WO2024106363A1 - Method and device for separating molecule binding to target molecule - Google Patents

Abstract

Provided is a simple method for separating a second molecule having a desired affinity for a first molecule by using a filter. The present invention relates to a method for separating a second molecule having a desired affinity (Kd}) for a first molecule, the method comprising: generating a complex in which the first molecule and the second molecule are bound, in a container having a filter; washing the container by causing a liquid to flow inside the container; passing, through a filter, molecules that do not bind to the first molecule and molecules that bind to the first molecule while having an affinity less than the desired affinity; collecting the complex in which the second molecule is bound to the first molecule and the free first molecule; and dissociating the second molecule from the first molecule in the complex, wherein in the passing step, with respect to an initial first molecule concentration [R]⁰ in the container, an association rate constant k_on of the first molecule and the second molecule, and the dilution ratio DC expressed by the flow rate FC of the liquid in the container/volume V of the container, k_on[R]⁰≪D_C is satisfied, and Kd=(dissociation rate constant k_off of first molecule and second molecule)/(association rate constant k_on of first molecule and second molecule).

Description

Method and apparatus for isolating molecules that bind to target molecules

The present invention relates to a method for separating molecules that bind to target molecules, and to an apparatus and container used therefor.

Two molecules, such as an antigen and an antibody or its antigen-binding fragment, a glycan and a lectin, an enzyme and its substrate or coenzyme, a protease and its proteinaceous protease inhibitor, a physiologically active substance such as a hormone and its receptor or transport protein, a nucleic acid and its complementary polynucleotide, are known to form complexes based on affinity.

The affinity Kd is the value expressed as the dissociation rate constant (k _off )/association rate constant (k _on ).

When the target molecule is an antigen, the antigen, which is the target molecule in a sample, is separated using a molecule such as an antibody that binds to the antigen. A B/F (Bound/Free) separation method is known that separates target molecules bound to antibodies from excess antibodies (Patent Document 1).

However, no method is known for easily separating molecules that have a specific affinity for a certain molecule using a filter. In particular, no method has been found for obtaining antibodies with the desired affinity in an open system and for selecting out antibodies that do not have the desired affinity.

Japanese Patent Application Laid-Open No. 11-248707

The objective of the present invention is to provide a simple method for separating a second molecule that has a desired affinity for a first molecule using a filter.

A first aspect of the present invention is a method for isolating a second molecule having a desired affinity (Kd) for a first molecule using a filter, the method comprising:
Providing a sample containing a second molecule to be separated and a first molecule that binds to the second molecule;
contacting a sample containing a second molecule with a first molecule in a container having a filter to generate a complex in which the first molecule and the second molecule are bound;
a liquid is passed through the vessel to wash and filter out molecules that do not bind to the first molecule and molecules that bind to the first molecule with less than a desired affinity;
Here, the permeation from the filter is expressed by the following equation, where ^R is the initial concentration of the first molecule in the container, k _on is the binding rate constant between the first molecule and the second molecule, and D _C is the dilution rate represented by F _C /V, the volume of the container.
k _on [R] ⁰ _≪D
where Kd=(dissociation rate constant k _off between the first molecule and the second molecule)/(association rate constant k _on between the first molecule and the second molecule),
collecting a complex in which the second molecule is bound to the first molecule and a free first molecule, and dissociating the second molecule from the first molecule in the complex;
a) isolating a second molecule having a desired affinity, said second molecule comprising:
The present invention provides a method for determining whether k _on [R] ⁰ << _DC
It has been found that by pouring a liquid into a container having a filter so as to fill the filter, the possibility of rebinding of dissociated molecules can be made negligible, and thus the second molecule bound to the first molecule with the desired affinity Kd can be allowed to remain in the container.

The present invention makes it possible to easily separate a second molecule that has a desired affinity for a first molecule.

FIG. 1 is a schematic diagram illustrating the concept of the present invention. FIG. 1 is a block diagram showing an apparatus according to an embodiment of the present invention. 1 is a graph showing the relationship between the binding rate constant k _on between a first molecule and a second molecule, the washing time (minutes), and the residual rate Φ. FIG. 1 is a schematic diagram of an apparatus according to an embodiment of the present invention. FIG. 1A is a top view, FIG. 1B is a side cross-sectional view, FIG. 1C is a side view, and FIG. 1A and 1B are a top cross-sectional view and a side cross-sectional view, respectively, of a first modified example of a reaction vessel. 1A is a top cross-sectional view of a second modified example of a reaction vessel, and FIG. 1B is a side cross-sectional view of the second modified example of the reaction vessel. 1A is a top sectional view of a third modified example of a reaction vessel, and FIG. 1B is a top sectional view of a fourth modified example of the reaction vessel. FIG. 1 shows band intensity of electrophoretic mobility in Example 1. This is a figure comparing the band intensities of BDA Elute (Sup(3)) in Example 1.

The present invention relates to a method for separating a second molecule having a desired affinity Kd for a first molecule using a filter. Examples of combinations of the first molecule and the second molecule include antigens and antibodies or antigen-binding fragments thereof, sugar chains and lectins, enzymes and their substrates or coenzymes, proteases and their proteinaceous protease inhibitors, physiologically active substances such as hormones and their receptors or transport proteins, nucleic acids and their complementary polynucleotides, etc.

The present invention relates to a method for easily obtaining a second molecule, which is a target molecule having a desired affinity for a first molecule, by separating, using a filter, a complex formed in a solution between a first molecule and a second molecule, which is a target molecule having a desired affinity for the first molecule, and a free first molecule, from molecules that bind to the first molecule with less than the desired affinity and molecules that do not bind to the first molecule (hereinafter also referred to as third molecules).

