KR100859791B1 - Simultaneous phase-separation and target molecule recovery using affinity ligand conjugated phase-separating molecule - Google Patents

Abstract

A method for separating a target material is provided to perform phase formation and molecule-specific separation at the same time using a PEGylated antibody, thereby conveniently and efficiently separating biomolecules including stem cells. A method for separating a target material comprises the steps of: (a) conjugating a molecular recognizing ligand having a molecular-specific binding capability of being bound to the target material into a phase forming material to prepare a molecular recognizing ligand-phase forming material conjugate; (b) mixing the conjugate with a phase constituting material containing the target material to form an aqueous two-phase system and to separate the target material from the molecular recognizing ligand-phase forming material conjugate layer; and (c) purifying the target material from the molecular recognizing ligand-phase forming material conjugate layer, wherein the molecular recognizing ligand is one selected from the group consisting of polypeptide, polynucleotide, aptamer, cell, metal, saccharide and lipid, the phase forming material is polyethylene glycol(PEG) or a derivative thereof and the phase constituting material is one selected from the group consisting of potassium phosphate buffer solution, dextran, polyvinylalcohol, polyethylene polypropylene block copolymer, oligosaccharide, potassium phosphate, sodium phosphate, ammonium sulfate and magnesium sulfate. Further, the derivative is one selected from mPEG-aldehyde, carboxylated-mPEG, amine-mPEG, mPEG-N-hydroxysuccinimide, mPEG-o-pyridyl disulfide, mPEG-maleimide, mPEG-thiol, mPEG-o-pyridyl thioster and mPEG-Carboxymethyl-Hydroxy benzoic acid-N-hydroxysuccinimide.

Description

Simultaneous phase-separation and target molecule recovery using affinity ligand conjugated phase-separating molecule

1 is a diagram showing the selective recovery mechanism of the antibody by molecular specific binding in the aqueous phase in which PEGylated antibody conjugated with polyethylene glycol (PEG, polyethylene glycol) is present,

Figure 2 is a diagram showing the structural formula and names of eight representative forms of polyethylene glycol (PEG, polyethylene glycol) derivatives,

3 is a drawing of a lab-on-a-chip with two injection holes and a microchannel at the center part in a straight line,

FIG. 4 is a drawing of a lab-on-a-chip with three inlets and increased lengths by bending the microchannels at the center.

      The present invention utilizes the molecular specific binding force of the molecular recognition ligand while simultaneously forming two phases which are not mixed with each other by dissolving a conjugate of the molecular recognition ligand and polyethylene glycol (PEG, polyethylene glycol) together with a salt or a polymer material in an aqueous solution. The present invention relates to a method for selectively separating a target substance from a sample in which various substances are dissolved.

     In order to measure the concentration of a biological material of interest in a biological sample as well as a sample in the mass production of useful biological materials, a technique capable of selectively recovering only a target material is essential. Biological samples are mixed with a number of impurities that have similar physical and chemical properties as the target material, and the availability of appropriate techniques to isolate them determines the efficiency of mass production and sample analysis.

     An aqueous phase system refers to the formation of two layers which are not mixed by the polymers which are equal to each other, the two polymers dissolved in water or the electrostatic repulsive force of the salt solution and the polymer solution. When used in a separation process, the technique is based on the selective distribution of the target substance in two phases that do not mix with each other. Mainly used polymer materials include polyethylene glycol (PEG, polyethylene glycol), polyviny alcohol, dextran, and the salts include potassium phosphate, sodium phosphate, Ammonium sulfate and magnesium sulfate. In general, they are mixed based on the mass ratio, not the volume ratio, and the volume ratio of the upper and lower layers varies depending on the molecular weight and mass ratio. However, the mass ratio is based on the volume ratio as a method for solving a problem that it is difficult to calculate the actual concentration after mixing because the density varies depending on the concentration of each substance.

     Aqueous phase extraction is a technique that separates a target substance using two or more polymer solutions or salt solutions that are not mixed with each other. In the aqueous phase system, since 65% or more of each phase is composed of an aqueous solution, it is a great advantage to provide high stability to biological materials as compared to using a general organic solvent. In addition, the aqueous phase system can be configured by changing the type, molecular weight and concentration of the polymer material and salt, pH of the solution, and the like, and thus, various systems can be configured.

