Nanobody exchange chromatography

Abstract

The present invention relates to the field of affinity purification and provides means and methods for applying protein binders that compete for a target protein for use as a capture / elution tool, where the elution agent comprises an immunoglobulin single variable domain (ISVD) and is capable of displacing the capture binder. More specifically, the displacement efficiency of the ISVD-containing protein binder is determined by the dissociation rate constant (k off ) is driven by its dissociation kinetics, which is equal to or lower than that of the capture agent. Furthermore, protein binding agents can be effectively used in high-throughput purification or for capturing protein complexes from complex mixtures, thereby facilitating structural, biochemical, and physicochemical analyses of target proteins.

Description

Technical Field

[0001] The present invention relates to the field of affinity purification and provides means and methods for applying a protein binder that competes for a target protein for use as a capture / elution tool, where the eluent contains an immunoglobulin single variable domain (ISVD) and is capable of displacing the capture binder. More specifically, the displacement efficiency of the ISVD-containing protein binder is driven by its dissociation reaction rate, where the dissociation rate constant (k off ) is equal to or lower than that of the capture agent. Furthermore, the protein binder is usefully available in high-throughput purification from complex mixtures or for capture protein complexes, thereby facilitating structural, biochemical, and physicochemical analysis of the target protein.

Background Art

[0002] As an affinity-based technique, immunoaffinity purification presents several advantages over chromatography methods based on chemical and physical properties. Immunoaffinity purification can simplify the purification of proteins from complex multi-step procedures to a single-step protocol, reducing costs and time. As a result, immunoaffinity purification can improve yields and limit potential product degradation. However, immunoaffinity purification performed with conventional antibodies often requires extreme elution conditions that can damage the product being purified. Single-domain antibody fragments such as VHHs or nanobodies (Nb) are used in affinity chromatography (AC) and are quite stable and easy to produce under different elution conditions, making them suitable tools in immunoaffinity chromatography (e.g., Verheesen et al., 2003). VHH-based affinity columns may also enable higher yields compared to long-chain antibody constructs and full IgG, perhaps due to their high density of binding to the matrix (Aliprandi et al., 2010). The suitability of VHHs for affinity purification has been further recognized in the development and general application of numerous VHH-based affinity resins, such as Capture Select resin (http: / / www.captureselect.com), Chromotek nanotrap for the purification of GFP fusion proteins from cell homogenates (Rothbauer et al., 2008; US10,125,166B2), among other resins (e.g., Pabst et al., 2016). Nanotag (TM) Biotechnologies (Gotzke et al., 2019), a technology equivalent to the EPEA tag, a C-terminal tag detected by the Nb-based CaptureSelect resin (US9518084B2; EP2576609B1), has developed a new alpha-peptide tag-based purification technique that applies an alpha-specific Nb combined with a high-affinity peptide for the elution of an ALFA-tagged target protein bound to the Nb.

[0003] Single domain antibody fragments, VHHs or nanobodies are often chemically conjugated to an insoluble matrix and the bound protein complex is eluted under conditions that disrupt non-covalent interactions between proteins. Instead, Pleiner et al. (2015, eLife; 4:e11349) used an enzyme that hydrolyzes the bond to enable detachment of the VHH from the matrix (along with any bound protein complex), thereby eluting the bound protein complex containing the VHH, allowing further studies such as structural analysis or physicochemical characterization. The drawback is that specific concentrations of the enzyme and specific conditions are required for optimal enzyme activity in the sample. Furthermore, the protease used is also detrimental to the bound target protein and the column or matrix can only be used once. A method of combining nanobody-based resources in competitive immunoassays, in which Nbs that bind to epitopes on significantly different targets are combined, has also been disclosed for use in protein detection by ELISA (Caljon et al., 2015).

[0004] Alternatively, in sample displacement chromatography (SDC), the sample is introduced onto the column and then displaced by the injection of a constant displacement fluid. Displacement chromatography of proteins and peptides is typically carried out in ion-exchange mode, although hydrophobic interaction mode and affinity mode are also used. The affinity of the displacer for the stationary phase must be higher than that of any feed component. SDC incorporated into a small analytical column for the efficient separation of microgram amounts of protein from human plasma can be applied as a sample preparation step for subsequent analysis of the separated protein by mass spectrometry (MS). However, reverse-phase mode (RPC) remains the fundamental method for separating target peptides after synthesis for removing trace impurities and is the most commonly applied chromatography step for separating proteolytic digests of proteins prior to MS analysis. However, the development of rapid, simple, reproducible, and cost-effective methods for the efficient purification of proteins and peptides from complex mixtures remains a challenge. Supports and devices for chromatography have been further improved, but there is still a need for the development of new, complementary methods for separating complex mixtures of proteins and peptides at both the analytical and preparative scales. Affinity assays or immunosubstitution assays are applicable under mild conditions and have been described as sophisticated antibody-based chromatography tools. For example, Abdiche et al. (2017) applied the "waterfall" mechanism of monoclonal antibodies with adjacent or minimally overlapping epitopes for the target molecule to displace and specifically elute the target. The drawback of using monoclonal antibodies for displacement is that they cross-block rather than displace each other when there is low epitope diversity, and a large number of highly specific, significantly different antibodies characterized by different association rates for non-overlapping or minimally overlapping epitopes are required to obtain optimal displacement.

Prior Art Documents

Patent Documents

[0005] [Patent Document 1] U.S. Patent No. 10,125,166 [Patent Document 2] U.S. Patent No. 9,518,084 [Patent Document 3] European Patent No. 2,576,609 [Non-Patent Document]

[0006] [Non-Patent Document 1] Verheesen et al. (2003), "Beneficial properties of single-domain antibody fragments for application in immunoaffinity purification and immuno-perfusion chromatography", Biochim Biophys Acta, 1624: 21 - 28 [Non-Patent Document 2] Aliprandi et al. (2010), "The availability of a recombinant anti-SNAP antibody in VHH format amplifies the application flexibility of SNAP-tagged proteins", J Biomed Biotechnol, ID 658954 [Non-Patent Document 3] http: / / www.captureselect.com [Non-Patent Document 4] Rothbauer et al. (2008), "A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins", Mol Cell Proteomics, 7: 282 - 289 [Non-Patent Document 5] Gotzke et al. (2019), "The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications", Nat. Comms., 10:4403.

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

Problems to be Solved by the Invention

[0007] The direct isolation of proteins or protein complexes from complex mixtures, under conditions suitable for analytical purposes such as structural biology, mass spectrometry, or proteomics, by a concise, rapid, and easy downscaling of protein purification is currently achievable using affinity purification, but is not suitable for high-throughput purposes. There is also no possibility of applying a general-purpose binder at specific binding sites or epitopes, and the determination of significantly different binders for each target is cumbersome, but a more general-purpose approach is really desired. Thus, there is a need for a concise, high-purity, small-scale purification method that enables direct analysis for testing small amounts of target proteins derived from complex mixtures.

Means for Solving the Problems

[0008] (Summary of the Invention) The present invention relates to a new method of nanobody-based displacement or competition-based exchange chromatography, in which, for binding to a target protein, perhaps a pair of Nbs that compete for the same epitope or overlapping, highly epitopes on the target compete with a target protein (or "target protein" used interchangeably herein) for purification from a complex mixture in a single step. The purification method enables the displacement of a competitive binder for the target protein, and when using a nanobody or even an antigen-binding domain of an immunoglobulin single variable domain (ISVD) as a displacer, the displacement reaction rate is different compared to the displacement reaction rate predicted for the antigen-binding domain of a conventional antibody. Furthermore, other advantages of ISVD-based binders, such as their ability to bind conformational epitopes and deep crevices, their high stability and ease of production, present the method described herein as a method suitable for affinity purification for high-throughput analysis. The method thereby results in a small amount of highly pure protein bound to a high-affinity Nb, which may be labeled, may be functionalized, or may be used as a chaperone for structural or biochemical analysis. In nanobody exchange chromatography (abbreviated herein as NANEX), the target protein or target protein is captured by a first (immobilized) nanobody called a nano-capturer or capturer, followed by binding to a second soluble nanobody called a nano-stripper or stripper that competes with the capturer for binding to the target protein but has dissociation reaction rate characteristics suitable for establishing efficient displacement and elution. More specifically, the reaction rate is determined by the dissociation rate constant for an ISVD-containing stripper, and a slow dissociation rate (or low k off ) and / or high affinity (or low K D) and / or requires high avidity. This step results in the quantitative and rapid elution of a highly pure complex of the protein of interest bound to the releasing agent under physiological conditions. Optionally, the nano-releasing agent may be functionalized as a chaperone, stabilizer or antigen-binding chimeric protein known as MegaBody™, or alternatively, has a detectable label or property that facilitates subsequent analysis.

[0009] In a first aspect, the invention is a method for purifying a target protein, comprising: a) contacting a first protein binder that specifically binds to an epitope of the target protein with a sample containing the target protein; b) mixing the sample with a second protein binder that competes for binding to the target protein in the presence of the first protein binder, such that the second protein binder replaces the first binder on the target protein and releases the first binder from the target protein; and c) eluting the mixture containing the second protein binder bound to the target protein. comprising wherein at least the second protein binder comprises an immunoglobulin single variable domain (ISVD) or a functional variant thereof that specifically binds to the target protein, and in this case, the dissociation rate constant (or k off value) of the second protein binder is low or the same as or the dissociation rate is slower or the same as that of the first protein binder compared to the first protein binder. The second protein binder may compete via binding to the same or mostly overlapping epitopes on the target protein. Alternatively, the second binder may bind to different epitopes or epitopes that overlap in minimal parts, but competes allosterically and / or kinetically for binding to the target protein driven by its dissociation rate constant. In a further embodiment, the second protein binder has a higher affinity for the epitope, i.e., a lower KD has a value. More specifically, the K D value for the epitope of the target protein is in the range of low micromolar concentration to nanomolar concentration for the first protein binder, and in the range of low nanomolar concentration to picomolar concentration for the second protein binder. Preferably, the relative affinity is at least 2-fold or preferably at least 10-fold, 20-fold or 100-fold compared to the K D value for the first protein binder, as defined by the K D value.

[0010] In another embodiment, the method described herein includes a step of washing the mixture of step a) to remove impurities and provide appropriate buffer conditions before adding the second protein binder.

[0011] Another embodiment is a method for purifying the target protein described herein, further comprising repeating or varying steps a) to c) using a third protein binder and a fourth protein binder instead of or in addition to each of the first protein binder and the second protein binder, wherein the third binder and the fourth binder specifically bind to the same target protein in steps a) to c) but to an epitope different from the epitope to which the first binder and the second binder bind. The tandem purification method specifically relates to the purification from a complex sample to obtain high purity. Optionally, a washing step may be included in the method to remove unbound protein or excess binder.

[0012] Furthermore, the methods disclosed herein use a first binder and a second binder that compete for binding to a target protein present in a sample, where the binders specifically recognize an epitope on a tag of the target protein, preferably as part of a fusion protein. Preferably, in this case, the tag may be an affinity tag, an epitope tag, a reporter tag or another synthetic and / or commercially available tag. The tag of the target protein may be selected from the group of fluorescent proteins (such as green fluorescent protein (GFP) or mCherry), and among those further listed herein, may be glutathione-S-transferase (GST), SUMO (small ubiquitin-like modifier), SMT3, C-terminal peptide EPEA. Alternatively, the epitope of the target protein recognized by the protein binder includes a specific epitope present on the native or endogenous protein or an epitope conferred by the protein in nature. Furthermore, the epitope can also be established by a post-translational modification (PTM) on the target protein to which a protein binder that recognizes the PTM specifically binds. A further alternative relates to the method of the invention where the epitope of the target protein is defined by a specific binding site on the scaffold protein domain of an antigen-binding chimeric protein, namely MegaBody™, as defined in Steyaert et al. (WO2019 / 086548A1). In a preferred embodiment, the epitope is specific for the scaffold protein domain of an antigen-binding chimeric protein comprising a scaffold derived from HopQ or a scaffold derived from Ygjk as disclosed in WO2019 / 086548A1.

[0013] Another embodiment relates to a method comprising a second protein binder that comprises a first protein binder in a multivalent format or a multispecific format. The methods described herein comprise a second protein binder that comprises an ISVD as defined herein as a domain with four framework regions (FR) and three complementarity determining regions (CDR) according to the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 format that is sufficient for binding to a target protein or antigen. Alternatively, functional variants of the ISVD are contemplated herein, and relate to ISVD-containing moieties that are capable of binding to an antigen in a manner similar to the ISVD. The method may further utilize a first protein binder that specifically binds to a target epitope and for which an ISVD that competes with the second binder for this is also included.

[0014] The methods disclosed herein further relate to competition to a first protein binder and a second protein binder, wherein the dissociation rate of the second binder for binding to the target is slow compared to the dissociation rate of the first binder, where the first binder comprises a mutant ISVD as compared to a second protein binder comprising an ISVD, or vice versa, and the binder comprising the first ISVD has a rapid dissociation rate and / or low affinity (or high K D ) compared to the binder comprising the second ISVD, caused by a change within its binding region or paratope.

[0015] An alternative embodiment provides a method as described herein, wherein the first binder and the second binder specifically bind to the target protein with a k of at least 0.0001 seconds -1 or more, and comprise the same ISVD binding moiety. off

[0016] A further embodiment relates to a method as described herein, wherein the protein binder comprises a functional moiety or a detectable label.

[0017] In a specific embodiment, the first protein binder and / or the second protein binder of the methods described herein are in a functionalized format, i.e., in addition to binding to an epitope, they have a format with a specific function. For example, the functionalized format can include an antigen-binding chimeric protein, in particular, MegaBody (trademark) as disclosed in Steyaert et al. (WO2019 / 086548A1). The MegaBody referred to herein includes an antigen-binding domain in the format of an ISVD functional variant D that specifically binds to an epitope of a target protein via an ISVD, where the antigen-binding domain of the ISVD is non-flexibly fused to a scaffold protein domain. In a preferred embodiment, the scaffold protein includes or is derived from a HopQ protein or a Ygjk protein. MegaBody is known to provide a function as a novel chaperone-type binder for the improvement of cryo-EM structural analysis of target proteins.

[0018] One embodiment relates to the methods described herein where the first protein binder is immobilized on a surface and the second protein binder is in solution, which means that the second binder is soluble under appropriate purification conditions. Preferably, the surface of the first protein binder is a resin and the appropriate conditions are physiological conditions. The resin or matrix may be suitable for purification for preparation or for purification for analysis, the latter preferably involving a low volume of a few microliters. An alternative aspect of the invention actually involves a first protein binder in an immobilized form on a surface and, for use of a chip in the methods described herein, thus, in combination with a solution, relates to a chip or microcolumn prepared to provide a second protein binder as described in the methods herein.

[0019] The method of the invention provides for the purification of a target protein or molecule from a sample, which sample can be a biological sample, a complex mixture, a cell sample or an in vitro sample.

[0020] Another aspect relates to a kit comprising a first protein binder and a second protein binder for use in the methods described herein. Further embodiments relate to a kit comprising a first protein binder and a second protein binder according to the present invention, wherein the first agent or the second agent is present on a surface, matrix or resin or the kit comprises a microchip described herein. More specifically, the kit can comprise a first protein binder and a second protein binder that compete for binding to a tag of a target protein, where the tag is selected from the group of tags containing GFP, mCherry, GST, SMT3 or EPEA, and the agent is selected from the group of proteins depicted in any of SEQ ID NOs: 1-6, 18 or 19, 20, 21, 23, 24, 26, 27 or 28 or a sequence with at least 90% identity thereto or a sequence without a His tag and / or an EPEA tag and optionally containing another (small) tag. In a specific embodiment, the first protein binder and the second protein binder of the kit comprise different sequences selected from the group. In another specific embodiment, the first protein binder and the second protein binder of the kit can comprise the same sequence selected from the group, and K D is 0.1 nM or greater.

[0021] Another aspect relates to a protein complex comprising a second or fourth protein binder and a target protein, as disclosed in the methods for purification herein. In certain embodiments, the target protein can be selected from the group consisting of GFP, mCherry, GST, SMT3, or EPEA. In one embodiment, the protein complex can be crystalline. The protein complex as defined herein can further comprise one or more additional proteins bound to the target protein. The additional complex can result in the use in the identification or characterization of protein - protein complexes or protein - protein interactions, which can be transient or conformation - specific. Finally, the protein complex disclosed herein can be useful for structural analysis, structure - based drug design, drug discovery, analysis by mass spectrometry, or alternative biochemical or physicochemical analysis.

[0022] Another alternative aspect described herein relates to high - resolution three - dimensional structure representation in the atomic resolution of a protein complex formed by a second (or fourth) protein binder and a target protein. For the crystal complexes disclosed herein, one embodiment, in particular, relates to crystals of GFP and GFP - specific Nb crystals characterized at points within the space group P212121, which is associated with unit cell parameters: a = 74.497 Å ± 5%, b = 103.450 Å ± 5%, c = 209.774 Å ± 5%, α = 90.00°, β = 90.00°, γ = 90.00°. The 3D structure or crystal discloses the specific epitope of the binding site for a protein binder specific for GFP as disclosed herein, where the binding site consists of a subset of atomic coordinates, and in this case, presents an embodiment where the binding site consists of the amino acid residues: PRO89, GLU90, GLU111, LYS113, PHE114, GLU115, GLY116 of the GFP protein depicted in SEQ ID NO: 16.

[0023] The drawings described are very schematic and non-limiting. In the drawings, the sizes of parts of elements are exaggerated and may not be drawn to scale for illustrative purposes.

Brief Description of the Drawings

[0024]

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Modes for Carrying Out the Invention

[0025] The present invention is described with reference to certain specific drawings in connection with certain embodiments, but the invention is not limited thereto and is limited only by the claims. Any reference signs within the claims shall not be construed as limiting the scope. It will be understood, of course, that all aspects or advantages may not necessarily be achieved in accordance with any particular embodiment of the invention. Thus, for example, those skilled in the art will recognize that the invention may be implemented or carried out in a form that achieves or optimizes one advantage or group of advantages taught herein without necessarily achieving other aspects or advantages that may be taught or suggested herein. The invention, together with its features and advantages, as well as the organization and method of operation, can be best understood by reference to the following "Detailed Description of the Invention" when read in conjunction with the accompanying drawings. The aspects and advantages of the invention will become apparent from and will be elucidated with reference to the embodiments described hereinafter. Throughout this specification, references to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, although they may.

[0026] Definitions When referring to a singular noun and using an indefinite or definite article, such as "a" or "an", "the", this includes the plural form of the noun unless something else is specifically stated. In this description and the claims, when the term "comprising" is used, this does not exclude other elements or steps. Further, in this description and the claims, terms such as "first", "second", "third", etc. are used to identify similar elements, but are not necessarily used to describe a sequential or chronological order. Terms used in this way are interchangeable in appropriate circumstances, and it is to be understood that embodiments of the invention described herein can operate in an order other than the arrangements described or illustrated herein. The following terms or definitions are presented only to assist in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they have to those skilled in the technical field of the invention. Practitioners are directed to the definitions and terms of the art, particularly Sambrook et al., "Molecular Cloning: A Laboratory Manual", 4th Edition, Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., "Current Protocols in Molecular Biology", (Supplement 114), John Wiley & Sons, New York (2016). Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., molecular biology, biochemistry, structural biology, and / or mathematical biology).

[0027] In this specification, the terms "protein", "polypeptide", and "peptide" are used interchangeably to refer to polymers of amino acid residues, as well as variants and synthetic analogs thereof. For example, a partial amino acid sequence derived from its original protein after trypsin digestion may also be referred to as a "peptide". Thus, these terms apply to both naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are synthetic, non-naturally occurring amino acids, such as chemical analogs of the corresponding naturally occurring amino acids. The term also includes, inter alia, post-translational modifications of polypeptides, such as glycosylation, phosphorylation, ubiquitination, sumoylation, and acetylation, which are known in the art. Based on the amino acid sequence and modifications, the atomic or molecular weight of a polypeptide is expressed in units of (kilo)daltons (kDa). "Isolated" or "purified" means a material that is substantially or essentially free of the components that normally accompany it in its native state. For example, an "isolated polypeptide" or "purified polypeptide" refers to a polypeptide that has been purified from the molecules that flank it in its natural state, e.g., an antibody or nanobody identified and disclosed herein that has been removed from the molecules present in a sample or mixture, such as a production host that flanks the polypeptide. An isolated protein or isolated peptide may be produced by chemical synthesis of amino acids or by purification from recombinant production or complex samples.

[0028] "Homolog" or "homologs" of a protein include peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions compared to the unmodified protein in question and have the same biological functional activity as the unmodified protein from which they are derived. As used herein, the term "amino acid identity" refers to the degree to which sequences are identical on a one-to-one amino acid correspondence basis over a comparison window. Thus, the "percent sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met, also denoted herein by their one-letter codes) occur in both sequences, resulting in the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percent sequence identity. As used herein, "substitution" or "mutation" or "variant" results from the replacement of one or more amino acids or nucleotides, respectively, with different amino acids or nucleotides as compared to the amino acid sequence or nucleotide sequence of the parent protein or a fragment thereof. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions that do not substantially affect the activity of the protein.

[0029] "Binding to" means any interaction, whether direct or indirect. Direct interaction implies contact between the binding partners. Indirect interaction means any interaction where the interacting partners interact within a complex of more than two molecules. The interaction can be completely indirect or partially indirect, with the help of one or more cross-linking molecules, in which case there is still direct contact between the partners, and these are stabilized by further interactions of one or more molecules. As used herein, the term "specifically binds to" means a binding domain that recognizes a specific target protein or a component or molecule of a specific target, but does not substantially recognize or bind to other molecules in the sample. Specific binding does not mean exclusive binding. However, specific binding means that the protein has increased the affinity for one or more of its binders to some extent or has a preference for them. As used herein, the term "affinity" generally refers to the degree to which a ligand, chemical, protein or peptide binds to another (target) protein or peptide such that the equilibrium by a single protein monomer is shifted towards the existence of the complex formed by their binding. Affinity is the strength of binding of a single molecule to its ligand. Affinity is typically measured and reported by the equilibrium dissociation constant (K D ). The binding of an antibody to its antigen is a reversible process, and the binding reaction rate is proportional to the concentration of the reagent. At equilibrium, the rate of complex formation between [antibody]-[antigen] is equal to the dissociation rate to its components, [antibody] + [antigen]. Measurement of the reaction rate constant can be used to define the equilibrium constant or affinity constant (1 / K D ). Briefly, the smaller the K D value, the greater the affinity of the antibody for its target. The rate constants for both directions of the reaction are named as follows: The association reaction rate constant (K on ) is the "on rate" (K onis the reaction term used to calculate it and is a constant used to characterize how rapidly an antibody binds to its target. Conversely, the dissociation reaction rate constant (K off ) is the reaction term used to calculate the "off-rate" (K off ) and is a constant used to characterize how rapidly an antibody dissociates from its target. In the measurements presented herein, the flatter the slope, the slower the off-rate or the stronger the binding of the antibody. Conversely, a sharp drop indicates a rapid off-rate and weak binding of the antibody. The ratio of the experimentally measured off-rate and on-rate (K off / K on ) is used to calculate the K D value. To those skilled in the art, several determination methods for measuring the on-rate and off-rate and calculating K D (see below and the examples) are known, and these are therefore considered as values that take into account the standard error and are independent of the assay used.

[0030] As used herein, the terms "protein complex" or "complex" or "assembled protein" refer to a group of two or more associated macromolecules in which at least one of the macromolecules is a protein. As used herein, a protein complex typically refers to an assembly of macromolecules that can be formed under physiological conditions. The individual members of a protein complex are linked by non-covalent interactions. A protein complex can be a non-covalent interaction complex consisting only of proteins, for example, a non-covalent interaction complex formed by two proteins, three proteins, four proteins, etc. More specifically, it can be a complex of a protein binder and a target protein to which, optionally, another protein or compound is bound thereto, and thus can be referred to as an inter-protein complex.