Specifically, a method for isolating a second molecule having a desired affinity (Kd) for a first molecule using a filter, the method comprising the steps of:
Providing a sample containing a second molecule to be separated and a first molecule that binds to the second molecule;
contacting a sample containing a second molecule with a first molecule in a container having a filter to generate a complex in which the first molecule and the second molecule are bound;
a liquid is passed through the vessel to wash and filter out molecules that do not bind to the first molecule and molecules that bind to the first molecule with less than a desired affinity;
Here, the permeation from the filter is expressed by the following equation, where ^R is the initial concentration of the first molecule in the container, k _on is the binding rate constant between the first molecule and the second molecule, and D _C is the dilution rate represented by F _C /V, the volume of the container.
k _on [R] ⁰ _≪D
where Kd=(dissociation rate constant k _off between the first molecule and the second molecule)/(association rate constant k _on between the first molecule and the second molecule),
collecting a complex in which the second molecule is bound to the first molecule and a free first molecule, and dissociating the second molecule from the first molecule in the complex;
and isolating a second molecule having a desired affinity, comprising:

The sample is not particularly limited as long as it is a liquid or solid that contains or may contain the second molecule to be separated.

Examples of combinations of the first molecule and the second molecule include an antigen and an antibody or an antigen-binding fragment thereof, a glycan and a lectin, an enzyme and its substrate or coenzyme, a protease and its proteinaceous protease inhibitor, a physiologically active substance such as a hormone and its receptor or transport protein, a nucleic acid and its complementary polynucleotide, etc. Thus, in one embodiment of the present invention, the second molecule is a polypeptide chain such as an antibody or an antigen-binding fragment.

The antibody may be a monoclonal or polyclonal antibody, but is preferably a monoclonal antibody. The antigen-binding fragment includes F(ab') ₂ , Fab', Fab, single chain antibody (single chain Fv: scFv), and VHH antibody (Variable domain of Heavy chain of Heavy chain antibody), etc.

The sample and the liquid containing the first molecule are mixed in a reaction vessel 110 having a filter 112 to form a mixture 116. Specifically, the mixture 116 in the reaction vessel 110 is a mixture of the first molecule, the second molecule, a molecule that binds to the first molecule with less than the desired affinity, and a molecule that does not bind to the first molecule (hereinafter referred to as the third molecule), and over time forms a mixture of a complex of the first molecule and the second molecule (hereinafter also referred to as the first complex), a complex of the first molecule and the third molecule (hereinafter also referred to as the second complex), a third molecule that does not bind to the first molecule, and free first molecules.

To efficiently form a complex (first complex) in which the first molecule and the second molecule are bound, a sample that may contain the first molecule and the second molecule may be shaken mechanically or non-mechanically as appropriate.

The incubation time until the first molecule and the second molecule form a complex can be appropriately selected depending on the first molecule and the second molecule, but when the first molecule is an antigen and the second molecule is an antibody, it is, for example, 1 to 360 minutes, preferably 10 to 180 minutes, and more preferably 30 to 120 minutes. The incubation temperature is preferably 10 to 50°C, more preferably 20 to 40°C, and even more preferably 25 to 30°C.

After the complex between the first molecule and the second molecule is formed in the reaction vessel 110, a liquid is added to the reaction vessel 110 in such a manner that k _on [R] ⁰ ≪D _C
The reaction vessel 110 is filled with the liquid, and substances (third molecules) that do not bind to the first molecules with a specific affinity pass through a filter 112 attached to the reaction vessel 110 and are collected in a collection vessel 100 as necessary.

The filter allows the complex in which the second molecule is bound to the first molecule and the free first molecule to remain in the reaction vessel 110. The filter has a separation ability that is used to elute molecules that are bound to the first molecule with less than the desired affinity and molecules that do not bind to the first molecule (third molecules). There are no particular limitations on the filter, so long as it is possible to separate the complex in which the second molecule is bound to the first molecule and the free first molecule from the molecules that are bound to the first molecule with less than the desired affinity and molecules that do not bind to the first molecule (third molecules) by utilizing the size difference between the two.

The pore size of the filter can be appropriately determined taking into consideration the size of the complex between the first molecule and the second molecule, or, if the first molecule is bound to a solid phase such as beads, the size of the beads. Examples of lower limits of the pore size include 0.05, 0.1, 0.2, 0.3, 0.5, and 1.0 μm. Examples of upper limits of the pore size include 100, 80, 50, 20, 10, 5, and 2 μm.

For example, when the first molecule is bound to magnetic beads, a filter having a pore size of 0.05 μm to 10 μm can be selected. Preferably, it is 0.1 μm to 5 μm, more preferably 0.2 to 1 μm, and even more preferably about 0.65 μm. (Examples of the molecular weight cutoff include filters with a molecular weight cutoff of 10,000, 30,000, 50,000, or 100,000 KDa.)

The first molecule can also be bound to a spherical structure having a diameter of 0.1 to 10 μm. An example of a spherical structure is a liposome.

An example of the filter 112 is a microfiltration membrane. The microfiltration membrane is not particularly limited as long as it is one that is generally used in microfiltration, but examples include membranes made of polypropylene, polyvinyl chloride, polytetrafluoroethylene, polyvinylidene fluoride, acrylic acid copolymers, polyamide, polysulfone, Teflon (registered trademark), cellulose acetate, nitrocellulose, a mixed ester of cellulose acetate and nitrocellulose, or regenerated cellulose.

The liquid to be passed through the reaction vessel 110 equipped with the filter 112 may be Good's buffer, Tris, PBS, etc. Those skilled in the art can select an appropriate buffer depending on the properties of the first molecule and the second molecule. When the first molecule is an antigen and the second molecule is an antibody, preferred liquids are PBS, Tris, etc., and PBS is more preferred.

In order to efficiently obtain a second molecule having a specific affinity Kd, the liquid flowing through the reaction vessel 110 equipped with the filter 112 is expressed as follows, where ^R is the initial first molecule concentration in the vessel, k _on is the binding rate constant between the first molecule and the second molecule, and D _C is the dilution rate expressed by F _C /V, the flow rate of the liquid in the vessel.
k _on [R] ⁰ ≪D _C (hereinafter also referred to as formula (1))
By flowing the liquid into the reaction vessel at _{a dilution rate D C that} satisfies formula (1), the possibility of recombination of the dissociated first molecule and second molecule can be made negligible.