     The aqueous phase system has been widely used as a biomaterial-friendly approach by using a partition between two liquids in the separation and purification of biological materials. The aqueous phase system does not use an organic solvent and has a water content of about 65 to 90%, thus providing an environment favorable for cells and organelles and substrates having biological activity. This means that the high interfacial surface tension and low interfacial surface tension minimize the inactivation of the material to be used, and high separation rate and yield can be obtained for the recovery of the biological material. In addition, it has the advantage of high capacity, easy scale-up, continuous process even with a simple device, which can be used for mass separation, and is safer and cheaper than organic solvent.

     The measure of efficiency in the separation of biomaterials using an aqueous phase system can be expressed by the partition coefficient (K), which is defined as the concentration ratio of the material present in the upper layer and the material present in the lower layer. In order to facilitate the distribution, methods such as changing the molecular weight and concentration of the polymer material and adding additional salts are used, but this may cause denaturation of the target substance by the salt, and distribution of impurities may also be affected. It has the disadvantage that the specific separation is difficult.

Various attempts have been made because of the merits of aqueous phase systems with high stability to biological materials. Recently, separation and purification techniques using affinity between biological materials in aqueous phase systems have been proposed. An example is the isolation and purification of monoclonal antibodies from cell culture solutions using an aqueous phase system (Sulk B, et al., J Immunol Methods , 149, 165-71, 1992).

     Aqueous phase-based constituents include ionic liquids as well as polyethylene glycol (PEG), dextran, polyethylene glycol (PEG) and salts And all materials capable of forming an aqueous phase system in an aqueous solution such as salt). In addition, ligands that can be applied to separation techniques include all of affinity separation polypeptides (enzymes, antigens and antibodies), polynucleotides, aptamers, cells, metals, sugars and lipids.

Polyethylene glycol (PEG), which is widely used in the composition of aqueous phase systems, is harmless to the human body, and has a high interest in the pharmaceutical field because it has an effect of increasing the residence time in the body and reducing antigenicity in the body when combined with antibodies . Therefore, an example of applying an aqueous phase system to separate a conjugate of an organism including an antibody and polyethylene glycol (PEG) has been reported (Karr LJ, et al., J Chromatography , 354, 269-282, 1986).

Examples of attempting affinity separation using antibodies include material separation using antibodies conjugated with ligands (Sharp KA, et al., Anal . Biochem . 154, 110-117, 1985), polyethylene glycol (PEG) and Cell separation using PEGylated antibodies conjugated with antibodies (Karr LJ, et al., J. Chromatography , 442, 219-227, 1988), separation by interfacial concentration, and the like. Among these, in the case of affinity separation using antibody-ligand conjugates such as immunoprecipitation, the ligand should be easily conjugated with the antibody, and the properties of the antibody should not be changed after binding with the ligand. Recently, in order to overcome these points, magnetic beads (magnetic beads) as ligands (Tsai HY, et al., Journal of Chromatography A 1130, 227-231, 2006), smart polymers (A. Kumar, et al., Biotechnology and Bioengineering 75, 570-580, 2001) have been reported examples of material separation attempts.

     However, these techniques are examples of the use of PEGylated materials as affinity ligands in aqueous phase systems preformed with polyethylene glycol (PEG) and dextran, which directly target the target material in the aqueous phase system already formed. It was an attempt to change its distribution characteristics by transforming it. As an example of the present invention to form a phase by using polyethylene glycol (PEG, polyethylene glycol) in the binder bonded to the polymer material, and recovering the target material using the molecular specific binding strength of the material bound to the polymer material No method has been reported.

Accordingly, the present inventors focused on the fact that the PEGylated antibody was used in the composition of the aqueous phase system, and thus the PEGylated molecular recognition ligand was separated from the phase by the polyethylene glycol (PEG). We devised a technology that can selectively recover the material to be separated by molecular specific binding force, and implemented the method on a lab on a chip, and as a result, it is possible to separate the target material with high purity in a short time. The present invention has been completed by confirming that it is possible.