[0031] "Binding agent" relates to a molecule capable of binding to another molecule, where the binding is preferably a specific binding that recognizes a defined binding site, pocket or epitope. The binding agent can be a binding agent of any nature or type and is independent of its origin. The binding agent may be chemically synthesized, may occur naturally, may be produced (and purified) recombinantly, or may be designed and produced synthetically. Thus, the binding agent can be a small molecule, chemical substance, peptide, polypeptide, antibody or, in particular, a peptidomimetic, antibody mimetic, active fragment, chemical derivative, etc., any derivative thereof. Preferably, the binding agent is a protein binding agent in the methods described herein. The terms "binding pocket" or "binding site" refer to a region of a molecule or molecular complex that preferentially associates with another chemical entity, compound, protein, peptide, antibody, ISVD or Nb as a result of its shape and charge. The term "pocket" includes but is not limited to a cleft, channel or site. The terms "portion of the binding pocket / site / epitope" or "overlapping epitope", used interchangeably herein, refer to amino acid residues less than all of the amino acid residues that define the binding pocket or binding site or epitope. For example, a portion of a residue may be a key residue that plays a role in binding to a ligand, or a residue that is spatially related and defines the three-dimensional compartment of the binding pocket. Residues may be contiguous or non-contiguous in the primary sequence. For antibody-like molecules, the term "epitope" is also used to describe the binding site used interchangeably herein. As used herein, "adjacent" binding sites or "overlapping in the minimal portion" binding sites refer to "non-overlapping (but binding to adjacent sites) amino acids" or up to about 30% overlap within the binding amino acid residues, respectively. "Epitope" refers to an antigenic determinant of a polypeptide that constitutes a binding site or binding pocket on a target protein molecule that is an accessible epitope or binding site outside the cell.An epitope can include three amino acids within a spatial conformation that is unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, 7 such amino acids, and more typically, at least 8, 9, 10 such amino acids. In the art, methods for determining the spatial conformation of amino acids are known, for example, X-ray crystallography, multidimensional nuclear magnetic resonance, cryo-EM hydrogen-deuterium exchange (HDX)-MS as well as cross-linking mass spectrometry (XL-MS), epitope binning or, less commonly, neutron scattering, X-ray free electron laser (XFEL) or small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) techniques are also included. As used herein, "conformational epitope" refers to an epitope that includes amino acids within a spatial conformation that is unique to the three-dimensional conformation of a folded polypeptide. Generally, conformational epitopes consist of amino acids that are discontinuous in the linear sequence but are integrated within the folding structure of the protein. However, conformational epitopes can also consist of a linear sequence of amino acids that adopt a conformation (and do not exist in the denatured state) that is unique to the three-dimensional conformation of the folded polypeptide. In a protein complex, conformational epitopes consist of amino acids that are discontinuous in the linear sequences of one or more polypeptides but become integrated when the different polypeptides that fold associate in their native quaternary structure. Similarly, conformational epitopes can also consist of a linear sequence of amino acids of one or more polypeptides that become integrated and adopt a conformation that is unique to the quaternary structure. The terms "conformation" or "conformational state" of a protein generally refer to a range of structures that a protein can adopt at any given instant. Those skilled in the art will recognize that the determinants of conformation or conformational state include the primary structure of the protein, which is reflected in the amino acid sequence (including modified amino acids) of the protein, and the environment surrounding the protein.The conformation or conformational state of a protein also relates to the secondary structure of the protein (e.g., among others, α - helix, β - sheet), the tertiary structure (e.g., the three - dimensional folding of the polypeptide chain), and the quaternary structure (e.g., the interaction of the polypeptide chain with other protein subunits), such as structural features. In particular, post - translational modifications and other modifications to the polypeptide chain, such as the binding of a ligand, phosphorylation, sulfation, glycosylation, ubiquitination, etc., or the attachment of hydrophobic groups, can affect the conformation of the protein. Further, environmental factors or conditions, such as, among others, the temperature, pH value, salt concentration, ionic strength, and osmotic pressure of the surrounding solution, as well as the interaction with other proteins and cofactors, can influence the conformation and binding properties of the protein. The conformational state of a protein may be determined by functional assays for activity or binding to another molecule, among other methods, and may also be determined by physical methods, such as X - ray crystallography, NMR, or spin labeling. For a general discussion of the conformation and conformational state of proteins, see Cantor and Schimmel, "Biophysical Chemistry", Part I: "The Conformation of Biological Macromolecules", W.H.Freeman and Company, 1980; and Creighton, "Proteins: Structures and Molecular Properties", W.H.Freeman and Company, 1993.

[0032] As used herein, the terms "antibody," "antibody fragment," and "active antibody fragment" refer to a protein containing an immunoglobulin (Ig) domain or antigen-binding domain that is capable of specifically binding to an antigen or target protein epitope. An "antibody" may further be an intact immunoglobulin derived from a natural or recombinant source and may be the immunoreactive portion of an intact immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. The term "active antibody fragment" refers to any portion of an antibody or antibody-like structure that, by itself, has a high affinity for an antigenic determinant or epitope and contains one or more CDRs that contribute to such specificity. Non-limiting examples include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy chain-light chain dimers, immunoglobulin single variable domains (ISVDs), nanobodies, domain antibodies, and single-chain structures such as full light or full heavy chains. A further requirement for "activity" of a fragment in the context of the present invention is that the fragment be capable of specifically binding to the target epitope. The term "immunoglobulin (Ig) domain," or more specifically, "immunoglobulin variable domain" (abbreviated "IVD"), as used in the art and hereinafter in this specification, refers to an immunoglobulin domain consisting essentially of four "framework regions" designated, respectively, "framework region 1" or "FR1"; "framework region 2" or "FR2"; "framework region 3" or "FR3" and "framework region 4" or "FR4," and interrupted by three "complementary determining regions" or "CDRs" designated, respectively, "complementary determining region 1" or "CDR1"; "complementary determining region 2" or "CDR2" and "complementary determining region 3" or "CDR3" in the art and hereinafter in this specification. Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable domain (IVD) that confers specificity for an antigen to an antibody by possessing an antigen-binding site.Typically, in conventional immunoglobulins such as monoclonal antibodies, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form an antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both VH and VL contribute to the antigen-binding site, i.e., a total of six CDRs are involved in the formation of the antigen-binding site. Keeping the above definition in mind, the antigen-binding domains of conventional four-chain antibodies (such as IgG, IgM, IgA, IgD, or IgE molecules known in the art) or Fv fragments, such as Fab fragments, F(ab’)2 fragments, disulfide-linked Fv fragments, scFv fragments, or diabodies (all known in the art) derived from such conventional four-chain antibodies, bind to the respective epitopes of an antigen by pairs of (associated) immunoglobulin domains, i.e., the VH-VL pair of immunoglobulin domains, which together bind to the respective epitopes of the antigen. As used herein, an immunoglobulin single variable domain (ISVD) refers to an amino acid sequence containing four framework regions (FRs) and three complementarity-determining regions (CDRs) according to the format of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, containing a protein domain sufficient for binding to an antigen or epitope, and thus, a protein with an amino acid sequence that requires only three CDR loop regions for interaction with its target epitope. The "active fragment" of an ISVD described herein is defined as a portion of the ISVD that is sufficient to specifically bind to an epitope in the same or a similar form as the ISVD from which the fragment is derived. The "immunoglobulin domain" of the present invention also refers to an "immunoglobulin single variable domain" (abbreviated as "ISVD"), which is synonymous with the terms "single variable domain" and "single-domain antibody", and defines a molecule in which the antigen-binding site is present on a single immunoglobulin domain and is formed thereby. This defines the immunoglobulin single variable domain separately from "conventional" immunoglobulins or fragments thereof in which two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site.The binding site of an immunoglobulin single variable domain is formed by a single VH / VHH domain or VL domain. Thus, the antigen-binding site of an immunoglobulin single variable domain is formed by three or fewer CDRs. Thus, a single variable domain can form a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, such that a single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit), and can be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof, or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof, as long as it is capable of forming such a unit.

[0033] In particular, an immunoglobulin single variable domain can be a nanobody (as defined herein) or a suitable fragment thereof. Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V. (Sanofi Company). For a general description of nanobodies, reference is made to the further description below and to the prior art cited herein, such as the prior art described in, for example, WO2008 / 020079. The "VHH domain", also known as VHH, VHH domain, VHH antibody fragment and VHH antibody, was originally described as the antigen-binding immunoglobulin (Ig) (variable) domain of "heavy chain antibodies" (i.e., "antibodies lacking a light chain"; Hamers-Casterman et al. (1993), Nature, 363:446-448). The term "VHH domain" has been chosen to distinguish these variable domains from the heavy chain variable domain (referred to herein as the "VH domain") present in conventional four-chain antibodies and the light chain variable domain (referred to herein as the "VL domain") present in conventional four-chain antibodies.For further description of VHHs and nanobodies, see the review article by Muyldermans (Review in Molecular Biotechnology, 74:277-302, 2001) and the following patent applications which are mentioned as general background art: WO94 / 04678, WO95 / 04079 and WO96 / 34103 by Vrije Universiteit Brussel; WO94 / 25591, WO99 / 37681, WO00 / 40968, WO00 / 43507, WO00 / 65057, WO01 / 40310, WO01 / 44301, EP 1134231 and WO02 / 48193 by Unilever; WO97 / 49805, WO01 / 21817, WO03 / 035694, WO03 / 054016 and WO03 / 055527 by Vlaams Instituut voor Biotechnologie (VIB); WO03 / 050531 by Algonomics N.V. and Ablynx N.V.; WO01 / 90190 by National Research Council of Canada; WO03 / 025020 (= EP 1433793) by Institute of Antibodies; WO04 / 041867, WO04 / 041862, WO04 / 041865, WO04 / 041863, WO04 / 062551, WO05 / 044858, WO06 / 40153, WO06 / 079372, WO06 / 122786, WO06 / 122787 and WO06 / 122825 by Ablynx N.V. and further patent application publications by Ablynx N.V. are referred to. As described in these references, nanobodies (in particular, VHH sequences and partially humanized nanobodies) can be characterized, in particular, by the presence of one or more "hallmark residues" in one or more of the framework sequences. Further description of humanization and / or camelization and other modifications, parts or fragments, derivatives or "nanobody fusions", multivalent constructs or bispecific constructs (including non-limiting examples of linker sequences) of nanobodies and different modifications for extending the half-life of nanobodies and preparations thereof can be found, for example, in WO08 / 101985 and WO08 / 142164.Nanobodies form the smallest antigen-binding fragments that fully retain the binding affinity and binding specificity of full-length antibodies. Nbs possess an exceptionally long complementarity-determining region 3 (CDR3) loop and a convex paratope that enables them to penetrate into hidden cavities of the target antigen. Immunoglobulin single variable domains, such as domain antibodies and Nanobodies® (including VHH domains), can be humanized, i.e., the degree of sequence identity with the most closely related human germline sequences can be increased. In particular, humanized immunoglobulin single variable domains, such as Nanobodies® (including VHH domains), are those that are humanized substitutions (as further defined herein) and / or corresponding to which there are at least one amino acid residue (and in particular at least one framework residue). Furthermore, additional suitable mutations, in particular substitutions, may be introduced, for example, at least at one of positions: 11, 13, 14, 15, 40, 41, 42, 82, 82a, 82b, 83, 84, 85, 87, 88, 89, 103 or 108, so as to create polypeptides with reduced binding to existing antibodies present in human or animal cells (see, for example, WO2012 / 175741 and WO2015 / 173325). The amino acid sequences and / or VHHs of the present invention can be suitably humanized at any framework residue, such as one or more hallmark residues (defined herein) or one or more other framework residues (i.e., non-hallmark residues) or any suitable combination thereof. Depending on the host organism used to express the amino acid sequences, VHHs or polypeptides of the present invention, such deletions and / or substitutions can also be designed in such a way that one or more sites for post-translational modifications (such as one or more glycosylation sites) are removed, as is within the ability of the person skilled in the art. Alternatively, substitutions or insertions can be designed so as to introduce one or more sites (described herein) for the conjugation of functional groups that enable the conjugation of labels, such as site-specific PEGylation or biotinylation or fluorophores, for example.

[0034] As used herein, the terms "determining", "measuring", "assessing", "identifying", "screening for", and "assaying for" are used interchangeably and include both quantitative and qualitative determinations.

[0035] DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the purification of proteins by affinity chromatography. In particular, pairs of target-specific protein binders that specifically and competitively bind to an epitope on a target are used in the context of complementary reaction kinetics. Such pairs of binders that are competitive in their binding to the target but have binding sites at non-overlapping or different epitopes are seen to act via a transient sandwich complex within defined dose-response and reaction rate relationships in affinity displacement. However, whether cross-blocking or displacement can occur when pairs of a first protein binder (capture agent) having the same or overlapping epitopes as a second binder are used depends on the nature of the binding. Using ISVD, or more specifically Nb as a displacer, the inventors have found that even when pairs targeting the same epitope on a target protein are combined under certain reaction rate requirements, and more specifically when the dissociation rate constant is higher for the first protein binder than for the second protein binder, and when the second protein binder, known as a displacing or stripping agent, includes ISVD-specific or nanobody-specific binding, displacement is efficiently established. Thus, for the ISVD binder used as a displacer, k off is thought to drive the displacement efficiency. This purification technique has a high off-rate (or k offA immobilized protein binder (capture agent) with low affinity and / or slow dissociation rate is used, such that the second protein binder has been shown to function optimally when it has a low off-rate and / or high affinity for the target protein. In fact, in the art, it is known that antibodies or antibody domains, including ISVDs and nanobodies, compete for binding to their targets when they interact with similar or overlapping epitopes. Purely based on the competitive nature, it is expected that competition will enable obtaining a satisfactory yield of purified protein in the elution fraction, and thus the same binder, such as a nanobody, can be used for binding (or capture) and elution (or stripping) to purify the target. Therefore, a binder with a dissociation rate enabling such "equivalent" competition (i.e., not too low k off or not too high affinity; see below) results in a certain amount of protein eluted using the same binder or Nb as both the capture agent and the elution agent. However, in this case, the purification does not yield optimal results because the equally competing capture agent retains some of the protein bound to the immobilized surface and the elution yield is sub-optimal. Those skilled in the art seeking a binder capable of completely abolishing the binding of the capture agent to the target by competition, as desired in high-throughput applications, have found that most conventional antibody binders to the same epitope block the substitution reaction (e.g., as demonstrated for monoclonal antibodies in Abdiche et al., 2017), and rather, protein binders such as antibodies that bind to adjacent or minimally overlapping epitopes compared to the capture agent are used to avoid such blocking of the epitope by competition. The use of an ISVD that binds to the same epitope, substantially the same epitope, or a large overlapping epitope as the first protein binder as the second protein binder, where the second protein binder comprising the ISVD has a low dissociation rate constant (k off) having, the use shows that ISVD can efficiently replace the first protein binder, thereby competitively eliminating the binding to the epitope of the same target protein and enabling the elution of the target protein in high yield. Thus, the present invention results in a highly pure elution (shown in the examples) of the protein complex of the second protein binder containing ISVD with the target, "ISVD-based substitution", or more specifically, "nanobody exchange" or "nanobody exchange chromatography" or "NANEX", which are used interchangeably herein. Even when a binding pair is used for substitution on a target where the epitopes do not overlap or overlap only in a minimal part, the binding property of ISVD, which is a substitution reaction, is considered to function according to the substitution reaction rate driven by the difference in k off between the capture agent and the eluent.

[0036] When using purification by NANEX, the eluted complex thus contains a stripping agent or a substitution agent, which has the advantage of allowing the application of a second protein binder containing ISVD (a stripping agent or, in the case of a nanobody, called a nano-stripping agent), which is further functionalized, i.e., provides a specific function to the eluted protein complex. Such functionalization may relate to the visualization of the protein complex (via fluorescence or drug labeling), and in other examples, may relate to the function as a chaperone protein or an adapter protein (including, but not limited to, MegaBody) that elutes the target in the functionalized complex. Furthermore, following the elution step and the regeneration procedure, an affinity matrix, which can be any type of surface such as beads, columns or resins, is prepared for the next affinity purification cycle and can be used in high-throughput platforms such as screening platforms, chips or microfluidic setups or microfluidic devices.

[0037] By using this next-generation affinity purification technology, called NANEX or nanobody exchange chromatography, a leap in high-throughput platforms or screening applications can be envisioned, such as purification for analysis and screening assays, structure-based drug design, discovery of new compounds, and structure-based screening. In fact, for stabilizing a target in a functional conformation, such as an active conformation, a protein binder with conformational selective recognition of an antigen or target can be selected, more specifically, a conformation of an agonist, partial agonist, or biased agonist can be selected. Due to the rapid progress of such technologies in biotechnology, the present invention is expected to affect the efficiency and potential of new therapeutic drug screening, as well as the increase in throughput and the potential of proteomics, MS-based analysis methods, and other analysis methods.

[0038] A first aspect is a purification process for a target protein present in a sample, a) mixing a first protein binder that specifically binds to an epitope on the target protein with a sample containing the target protein; b) adding a second protein binder that competes with the binding by the first binder for binding to the target protein; and c) eluting a protein complex containing the target protein and the second protein binder, comprising When the target protein is exchanged or replaced from the first protein binder to the second protein binder, the eluted protein complex contains the target protein and the second protein binder, and the second protein binder contains an ISVD or a functional variant thereof that specifically binds to the epitope of the target protein, and the dissociation rate (or "dissociation rate constant" or "k off ") for the second protein binder is slow or equal compared to k off of the first protein binder (or k offis the k of the first protein binder off as follows) Regarding the step. In further applications, the method may also include a washing step before the addition of the second protein binder.

[0039] The term "functional variant" of ISVD is defined herein, for binding to a target protein, as any polypeptide that may differ in its sequence or composition but retains its functionality in binding to the target protein by the same binding region as ISVD, i.e., containing a binding region or paratope identical to the binding region or paratope of ISVD. In particular, this paratope or binding region of ISVD very often includes at least the CDR3 region, preferably the three CDRs and, optionally, also a part of the FR region.

[0040] The feature of "competing for binding to the target" may be interpreted as competing for the same epitope, or may mean competing in a different manner, such as kinetically or allosterically. Thus, in one embodiment, the stripping agent may compete for binding by targeting overlapping or adjacent epitopes in the minimal portion, or alternatively, the stripping agent may still bind to an allosteric site on the target and disrupt the interaction between the capture agent and the target by inducing a conformational change in the target.

[0041] Competitive binders can be established using several methods known in the art, such as, but not limited to, competitive ELISA, alphalisa, measurements by Octet or Biolayer Interferometry (BLI), SPR Biacore, Microscale Thermophoresis (MST).

[0042] More specifically, in order to obtain efficient substitution in the purification method, for example, by binding to adjacent epitopes or by binding to allosteric sites that induce conformational changes that enhance substitution, through binding to overlapping or non-overlapping epitopes in the minimal portion, compared to competing binding pairs for the target, differences in requirements can be considered for competing binding pairs for the target through binding to the same or overlapping epitopes in the majority. Substitution reactions using a first type of pair where the ISVD-containing releasing agent binds to similar epitopes are driven by differences in k off and thus also often result in a releasing agent with a higher affinity than the capture agent, requiring a displacing agent with a lower k off value compared to the capture agent. Thus, in this specification, substitution is not driven by the association rate constant.

[0043] Substitution reactions using a second type of pair where the ISVD-containing releasing agent binds to different or overlapping epitopes in the minimal portion are also driven by differences in k off or affinity, but allow for milder differences, for example, it has been shown to be driven by a difference of only 2-fold to allow substitution. In a specific embodiment, the binding agent is not a monoclonal antibody or a conventional antibody as it requires different substitution reaction rates.

[0044] In the purification method, as used herein, a binder having the "same" epitope is defined as a binder in which the amino acid residues of the target protein that interact with the binder are identical, where "interact with" or "contact with" the binder is described as being within less than 3 Å proximity from the residue (or atom) at the time of binding of the binder to the target protein at the epitope. The terms "substantially the same" epitope or "largely overlapping" epitope as described herein mean that the number of identical amino acid residues in both epitopes is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of the total or maximum number of amino acid residues of the epitope. Most preferably, a "substantially the same" epitope means that the number of identical amino acid residues in both epitopes is at least 85 - 99% of the maximum number of amino acid residues of the epitope. Most preferably, a "largely overlapping" epitope means that the number of identical amino acid residues in both epitopes is at least 50 - 84% of the maximum number of amino acid residues of the epitope. Further, the purification method provides optimal results when the first binder or capturer has a high dissociation rate or a low or equal affinity compared to the second binder and vice versa, i.e., when the second binder has a lower dissociation rate and / or the same or higher affinity for the epitope compared to the first binder. From the latest knowledge in the art, the dissociation rate constant (or off-rate or k off ) and the association rate constant (or on-rate or k on ) are related to each other as K D =k off / k on [wherein K D is defined as the dissociation constant, which is inversely correlated with the affinity of the binder for its target, as also described in detail in the above definition]. Thus, when the dissociation constant K D value is low, the affinity is high (when k on is the same). Alternatively, kon is high, K D is low, and the affinity is high (k off is the same).

[0045] Thus, for the purification method of the present invention, the protein binders described herein have a k off and / or an affinity (or K D ) for the same epitope, substantially the same epitope, or an epitope that overlaps in most parts, which are relatively different. The methods described herein more specifically refer to the k off of a second binder for the same epitope, substantially the same epitope, or an epitope that overlaps in most parts of the target protein being lower than the k off of the first binder. As used herein, "low" refers to a value that is at least 1 / 2, 1 / 5, or 1 / 10, or at least 1 / 30, or at least 1 / 100, or at least 1 / 200, at least 1 / 300, at least 1 / 400, or at least 1 / 500. More preferably, the k off value of the second binder is within the range of at least 1 / 2 to at least 1 / 10, or at least 1 / 5 to at least 1 / 20, or at least 1 / 10 to at least 1 / 30, or at least 1 / 100 compared to the k off value for the first protein binder.

[0046] Similarly, the affinity of the second binder for the epitope of the target protein can be equal to or higher than the affinity of the first binder. In this case, "high affinity" means that the "K D value" of the second protein binder is at least 1 / 2, or at least 1 / 5, 1 / 10, 1 / 20, or 1 / 100, or within the range of at least 1 / 2 to at least 1 / 2000 compared to the K D value for the first protein binder, referring to the K D value.

[0047] In a preferred embodiment, the purification method described herein has a K value in the range of 1 mM to about 1 nanomolar for an epitope of a target protein D and discloses a first binder, and optionally a second protein binder having a K value of 1 nanomolar or less, optionally down to 1 picomolar, for an epitope that is substantially the same or overlapping in most of the target D . More preferably, the first binder has a K value in the range of nanomolar to micromolar (i.e., 10 -9 to 10 -3 ), and the second binder has a K value in the range of femtomolar to micromolar (i.e., 10 D -12 to 10 -6 ), and most preferably, the relative difference between the first binder and the second binder is at least 2-fold. In one embodiment, the K value for the first protein binder is at least 2-fold that of the K value of the displacer, which is driven, inter alia, by the difference in k values when the displacer binds to the same or overlapping epitopes in most D D . This is a difference D off .