The complex of the first molecule and the second molecule, and the free first molecule are collected in the reaction vessel 110 or in a separately provided collection section 140. Collection methods include centrifugation, gravitational sedimentation, etc. If the first molecule is bound to magnetic beads, it can be collected using magnetism.

Then, the second molecule is dissociated from the collected complex of the first molecule and the second molecule. Dissociation may be performed in the reaction vessel 110, or in a separately provided collection section 140 or separation section 160. The dissociation method is not particularly limited as long as it does not decompose the target second molecule and maintains its activity. In addition to a physical method in which the washing time t is adjusted by pouring a liquid into the reaction vessel, the second molecule can be dissociated from the complex by a chemical method such as contacting it with an organic solvent such as acetonitrile or an acid such as hydrochloric acid.

Below, we will explain in more detail the examples of how to implement the present invention with reference to the drawings.

FIG. 1 is a schematic diagram showing the concept of the present invention. A mixture of a first molecule 10, a second molecule 20 to be separated, and a third molecule that is not to be separated is inserted into a reaction vessel 110. The second molecule 20 binds to the first molecule 10 to form a complex 40. The second molecule 20 that binds to the first molecule to form the complex 40 has a desired affinity for the first molecule. The third molecule 30 that binds to the first molecule to form the complex 40 has an affinity for the first molecule that is less than the desired affinity. Therefore, the third molecule may bind to the first molecule 10 to form a complex 40, or may not bind to the first molecule 10 to form a complex 40. Here, the complex of the first molecule 10 and the second molecule 20 may be referred to as the first complex, and the complex of the first molecule 10 and the third molecule 30 may be referred to as the second complex.

In the reaction vessel 110, the initial first molecule concentration [R] ⁰ in the vessel, the binding rate constant k _on (mol/dm ³ s) between the first molecule and the second molecule, and the dilution rate D _C expressed as the flow rate of the liquid in the vessel F _C (L/sec)/the volume of the vessel V (L) are calculated as follows:
k _on [R] ⁰ ≪D _C (hereinafter also referred to as formula (1))
When the liquid is passed through the reaction vessel 110 at a dilution rate D _C that satisfies the above, the third molecule bound to the first molecule with an affinity less than the desired affinity in the second complex is released from the first molecule, and the third molecule dissociates from the first molecule. The third molecule 30 dissociated from the first molecule 10 and the third molecule 30 not bound to the first molecule flow out of the reaction vessel 110 through the filter 112. As a result, the reaction vessel 110 contains a complex in which the second molecule having the desired affinity is bound to the first molecule, i.e., the first complex, and a free first molecule, so that the second molecule having the desired affinity can be recovered. That is, the third molecule can be separated from the mixture in the vessel by the filter.

2 is a block diagram showing an apparatus according to this embodiment. A mixture 116 contains a mixture of a first molecule 10, a second molecule 20, and a third molecule 30, and is injected into a reaction vessel 110. In order to efficiently obtain a second molecule having a specific affinity Kd, the liquid supply unit 120 calculates a dilution ratio D C expressed as follows, where ^R is the initial first molecule concentration in the vessel, k _on is the binding rate constant between the first molecule and the second molecule, and F _{C is} the flow rate of the liquid in the vessel/ _V is the volume of the vessel:
k _on [R] ⁰ ≪D _C (Equation (1))
A liquid is supplied to a reaction vessel 110 equipped with a filter 112 at a dilution rate D _C that satisfies formula (1). By flowing the liquid into the reaction vessel at a dilution rate D _C that satisfies formula (1), the possibility of recombination of the dissociated first molecule and second molecule can be made negligible.

Figure 3 is a graph showing the relationship between the desired affinity Kd between the first and second molecules, the washing time (minutes), and the residual ratio Φ. The details of this system are described below.

The concentration of the unbound first molecule is [R], the concentration of the unbound second molecule is [Ls], and the concentration of the complex of the first and second molecules is [Ls・R]. The dilution rate Dc of this system is defined as Fc/V. Here, Fc is the volume of the washing buffer (washing liquid) flowing into the system per unit time, and V is the volume of the system.

で与えられ、この系の第２分子についての速度式は、

で与えられる。ここで、ｋ_ｏｎは、第１分子と第２分子との結合速度定数であり、ｋ_ｏｆｆは、第１分子と第２分子との解離速度定数である。 If there are binding and dissociation reactions in the system, the rate equation for the complex in this system is:

and the rate equation for the second molecule in the system is given by

where k _on is the association rate constant between the first molecule and the second molecule, and k _off is the dissociation rate constant between the first molecule and the second molecule.

Assuming that the supernatant liquid in the system is replaced promptly after the start of washing, the initial conditions are that the concentration of the complex [Ls·R] ₍₀₎ is arbitrary, and the concentration of the second molecule [Ls] ₍₀₎ =0.

となり、第２分子の濃度［Ｌｓ］_（ｔ）が、

となる。 In this case, the exact solution is that the concentration of the complex [Ls · R] _(t) is

The concentration of the second molecule [Ls] _(t) is

It becomes.

であり、Θ_１、Θ_２は、

で定義される。 here,

and Θ ₁ and Θ ₂ are

is defined as:

と定義する。 Next, the "residual rate of the second molecule bound to the first molecule" Φ after a certain washing time is defined as a function Φ(k _off , t) of the washing time t and the dissociation rate constant k _off between the first molecule and the second molecule, as follows:

It is defined as:

The parameters that can be controlled in the experiment are the dilution rate Dc and the concentration of the first molecule [R] ⁰ .

The binding rate constant k _on between the first and second molecules has little sequence dependence and is approximately 10 ⁴ to 10 ⁵ M/sec (molar per second).

The residual rate Φ when formula (1) is satisfied is expressed by the following function: (a) k _on [R] ⁰ << Dc (recombination rate of the second molecule < outflow rate of the molecule)
When k _on [R] ⁰ <<Dc (recombination rate of the second molecule<rate of escape out of the form), in the above equations for Θ ₁ and Θ ₂ , Λ+Dc≈k _off +Dc.

