JP2017209604A - Composite stimulation-responsive affinity controlling polymer, adsorbent, and purification method of target material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composite stimulation-responsive affinity controlling polymer which has a bonding force sufficient for efficiently adsorbing a target material present in a solution, especially an antibody, in a short time, and from which the adsorbed target material can be desorbed with a condition mild for the target material, that is, with a non-invasive stimulus.SOLUTION: A composite stimulation-responsive affinity controlling polymer is able to adsorb or desorb a target material, and has a polymer chain responding to a first stimulus so as to change the conformation thereof and a plurality of kinds of ligands bonded to the polymer chain, in which at least one kind of the plurality of kinds of ligands has a bonding force to the target material changing corresponding to a second stimulus.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a complex stimulus-responsive affinity control polymer, an adsorbent, and a method for purifying a target substance.

  The process of recovering and purifying target substances, particularly biomolecules typified by proteins, from solutions is becoming increasingly important as a basic step of bioprocesses. In particular, with the advancement of antibody drugs, there is a need for a technique for recovering and purifying a target antibody from a culture solution with high efficiency and low cost without degrading the antibody.

  Affinity purification based on the principle of adsorbing a target substance to a solid phase due to the affinity between the ligand and target substance of the adsorbent is the adsorption / non-adsorption component of the target substance present in the solution to the adsorbent. Cleaning, and desorption of the target substance from the adsorbent by the eluent. At this time, a strong acid or a basic solution is often used as the eluent. This is because it is necessary to change the ionization state of the ligand and the target substance and weaken the interaction between the ligand and the target substance by charge repulsion. However, in harsh pH environments such as strong acids and strong bases, biomolecules are often unstable and the purified target substance is problematic. There is also a concern about deterioration of the ligand and column material.

  In view of this, an adsorbent material for affinity columns has been developed that improves the ligand and makes it possible to purify biomolecules under mild pH conditions. In Patent Document 1, protein A is modified to respond with a mild acid by gene recombination. Thus, when the ligand is a protein, it is relatively easy to adjust the responsive pH value by applying a technique such as gene recombination. However, even under mildly acidic conditions, when the target substance is an antibody, the antibody may associate with a small amount. Therefore, in the purification process of an antibody drug, a process for removing the associated antibody after purification is often required.

  Furthermore, compared to protein-derived ligands, low-molecular ligands, which are lower in cost, have fewer sites that interact with the target substance, so there are fewer sites that can modify the chemical structure to adjust the responsive pH value, The degree of freedom for adjusting the responsiveness is low (Non-Patent Document 1). Although there is a report of perturbing local pH in the vicinity of a ligand by copolymerizing a low molecular ligand and an amine (Non-patent Document 2), this is not a generally applicable technique.

  A technique for controlling the binding and desorption of a target substance in response to temperature by binding a ligand to a polymer chain whose conformation changes in response to temperature has also been reported. This technique may be able to be operated under an almost neutral pH condition without changing the pH condition, and may possibly avoid the risk of deterioration of the target substance. For example, Patent Document 2 uses a temperature-responsive polyamino acid, Non-Patent Document 3 uses a temperature-responsive polymer chain poly (N-isopropylacrylamide) (PNIPAM) on the microparticle surface, and Protein A as a ligand. An immobilized adsorbent material is disclosed. Non-Patent Document 3 discloses an adsorbing material formed by binding 4-mercaptoethylpyridine as a low molecular ligand. In either case, the target antibody can be adsorbed under low temperature conditions with a conformation in which the polymer chain is extended, and the adsorbed antibody can be desorbed by changing the conformation to an aggregated state due to temperature rise. It is shown.

  This method shows that the conformation of the polymer chain due to temperature change restricts the steric arrangement of the ligand and the antibody, that is, increases the steric hindrance, thereby dissociating the binding between the ligand and the antibody. It is the principle of operation. That is, the binding energy between the ligand and the target material is basically constant throughout the adsorption process and the desorption process. For this reason, when the binding energy between the ligand and the target substance is larger than the change in conformational energy associated with the temperature change, it is difficult to desorb the bound target substance. On the other hand, if a ligand with a low binding force to the target substance is selected so that desorption is possible, the target substance present in the solution cannot be sufficiently adsorbed, and there is a problem that it takes a long time for the adsorption. As a result, there is a problem that the efficiency of the purification process is low.

JP 2010-81866 A JP 2014-219245 A

Journal of Chromatography B, 2000, 740, 1-15. Chemical Science, 2012, 3, 1467-1471. Biomacromolecules, 2016, 17, 280-290.

  In the conventional method, there is a problem that a target substance existing in a solution, particularly an antibody useful as a medicine, cannot be efficiently recovered and purified without damaging the target substance. As described above, the method using a temperature-responsive polymer chain can be purified by immobilizing, purifying, and desorbing without degrading the target substance. It is difficult to achieve both detachability.

  The present invention has been made in view of the above problems, and the object is to have a binding force sufficient to efficiently adsorb a target substance, particularly an antibody, present in a solution in a short time, Another object of the present invention is to provide a complex stimulus-responsive affinity controlled polymer capable of desorbing an adsorbed target substance under mild conditions with respect to the target substance, that is, non-invasive stimulation. Another object of the present invention is to provide an adsorbent using the polymer and a method for purifying a target substance with high efficiency using the adsorbent.

  As a result of intensive studies, the present inventors have arranged a plurality of ligands that associate with a target substance on a polymer chain whose conformation changes in response to a stimulus, and one of the plurality of ligands is as described above. Discovered that the above problems could be solved by a multi-ligand, multi-response complex stimulus-responsive affinity-controlling polymer, which is a ligand whose binding force to the target substance is changed by another stimulus. did.

  That is, the present invention is a composite stimulus-responsive affinity control-type polymer capable of adsorbing and desorbing a target substance, a polymer chain whose conformation changes in response to a first stimulus, and the polymer chain. And at least one of the plurality of ligands has a binding force that changes with the target substance by a second stimulus. In addition, the present inventors have completed the invention of an efficient purification process of a target substance using an adsorbent in which the above-mentioned composite stimulus-responsive affinity control polymer is immobilized on a carrier, and a column packed with the adsorbent. It came to do.

  By using the complex stimulus responsive affinity control polymer according to the present invention, the target substance in the solution can be efficiently adsorbed and immobilized, and it is fixed by a mild stimulus that does not affect the target substance. It is possible to desorb, elute and collect the target substance. Problems, configurations, and effects other than those described above will be clarified by the following description of the embodiments.

It is the figure which showed typically the structure of the composite stimulus responsive affinity control type polymer which concerns on one Embodiment of this invention in the state couple | bonded with the support | carrier. It is the schematic diagram which showed the process of capturing, adsorbing, and desorbing a target substance (antibody) by the composite stimulus responsive affinity control polymer according to one embodiment of the present invention. It is the figure which showed typically the structure of the adsorbent formed by the composite stimulus responsive affinity control type polymer which concerns on one Embodiment of this invention couple | bonding with the surface. It is the figure which showed typically the purification column with which the adsorbent which concerns on one Embodiment of this invention was filled. The schematic diagram which showed the process of collect | recovering and refine | purifying a target substance (antibody) using the refinement | purification column with which the composite stimulus responsive affinity control type polymer which concerns on one Embodiment of this invention couple | bonded with the surface was packed. It is. It is a synthetic scheme of the compound stimulus responsive affinity control type polymer in an Example. It is an adsorption / desorption curve of a complex stimulus-responsive affinity controlled polymer (Polymer 5) with respect to a human IgG fixed substrate, measured by SPR method. It is a graph which shows the influence which the ligand kind which a composite stimulus responsive affinity control type polymer has, and the average degree of polymerization of PNIPAM have on dissociation constant _Kd with human IgG. It is a graph which shows the influence of temperature and glucose addition on the dissociation constant _Kd of the compound stimulus responsive affinity control type | mold polymer | macromolecule which has phenyl boronic acid and PAM2 as a ligand, and human IgG.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings as appropriate. The following description shows specific examples of the contents of the present invention, and the present invention is not limited to these descriptions. Various changes and modifications are within the scope of the technical idea disclosed in this specification. Is possible. In all the drawings for explaining the present invention, components having the same function are denoted by the same reference numerals, and repeated description thereof may be omitted. In the following description, an antibody is taken as an example of the target substance, but the target substance targeted by the present invention is not limited to this. If it is a biomolecule or a molecule that is unstable under severe pH conditions, the features of the present invention can be utilized. Further, in order to clarify the concept of the present invention, in the following description and drawings, a dual ligand type in which two kinds of ligands are bonded to a polymer chain is used as a composite stimulus-responsive affinity control type polymer. A preferred example is given below. However, the chemical structure of the composite stimulus responsive affinity control polymer according to the present invention is not limited thereto.