[0048] ​​​The method of the present invention comprises a second protein binder that is in solution and is soluble under elution conditions. The elution conditions preferably relate to physiological conditions known to those skilled in the art. As used herein, the term "soluble" refers to the fact that the protein binder is in a functional form and means that the protein binder can specifically bind to its target within the range predicted for its affinity for the epitope. The purification method may include a first protein binder that is present as a free protein binder, a labeled protein binder, or a covalently bound protein binder in a solute for mixing with a sample of interest. The first binder may be coupled to beads, such as agarose beads or magnetic beads, and more specifically, may be an affinity column that may be suitable for the purification scale for preparation and analysis, more specifically, a microcolumn on the order of a column volume of less than 1 mL or a volume of less than micromolar, and may be present on the surface or matrix packed as an affinity column, and may also be presented on a chip using microfluidic technology. The first protein binder is preferably immobilized for the method of the present invention, and in this case, the first protein binder can be immobilized on the surface via covalent means or other coupling means. Most preferably, the first protein binder is immobilized on a solid support or resin. As used interchangeably herein, "resin" or "affinity resin" is an activated affinity chromatography support for the immobilization of biomolecules, such as ISVD or other protein binders. In a specific embodiment, the first protein binder comprises ISVD and is coupled to the resin using coupling methods known in the art (see examples).

[0049] The purification method described herein optionally includes the step of mixing an immobilized first protein binder with the sample in step a), where the sample contains a target protein that specifically binds to the first protein binder. In a specific embodiment, the sample can relate to a biological sample, a biopsy sample, a cell-containing sample or a tissue-containing sample, a cell lysate, a cell mixture or a complex mixture, a solvent or lysate containing non-specific components. Other embodiments relate to a sample containing a synthetic compound or a non-natural compound, a complex sample or other in vitro samples.

[0050] Depending on the nature of the sample, the optional column washing after step a) of the method of the present invention may require neutral conditions or extremely mild conditions or, conversely, harsh conditions, and may require repetitions to remove any abundant components that did not bind. Depending on the nature of the sample and the amount of target protein present in the sample and the first protein binder according to step a) or b) of the method of the present invention, the elution in step c) using a second protein binder may be repeated and may require a large volume of elution to enable complete elution of the target protein. Since the method presented herein uses physiological conditions during elution and optionally includes a mild regeneration step, the affinity column (i.e., the first protein binder immobilized on the resin) has the advantage of being reusable for further purification from further samples.

[0051] If the eluate of step c) of the method of the present invention requires a higher purity than the eluate obtained after the purification step of the method described herein, a further purification step is preferred, in which case both bind to the epitope of the same target protein and the epitope does not overlap, is adjacent to, or is different from the epitope to which the first protein binder and the second protein binder bind, and the same steps of the purification method described herein are repeated using a third protein binder and a fourth protein binder. The method for tandem purification of a target protein is a) Mixing a first protein binder that specifically binds to an epitope of a target protein with a sample containing the target protein; b) Adding a second protein binder that competes with the first binder for specifically binding to the target protein to displace the first binder from the target protein; c) Recovering an elution complex containing the second protein binder bound to the target protein; and d) Repeating steps a) - c) using a third protein binder and a fourth protein binder instead of the first protein binder and the second protein binder, respectively, wherein the third binder and the fourth binder specifically bind to an epitope of the target protein that is different compared to the epitopes for the first binder and the second binder. comprising wherein the second protein binder (and / or the fourth protein binder) comprises an immunoglobulin single variable domain (ISVD) that specifically binds to the epitope or an active fragment thereof, and the dissociation rate constants (k off values) of the second protein binder and the fourth protein binder are lower than or equal to the k off values of the first protein binder and the third protein binder, respectively.

[0052] Indeed, such tandem NANEX affinity purification methods are also suitable for purifying protein complexes containing more than one target protein, as is classical tandem affinity purification (TAP) using a TAP tag. Indeed, the epitopes recognized by the third and fourth protein binders can also be present on a target protein different from the target protein that binds to the first and second binders, which enables the capture and purification of the complex formed between the first target protein and the second target protein, and is also an epitope for purifying such protein-protein interactions from a highly complex matrix when a one-step purification step is not sufficient. The advantage of this tandem affinity purification method over the TAP method known in the art is that the purification does not require enzymatic cleavage (of the tag) and no protease remains in the eluate. Further advantages of this method include the absence of a concentration step or the need for dialysis against certain buffer conditions, and the fact that an excess of the second protein binder or eluent Nb can be removed in the second step of the tandem method. Alternatively, a tandem NANEX can also be envisioned, where the first and third binders are both coupled on the same column or resin, and the second and fourth binders are used simultaneously or subsequently as eluents, optionally allowing for an intervening washing step, to perform the tandem as a type of multiplex reaction.

[0053] Finally, the purification methods described herein preferably apply a capture agent / stripping agent pair, in which case any of the protein binders are of this type of protein binder, which are highly specific, have the advantageous properties of good expression in E. coli / Pichia, high (thermal) stability, allow for selection regarding salt / pH tolerance of binding affinity, and all bind to their targets via a single variable domain as described herein, and for this class of ISVD-containing stripping agents, apply a similar substitution reaction rate, so that they include an ISVD or more specifically, a VHH, or even more specifically, a nanobody.

[0054] Another embodiment relates to a method of purifying a target protein disclosed herein, wherein the second (or fourth) protein binder comprises a label or a detectable label. The term "detectable label" or "labeling" refers to a detectable label or detectable tag that enables the detection, visualization and / or isolation, further purification and / or immobilization of an isolated or purified (poly)peptide or complex as described herein, and is intended to include any label / tag known in the art for these purposes. In particular, fluorescent labels or fluorescent tags (i.e., fluorescent dyes / fluorophores) such as fluorescent proteins (e.g., GFP, YFP, RFP, etc.) and fluorescent dyes (e.g., FITC, TRITC, coumarin and cyanine); luminescent labels or luminescent tags such as luciferase; and (other) enzyme labels (e.g., peroxidase, alkaline phosphatase, beta-galactosidase, urease or glucose oxidase) are preferred. Also included are affinity tags such as chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), poly(His) (e.g., 6×His or His6), Strep-tag®, Strep-tag II® and Twin-Strep-tag®; solubilizing tags such as thioredoxin (TRX), poly(NANP) and SUMO; chromatography tags such as FLAG tag; and epitope tags such as V5 tag, myc tag and HA tag. Also included are any combinations of the aforementioned labels or tags. The second protein binder (or fourth protein binder) may, for example, be fused or conjugated to a half-life extension module or may function as a half-life extension module itself. Such modules are known to those skilled in the art and include, for example, albumin, albumin-binding domain, Fc region / domain of immunoglobulin, immunoglobulin-binding domain, FcRn-binding motif and polymers. In particular, preferred polymers include polyethylene glycol (PEG), hydroxyethyl starch (HES), hyaluronic acid, polysialic acid and PEG-mimetic peptide sequences.Modifications that prevent aggregation of isolated (poly)peptides are also known to those skilled in the art and include, for example, substitution of one or more hydrophobic amino acids, preferably surface-exposed hydrophobic amino acids, with one or more hydrophilic amino acids.

[0055] In further embodiments, protein binders that specifically bind to epitopes on different types of target proteins, which may or may not include tags, such as those present on fusion proteins, are described. Examples of tags herein include, but are not limited to, affinity tags such as commonly used polyhistidine (His), glutathione transferase (GST), maltose binding protein (MBP), calmodulin binding peptide (CBP), intein-chitin binding domain (intein-CBD), streptavidin / biotin-based tags, His-Patch Thio Fusion (thioredoxin-based), EPEA (CaptureSelect C-tag; US9518084B2), ubiquitin or SUMO (small ubiquitin-like modifier), yeast SUMO or SMT3 or Halo tag. Additionally, tags may constitute epitope tags such as HA, FLAG or cMyc or the latter, while not as preferred for affinity purification, may constitute reporter tags such as HRP, or alkaline phosphatase. Numerous non-limiting examples are presented, for example, in Kimple et al. (2015, Table 9.9.1).

[0056] Alternatively, the protein binders for the purification methods described herein specifically bind to epitopes of native proteins, naturally occurring proteins, and / or endogenous proteins and do not require fusion to a tag. Another alternative protein binder is a protein binder where the epitope is present on a recombinantly produced exogenous protein and does not require a tag. Such pairs of protein binders can also be screened and selected to result in pairs of competitive binders such that they compete for the same target, and can also be designed for high affinity / low affinity pairs of protein binders. Indeed, the use of the 3D structure of a displacer or a second protein binder bound to the target can result in mutations within the binding site of the protein binder that result in a conforming capturer or a first protein binder. Additionally, a simpler method that does not require structural information can also make it possible to determine pairs based on a single binder if the sequence is known. In the examples, for the mCherry target protein, as shown in a non-limiting form, based on screening for different binders, their competitiveness is analyzed by epitope mapping using BLI or alternatively, within the CDR3 region, which is known to be most important in defining the binding reaction rate based on the sequence of the nanomolar concentration binder or displacer, a new pair with a low dissociation rate constant was identified to function as a capturer in the NANEX method. Thus, simply by introducing one or more mutations, pairs of protein binders that bind to the same epitope with different k off or affinity can be obtained. off

[0057] ​In a specific embodiment of a method involving an ISVD, not only the second protein binder, but also the first protein binder (and optionally also the third and fourth protein binders), the "monovalent" format may be used as a capture agent (the first binder or the third binder), and the multivalent format has a higher avidity compared to the monovalent form and results in an optimal elution yield and target protein purity (see Example 12) with a high k off and thus the "multivalent" format can be used in combination as an eluent (the second binder or the fourth binder). The term "monovalent format" as used herein refers to an ISVD used herein that can recognize only one antigenic determinant, whereas the term "multivalent" format refers to an ISVD used herein that can recognize more than one antigenic determinant, such as but not limited to a bivalent, trivalent or tetravalent format. Furthermore, instead of a multivalent eluent, the eluent may include the binding of at least one or more components that may be identical and bind to the same antigenic determinant, or different and bind to the same or another epitope on the target protein, or alternatively bind to an epitope on another target protein complexed with the first target protein. A multiparatope or multispecific eluent is also envisioned.

[0058] In a further embodiment, a method for purifying a target protein applies a MegaBody as a first protein binder and / or a second protein binder. As used herein, the term MegaBody refers to a novel fusion protein disclosed in Steyaert et al. (WO2019 / 086548A1), also referred to herein as an antigen-binding chimeric protein, which is coupled to an antigen-binding domain at one or more amino acid sites that are accessible or exposed on the surface of the domain and connected to a scaffold protein that results in disruption of the topology of the antigen-binding domain, and refers to a fusion protein comprising an antigen-binding domain. The antigen-binding chimeric protein is further characterized in that it retains its antigen-binding functionality compared to an antigen-binding domain that is not fused to a scaffold protein. The MegaBody described herein is related to a specific MegaBody or antigen-binding chimeric protein in which the antigen-binding domain is fused or connected to a scaffold protein at an accessible surface (β-turn or loop excluding CDR) of the ISVD domain, resulting in disruption of the topology of the antigen-binding domain and retaining its antigen-binding functionality, i.e., an immunoglobulin single variable domain (ISVD) or nanobody that retains specific epitope recognition. In a specific embodiment, the second protein binder relates to a MegaBody or antigen-binding chimeric protein comprising an ISVD connected to a scaffold protein via an insertion of the scaffold protein in the first β-turn that connects β-strand A to B of the ISVD (defined according to the IMGT terminology and as defined in WO2019 / 086548A1). In an even more specific embodiment, the scaffold protein used herein is a HopQ scaffold protein or a Ygjk scaffold protein in which the fusion of the scaffold disrupts the topology of the ISVD but does not disrupt its overall 3D structure or its epitope-binding specificity.As used herein, the "HopQ" scaffold or "HopQ-derived" scaffold also relates to the protein scaffold of the adhesin domain of type 1 HopQ (Protein Database: PDB 5LP2) of Helicobacter pylori strain G27, also called cHopQ or c7HopQ (see also WO2019 / 086548A1), or its circularly permuted protein. As used herein, the "Ygjk" scaffold or "Ygjk-derived" scaffold also relates to the protein scaffold of Escherichia coli K12 YgjK (PDB 3W7S) or its circularly permuted gene encoding the protein, also called cYgjk (see also WO2019 / 086548A1).

[0059] Certain embodiments relate to a method in which the second protein binder is a MegaBody that specifically binds to an epitope of a target protein via its antigen-binding domain of the ISVD, resulting in elution of the target protein bound to the MegaBody. As disclosed in Steyaert et al. (WO2019 / 086548A1) and Laverty et al. (2019), Masiulis et al. (2019) and Uchanski et al. (2019), these exemplary MegaBodies act as a new class of nanobody-based chaperones for improving structural resolution in cryo-EM analysis. Thus, the purification method applying the MegaBodies described herein as the second protein binder or the fourth protein binder, i.e., the releasing agent, is advantageous for the concise preparation and purification of complex samples for cryo-EM analysis or other structural analysis. The k of the ISVD contained in the MegaBody off constitutes another protein binder that binds to the same epitope, substantially the same epitope or an epitope that overlaps mostly, and is lower than the k of the first protein binder. off Lower.

[0060] In fact, a specific embodiment is a method for purifying a target protein, comprising: a) mixing a first protein binder comprising an ISVD that specifically binds to an epitope of the target protein with a sample containing the target protein, and optionally washing the mixture of step a) to remove unbound sample components; and b) adding a second protein binder that recognizes an epitope of the target protein that is the same as or substantially overlapping with the first binder and specifically binds to the target protein to dissociate the first binder from the target protein; and c) recovering an eluate containing the target protein bound to the second protein binder, wherein the second protein binder comprises an ISVD that specifically binds to the same epitope, substantially the same epitope or a substantially overlapping epitope as the first protein binder, and comprises a MegaBody described herein, and the dissociation rate constant (k off value) is low and its affinity is equal to or higher than that of the binder containing the first ISVD. In a specific embodiment, the ISVD of the first binder is a mutant ISVD of the ISVD contained within the MegaBody. The purification method ultimately provides single-step purification from complex cell samples or small sample mixtures, for example, for use in structural biology and physicochemical characterization or analytical studies.

[0061] Another embodiment relates to a method for purifying a target protein described herein, wherein the epitopes recognized by the first protein binder and the second protein binder are protein-binding sites or epitopes present on the scaffold protein contained within the MegaBody. The MegaBody can preferably be constructed using a scaffold protein derived from the HopQ protein or the Ygjk protein, thus using a HopQ protein scaffold or a Ygjk protein scaffold containing an epitope that specifically binds to the protein binder of the method. A pair of protein binders that specifically bind to the scaffold protein epitopes present on the MegaBody disclosed herein or disclosed in Steyaert et al. (WO2019 / 086548A1) or described elsewhere has the further advantage that the purification method can be applied to capture or scavenge the target protein bound to the MegaBody from a complex mixture.

[0062] Furthermore, when a protein binder that specifically binds to an epitope of the HopQ scaffold protein or the Ygjk scaffold protein is fused to another protein or binds to HopQ and Ygjk in addition to the MegaBody fusion via other fusion formats, a pair of HopQ-specific protein binders and / or Ygjk-specific protein binders can be used in a method for further applications that requires the purification of HopQ-based fusion proteins or Ygjk-based fusion proteins in a similar manner as for the tags discussed herein.

[0063] In addition, the methods presented herein further enable, for example, on a microfluidic chip, miniaturization of the method to allow for applications in mass spectrometry and structural analysis for HTP analysis, a complete toolbox for the analysis of endogenous (often present in small amounts) and difficult-to-isolate proteins, as well as an analysis tool for tagged proteins, which constitutes another aspect of the present invention for the binder pair itself. Thus, a second aspect of the present invention relates to a kit for purifying a target protein, comprising a first protein binder and a second protein binder, wherein the second protein binder comprises ISVD or a functional variant thereof, the dissociation rate constant of the second protein binder is equal to or lower compared to the first binder, and which competes for the target protein. The kit may further comprise a buffer or resin for solubilizing, washing or eluting. Further, instructions or protocols for performing the purification method may also be provided in the kit. The first protein binder and the second protein binder of the kit relate to protein binders, wherein the second protein binder is defined as a protein binder with a low dissociation rate or, alternatively, with high affinity. If more than two protein binders are present in the kit, these multiple protein binders may specifically bind to the same epitope, substantially the same epitope or mostly overlapping epitopes or different epitopes, and / or, in particular, may differ in format (format including ISVD and / or its functional variant) and antibody, peptide between the first binder / the third binder. Alternatively, a kit comprising multiple protein binders for the purification method of a target protein described herein may comprise protein binders that bind to different epitopes or different proteins (e.g., of a protein complex) so as to enable the purification of a mixture of proteins or protein complexes. However, the multiple protein binders shall be present at least in the pairs described herein so as to enable their use in the purification methods or tandem purification methods described herein.In another embodiment, a kit comprising a binder can include a first binder or capture agent in an immobilized format that is present on a solid structure such as beads, on a resin, or within a chip.

[0064] In a specific embodiment, the kit includes a first protein binder and a second protein binder for use in a method for purifying a GFP-tagged protein, where the protein binder is selected from the group of SEQ ID NOs: 1-6, 18, or 19 (or any of these sequences without the his-EPEA tag) or a sequence with a homologous amino acid sequence having at least 70% or at least 80% or at least 90% or at least 95% or at least 99% identity thereto, and when the KD of the ISVD is below 0.1 nM, or more specifically, when SEQ ID NO: 1 is selected, the first binder and the second binder must not be identical within the selected sequence. Similarly, the kit can include other protein binders for use in the methods described herein, where the agent specifically recognizes commercially available tags for proteins such as GST, EPEA, mCherry, ubiquitin, SMT3, or other tags exemplified and described herein.

[0065] Another embodiment relates to the use of a kit for the methods described herein for purposes of purification or analysis and, for example, for use in a screening assay where the binder is screened for a pharmaceutically viable conformation.

[0066] A further aspect of the present invention relates to a protein complex comprising a second (or fourth) protein binder of a purification method, bound to a target protein. The protein complex is prepared in the elution step c) (or iteration of c) in step d)) of the method described herein for preparing a sample containing the protein complex of interest for further analysis. Indeed, the (second or fourth) protein binder complexed and eluted with the target protein can also provide a stabilizing chaperone required for high-resolution structural analysis and / or can provide a stabilizing effect on a particular target protein conformation and / or can provide an increase in protein mass required for imaging by atomic-resolution cryo-EM microscopy, for example, can provide a visually detectable complex when a labeled protein binder is used, and can provide alternative applications such as mass spectrometry, but is not limited to the examples presented herein. The elution fraction in step c) (or iteration of c) in step d)) of the method described herein yields at least a protein complex, but may also contain additional buffer components, residual impurities and / or components added to the eluate to provide analytical conditions suitable for the protein complex. Furthermore, the complex comprising the target protein of interest and the substitute may also contain additional proteins bound to the target protein as part of the protein-protein complex isolated from the sample via purification by NANEX.

[0067] Specific embodiments relate to protein complexes in which the epitope of the target protein recognized and bound by the second or fourth protein binder comprises or consists of a tag selected from the list of tags enumerated herein, including GFP, mCherry, GST, EPEA, SMT3. Thus, the protein complex comprises a second (or fourth) protein binder and a target protein comprising or consisting of a tag selected from the group. Furthermore, the protein complex can be a crystal complex or a crystal.

[0068] Further aspects relate to the use of the protein complexes described herein for structural analysis, structure-based drug design, drug discovery, mass spectrometry, but also for use as a diagnostic tool or for in vivo imaging. In fact, quantitative mass spectrometry for rare proteins present in complex mixtures is often difficult and uncertain due to the low signal-to-noise ratio. Thus, the purification methods described herein can be advantageous in providing highly pure analytical samples for MS profiling of such target proteins or for the identification of interacting protein partners.

[0069] Furthermore, the protein complexes described herein can provide a three-dimensional structure representation in the atomic resolution of the complex, preferably at a high resolution with a resolution between 0.1 and 3 Å obtained by cryo-EM structural analysis. In an alternative embodiment, the crystalline protein complexes described herein may specifically relate to crystals of a GFP-specific nanobody and a target protein that is GFP, in which case the GFP-specific nanobody is depicted in a sequence having at least 90%, at least 95% or at least 99% identity with SEQ ID NO: 1, and the GFP protein is depicted in a sequence having at least 90%, at least 95% or at least 99% identity with SEQ ID NO: 16, and the crystal is characterized in that the crystal lattice constants are a = 74.497 Å ± 5%, b = 103.450 Å ± 5%, c = 209.774 Å ± 5%, α = 90.00°, β = 90.00°, γ = 90.00° (space group: P212121).

[0070] The binding site of the Nb depicted in SEQ ID NO: 1 or the epitope on the GFP protein interacts with and consists of a subset of atomic coordinates, resulting in a binding site consisting of amino acid residue numbers: PRO89, GLU90, GLU111, LYS113, PHE114, GLU115, GLY116 of SEQ ID NO: 16 from the crystal structure.

[0071] Binding sites or epitopes determined by 3D structural representations of crystals can further reduce their affinity or increase their k off as compared to protein binders or ISVDs present within the protein complex, such as mutant ISVDs, to enable the design and creation of mutant protein binders. To design such mutants, which differ from the protein binders present in the complex in at least one amino acid, one of ordinary skill in the art relies on (computer-aided) methods available in the art to obtain high k off and / or low affinity for the epitopes on the target protein as defined herein. Using the 3D structure of the protein complex described herein, one of ordinary skill in the art can use computer-aided methods to create mutations within the binder from the 3D structure of its binding domain, and further enable one of ordinary skill in the art to present the superposition of the model of the mutated binding domain on the three-dimensional model, and finally enable one of ordinary skill in the art to evaluate whether the mutated binding domain results in high k off and / or low affinity for the target protein. As presented in Examples 1 and 2 herein, GFP binders are designed similarly.

[0072] Finally, the application of the methods and means of the present invention for use in structure-based drug design, drug discovery and structure-based drug screening is also encompassed herein. The iterative process of structure-based drug design often involves multiple cycles before an optimized lead compound progresses to Phase I clinical trials. The first cycle involves cloning, purifying the target protein or nucleic acid and determining its structure by one of three main methods: X-ray crystallography, NMR or homology modeling. Using computer algorithms, database-derived compounds or fragments of compounds are docked into selected regions of the structure. The purification methods and ISVD-based protein binders of the present invention can be used to purify, immobilize and / or stabilize a particular structural conformation of the target. The selected compounds are evaluated and ranked based on their steric and electrostatic interactions with their target sites, and the best compounds are examined by biochemical assays. In the second cycle, the determination of the structure of the target complexed with a promising lead compound from the first cycle, which is a compound with at least micromolar inhibition in vitro, can reveal sites on the compound that can be optimized to increase potency. The purified protein complexes of the present invention can also be involved in this regard as they facilitate the structural analysis of the target in a particular conformational state. Further cycles involve the synthesis of the optimized lead compound, the determination of the structure of the new target:lead compound complex and further optimization of the lead compound. After several cycles of the drug design process, the optimized compounds typically show a marked improvement in binding and often show specificity for the target. Structural information for structure-activity relationship analysis and medicinal chemistry are essential for libraries that screen hit lead compounds that are further developed into lead compounds.The selection for a presented target using a nanobody or MegaBody, and thus the application of protein binders described herein, including ISVDs such as nanobodies or Megabodies, to reveal mainly binders to conformational epitopes, provides an additional advantage for drug discovery methods in that only an average image of the properly folded target protein is included. According to a particularly preferred embodiment, the method described above for identifying a conformationally selective compound is carried out by a ligand binding assay or a competition assay, and even more preferably, by a radioligand binding assay or a competition assay. Most preferably, the method described above for identifying a conformationally selective compound is carried out in a comparative assay, more specifically, a comparative ligand competition assay, and even more specifically, a comparative radioligand competition assay. The test compound can be any small chemical compound or macromolecule such as a protein, sugar, nucleic acid or lipid. Typically, the test compound is a small chemical compound, a peptide, an antibody or a fragment thereof. In some cases, it is understood that the test compound can be a library of test compounds. In particular, high-throughput screening assays for therapeutic compounds such as agonists, antagonists or inverse agonists and / or modulators form part of the present invention. Methods for preparing and screening such libraries are known to those skilled in the art. The test compound may optionally be covalently or non-covalently linked to a detectable label. Suitable detectable labels and techniques for conjugating, using and detecting them will be apparent to those skilled in the art and include, but are not limited to, any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Means for detecting such labels are well known to those skilled in the art. Thus, for example, a radiolabel may be detected using a photographic film or a scintillation counter, and a fluorescent marker may be detected using a photodetector that detects luminescence intensity.Enzyme labels are typically detected by supplying a substrate to the enzyme and detecting the reaction product generated by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label. Test compounds used in any of the above screening methods include polypeptides, peptides, small molecules, natural products, peptidomimetics, nucleic acids, lipids, lipopeptides, carbohydrates, Fab, Fab’, and F(ab’)2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (dsFv) and fragments containing VL or VH domains, heavy-chain antibodies (hcAb), single-domain antibodies (sdAb), minibodies, variable domains (VHH or nanobodies) derived from camelid heavy-chain antibodies, variable domains of novel antigen receptors (VNAR) derived from shark antibodies, etc., antibodies or any fragments derived therefrom, alpha bodies, protein A, protein G, DARPins (designed ankyrin-repeat domains), fibronectin type III repeats, anticalins, knottins, engineered CH2 domains (nanobodies) defined in the foregoing of this specification, and are selected from the group containing protein scaffolds. In a preferred embodiment, the high-throughput screening method involves a combinatorial chemical library or peptide library containing a large number of potential therapeutic ligands. Such a "combinatorial library" or "compound library" is then screened in one or more assays described herein to identify library members (specific chemical molecular species or chemical subclasses) that exhibit the desired characteristic activity. A "compound library" is typically a collection of stored chemical substances that are ultimately used in high-throughput screening. A "combinatorial library" is a collection of diverse chemical compounds created by chemical synthesis or biological synthesis by combining a large number of chemical "building blocks". The preparation and screening of combinatorial libraries are well known to those skilled in the art.Compounds identified in this way may be used as conventional "lead compounds", and these themselves may be used as potential or actual therapeutic agents.