      The present invention relates to a method for simultaneously performing phase formation and molecular specific separation using a molecular recognition ligand bound to an image forming material. Specifically, a conjugate of a molecular recognition ligand and polyethylene glycol (PEG) Dissolving in a solution with a salt or a polymer material to form two phases that do not mix with each other, and at the same time using a molecular specific binding force of the ligand to selectively separate only the desired target material from the sample in which various substances are dissolved; and Furthermore, the present invention relates to a method of selectively separating a target material by grafting a phase-forming material and a salt or a polymer material to which the molecular recognition ligand is bound to a lab on a chip.

      The present invention dissolves a conjugate of molecular recognition ligand and polyethylene glycol (PEG, polyethylene glycol) together with a salt or a polymeric material in an aqueous solution to form two phases that do not mix with each other and at the same time utilizes the molecular specific binding force of the ligand. Provided is a method for selectively separating only a desired target substance from a sample in which a substance is dissolved.

     In the present invention, the PEGylated antibody refers to a conjugated antibody to polyethylene glycol (PEG, polyethylene glycol) or derivatives thereof, and the derivatives thereof are M polyethylene glycol aldehyde (mPEG-aldehyde), carboxylated M polyethylene glycol (carboxylated-m PEG ), Amine M polyethylene glycol (amine-mPEG), m polyethylene glycol n-hydroxysuccinimide, m polyethylene glycol olso-pyridyl disulfide, m polyethylene Glycol maleimide, mPEG-thiol, M polyethylene glycol-ol-pyridyl thioester or m polyethylene glycol carboxymethyl hydroxy benzoic acid N-hydroxy cyanide (mPEG-Carboxymethyl-Hydrox ybenzoic acid-N-hydroxysuccinimide) and the like can be used.

     In addition, in the present invention, the phase forming material refers to a material conjugated with an antibody among the materials constituting the aqueous phase system.

     In addition, in the present invention, the phase constituent refers to a substance other than the phase constituent among the substances constituting the aqueous phase system.

       Hereinafter, the present invention will be described in detail.

       The present invention

       i) preparing a molecular recognition ligand-phase former conjugate by conjugating the phase former to a ligand having a molecular specific binding ability to bind the target substance;

      ii) mixing the conjugate of step i) with a phase constituent containing the target material to form an aqueous phase and simultaneously separating the target material into a molecular recognition ligand-phase former conjugate layer; And

      iii) purifying the target material from the molecular recognition ligand-phase former conjugate layer of step ii).

      In the separation method, the ligand having a molecular specific binding capacity in step i) is a polypeptide (antibody, antigen and enzyme), polynucleotide, aptamer, cell, metal which can bind to a target substance and can be affinity separated , Sugars and lipids are preferred, and antibodies are more preferred. In the embodiment of the present invention, a mouse-derived human antibody (Mouse anti-human IgG) was used.

     In addition, the phase forming material of step i) is preferably polyethylene glycol (PEG, polyethylene glyc ol) or derivatives thereof, and the derivatives are m polyethylene glycol aldehyde (mPEG-aldehyde), carboxylated em polyethylene glycol (carboxylated-mPEG). ), Amine M polyethylene glycol (amine-mPEG), m polyethylene glycol N-hydroxysuccinimide, m polyethylene glycol-ol-pyridyl disulfide, m Polyethylene glycol maleimide (mPEG-maleimide), M polyethylene thiol (mPEG-thiol), M polyethylene glycol olso-pyridyl thioester (mPEG-o-pyridyl thioester) and M polyethylene glycol carboxymethyl hydroxy benzoic acid It is preferable that it is any one selected from the group consisting of en hydroxy cyanide (mPEG-Carboxymethyl-Hydroxybenzoicacid-N-hydroxysuccinimide), It is not limited to this.

