Screening method

Abstract

Disclosed herein is a screening method comprising: Providing a capillary (1) and a magnetic field gradient generator (2) being configured to generate a magnetic field gradient inside the capillary (1); Forming at least one hit-target pair by exposing a binder complex library to at least one target particle, wherein the binder complex library comprises a plurality of different binder complexes, wherein each binder complex comprises a binder and a unique identifier, and wherein the at least one hit-target pair comprises a target particle being bound to a magnetic bead and being bound to at least one binder complex of the binder complex library; Isolating the at least one hit-target pair inside the capillary (1) by retaining the at least one hit-target pair inside the capillary under application of the magnetic field gradient and flushing the capillary; Analyzing the unique identifier of the binder complex of the isolated at least one hit-target pair.

Description

[0001] P29176PC00

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[0003] Screening Method

[0004] FIELD OF DISCLOSURE

[0005] The present disclosure pertains to the field of screening methods, in particular pharmaceutical and / or biochemical screening methods, such as those used for identifying drug candidates.

[0006] BACKGROUND, PRIOR ART

[0007] Generally, screening methods, such as methods for screening new drugs can be divided into two strategies, namely phenotypic screenings and target-based screenings. In targetbased screenings, a specific target is known to play a major role in a disease pathway and the screening aims to identify a binder which can bind to the target to induce a beneficial effect on the disease progression or treatment. A major advantage of such screenings is their cost effectiveness and their general efficiency. Importantly, if a specific known target is selected, optimization of a first in class binder is significantly enhanced as the target structure is known thereby enabling to tailor the molecular architecture of the binder. A targeted screening approach is limited to targets which can be expressed at liters scale and which are physically stable enough to be isolated in sufficient homogeneity and yield. Screenings with recombinant-synthetic target material build on the fundamental assumption to represent the wider pathogenic context and complexity of the disease in patients well. On the other hand, phenotypic screenings do not focus on a specific target, but instead observe the biological effect of a binder on cells, tissues or even whole organisms. An advantage of such screenings is that yet unknown effects and interactions can be revealed, which would otherwise have been missed. However, cellular screens are delicate, the throughput is limited and often it is difficult to optimize hits later on, because the target can be unknown.P29176PC00

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[0009] To overcome the limitations of sequential one-by-one compound screening, a variety of different display technologies have been developed which allow to identify multiple enriched hits from (ultra) large molecular repertoires in parallel. Display technologies are powerful methods used in molecular biology, biochemistry, and drug discovery to isolate and identify molecules, such as antibodies, peptides, aptamers, or small molecules, that bind specifically to a target of diagnostic or pharmaceutical interest. These techniques leverage the concept of linking a molecule’s phenotype (its functional binding ability) to its genotype (the genetic information encoding the molecule). Key display technologies include antibody phage display, ribosome display, yeast display, mRNA display or DNA-encoded chemical libraries (DECLs). Screening such libraries involves identifying specific molecules that bind to a target of interest through iterative binding, washing, and selection cycles. First, a diverse library of potential binders (for example, proteins, peptides, aptamers, or small molecules) is prepared, with each member linked to a unique identifier (genetic or chemical). The library is exposed to an immobilized or solution-phase target, allowing specific binders to interact while non-binders are washed away. Target-bound binders (hits) are eluted, amplified, and enriched through multiple rounds to select and isolate high-affinity candidates (hits). Thus, the barcodes are amplified by polymerase-chain reaction (PCR) and the identities of the binders (hits) are determined via sequencing or barcode analysis, followed by validation of their binding affinity, specificity, and biological activity through functional assays. High-throughput systems and automation enhance efficiency, enabling rapid screening and optimization of binders for therapeutic or diagnostic applications.

[0010] For screening small organic molecules (that is as generally used herein, molecules with a molecular mass of less than 2’000 Dalton), for example, as drug candidates, a similar principle has been developed for parallel screening, namely DNA-encoded chemical library (DECL) screening. DECLs are vast collections of small molecules, each attached to a unique DNA sequence that acts as a barcode to identify the molecule. These libraries are exposed to an immobilized target (for example, a protein), and molecules that bind to the target are isolated while non-binders are washed away. The DNA barcodes of the bound molecules are amplified by PCR and sequenced to identify the small molecules that interacted with the target. This approach allows for the rapid and efficient screening ofP29176PC00

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[0012] millions to billions of compounds in a single well and experiment, significantly accelerating drug discovery.