[0073] In this specification, specific embodiments, specific configurations, and materials and / or molecules have been discussed for the methods, samples, and biomarker products according to the present disclosure. It is understood that various changes or modifications in form and detail can be made without departing from the scope of the invention. The following examples are presented to illustrate specific embodiments more and are not considered to limit this application. This application is limited only by the claims.

Examples

[0074] General Introduction Nanobody Exchange Chromatography (NANEX) is described for the first time herein and is specifically developed herein for binders that compete for binding to a target protein in exchange chromatography and use an ISVD-containing displacer, based on the principle of affinity displacement chromatography. Figure 1 illustrates the principle of competitive affinity exchange by the use of nanobodies as binders for a protein of interest (or target protein used interchangeably herein). The protein of interest is first optionally captured by an immobilizable capture agent antigen-binding protein or, in particular, a nanobody, followed by a washing step, and then specifically eluted by the addition of an elution buffer containing a second binder that acts as a displacer or eluent and specifically binds to the antigen via its immunoglobulin single variable domain that recognizes the epitope of the target protein. The eluted complex is obtained through a kinetic competition with the first binder or capture agent and, as shown herein, competes by binding to the same epitope or an epitope that largely overlaps or binds to an epitope that minimally overlaps or is different in the smallest part via an allosteric difference or kinetic difference, enabling the application of a displacer that competes by binding to the target. The main kinetic requirement for using an ISVD or its functional variant as a displacer is preferably lower than the dissociation rate constant (k off ) of the capture agent. This often results in a higher affinity for the displacer compared to the capture agent. Dose dependence also enables displacement even when the same binder is used for capture and elution and thus the k off values for the reaction are equal, provided that the dissociation rate constant (k off ) results in a half-life of less than 2 hours that enables good capture and elution, which results in a relatively efficient reaction only when it is at least 0.0001 seconds -1 . More specifically, k off is also 0.0001 seconds off lower dissociation rate constant (k off ). This often results in a higher affinity for the displacer compared to the capture agent. Dose dependence also enables displacement even when the same binder is used for capture and elution and thus the k off values for the reaction are equal, provided that the dissociation rate constant (k off ) results in a half-life of less than 2 hours that enables good capture and elution, which results in a relatively efficient reaction only when it is at least 0.0001 seconds -1 . More specifically, k off is also 0.0001 seconds-1 Lower values ​​are possible, resulting in displacement, but with longer dissociation half-lives and therefore less preferred. off is at least 0.00005 or 0.00001 seconds -1 In another embodiment, k off 0.0001 to approx. 0.05 seconds -1 The range may be:

[0075] In general, due to the addition of this second, competitive Nb to a mixture / column where the first binder binds to the target, the binding of the target to the first Nb is perturbed and the target protein is released from the first protein binder or Nb via competitive binding to the second protein binder or Nb, resulting in elution of the protein of interest. As shown for other antibodies, such as monoclonal antibodies, if the epitopes overlap to a large extent, it can be expected that competition may result in blocking of the first binder to the epitope of the second binder, but the kinetic mechanism for ISVD-driven displacement to the target has been shown to be different. Furthermore, the inventors have shown that k off It has been shown that the optimal reaction rate ratio of the binding agent for capture and the binding agent for release, as defined by, results in a robust and sophisticated displacement and purification method. Moreover, this novel technique allows for mild purification conditions that result in high recovery and purity in a single rapid purification step, even when starting from complex mixtures. Moreover, the method is applicable even to very small sample sizes due to the high affinity and specificity of the applied binding agents.

[0076] The examples provide support for the development, application, and optimization of NANEX technology, where several aspects are emphasized. As mentioned herein, the binders are related to the ISVD or nanobody depicted herein by "CA" numbering for the sequence information of specific Nb clones that are associated through the sequence numbers presented herein throughout most of the examples. The examples given herein are not limiting and present a separable approach so that those skilled in the art can apply the NANEX technology for multiple purposes.

[0077] As an overview, the examples first present a proof of concept by targeting the green fluorescent protein (GFP), a well-known and easily traceable target protein. Here, more specifically, a subset of Nbs was created for designing the optimal method. In particular, in Examples 1 and 2, based on the crystal structures of high (low picomolar concentration) affinity Nbs that bind to GFP, a number of capture / eluent paired Nbs were designed, made, purified, and analyzed by BLI to determine the reaction rate constants through mutagenesis of paratope residues. In Examples 1 - 6, these Nb binders that target the same epitope on GFP with different reaction rate constants were examined in several combinations for their relative substitutability, which allows binding pairs that target the same epitope of the target to be used for substitution, provided mainly with a certain specific reaction rate limitation specified by the relative difference in k off values. Another way to create an appropriate capture / eluent pair relates to the increase in the avidity of the eluent compared to the target. This is shown in Example 12 where the eluent is a bivalent ISVD and the capture agent is a monovalent ISVD that targets the EPEA tag fused to the GFP protein.

[0078] In addition to using Nb pairs that compete for the same epitope, nanobody binding pairs that compete for targets that are epitopes with respect to reaction rate, but which only partially overlap (or do not overlap to a large extent), are also shown in Example 15 to enable substitution for Factor IX and the like.

[0079] As shown in Examples 9, 10, and 16, it is possible to be used as a releasing agent, or as shown in Example 24, it is also possible to be used as a capturing agent. Similar to the case of MegaBody targeting GFP, as shown herein, in addition to pure Nbs, functional variants of ISVD, such as functionalized forms, can also be used as capturing or releasing agents.

[0080] As shown in Examples 1 to 10, 18 for GFP, Example 12 for EPEA, Example 19 for GST, Example 20 for SMT3, and Examples 21 and 22 for mCherry herein, additional capturing agent / releasing agent pairs targeting commercially available tags have also been developed. Based on the structural information of the complex between the binder and the target, in addition to designing and creating the pairs, Examples 21 and 22 also demonstrate that simpler tools are available for obtaining appropriate pairs.

[0081] Furthermore, the target protein does not require a tag for NANEX and can also be recognized at natural epitopes or specifically presented endogenous epitopes, as shown in Example 13 for synaptojanin, Examples 14, 15, 16, as well as 23 and 24 for Factor IX.

[0082] Purification using NANEX is also supported in another application in the field of protein-protein interactions (PPIs) as it enables the identification and purification of PPI complexes. This was demonstrated for eGFP-GR (glucocorticoid receptor) (Example 25) and eGFP-ARb (androgen receptor) (Example 26) purified by a GFP capturer / stripper pair, revealing the copurification of HSP proteins known to form complexes with these nuclear receptors.

[0083] Regarding the possibility of designing kits and products for applying NANEX, the inventors have supported several approaches of immobilizing the capturer to further apply the stripper in solution. For example, in Examples 1 to 5, agarose beads were used to immobilize the capturer, and in Example 27, magnetic beads were used. Further, for example, in Examples 6 to 10, the immobilization of Nb as the capturer was obtained by coupling to a resin and packing in a column. To miniaturize the system, in Example 11, a microcolumn was applied, and in Example 28, a prototype of a microchip was investigated.

[0084] Finally, herein, for example, bacterial lysates (e.g., in Example 1 and further examples), yeast extracts (e.g., in Examples 17, 27), human plasma (in Examples 23 and 24), and HEK cell lysates (in Examples 25, 26), all complex mixtures provide a one-step purification option using NANEX, so the diversity of samples that can be used for purification is evident from the examples. Further, if a second purification step is desired, tandem purification using NANEX sequentially and / or using multiple capturer / stripper pairs can also be done (e.g., Example 16).

[0085] [Example 1] Purification of GFP protein spiked in bacterial lysate by NANEX chromatography using Nb CA15816 as an immobilized capture agent and Nb CA12760 as an eluent on a HiTrap NHS-activated Sepharose HP column To demonstrate the proof of concept that proteins can be purified by nanobody exchange chromatography (NANEX), the inventors selected two nanobodies that bind to substantially the same epitope or overlapping epitopes on GFP and used these nanobodies to purify the GFP protein according to FIG. 1.

[0086] First, the inventors selected a high-affinity nanobody for GFP according to standard procedures (Pardon et al., 2014) to be used as an eluent. CA12760 was selected as a nanobody that binds to the GFP protein with low picomolar affinity. The biolayer interferometry (BLI) assay showed that the affinity (K D ) was 40 pM, k on was 2.56×10 5 (M -1 × s -1 ), and k off was 0.032×10 -3 (s -1) was identified. To perform NANEX chromatography, the inventors next designed, by site-directed mutagenesis, a nanobody that binds to substantially the same epitope or an epitope that mostly overlaps on GFP, which is immobilized on a solid phase as a capture agent. NANEX has a low affinity and / or a high off-rate compared to the nanobody, which is a stripping agent used to competitively elute the target, and conversely, maximum elution could be obtained when used with a nanobody that is an immobilized capture agent having a high affinity and / or a low off-rate for the target protein. Based on the structure of the GFP·CA12760 protein complex (Figure 3, Example 2), the inventors designed CA15816 as a modified form of CA12760 with a low affinity and a high off-rate. In fact, CA15816 was designed by mutating two residues (T54A, V55A) contained within the paratope so as to reduce its affinity compared to CA12760.

[0087] The BLI assay showed that the affinity (K D ) of CA15816 for the epitope of GFP was 2.7 nM, and k on was 8.35×10 5 (M -1 × sec -1 ), and k off was 2.25×10 -3 (sec -1) was identified. 1 mg (66.67 nmol) of CA15816 Nb was immobilized onto a HiTrap NHS-activated Sepharose HP column (GE) according to the supplier's recommendation. 10 mL of bacterial lysate (equivalent to the cell pellet of 0.5 L of E. coli culture grown in LB) was spiked with 2 mg of GFP. The spiked lysate was loaded onto the CA15816 column using a syringe and washed twice with 10 mL (10 column volumes (CV)) of buffer (100 mM Hepes pH 7.5, 150 mM NaCl). Then, the column was connected to an Akta pure FPLC system (GE) for elution using the eluent in 8 CV of elution buffer (100 mM Hepes pH 7.5, 150 mM NaCl, 66.67 μM eluent Nb (1 mg / mL)) at a flow rate of 0.1 mL / min. The eluate was collected in 500 μl fractions and the main elution peak was analyzed by SDS / PAGE gel (Figure 2). Column regeneration was obtained with 8 CV of 200 mM glycine buffer at pH 2.3.

[0088] By measuring the absorbance at 280 nm (absorbance by protein) and 488 nm (absorbance by GFP fluorophore), the purification of GFP protein using CA15816 as the immobilized capture agent on a HiTrap NHS-activated Sepharose HP column, eluted from the column by CA12760 as the eluent, was monitored. From the elution profile, the inventors could conclude that the washing was sufficient to remove most of the impurities and that elution fractions 2 and 3 contained the eluent Nb and the GFP protein detected within the main peak. The small peak at 280 nm represents the amount of sample (or GFP protein) remaining on the column washed off by the regeneration buffer.

[0089] Nanobody exchange chromatography (NANEX) using binders for the same epitope requires that the immobilized nanobody (capture agent) has a high off-rate and / or low affinity, while the nanobody (elution agent) used to competitively elute the target has a low off-rate and / or high affinity for the target protein, resulting in optimal yields and purity. To demonstrate this principle, in Examples 2-8, we performed NANEX experiments with different capture agent-elution agent pairs that target the same GFP epitope but have different affinities and off-rates (k off ).

[0090] [Example 2] Purification of GFP protein using Nb CA12760 as the immobilized capture agent and CA12760, CA15818, CA15816, CA15861 as the elution agents on NHS-activated agarose beads In this example, we show that a high-affinity capture agent cannot be conveniently combined with a low-affinity elution agent.

[0091] This example describes the affinity purification of GFP using CA12760 (SEQ ID NO: 1) as the capture agent, a nanobody that specifically binds to GFP with a low picomolar affinity. The BLI assay identified that the affinity (K D ) was 40 pM, k on was 2.56×10 5 (M -1 × sec -1 ), and k off was 0.032×10 -3 (sec -1 ).

[0092] To identify the epitope and paratope of GFP·CA12760, the inventors elucidated the structure of this complex by X-ray crystallography (Figure 3). Crystals containing GFP (depicted in SEQ ID NO: 16) and the GFP-nanobody (SEQ ID NO: 1) are in the space group P212121 with the following lattice constants: a = 74.497 Å ± 5%, b = 103.450 Å ± 5%, c = 209.774 Å ± 5%, α = 90°, β = 90°, γ = 90°. These crystals were able to further delineate a binding site, defined herein as being formed by the amino acid residues (SEQ ID NO: 16) of the GFP protein, which are hydrogen-bonded to Nb CA12760 and identified as amino acids Pro89, Glu90, Glu111, Lys113, Phe114, Glu115, and Gly116 of SEQ ID NO: 16. Based on the structure of GFP·CA12760, the inventors identified three key residues (Thr54, Val55, Phe103) that can be mutated within the paratope region of CA12760, a GFP-specific Nb, to reduce the affinity of CA12760 (Figure 4).

[0093] ·CA15818 Nb has one residue (F103A) mutated to reduce its affinity compared to CA12760. The BLI assay identified that the affinity (K D ) of CA15818 is 1.5 nM, k on is 3.87×10 5 (M -1 × sec -1 ), and k off is 0.29×10 -3 (sec -1 ) (Figure 5 and Table 1).

[0094] ·CA15816 was designed based on the structure of GFP·CA12760 and has two residues (T54A, V55A) mutated to reduce its affinity compared to CA12760. The BLI assay identified that the affinity (K D ) of CA15816 is 2.7 nM and k onis 8.35×10 5 (M -1 × second -1 ), and k off is identified as 2.25×10 -3 (second -1 ).

[0095] ·CA15861 is designed based on the structure of GFP·CA12760 and mutated at three residues (T54A, V55A, F103) to lower its affinity compared to CA12760. The BLI assay identifies that the affinity (K D ) of CA15861 is 111 nM, and k on is 28.85×105 (M -1 × second -1 ), and k off is identified as 45.8×10 -3 (second -1 ).

[0096]

Table 1

[0097] The half-life is defined in the function of k off (t 1 / 2 =ln2 / k off ), and the half-life ratio is defined herein compared to the wt Nb and the K D ratio.

[0098] To purify GFP, the inventors covalently immobilized 4 mg of the capture agent Nb CA12760 onto 500 μl of NHS-activated agarose beads (Thermo Fisher Scientific) according to the supplier's recommendations. 200 μg (7.69 nanomoles) of purified GFP was loaded onto 50 μl of CA12760 Nb-coupled agarose beads in 100 mM Hepes buffer pH 7.5, 150 mM NaCl at a final volume of 1 mL. To specifically elute the GFP protein from this affinity matrix, the inventors used CA12760, CA15818 (SEQ ID NO: 2), CA15816 (SEQ ID NO: 3), and CA15861 (SEQ ID NO: 4) as eluting agents.

[0099] Purification of GFP using CA12760 as an immobilized capture agent on NHS-activated agarose beads and CA12760, CA15818, CA15816, and CA15861 as eluting agents was carried out in elution buffer (100 mM Hepes pH 7.5, 150 mM NaCl, 53 μM eluting agent Nb (800 μg / mL)), and monitored by measuring the absorbance at 488 nm (indicating the amount of GFP fluorescent protein) at five different time points (0, 15, 30, 60, 120 minutes later) (Figure 6). From the absorbance profile of the GFP fluorophore at elution, the inventors were able to conclude that using the same Nb as an eluting agent in combination with a high-affinity capture agent Nb with picomolar affinity or using a low-affinity Nb (compared to the capture agent) as an eluting agent does not result in rapid and quantitative elution of the GFP protein from the beads.

[0100] [Example 3] Purification of GFP using Nb CA15818 as an immobilized capture agent on NHS-activated agarose beads and CA12760, CA15818, CA15816, and CA15861 as Nb eluting agents In this example, a medium-affinity capture agent was combined with each of a high-affinity elution agent and a low-affinity elution agent.

[0101] This example describes the affinity purification of GFP protein using CA15818 (see Example 2 and Table 1) as a capture agent. To purify GFP, the inventors covalently immobilized 4 mg of the capture agent CA15818 on 500 μl of NHS-activated agarose beads (Thermo Fisher Scientific) according to the supplier's recommendations. 200 μg (7.69 nanomoles) of purified GFP was loaded onto 50 μl of CA15818 Nb-coupled agarose beads in 100 mM Hepes buffer pH 7.5, 150 mM NaCl at a final volume of 1 mL. To specifically elute GFP from this affinity matrix, the inventors used CA12760, CA15818, CA15816, CA15861 (see Table 1) as elution agents.

[0102] Purification of GFP protein using Nb CA15818 as an immobilized capture agent and CA12760, CA15818, CA15816, and CA15861 as eluting agents on NHS-activated agarose beads was carried out in elution buffer (100 mM Hepes pH 7.5, 150 mM NaCl, 53 μM eluting agent Nb (800 μg / mL)), and monitored by measuring the absorbance at 488 nm at five different time points (0, 15, 30, 60, and 120 minutes later) (Figure 7). From the absorbance profile of the GFP fluorophore for elution, the inventors were able to conclude that using Nb, a capture agent with nanomolar affinity, in combination with Nb, an eluting agent with picomolar affinity, which is approximately 35-fold more affinity than the capture agent (CA12760 Nb), enables good elution of GFP as compared to the results in Example 2. Furthermore, the inventors confirmed, as in Example 2, that the use of Nb, a capture agent with higher affinity than the eluting agent Nb, does not result in rapid and quantitative elution of GFP protein from the beads.

[0103] [Example 4] Purification of GFP protein using Nb CA15816 as an immobilized capture agent and CA12760, CA15818, CA15816, and CA15861 as eluting agents, which are Nb In this example, the inventors combined a low-affinity capture agent with each of a high-affinity eluting agent and a low-affinity eluting agent.

[0104] This example describes the affinity purification of GFP using CA15816 Nb as a capture agent (see Example 2 and Table 1). To purify GFP, the inventors covalently immobilized 4 mg of the capture agent CA15816 Nb onto 500 μl of NHS-activated agarose beads according to the supplier's recommendations. 200 μg (7.69 nanomoles) of purified GFP was loaded onto 50 μl of CA15816 Nb-coupled agarose beads in 100 mM Hepes buffer pH 7.5, 150 mM NaCl at a final volume of 1 mL. To specifically elute the GFP protein from this affinity matrix, the inventors used Nbs CA12760, CA15818, CA15816, and CA15861 (see Example 2, Table 1) as eluents. The purification of GFP using CA15816 Nb as the immobilized capture agent on NHS-activated agarose beads and Nbs CA12760, CA15818, CA15816, CA15861 as eluents was performed in elution buffer (100 mM Hepes pH 7.5, 150 mM NaCl, 53 μM eluent Nb (800 μg / mL)), and monitored by measuring the absorbance at 488 nm at five different time points (0, 15, 30, 60, 120 minutes later) (Figure 8). From the absorbance profile of the GFP fluorophore for elution, the inventors were able to conclude that using Nb (CA15816), a capture agent with nanomolar affinity, in combination with Nb (CA12760), a picomolar eluent with approximately 200-fold higher affinity than the capture agent, enables rapid and quantitative elution of GFP from the column. Furthermore, elution of the beads with slightly higher affinity Nb (CA15818) or equal affinity Nb (CA15816), which is only 2-fold higher, enables almost complete elution of GFP, whereas the use of an eluent Nb with approximately 1 / 100th lower affinity compared to the capture agent does not allow for the recovery or elution of the GFP protein.

[0105] [Example 5] Purification of GFP protein using Nb CA15861 as an immobilized capture agent on NHS-activated agarose beads and Nb CA12760, CA15818, CA15816, CA15861 as eluents In this example, the inventors combined a low-affinity capture agent with each of the high-affinity eluents.

[0106] This example describes the affinity purification of GFP using CA15861 Nb as a capture agent (see Example 2 and Table 1). To purify GFP, the inventors covalently immobilized 4 mg of the capture agent CA15861 Nb onto 500 μl of NHS-activated agarose beads according to the supplier's recommendations. 200 μg (7.69 nanomoles) of purified GFP protein was loaded onto 50 μl of CA15861 Nb-coupled agarose beads in 100 mM Hepes buffer pH 7.5, 150 mM NaCl at a final volume of 1 mL. To specifically elute GFP from this affinity matrix, the inventors used Nb CA12760, CA15818, CA15816, and CA15861 (see Example 2 and Table 1) as eluents. The purification of GFP using CA15861 as an immobilized capture agent on NHS-activated agarose beads and CA12760, CA15818, CA15816, CA15861 as eluents was performed in elution buffer (100 mM Hepes pH 7.5, 150 mM NaCl, 53 μM eluent Nb (800 μg / mL)), and monitored by measuring the absorbance at 488 nm at five different time points (0, 15, 30, 60, 120 minutes later) (Figure 9). The inventors were able to conclude that the affinity (micromolar concentration range) of the capture agent Nb (CA15861) was too low to capture GFP on the beads.