In addition, the molecular recognition ligand-phase former conjugate of step i) is preferably formed using a PEGylation method. The method of PEGylation depends on the active PEG used, but conventional PEGylation methods can be used. More specifically, polyethylene glycol N-hydroxysuccinimide derivative and protein amine were simultaneously dissolved in 50 mM Phosphate buffer (pH 7.2, 4 ° C). Wait for 2 hours or dissolve in 50 mM Borate-phosp hate buffer (pH 8.0, 25 ℃) and wait for 2 hours, simultaneously with polyethylene glycol aldehyde derivative and amine protein Dissolve in sodium cyanoborohydride (4 ℃) and wait for 20 hours. Polyethylene glycol aldehyde (PEG-aldehyde) derivative and cysteine protein (protein cystein) at the same time 100 mM Phosphate buffer (pH 6.5, 4 4 hours after dissolving in polyethylene glycol amine (PEG-amine) derivatives and carboxylate (protein carboxylates) at the same time 50 mM Phosphate buffer (pH 7.2, WSC, 4 ℃) How to wait 10 hours after dissolving, with polyethylene glycol-para-nitrophenylcar bonate derivative Dissolve the amine protein in 50 mM boron-phosphate buffer (pH 8.0 ~ 8.3, room temperature) at the same time and wait 1 hour or M polyethylene glycol alcohol (mPGE-OH) and amine protein (protein) Amine is simultaneously dissolved in 0.1 M boron buffer (pH 9.2, 4 ° C.) and then waited for 1 hour.

     In addition, the phase constituents of step ii) are potassium phosphate buffer, dextran, polyvinylalcohol, polyethylene polypropylene block copolymer, oligosaccharides, potassium phosphate Any substance capable of phase forming with polyethylene glycol (PEG) or its derivatives, such as potassium phosphate, sodium phosphate, ammonium sulfate, and magnesium sulfate, can be used. It is preferable to use dextran. In the embodiment of the present invention, polyethylene glycol (PEG, polyethylene glycol) as the phase forming material and dextran (dext ran) as the phase forming material were used.

       The present invention

      i) preparing a lab-on-a-chip having a molecular recognition ligand-phase former conjugate inlet, a mixture inlet containing a phase constituent and a target substance, and a channel connecting the inlet;

      ii) injecting a molecular recognition ligand-phase former conjugate and a mixture containing a phase constituent and a target substance into each inlet of step i);

      iii) mixing the conjugate of step ii) with the phase constituent containing the target material within the channel of the lab-on-a-chip to form an aqueous phase system and simultaneously separating the target material into a molecular recognition ligand-phase former conjugate layer. Doing; And

      iv) a method of separating a target substance using a lab-on-a-chip comprising purifying the target substance from the molecular recognition ligand-phase former conjugate layer of step iii).

      In the separation method, the number of injection holes of the lab-on-a-chip of step i) is preferably two or three, and the location is preferably at both ends and the center, but the number and location of the injection holes is not limited thereto.

      In addition, the channel connecting the injection hole of the lab-on-a-chip of step i) is preferably used to increase the length by giving a straight or curved shape, but is not limited thereto.

      In addition, the molecular recognition ligand of step ii) is to use polypeptides (antibodies, antigens and enzymes), polynucleotides, aptamers, cells, metals, sugars and lipids that can be affinity-separated by binding to a target substance. Preferably, it is more preferable to use an antibody. In the embodiment of the present invention, a mouse-derived human antibody (Mouse anti-human IgG) was used.

     In addition, the phase forming material of step ii) is preferably polyethylene glycol (PEG, polyethylene glycol) or derivatives thereof, and the derivatives are m polyethylene glycol aldehyde (mPEG-aldehyde), carboxylated empolyethylene glycol (carboxylated-mPEG). , Amine M polyethylene glycol (amine-mPEG), m polyethylene glycol n-hydroxysuccinimide, m polyethylene glycol-ol-pyridyl disulfide, m polyethylene Glycol Maleimide (mPEG-maleimide), M Polyethylene Thiol (mPEG-thiol), M Polyethylene Glycol Olso-Pyridyl Thioester (mPEG-o-pyridyl Thioester) and M Polyethylene Glycol Carboxymethyl Hydroxy Benzoic Acid En It is preferably one selected from the group consisting of hydroxy cyanide (mPEG-Carboxymethyl-Hydroxybenzoicacid-N-hydroxysuccinimide), It is not limited to this.