となる。 As a result, under the above assumptions, the survival rate Φ is

It becomes.

On the other hand, in the case of (b) Dc<<k _on [R] ⁰ (rate of escape out of the form<rate of recombination of the second molecule), the survival rate is as follows:

と近似でき、Θ_２は、

と近似できる。 When Dc<<k _on [R] ⁰ (rate of escape out of the form<rate of recombination of the second molecule), in the above formula for Θ ₁ and Θ ₂ , Λ+Dc≒Λ, and Θ ₁ is

and Θ ₂ can be approximated as follows:

can be approximated as follows.

と近似できる。 Here, when k _on [R] ⁰ << k _off , the survival rate Φ is, as above,

can be approximated as follows.

Here, the affinity (K _d ) of the second molecule for the first molecule is K _d =k _off /k _on , and the binding rate constant k _on between the first molecule and the second molecule is 10 ⁴ to 10 ^5. Therefore, assuming that k _on is 10 ⁴ , the above formula can be expressed using the desired affinity K _d as follows:
Φ( _Kd , t)≈exp( ⁻¹⁰⁴ · _Kd ·t)
This is expressed as:

3 shows that when the affinity parameter log ₁₀ K _d is −9 or less, that is, when the dissociation rate constant k _off between the first molecule and the second molecule is sufficiently small relative to the association rate constant k _on between the first molecule and the second molecule, the residual rate Φ hardly decreases even with a long washing time of 10 minutes or more. Therefore, under such conditions, that is, when k _on [R] ⁰ << k _off in the above (a) or (b), a second molecule having a desired affinity K _d can be obtained.

The liquid flowing into the reaction vessel may be, for example, a buffer. The collection vessel 100 collects the liquid and a third molecule that does not have the desired affinity for the first molecule. The flow rate may be measured, for example, by placing the reaction vessel on a weighing scale (not shown).

The collecting section 140 collects a complex in which a second molecule having a desired affinity is bound to a first molecule, and the released first molecule. The separating section 160 is configured to dissociate the second molecule of the complex from the first molecule and separate the second molecule having a desired affinity. By flowing a liquid at a dilution rate D _C that satisfies k _on [R] ⁰ ≪ D _C , the remaining rate Φ of the complex in which the first molecule and the second molecule are bound in the container is
It is expressed as Φ=exp(-k _off ×t).

Since the residual rate Φ in the container decreases by increasing the cleaning time t, dissociation of the first molecule and the second molecule can be achieved, for example, by adjusting t. The separation section 160 may be omitted, and dissociation processing may be performed in the reaction container 110 or the collection section 140.

The structure of the separated second molecule can be analyzed as appropriate using known methods.

FIG. 4 is a schematic diagram of the device according to this embodiment. The mixture 116 shown in FIG. 2 is supplied to the reaction vessel 110. The mixture is a mixture of a first molecule, a second molecule, and a third molecule, and over time forms a complex of the first molecule and the second molecule, a complex of the first molecule and the third molecule, a third molecule that does not bind to the first molecule, and a mixture of free first molecules. For example, the first molecule is an antigen, the second molecule and the third molecule are antibodies or antigen-binding fragments, and the liquid is, for example, a PBS buffer. In the figure, L1 indicates the liquid surface.

The liquid supply unit 120 uses, for example, a pump (not shown) to supply a liquid, for example, a PBS buffer, to the reaction vessel 110 via the injection tube 114 at a dilution rate D _C that satisfies k _on [R] ⁰ ≪D _C. The injection tube is configured to inject the liquid from the liquid supply unit 120 into the reaction vessel 110, and may be, for example, a silicone tube.

The filter 112 has a pore size that allows the third molecule to pass but not the first molecule. For example, when the first molecule is bound to a magnetic bead, the filter pore size may be, for example, 0.3-1.5 micrometers, preferably 0.5-1.0 micrometers, and more preferably 0.65 micrometers.

The mixture may be stirred to disperse the mixture in the container before filtering.

Figure 5 shows (a) a top view of the reaction vessel 110, (b) a side cross-sectional view taken along line A-A, (c) a side view, and (d) a top cross-sectional view taken along line B-B. As shown in Figure 5(d), the liquid injected from the injection tube circulates through the reaction vessel 110 at flow F.

The shape of the reaction vessel is preferably cylindrical, as this can prevent the filter from clogging. When the vessel is cylindrical, it is preferable to form a filter on at least one of the bottom surfaces. In FIG. 5(b), circular filters are formed on the two bottom surfaces of the vessel. In this case, the two filters do not necessarily have to be parallel to each other, and may be inclined. This can increase the filter area.

The shape of the reaction vessel is not limited to a cylinder, but may be any shape, such as a sphere, cube, rectangular parallelepiped, or cone. In this case, the shape of the filter can also be any shape, such as a hemisphere or square, to match the shape of the reaction vessel.

The cross-sectional area of the injection tube 114 is preferably smaller than the filter area. This is to facilitate circulation of the liquid within the reaction vessel. For example, when there is one filter, the cross-sectional area of the injection tube 114 is preferably 1/10 or less of the filter area, more preferably 1/20 or less, and even more preferably 1/40 or less. When there are two filters, the cross-sectional area of the injection tube 114 is preferably 1/20 or less of the filter area, more preferably 1/40 or less, and even more preferably 1/80 or less. In view of the above, when the vessel is cylindrical with a bottom diameter of 3 cm, the tube diameter (inner diameter) is preferably 1 cm or less, more preferably about 0.5 cm, and even more preferably about 0.3 cm.

The reaction vessel may further have a discharge tube (not shown) for connecting to the separation section 140 or the collection section 160. The discharge tube can be opened and closed, for example, by a valve. Note that when filtering the mixture, the discharge tube must be closed to prevent liquid or complexes from being discharged from the discharge tube.

FIG. 6 shows (a) a top cross-sectional view and (b) a side cross-sectional view of a first variant of a reaction vessel. The first variant has two injection tubes. The number of injection tubes is not limited to one or two, and may be three or more.