[0107] [Example 6] Purification of GFP protein by nanobody exchange chromatography using Nb CA12760 as an immobilized capture agent on a HiTrap NHS-activated Sepharose HP column and Nb CA12760, CA15818, CA15816, and CA15861 as eluents In this example, the inventors used a 1 mL column (1 mL CV) connected to an FPLC (AktaPure-GE) system, coupled to HiTrap NHS-activated Sepharose HP beads, and used CA12760 Nb (see Example 2 and Table 1) to combine a high-affinity capture agent with a low-affinity eluent (similar to Example 2, but using a capture agent immobilized on beads contained in a pre-packed column) for the affinity purification of GFP. To specifically elute GFP from this affinity matrix, the inventors used each of CA12760, CA15818, CA15816, and CA15861 as eluents (see Example 2 and Table 1). 1 mg (66.67 nmol) of CA12760 was immobilized on HiTrap NHS-activated Sepharose HP beads pre-packed in a 1 mL column (GE) according to the supplier's recommendations. 2 mg (76.92 nmol) of GFP was loaded onto the CA12760 nb-coupled column via an injection loop. 10 CV of 100 mM Hepes wash buffer pH 7.5, 150 mM NaCl was passed over the column to remove unbound material. Elution was performed using 8 CV of elution buffer (100 mM Hepes buffer pH 7.5, 150 mM NaCl, 66.67 μM eluent Nb (1 mg / mL)) at a flow rate of 0.1 mL / min. Elution peaks were collected in 500 μl fractions and analyzed in an SDS / PAGE gel (Figure 10). Column regeneration was obtained with 8 CV of 200 mM glycine buffer pH 2.3.

[0108] By measuring the absorbance at 280 nm (absorbance by protein) and 488 nm (absorbance by GFP fluorophore), the purification of GFP protein was monitored using CA12760, CA15818, CA15816, CA15861, where CA12760 is the immobilized capture agent and Nb is the eluent on a HiTrap NHS-activated Sepharose HP column (Figure 10). From the absorbance profile of the GFP fluorophore at elution and SDS-PAGE analysis of the elution fractions, the inventors found that using Nb, a capture agent with picomolar affinity, in combination with the same Nb as the eluent or using low-affinity Nb (compared to the capture agent) as the eluent does not result in rapid and quantitative elution of GFP from the column, whereas the peak eluted during the regeneration step indicates that most of the GFP protein still remains on the column and is eluted only when using a low pH (low pH abolishes the absorbance of the GFP fluorophore at 488 nm).

[0109] [Example 7] Purification of GFP Protein by Nanobody Exchange Chromatography Using CA12760, CA15818, CA15816, CA15861, Where Nb CA15816 Is the Immobilized Capture Agent and Nb Is the Eluent on a HiTrap NHS-activated Sepharose HP Column In this example, the inventors coupled CA15816 Nb (see Example 2, Table 1) to HiTrap NHS-activated Sepharose HP beads pre-packed in a 1 mL column (GE) connected to an FPLC (AktaPure - GE) system, and combined a low-affinity capture agent with a high-affinity eluent and a low-affinity eluent (similar to Example 4, but using a capture agent immobilized on beads contained in a pre-packed column) for the affinity purification of GFP. To specifically elute GFP from this affinity matrix, the inventors used CA12760, CA15818, CA15816, and CA15861 (see Example 2, Table 1) respectively as eluents. 1 mg (66.67 nmol) of CA15816 Nb was immobilized on HiTrap NHS-activated Sepharose HP beads pre-packed in a 1 mL column (1 mL CV) (GE) according to the supplier's recommendation. 2 mg (76.92 nmol) of GFP protein was loaded onto the CA15816 Nb coupling column via an injection loop. 10 CV of wash buffer (100 mM Hepes pH 7.5, 150 mM NaCl) was passed over the column, followed by 8 CV of elution buffer (100 mM Hepes pH 7.5, 150 mM NaCl, 66.67 μM eluent Nb (1 mg / mL)) at a flow rate of 0.1 mL / min to remove unbound material. The elution peak was collected in 500 μl fractions and analyzed in an SDS / PAGE gel (Figure 11). Column regeneration was obtained with 8 CV of 200 mM glycine buffer pH 2.3.

[0110] The purification of GFP protein was monitored by measuring the absorbance at 280 nm (absorbance by protein) and 488 nm (absorbance by GFP fluorophore), using CA12760, CA15818, CA15816, CA15861, where CA15816 is the immobilized capture agent and Nb (CA12760) is the eluent on a HiTrap NHS-activated Sepharose HP column (Figure 11). From the absorbance profile of the GFP fluorophore at elution and SDS-PAGE analysis of the elution fractions, the inventors concluded that since no protein elutes during column regeneration, using the capture agent Nb (CA15816) with nanomolar affinity in combination with the eluent Nb (CA12760) with picomolar affinity, which has approximately 200-fold higher affinity than the capture agent, enables rapid and quantitative elution of GFP from the column. Furthermore, since the 280 nm peak during regeneration is extremely low, elution of the column with the slightly higher affinity Nb (CA15818) with only 2-fold higher affinity or Nb (CA15816) with equal affinity also enables almost complete elution of GFP, whereas the use of the eluent Nb (CA15861), which has approximately 1 / 100 lower affinity compared to the capture agent, does not allow recovery or elution of the GFP protein.

[0111] [Example 8] Purification of GFP Protein by Nanobody Exchange Chromatography Using Nb CA15861 as the Immobilized Capture Agent and Nb CA12760, CA15818, CA15816, CA15861 as the Eluent on a HiTrap NHS-activated Sepharose HP Column In this example, the inventors used a low-affinity capture agent in combination with a high-affinity elution agent (similar to Example 5 but using a capture agent immobilized on beads contained in a pre-packed column) for the affinity purification of GFP using CA15861 Nb coupled to HiTrap NHS-activated Sepharose HP beads and using a 1 mL column (see Example 2, Table 1) connected to an FPLC (AktaPure - GE) system. To specifically elute GFP from this affinity matrix, the inventors used each of CA12760, CA15818, CA15816, and CA15861 (Table 1) which are Nbs as elution agents. 1 mg (66.67 nanomoles) of CA15861 was immobilized on HiTrap NHS-activated Sepharose HP beads pre-packed in a 1 mL column (1 mL CV; GE) according to the supplier's recommendations. 2 mg (76.92 nanomoles) of GFP was loaded onto the CA15861 column via an injection loop. Following the passage of 10 CV of wash buffer (100 mM Hepes pH 7.5, 150 mM NaCl) over the column, 8 CV of elution buffer (100 mM Hepes pH 7.5, 150 mM NaCl, 66.67 μM elution agent Nb (1 mg / mL)) was used at a flow rate of 0.1 mL / min to remove unbound material. The elution peaks were collected in 500 μl fractions and analyzed in an SDS / PAGE gel (Figure 12). Column regeneration was obtained with 8 CV of 200 mM glycine buffer pH 2.3. The purification of GFP using CA15861 Nb as the immobilized capture agent and Nbs as elution agents, CA12760, CA15818, CA15816, CA15861, on a HiTrap NHS-activated Sepharose HP column was monitored by measuring the absorbance at 280 nm and 488 nm (Figure 12).From the absorbance profile of the GFP fluorophore during elution and SDS-PAGE analysis of the elution fractions, the inventors were able to conclude that using the capture agent Nb(CA15861), which has an affinity in the nanomolar to micromolar range, in combination with the high-affinity eluent Nb enables the recovery or elution of GFP protein. Considering that the capture agent has a very low affinity, the yield is likely to be somewhat low because not all of the GFP loaded onto the column was retained.

[0112] [Example 9] Purification of GFP protein spiked in bacterial lysate by NANEX chromatography using Nb CA15816 as the immobilized capture agent on a HiTrap NHS-activated Sepharose HP column and eluting with CA15621 MegaBody Mb as the eluent CA12760Nb cHopQ One major advantage of NANEX is that the eluent can be a functionalized or engineered nanobody (e.g., a MegaBody fluorescently labeled for imaging and biotin-coupled for detection) that elutes the target from the functionalized complex. To verify these options, the inventors purified GFP from bacterial lysate by nanobody exchange chromatography (NANEX) and eluted the GFP as a GFP·MegaBody complex. Example 9 describes the affinity purification of GFP protein spiked in bacterial lysate using a HiTrap NHS-activated Sepharose HP column coupled with CA15816 Nb (see Table 1) and connected to an FPLC (AktaPure - GE) system. To specifically elute the GFP protein from this affinity matrix, the inventors used CA15621 Mb as the eluent. CA15621 is a MegaBody or an antigen-binding chimeric protein described herein, with the fusion disclosed in WO2019 / 086548A1, specifically Mb​CA12760Nb cHopQ It is composed of a non-flexible fusion of CA12760, which is a GFP-specific Nb, with the cHopQ scaffold.

[0113] Specifically, the 58 kDa MegaBody described in this example is a chimeric polypeptide concatenated from a single-domain immunoglobulin portion and a cHopQ scaffold protein portion. In this example, the immunoglobulin domain used is the GFP-binding nanobody depicted in SEQ ID NO: 1. The scaffold protein is the adhesin domain of Helicobacter pylori strain G27 (PDB: 5LP2, SEQ ID NO: 17), called HopQ (Javaheri et al., 2016). The N-terminus and C-terminus of HopQ were connected to enable the creation of a circular permutation variant of HopQ called cHopQ, where the cleavage of the sequence was performed at another position within this sequence. Mb NbCA12760 cHopQ To design the construct, following the order by peptide bonds, all parts were connected to each other from the amino terminus (N-terminus) to the carboxy terminus (C-terminus) as follows: β-strand A of the anti-GFP nanobody (residues 1 - 13 of SEQ ID NO: 1), the C-terminal portion of HopQ (residues 192 - 414 of SEQ ID NO: 17), a short peptide linker connecting the C-terminus and N-terminus of HopQ to yield cHopQ, which is a circular permutation variant of the scaffold protein, the N-terminal portion of HopQ (residues 14 - 186 of SEQ ID NO: 17), β-strands B - G of the GFP-binding nanobody (residues 16 - 126 of SEQ ID NO: 1), 6×His tag.

[0114] For coupling, 1 mg (66.67 nmol) of CA15816 Nb was immobilized onto a HiTrap NHS-activated Sepharose HP column (1 mL; GE) following the supplier's recommendations. 10 mL of bacterial lysate (equivalent to the cell pellet of 0.5 L of E. coli culture grown in LB) was spiked with 2 mg of GFP protein. The lysate was loaded onto the CA15816 Nb coupling column using a syringe and washed twice with 10 CV of wash buffer (100 mM Hepes pH 7.5, 150 mM NaCl). The column was then connected to an Akta system (GE) and eluted with 8 CV of elution buffer (100 mM Hepes pH 7.5, 150 mM NaCl, 68.18 μM eluent (4.5 mg / mL)) at a flow rate of 0.1 mL / min. The elution peak was collected in 500 μl fractions and analyzed in an SDS / PAGE gel (Figure 13). Column regeneration was obtained with 8 CV of 200 mM glycine buffer, pH 2.3. By measuring the absorbance at 280 nm (absorbance by protein) and 488 nm (absorbance by GFP fluorophore), CA15816 Nb as an immobilized capture agent and CA15621 Mb as an eluent on the HiTrap NHS-activated Sepharose HP column CA12760Nb cHopQ were used to monitor the purification of GFP (Figure 13). From the absorbance profile of the GFP fluorophore at elution and the SDS-PAGE analysis of the elution fractions, the inventors were able to conclude that when the capture agent Nb (CA15816) was used in combination with the high-affinity eluent Nb in its functionalized form as MegaBody, the washing was sufficient to remove most of the impurities and elution fractions 2 and 3 contained the eluent Mb complexed with the GFP protein detected within the main peak. Thus, the inventors were able to conclude from this that Mb functions equally well as an eluent in NANEX, eluting GFP quantitatively and rapidly, compared to its parental Nb, CA12760.

[0115] [Example 10] Purification of GFP spiked in bacterial lysate by nanobody exchange chromatography using Nb CA15816 as the capture agent immobilized on a HiTrap NHS-activated Sepharose HP column and eluting with CA15616 MegaBody Mb as the eluent CA12760Nb Ygjk Example 10 further describes the affinity purification of GFP protein spiked in bacterial lysate using a HiTrap NHS-activated Sepharose HP column coupled with CA15816 Nb (see Table 1) and connected to an FPLC (AktaPure - GE) system, and specifically eluting using CA15816 Mb as the eluent. CA15816 is a MegaBody or an antigen-binding chimeric protein described herein, with the fusion disclosed in WO2019 / 086548A1, and in particular, Mb CA12760Nb Ygjk is composed of a non-flexible fusion of CA12760, a GFP-specific Nb, with the Ygjk scaffold.

[0116] ​Specifically, the 100 kDa MegaBody is a chimeric polypeptide concatenated from a single-domain immunoglobulin portion and a scaffold protein portion linked by a short-chain polypeptide linker. The immunoglobulin used is the GFP-binding nanobody depicted in SEQ ID NO: 1. The alternative scaffold protein used was YgjK, an 86 kDa periplasmic protein of E. coli (PDB: 3W7S, SEQ ID NO: 32). Following the order by peptide bond, all parts were connected to each other from the amino terminus to the carboxy terminus as follows: β-strand A of the anti-GFP nanobody (residues 1-12 of SEQ ID NO: 1), a peptide linker of 1 or 2 amino acids with a random composition, the C-terminal portion of YgjK (residues 464-760 of SEQ ID NO: 32), a short-chain peptide linker connecting the C-terminus and the N-terminus of YgjK to result in a circular permutation variant of the scaffold protein, the N-terminal portion of YgjK (residues 1-461 of SEQ ID NO: 32), a peptide linker of 1 or 2 amino acids with a random composition, β-strands B-G of the anti-GFP nanobody (residues 17-126 of SEQ ID NO: 1), and a 6×His tag.

[0117] For coupling, 1 mg (66.67 nmol) of CA15816 was immobilized onto a HiTrap NHS-activated Sepharose HP column (1 mL CV; GE) according to the supplier's recommendations. 10 mL of bacterial lysate (equivalent to the cell pellet of a 0.5 L E. coli culture grown in LB) was spiked with 2 mg of GFP. The lysate was loaded onto the CA15816 Nb coupling column using a syringe and washed twice with 10 CV of wash buffer (100 mM Hepes pH 7.5, 150 mM NaCl). The column was then connected to an Akta system (GE) and eluted with 8 CV of elution buffer (100 mM Hepes pH 7.5, 150 mM NaCl, 48.54 μM eluent (5 mg / mL)) at a flow rate of 0.1 mL / min. The elution peak was collected in 500 μl fractions and analyzed in an SDS / PAGE gel (Figure 14). Column regeneration was obtained with 8 CV of 200 mM glycine buffer, pH 2.3. By measuring the absorbance at 280 nm (absorbance by protein) and 488 nm (absorbance by GFP fluorophore), CA15816 Nb as an immobilized capture agent and CA15616 Mb as an eluent on the HiTrap NHS-activated Sepharose HP column CA12760Nb Ygjk were used to monitor the purification of GFP (Figure 14). From the absorbance profile of the GFP fluorophore at elution and the SDS-PAGE analysis of the elution fractions, the inventors conclude that when the capture agent Nb (CA15816) was used in combination with the high-affinity eluent Nb in its functionalized form as MegaBody, the washing was sufficient to remove most of the impurities and elution fractions 2 and 3 contained the eluent Mb complexed with the GFP protein detected within the main peak. Thus, the inventors can conclude from this that Mb functions equally well as an eluent in NANEX, eluting GFP quantitatively and rapidly, compared to its parental Nb, CA12760.

[0118] [Example 11] Purification of GFP Protein by Nanobody Exchange Chromatography Using Nb CA15816 as an Immobilized Capture Agent on NHS-Activated Agarose Beads and Nb CA12760 as a Stripping Agent for Application in a Custom-Made 75 μL Microcolumn This example describes the affinity purification of GFP protein on an affinity microcolumn connected to an FPLC (AktaPure - GE) system. The inventors covalently immobilized 4 mg of the capture agent CA15816 Nb (see Table 1) on 500 μl of NHS-activated agarose beads. 75 μl of the agarose beads were packed into a custom-made microcolumn using commercially available parts that can be connected to a general experimental setup (Figure 15). In this example, CA12760 Nb was used as the stripping agent (Table 1). A 0.1 mg (3.85 nanomoles) GFP sample was loaded onto the CA15816 Nb-coupled microcolumn (75 μl CV) via a 500 μl injection loop. Following 5 mL (66 CV) of wash buffer (100 mM Hepes pH 7.5, 150 mM NaCl), 106 CV (8 mL) of elution buffer (100 mM Hepes pH 7.5, 150 mM NaCl, 13.33 μM stripping agent Nb (0.2 mg / mL) with a volume of 500 μl) at a flow rate of 0.1 mL / min was passed through the column to remove unbound material. The elution peak was collected in 500 μl fractions and analyzed in an SDS / PAGE gel (Figure 15). Column regeneration was obtained with 106 CV of 200 mM glycine buffer, pH 2.3. The purification of GFP using CA15816 Nb as an immobilized capture agent and CA12760 as a stripping agent on the custom-made microcolumn was monitored by measuring the absorbance at 280 nm (absorbance by protein) and 488 nm (absorbance by GFP fluorophore) (Figure 15).

[0119] Similar to the use of the large column (see Example 7, Figure 11), the GFP protein was also eluted rapidly and quantitatively from this microcolumn.

[0120] [Example 12] Purification of GFP-EPEA protein by nanobody exchange chromatography using synuclein-2 specific Nb CA4375 as the immobilized capture agent on a HiTrap NHS-activated Sepharose HP column and eluting with Nb CA4375 in its monovalent and divalent forms, respectively, as the eluent Nanobody exchange chromatography (NANEX) requires that the immobilized nanobody (capture agent) has low affinity and / or a high off-rate, while the nanobody (eluent) used to elute the target competitively has high affinity and / or a low off-rate for the target protein, resulting in optimal yield and purity. To demonstrate this, the inventors performed NANEX experiments with different capture agent-eluent pairs with varying affinities and off-rates. Here, the inventors also describe the EPEA tag-specific Nb as the capture agent and the same EPEA-specific Nb in its monovalent and divalent forms as the eluent. This example describes the affinity purification of the GFP-EPEA protein. CA4375 (SEQ ID NO: 7) is a nanobody that binds to the C-terminal linear epitope (EPEA) selected against human alpha-synuclein (UniProtKB: P37840). CA4394 (SEQ ID NO: 8) is a divalent format of the CA4375 Nb that binds to the C-terminal linear epitope (EPEA) selected against human alpha-synuclein (UniProtKB: P37840).

[0121] The BLI assay showed that the affinity (K D ) of CA4375 was 60 nM and the k on was 6.16×10 5 (M -1 × sec -1 ) and the k offis 0.324×10 -3 (seconds -1 ) was identified. 1 mg (69.89 nmol) of monovalent Nb of CA4375 was immobilized on a HiTrap NHS-activated Sepharose HP column (1 mL CV; GE) according to the supplier's recommendation. 10 mL of bacterial lysate (0.5 L of culture) was spiked with 2 mg of GFP (76.92 nmol). The lysate was loaded onto the CA4375 Nb coupling column using a syringe and then washed twice with 10 CV of buffer (100 mM Hepes pH 7.5, 150 mM NaCl). The column was then connected to an Akta system (GE), followed by elution with 8 CV of elution buffer (25 mM HEPES pH 7.5, 150 mM NaCl, with the concentration of the eluent CA4375 being 69.93 μM (1 mg / mL) and that of CA4394 being 33.57 μM (0.95 mg / mL)) at a flow rate of 0.1 mL / min. The elution peaks were collected in 500 μl fractions and analyzed in an SDS / PAGE gel (Figures 16 and 17). Column regeneration was obtained with 8 CV of 200 mM glycine buffer, pH 2.3.

[0122] Purification of GFP-EPEA using monovalent Nb of CA4375 as the immobilized capture agent and divalent CA4394 as the eluent on a HiTrap NHS-activated Sepharose HP column was monitored by measuring the absorbance at 280 nm and 488 nm (Figures 16 and 17). From the absorbance profile of the GFP fluorophore at elution and SDS-PAGE analysis of the elution fractions, the inventors conclude that when the monovalent capture agent Nb (CA4375) was used in combination with the same but divalent Nb (CA4394) as the eluent, the washing was sufficient to remove most of the impurities, and elution fractions 2 - 5 contained the eluent Nb and (part of) the GFP-EPEA protein detected within the main peak.

[0123] In contrast, when monovalent Nb was used as an eluent, the inventors found that monovalent Nb only enabled the elution of a small amount of GFP-EPEA protein. Therefore, polyvalent eluents are considered to have a high (apparent) affinity (due to the avidity effect) and act as strong eluents.

[0124] [Example 13] Purification of recombinant human synaptophysin protein by NANEX using Nb CA13016 as an immobilized capture agent on a HiTrap NHS-activated Sepharose HP column and synaptophysin-specific Nb CA13080 as an eluent In this example, the inventors further confirmed that a low-affinity synaptophysin-specific Nb can be used as a capture agent in combination with a non-related, high-affinity synaptophysin-specific Nb that competes for the same epitope as an eluent to isolate synaptophysin from cells or cell extracts. This example describes the purification of recombinant human synaptophysin 1 (amino acids 528 - 873 of UniProtKB: O43426) in E. coli cell extracts by NANEX chromatography. CA13016 Nb (SEQ ID NO: 8) and CA13080 Nb (SEQ ID NO: 9) are nanobodies selected against human synaptophysin 1 (528 - 873).

[0125] ·The BLI assay identified that the affinity (K D ) of CA13016 was 1.06 μM, k on was 3.8×10 4 (M -1 × s -1 ), and K off was 5×10 -2 (s -1 ).

[0126] ·The BLI assay showed that the affinity (K D ) of CA13080 was 3.2 nM, k on was 1.8×10 5 (M-1 × seconds -1 ) and K off is 4 × 10 -3 (seconds -1 ) was identified.

[0127] CA13016 Nb and CA13080 Nb have different CDRs but bind to the same epitope on human synaptophysin 1 and are mutually exclusive. 1 mg (66.57 nanomoles) of CA13016 Nb was immobilized on a HiTrap NHS-activated Sepharose HP column (1 mL CV; GE) according to the supplier's recommendations.

[0128] Recombinant human synaptophysin 1 (528 - 873) was expressed using the pET28a expression vector that transforms the expression strain BL21(DE3)-T1 R and the cells were grown in TB medium until OD 600 = 0.6 and induced with 1 mM IPTG at 20 °C overnight. The cells were harvested by centrifugation and resuspended in 25 mM Hepes (pH 7.5), 300 mM NaCl, 10% glycerol, 5 mM MgCl2, 1 mM DTT (supplemented with DNase and protease inhibitors), and then lysed using a cell disruptor. The crude extract was clarified by centrifugation, the supernatant was recovered and filtered (0.45 μm filter). Using a syringe, 10 mL of the lysate (equivalent to the cell pellet of a 0.6 L E. coli culture grown in LB) was loaded onto a HiTrap NHS-activated Sepharose HP column coated with CA13016 Nb and the flow-through was recovered. Using a syringe, washing was performed with 10 mL of washing buffer containing 25 mM Hepes (pH 7.5), 300 mM NaCl, 10% glycerol, 5 mM MgCl2, 1 mM DTT (performed twice) and this was recovered. Then, for elution, the affinity column was connected to an FPLC (AktaPure - GE) system.

[0129] Dissolution conditions (25 mM Hepes (pH 7.5), 300 mM NaCl, 10% glycerol, 5 mM MgCl2, 1 mM DTT, volume 1 mL, concentration of eluent (CA13080): 66.97 μM (1 mg / mL), flow rate 0.1 mL / min). Subsequently, 8 mL (8 CV) of elution buffer is exchanged with 8 mL (8 CV) of regeneration buffer (200 mM glycine, pH 2.3).