  With reference to FIG. 1 as appropriate, the composite stimulus-responsive affinity controlled polymer 10 of the present invention will be described. FIG. 1 is a diagram schematically showing the structure of a composite stimulus responsive affinity controlled polymer 10. As shown in FIG. The composite stimulus-responsive affinity control polymer 10 is constituted by binding a plurality of types of ligands having the ability to bind to a target substance typified by an antibody to a polymer chain 11. In addition, the composite stimulus responsive affinity control polymer 10 has a fixing portion 14 that binds to the carrier 20. The composite stimulus-responsive affinity control type polymer 10 is immobilized on the carrier 20 so as to function as an adsorbent as a whole.

  The present embodiment is characterized in that a plurality of types of ligands that bind to a target substance via different binding mechanisms are bonded to the same polymer chain 11, that is, are multi-ligand types. FIG. 1 illustrates a dual-ligand complex stimulus-responsive affinity controlled polymer 10 in which a first ligand 12 and a second ligand 13 of different types are each bonded to a polymer chain 11. However, the number of ligand species and the number of ligands that bind to the polymer chain 11 are not particularly limited, and may be appropriately selected so as to correspond to the type and arrangement of the binding site of the target substance. In general, biomaterials such as proteins and antibodies have a specific chemical / stereostructure, and a binding site having a binding ability with a ligand is uniquely arranged. Therefore, it is desirable to determine the ligand type, number, and arrangement of the polymer chain so as to be opposite to the binding site of the target substance and its arrangement.

  The polymer chain 11 is composed of a stimulus-responsive polymer chain having a mechanism that changes its conformation in response to a stimulus. Stimulation is not particularly limited as long as it does not degrade the target substance or is mild in that it does not affect the target substance. Physical stimulation such as light, magnetic field, electric field, ultrasonic wave, temperature, pH, salt A chemical stimulus such as concentration is exemplified as a suitable stimulus. In particular, it is desirable to use a temperature change because it is easy to apply a stimulus.

  Hereinafter, the case where the stimulus for changing the conformation of the polymer chain 11 is set as an example will be described. However, in the present invention, the stimulus for changing the conformation of the polymer chain 11 is not limited to the temperature. In the case of applying another stimulus, it can be used in the same manner if the chemical structure of the polymer chain 11 is determined so as to respond to the stimulus.

  The hydrophilic polymer has a conformation in which the polymer chain is spread by hydration in an aqueous solution, that is, a so-called coiled conformation. On the other hand, the hydrophobic polymer chain aggregates due to dehydration, resulting in a so-called globule-like conformation. The temperature-responsive polymer chain is a polymer that causes conformational transition by changing physicochemical properties such as hydrophilicity / hydrophobicity in response to a change in temperature in an aqueous solution. Temperature-responsive polymers are classified into a low critical solution temperature (LCST) type and a high critical solution temperature (UCST) type according to their temperature transition characteristics. A polymer having LCST-type temperature characteristics changes from a hydrophilic conformation to a hydrophobic conformation when passing through the LCST from a temperature lower than the LCST. A polymer having a temperature characteristic of the UCST type changes from a hydrophobic conformation to a hydrophilic conformation when the temperature passes through the UCST from a temperature lower than the UCST. The exact values of LCST and UCST are known to depend on the polymer chain and other application conditions (solvents, other solutes, etc.). When used in the present invention, the characteristics of the temperature-responsive polymer chain may be either LCST type or UCST type.

  As temperature-responsive polymer chains, those having various chemical structures are known, but when applied to the present invention, the critical solution temperature is within a temperature range in which the target substance, that is, the antibody or the like does not deteriorate, that is, It is desirable to be within the range of the non-freezing temperature to 50 ° C. Examples of the temperature-responsive polymer chain having such characteristics include poly (N-substituted acrylamide) including poly (N-isopropylacrylamide) and poly (N-isopropylmethacrylamide) and other poly (N-isopropylmethacrylamide). N-substituted methacrylamide), poly (N, N-disubstituted acrylamide), poly (N, N-disubstituted methacrylamide), polymethyl vinyl ether, poly (ethylene oxide-propylene oxide) copolymer, partially saponified polyvinyl alcohol. Examples thereof include polyvinyl alcohol derivatives including cellulose derivatives and cellulose derivatives including methyl cellulose. In particular, poly (N-isopropylacrylamide) (PNIPAM) has been studied in detail, and since the critical solution temperature is 33 ° C., it is mentioned as a suitable polymer chain in terms of easy temperature control.

  Next, the ligand will be described. A ligand is a group of atoms having chemical / physical properties and structures that are specifically adsorbed to a binding site of a target substance. There are various types of ligands depending on the binding site, and they are roughly classified into those derived from proteins or modified proteins and those composed of chemically synthesized low-molecular compounds. Protein-derived ligands are so specific that they are generally described as a relationship between a key and a keyhole, and bind tightly to a target substance. On the other hand, the bond dissociation often needs to be performed under severe conditions such that the ligand is denatured, and there is a concern about the deterioration of the target substance. On the other hand, low molecular ligands are often less specific than protein-derived ligands, but those that dissociate binding by mild stimulation can be synthesized by molecular design. In general, it is less expensive than protein-derived ligands. The ligand applied to the present invention is not particularly limited, but it is desirable to use a low molecular weight ligand in that it can be recovered without degrading the target substance. There are two types of low-molecular ligands: one that binds to the binding site of the target substance via a non-covalent bond typified by physical binding, and one that binds by forming a covalent bond.

  Examples of the low molecular weight ligand that binds to the target substance via a non-covalent bond include 3-thia-6-pyridylhexyl vinyl sulfone, 3-thia-6-arylhexyl vinyl sulfone, and 3-oxa-6-pyridylhexyl vinyl. Sulfone, 3-oxa-6-arylhexyl vinyl sulfone, 3-aza-6-pyridylhexyl vinyl sulfone, 3-aza-6-arylhexyl vinyl sulfone, pyridyl oligoethylene glycol vinyl sulfone, aryl oligoethylene glycol vinyl sulfone, 3-hydroxyphenylethylamino) (4-hydroxyphenylethylamino) triazine derivative, (4-hydroxyphenylethylamino) (phenylamino) triazine derivative, (4-hydroxy-1-naphthylamino) (3-hydroxy Nenylamino) triazine derivatives, mimetic red, mimetic orange, mimetic yellow, mimetic green, mimetic blue, TG19318, polypeptides, peptide dendrimers, protein A partial structure, protein A mimetic structure, protein G partial structure, Examples include protein G mimetic structures, lectins, sugar derivatives and the like.

  Examples of the low molecular weight ligand that binds to a target substance via a covalent bond include phenylboronic acid that covalently binds to a sugar chain on an asparagine residue, retinal that covalently binds to rhodopsin, and the like.

  The composite stimulus-responsive affinity control polymer 10 according to this embodiment is characterized by having a plurality of types of ligands having different binding mechanisms with a target substance. Here, each of the two types of ligands has one. The description will be continued by taking the dual ligand type as an example. As the first ligand 12 and the second ligand 13 bound to the polymer chain 11, it is desirable to adopt one having the ability to bind to the target substance by different binding mechanisms. In general, it is known that a biomolecule represented by a protein has a uniquely determined higher order structure, and a specific site in the molecule specifically binds to a specific ligand. The binding mechanism between the binding site of the target substance and the ligand includes chemical bonds, covalent bonds, non-covalent physical hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic bonds. Etc. In the present embodiment, each of the binding between the first ligand 12 and the target substance and between the second ligand 13 and the target substance has a different main binding mechanism. The combination of the main coupling mechanisms is not particularly limited, but it is desirable that at least one coupling mechanism is a coupling whose main factor is a covalent bond. This is because a covalent bond has a larger binding energy than a non-covalent bond and has the ability to bind strongly to a target substance.