[0013] Display technologies often rely on in vitro conditions that may not accurately replicate the physiological environment, potentially leading to the identification of binders that fail to perform effectively in vivo. This discrepancy can result in binders that exhibit strong affinity in controlled settings but lack efficacy or stability under biological conditions, within a competitive binding environment. Additionally, preparing targets for screening, such as membrane proteins or complex receptors, can be labor-intensive and costly and induce artefacts, for example because glycosylation sites or post translational modifications are not sufficiently well represented. Targets often require recombinant expression, purification, or stabilization, which introduces logistical and technical challenges. These factors limit targeted screening approach to materials which fulfill the foundational boundary condition to be synthetically available, isolable, homogenous and size similar to be pure, but still disease relevant. It would be desirable to have a screening method that allows one to identify the target protein directly from a patient’s body fluid sample. This has the advantage that one would be able to move the screening away from a target-focused approach to a disease-focused approach.

[0014] In addition, many screening methods are relatively laborious. For example, they require handling relatively large volumes and quantities. That is, the target protein must, for example, be provided in large amount in an immobilized manner, e.g. on a solid support. Flushing the mixture to remove non-binding partners then also requires large solution volumes and, in some cases, multiple vessel or vial transfer steps. The use of large solution volumes is undesirable, because this typically requires concentration steps where an excess of solvent has to be removed. Another issue observed is the fact that the target proteins are usually synthesized in a relatively time and resource-consuming manner. It would be desirable to have a screening method which is miniaturized and / or allows to identify the target protein directly from a patient’s body fluid sample. This has the advantage that one would be able to move the screening away from a target-focused approach to a disease-focused approach.P29176PC00

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[0016] SUMMARY OF DISCLOSURE

[0017] It is the general object of the present disclosure to advance the state of the art in the field of screening methods and advantageously to overcome one or more of the disadvantages of the prior art fully or at least partly. In advantageous embodiments, a screening method is provided which is faster and / or easier to perform and / or is operationally simpler to perform. In further advantageous embodiments, methods are provided which allow to directly isolate target particles from a patient’s body fluid sample, in particular in a fast and easily performable manner.

[0018] The general object is achieved by the subject-matter of the independent claims. Favorable embodiments follow from the dependent claims and the overall disclosure.

[0019] In a first aspect, the disclosure relates to a screening method. The screening method may also be considered as a method for identifying a binder complex or a binder from a binder complex library. The identified binder complex or binder may be configured to bind to a specific target particle. The method may for example be an in-vitro screening method. That is, even in embodiments in which an endogenous sample, e.g. a patient’s body fluid sample is used, the screening method is performed in-vitro. A patient’s body fluid sample may for example be a blood, urine, saliva, cerebral or spinal sample or a cell lysate. Typically, the patient’s body fluid sample is an endogenous patient’s body fluid sample. The screening method may be a microfluidic screening method. In such a microfluidic method, volumes used in each step may be less than 1 mL, in particular less than 1 pL. In some embodiments, the screening method is a screening method for identifying a binder which binds to a target, in particular an endogenous target originating from a specific patient. In some embodiments, the screening method may be a DNA-encoded library (DEL) screening method, such as a DNA-encoded chemical library (DECL) screening method. In such screening methods, a DEL, respectively a DECL is used as binder complex library.

[0020] In some embodiments, the screening method is both an isolation method for isolating an endogenous target particle, such as an endogenous target from a patient’s body fluidP29176PC00

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[0022] sample derived from a specific patient, and a screening method for identifying a binder which binds to the isolated target particle.

[0023] The screening method may, in some embodiments, comprise the step of providing a magnetic field gradient generator. In some embodiments, the screening method may comprise the step of providing a capillary and a magnetic field gradient generator. The magnetic field gradient generator may in some embodiments be configured to generate or provide a magnetic field gradient, respectively a magnetic field, such as, for example, inside the capillary or in a vessel, in particular in a magnetic trap section. The vessel may be any vessel which can be used to perform the screening method.

[0024] The screening method may, in some embodiments, comprise the step of forming at least one hit-target pair. This may, for example, be achieved by exposing a binder complex library to at least one target particle. The binder complex library may comprise a plurality of different binder complexes, wherein each binder complex comprises a binder and a unique identifier. The hit-target pair may be considered as a binder complex - target complex pair. This is because the binder, which is able to bind a target complex, respectively a target particle, is considered as a hit. For example, a binder of a binder complex from a binder complex library which can bind to the target, respectively target particle may generally be considered a “hit”. Accordingly, the binder complex from a binder complex library whose binder can bind to the target may be considered a “hit complex”.