[0130] By measuring the absorbance at 280 nm, the purification of recombinant human synaptophysin 1 (528 - 873) using CA13016 Nb as the immobilized capture agent and CA13080 Nb as the eluent on a HiTrap NHS-activated Sepharose HP column was monitored. The elution peaks were collected in 500 μl fractions and analyzed in an SDS / PAGE gel (Figure 18). From the absorbance profile for the elution chromatogram and the SDS-PAGE analysis of the elution fractions, the inventors concluded that when using Nb (CA13016), a capture agent with micromolar affinity, in combination with Nb (CA13080), a high-affinity eluent, the washing was sufficient to remove most of the impurities, and elution fractions 2 and 3 contained the eluent Nb and human synaptophysin (synaptophysin dimer was also visible on the SDS-PAGE gel) detected within the main peak. Thus, the inventors were able to conclude from this that the use of Nb pairs with different affinities that bind to overlapping epitopes enables the purification of overexpressed recombinant proteins from bacterial lysates.

[0131] [Example 14] Purification of recombinant human factor IXa by NANEX using Nb CA11138 as the immobilized capture agent on a HiTrap NHS-activated Sepharose HP column and eluting with Nb CA10304 as the eluent For further proof of concept, the inventors performed NANEX experiments with a recombinant human coagulation factor IXa-specific capture agent and eluted factor IXa with a high-affinity eluent. This example describes nanobody-exchange chromatography for recombinant human coagulation factor IXa (light chain residues: 134-191, heavy chain residues: 227-461, UniProt accession number UniProtKB:P00740) expressed in E. coli. CA11138 (SEQ ID NO: 11) and CA10304 (SEQ ID NO: 12) are nanobodies selected against human coagulation factor IXa.

[0132] · The BLI assay identified that the affinity (K D ) of CA11138 was 141 nM, k on was 3.4×10 4 (M -1 × sec -1 ), and k off was 4.2×10 -3 (sec -1 ).

[0133] · The BLI assay identified that the affinity (K D ) of CA10304 was 46 nM, k on was 8.8×10 4 (M -1 × sec -1 ), and k off was 3.5×10 -3 (sec -1 ).

[0134] CA11138 and CA10304 have different CDRs but bind very partially to the same epitope on human coagulation factor IXa and are mutually exclusive. 1 mg (67.25 nanomoles) of CA11138 was immobilized on a HiTrap NHS-activated Sepharose HP column (GE) according to the supplier's recommendations. Human coagulation factor IXa was fluorescently labeled using Dylight-647 to follow purification by measuring the absorbance at 650 nm. 0.4 mg of fluorescently labeled (Dylight-647) human coagulation factor IXa was loaded onto the CA11138 column via an injection loop. 10 mL (10 CV) of buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 2.5 mM CaCl2) was passed over the column to wash unbound material. Elution conditions (20 mM Hepes pH 7.5, 150 mM NaCl, 2.5 mM CaCl2, volume 1 mL, concentration of CA10304 eluent: 468.7 μM (7 mg / mL), flow rate 0.1 mL / min). Subsequently, 8 mL (8 CV) of elution buffer was exchanged for 8 ml (8 CV) of regeneration buffer (200 mM glycine, pH 2.3). The purification of human coagulation factor IXa using CA11138 as the immobilized capture agent and CA10304 as the eluent on a HiTrap NHS-activated Sepharose HP column was monitored by measuring the absorbance at 280 nm and 650 nm. Elution peaks were collected in 500 μL fractions and analyzed in an SDS-PAGE gel (Figure 19). From the absorbance profile for the elution chromatogram and the SDS-PAGE analysis for the elution fractions, the inventors conclude that when the capture agent Nb(CA11138) was used in combination with the high-affinity eluent Nb(CA10304), the wash was sufficient to remove most of the impurities and elution fractions 2, 3, and 4 contained the eluent and human coagulation factor IXa detected within the main peak. Thus, the inventors can conclude from this that the use of competing Nb pairs with different affinities that bind to partially overlapping epitopes enables the purification of human coagulation factor IXa.

[0135] [Example 15] Purification of recombinant human coagulation factor IXa·CA10304 complex by NANEX using CA10502 as an immobilized capture agent on a HiTrap NHS-activated Sepharose HP column and eluting with CA10309 as an eluent To further demonstrate that sequential purification using dual NANEX is possible, the inventors performed NANEX experiments with the recombinant human coagulation factor IXa·CA10304 complex eluted in Example 14 for another NANEX purification using different pairs of capture and elution agents that bind to different epitopes compared to the binding agent of Example 14.

[0136] CA10502 (SEQ ID NO: 13) and CA10309 (SEQ ID NO: 14) are nanobodies selected against human coagulation factor IXa. The BLI assay identified that the affinity (K D ) of CA10502 was 74 nM, k on was 7.0×10 4 (M -1 ×sec -1 ) and K off was 4.0×10 -3 (sec -1 ). The BLI assay identified that the affinity (K D ) of CA10309 was 21 nM, k on was 6.2×10 4 (M -1 ×sec -1 ) and K off was 7.3×10 -4 (sec -1 ). CA10502 and CA10309 have different CDRs but partially bind to the same epitope on human coagulation factor IXa and are mutually exclusive.

[0137] 1 mg (73.33 nanomoles) of CA10502 was immobilized on a HiTrap NHS-activated Sepharose HP column (GE) according to the supplier's recommendation. Human coagulation factor IXa was fluorescently labeled using Dylight-647 to follow purification by measuring the absorbance at 650 nm. 2 mL of the recombinant human coagulation factor IXa (Dylight-647)·CA10304 complex eluted by Example 14 was loaded onto the CA10502 column via an injection loop. 10 mL (10 CV) of buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 2.5 mM CaCl2) was passed over the column to wash unbound material.

[0138] Dissolution conditions (20 mM Hepes pH 7.5, 150 mM NaCl, 2.5 mM CaCl2, volume 1 mL, concentration of CA10309 stripping agent: 52.42 μM (0.75 mg / mL), flow rate 0.1 mL / min). Thereafter, 8 mL (8 CV) of elution buffer is exchanged with 8 mL (8 CV) of regeneration buffer (200 mM glycine, pH 2.3). By measuring the absorbance at 280 nm and 650 nm, the purification of human coagulation factor IXa·CA10304 complex using CA10502 as the immobilized capture agent and CA10309 as the stripping agent on a HiTrap NHS-activated Sepharose HP column was monitored. The elution peak was collected in 500 μl fractions and analyzed in an SDS / PAGE gel (Figure 20). From the absorbance profile for the elution chromatogram and the SDS-PAGE analysis for the elution fractions, the inventors found that when the capture agent Nb(CA10502) was used in combination with the high-affinity stripping agent Nb(CA10309), the washing was sufficient to remove most of the impurities and the excess stripping agent Nb (CA10304 used in Example 14), and it can be concluded that elution fractions 2, 3, and 4 contained both stripping agents (CA10309 and CA10304) and human coagulation factor IXa detected within the main peak. The small peaks at 280 nm and 650 nm after 10 CV of elution represent the amount of sample remaining on the column washed off by the regeneration buffer.

[0139] Thus, the inventors can conclude that the use of Nb pairs with different affinities that bind to minimally or partially overlapping epitopes can purify human coagulation factor IXa complexed with CA10304.

[0140] [Example 16] Purification of Recombinant Human Coagulation Factor IXa by tandem nanobody exchange chromatography using CA11138 and CA10502 as immobilized capture agents on a HiTrap NHS-activated Sepharose HP column and eluting with CA10304 and CA14208 as eluents To demonstrate the proof of concept that a protein can be purified by tandem nanobody exchange chromatography (tandem NANEX), the inventors selected two pairs of nanobodies that compete as pairs for two different epitopes on human coagulation factor IXa according to the scheme described in Figure 21.

[0141] To perform tandem NANEX, the inventors connected a first column (the CA11138 column used in Example 14) to a second column (the CA10502 column used in Example 15).

[0142] The first NANEX pair consists of CA11138 as capture agent 1 and CA10304 as eluent 1, and the second NANEX pair consists of CA10502 as capture agent 2 and CA14208 (SEQ ID NO: 15) as eluent 2. CA14208 is a functionalized nanobody derived from CA10309 made on the YgjK scaffold for generating MegaBody. Human coagulation factor IXa was fluorescently labeled using Dylight-647 to follow the purification by measuring the absorbance at 650 nm.

[0143] Fluorescently labeled (Dylight-647), 0.4 mg of human coagulation factor IXa was injected into the column. 5 mL (2.5 CV) of buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 2.5 mM CaCl2) was passed over the column to wash away unbound material. 200 μM of eluent 1 (CA10304) was injected from a 1.5 mL loop pre-rinsed with buffer, followed by 5 mL (2.5 CV) of buffer. Then, 24 μM of eluent 2 (CA14208) was injected onto the column, followed by 10 mL (5 CV) of buffer. Next, 10 mL of regeneration buffer (200 mM glycine, pH 2.3) was applied to remove all proteins from the column. Tandem NANEX was monitored by measuring the absorbance at 280 nm and 650 nm. Elution peaks were collected in 500 μL fractions and analyzed in an SDS / PAGE gel (Figure 22). From the SDS-PAGE analysis of the elution fractions, the inventors conclude that factor IXa can be purified using tandem NANEX by connecting the first column (CA11138 column used in Example 14) to the second column (CA10502 column used in Example 15) according to Figure 21 when using Nb CA10304 and Nb CA14208 as eluents in a stepwise manner.

[0144] [Example 17] Purification of Yeast 60S Ribosomal Subunit Containing RPP1A-GFP Fusion Protein from Yeast Extract by NANEX Chromatography Using Nb CA15816 as an Immobilized Capture Agent and Nb CA12760 as an Eluent Nanobody exchange chromatography (NANEX) is a method where immobilized nanobodies (capture agents) have low affinity and / or high off-rates, while the nanobodies used to competitively elute the target (elution agents) have high affinity and / or low off-rates for the target protein, resulting in optimal yields and purity. To demonstrate that it works quickly and quantitatively, the inventors purified Saccharomyces cerevisiae 60S acidic ribosomal protein P1 alpha (RPP1A, YDL081C, UniProtKB:P05318) containing GFP fused to its carboxy terminus, derived from Saccharomyces cerevisiae extract, by NANEX. The yeast clone (reference number: GFP+35:G8, ThermoFisher Yeast GFP Clone Collection; Hugh et al., 2003) is from a collection of S. cerevisiae yeast strains that express the full-length ORF containing the Aequorea victoria GFP (S65T) tag (Tsien, 1998) at the C-terminus. The GFP fusion protein is integrated into the yeast chromosome via homologous recombination and expressed using the endogenous promoter (Huh et al., 2003).

[0145] 1 mg (66.67 nmol) of CA15816 Nb was immobilized onto a HiTrap NHS-activated Sepharose HP column (GE) according to the supplier's recommendations. 20 mL of clarified yeast lysate (equivalent to the cell pellet of 6 L culture of yeast clone GFP+35:G8 grown in YPD) was loaded onto the CA15816 column using a syringe and washed twice with 10 mL (10 column volumes (CV)) of buffer (100 mM Hepes pH 7.5, 150 mM NaCl). Subsequently, the column was connected to an Akta pure FPLC system (GE) for elution using the elution buffer (100 mM Hepes pH 7.5, 150 mM NaCl, 66.67 μM of the eluent Nb CA12760 (1 mg / mL)) at a flow rate of 0.1 mL / min. The eluate was collected in 100 μl fractions and the major elution peak was analyzed by SDS / PAGE gel (Figure 23). Column regeneration was obtained with 8 CV of 200 mM glycine buffer at pH 2.3.

[0146] By measuring the absorbance at 280 nm (absorbance by protein) and 488 nm (absorbance by GFP fluorophore), the purification of RPP1A-GFP was monitored using CA15816 as the immobilized capture agent and CA12760 as the eluent on a HiTrap NHS-activated Sepharose HP column (Figure 23). From the absorbance profile of the GFP fluorophore at elution and SDS-PAGE analysis of the elution fractions, the inventors were able to conclude that in NANEX, CA15816 can be used as a capture agent in combination with CA12760 as an eluent to purify RPP1A-GFP from yeast lysates. Furthermore, elution fractions 2-11 contained the eluent Nb complexed with RPP1A-GFP and other components of the yeast ribosome, detected within the main peak. Indeed, the inventors analyzed fraction 6 of the elution peak by negative staining electron microscopy (Figure 24) and observed large single particles corresponding to the 60 ribosomal subunit of yeast. Thus, the inventors conclude from this example that NANEX can be used to purify (endogenous) protein complexes from cell lysates by using pairs of Nbs (CA15816 and CA12760, also used in Examples 1 and 11) with different affinities that bind to overlapping epitopes contained within one of the constituent proteins of the multi-protein complex.

[0147] [Example 18] Purification of GFP using Nb CA16695 as the immobilized capture agent and Nb CA16047 as the eluent on HiTrap NHS-activated Sepharose HP For NANEX, the eluent is required to disrupt the interaction between the capture agent and the target and replace the capture agent. This can be achieved for an eluent that binds to the same or overlapping epitope as the capture agent on the target, but with high affinity or low k off and binds (high affinity).

[0148] To further confirm that any eluent-capture agent pair that competitively binds to an epitope on the target can in principle be used for NANEX, the inventors also identified eluent-capture agent pairs (Nb CA16047, Nb CA16695) that competitively bind to different epitopes on GFP, compared to Example 1 (Nb CA12760, Nb CA15816).

[0149] This example describes the affinity purification of recombinant GFP using CA16695 (SEQ ID NO: 19) as a capture agent with nanomolar affinity and CA16047 (SEQ ID NO: 18) as an eluent with low nanomolar affinity.

[0150] To identify the epitope and paratope within the GFP·CA16047 complex, the inventors elucidated the structure of this complex by X-ray crystallography (Figures 25 and 26). The complex containing GFP (depicted in SEQ ID NO: 16) and the GFP-nanobody (SEQ ID NO: 18) crystallized within the space group C121 with the following lattice constants: a = 126.91 Å ± 5%, b = 50.35 Å ± 5%, c = 83.13 Å ± 5%, α = 90°, β = 130.45°, γ = 90°. The crystal structure enabled further delineation of the binding of the eluent-capture agent pair to the epitope, defined herein as formed by all amino acid residues (SEQ ID NO: 16) of the GFP protein that make hydrogen bonds with Nb CA16047. This epitope contains the amino acids Tyr151, Lys156, Lys158, Lys162, Val163, Asn164, Lys166, Asp180, Tyr182 of SEQ ID NO: 16. Based on the crystal structure of GFP·CA16047, the inventors also identified one key binding residue within the paratope of the GFP-specific Nb (Tyr119) that can be mutated for the purpose of reducing the affinity of CA16047 (Figure 27).

[0151]

Table 2

[0152] Based on the structure of GFP·CA16047, Tyr119 of the releasing agent (CA16047) was mutated to Phe (Tyr119phe) to prepare a capturing agent designated as CA16695.

[0153] ·The BLI assay identified that the affinity (K D ) of CA16047 was 6.15 nM, k on was 2.3×10 4 (M -1 × s -1 ), and K off was 9.8×10 -4 (s -1 ) (Figure 27).

[0154] ·The BLI assay identified that the affinity (K D ) of CA16695 was 8.53 nM, k on was 2.0×10 5 (M -1 × s -1 ), and K off was 1.7×10 -3 (s -1 ) (Figure 27).

[0155] Next, the inventors purified GFP by nanobody exchange chromatography (NANEX) using CA16047 and Nb CA16695 as the releasing agent-capturing agent pair, and eluted GFP as a GFP-nanobody complex. Affinity purification of the GFP protein was performed using Nb CA16695 coupled to a HiTrap NHS-activated Sepharose HP column connected to an FPLC (AktaPure-GE) system. The inventors used CA16047 Nb as the releasing agent to specifically elute the GFP protein from this affinity matrix.

[0156] For coupling, 1 mg (66.13 nmol) of CA16695 Nb was immobilized onto a commercially available HiTrap NHS-activated Sepharose HP column (1 mL; GE) according to the supplier's recommendation. 2 mg of GFP was loaded onto the CA16695 Nb coupling column using a syringe. The column was washed twice with 10 CV of wash buffer (100 mM Hepes pH 7.5, 150 mM NaCl). The column was then connected to an Akta system (GE). GFP was eluted with 8 CV of elution buffer (100 mM Hepes pH 7.5, 150 mM NaCl) containing 66.06 μM of the eluent Nb CA16047 (1 mg / mL) at a flow rate of 0.1 mL / min. The purification of GFP using CA16695 Nb as the immobilized capture agent and CA16047 as the eluent on the HiTrap NHS-activated Sepharose HP column was monitored by measuring the absorbance at 280 nm (absorbance by protein) and 488 nm (absorbance by the GFP fluorophore) (Figure 28). The elution peaks were collected in 500 μl fractions and analyzed in an SDS / PAGE gel (Figure 28). The column was regenerated with 8 CV of 200 mM glycine buffer at pH 2.3. From the absorbance profile of the GFP fluorophore at elution and the SDS-PAGE analysis of the elution fractions, the inventors conclude that using the capture agent Nb(CA16695) in combination with the high-affinity eluent Nb(CA16047) enables the purification of the GFP protein. The fact that elution fractions 3 - 9 contained the eluent Nb complexed with the GFP protein, detected within the main peak, indicates that this Nb pair (CA16695 as the capture agent and CA16047 as the eluent) enables the quantitative and rapid purification of GFP and GFP-tagged proteins by NANEX.

[0157] [Example 19] Purification of GST Protein Using Nb CA16240 as an Immobilized Capture Agent and Nb CA16239 as an Eluent on a HiTrap NHS-activated Sepharose HP Column Glutathione S-transferase (GST) is often used as a GST tag for separating and purifying proteins containing GST fusion proteins. This example describes the affinity purification of GST using CA16240 (SEQ ID NO: 21) as a capture agent and CA16239 (SEQ ID NO: 20) as an eluent, which are nanobodies that specifically bind to GST at nanomolar concentrations with low affinity.

[0158] To identify the epitope and paratope within the GST·CA16239 interaction, the inventors elucidated the structure of this complex by X-ray crystallography (Figs. 29 and 30). Crystals containing GST (depicted in SEQ ID NO: 22) and the GST-nanobody (SEQ ID NO: 20) are in the space group C121 with the following lattice constants: a = 69.866 Å ± 5%, b = 72.451 Å ± 5%, c = 79.710 Å ± 5%, α = 90°, β = 93.61°, γ = 90°. This crystal structure made it possible to further delineate an epitope defined herein as being formed by the amino acid residues of the GST protein (SEQ ID NO: 22) that make hydrogen bonds with Nb CA16239. This epitope contains the amino acids Asp160, Tyr164, Pro167, Leu170, Asp171, Lys181, Glu184, His215 of SEQ ID NO: 22. Based on the crystal structure of GST·CA16239, the inventors also identified one key residue (Tyr109) within the paratope region of the GST-specific Nb that can be mutated for the purpose of reducing the affinity of CA16239 (Fig. 30).

[0159] [Table 3]

[0160] Based on the structure of GST·CA16239, one residue (Y109) of the releasing agent (CA16239) was mutated to alanine (Y109A) to prepare a capturing agent designated as CA16240.

[0161] ·The BLI assay identified that the affinity (K D ) of CA16239 was 4.9 nM, k on was 7.2×10 5 (M -1 × s -1 ), and K off was 3.1×10 -3 (s -1 ) (Figure 31).

[0162] ·The BLI assay identified that the affinity (K D ) of CA16240 was 659 nM, k on was 5.1×10 5 (M -1 × s -1 ), and K off was 2.5×10 -1 (s -1 ) (Figure 31).

[0163] Next, the inventors purified GST using nanobody exchange chromatography (NANEX) with CA16239 and CA16240 as the releasing agent-capturing agent pair, and eluted GST as a GST-nanobody complex. Affinity purification of the GST protein was performed using Nb CA16240 coupled to a HiTrap NHS-activated Sepharose HP column c connected to an FPLC (AktaPure-GE) system. The inventors used CA16239 Nb as the releasing agent to specifically elute the GFP protein from this affinity matrix.

[0164] For coupling, 5 mg (326.5 nmol) of CA16240 Nb was immobilized onto a commercially available HiTrap NHS-activated Sepharose HP column (1 mL; GE) according to the supplier's recommendations. 2 mg of GST protein was loaded onto the CA16240 Nb coupling column using a syringe and washed twice with 10 column volumes (CV) of wash buffer (100 mM Hepes pH 7.5, 150 mM NaCl). The column was then connected to an Akta system (GE) and eluted with 8 CV of elution buffer (100 mM Hepes pH 7.5, 150 mM NaCl) containing 129.82 μM of the eluent Nb CA16239 (2 mg / mL) at a flow rate of 0.1 mL / min. The purification of GST using CA16240 Nb as the immobilized capture agent and CA16239 as the eluent on the HiTrap NHS-activated Sepharose HP column was monitored by measuring the absorbance at 280 nm (absorbance by protein) (Figure 32). The elution peaks were collected in 500 μl fractions and analyzed in an SDS / PAGE gel (Figure 32). The column was regenerated with 8 CV of 200 mM glycine buffer, pH 2.3. From the absorbance profile and SDS-PAGE analysis of the elution fractions, the inventors conclude that using the Nb pair (CA16240 as the capture agent and CA16239 as the eluent), which has a much lower k off but nearly equal k on in combination with the high-affinity eluent Nb(CA16239) enables the purification of GST protein. The fact that elution fractions 3 - 7 contained the eluent Nb complexed with the GST protein, detected within the main peak, indicates that this Nb pair (CA16240 as the capture agent and CA16239 as the eluent) enables the quantitative and rapid purification of GST or GST-tagged proteins by NANEX.

[0165] [Example 20] Purification of SMT3 protein using Nb CA16687 as an immobilized capture agent and Nb CA15839 as an eluent on a HiTrap NHS-activated Sepharose HP column The ubiquitin-like protein SMT3 (which is smt3, the yeast SUMO protein) is often used as an smt3 tag to separate and purify proteins containing smt3 fusion proteins.

[0166] This example describes the affinity purification of SMT3 using CA16687 (SEQ ID NO: 24) as a capture agent and CA15839 (SEQ ID NO: 25) as a low-affinity eluent at nanomolar concentrations, which are nanobodies that specifically bind to SMT3 with low affinity at nanomolar concentrations.

[0167] To identify the epitope and paratope within the SMT3·CA15839 interaction, the inventors elucidated the structure of this complex by X-ray crystallography (Figs. 33 and 34). Crystals containing SMT3 (depicted in SEQ ID NO: 25) and the SMT3-nanobody (SEQ ID NO: 23) are in the space group P1211 with the following lattice constants: a = 45.71 Å ± 5%, b = 90.66 Å ± 5%, c = 57.75 Å ± 5%, α = 90°, β = 112.36°, γ = 90°. This crystal structure made it possible to further delineate the epitope defined herein as being formed by the amino acid residues of the SMT3 protein that make hydrogen bonds with Nb CA15839. This epitope contains the amino acids His21, Asn23, Phe34, Lys36, Lys38, Arg45, Asn84 of SEQ ID NO: 25.

[0168] Based on the structure of SMT3·CA15839, the inventors also identified one key residue (Asp50) within the paratope of the SMT3-specific Nb that can be mutated for the purpose of reducing the affinity of CA15839 (Fig. 34).

[0169]

Table 4

[0170] Based on the structure of SMT3·CA15839, one residue (Asp50) of the release agent (CA15839) was mutated to alanine (D50A) to produce a capture agent called CA16687. The capture agent thus produced was used.

[0171] ·The BLI assay identified that the affinity (KD) of CA15839 was 6.7 nM, k on was 3.2×105 (M -1 ×sec -1 ), and K off was 2.1×10 -3 (sec -1 ) (Figure 35).

[0172] ·The BLI assay identified that the affinity (KD) of CA16687 was 29 nM, k on was 3.9×105 (M -1 ×sec -1 ), and K off was 8.0×10 -3 (sec -1 ) (Figure 35).