FIG. 7 shows (a) a top cross-sectional view and (b) a side cross-sectional view of the reaction vessel variant 2. Variation 2 has one injection tube. The offset angle, which is the angle of the longitudinal axis of the injection tube with respect to the center line X of the reaction vessel, is approximately 30°. This configuration makes it easier for the flow of liquid F in the reaction vessel to circulate, effectively preventing clogging of the filter. The offset angle may be any angle greater than 0° and less than or equal to 90°.

FIG. 8 shows (a) a top cross-sectional view of reaction vessel variant 3 and (b) a top cross-sectional view of reaction vessel variant 4. Variation 3 has two injection tubes. The offset angle, which is the angle of the longitudinal axis of the injection tube with respect to the center line X of the vessel, is approximately 30°. Variation 4 has three injection tubes. Even with this configuration, the flow F of liquid in the reaction vessel is easier to circulate, and clogging of the filter can be effectively suppressed.

The above-described embodiments are illustrative of the following aspects.
(Aspect 1)
1. A method for isolating a second molecule having a desired affinity (Kd) for a first molecule using a filter, the method comprising:
Providing a sample containing a second molecule to be separated and a first molecule that binds to the second molecule;
contacting a sample containing a second molecule with a first molecule in a container having a filter to generate a complex in which the first molecule and the second molecule are bound;
a liquid is passed through the vessel to wash and filter out molecules that do not bind to the first molecule and molecules that bind to the first molecule with less than a desired affinity;
Here, the permeation from the filter is expressed by the following equation, where ^R is the initial concentration of the first molecule in the container, k _on is the binding rate constant between the first molecule and the second molecule, and D _C is the dilution rate represented by F _C /V, the volume of the container.
k _on [R] ⁰ _≪D
where Kd=(dissociation rate constant k _off between the first molecule and the second molecule)/(association rate constant k _on between the first molecule and the second molecule),
collecting a complex in which the second molecule is bound to the first molecule and a free first molecule, and dissociating the second molecule from the first molecule in the complex;
a second molecule having a desired affinity, comprising:
(Aspect 2)
k _on is 10 ⁴ to 10 ⁵ M/sec (molar per second),
The remaining rate Φ of the complex formed by binding the first molecule and the second molecule in the container is
Φ=exp( _-koff ×t), where t is the washing time (seconds), _koff is the dissociation rate constant between the first molecule and the second molecule, and is expressed as the product of the affinity and the initial binding rate constant _kon .
The method according to aspect 1.
(Aspect 3)
The method according to claim 1, wherein the pore size of the filter is from 0.1 to 100 μm.
(Aspect 4)
2. The method of embodiment 1, wherein the second molecule is a polypeptide chain such as an antibody or antigen-binding fragment.
(Aspect 5)
2. The method of embodiment 1, wherein the first molecule is bound to a magnetic bead.
(Aspect 6)
2. The method of embodiment 1, wherein the first molecule is bound to a spherical structure having a diameter of 0.1 to 10 μm.
(Aspect 7)
1. An apparatus for separating a second molecule having a desired affinity Kd for a first molecule using a filter, the apparatus comprising:
A container having at least one filter and an inlet port is configured to contact a sample containing a second molecule with a first molecule to generate a complex in which the first molecule and the second molecule are bound, and the device is configured to flow a liquid through the container to wash the container and to allow molecules that do not bind to the first molecule and molecules that bind to the first molecule with less than a desired affinity to permeate through the filter, and the permeation through the filter is expressed as follows, where [R] ⁰ is an initial first molecule concentration in the container, k _on is a binding rate constant between the first molecule and the second molecule, and D _C is a dilution rate represented by F _C /V, the flow rate of the liquid in the container,
k _on [R] ⁰ _≪D
where Kd=(dissociation rate constant k _off between the first molecule and the second molecule)/(association rate constant k _on between the first molecule and the second molecule);
A collection section that collects a complex in which the second molecule is bound to the first molecule and free first molecules;
a separation unit that dissociates a second molecule from a first molecule in the complex and separates the second molecule of the complex;
An apparatus comprising:
(Aspect 8)
k _on is 10 ⁴ to 10 ⁵ M/sec (molar per second),
The remaining rate Φ of the complex formed by binding the first molecule and the second molecule in the container is
Φ=exp( _-koff ×t), where t is the washing time (seconds), _koff is the dissociation rate constant between the first molecule and the second molecule, and is expressed as the product of the affinity and the binding rate constant _kon .
8. The apparatus according to aspect 7.
(Aspect 9)
8. The device according to aspect 7, wherein the pore size of the filter is between 0.1 and 100 μm.
(Aspect 10)
8. The apparatus of aspect 7, further comprising a collection vessel configured to collect the third molecule released from the second complex.
(Aspect 11)
1. A container for use in an apparatus for separating a second molecule having a desired affinity Kd for a first molecule, the container comprising:
at least one filter for separating the second molecule;
an inlet port;
the container is configured to contact a sample containing a second molecule with a first molecule to generate a complex in which the first molecule and the second molecule are bound to each other;
the container is configured to transmit through the at least one filter, via the inlet port, molecules that do not bind to the first molecule and molecules that bind to the first molecule with less than a desired affinity;
The permeation rate from the at least one filter is expressed by the following equation, where ^R is the initial concentration of the first molecule in the container, k _on is the binding rate constant between the first molecule and the second molecule, and D _C is the dilution rate represented by F _C /V, the volume of the container:
k _on [R] ⁰ _≪D
where Kd=(dissociation rate constant k _off between the first molecule and the second molecule)/(association rate constant k _on between the first molecule and the second molecule).
(Aspect 12)
k _on is 10 ⁴ to 10 ⁵ M/sec (molar per second),
The remaining rate Φ of the complex formed by binding the first molecule and the second molecule in the container is
The container of claim 11, wherein Φ=exp(-k _off ×t), where t is the washing time (seconds), k _off is the dissociation rate constant between the first molecule and the second molecule, and is expressed as the product of the affinity and the binding rate constant k _on .
(Aspect 13)
12. The container of aspect 11, wherein the pore size of the at least one filter is between 0.1 and 100 μm.
(Aspect 14)
12. The container of aspect 11, wherein the container is cylindrical and at least one bottom surface thereof is provided with the at least one filter.
(Aspect 15)
13. The container of aspect 12, wherein the inlet port is a first inlet port, and the container further comprises a second inlet port separate from the first inlet port.
(Aspect 16)
A container according to any one of aspects 11-15, wherein the cross-sectional area of the injection tube is no more than 1/10 of the area of the filter.
The present invention will be described below based on examples. However, the following examples are for the purpose of illustrating the present invention and are not intended to limit the present invention.

