

  In fields such as biochemistry and medicinal chemistry, liquid chromatography is used as an analysis method using proteins, nucleic acids, and the like as samples to be analyzed. There are various methods for liquid chromatography, such as ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, etc., using a column packed with a suitable packing material to perform each method. Perform analysis.

  A column used for liquid chromatography is one in which both ends of a column housing filled with a packing material are sealed with a column end equipped with a filter. Metal (stainless steel, titanium), glass, polyether ether ketone (PEEK), polytetrafluoroethylene (Teflon: registered trademark), polyethylene, etc. are known (For example, see Patent Documents 1 to 8).

  Patent Document 9 shows a porous glass filter modified with various silane compounds, and Patent Documents 10 and 11 show a technique for introducing a silicone film into the filter. Further, Non-Patent Documents 4 and 5 disclose a technique for introducing 3-Glyoxypropyltrimethylsilane, a silane treatment agent, onto a metal surface to prevent protein adsorption on stainless steel and introducing various functional groups into the introduced epoxy group. Yes.

  On the other hand, as a method for preventing non-specific adsorption of proteins or the like 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. In recent years, a method 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 packed packing from flowing away, but does not easily clog, and the solid content in the sample or eluent flows into the column. It is a porous body used for the purpose of preventing this and protecting the filler. In recent years, a packing material having a particle diameter of 2 microns or less has been put into practical use, and the pressure when packing the packing material into the column and the operating pressure during analysis (pressure for feeding the liquid to be analyzed) are 100 MPa. Has reached close. For this reason, high pressure resistance is required for column filters, and the filter members used are limited in terms of pressure resistance, such as glass, tetrafluoroethylene, polyethylene, etc. Has no pressure resistance that can withstand 100 MPa. A product made of polyetheretherketone that can withstand 100 MPa has been put to practical use, but has a drawback of high hydrophobicity.

  A column filter using a sintered stainless steel or porous titanium has high pressure resistance because it is a filter in which metal is fused. However, since these filters are metal, it is known that basic proteins and metal-coordinating proteins are adsorbed, and high molecular weight proteins such as antibody aggregates are adsorbed to the column at the beginning of injection. A 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 (hereinafter this method is referred to as “conditioning”), initial adsorption may occur again when the column is stored. It is necessary to confirm the presence or absence of initial adsorption before analysis.

  In order to satisfy these requirements, a filter for a liquid chromatography column is required to have mechanical strength that can withstand high pressure, and it is difficult to adsorb proteins and nucleic acids that are samples to be analyzed. It was sought after.

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

  If a biopolymer such as a protein is used to prevent nonspecific adsorption of a filter for a liquid chromatography column, there is a concern about the generation of bacterial cells depending on the storage conditions of the column. In addition, there is a concern that a biopolymer such as a protein used for preventing filter adsorption is mixed in the fractionated sample, and there is a concern that the fractionated sample is used for further examination. When synthetic polymers are used, there is no concern like biopolymers. However, synthetic polymers often have a hydrophobic part in the polymer main chain, so there is concern about interaction with the measurement sample. Arise. Further, when the synthetic polymer is eluted in the fractionated sample, there is a concern about the influence of its toxicity.

  The method of coating the filter with a silicone film is not suitable because the base polymer is hydrophobic and proteins adsorb hydrophobically. Further, in the method of modifying the metal surface with a silane treating agent described in Non-Patent Documents 4 and 5, the metal surface must be treated in advance, and the silane treating agent is introduced in a water-insoluble organic solvent such as toluene. A complicated procedure is required because it is necessary and a functional group needs to be further introduced into the introduced epoxy group.

JP 2000-081424 A JP 2000-131302 A JP 2001-249120 A JP 2005-508398 A JP 2006-138724 A JP 2006-227002 A Japanese Patent Laid-Open No. 06-242094 Japanese Patent Application Laid-Open No. 07-260763 JP 1998-282078 Japanese Patent No. 3990503 Japanese Patent No. 3857439

Immunodiagnostics Oxford University Press, Oxford, UK 1999 Surface Modification of Polymer Biomaterials, Plenum, New York, N .; Y. (1997) Biointerface, bioconjugation, and biomatrix based on biospired 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 organicsilaner

  An object of the present invention is to provide a method for producing a filter for a liquid chromatography column, which has a mechanical strength that can withstand high pressure and is difficult to non-specifically adsorb proteins and nucleic acids that are analytes. is there.