[0173] Next, the inventors purified SMT3 using nanobody exchange chromatography (NANEX) using CA15839 and CA16687 as the release agent-capture agent pair, and eluted SMT3 as an SMT3·nanobody complex. Affinity purification of the SMT3 protein was performed using Nb CA16687 coupled to a HiTrap NHS-activated Sepharose HP column connected to an FPLC (AktaPure-GE) system. To specifically elute the SMT3 protein from this affinity matrix, the inventors used CA15839 Nb as the release agent.

[0174] For coupling, 1 mg (69.58 nanomoles) of CA16687 Nb was immobilized on a HiTrap NHS-activated Sepharose HP column (1 mL; GE) according to the supplier's recommendations. 2 mg of SMT3 protein was loaded onto the CA16687 Nb coupling column using a syringe and washed twice with 10 column volumes (CV) of wash buffer (100 mM Hepes pH 7.5, 150 mM NaCl). The column was then connected to an Akta system (GE), and elution was performed with 8 CV of elution buffer (100 mM Hepes pH 7.5, 150 mM NaCl) containing 138.73 μM of the eluent Nb CA15839 (2 mg / mL) at a flow rate of 0.1 mL / min. The purification of SMT3 using CA16687 Nb as the immobilized capture agent and CA15839 as the eluent on the HiTrap NHS-activated Sepharose HP column was monitored by measuring the absorbance at 280 nm (absorbance by protein) (Figure 36). The elution peaks were collected in 500 μl fractions and analyzed in an SDS / PAGE gel (Figure 36). Column regeneration was obtained with 8 CV of 200 mM glycine buffer, pH 2.3. From the absorbance profile and SDS-PAGE analysis of the elution fractions, the inventors concluded that using the Nb (CA16687) capture agent in combination with the high-affinity eluent Nb (CA15839) with a low k off but with a nearly equal k on enabled the purification of the SMT3 protein. The fact that elution fractions 3 - 8 contained the eluent Nb complexed with the SMT3 protein, detected within the main peak, indicates that this Nb pair (CA16687 as the capture agent and CA15839 as the eluent) enables the quantitative and rapid purification of SMT3 and SMT3-tagged proteins by NANEX. Thus, the SMT3-NANEX pair can be used to specifically purify post-translationally modified proteins from yeast.

[0175] [Example 21] Purification of mCherry-fusion protein using Nb CA16964 as an immobilized capture agent and Nb CA17302 as an eluent on a HiTrap NHS-activated Sepharose HP column mCherry is a member of the mFruits family of monomeric red fluorescent proteins, derived from DsRed of the sea anemone, Discosoma sea. Similar to GFP, mCherry is often used to tag intracellular proteins and can be studied using fluorescence spectroscopy and fluorescence microscopy. This example describes the affinity purification of mCherry-fusion protein using Nb CA16964 (SEQ ID NO: 26) as a capture agent and Nb CA17302 (SEQ ID NO: 27) as an eluent, which are nanobodies that specifically bind to mCherry with low affinity at nanomolar concentrations.

[0176] CA16964 and CA17302, which are Nbs, are two non-related nanobodies derived from different sequence families, generated by immunizing llamas with mCherry and selected by phage display against this antigen following standard procedures (Pardon, 2014). Epitope mapping using BLI indicated that CA17302 and CA16964 compete for overlapping epitopes on mCherry and bind to this fluorescent protein in a mutually exclusive manner (Figure 37). Each Nb was further characterized by BLI (Figure 38) and the following values were obtained.

[0177] [Table 5]

[0178] ·The BLI assay showed that the affinity (K D ) of CA17302 was 1.7 nM and k on was 6.2×10 5 (M -1 ×sec-1 ) and K off was identified to be 1×10 -3 (seconds -1 ) (Figure 38).

[0179] · The BLI assay showed that the affinity (K D ) of CA16964 was 6.53 nM, and k on was 6.94×10 5 (M -1 × second -1 ), and K off was 4.4×10 -3 (seconds -1 ) (Figure 38).

[0180] Next, the inventors purified FmlH_lectin_mCherry_his (SEQ ID NO: 29), a fusion protein containing mCherry, FmlH lectin derived from uropathogenic E. coli strain, and a short-chain His tag, by nanobody exchange chromatography (NANEX) using Nb CA17302 and Nb CA16964 as the stripping agent-capture agent pair, and eluted the FmlH_lectin_mCherry_his·nanobody complex. Affinity purification was performed using Nb CA16964 coupled to HiTrap NHS-activated Sepharose HP, which was connected to an FPLC (AktaPure-GE) system. To specifically elute the FmlH_lectin_mCherry_his fusion protein from this affinity matrix, the inventors used CA17302 Nb as the stripping agent.

[0181] For coupling, 1 mg (68 nmol) of CA16964 Nb was immobilized on a HiTrap NHS-activated Sepharose HP column (1 mL; GE) according to the supplier's recommendations. 50 mL of lysate (derived from 2 L of bacterial culture of overexpressed recombinant FmlH_lectin_mCherry_his) was loaded onto the CA16964 Nb coupling column using a syringe and washed twice with 10 CV of wash buffer (100 mM Hepes pH 7.5, 150 mM NaCl). The column was then connected to an Akta system (GE), followed by elution with 8 CV of elution buffer (100 mM Hepes pH 7.5, 150 mM NaCl) containing 65.5 μM of the eluent Nb CA17302 (1 mg / mL) at a flow rate of 0.1 mL / min. The purification of FmlH_lectin_mCherry_his using CA16964 Nb as the immobilized capturer and CA17302 as the eluent was monitored by measuring the absorbance at 280 nm (absorbance by protein) (Figure 39). The elution peaks were collected in 500 μl fractions and analyzed in an SDS / PAGE gel (Figure 39). The regeneration of the column was obtained with 8 CV of 200 mM glycine buffer, pH 2.3. From the SDS-PAGE analysis of the elution chromatogram and elution fractions, we conclude that the pair of Nbs (CA16964 as the capturer and CA17302 as the eluent), with the capturer Nb having a low k off accompanied by, but with a nearly equal k on accompanied by, the high-affinity eluent Nb(CA17302) enables the purification of the FmlH_lectin_mCherry_his protein. The fact that elution fractions 4 - 8 contained the eluent Nb complexed with the FmlH_lectin_mCherry_his fusion protein detected within the main peak indicates that this pair of Nbs (CA16964 as the capturer and CA17302 as the eluent) enables the quantitative and rapid purification of the mCherry fusion protein by NANEX.

[0182] [Example 22] Purification of mCherry-fusion protein using Nb CA17341 as an immobilized capture agent and Nb CA17302 as an eluent on a HiTrap NHS-activated Sepharose HP column This example describes the affinity purification of FmlH_lectin_mCherry_his fusion protein using Nb CA17341 (SEQ ID NO: 28) as a capture agent and Nb CA17302 (SEQ ID NO: 27) as an eluent with low affinity in nanomolar concentration, which is a nanobody that specifically binds to mCherry with low affinity in nanomolar concentration.

[0183] Nb CA17341 was obtained by performing an alanine scan on the CDR3 of Nb CA17302 starting from Nb CA17302. The mutation of Ile101 to alanine enabled the conversion of the eluent to a capture agent without prior structural information on the Nb-antigen interaction. CA17341 was further characterized by BLI (Figure 40), and the following values were obtained.

[0184] [Table 6]

[0185] ·The BLI assay identified that the affinity (K D ) of CA17302 was 1.7 nM, k on was 6.2×10 5 (M -1 ×sec -1 ) and K off was 1×10 -3 (sec -1 ) (Figure 40).

[0186] ·The BLI assay identified that the affinity (K D ) of CA17341 was 4.1 nM, k on was 6×10 5 (M -1 ×sec -1 ) and Koff was identified as 2.3×10 -3 (seconds -1 ) (Figure 40).

[0187] Next, the inventors purified the FmlH_lectin_mCherry_his fusion protein by nanobody exchange chromatography (NANEX) using Nb CA17341 and Nb CA17302 as the release agent-capture agent pair, and eluted the target as the FmlH_lectin_mCherry_his·nanobody complex. Affinity purification was performed with Nb CA17341 immobilized on a HiTrap NHS-activated Sepharose HP column connected to an FPLC (AktaPure-GE) system. To specifically elute the FmlH_lectin_mCherry_his fusion protein from this affinity matrix, the inventors used CA17302 Nb as the release agent.

[0188] For coupling, 1 mg (65.69 nmol) of CA17341 Nb was immobilized onto a HiTrap NHS-activated Sepharose HP column (1 mL; GE) according to the supplier's recommendations. 50 mL of cell lysate (collected from 2 L of a culture of E. coli overexpressing the FmlH_lectin_mCherry_his fusion protein) was loaded onto the CA16964 Nb coupling column. The column was washed twice with 10 column volumes (CV) of wash buffer (100 mM Hepes pH 7.5, 150 mM NaCl) and connected to an Akta system (GE). Next, the fusion protein was eluted from the column with 8 CV of elution buffer (100 mM Hepes pH 7.5, 150 mM NaCl) containing 65.5 μM of the eluent Nb CA17302 (1 mg / mL) at a flow rate of 0.1 mL / min. The purification of FmlH_lectin_mCherry_his using CA17341 Nb as the immobilized capture agent and CA17302 as the eluent on the HiTrap NHS-activated Sepharose HP column was monitored by measuring the absorbance at 280 nm (absorbance by protein) and at 585 nm (absorbance by mCherry) (Figure 41). The elution peaks were collected in 500 μl fractions and analyzed in an SDS / PAGE gel (Figure 41). Column regeneration was obtained with 8 CV of 200 mM glycine buffer, pH 2.3. From the SDS-PAGE analysis of the elution fractions, the inventors could conclude that using the capture agent Nb (CA17341) in combination with the high-affinity eluent Nb (CA17302) enables the purification of the FmlH_lectin_mCherry_his protein. The fact that elution fractions 4 - 8 contained the eluent Nb complexed with the FmlH_lectin_mCherry_his fusion protein, detected within the main peak, indicates that this Nb pair (CA17341 as the capture agent and CA17302 as the eluent) enables rapid and quantitative purification of the mCherry fusion protein by NANEX.This example also shows that a simple alanine scan for CDR3 is a rapid and easy way to convert a release agent into a capture agent without the need for structural information.

[0189] [Example 23] Purification of native human coagulation factor IX using Nb CA11143 as an immobilized capture agent on HiTrap NHS-activated Sepharose HP and CA16383 (an engineered antigen-binding protein derived from Nb CA14208) as a release agent The concept of nanobody exchange chromatography (NANEX) is not limited to the use of nanobodies as release agents and / or capture agents. In fact, any protein pair that competes for binding to the same epitope of a target: antibody, megobody, darpin, synthetic binding protein, etc. can be used to purify this target according to the principle of NANEX. In this example, the inventors show that a certain nanobody can be used in combination with a megobody to purify human coagulation factor IX according to the principle of NANEX. Megobodies are engineered antigen-binding proteins obtained by grafting nanobodies onto a selected protein scaffold so as to increase their molecular weight while retaining their complete antigen-binding specificity and affinity, as previously described. This example is the same as Example 16, except that the inventors used a megobody (CA16383) derived from Nb CA14208 as a release agent. In this way, the inventors used NANEX to purify native protein from its native source (human blood) to prepare for structural characterization by cryo-EM from human plasma complexed with megobody.

[0190] More specifically, this example describes the affinity purification of human coagulation factor IX from plasma derived from human whole blood treated with an ACD anticoagulant, using CA11143 (SEQ ID NO: 31), which is an immobilized nanobody as a capture agent, and CA16383 (SEQ ID NO: 30), which is a MegaBody as a release agent.

[0191] For coupling, 3.7 mg (250.97 nanomoles) of CA11143 Nb was immobilized onto a HiTrap NHS-activated Sepharose HP column (1 mL; GE) according to the supplier's recommendations. 30 mL of plasma derived from human whole blood treated with ACD anticoagulant (Tebu-Bio, SER-PLE200ML-ACD) was loaded onto the CA11143 Nb coupling column by recirculation over 120 minutes using a peristaltic pump. Next, the column was washed with 15 column volumes (CV) of wash buffer (20 mM Hepes pH 8.0, 150 mM NaCl, 5 mM CaCl2). The column was then connected to an Akta system (GE), and factor IX was eluted with 1 mL of buffer (20 mM Hepes pH 8.0, 150 mM NaCl, 5 mM CaCl2) containing 9.92 μM CA16383, a MegaBody that was used as an eluent at a flow rate of 0.05 mL / min. The purification process was monitored by measuring the absorbance at 280 nm (absorbance by protein) (Figure 42). The elution peak was collected in 750 μl fractions, and the fractions were analyzed in an SDS / PAGE gel and by Western blot containing commercially available human coagulation factor IX as a positive control (Figure 42). Column regeneration was obtained with 5 CV of 200 mM glycine buffer, pH 2.3. From the SDS-PAGE analysis of the elution fractions, the inventors could conclude that using the capture agent Nb (CA11143) in combination with the functionalized eluent Nb (CA16383) enabled the purification of human coagulation factor IX protein from plasma. That fractions 4 and 5 corresponding to the main elution peak contained purified human coagulation factor IX protein complexed with the MegaBody indicates that a MegaBody can be combined with a nanobody as an eluent-capture agent pair to purify native proteins from native complex sources.

[0192] [Example 24] CA16388, a MegaBody as an immobilized capture agent on HiTrap NHS-activated Sepharose HP and a functionalized nanobody as an eluent (MegaBody MbCA10309 YgjK Purification of native human coagulation factor IX using Nb CA16383, which is ()) As shown below in Example 23, a nanobody can be used as a capture agent in combination with MegaBody to strip the target from the functionalized resin. In Example 24, the inventors used MegaBody as a capture agent in combination with another MegaBody as a stripping agent to purify human coagulation factor IX from human serum. To demonstrate this principle, the inventors used NANEX to purify human coagulation factor IX complexed with MegaBody CA16383 using MegaBody CA16388 as a capture agent and MegaBody CA16383 as a stripping agent from human plasma treated with ACD anticoagulant. A NANEX experiment was conducted to purify native human coagulation factor IX.

[0193] For coupling, 1.1 mg (10.87 nanomoles) of CA16388, which is MegaBody, was immobilized onto a HiTrap NHS-activated Sepharose HP column (1 mL; GE) according to the supplier's recommendations. 30 mL of plasma derived from human whole blood treated with ACD anticoagulant (Tebu-Bio, SER-PLE200ML-ACD) was loaded onto the CA16388 Nb coupling column by recirculation over 60 minutes using a peristaltic pump. The column was washed with 15 column volumes (CV) of wash buffer (20 mM Hepes pH 8.0, 150 mM NaCl, 5 mM CaCl2). The washed column was then connected to an Akta system (GE), and factor IX was eluted with 1 mL of buffer (20 mM Hepes pH 8.0, 150 mM NaCl, 5 mM CaCl2) containing 9.92 μM of CA16383, which is MegaBody, as an eluent at a flow rate of 0.05 mL / min. The purification process was monitored by measuring the absorbance at 280 nm (absorbance by protein) (Figure 43). The elution peak was collected in 750 μl fractions and analyzed in an SDS / PAGE gel and by Western blot containing commercially available human coagulation factor IX as a control (Figure 43). Column regeneration was obtained with 5 CV of 200 mM glycine buffer, pH 2.3. From the SDS-PAGE analysis and Western blot analysis of the elution fractions, the inventors were able to conclude that MegaBody CA16388 and MegaBody CA16383 as a capture agent-eluent pair enabled the purification of human coagulation factor IX protein from plasma by NANEX. Elution fractions 4 and 5 contained the eluent Nb complexed with the human coagulation factor IX protein detected within the main peak.

[0194] [Example 25] Purification of human GFP-tagged glucocorticoid receptor (GFP-GR) complexed with its native molecular chaperone from HEK293T cell lysates using Nb CA15816 as an immobilized capture agent and Nb CA12670 as an eluent on HiTrap NHS-activated Sepharose HP Since the NANEX technology does not require high salt concentrations or extreme pH conditions for elution and can be carried out completely under natural conditions (pH, buffer composition, temperature, ···), this method is also applicable to the purification of (transient) protein - protein complexes from natural sources. To demonstrate this principle, the inventors tagged the human glucocorticoid receptor (GR) with GFP, expressed this fusion protein in a human cell line, and complexed the purified receptor with its molecular chaperones, Hsp70 and Hsp90 (heat shock proteins). From the literature, it is known that apo - GR is mainly cytoplasmic and associates with heat shock proteins and immunophilins within the so - called foldosome under its resting state (Pratt et al., 1997). Thus, the inventors used CA15816 (SEQ ID NO: 3) as an immobilized capture agent (a medium - affinity capture agent for GFP) on a HiTrap NHS - activated Sepharose HP column and Nb CA12670 (SEQ ID NO: 1) as a high - affinity eluent for the GFP - tagged GR receptor complexed with its molecular chaperones to purify the eGFP - tagged recombinant human glucocorticoid receptor (SEQ ID NO: 34) complexed with Hsp70 and Hsp90 from human HEK293T cells.

[0195] For coupling, 1 mg (66.955 nanomoles) of CA15816 Nb was immobilized onto a HiTrap NHS-activated Sepharose HP column (1 mL; GE) according to the supplier's recommendations. Recombinant human eGFP-6His-TEV-GR was expressed using a pcDNA3.1+N-eGFP vector transfected into human HEK239T cells grown in 150×21 mm dishes (Nunclon(™) Delta) using X-tremeGENE(™) 9 DNA Transfection Reagent (XTG9-RO Roche) for transient expression. After transfection, HEK239T cells were grown at 37 °C under 5% CO2 for 48 hours. Cells from three plates were harvested by pipetting and centrifugation, washed with PBS, and resuspended in 10 mL of lysis buffer containing 10 mM sodium phosphate pH 8, 5 mM DTT, 0.1 mM EDTA, 10 mM Na2MoO4, 10% glycerol (supplemented with protease inhibitors) and then lysed using a Dounce homogenizer. The lysate was clarified by centrifugation, the supernatant was recovered, filtered (0.45 μm filter), and loaded onto a HiTrap NHS-activated Sepharose HP column coated with CA15816 Nb using a syringe. Using a syringe, 10 CV of wash buffer (50 mM Hepes pH 7.5, 150 mM NaCl) was applied onto the column. The affinity column was then connected to an FPLC (AktaPure-GE) system and the foldosome was eluted with 1 mL of elution buffer containing 66.7 μM CA15816 Nb eluent (1 mg / mL) in 50 mM Hepes pH 7.5, 150 mM NaCl at a flow rate of 0.1 mL / min. Column regeneration was obtained using 5 CV of 200 mM glycine buffer, pH 2.3. The purification process was monitored by measuring the absorbance at 280 nm (absorbance by protein) and 488 nm (eGFP) (Figure 44).The elution peak was recovered in a 500 μL fraction and analyzed in an SDS / PAGE gel and by Western blot using a commercially available anti-human glucocorticoid receptor antibody (anti-GRG-5, Santa Cruz) (Figure 44). The three major bands observed in the SDS-PAGE analysis of the elution fraction were excised and analyzed by mass spectrometry. The major bands were identified as human glucocorticoid receptor, HSP90, and HSP70 proteins, respectively (Figure 44). Hsp70 and Hsp90 are molecular chaperones known to form a complex (foldosome) with nuclear receptors such as the human glucocorticoid receptor in the cytoplasm, and NANEX enables the rapid and easy purification of native protein complexes containing eGFP-tagged GR, Hsp70, Hsp90, and the eluent, especially from transfected HEK293T cells.

[0196] [Example 26] Purification of recombinant human GFP-tagged androgen receptor (GFP-ARb) complexed with molecular chaperones from HEK293T cell lysate using Nb CA15816 as an immobilized capturer and Nb CA12670 as an eluent on HiTrap NHS-activated Sepharose HP As demonstrated in Example 25, NANEX can also be used to purify (transient) protein complexes. To further demonstrate this principle, the inventors also purified the human androgen receptor complexed with its molecular chaperones Hsp70 and Hsp90 and immunophilin in the so-called foldosome from HEK293T cells (Pratt et al., 1997).

[0197] This example describes the purification of eGFP-tagged recombinant human androgen receptor (SEQ ID NO: 35) complexed with Hsp70 and Hsp90 from human HEK293T cell lysates using CA15816 (SEQ ID NO: 3) as an immobilized capture agent (medium affinity capture agent for GFP) and Nb CA12670 (SEQ ID NO: 1) as an elution agent (high affinity elution agent for GFP) by NANEX.

[0198] For coupling, 1 mg (66.955 nanomoles) of CA15816 Nb was immobilized onto a HiTrap NHS-activated Sepharose HP column (1 mL; GE) according to the supplier's recommendations.

[0199] Recombinant human eGFP-6His-TEV-AR was expressed using the pcDNA3.1+N-eGFP vector transfected into human HEK239T cells. In 150×21 mm dishes (Nunclon™ Delta), cells were transfected using X-tremeGENE™ 9 DNA Transfection Reagent (XTG9-RO Roche) with a μL ratio of 3:1 per μg of DNA. After transfection, HEK239T cells were grown for 48 hours at 37 °C and 5% CO2. Cells from three plates were collected by pipetting and centrifugation, washed with PBS, and resuspended in 10 mL of lysis buffer containing 10 mM Hepes 7.5, 2.5 mM DTT, 1 mM EDTA, 20 mM Na2MoO4, 10% glycerol (supplemented with protease inhibitors), and then lysed using a Dounce homogenizer. The lysate was clarified by centrifugation, the supernatant was collected, filtered (0.45 μm filter), and loaded onto a HiTrap NHS-activated Sepharose HP column coated with CA15816 Nb using a syringe. Next, the column was washed with 10 CV of wash buffer (10 mM Hepes 7.5, 2.5 mM DTT, 1 mM EDTA, 20 mM Na2MoO4, 10% glycerol) using a syringe. Then, the affinity column was connected to an FPLC (AktaPure-GE) system for elution with 1 mL of elution buffer containing 66.7 μM of the eluent (1 mg / mL), which is CA15816 Nb, in 10 mM Hepes 7.5, 2.5 mM DTT, 1 mM EDTA, 20 mM Na2MoO4, 10% glycerol at a flow rate of 0.1 mL / min over 6 mL (6 CV). Column regeneration was obtained with 5 CV of 200 mM glycine buffer, pH 2.3. The purification process was monitored by measuring the absorbance at 280 nm (absorbance by protein) and 488 nm (absorbance by eGFP) (Figure 45).The elution peak was recovered within a 500 μl fraction and analyzed on an SDS-PAGE gel and by Western blot developed with a commercially available anti-GFP antibody (Figure 45). Three of the most prominent bands from the SDS-PAGE analysis of the elution fraction were excised and analyzed by mass spectrometry. The bands were identified as human androgen receptor (ARb) protein, Hsp90 protein, and Hsp70 protein (Figure 45). Hsp70 and Hsp90 are molecular chaperones known to form complexes with nuclear receptors such as the human androgen receptor in the cytoplasm. This example confirms that NANEX enables the rapid and easy purification of native protein complexes containing eGFP-tagged androgen receptor, Hsp70, Hsp90, and the eluent, especially from transfected HEK293T cells.

[0200] [Example 27] Purification of diverse GFP-tagged fusion proteins from yeast (S. cerevisiae) cell lysates by high-throughput nanobody exchange chromatography (NANEX) using Nb CA15816 as an immobilized capturer on magnetic tosyl-activated Dynabeads (registered trademark) and Nb CA12760 as an eluent Nanobody exchange chromatography (NANEX), which mixes and separates a solid phase with a liquid phase, is not limited to the purification of proteins on beads packed in a column. In Example 27, the inventors used magnetic beads in combination with a magnet to mix and exchange a solid and a liquid in an automated high-throughput method prepared to purify 12 different proteins in parallel within a standard 96-well plate on a KingFisher Flex (ThermoFisher) instrument.