[0025] The formed hit-target pair may comprise a target particle which is bound to, respectively attached to, a magnetic bead. A target particle being bound to one or more magnetic beads may be considered a target complex. The target particle of the formed hit-target pair may further be bound, in particular be directly bound, to at least one binder complex of the binder complex library. For example, the binder complex library may have been exposed to the target particle to form the hit-target pair. In some embodiments, the formed hit-target pair may therefore have the general structure “magnetic bead - target particle - binder complex”. The at least one hit-target pair may be formed by exposing a binder complex library to at least one target particle (which may be part of a target complex). The at leastP29176PC00

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[0027] one target particle (or the at least one target complex) may for example be present in a patient’s body fluid sample.

[0028] In some embodiments, the target particle of the target complex may be unknown prior and optionally during the screening method. It may be possible that the target particle of the target complex may optionally be defined by the selected molecular recognition element or the molecular recognition element library. For example, the target particle and / or the target complex may be comprised in a patient’s fluid sample. In some embodiments, forming the at least one hit-target pair may therefore comprise exposing the binder complex library to a patient’s fluid sample which contains the at least one target particle and optionally a plurality of other endogenous elements, such as proteins, peptides, nucleic acids, cellular components. In some embodiments, the patient’s fluid sample may have an unknown composition, e.g. prior and optionally during the screening method.

[0029] The binder complex library may comprise, or consist of, a plurality of different binder complexes, such as for example at least 106, or at least 107, or at least 108, or at least 109different binder complexes. Each binder complex of the binder complex library may comprise, or consist of, a binder and a unique identifier. By virtue of the unique identifier, every binder complex of the binder complex library may be different from the other binder complex of the binder complex library.

[0030] In some general embodiments, each binder complex may be present at least 30 times, e.g. at least 102times, e.g. at least 103times, e.g. at least 104times, in the binder complex library. That means that multiple copies of each of the binder complexes is present in the binder complex library.

[0031] The magnetic bead may, for example, generally comprise in some embodiments a molecular recognition element that is configured to bind to the at least one target particle. The molecular recognition element may in some embodiments be configured to bind, in particular selectively, to the at least one target particle. Thus, the magnetic bead may be bound to the at least one target particle via its molecular recognition element.P29176PC00

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[0033] The screening method may in some embodiments further comprise an isolation step. In some embodiments, the isolation step may comprise isolating the at least one hit-target pair by retaining the at least one hit-target pair under application, respectively under action or in presence of, the magnetic field gradient as a retaining position, such as in the magnetic trap section. In some embodiments, the isolation step may comprise isolating the at least one hit-target pair inside the capillary by retaining the at least one hit-target pair inside the capillary under application, respectively under action or in presence of, the magnetic field gradient. The term “isolation” or “isolating” means in this respect that the at least one hittarget pair may be separated, e.g. from other components, such as non-binding binder complexes of the library or from any remains of the patient’s body fluid sample.

[0034] It is understood that the magnetic field gradient, respectively the magnetic field, is generated inside the capillary by the magnetic field gradient generator. In some embodiments, the isolation step may further comprise flushing the capillary, e.g. with a washing solution. The washing solution may be selected such that any non-binding binder complexes are flushed away from the retained at least on hit-target pair, in particular out of the capillary. Flushing therefore separates binder complexes of the binder complex library that bind to the at least one target particle from binder complexes of the library that are non-binding or do not bind to the at least one target particle.

[0035] The screening method may in some embodiments comprise the step of analyzing the unique identifier of the binder complex of the isolated at least one hit-target pair. Analyzing the unique identifier can comprise, or consist of, different process, such as, but not limited to one or more of: reading a sequence, such as a nucleic acid sequence of the identifier, sequencing the identifier and determining its sequence, amplifying the identifier, such as by PCR, or by introducing it into cells, etc. It may also comprise or consist of identifying the unique identifier and in particular identifying its nucleic acid sequence. Analyzing may, for example, result in identification of the unique identifier, e.g. by sequencing the unique identifier. After the unique identifier has been identified, a database comparison with a database which associates each unique identifier with a binder to which it is bound in theP29176PC00

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[0037] binder complex library allows to identify which binder is present in the at least one isolated hit-target pair.