  At least one of the first ligand and the second ligand is characterized in that the binding force to the target substance is changed by a stimulus different from the stimulus that induces a conformational change of the polymer chain 11. . For example, when a temperature-responsive polymer whose conformational change is induced by temperature stimulation is applied as the polymer chain 11, at least one of the first ligand and the second ligand is a stimulus other than temperature, for example, a chemical substance. It is desirable to apply a substance whose binding state with the target substance changes depending on the salt concentration or pH. In this way, by making the composite stimulus responsive affinity control polymer 10 a multi-response type that responds to a plurality of stimuli, the target substance that has been firmly bound can be efficiently removed by the combined action of mild stimuli. Can be separated.

The combination of the binding site of the ligand and the target substance will be described in further detail, taking as an example the case where immunoglobulin (IgG) is the target substance. IgG is known to consist of a Fab site and an Fc site. Here, it is known that protein A, which is a modified protein ligand, binds to the C _H 2 -C _H 3 region of the Fc site of IgG by noncovalent bonding due to physical interaction. Therefore, triazine derivative low-molecular ligands that mimic the structure of protein A also bind to this region by non-covalent bonds. Also, sugar chains bound to asparagine residues present N297 site of C _{H 2} region of an IgG, an ester bond, i.e. capable of forming a bond with the ligand by chemical bonds. Here, boronic acid such as phenylboronic acid can be exemplified as a suitable ligand that binds to a sugar chain on an asparagine residue. Phenylboronic acid not only forms a boronic acid ester bond with a sugar chain, but the bond can be dissociated by adding a compound having a diol to the system, for example, a saccharide such as glucose as a substitute substance.

  As described above, a temperature-responsive polymer chain 11, a triazine derivative low-molecular ligand having a protein A mimetic structure as the first ligand 12, and a sugar chain and an ester bonded to the asparagine residue as the second ligand 13 A dual-ligand complex stimulus-responsive affinity-controllable polymer 10 to which a phenylboronic acid group that forms a bond is bonded includes a temperature stimulus that changes the conformation of the polymer chain, a second ligand 13 and a target substance. In response to the addition of a substitution substance that cleaves the chemical bond with IgG, a multi-response characteristic in which the binding force with IgG changes is exhibited.

  As schematically shown in FIG. 1, the first ligand 12 and the second ligand 13 are bonded to the polymer chain 11 by a chemical bond. Here, when the first ligand 12 and the second ligand 13 bind to the target substance, the complex stimulus responsiveness is set so as to be opposed to the arrangement of the binding site of the target substance to which each ligand selectively binds. It is desirable to design the molecular structure of the affinity controlled polymer 10. That is, in the state of being bound to the target substance, the polymer chain 11 is set such that the distance between the first ligand 12 and the second ligand 13 in the polymer chain 11 is equal to the distance between the binding sites in the target substance. It is preferable to adjust the molecular weight and the arrangement of each ligand with respect to the polymer chain 11. For example, as described above, when a triazine derivative-based small molecule is used as the first ligand and a phenylboronic acid group is used as the second ligand, the binding site of IgG to which each ligand binds is specified. Therefore, based on the three-dimensional structure analysis data of IgG, a triazine derivative low molecule and a phenylboronic acid group may be arranged on the polymer chain 11 so that both binding sites and both ligands are stably associated with each other. desirable. By adopting such a molecular structure, the free energy when the target substance and the complex stimulus responsive affinity controlled polymer 10 are bonded via the first ligand 12 and the second ligand 13 is reduced, Strong binding to the target substance becomes possible. In addition, the conformation of the polymer chain in the aqueous solution has a coiled conformation, and the distance between the ligands is not necessarily the value in the state where the polymer chain is extended. It is necessary to pay attention to this point when designing the molecular structure of the composite stimulus-responsive affinity controlled polymer 10.

  The composite stimulus-responsive affinity control polymer 10 desirably has a fixing portion 14 that can be bonded to the carrier 20. The chemical structure of the fixing part 14 can be appropriately selected according to the material of the carrier 20 and the fixing mechanism. For example, when the carrier 20 is an inorganic substance typified by glass, an alkoxysilane group or a hydroxyl group can be used, and when the carrier 20 is a metal such as gold, a thiol group can be used. When the carrier 20 is an organic substance, a functional group that forms a covalent bond with the chemically activated carrier surface can be used. For example, when the fixing part 14 is an amino group, it is possible to activate the carrier surface to introduce a carboxylic acid group and bond it via carbodiimide. In addition, click bonding using a thiol-ene reaction can also be used. In the composite stimulus-responsive affinity control polymer 10 shown in FIG. 1, the fixing part 14 is illustrated to be present at the end of the polymer chain 11, but the position of the fixing part 14 is not limited to this. Absent. The fixing part 14 may exist not in the end part of the polymer chain 11 but in the intermediate part. Further, there is no problem even if it is a so-called star structure that is indirectly bonded to the polymer chain 11 via a linker, a second polymer chain, or the like. At this time, the second polymer chain does not need to be stimuli-responsive.

  The composite stimulus-responsive affinity control polymer 10 may be synthesized by an appropriate polymerization method, but the molecular weight distribution of the main chain is as small as possible and the number of ligands or immobilization parts is as much as possible so as to correspond to the structure of the target substance. -It is desirable to apply a precision polymerization method that can obtain a polymer whose arrangement is uniquely determined. With precision polymerization, it is possible to synthesize functional polymers whose structure is uniquely determined. Especially when living polymerization is used, not only can the molecular weight distribution of the polymer main chain be small, but also the introduction and function of a wide range of functional groups. Even the position control of the group is possible. For example, when the polymer chain 11 is PNIPAM, a monodisperse polymer having a very small molecular weight distribution can be obtained by using an atom transfer radical polymerization method (Atom Transfer Radical Polymerization method) which is a kind of living polymerization method. Moreover, a ligand and a fixing | fixed part can be uniquely introduce | transduced by utilizing a polymerization reaction initiator, a reactive living polymer terminal, etc. suitably. In the case of a temperature-responsive polymer, if the molecular weight distribution of the main chain is reduced, there is also an advantage that the conformational transition accompanying the temperature change appears more sharply. Such behavior is preferable because the adsorption and desorption control becomes clear when the target substance is adsorbed and desorbed by temperature stimulation. Therefore, the ratio of the weight average molecular weight Mw to the number average molecular weight Mn of the polymer chain 11 is 1.01 to 1.40, preferably 1.01 to 1.30, particularly 1.01 to 1.20. It is desirable to be.

  Next, referring to FIG. 2 as appropriate, the process of capturing, binding, and desorbing the target substance using the adsorbent in which the composite stimulus responsive affinity control polymer 10 is immobilized on the carrier 20 will be continued. A case where the composite stimulus responsive affinity control polymer 10 is applied will be described as an example. In the following description, the composite stimulus-responsive affinity control polymer 10 is physically bonded to the polymer chain 11 whose conformation changes in response to temperature stimulation and the first adsorption site 31 of the target substance 30 (antibody). The first ligand 12 and the second ligand 13 that is bonded to the second adsorption site 32 of the target substance 30 by a chemical covalent bond and can be cleaved by the addition of the substitution substance 43 are assumed. In addition, this embodiment is illustrated as a suitable example in order to clarify the feature of the adsorption / desorption process of the target substance in the present invention, and the configuration of the composite stimulus responsive affinity control type polymer 10 according to the present invention is limited. Not what you want.

  First, a process for capturing and adsorbing the target substance 30 dissolved in the aqueous solution on the surface of the carrier 20 using the composite stimulus responsive affinity control polymer 10 will be described. The trapping process is performed under temperature conditions where the polymer chain 11 having temperature responsiveness is spread by hydration, that is, has a coiled conformation. For example, when the polymer chain 11 has an LCST type phase diagram, the target substance is captured at a critical solution temperature or lower. In the coiled state, which is a conformation in which the polymer chain 11 is hydrated and spread, the main chain undergoes molecular motion due to thermal fluctuation and has a high degree of freedom. Therefore, the ligand possessed by the composite stimulus responsive affinity control polymer 10 easily forms a bond with the adsorption site of the target substance 30 existing in the vicinity of the composite stimulus responsive affinity control polymer 10, so that the target substance 30 Captured on the surface of the carrier 20. Note that there may be a difference in the critical solution temperature between the bulk state where the composite stimulus responsive affinity control polymer 10 alone is dissolved in the aqueous solution and the state where the complex stimulus responsive affinity control polymer 10 is immobilized on the carrier surface and in contact with the aqueous solution. This is because the pH and salt concentration locally differ from the bulk near the carrier surface. This should be noted when setting the temperature at the time of capture.