[0201] For this experiment, a set of 12 yeast clones was selected from the yeast GFP clone collection (Huh et al., 2003). Since each clone expresses a different protein as a fusion with GFP, it can be purified by NANEX using Nb CA15816 as the capture agent and Nb CA12760 as the eluent.

[0202] The 96-well format can be used to grow small cultures (1 mL) of yeast cells, which allows for the possibility of transforming, transfecting, inducing, or lysing these in parallel with different cell lines. This enables rapid and high-throughput processes for downstream applications such as protein expression, protein purification, ELISA, functional assays, etc.

[0203] To demonstrate the feasibility of this principle, the inventors performed NANEX experiments to purify 12 different yeast proteins expressed in 1 mL cultures and lysed and purified in parallel in standard 96-well plates. As the solid support, the inventors used tosyl-activated Dynabeads® coated with Nb CA15816 (SEQ ID NO: 3) as the capture agent. The purification process was carried out in 96-well plates on a KingFisher Flex (ThermoFisher) instrument and included an elution step using Nb CA12760 (SEQ ID NO: 1) as the eluent.

[0204] Given that these clones were not overexpressed and were made at their physiological expression levels, the inventors were able to select yeast housekeeping proteins expressed at high levels, but also proteins expressed at low levels within viable cells (Figure 46).

[0205] A set of 12 selected clones expressing GFP-tagged proteins at different levels (Figure 46) was grown in 1 mL of YPD medium in a 96-well deep-well plate for 72 hours. The pellets were lysed in Y-PER™ Plus (ThermoFisher) for 1 hour, frozen, thawed, and then spun. The recovered lysate was used as a source for the purification of GFP fusion proteins by NANEX with a KingFisher Flex device, where Kingfisher is a multi-purpose, benchtop, automated extraction device capable of processing magnetic beads in a 96-well format. Tosyl-activated magnetic Dynabeads® were coupled with the capture agent Nb CA15816 at a concentration of 40 mg of capture agent per mg of beads according to the manufacturer's instructions. 5 μL of beads per mL of solution per well (corresponding to 20 mg of coupled capture agent CA15816) were used to immobilize and purify the proteins from individual clones in high-throughput mode. The parallel purification procedure involved an automated 30-second incubation of the beads with 100 mL of pre-equilibrated PBS buffer, a 30-minute incubation with the lysate (for target binding), and three 1-minute washes with PBS buffer. 40 μL of the eluent Nb CA12760 was used at a concentration of 0.5 mg / mL (33.35 μM) in PBS buffer to elute the proteins from the magnetic beads for 15 minutes. From SDS-PAGE analysis and Western blot analysis for different steps of the purification, the inventors conclude that the endogenously expressed GFP fusion proteins can be captured on Nb CA15816-beads and these beads can be specifically eluted by using an appropriate eluent (Figure 47).

[0206] [Example 28] Purification of GFP by nanobody exchange chromatography using Nb CA15816 as an immobilized capture agent on magnetic NHS-activated agarose beads packed in a microfluidic column of less than 1 microliter (<1 μL) and using Nb CA12760 as a releasing agent High-throughput applied proteomics research and single-cell research require protein purification methods that require small devices (downscaling) to separate proteins from small amounts of sample, among other things. Example 28 illustrates how the inventors designed a microfluidic chip to downscale their NANEX technology to a column volume of less than 1 microliter (<1 μL) and reduce the amount of (immobilized) capture agent and releasing agent required to purify the protein of interest from a small amount of sample.

[0207] On the microfluidic device, to purify GFP, the inventors covalently immobilized the capture agent Nb CA15816 (see Example 1) onto 50 μl of commercially available magnetic NHS-activated agarose beads (30 μm in diameter, Cube Biotech) according to the supplier's recommendations. 0.7 μl of these agarose beads were packed into a small chamber (<1 μL) of a custom-made microfluidic chip, detailed in Figure 48. This 30×30×6 mm microfluidic chip was fabricated by milling two 0.420 mm deep channels and a 0.420 mm deep chamber into a 3 mm PMMA plate with a milling machine (Datron model M7). Next, a 3 mm PMMA cover plate was sealed with (-)-butyl L-lactate (Sigma-Aldrich), and an inlet capillary (fused silica, OD: 0.360 mm, ID: 0.250 mm, CM Scientific) and a small-diameter outlet capillary (fused silica, OD: 0.360 mm, ID: 0.075 mm, CM Scientific) were inserted into the channels and cured with an adhesive (Norland Optical Adhesive 85) and UV light. The dimensions of the chamber and channels with the inserted capillaries are depicted in Figure 48. The inlet capillary was connected to a 50 μL syringe (Hamilton Company) actuated by a syringe pump (World Precision Instruments model SP100iZ), and all different reagents (washing buffer, sample, elution buffer, and glycine buffer) were injected into the chip. A 0.7 μl slurry containing the capture agent-functionalized magnetic beads was injected via a wide-diameter capillary. Since the beads do not pass through the outlet capillary (ID: 0.075 mm), the beads fill the small chamber, constituting a small chromatography device (<1 μL), and the two capillaries function as the inlet and outlet of the column (Figure 48).

[0208] This CA15816 Nb coupling microfluidic column (with 1CV being 0.7 μl) was first washed with 50 μl (70 CV) of washing buffer (PBS) at a flow rate of 10 μL / min. Next, 40 μl of a sample containing GFP (25 mM Hepes pH 7.4, 150 mM NaCl, 4.8 μg of GFP (0.12 mg / mL)) was injected at 10 μL / min. Next, 90 μl of washing buffer (PBS) was passed through the column at 10 μL / min to remove unbound material. Next, 52 μl (74 CV) of elution buffer containing an eluent (25 mM HEPES pH 7.4, 150 mM NaCl, 26 μg of the eluent Nb CA12760 (0.5 mg / mL)) was applied to elute affinity-captured GFP from the microfluidic device. After washing with 80 μl of washing buffer, regeneration of the column was obtained by injecting 50 μl of glycine buffer (200 mM glycine buffer, pH 2.3 at 10 μL / min). The capture of GFP on the microcolumn using Nb CA15816, the elution of GFP from the microcolumn by Nb CA12760, and the regeneration of the microcolumn by glycine were monitored using an inverted fluorescence microscope (Olympus Corporation, IX71 model IX71S1F-3) (Figure 49). Also, the first wash solution, the flow-through of the sample, the second wash solution, the eluent solution, the third wash solution, and the glycine eluate were also collected from the outlet capillary into 1.5 mL Eppendorf tubes, placed in a blue light transilluminator, and the presence or absence of GFP in different fractions was visualized (Figure 49). In accordance with the principle of NANEX, it was confirmed that GFP was actually captured and eluted from this microfluidic column using a minimum amount of capture agent and eluent.

[0209] Expression and purification of nanobody Following the nanobody containing a C-terminal His6 tag, the nanobody containing an EPEA tag was routinely expressed and purified from the periplasm of the E. coli strain WK6 (Pardon et al., 2014).

[0210] Sequence listing >SEQ ID NO:1: GFP-Nb207, CA12760 (containing C-terminal 6×His+EPEA tag) >SEQ ID NO:2: GFP-NbF103A, CA15818 (mutated residues are bold and underlined; C-terminal 6×His+EPEA) QVQLVESGGGLVQAGGSLRLSCAASGRTFSTAAMGWFRQAPGKERDFVAGIYWTVGSTYYADSAKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCAARRRG A TLAPTRANEYDYWGQGTQVTVSSHHHHHHEPEA >SEQ ID NO:3: GFP-NbT54A / V55A, CA15816 (mutated residues are bold and underlined; C-terminal 6×His+EPEA) QVQLVESGGGLVQAGGSLRLSCAASGRTFSTAAMGWFRQAPGKERDFVAGIYW AA GSTYYADSAKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCAARRRGFTLAPTRANEYDYWGQGTQVTVSS HHHHHHEPEA >SEQ ID NO:4: GFP-NbT54A / V55A / F103A, CA15861 (mutated residues are bold and underlined; C-terminal 6×His+EPEA) QVQLVESGGGLVQAGGSLRLSCAASGRTFSTAAMGWFRQAPGKERDFVAGIYW AA GSTYYADSAKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCAARRRG A TLAPTRANEYDYWGQGTQVTVSS HHHHHHEPEA >SEQ ID NO:5: Mb Nb207 cHopQ CA15621 (C-terminal 6×His) >SEQ ID NO:6: Mb Nb207 YgjK CA15616 (C-terminal 6×His) >SEQ ID NO:7: EPEA-NbSyn2, CA4375 (C-terminal 6×His) >SEQ ID NO:8: Di-valent EPEA-NbSyn2 EPEA, CA4394 (C-terminal 6×His) >SEQ ID NO:9: Synaptophysin-specific Nb CA13016 (capture agent; C-terminal 6×His + EPEA) >SEQ ID NO:10: Synaptophysin-specific Nb CA13080 (stripping agent; C-terminal 6×His + EPEA) >SEQ ID NO:11: FIXa-specific Nb CA11138 (capture agent 1; C-terminal 6×His + EPEA) >SEQ ID NO:12: FIXa-specific Nb CA10304 (stripping agent 1; C-terminal 6×His + EPEA) >SEQ ID NO:13: FIXa-specific Nb CA10502 (capture agent 2; C-terminal 6×His + EPEA) >SEQ ID NO:14: FIXa-specific Nb CA10309 (stripping agent 2; C-terminal 6×His + EPEA) >SEQ ID NO:15: FIXa-specific MegaBody Mb NbFIXa cHopQ which is CA14208 (stripping agent 2; C-terminal 6×His + EPEA) >SEQ ID NO:16: GFP protein >SEQ ID NO:17: Adhesin domain protein of Helicobacter pylori G27 HopQ strain (PDB 5LP2) >SEQ ID NO:18: CA16047 (including C-terminal 6×His + EPEA tag), which is the second GFP nanobody stripping agent >SEQ ID NO:19: CA16695 (Y119F) (including C-terminal 6×His + EPEA tag), which is the second GFP nanobody capture agent >SEQ ID NO:20: CA16239 (including C-terminal 6×His + EPEA tag), which is the GST nanobody stripping agent >SEQ ID NO:21: CA16240 (Y109A) (including C-terminal 6×His + EPEA tag), which is the GST nanobody capture agent >SEQ ID NO:22: GST (glutathione S-transferase) >SEQ ID NO:23: CA15839 (including C-terminal 6×His + EPEA tag), which is the SMT3 nanobody stripping agent >SEQ ID NO:24: CA16687 (D50A) (containing C-terminal 6×His+EPEA tag), an SMT3 nanobody capture agent >SEQ ID NO:25: SMT3 (YDR510W) >SEQ ID NO:26: CA16964 (containing C-terminal 6×His+EPEA tag), an mCherry nanobody capture agent >SEQ ID NO:27: CA17302 (containing C-terminal 6×His+EPEA tag), an mCherry nanobody stripping agent >SEQ ID NO:28: CA17341 (I103A mutant of CA17302) (containing C-terminal 6×His+EPEA tag), an mCherry nanobody capture agent >SEQ ID NO:29: mCherry (FmlH_lectin_mCherry_his) (CA17337) >SEQ ID NO:30: CA16383 (MegaBody Mb containing C-terminal 6×His+EPEA tag), a FIX-functionalized nanobody stripping agent CA10309 YgjK ) >SEQ ID NO:31: CA11143 (containing C-terminal 6×His+EPEA tag), a FIX nanobody capture agent >SEQ ID NO:32: E. coli Ygjk protein (PDB 3WFS) >SEQ ID NO:33: CA16388 (MegaBody Mb containing C-terminal 6×His+EPEA tag), a FIX-functionalized nanobody capture agent CA11143 YgjK ) >SEQ ID NO:34: P04150 (eGFP-6His-TEV-GR), an eGFP-tagged human recombinant glucocorticoid receptor by CA16607 >SEQ ID NO:35: P10275 (eGFP-6His-TEV-ARb), an eGFP-tagged human recombinant androgen receptor by CA16976

[0211] Aspects of the present disclosure: A method for purifying a target protein, comprising: · Mixing a first protein binder that specifically binds to an epitope of a target protein with a sample containing the target protein; · Adding to the mixture of a) a second protein binder that recognizes an epitope of the target protein that is the same as or mostly overlapping with the first binder and specifically binds to the target protein to displace the first binder from the target protein; and · Recovering the second protein binder that complexes with the target protein and elutes. comprising The second protein binder comprises an immunoglobulin single variable domain (ISVD) that specifically binds to the epitope or an active fragment thereof; The dissociation rate constant (k off value) of the second protein binder is lower compared to the k off value of the first binder Method.

[0212] The method described herein, wherein the second protein binder has a lower or higher affinity for the epitope compared to the first protein binder.

[0213] The K D value for the epitope of the target protein is in the range of 1 μM to 1 nM for the first protein binder and in the range of 1 nM to 1 pM for the second protein binder, the method described herein.

[0214] The K D value for the first protein binder is 200 to 5000 times compared to the K D value for the second protein binder, the method described herein.

[0215] The method as any of the methods described herein, wherein the first protein binder is immobilized and the second protein binder is in solution.

[0216] A method as any of the methods described herein, wherein the second protein binder comprises a functional moiety or a detectable label.

[0217] A method as any of the methods described herein, wherein the sample is a biological sample, a complex mixture, a cell sample or an in vitro sample.

[0218] A method as any of the methods described herein, wherein the epitope of the target protein comprises a tag, preferably the tag is selected from the group consisting of GFP, GST, SUMO, ubiquitin and EPEA.

[0219] A method as any of the methods described herein, wherein the epitope of the target protein comprises a specific epitope present on a native protein or an endogenous protein.

[0220] A method as any of the methods described herein, wherein the epitope of the target protein comprises a protein-binding site on a scaffold protein domain of MegaBody, preferably on a scaffold protein domain comprising HopQ or Ygjk.

[0221] A method as any of the methods described herein, wherein the first protein binder is a mutant of the second protein binder and has a lower affinity compared to the second protein binder.

[0222] A method as any of the methods described herein, wherein the first protein binder comprises an ISVD or an active fragment thereof that specifically binds to an epitope.

[0223] A method as any of the methods described herein, wherein the ISVD comprises four framework regions (FR) and three complementarity-determining regions (CDR) according to the format of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0224] Any of the methods described herein, wherein the second protein binder is a multivalent form of the first protein binder.

[0225] Any of the methods described herein, wherein the second protein binder is an antigen-binding chimeric protein comprising an antigen-binding domain of an ISVD that specifically binds to an epitope and a scaffold protein, preferably a scaffold protein comprising HopQ, Ygjk or a derivative thereof, in particular MegaBody™.

[0226] A kit comprising a first protein binder and a second protein binder for any of the methods described herein.

[0227] A kit wherein the first protein binder is immobilized on a surface.

[0228] A kit comprising a first protein binder and a second protein binder selected from the group of proteins depicted in SEQ ID NOs: 1-6 or a sequence having at least 70% amino acid identity thereto, wherein the first binder and the second binder specifically bind to an epitope of GFP.

[0229] Further comprising repeating the steps of any of the methods described herein using a third protein binder and a fourth protein binder in place of the first protein binder and the second protein binder, respectively, wherein the third binder and the fourth binder specifically bind to an epitope of a target protein that is different as compared to the epitope for the first binder and the second binder, wherein the fourth protein binder has a lower dissociation rate constant (k off value) as compared to the k off value for the third protein binder. A method for purifying a target protein as any of the methods described herein.

[0230] A protein complex comprising a second protein binder of the method described herein or a fourth protein binder of the method described herein and a target protein.

[0231] A protein complex in which the target protein comprises a tag selected from the group consisting of GFP, GST, SUMO, ubiquitin, and EPEA.

[0232] A protein complex that is crystalline.

[0233] Use of any of the protein complexes for structural analysis, structure-based drug design, analysis by mass spectrometry, or proteomics.

[0234] Three-dimensional structure display in the atomic resolution of the protein complex described herein, with a resolution corresponding to 0.1 to 3 Å.

[0235] Comprising a protein complex described herein, including GFP as a target protein and a GFP-specific nanobody as a second protein binder, wherein GFP is depicted within a sequence having at least 90% amino acid identity with SEQ ID NO: 16 or this sequence, the GFP-specific nanobody is depicted within a sequence having at least 90% amino acid identity with SEQ ID NO: 1 or this sequence, and the crystal is further characterized at a point within the space group P212121, with the following crystal lattice constants: a = 74.497 Å ± 5%, b = 103.450 Å ± 5%, c = 209.774 Å ± 5%, α = 90°, β = 90°, γ = 90°.

[0236] A binding site consisting of a subset of atomic coordinates present in the crystal described herein, and consisting of the amino acid residues Pro89, Glu90, Glu111, Lys113, Phe114, Glu115, and Gly116 of the GFP protein depicted in SEQ ID NO: 16.

[0237]

Table 7

Claims

1. A method for purifying a target protein, comprising: a) mixing a first protein binder that specifically binds to the target protein with a sample containing the target protein; b) adding to the mixture of a) a second protein binder that competes with the first binder for binding to the target protein and specifically binds to the target protein to displace the first binder from the target protein; and c) recovering the eluted second protein binder that is bound to the target protein. The method includes: wherein the second protein binder includes an immunoglobulin single variable domain (ISVD) that specifically binds to the target protein; The dissociation rate constant (k ｏｆｆ value) of the second protein binder is lower than or equal to the k ｏｆｆ value of the first binder, A method.

2. The method according to claim 1, wherein the second protein binder recognizes the same epitope as the first binder or an epitope that overlaps substantially.

3. K for binding to the target protein Ｄ The method according to claim 1 or 2, wherein the K value for binding to the target protein is in the range of 1 mM to 1 nM for the first protein binder and is less than 1 μM for the second protein binder.

4. The K for the first protein binder Ｄ value is at least twice that of the K Ｄ value for the second protein binder, the method according to claim 3.

5. The method according to any one of claims 1 to 4, wherein the binder specifically binds to a tag on the target protein.

6. The method according to claim 5, wherein the tag is selected from the group consisting of GFP, mCherry, GST, SMT3, and EPEA.

7. The method according to any one of claims 1 to 6, wherein the binder specifically binds to a post-translational modification on the target protein.

8. The method according to any one of claims 1 to 4, wherein the binder specifically binds to a scaffold protein domain of a target protein, the binder comprising an antigen-binding chimeric protein defined as an ISVD fused to a scaffold protein via at least two sites.

9. The method according to claim 8, wherein the scaffold protein domain includes HopQ, Ygjk, or derivatives thereof.

10. The method according to any one of claims 1 to 9, wherein the second protein binder is in a multivalent or multi-paratope form of the first protein binder.

11. The method according to any one of claims 1 to 10, wherein the first protein binder includes an ISVD that specifically binds to the target protein.

12. The first protein binder comprises an ISVD mutated within the binding region to the target protein as compared to the ISVD which is the second protein binder, and the first protein binder has a higher k ｏｆｆ The method according to claim 11, which has.

13. The first binder and the second binder contain the same ISVD, and the ISVD specifically binds to the target protein for 0.0001 seconds or more －１ k ｏｆｆ The method according to claim 11, wherein the binding occurs at

14. The method according to any one of claims 1 to 13, wherein the first protein binder and / or the second protein binder includes a functional moiety or a detectable label.

15. The method according to claim 14, wherein the first protein binder and / or the second protein binder comprises a functional portion characterized in that the functionalized binder is an antigen-binding chimeric protein in which an ISVD is fused to a scaffold protein via at least two sites, and the ISVD specifically binds to a target protein.

16. The method according to claim 15, wherein the scaffold protein comprises HopQ, Ygjk or a derivative thereof.

17. The method according to any one of claims 1 to 16, wherein the first protein binder is immobilized and the second protein binder is in solution.

18. The method according to any one of claims 1 to 17, wherein the sample is a biological sample, a complex mixture, a cell sample or an in vitro sample.

19. Instead of or in addition to each of the first protein binder and the second protein binder, repeating steps a) to c) of the method according to claims 1 to 18 using a third protein binder and a fourth protein binder, wherein the third binder and the fourth binder specifically bind to different epitopes of the target protein as compared to the epitopes of the first binder and the second binder, the fourth protein binder comprises ISVD, and the dissociation rate constant (k ｏｆｆ value) of the third protein binder is low or equal as compared to the ｏｆｆ k value), the method according to any one of claims 1 to 18.

20. A microchip or microcolumn comprising the immobilized first protein binder of the method according to claim 17 and configured for use in the method according to any one of claims 1 to 19, wherein the first protein binder is a. a sequence comprising SEQ ID NO: 1-6, 18 or 19, or the sequences of CDR1-3 of SEQ ID NO: 1-6, 18 or 19, for specific binding to GFP, and having at least 90% amino acid identity to SEQ ID NO: 1-6, 18 or 19; b. a sequence comprising SEQ ID NO: 20 or 21, or the sequences of CDR1-3 of SEQ ID NO: 20 or 21, for specific binding to GST, and having at least 90% amino acid identity to SEQ ID NO: 20 or 21; c. a sequence comprising SEQ ID NO: 23 or 24, or the sequences of CDR1-3 of SEQ ID NO: 23 or 24, for specific binding to SMT3, and having at least 90% amino acid identity to SEQ ID NO: 23 or 24, and d. a sequence comprising SEQ ID NO: 26, 27 or 28, or the sequences of CDR1-3 of SEQ ID NO: 26, 27 or 28, for specific binding to mCherry, and having at least 90% amino acid identity to SEQ ID NO: 26, 27 or 28 and comprising a protein sequence selected from the group consisting of, or comprising any of these sequences without an N-terminal His-EPEA tag. A microchip or microcolumn.

21. A kit comprising a first protein binder and a second protein binder of the method according to any one of claims 1 to 19, wherein the first protein binder is immobilized on a surface by the method according to claim 17 and / or provided as a microcolumn or microchip according to claim 20.

22. A kit comprising a first protein binder and a second protein binder of the method according to claim 6, the first protein binder and the second protein binder competing for a target protein comprising a tag, the binder being a. comprising the sequence of SEQ ID NO: 1-6, 18 or 19, or the CDR1-3 sequences of SEQ ID NO: 1-6, 18 or 19, for specific binding to GFP, a sequence having at least 90% amino acid identity to SEQ ID NO: 1-6, 18 or 19; b. comprising the sequence of SEQ ID NO: 20 or 21, or the CDR1-3 sequences of SEQ ID NO: 20 or 21, for specific binding to GST, a sequence having at least 90% amino acid identity to SEQ ID NO: 20 or 21; c. comprising the sequence of SEQ ID NO: 23 or 24, or the CDR1-3 sequences of SEQ ID NO: 23 or 24, for specific binding to SMT3, a sequence having at least 90% amino acid identity to SEQ ID NO: 23 or 24, and d. comprising the sequence of SEQ ID NO: 26, 27 or 28, or the CDR1-3 sequences of SEQ ID NO: 26, 27 or 28, for specific binding to mCherry, a sequence having at least 90% amino acid identity to SEQ ID NO: 26, 27 or 28 selected from the group of sequences, or comprising any of these sequences without an N-terminal His-EPEA tag. When the binder has a K below 0.1 nM Ｄ A kit in which the first protein binder and the second protein binder are not identical.

23. A protein complex comprising a second protein binder according to any of claims 1 to 18 or a fourth protein binder according to claim 19 and a target protein, wherein the target protein comprises a tag selected from the group consisting of GFP, GST, SMT3, EPEA and mCherry.

24. The protein complex according to claim 23, which is crystalline.

25. The protein complex according to claim 23 or 24, further comprising a protein that interacts with the target protein.

26. Use of the protein complex according to any one of claims 23 to 25 for structural analysis, structure-based drug design and drug discovery, analysis by mass spectrometry or proteomics.

27. Use of the protein complex according to claim 25 for the identification of protein-protein complexes involving a target protein.

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