  The present inventor has conducted extensive research to solve the problems related to 1) non-specific adsorption of 1) liquid chromatography columns using porous metal filters having high pressure resistance, which have been used conventionally. The present invention has been completed. In other words, the present invention reduces the adsorption of basic proteins and metal-coordinating proteins, which has been a drawback of porous metal filters, by preconditioning a liquid chromatography column with polylysine in advance. This is a method for preventing the adsorption of a column for liquid chromatography using a porous metal filter, which also reduces the initial adsorption. Hereinafter, the present invention will be described in detail.

  The present invention relates to a method for reducing basic protein, metal coordinating protein adsorption, and particularly high-molecular protein initial adsorption in a liquid chromatography column using a porous metal filter, and performs conditioning using polylysine. Is the method. The liquid chromatography column conditioned using the polylysine of the present invention improves the elution peak shape of basic proteins and metal coordination proteins, and reduces the initial adsorption of proteins. It is particularly suitable for analysis of biopolymers such as proteins and nucleic acids.

  Polylysine having various molecular weights can be used for conditioning. However, the molecular weight is preferably large and the molecular weight is preferably 100,000 or more because the effect may be reduced during use if the molecular weight is low. .

  As a conditioning method, it can be easily conditioned by injecting a predetermined amount of polylysine into the column while passing a solvent through the column for liquid chromatography using the same method and apparatus as in liquid chromatography analysis. As the solvent to be used, a solvent that does not adsorb to the filler is used when the interaction between polylysine is expected, such as a cation exchanger filler, without inhibiting the binding between the porous metal filter and polylysine. An example of a solvent that inhibits the binding between the porous metal filter and polylysine is a solvent containing a metal coordinating low molecular compound such as imidazole or ethylenediaminetetraacetic acid. Further, as an example of adsorbing polylysine to the filler, when a silica filler is used as the filler, for example, by using a sodium phosphate buffer solution having a concentration of 100 mmol / L or more, the polylysine is adsorbed onto the filler. Adsorption can be suppressed. Moreover, when using the filler which has ion exchange groups, such as carboxylic acid and a sulfonic acid, the solvent containing 0.5 mol / L or more sodium chloride is used, for example.

  The amount of polylysine used is preferably 0.5 mg / cm 2 or more, particularly preferably 1.0 mg / cm 2 or more per column cross-sectional area. There is no difference in effect as long as the amount used is above a certain level.

The method for preventing adsorption of a liquid chromatography column of the present invention is as follows. 1) A polymer biological sample is used in a liquid chromatography column using a porous metal filter having high pressure resistance, and further conditioned using polylysine. 2) This is a technique for reducing non-specific adsorption of proteins, nucleic acids and the like.

  Hereinafter, although the Example demonstrates the adsorption | suction prevention method of a liquid chromatography column in detail, this invention is not limited to these.

Example 1
The packing material was extracted from TSKgel UP-SW3000 manufactured by Tosoh Corporation and packed in a liquid chromatography column having a diameter of 4.6 mm and a length of 15 cm using a commercially available sintered stainless steel filter. The packed column was connected to a liquid chromatography apparatus, and a Gondi column was injected by injecting a poly-L-lysine solution (Sigma P4832 mol wt 150,000-300,000 0.01%) under the conditions described below. We performed a shot.

<Conditioning conditions>
Eluent: 100 mmol / L sodium sulfate in 100 mmol / L phosphate buffer (pH 6.7)
Flow rate: 0.35 mL / min
Temperature: 25 ° C
Injection volume: 150 uL
Samples were sequentially injected into the conditioned column under the following conditions, the peak shape was measured, and the degree of adsorption was observed.