  In a state where the target substance 30 is trapped by the complex stimulus responsive affinity control polymer 10, the first adsorption site 31 of the target substance 30 is the first ligand 12, and the second adsorption site 32 is the second. It is in a state of being selectively bound to the ligand 12. That is, the target substance 30 is captured through bonds derived from the complex stimulus-responsive affinity control polymer 10 and two different mechanisms, and the binding between the two is not only strong, Compared with the coupling by this mechanism, it is possible to achieve more stable capture against changes in the surrounding environment. In addition, the conformation of the polymer chain 11 in the bound state is constrained by the binding with the target substance 30, and the conformational energy is generally higher than that before the capture. In order to realize stronger bonding, it is desirable to reduce the conformational energy at the time of bonding as much as possible. For this purpose, a molecular design in which the first ligand 12 and the second ligand 13 are arranged on the polymer chain 11 so that the polymer chain 11 can maintain a relaxed conformation as much as possible in a state where it is bound to the target substance 30; It is important to.

  The target substance 30 can be released from the surface of the carrier 20 by applying a stimulus to the system to dissociate the bond between the ligand and the binding site. The present embodiment is characterized in that a strong fixation is dissociated by a mild stimulus by using a plurality of types of stimuli at the same time. First, the first stimulus uses a stimulus having an action of changing the conformation of the main chain. That is, when the polymer chain 11 has temperature responsiveness, it is preferable to cause a temperature change to act as the first stimulus. When the temperature is changed across the critical solution temperature, the conformation of the polymer chain 11 transitions from a hydrated and expanded coil state to a dehydrated and aggregated globule state. At this time, tension acts between the first ligand and the second ligand, and acts to dissociate the bond between each ligand and the adsorption site of the target substance. Further, a substance having an action of dissociating the bond between the second ligand and the second adsorption site that are firmly bonded by covalent bonds is added. Due to the breakage of the bond by the second stimulus, the conformational change of the polymer chain 11 is further promoted, and the target substance 30 is finally dissociated from the complex stimulus-responsive affinity control-type polymer 10, whereby the surface of the carrier 20. Leave.

  The second stimulus can be applied by adding a substitute substance 43 to the system. For example, when the ligand is designed so that the second ligand and the second adsorption site form an ester bond, an ester exchange reaction by adding the substitution substance 43 can be used. When a boronic acid such as phenylboronic acid as described above is used as a ligand, the bond between the phenylboron group and the binding site can be replaced by using a chemical substance having a diol as the substitution substance 43. In particular, using a saccharide as a chemical substance having a diol is an excellent method in that the influence on the target substance is minimal.

  Even if the polymer chain 11 is characterized by having temperature responsiveness, the hydration state of the main chain may be influenced not only by temperature but also by pH, salt concentration, and the like. Therefore, the first stimulus that changes the hydration state such as pH and salt concentration can also be used. Furthermore, even when temperature stimulation is applied, the critical solution temperature can be adjusted to an appropriate value by appropriately adjusting values such as pH and salt concentration. In the case where the temperature range where the target substance exists stably is limited, it is desirable to adjust the temperature at which the capture / adsorption process and the desorption process are performed by such a method.

  In order to use the composite stimulus responsive affinity control polymer 10 according to the present invention for purification of a target substance by affinity chromatography, as shown schematically in FIG. It is desirable that the adsorbent 50 be fixed to the carrier 20 made of an insoluble material. As the carrier 20, it is desirable that the surface can be activated so as to chemically bind the complex stimulus-responsive affinity controlled polymer 10, no non-specific adsorption is present, and that it is physically and chemically stable. . As materials having such properties, organic materials such as polystyrene and agarose, and inorganic materials such as silica gel and porous glass are known, and may be appropriately applied depending on the purpose. The shape of the carrier is desirably a shape that can be filled in the container with high density, that is, a bead shape. Furthermore, in order to improve the bonding opportunity with the target substance, it is desirable that the surface area of the carrier is large. For this purpose, it is desirable that the carrier is porous having micropores of a size that allows the target substance to enter.

  FIG. 4 shows a purification column 60 packed with an adsorbent 50. The purification column 60 is configured by filling a large number of adsorbents 50 in a container.

  Referring to FIG. 5 as appropriate, with respect to a method for purifying a target substance in a solution using a purification column 60 packed with an adsorbent according to the present invention, a dual-ligand type composite in which the polymer chain 11 continues to have temperature responsiveness. The case where the stimulus-responsive affinity control polymer 10 is applied will be described below as an example. The target substance purification method according to the present embodiment includes a step of adsorbing a target substance on an adsorbent in the purification column 60, a step of cleaning the inside of the purification column to remove impurities, and a target substance from the purification column in combination with a plurality of stimuli. And desorbing and recovering.

  First, in the adsorption step, the solution 70 in which the target substance 30 and the impurities 71 are mixed is introduced into the purification column 60 packed with the adsorbent 50. At this time, the temperature in the purification column 60 is the temperature at which the composite stimulus-responsive affinity control polymer 10 fixed on the surface of the adsorbent 50 captures the target substance 30, that is, the polymer chain 11 is hydrated and spreads. The temperature is set to a coil state that is a conformation. When the polymer chain 11 included in the composite stimulus responsive affinity control polymer 10 has LCST type characteristics, the temperature may be set to a temperature lower than the critical solution temperature. In many cases, the critical solution temperature varies under the influence of chemical composition such as salt concentration and pH of the solution. Therefore, it is desirable that the set temperature has a width so that it can cope with a change in the chemical composition of the solution. Since the complex stimulus-responsive affinity control polymer 10 has a property of specifically adsorbing the target substance 30, most of the impurities 71 mixed in the solution 70 pass through the purification column 60 and are discharged. However, some impurities may remain in the purification column. This phenomenon is particularly noticeable when the impurity concentration is high.

  In order to remove the impurities 71 remaining in the purification column 60, it is desirable to perform a washing step after the adsorption step. If the amount of impurities 71 remaining in the purification column 60 is small, this step can be omitted. In the washing process, the washing liquid 72 is passed through the purification column 60 that has adsorbed the target substance 30 in the adsorption process, and is not specific to the impurities 71 contained in the solution 70 remaining in the voids in the purification column or the adsorbent 50. The adsorbed impurities 71 are removed by washing. As the cleaning solution 72, a buffer solution using water as a solvent is preferably used. However, the solution is not particularly limited as long as it does not degrade the target substance, and may contain a solvent other than water. The washing step is performed at a temperature at which the composite stimulus-responsive affinity control polymer 10 can capture and maintain the target substance 30, that is, at a temperature at which the polymer chain 11 is in a coiled state that is a hydrated and spread conformation. The multi-ligand type complex stimulus responsive affinity controlled polymer 10 according to the present invention binds firmly to the target substance 30. Therefore, the target substance 30 is maintained in a state of being bonded to the surface of the adsorbent 50 even after a sufficient amount of the cleaning liquid 72 is passed through the purification column 60 to remove the impurities 71.

  In the desorption step, a plurality of stimuli are given to the purification column 60, and the target substance 30 bound to the surface of the adsorbent 50 in the purification column 60 is desorbed and eluted to be recovered. First, in the desorption step, a first stimulus that induces a conformational change of the temperature-responsive polymer chain 11 is applied. That is, the temperature of the system is changed from a temperature at which the polymer chain 11 is in a coiled state, which is a hydrated and expanded conformation, to a temperature at which the polymer chain 11 aggregates and becomes a globule state by dehydration. When the polymer chain 11 has LCST-type temperature responsiveness, the temperature set to be lower than the critical solution temperature during the adsorption and washing steps may be heated to a temperature higher than the critical solution temperature. Even if the polymer chain 11 is characterized by having temperature responsiveness, the hydration state of the main chain may be influenced not only by temperature but also by pH, salt concentration, and the like. Therefore, the first stimulus that changes the hydration state such as pH and salt concentration can also be used. By the first stimulus (temperature stimulus), the cohesive force acts on the polymer chain 11 of the composite stimulus-responsive affinity controlled polymer 10 that is bound to the target substance at two points of the first ligand and the second ligand. A tension acts between the coupling points. This tension acts to dissociate the bond between the ligand and the adsorption site, particularly a physical bond with a relatively low binding force.