[Example 1]
Creating a device that uses a filter to separate a second molecule that has a desired affinity for a first molecule

A 50 mL centrifuge tube with the lid removed was cut into slices about 8 mm long, approximately 1.3 cm from the opening, and two holes with a diameter of 4.8 cm were drilled on the side using a drill press. A polyvinylidene fluoride (PVDF) membrane filter (Durapore® DVPP04700, Merck) was then glued to both ends using a glue gun (HOT BOND HB-80, Taiyo Denki). Silicone tubes (outer diameter/inner diameter: 5 mm/3 mm) were then inserted and fixed with instant adhesive (Super Multipurpose 2, 3M). Before use, the tubes were washed with PBS buffer, soaked in 200 mL of blocking buffer for at least 30 minutes, and washed again with PBS buffer. The apparatus created is shown generally in Figures 4 to 7.

[Example 2]
(1) mRNA transcription
The DNA was transcribed using the T7 RiboMAX™ Express Large Scale RNA Production System to obtain mRNA. The composition and conditions of the reaction solution are as follows:

The resulting solution, with a total volume of 10 μL, was incubated at 37°C for 30 minutes, after which 1 μL of RQ1 Rnase-Free Dnase was added and incubated at 37°C for 15 minutes. After incubation, purification was immediately performed using RNAClean(trademark) XP. The method of use was as described in the attached manual.

The template DNA sequences for BDA and PDO are as shown in SEQ ID NOs: 1 and 2 below.

SEQ ID NO:1:
Sequence name: T7Ω-BDA-His-Ytag
Sequence (367mer)
5'-GATCCCGCGAAATTAATACGACTCACTATAGGGGAAGTATTTTTACAACAATTACCAACAACAACAACAAACAACAACAACATTACATTTTACATTCTACAACTACAAGCCACCATGGATAACAAATTCAACAAAGAACAACAAAATGCTTTCTATGAAATCTTACATTTACCTAACTTAAACGAAGAACAACGCAATGGTTTCATCCAAAGCCTAAAAGATGACCCAAGCCAAAGCGCTAACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCTCAAGCACCAAAAGCTGACAACAAATTCAACGGGGGAGGCAGCCATCATCATCATCATCACGGCGGAAGCAGGACGGGGGGCGGCGGGGAAA-3'

SEQ ID NO:2:
Sequence name: T7Ω-PDO-His-Ytag
Sequence (391mer)
5'-GATCCCGCGAAATTAATACGACTCACTATAGGGGAAGTATTTTTACAACAATTACCAACAACAACAACAAACAACAACAACATTACATTTTACATTCTACAACTACAAGCCACCATGGACCTTGAGGAGCTTGAGCAGTTTGCCAAGACCTTCAAACAAAGACGAATCAAACTTGGATTCACTCAGGGTGATGTTGGGCTCGCTATGGGGAAACTATATGGAAATGACTTCAGCCAAACTACCATCTCTCGATTTGAAGCCTTGAACCTCAGCTTTAAGAACATGGCTAAGTTGAAGCCACTTTTAGAGAAGTGGCTAAATGATGCAGAGGGGGGAGGCAGCCATCATCATCATCATCACGGCGGAAGCAGGACGGGGGGCGGCGGGGAAA-3'

Newleft of sequence number 3 was used as a primer for PCR of template DNA of BDA or PDO.

SEQ ID NO:3:
Sequence name: Newleft
Sequence (33mer)
5'-GATCCCGCGAAATTAATACGACTCACTATAGGG-3'

(2) Ligation reaction of linker and RNA
The cnvK polyA Linker and the mRNA synthesized in (1) were hybridized by annealing. Then, a ligation reaction was carried out by photocrosslinking with UV irradiation. This resulted in a linker-mRNA conjugate. The composition of the reaction solution and reaction conditions are as follows.

The resulting solution, with a total volume of 20 μL, was annealed at 90-25°C/46 minutes using a T3 Thermocycler (Biometra), and then irradiated with UV (366 nm, 405 mJ).

(3) Translation: The linker-mRNA conjugate synthesized in (2) was used to synthesize a linker-protein conjugate using a lysate of rabbit reticulocyte cells (a cell-free translation system, treated with nuclease). The composition and conditions of the reaction solution are as follows:

A total of 150 μL of the solution was incubated at 30 ° C for 20 min, after which 72 μL of 3 M KCl was added, followed by 18 μL of 1 M MgCl ₂ , and then incubated at 37 ° C for 60 min. Then, 54 μL of 0.5 M EDTA was added and incubated at 37 ° C for 5 min.

(4) Creation of cDNA display An equal amount of 2x SA Binding Buffer was added to the Linker-Protein conjugate, which is a sample after translation, and added to magnetic beads (MyOne Streptavidin C1) washed with 1x SA Binding Buffer. Then, the reaction was carried out at room temperature for 30 minutes while stirring using a cooled thermoblock rotator (Nisshin Rika). Here, the magnetic beads were 60 μL for 6 pmol of linker. Then, the mixture was placed on a magnetic stand and the supernatant was removed. Next, 300 μL of 1x SA Binding Buffer was added, tapped, placed on a magnetic stand, and the supernatant was removed. This operation was repeated twice, and then 300 μL of 1x ReverTra Ace Buffer was added and the supernatant was removed as before. Then, the following reverse transcription reaction solution was added, and the reaction was carried out at 42 ° C for 30 minutes while stirring using a cooled thermoblock rotator (Nisshin Rika), and cDNA synthesis was performed. The composition of the reaction solution is as follows.