<Measurement condition>
Eluent: 100 mmol / L sodium sulfate in 100 mmol / L phosphate buffer (pH 6.7)
Flow rate: 0.35 mL / min
Temperature: 25 ° C
Sample: 1. 0.01 g / L 4-Aminobenzoic acid
2. 1.5 g / L Lysozyme (Wako Pure Chemicals 120-02674)
3. 1.5 g / L Cytochrome C (Wako Pure Chemicals 033-22414)
4). 1.5 g / L Trypsin inhibitor from soybean (Wako Pure Chemicals 208-09223)
5. 1.5 g / L Albumin from chicken egg (Wako Pure Chemical 016-17073)
6). 1.5 g / L mouse monoclonal IgG1 5uL each
Table 1 shows the elution time, peak plate number and asymmetry coefficient of each sample. Lysozyme (pI 11.2) that is basic and strongly hydrophobic, Cytochrome C (pI10.2) that is basic and coordinates to metal, Trypsin inhibitor (pI 4.6), which is an acidic protein, and Albumin (pI4. All the elution peaks of 7) were eluted without tailing, and no adsorption phenomenon was observed. In addition, the aggregate component of the mouse antibody containing about 20% of the aggregate was eluted without being adsorbed.

Comparative Example 1
In the same manner as in Example 1, a 4.6 mm inner diameter × 15 cm long liquid chromatography column using a commercially available sintered stainless steel filter was prepared. The peak shape was measured in the same manner as in Example 1 without conditioning, and the degree of adsorption was observed. The results are shown in Table 1. Although acidic protein was eluted without adsorption, tailing of basic protein was observed, and the asymmetry coefficient was larger than that of Example 1. Antibody aggregates were not eluted at all.

Comparative Example 2
In the same manner as in Example 1, a 4.6 mm inner diameter × 15 cm long liquid chromatography column using a commercially available sintered stainless steel filter was prepared. As a conditioning, 150 μL of 0.1% concentration of Albumin Bovine Serum (Hiroshima Wako 016-15091) was injected. Subsequently, the peak shape was measured in the same manner as in Example 1 to observe the degree of adsorption. The results are shown in Table 1. The acidic protein was eluted without adsorption, but the basic protein showed some tailing, and the asymmetry coefficient was larger than that of Example 1. Although the antibody aggregate was eluted, the peak area was smaller than that in Example 1.

Comparative Example 3
In the same manner as in Example 1, a 4.6 mm inner diameter × 15 cm long liquid chromatography column using a commercially available sintered stainless steel filter was prepared. As a conditioning, 150 uL of 0.1% concentration Casein (Sigma C7078) was injected. Subsequently, the peak shape was measured in the same manner as in Example 1 to observe the degree of adsorption. The results are shown in Table 1. The acidic protein was eluted without adsorption, but the basic protein showed some tailing, and the asymmetry coefficient was larger than that of Example 1. Little antibody aggregates were eluted.

Comparative Example 4
In the same manner as in Example 1, a 4.6 mm inner diameter × 15 cm long liquid chromatography column using a commercially available sintered stainless steel filter was prepared. As conditioning, 150 uL of 0.1% concentration Dextran70 (Sigma 31290) was injected. Subsequently, the peak shape was measured in the same manner as in Example 1 to observe the degree of adsorption. The results are shown in Table 1. The acidic protein was eluted without adsorption, but the basic protein showed some tailing, and the asymmetry coefficient was larger than that of Example 1. Little antibody aggregates were eluted.

Comparative Example 5
In the same manner as in Example 1, a 4.6 mm inner diameter × 15 cm long liquid chromatography column using a commercially available sintered stainless steel filter was prepared. As a conditioning, 150 μL of 0.1% concentration of polyacrylamide (Sigma 43494-9) was injected. Subsequently, the peak shape was measured in the same manner as in Example 1 to observe the degree of adsorption. The results are shown in Table 1. The acidic protein was eluted without adsorption, but the basic protein showed some tailing, and the asymmetry coefficient was larger than that of Example 1. No antibody aggregates were eluted.

Claims (4)

A method for producing a column for liquid chromatography, which comprises conditioning a column for liquid chromatography using a porous metal filter with polylysine. 2. The method for producing a column for liquid chromatography according to claim 1, wherein the porous metal filter is sintered stainless or titanium. The method for producing a column for liquid chromatography according to claim 1, wherein the molecular weight of polylysine is 100,000 or more. The method for producing a column for liquid chromatography according to claim 1, wherein the amount of polylysine used is 0.5 mg / cm2 or more per column cross-sectional area.