  Further, a second stimulus is applied to change the binding force between the ligand and the target substance adsorption site. For example, as described above, when IgG is a target substance, a triazine derivative low molecule is used as the first ligand, and a phenylboronic acid group is used as the second ligand, diol is included as the second stimulus. What is necessary is just to let the detachment | desorption liquid 73 containing a compound as a substituted substance pass in the purification column 60. FIG.

  By the above composite stimulation, the target substance 30 fixed on the surface of the adsorbent 50 can be efficiently dissolved and recovered in the desorbing liquid 73 only by mild stimulation such as temperature and chemical substance. In addition, in this method, there is no restriction | limiting in particular in the order which provides a 1st stimulus and a 2nd stimulus. Either one may be given first, or both may be applied at once.

  The characteristics of the purification process using the adsorbent 50 can be utilized by applying it to the purification of a target substance that decomposes or generates aggregates at a low pH, for example. Further, since the desorbed target substance is obtained in a state of being dissolved in a buffer solution in a pH range close to neutrality, it is also advantageous that a neutralization step after purification is not required. In addition, although the substituted substance 43 contained in the detachment liquid 73 may remain in the collected solution, the influence can be reduced by applying a saccharide such as glucose as the substituted substance 43.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to these Examples.

  PNIPAM having LCST type temperature responsiveness as polymer chain 11 having temperature responsiveness, one triazine derivative protein A mimetic molecule 22/8 (hereinafter referred to as PAM2) as second ligand, second ligand An example in which the compound stimulus responsive affinity control polymer 10 to which one phenylboronic acid is bonded as a target substance and applied to the purification of human IgG (gamma globulin intramuscular injection) as a target substance will be described in detail below together with a comparative example. .

<Synthesis of complex stimulus-responsive affinity control polymer>
According to the scheme shown in FIG. 6, PNIPAM having LCST type temperature responsiveness as the polymer chain 11, one triazine derivative protein A mimic molecule 22/8 (hereinafter referred to as PAM2) as the first ligand, A monodisperse complex stimulus-responsive affinity controlled polymer (“Polymer 5” in FIG. 6) was synthesized in which one phenylboronic acid was bound as a ligand. In addition, as a comparison object, a complex stimulus-responsive affinity controlled polymer having only PAM2 or only phenylboronic acid as a ligand was synthesized in PNIPAM having the same molecular weight.

A molecular design for precisely synthesizing PNIPAM while introducing PAM2 and a phenylboronic acid group onto the PNIPAM main chain is shown below.
-Chlorine bound to triazine of PAM2 can react with primary amines. Therefore, an amine is placed at a clear position (terminal) of PNIPAM, and PAM2 is introduced.
-As the phenylboronic acid, 4-vinylphenylboronic acid (BASt), which is a corresponding vinyl monomer, is known. However, since boronic acid adversely affects polymerization control, a monomer (B (pin) St) protected with pinacol is polymerized. After polymerization, it is deprotected and converted to boronic acid.
PNIPAM is synthesized by NIPAM atom transfer radical polymerization (ATRP, one of controlled radical polymerization).

  Specifically, an ATRP initiator protected with an amine was designed and ATIP of NIPAM was performed. After NIPAM was polymerized to some extent, B (pin) St was (co) polymerized from the end. The polymer was subjected to deprotection of boronic acid, deprotection of the protecting group of amine, and PAM reaction to the amine to obtain PNIPMA having PAM2 at one end and phenylboronic acid at one end. Since PAM2 is introduced through an initiator, if the initiator efficiency is 100% and the introduction reaction is quantitative, one unit is introduced into all polymer chains. On the other hand, since phenylboronic acid is introduced as a monomer, it is statistically introduced.

  Here, the order in which the deprotection of the boronic acid, the deprotection of the protecting group of the amine, and the PAM2 reaction to the amine are performed becomes important. As a result of various examinations of the conditions, it was found that the target polymer was obtained by performing (1) deprotection of the protecting group of amine, (2) PAM2 reaction with amine, and (3) deprotection of boronic acid. did. In the synthesis scheme of FIG. 6, Polymer 5 (PAM2 and phenylboronic acid at the terminal) is the target composite stimulus-responsive affinity controlled polymer, but Polymer 1 produced in the middle (all protected) Polymer 3 (with amine at the end), Polymer 4 (with PAM 2 at the end), and Polymer 2 (with phenyl boronic acid at the end) were used as comparative samples.

Hereinafter, each synthesis step will be described in detail.
Synthesis of Initiator 1 Under an argon atmosphere, tert-butyl (2-aminoethyl) carbamate (984 μL, 6.25 mmol) and 20 ml of THF were added to the flask, and Et ₃ N (0.91 ml, 6) was stirred at −78 ° C. .6 mmol) was added. Thereafter, 2-chloropropanoyl chloride (0.653 ml, 6.6 mmol) was added dropwise over about 10 minutes, and the mixture was returned to room temperature and stirred for 18 hours. The precipitate was removed by filtration and the solvent was evaporated by evaporation under reduced pressure. The obtained white solid was dissolved in 10 ml of methanol and added dropwise to 300 ml of saturated Na ₂ CO ₃ to produce a white precipitate. The precipitate was filtered to remove the solvent, and the resulting solid was dissolved with methanol. Insoluble material was removed by filtration. Removal of the solvent and drying overnight gave 670 mg of initiator 1 as a white solid. The structure was assigned by ¹ H NMR.

Synthesis of B (pin) St 1 g of powdered molecular sieves 4A was placed in a 200 ml flask and heated with a blaster under reduced pressure to activate the molecular sieves. After returning to room temperature and replacing with Ar, 50 ml of degassed methylene chloride was added. Under an Ar atmosphere, BASt (3.01 g, 20.3 mmol) and pinacol (2.53 g, 21.4 mmol) were added and allowed to stir for 18 hours. When the precipitate was removed by Celite filtration and the solvent was removed, 3.7 g of B (pin) St was obtained as a colorless transparent high-viscosity liquid. The structure was assigned by ¹ H NMR.

The synthetic operation of NIPAM and B (pin) St copolymer (Polymer 1) was performed in an argon atmosphere. NIPAM (4.500 g, 4.0 × 10 ⁻² mol) and CuCl (39.36 mg, 4.0 × 10 ⁻⁴ mol) were taken into Schlenk tube A, a stir bar was added, and a three-way cock was attached. . Here, 7.5 ml of 2-propanol as a solvent, 0.12 ml of tetralin as an internal standard for measuring the polymerization rate, Me ₆ TREN (0.107 ml, 4.0 × 10 ⁻⁴ mol) as a catalyst ligand ) And stirred for 20 minutes or more to prepare a solution. In another Schlenk tube B, initiator 1 (100.3 mg, 4.0 × 10 ⁻⁴ mol, 1/100 equivalent with respect to NIPAM monomer) was taken, a stirrer was placed, a three-way cock was attached, and a solvent was added. 2.78 ml of 2-propanol was added and stirred well to prepare an initiator solution. All of this solution was added to Schlenk tube A with a syringe to prepare a polymerization solution. At this point, the amount of NIPAM relative to the internal standard (tetralin) was evaluated by ¹ H NMR.

This solution was allowed to stir at 25 ° C. for 1 hour, and ¹ H NMR was measured for a part of the polymerization solution, and the amount of residual NIPAM was evaluated based on the tetralin peak. By comparing with the value before polymerization, the polymerization rate (conversion rate) of NIPAM was calculated to be 43%. Since polymerization is performed under conditions of 100 equivalents, it is considered that 43 units of NIPAM are averaged per one polymer chain produced.