[0038] In some general embodiments, the step of forming at least one hit-target pair by exposing the binder complex library to the at least one target particle and the step of isolating the at least one hit-target pair, may be repeated once or more, in particular before the unique identifier is analyzed. Every repetition of these two steps may be referred to as a screening cycle.

[0039] Typically, in such embodiments, for at least all screening cycles except the last one, or also for all screening cycles, the binder complex of the isolated hit-target pair may be detached from the target particle. The thereby detached one or more binder complexes may then be exposed again to the target particle. The target particle may for example be a fresh target particle having the same structure and / or properties as the target particle from previous screening cycles. Thereby, better binders can be identified. The target particle may for example be part of a target complex. A second screening cycle following a first screening cycle may have a modified target particle to enrich or select a certain class of binders, such as a mutated or differently complexed target particles.

[0040] In some embodiments, different screening cycles may be performed under different conditions, such as performing one or both of the two steps at different temperatures and / or for different time intervals. This allows to alter the stringency of the selection process.

[0041] In some embodiments, the target particle, in particular the target complex, is exposed in different screening cycles to different conditions, such as different sterical conditions. Thereby, different epitopes may be covered in the screening. It may for example be possible to employ orthogonal immobilization in different screening cycles or to perform the one of the screening cycles additionally in presence of an allosteric modulator.

[0042] In some embodiments, different target complexes, in particular different target particles may be used in each screening cycle. As an example, a known anti-target complex or anti-targetP29176PC00

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[0044] particle can be used to remove binder complexes from the binder complex library which bind thereto to form a reduced binder complex library. In a subsequent screening cycle, a different (e.g. desired) target complex or target particle may be exposed to the reduced binder complex library to form at least one hit-target pair. This allows to fine-tune the selection process in terms of selectivity.

[0045] The capillary and the magnetic field gradient generator may in some embodiments be part of a screening apparatus. The capillary may typically be a straight and / or linear capillary. For example, it may have the shape of a hollow cylinder, in particular a rounded, e.g. circular hollow cylinder. The capillary may extend along a longitudinal capillary axis, which may for example be a linear axis. The capillary may be open on at least one side, or it may have two openings, such as two opposing openings. The capillary may for example be a tube or the like. In some embodiments, the capillary may be a microcapillary. The capillary is typically made from a diamagnetic material. The magnetic field gradient generated by the magnetic field gradient generator may in some embodiments be generated in a section of the capillary, in particular only in a section of the capillary (and thus not over the entire length of the capillary). The section of the capillary in which the magnetic field gradient is generated may be considered as the magnetic trap section. The capillary may comprise other sections, in which no magnetic field gradient or no magnetic field caused by the magnetic field generator is present. The magnetic field gradient generator may generate the magnetic field gradient essentially perpendicular to the longitudinal capillary axis. In some embodiments, the length of the magnetic trap section (i.e. its extension along the longitudinal capillary axis) may be 0.02 mm to 20 mm, or 0.02 mm to 5 mm in particular 1 mm to 20 mm. The inner diameter of the capillary may for example be 10 to 1000 pm, or 10 to 500 pm, or 10 to 100 pm. The magnetic field gradient generated by the magnetic field gradient generator inside the capillary is typically selected such that the magnetic bead, such as the target particle bound to the magnetic bead and / or the hit-target pair, can be retained within the magnetic trap section against external forces, for example against the gravitational force and optionally at least one of: Brownian motion, diffusion introduced by a pressure gradient and / or osmose.P29176PC00

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[0047] As the skilled person understands, a unique identifier is an identifier which is different from any other identifier comprised in the binder complex library. Thus, the unique identifier allows to unambiguously identify the binder to which it is bound. In some embodiments, every binder complex of the binder complex library is unique, i.e. different from any other binder complex of the binder complex library. While it may in some embodiments be possible that the binder of each binder complex is unique and thus different from any other binder, it may in some embodiments also be possible that multiple different binder complexes of the binder complex library may comprise the same binder but different (unique) identifiers. In some embodiments, the unique identifier may be directly bound to the binder.

[0048] The term “binding” may refer to establishing any bond, e.g. chemical bond, such as but not limited to one or more selected from: covalent bond hydrogen bond, van der Waals bond, ionic bond, and the like. Furthermore, a binding event may typically imply that the binding of the hit-target pair is sufficiently stable and selective to be distinguished from random and non-specific interactions.

[0049] The capillary may typically refer to a hollow tube. In its simplest form, the capillary may be a regular, hollow cylinder.