In addition, the molecular recognition ligand-phase former conjugate of step ii) is preferably formed using a PEGylation method. The method of PEGylation depends on the active PEG used, but conventional PEGylation methods can be used. More specifically, polyethylene glycol N-hydroxysuccinimide derivative and protein amine were simultaneously dissolved in 50 mM Phosphate buffer (pH 7.2, 4 ° C). Wait for 2 hours or dissolve in 50 mM Borate-phosp hate buffer (pH 8.0, 25 ℃) and wait for 2 hours, simultaneously with polyethylene glycol aldehyde derivative and protein amine Dissolve in sodium cyanoborohydride (4 ℃) and wait for 20 hours. Polyethylene glycol aldehyde (PEG-aldehyde) derivative and cysteine protein (protein cystein) at the same time 100 mM Phosphate buffer (pH 6.5, 4 4 hours after dissolving in polyethylene glycol amine (PEG-amine) derivatives and carboxylates (protein carboxylates) at the same time 50 mM Phosphate buffer (pH 7.2, WSC, 4 ℃) After 10 hours of waiting method, polyethylene glycol-p-nitrophenyl carbonate (PEG-p-Nitrophenylcar bonate) derivative and the Dissolve the amine protein in 50 mM boron-phosphate buffer (pH 8.0 ~ 8.3, room temperature) at the same time and wait 1 hour or M polyethylene glycol alcohol (mPGE-OH) and amine protein (protein) Amine is simultaneously dissolved in 0.1 M boron buffer (pH 9.2, 4 ° C.) and then waited for 1 hour.

      In addition, the phase constituents of step ii) are potassium phosphate buffer, dextran, polyvinylalcohol, polyethylene polypropylene block copolymer, oligosaccharides, potassium phosphate Any substance capable of phase forming with polyethylene glycol (PEG) or its derivatives, such as potassium phosphate, sodium phosphate, ammonium sulfate, and magnesium sulfate, can be used. It is preferable to use dextran. In the embodiment of the present invention, polyethylene glycol (PEG, polyethylene glycol) and dextran (dext ran) were used as phase constituents.

      Hereinafter, the present invention will be described in detail through examples. However, the following examples are only for illustrating the present invention, but do not limit the content of the present invention.

     In the embodiment of the present invention, a PEGylated antibody (PEGylate d antibody) in which an antibody, which is one of the affinity separation forms using the antibody, is conjugated to polyethylene glycol (PEG) is used. Using pegylated antibodies, solutions consisting of two types of polymers formed an aqueous phase that did not mix with each other, while at the same time allowing specific separation of specific biological materials.

< Example  1> Mercury phase system  Composition and In the aqueous phase  Affinity separation

      The present invention is a substance for performing separation by molecular specific binding at the same time as phase separation and polyethylene glycol (PEG, polyethylene glycol) and mouse anti-human IgG (Pierce Biotechnology, Inc., USA Dextran was selected as a polymer material in which the antibody is present in the aqueous phase with a PEGylated antibody combined with polyethylene glycol (PEG) to form two phases. It was. Samples of the mixture for molecular specific binding were goat-derived mouse antibodies (Goatanti-mouse IgG) (purchased from Pierce Biotechnology, Inc., USA) and goat-derived rabbit antibodies (Goat anti-rabbit IgG) (Pierce Biotechnology, Inc., Purchased from USA). However, this is an example selected to demonstrate the superiority of the present invention and does not limit the scope of the present invention.

     Mouse anti-human IgG CD34, which is an antibody to be used for isolation, was PEGylated using a polyethylene glycol (PEG, polyethylene glycol) derivative having a molecular weight of 10000 to 20,000 in Example 1, The antibody and dextran were used to construct an aqueous phase system, which could be used to separate affinity cells or antibodies from other materials with no affinity.

< Example  2> In the aqueous phase  With affinity separation Lab-on-a-chip

     Of three inputs of lab on a chip, pegylated antibody and dextran are injected at each end to form an aqueous phase system in the chip, and pegylated antibody in the middle of the chip. A mixture of the target material with and affinity with and without the affinity was injected to allow separation in the chip. In addition, a lab-on-a-chip having two inlets was made and the material to be separated was previously dissolved in a phase other than PEG to induce separation in the chip. When the present invention is applied to a lab on a chip, even if a small amount of sample is used, the target material can be easily separated within a short time, thereby reducing the cost and time.

The present invention utilizes pegylated antibodies to allow solutions consisting of two polymeric materials to form two phases, and at the same time enable molecular specific separation for specific materials. PEGylated antibody of the present invention has the advantage that can be used for simple and efficient biomolecular separation of biomolecules including stem cells. Formation of an aqueous phase system using the PEGylated antibody of the present invention and molecular specific separation technology can be applied to a lab on a chip, enabling rapid separation and detection using a small amount of sample, thereby ensuring stability of a target substance. And separation efficiency can be increased. These techniques can be applied to the analytical process as well as to the mass production of the target material, so the scope of application is expected to be huge.