When the above polymerization had passed for 1 hour, degassed B (pin) St (461 mg, 2.0 × 10 ⁻³ mol, 5 equivalents of initiator) was added. One hour after the addition of B (pin) St, the three-way cock of the Schlenk tube B was taken and exposed to air to deactivate the catalyst, and the polymerization reaction was stopped. ^When the conversion rates of NIPAM and B (pin) St were calculated from ¹ H NMR, they were 50% and 42%, respectively. It is considered that a polymer in which an average of 43 units of NIPAM was polymerized and an average of 7 units of NIPAM and an average of 2 units of B (pin) St were bonded together was formed.

  In order to purify the produced polymer, the solvent was distilled off under reduced pressure, and the residue was redissolved with ethanol. The catalyst was removed by adding KYOWARD 2000 which is a metal adsorbent, stirring and centrifuging. Thereafter, unreacted monomers were removed by dialyzing with ethanol. After dialysis, the solvent was distilled off under reduced pressure, dissolved in 1,4-dioxane, and lyophilized. When the produced polymer was analyzed by GPC using DMF as an eluent, 1.30 g of a polymer having Mn = 1,500, Mw = 15,000, and Mw / Mn = 1.45 was obtained in terms of PMMA. The polymer obtained here was used as Polymer 1 for the following conversion reaction.

Polymer 1 Boc group deprotection reaction (deprotection of terminal amine protecting group)
A stirrer, Polymer 1 (200 mg, 2.0 × 10 ⁻⁵ mol), and 4.2 ml of CH ₂ Cl ₂ were added to a 50 ml eggplant flask, and a three-way cock was attached and stirred well. The flask was immersed in an ice bath, and 4.2 ml of TFA was added dropwise over about 5 minutes. Then, it returned to room temperature and stirred for 5 hours. Distilled water (0.42 ml) was added, and the mixture was further stirred for 24 hours. After removing the solvent, the precipitated white solid was dissolved again in THF and dialyzed against THF. After removing the solvent, it was dissolved in 1,4-dioxane and lyophilized. 107 mg of a white solid (Polymer 3) was obtained. It was confirmed that the reaction proceeded by ¹ H NMR and HPLC.

PAM2 introduction reaction to terminal amine of Polymer3 A three-way cock was attached to one mouth of a 50 ml two-neck eggplant flask, a stirrer was placed, Polymer3 (250 mg, 2.5 × 10 ⁻⁵ mol), PAM2 (47.5 mg, 1.25 × 10 ⁻⁴ mol) and 8 ml of 1,4-dioxane was added and dissolved. A three-way cock was also attached to the other mouth, and the inside of the flask was placed in an Ar atmosphere. Et ₃ N (160 μl) was added, and the mixture was stirred in an oil bath at 100 ° C. for 21 hours. After allowing to cool, the solvent was removed, and the precipitated brown solid was dissolved again with ethanol. Dialyzed in ethanol. After dialysis, the solvent was removed, dissolved in 1,4-dioxane, and lyophilized. 220 mg of a light brown solid (Polymer 4) was obtained. It was confirmed that the reaction proceeded by ¹ H NMR and HPLC.

Polymer 4 pinacol deprotection (deprotection of boronic acid site)
Polymer 50 (190 mg, 1.9 × 10 ⁻⁵ mol) and NaIO ₄ (92.0 mg, 4.3 × 10 ⁻⁴ mol) were taken in a 50 ml eggplant flask, and 8 ml of THF and 2.67 ml of H ₂ O were added and stirred for 30 minutes. I let you. 0.067 ml of 1N HClaq was added and allowed to stir at room temperature for 17 hours. The solvent was removed, and the precipitated red solid was dissolved again in ethanol and dialyzed against ethanol. After removing the solvent, it was dissolved in 1,4-dioxane and lyophilized. 133 mg of a pink solid (Polymer 5) was obtained.

Polymer 1 pinacol deprotection reaction (deprotection of boronic acid site)
Polymer 1 (200 mg, 2.0 × 10 ⁻⁵ mol) and NaIO ₄ (96.2 mg, 4.5 × 10 ⁻⁴ mol) were placed in a 50 ml eggplant flask, and 8 ml of THF and 2.67 ml of H ₂ O were added and stirred for 30 minutes. I let you. 0.067 ml of 1N HClaq was added and allowed to stir at room temperature for 17 hours. The precipitate was removed by filtration, and the filtrate was dialyzed against THF / H ₂ O = 1/1 (v / v). After removing the solvent, it was dissolved in 1,4-dioxane and freeze-dried to obtain 150 mg of a white solid (Polymer 2). Progress of the reaction was confirmed by ¹ H NMR and HPLC.

  By the above method, a series of complex stimuli responsive affinity controlled polymers having an average degree of polymerization of 43 units of PNIPAM, namely Polymer 1 (protects all ligands), Polymer 2 (has phenylboronic acid as a ligand), Polymer 4 (With PAM2 as ligand) and Polymer5 (with phenylboronic acid and PAM2 as ligand) were obtained. Further, according to the same synthesis scheme as described above, a series of complex stimulus-responsive affinity controlled polymers having an average degree of polymerization of 76 units and 150 units of PNIPAM were synthesized.

<LCST evaluation of complex stimulus responsive affinity control polymer>
The LCST (lower critical solution temperature) of the synthesized series of complex stimulus responsive affinity controlled polymers was investigated. A 5 mM PBS aqueous solution (pH 7.4) was used as a solvent, and a polymer was dissolved at 0.1 wt% and passed through a filter. Using a UV / Vis spectrophotometer set at a wavelength of 670 nm, the transmittance was measured by raising / lowering the temperature at 1 ° C./min. At an initial temperature of 10 ° C., the transmittance was 100 to 90% in any polymer solution, but when the temperature was raised, it was confirmed that the transmittance suddenly decreased to 0% at about 30 ° C. It was confirmed that a series of synthesized polymers had an LCST type phase diagram. Generally, it is known that the LCST of PNIPAM is around 37 ° C., but all of the polymers exhibited LCST around 30 ° C. This was considered because a hydrophobic group was introduced into the PNIPAM main chain such as a boronic acid monomer and an initiator.

<Evaluation of binding constant with IgG by SPR method>
The adsorption / desorption behavior of the synthesized series of complex stimulus-responsive affinity controlled polymers and the antibody immobilized on the gold thin film was analyzed by the surface plasmon resonance method (SPR method). The SPR method uses a phenomenon in which evanescent waves generated at the solution interface under the condition of total reflection of the laser at the glass- (gold thin film) -solution interface forcibly vibrate free electrons on the gold thin film surface. Under conditions in which free electrons vibrate, the energy required for free electron vibration is absorbed from the evanescent wave, and the reflectivity of the laser decreases. Since the amount of change in laser reflectivity is known to be proportional to the amount of molecules adsorbed on the gold thin film surface, by measuring the amount of laser reflectivity change, the composite stimulus responsive affinity control adsorbed on the surface The amount of mold polymer can be measured.

Production of IgG-fixed gold surface The surface of the gold thin film was modified using the phenomenon that thiol molecules self-assemble on the gold thin film surface. First, the organic substance on the surface of the gold thin film for SPR measurement (formed on a glass substrate: film thickness 50 nm) was removed using a dry etcher. Thereafter, the gold thin film is immersed overnight in an ethanol solution in which the hydroxyl group or carboxyl group-containing thiol molecule is dissolved via polyethylene glycol at the alkyl chain terminal, and the terminal of the alkyl thin film having a hydroxyl group and a carboxyl group is terminated on the gold thin film surface. A mixed self-assembled film was prepared. Thereafter, the self-assembled film was washed with ethanol and pure water. The surface of the self-assembled film was designed so that a polyethylene glycol chain effective for suppressing nonspecific adsorption of antibodies was exposed. In addition, the carboxyl group was designed to be exposed on the surface of the self-assembled film, so that the antibody could be immobilized by an amide bond.

  Next, the antibody was immobilized on the surface of the self-assembled film by the following procedure. First, the self-assembled film was immersed in a 1 wt% aqueous solution (pH = 5.8) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and left at 37 ° C. for 2 hours to self-assemble. The carboxyl group on the surface of the membrane was activated for reaction with amino groups. The surface was washed with a phosphate buffer (pH = 5.8), immersed in a phosphate buffer solution (0.1 wt%, pH = 5.8) of the antibody, and allowed to stand at 37 ° C. for 2 hours. Thereafter, unreacted carboxyl groups were blocked with ethanolamine, and the surface was washed with a phosphate buffer solution (pH = 5.8) to obtain a surface on which human IgG was immobilized.