[0050] It is generally understood herein that the term “comprising” is interpreted as meaning that it includes those features following this term, but that it does not exclude the presence of other features, as long as they do not render the matter unworkable. On the other hand, if the wording "consist of" is used, then no further features are present apart from the ones following said wording.

[0051] The binder of each binder complex may in some embodiments be a protein, a peptide, an enzyme, an antibody, and antibody fragment, a nanobody, an aptamer, a small molecule (e.g. a small organic molecule) or an oligonucleotide, such as DNA or RNA.P29176PC00

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[0053] The target particle may also be considered as a target compound. The target particle may for example be or comprise a protein, such as a receptor, a supramolecular protein assembly or a fibrillar assembly of a plurality of proteins. In particular, the target particle may be endogenous and thus derived from a patient, in particular derived from a single patient for whom the screening method may be performed. That is, in some embodiments, the screening method may be a personalized screening method. In a personalized screening method, the target particle is endogenous and thus derived from the patient. In some general embodiments, this may be achieved by a method for isolating and / or obtaining a target particle from a patient’s body sample, as described in embodiments herein. In some embodiments, the target particle may be derived from a pool of patients.

[0054] The target particle may be bound to the magnetic beads by means of a molecular recognition element. Each magnetic bead may for example be bound to a molecular recognition element which is configured to selectively bind to the target particle. As a nonlimiting example, if the target particle comprises a biotin moiety, the magnetic beads may comprise streptavidin, such as a streptavidin coating to bind the biotin moiety. In some embodiments, the molecular recognition element may be a protein, a peptide, an enzyme, an antibody, and antibody fragment, a nanobody, an aptamer, a small molecule (e.g. a small organic molecule) or an oligonucleotide, such as DNA or RNA.

[0055] A “binder” as used herein can be any structure which is configured to bind, in particular specifically bind, a target. Non limiting examples include proteins, peptides, aptamers, or small molecules (e.g. molecules having a molar mass of less than 1’500 Da, e.g. 1’000 Da or less).

[0056] A “small molecule” is a compound which has a molar mass of less than 1’500 Da, e.g. 1’000 Da or less. Typically a small molecule is an organic molecule. An organic molecule may consist only of one or more atoms selected from H, C, N, O, P, F, Cl, Br, I and S.

[0057] A “binder complex” as used herein comprises, or consists of, a binder and a unique identifier.P29176PC00

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[0059] A “unique identifier” as used herein is an identifier which is different from any other identifier comprised in the binder complex library. Thus, the unique identifier allows to unambiguously identify the binder to which it is bound. In some embodiments, every binder complex of the binder complex library is unique, i.e. different from any other binder complex of the binder complex library. While it may be possible that the binder of each binder complex is unique and thus different from any other binder, it may in some embodiments also be possible that multiple different binder complexes of the binder complex library may comprise the same binder but different (unique) identifiers. The unique identifier may be directly bound to the binder. The unique identifier may in non-limiting examples be a unique amino acid sequence or a unique nucleic acid sequence.

[0060] A “library” as used herein refers to a collection of multiple elements, e.g. or multiple different elements. Every element is present in the library at least once, but it can also be the case that every element is present in the library multiple times.

[0061] A “hit-target pair” as used herein refers to a pair which is formed by binding a binder to a target particle. The binder may be part of a binder complex and then the binder complex may be part of the hit-target pair. Vice versa, the target particle may be part of a target complex and then the target complex may be part of the hit-target pair.

[0062] A “target complex” as used herein comprises, or may in some embodiments consist of, a target particle and a magnetic bead and optionally also a molecular recognition element. If a molecular recognition element is present, the molecular recognition element binds to the target particle and the magnetic bead. Thus, the molecular recognition element may be arranged between the target particle and the magnetic bead and / or may be a linker between them.

[0063] A “molecular recognition element” is an element which can bind, e.g. specifically bind, to a target particle and which can be bound to or is already bound to a magnetic bead. A molecular recognition element is typically different from the binders as also mentioned herein. Non-limiting examples of such a molecular recognition element may comprise orP29176PC00

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[0065] consist of an antibody, nanobody, antibody fragment, peptide, protein, enzyme, aptamer, a chelate forming transition element, such as Ni, a small molecule or oligonucleotide, such as DNA or RNA.