After the reaction, the cDNA display molecules were recovered by nucleic acid enzyme treatment. First, the sample was placed on a magnetic stand and the supernatant was removed, then 300μL of His Tag Binding Buffer was added, tapped, and the sample was placed on the magnetic stand and the supernatant was removed. Next, 58.5μL of His Tag Binding Buffer and 1.5μL of RNase T1 were added and the reaction was allowed to proceed with stirring at 37℃ for 15 minutes (cooled thermoblock rotator, Nisshin Rika). The cDNA display molecules were then subjected to His-tag purification. 60μL of the cDNA display sample recovered in 60μL of His Mag Sepharose Ni, washed with 300μL of His-tag Binding Buffer, was left overnight at 4℃, and the sample was placed on a magnetic stand and the supernatant was removed. Then, 300μL of His Tag Binding Buffer was added, pipetting was performed three times, and the supernatant was removed. This procedure was repeated twice, after which 60 μL of His-tag Elute Buffer was added and stirred at room temperature for 10 minutes using a microtube mixer (MT-360, TOMY), after which the supernatant was collected. Buffer exchange was then performed using Micro Bio-spin™ 6 Columns, and the supernatant solution was replaced with PBS. The method of use was as described in the attached manual.

(5) Immobilization of IgG on magnetic beads
IgG biotinylated using a biotinylation reagent (EZ-Link™ Sulfo-NHS-SS-Biotin) was immobilized on magnetic beads (Dynabeads M-270 Streptavidin). First, IgG was dissolved in PBS to a concentration of 1 mg/mL, and 1 μL of PBS in 10 mM EZ-Link™ NHS-SS-Biotin was added to 150 μL of the solution, which was then incubated at 25° C. for 30 minutes (Cool Stat 5200) to biotinylate the IgG. Next, buffer exchange was performed using Zeba™ spin desalting Columns (7K), and the solution was replaced with 1x SA Binding Buffer. The method of use was according to the attached manual. The Biotin-IgG solution was stored at -80° C. Then, 5 μL of Biotin-IgG solution and 45 μL of 1x SA Binding Buffer were added to 50 μL of Dynabeads M-270 Streptavidin that had been washed with 100 μL of 1x SA Binding Buffer, and the mixture was incubated with stirring at 25°C for 20 minutes (cooled thermoblock rotator, Nisshin Rika) to immobilize the IgG.

(6) _koff -Selection
To evaluate the device prepared in Example 1, model selection was performed using the BDA cDNA display targeting IgG and the negative control PDO cDNA display. 50 μL of IgG-Beads prepared in (5) was washed three times with 100 μL of PBS, and 20 μL of BDA cDNA display and 20 μL of PDO cDNA display prepared in (4) were added and stirred at 25°C for 60 minutes (cooled thermoblock rotator, Nisshin Rika) to bind IgG and BDA cDNA display. After that, it was placed on a magnetic stand and the supernatant was collected (Sup(1)), and then 200 μL of PBS was added and tapped, followed by washing for 5, 10 or 30 minutes at a dilution rate that satisfied k _on [R] ⁰ ≪ D _C using the device prepared in Example 1. After washing, the IgG-Beads solution inside the device was collected, placed on a magnetic stand, and 100 μL of the supernatant was collected (Sup(2)). Then, the SS bond between the IgG and biotin was cleaved with DTT, a reducing agent, and the IgG-BDA cDNA display complex was eluted by adding 50 μL of PBS in 100 mM DTT and tapping, followed by reaction with stirring at 50°C for 30 minutes (cooled thermoblock rotator, Nisshin Rika). The mixture was then placed on a magnetic stand and the supernatant was recovered (Sup(3)). 100 μL of PBS in 0.1% (V/V) SDS was then added and the mixture was stirred at room temperature for 10 minutes using a microtube mixer (MT-360, TOMY), after which the mixture was placed on a magnetic stand and the supernatant was recovered (Sup(4)).

(7) PCR for confirmation of k _off -Selection
To confirm the results of k _off -Selection by nucleic acid PAGE, the recovered cDNA display molecules were amplified by PCR. PCR was performed using a solution of Sup(1),(2),(3),(4) diluted 10-fold with UPDW as the DNA template, and the PCR product was analyzed by 8M Urea 4% PAGE. A 100 bp DNA ladder was used as a marker. The composition and conditions of the PCR reaction solution are as follows:

Step 1: 98℃ (30 seconds)
Step 2: 95°C (15 seconds)
Step 3: 68°C (5 seconds)
Step 4: 72°C (23 seconds)
Step 5: 72°C (2 min)
Step 6: 4℃ (Pause)
[Step 2 → 4, 30 cycles]

Here, New Y tag poly A for cnvK and T7omegaNew are sequences represented by the following sequence numbers 4 and 5, respectively.

SEQ ID NO:4:
Sequence name: New Ytag poly A for cnvK
Sequence (22mer)
5'-TTTCCCCGCCGCCCCCCGTCCT-3'

SEQ ID NO:5:
Sequence name: T7omegaNew:
Sequence (60mer)
5'-GATCCCGCGAAATTAATACGACTCACTATAGGGGAAGTATTTTTACAACAATTACCAACA-3'

As a result of electrophoresis, a band with the same electrophoretic mobility as the reference sample was detected, and when the band intensity of the DTT eluate was compared with the washing time, the band intensity attenuated with time, confirming that k _off -Selection was performed using the device prepared in Example 1.

The results are shown in Figure 9.
FIG. 10 shows the band intensity ratio, which is the percentage of the intensity of each band relative to the total intensity of all bands in Sup(3) (DTT eluate).