SPR measurement The amount of adsorbed complex stimulus-responsive affinity-controlling polymer by continuously acting a complex stimulus-responsive affinity control-type polymer solution of a predetermined concentration (10 to 50 μM) on the surface of a gold thin film on which human IgG is immobilized. The time change was measured. After a lapse of a predetermined time (t ₁ ), a buffer solution is continuously applied instead of the complex stimulus responsive affinity control polymer solution, and the temporal change in the amount of the complex stimulus responsive affinity control polymer released is measured. did. The adsorption / desorption curves thus measured were theoretically analyzed to quantify the adsorption rate constant and desorption rate constant of the complex stimulus-responsive affinity controlled polymer. For the measurement of the adsorption / desorption curve, a commercially available SPR measurement device was used, and the measurement was performed at a temperature condition where the solubility of the evaluation sample was ensured, that is, at 15 ° C., which was sufficiently lower than the LCST at pH 8.3.

The adsorption equilibrium between the ligand (L) and the antibody (A) can be described by the formula (1). In addition, the adsorption rate of the antibody can be described as in equation (2). Note that k _a and k _d in the formula (2) are the adsorption rate constant and the desorption rate constant in the formula (1), respectively.

In SPR measurement, assuming that the laser incident angle is constant, the change amount of the laser reflectivity at time t is R (t), and the change amount of the laser reflectivity when the polymer is adsorbed to all the antibodies is R _max , Equation (2) [LA] and [A] can be replaced by [LA] = R (t) and [A] = R _max −R (t) (equation (3)). In addition, since the density | concentration (C) of a composite stimulus responsive affinity control type | mold polymer solution does not change during a measurement, [L] is the fixed value C.

  Here, considering that R (0) = 0, equation (4) can be derived from equation (3).

  On the other hand, the desorption rate when the buffer is allowed to act after the adsorption to desorb the polymer can be described as the equation (5).

When the amount of change in laser reflectivity after time t ₁ second is R ₁ (R (t ₁ ) = R ₁ ), equation (6) can be derived from equation (5).

The adsorption / desorption curve of the polymer obtained by the experiment was fitted using the equations (4) and (6), and k _a , k _d , and R _max were obtained. Moreover, to calculate the dissociation constant _K d _{= k} d / k _a, which is expressed by the ratio of _{k d} and _{k a.} The K _d value decreases as the binding ability between the complex stimulus-responsive affinity control polymer and human IgG increases. Using this K _d value as a measure of the antibody adsorption capacity, the binding ability of the complex stimuli-responsive affinity controlled polymer and human IgG was compared.

Adsorption behavior evaluation FIG. 7 shows an adsorption / desorption curve of Polymer5 (having phenylboronic acid and PAM2 as ligands) having an average degree of polymerization of 43 units of PNIPAM as a sample on a human IgG fixed substrate. The measurement was carried out a plurality of times while changing the concentration C, and the obtained curve was fitted using the equations (4) and (6) to determine the association constant _Kd . Similarly, adsorption and desorption of PNIPAM with an average polymerization degree of 43 units of Polymer 1 (protecting all ligands), Polymer 2 (having phenylboronic acid as a ligand), and Polymer 4 (having PAM 2 as a ligand) The association constant _Kd was determined from the curve. Furthermore, in the same manner, the dissociation constant K _d of a series of 76 units and 150 units of a complex stimulus-responsive affinity controlled polymer for a human IgG fixed substrate was determined.

FIG. 8 shows a series of complex stimulus-responsive affinity controlled polymers of PNIPAM having an average degree of polymerization of 43 units, 76 units, and 150 units on a human IgG-immobilized substrate at a temperature of 15 ° C. and a pH of 8.3. The dissociation constant _Kd is shown. Large Firstly, in all polymerization degree, with a K _d is substantially constant composite stimulus responsive affinity control type polymer with a protected ligand (Polymer1), than the composite stimulus responsive affinity control type polymer having a ligand to be described below The value is shown. This indicates that Polymer 1 has a very weak adsorptive power to the surface on which human IgG is immobilized.

Next, the adsorption behavior of the complex stimulus-responsive affinity-controlling polymer having phenylboronic acid or PAM2 as a ligand, that is, Polymer2 and Polymer4, to the human IgG-immobilized surface will be considered. The K _d of Polymer 2 and Polymer 4 showed a small value compared to Polymer 1 and was found to have a large adsorptive power on the surface of human IgG. This is considered to be because the ligand possessed by the complex stimulus-responsive affinity-controlling polymer specifically bound to IgG according to the molecular design. Further, it was found that the K _d of Polymer 2 having phenylboronic acid is smaller than the K _{d of} Polymer 4 having PAM2. This is considered to be because phenylboronic acid is adsorbed by an ester bond that is a chemical interaction with IgG, whereas PAM2 is adsorbed by a physical interaction.

Next, the adsorption behavior of the complex stimulus-responsive affinity controlled polymer (Polymer 5) having both phenylboronic acid and PAM2 ligands on the surface immobilized with human IgG will be discussed. First, _{K d} in which the degree of polymerization is obtained the Polymer5 of 43 units as a sample of PNIPAM became almost the same value as the _{K d} obtained with Polymer4 having only PAM2. Also, _{K d} the degree of polymerization is obtained the Polymer5 of 76 units as a sample of PNIPAM is, _{K d} is significantly reduced as compared to Polymer4 having only PAM2, same as the _{K d} for Polymer2 having only phenylboronic acid The value was about. For 43 units and 76 units, no clear superiority of Polymer5 was observed, but it was found that the adsorption amount of Polymer5 to human IgG was large and had sufficient binding power. On the other hand, Polymer 5 with a degree of polymerization of 150 units had a marked decrease in _Kd compared to Polymer 2 and Polymer 4 having only phenylboronic acid or PAM2. This indicates that Polymer 5 having a degree of polymerization of 150 units has a larger amount of adsorption to human IgG than Polymer 2 and Polymer 4 having the same degree of polymerization having only phenylboronic acid or PAM2, and has the effect of having multiple types of ligands. This is a clearly recognized result.

  From the results obtained above, it was revealed that Polymer 5 having both phenylboronic acid and PAM2 ligands has sufficient binding power to human IgG. It was also found that the behavior of adsorption to human IgG is affected by the degree of polymerization of PNIPAM, that is, the distance between the phenylboronic acid group and the PAM2 group. Phenylboronic acid and PAM2 specifically bind to specific adsorption sites present in the Fc site of human IgG. From this, it is preferable that the complex stimulation-responsive affinity-controllable polymer is discretely arranged between the phenylboronic acid and PAM2 with sufficient distance so that they can be sterically opposed to the adsorption site of human IgG. it is conceivable that. The adsorption power of Polymer 5 with a polymerization degree of 43 units and 76 units remained at the same value as Polymer 2 with the same polymerization degree because the distance between phenylboronic acid and PAM2 was short, and the stronger the binding strength between phenylboronic acid and IgG. This is probably because PAM2 may not reach the binding site in a state where the phenylboronic acid adsorption site is bound. From the above results, it was shown that the amount of adsorption to the target substance can be further improved by arranging a plurality of ligands discretely so as to be sterically relative to the interaction site of the target substance.

Effect of Temperature and Glucose on Adsorption Properties Evaluation of the effect of temperature change and addition of glucose on the adsorption properties of a complex stimulus-responsive affinity control polymer having both phenylboronic acid groups and PAM2 on human IgG immobilized surface by SPR method did. For SPR measurement, Polymer5 having a polymerization degree of PNIPAM of 43 units and 150 units was used, and the dissociation constant _{Kd of} Polymer5 on the surface immobilized with human IgG was measured using the same method as described above. The measurement was performed under pH 7.4, which is a milder pH condition with respect to the target substance, human IgG. FIG. 9 shows the result.