Claims (12)

       i) conjugating the phase forming material to a molecular recognition ligand which has a molecular specific binding ability to bind the target material to prepare a molecular recognition ligand-phase forming material conjugate;       ii) mixing the conjugate of step i) with a phase constituent containing the target material to form an aqueous phase system and simultaneously separating the target material into the molecular recognition ligand-phase former conjugate layer; And       iii) purifying the target material from the molecular recognition ligand-phase former conjugate layer of step ii).       The method of claim 1, wherein the molecular recognition ligand is any one selected from the group consisting of polypeptides, polynucleotides, aptamers, cells, metals, sugars, and lipids.  The method of claim 2, wherein the polypeptide is an antibody.     The method of claim 1, wherein the phase forming material is polyethylene glycol (PEG, polyethylene glycol) or a derivative thereof. The method of claim 4, wherein the derivative is m polyethylene glycol aldehyde (mPEG-aldehyd e), carboxylated M polyethylene glycol (carboxylated-mPEG), amine M polyethylene glycol (amine-mPEG), M polyethylene glycol en-hydroxy susini MPEG-N-hydrox ysuccinimide, mPEG-o-pyridyl disulfide, mPEG-maleimide, M polyethylene thiol, mPEG-thiol, With mPEG-o-pyridyl thioester and m polyethyleneglycol carboxymethyl hydroxy benzoic acid n hydroxysuccinimide (mPEG-Carboxymethyl-Hydroxy benzoicacid-N-hydroxy succinimide) Separation method, characterized in that any one selected from the group consisting of.       The phase composition of claim 1, wherein the phase constituents are potassium phosphate buffer, dextran, polyvinylalcohol, polyethylene polypropylene block copolymer, oligosaccharide, Separation method, characterized in that any one selected from the group consisting of potassium phosphate, sodium phosphate, ammonium sulfate (mmonium sulfate) and magnesium sulfate (magnesium sulfate).       i) preparing a lab-on-a-chip comprising a first inlet for injecting a molecular recognition ligand-phase former conjugate, a second inlet for injecting a mixture containing a phase constituent and a target substance and a channel connecting the inlets; Doing;       ii) injecting the molecular recognition ligand-phase former conjugate and a mixture containing the phase constituent and the target substance into the first inlet and the second inlet of the lab-on-a-chip respectively;       iii) mixing the conjugate of step ii) with the phase constituent containing the target material within the channel of the lab-on-a-chip to form an aqueous phase and simultaneously separating the target material into a molecular recognition ligand-phase former conjugate layer. Doing; And       iv) purifying the target material from the molecular recognition ligand-phase former conjugate layer of step iii).       8. The method of claim 7, wherein the molecular recognition ligand is any one selected from the group consisting of polypeptides, polynucleotides, aptamers, cells, metals, sugars, and lipids.        The method of claim 8, wherein the polypeptide is an antibody.     8. The method of claim 7, wherein the phase forming material is polyethylene glycol (PEG, polyethylene glycol) or derivatives thereof.      The method of claim 10 wherein the derivative is m polyethylene glycol aldehyde (mPEG-aldehyde), carboxylated-m polyethylene glycol (carboxylated-mPEG), amine M polyethylene glycol (amine-mPEG), M polyethylene glycol en-hydroxy susini MPEG-N-hydrox ysuccinimide, mPEG-o-pyridyl disulfide, mPEG-maleimide, M polyethylene thiol, mPEG-thiol, With mPEG-o-pyridyl thioester and m polyethyleneglycol carboxymethyl hydroxy benzoic acid n hydroxysuccinimide (mPEG-Carboxymethyl-Hydroxy benzoicacid-N-hydroxy succinimide) Separation method, characterized in that any one selected from the group consisting of.        The method of claim 7, wherein the phase constituents are potassium phosphate buffer, dextran, polyvinylalcohol, polyethylene polypropylene block copolymer, oligosaccharide, Separation method characterized in that any one selected from the group consisting of potassium phosphate, sodium phosphate, ammonium sulfate (mmonium sulfate) and magnesium sulfate (magnesium sulfate).

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