Using the device created in Example 1, it was shown that the band intensity ratio decreases and second molecules with high affinity can be obtained in correlation with increasing the washing time shown on the horizontal axis from 5 minutes (300 seconds), 10 minutes (600 seconds), to 30 minutes (1800 seconds). Specifically, in this example, the B domain of Protein A (BDA, corresponding to the second molecule) with a Kd of about 10 nM for IgG (corresponding to the first molecule) is used, and this shows that the bound molecules peel off over time in correlation with the washing time, resulting in fewer remaining molecules. It can be seen that the smaller the Kd (the stronger the binding), the more likely the molecule is to remain even with a longer washing time. In other words, it is selected.

REFERENCE SIGNS LIST 10 First molecule 20 Second molecule 30 Third molecule 40 Complex 100 Collection vessel 110 Reaction vessel 112 Filter 114 Injection tube 116 Mixture 120 Liquid supply section 140 Collection section 160 Separation section Fc Flow rate L1 Liquid surface V Flow velocity

Claims (16)

1. A method for isolating a second molecule having a desired affinity (Kd) for a first molecule using a filter, the method comprising:
Providing a sample containing a second molecule to be separated and a first molecule that binds to the second molecule;
contacting a sample containing a second molecule with a first molecule in a container having a filter to generate a complex in which the first molecule and the second molecule are bound;
a liquid is passed through the vessel to wash and filter out molecules that do not bind to the first molecule and molecules that bind to the first molecule with less than a desired affinity;
Here, the permeation from the filter is expressed by the following equation, where ^R is the initial concentration of the first molecule in the container, k _on is the binding rate constant between the first molecule and the second molecule, and D _C is the dilution rate represented by F _C /V, the volume of the container.
k _on [R] ⁰ _≪D
where Kd=(dissociation rate constant k _off between the first molecule and the second molecule)/(association rate constant k _on between the first molecule and the second molecule),
collecting a complex in which the second molecule is bound to the first molecule and a free first molecule; and dissociating the second molecule from the first molecule in the complex.
a second molecule having a desired affinity, comprising: k _on is 10 ⁴ to 10 ⁵ M/sec (molar per second),
The remaining rate Φ of the complex formed by binding the first molecule and the second molecule in the container is
Φ=exp( _-koff ×t), where t is the washing time (seconds), _koff is the dissociation rate constant between the first molecule and the second molecule, and is expressed as the product of the affinity and the initial binding rate constant _kon .
The method of claim 1. The method according to claim 1, wherein the filter pore size is 0.1 to 100 μm. The method of claim 1, wherein the second molecule is a polypeptide chain, such as an antibody or an antigen-binding fragment. The method of claim 1, wherein the first molecule is bound to a magnetic bead. The method of claim 1, wherein the first molecule is bound to a spherical structure having a diameter of 0.1 to 10 μm. 1. An apparatus for separating a second molecule having a desired affinity Kd for a first molecule using a filter, the apparatus comprising:
A container having at least one filter and an inlet port is configured to contact a sample containing a second molecule with a first molecule to generate a complex in which the first molecule and the second molecule are bound, and the device is configured to flow a liquid through the container to wash the container and to allow molecules that do not bind to the first molecule and molecules that bind to the first molecule with less than a desired affinity to permeate through the filter, and the permeation through the filter is expressed as follows, where [R] ⁰ is an initial first molecule concentration in the container, k _on is a binding rate constant between the first molecule and the second molecule, and D _C is a dilution rate represented by F _C /V, the flow rate of the liquid in the container,
k _on [R] ⁰ _≪D
where Kd=(dissociation rate constant k _off between the first molecule and the second molecule)/(association rate constant k _on between the first molecule and the second molecule);
A collection section that collects a complex in which the second molecule is bound to the first molecule and free first molecules;
a separation unit that dissociates a second molecule from a first molecule in the complex and separates the second molecule of the complex;
An apparatus comprising: k _on is 10 ⁴ to 10 ⁵ M/sec (molar per second),
The remaining rate Φ of the complex formed by binding the first molecule and the second molecule in the container is
Φ=exp( _-koff ×t), where t is the washing time (seconds), _koff is the dissociation rate constant between the first molecule and the second molecule, and is expressed as the product of the affinity and the binding rate constant _kon .
8. The apparatus of claim 7. The device according to claim 6, wherein the filter pore size is 0.1 to 100 μm. The device of claim 7, further comprising a collection vessel configured to collect the third molecule released from the second complex. 1. A container for use in an apparatus for separating a second molecule having a desired affinity Kd for a first molecule, the container comprising:
at least one filter for separating the second molecule;
an inlet port;
the container is configured to contact a sample containing a second molecule with a first molecule to generate a complex in which the first molecule and the second molecule are bound to each other;
the container is configured to transmit through the at least one filter, via the inlet port, molecules that do not bind to the first molecule and molecules that bind to the first molecule with less than a desired affinity;
The permeation rate from the at least one filter is expressed by the following equation, where ^R is the initial concentration of the first molecule in the container, k _on is the binding rate constant between the first molecule and the second molecule, and D _C is the dilution rate represented by F _C /V, the volume of the container:
k _on [R] ⁰ _≪D
where Kd=(dissociation rate constant k _off between the first molecule and the second molecule)/(association rate constant k _on between the first molecule and the second molecule). k _on is 10 ⁴ to 10 ⁵ M/sec (molar per second),
The remaining rate Φ of the complex formed by binding the first molecule and the second molecule in the container is
The container of claim 11, wherein Φ=exp(-k _off ×t), where t is the washing time (seconds), k _off is the dissociation rate constant between the first molecule and the second molecule, and is expressed as the product of the affinity and the binding rate constant k _on . The container according to claim 11, wherein the pore size of the at least one filter is 0.1 to 100 μm. The container according to claim 11, wherein the container is cylindrical and at least one of the bottom surfaces is provided with the at least one filter. The container of claim 12, wherein the inlet port is a first inlet port, and the container further comprises a second inlet port separate from the first inlet port. The container according to any one of claims 11 to 15, wherein the cross-sectional area of the injection tube is 1/10 or less of the area of the filter.

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