First, the results of investigation on the influence of temperature will be described. The measurement was carried out at 20 ° C. below LCST of PNIPAM and 37 ° C. above LCST using a sample in which Polymer 5 was dissolved in a buffer solution of pH 7.4. With Polymer 5 with a polymerization degree of 43 units and 150 units, the dissociation constant K _d increases by about two orders of magnitude by changing the temperature from 20 ° C. below LCST to 37 ° C. above LCST, greatly increasing the adsorption capacity to the surface of human IgG. It turned out to fall. This is presumably because at higher temperatures than LCST, Polymer5's hydrophobicity increased and the polymer chains aggregated, resulting in a decrease in the degree of freedom of ligand arrangement and orientation.

Next, the measurement was carried out in the same manner at a temperature of 20 ° C. using a sample in which glucose was added at a concentration of 1.0 M to a buffer solution in which Polymer 5 was dissolved. As a result, it was found that the addition of glucose increased the dissociation constant _Kd by about two orders of magnitude compared to the sample without addition, and the adsorptivity to the surface of human IgG was greatly reduced. This is probably because the phenylboronic acid group possessed by Polymer5 and glucose formed an ester bond, and the ability to bind to the sugar chain on the asparagine residue, which is the binding site of human IgG, was lost.

  From the above results, it was shown that the binding between the complex stimuli-responsive affinity control polymer having both phenylboronic acid group and PAM2 and human IgG can be controlled by temperature and glucose.

<Fixation of bead-shaped adsorbent>
Next, an adsorbent was produced by immobilizing the composite stimulus-responsive affinity control polymer on a polymer bead as a carrier. The polymer beads were produced by the following method.

Chemical structure of complex stimulus-responsive affinity control polymer Both phenylboronic acid and PAM2 whose performance was demonstrated by the above-mentioned evaluation by the SPR method in order to realize immobilization of the complex stimulus-responsive affinity control polymer on the carrier Based on Polymer 5 having a polymerization degree of PNIPAM having 150 units, the chemical structure was partially changed. Specifically, the polymerization initiator 1 shown in FIG. 6 is changed from that based on a diamine derivative to one based on a lysine derivative having two amino groups and one carboxyl group, and protecting groups for both amino groups are changed. By appropriate use, PAM2 was introduced into one amine, and a functional group used for binding to a carrier was introduced into the other amine. Polymerization of NIPAM was carried out by the ATRP method in accordance with the method described above to obtain Polymer 5A.

Immobilization on Bead Surface The obtained composite stimulus-responsive affinity controlled polymer Polymer 5A was immobilized on the surface of Sepharose beads, which are commercially available agarose beads having an average particle diameter of 90 μm, by the following method. First, a solution (2.0 mL) in which Polymer 5A was dissolved in a pH 7.4, 20 mM PBS buffer to 1 wt% was added to NHS-activated Sepharose 4FF (1.0 mL) that had been washed with 1 mM dilute hydrochloric acid in advance. In addition, the reaction was allowed to proceed overnight at room temperature with shaking. The resulting beads were washed with deionized water. The amount of the Polymer 5A immobilized on the beads was determined by analyzing the residue of the reaction solution and the remaining amount in the washing solution using a reverse phase HPLC column and quantifying the amount of the unreacted Polymer 5A adsorbing material. As a reference, beads in which NHS ester of NHS-activated Sepharose 4FF was deactivated with 1M NaOH aqueous solution were also prepared.

<Adsorption and elution of IgG on bead-shaped adsorbent packed column>
The bead-shaped adsorbent on which Polymer 5A produced by the above method was immobilized was packed in a Tricorn 5/50 column at a first stage flow rate of 0.2 mL / min and a second stage flow rate of 1.2 mL / min.

  The retention time of human IgG was evaluated under the conditions of a temperature of 20 ° C. and a pH of 7.4 using a column packed with a bead-shaped adsorbent on which Polymer 5A was immobilized. For the measurement, pH 7.4, 5 mM PBS buffer solution was used as an eluent, and the solution was passed at a flow rate of 0.5 mL / min (linear flow rate of about 150 cm / h, contact time of about 2 minutes). The human IgG solution to be injected was 0.1 wt% solution, 0.5 mL. By measuring the absorbance of the solution flowing out from the column, the time change of the amount of human IgG eluted from the column was measured. As a result, it was found that the bead-shaped adsorbent on which Polymer5A was immobilized had a smaller peak area than that of the reference and had human IgG adsorption ability.

  Next, the column is washed by passing a pH 7.4, 5 mM PBS buffer solution at 20 ° C. through an agarose column in which human IgG is adsorbed on a bead-shaped adsorbent, and then the temperature is changed to 37 ° C. A buffer solution in which glucose was dissolved was passed through to a concentration of 1.0M. When the amount of eluted IgG was quantified, 85% of human IgG could be recovered.

  From the above results, by using an adsorbent in which a composite stimulus-responsive affinity-controlling polymer having PAM2 and phenylboronic acid is immobilized on the surface of a PNIPAM main chain whose conformation changes in response to temperature stimulation, human IgG is used. It was found that human IgG can be purified efficiently by mild temperature that is non-invasive and stimulation by sugar.

  The embodiment of the present invention has been described in detail above, but the specific configuration is not limited to this embodiment, and even if there is a design change or the like without departing from the gist of the present invention, It is included in the present invention.

DESCRIPTION OF SYMBOLS 10 Composite stimulus responsive affinity control type polymer 11 Polymer chain 12 1st ligand 13 2nd ligand 14 Immobilization part 20 Carrier 30 Target substance 31 1st adsorption site 32 2nd adsorption site 43 Substitution material 50 Adsorbent 60 Purification column 70 Solution 71 Impurity 72 Cleaning solution 73 Desorption solution

Claims (18)

A complex stimulus-responsive affinity controlled polymer capable of adsorbing and desorbing a target substance,
A polymer chain whose conformation changes in response to a first stimulus, and a plurality of types of ligands bound to the polymer chain,
At least one of the plurality of types of ligands changes the binding force with the target substance by a second stimulus,
The composite stimulus responsive affinity control polymer. Multiple types of ligands bind to target substances through different binding mechanisms,
One of the coupling mechanisms is a covalent bond,
The composite stimulus-responsive affinity-controlling polymer according to claim 1.   The composite stimulus-responsive affinity-controlling polymer according to claim 2, wherein the covalent bond is an ester bond.   The complex stimulus-responsive affinity-controlling polymer according to claim 3, wherein the ligand bonded through an ester bond is boronic acid.   The complex stimulus-responsive affinity-controlling polymer according to claim 1, wherein one of the plurality of ligands is a triazine derivative.   The complex stimulus-responsive affinity-controlling polymer according to claim 4, wherein one of the plurality of ligands other than boronic acid is a triazine derivative.   The complex stimulus-responsive affinity-controlling polymer according to claim 1, wherein the ligand is of two types.   The complex stimulus-responsive affinity-controlling polymer according to claim 1, which has one each ligand.   The composite stimulus-responsive affinity controlled polymer according to claim 1, wherein a plurality of types of ligands are discretely bound to the polymer chain.   The complex stimulus-responsive affinity controlled polymer according to claim 1, wherein the first stimulus is a temperature change.   The complex stimulus responsive affinity controlled polymer according to claim 1, wherein the second stimulus is a chemical substance.   The composite stimulus-responsive affinity controlled polymer according to claim 4, wherein the second stimulus is a chemical substance having a diol in its structure.   The polymer chain is selected from the group consisting of poly (N-substituted acrylamide), poly (N-substituted methacrylamide), poly (N, N-disubstituted acrylamide) and poly (N, N-disubstituted methacrylamide). The complex stimulus-responsive affinity-controlling polymer according to claim 1, comprising a chemical structure.   The complex stimulus-responsive affinity-controlling polymer according to claim 1, wherein the target substance is an antibody.   An adsorbent formed by binding the composite stimulus-responsive affinity control polymer according to any one of claims 1 to 14 to a carrier. An adsorption step of passing the solution containing the target substance and impurities through a column packed with the adsorbent according to claim 15, and binding the target substance to the adsorbent;
A desorption step of desorbing the target substance by applying a first stimulus to change the conformation of the polymer chain and applying a second stimulus to change the binding force between the ligand and the target substance; A method for purifying a target substance.   The method for purifying a target substance according to claim 16, wherein the first stimulus is a temperature change, and the second stimulus is a flow of an eluate containing sugar.   The method for purifying a target substance according to claim 16, wherein the target substance is an antibody.

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