[0066] One advantage of the present approach is that an endogenous patient’s body fluid sample containing a target particle or target complex which may optionally be unknown can be directly used without prior purification and screened against a large binder complex library. Often, such proteins are scarce, as they originate directly from a patient. For example, after forming the target complex, the whole endogenous patient’s body fluid sample can be exposed to the binder complex library. After isolation, suitable binders are identified. This has the additional benefit that the screening is performed in the endogenous environment which closely resembles the actual environment in nature, such as within an organism, e.g. a patient. Thereby, the present approach can be seen as a combination of a target-based and phenotype screening. The target may be defined by the recognition element, however, the disease associated target context is widely retained. This is because the target particle within the patient’s body fluid sample can be known and / or can be defined by the engaging molecular recognition element, but since the endogenous patient’s body fluid sample (of often unknown composition) can be exposed to the binder complex library, phenotype screening benefits can be exploited as well, such as the discovery of unexpected or unknown interactions. Such interactions may be peripheral protein-protein interactions, post translational modifications, various conformational-constitutional states. A non-limiting example for such an unexpected interaction may be that a first binder binds to the target particle and induces a conformational change, which only enables a different second binder to bind to the target particle. Generally, it is clear to the skilled person that the endogenous patient’s body fluid sample does not only comprise the target particle, but is commonly includes a plethora or a plurality of various different elements, such as proteins, peptides, nucleic acids, hormones, cellular components, for example cell wall or fragments, lysosomes, etc., and also body fluid. In other words, the endogenous patient’s body fluid sample is highly heterogenous. This is also beneficial if the target particle is a complex-heterogenous aggregate of proteins, as it is the case in many proteinopathies. One mayP29176PC00

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[0068] know how to anchor such an aggregate (i.e. by which molecular recognition element), but it is not clear yet what binders may bind against such an aggregate.

[0069] Another advantage of the method according to the present disclosure is that instead of exposing the while endogenous patient’s body fluid sample directly to the binder complex library (as described above), it is possible to first capture, and optionally isolate, a specific target particle from the endogenous patient’s body fluid sample and then directly screen a binder complex library against this isolated target particle. In some embodiments, the isolation separates the binder particle from the rest of the endogenous patient’s body fluid sample, such as from the various different elements, such as proteins, peptides, nucleic acids, hormones, cellular components, for example cell wall or fragments, lysosomes, etc., and also body fluid. Such methods may therefore be considered a capturing, and optionally isolation, and screening method. An important benefit is that the capturing, and optionally isolation, of the target particle and the screening of binder complexes against it can be done in the same experiment.

[0070] Generally, the method may in some embodiments comprise:

[0071] Providing the capillary and the magnetic field gradient generator being configured to generate a magnetic field gradient inside the capillary;

[0072] Providing an endogenous patient’s body fluid sample comprising a target particle and optionally a plurality of different elements, e.g. biological elements (such as for example proteins, peptides, nucleic acids, hormones, cellular components, for example cell wall or fragments, lysosomes, etc., and also body fluid);

[0073] Forming at least one target complex by exposing a molecular recognition element or a molecular recognition element library (which library may comprise a plurality of different molecular recognition elements) to the endogenous patient’s body fluid sample and thus also to the at least one target particle being present therein, and wherein the at least one target complex comprises, or consists of, a target particle being bound to a magnetic bead via the molecular recognition element or via one molecular recognition element of the molecular recognition element library.P29176PC00

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[0075] Such a method may be referred to as a method for obtaining a target complex.

[0076] The last step mentioned above therefore allows to selectively bind a target particle which is initially present in an endogenous patient’s body fluid sample to a magnetic bead by means of the molecular recognition element. The molecular recognition element library may therefore be a library which comprises multiple different potential molecular recognition element candidates which may bind to the target particle. This molecular recognition element library is usually different from the binder complex libraries mentioned herein as it serves ultimately to immobilize the target particle on a magnetic bead via a suitable molecular recognition element being able to bind the target particle. For example, the molecular recognition element or the molecular recognition elements of the first binder library may bind to a first epitope of the target particle, while the binder complexes of the binder complex library mentioned herein may bind to a second epitope of the target particle being different from the first epitope. The molecular recognition element or the molecular recognition elements of the molecular recognition element library may therefore be seen as anchor elements to anchor the target particle to a magnetic bead and thereby immobilize them for a subsequent screening. Generally, the molecular recognition element(s) are each bound to a magnetic bead.

[0077] As an illustrative, non-limiting example: a screen with a library of recognition elements consisting of polyclonal IgGs may enable finding various different suitable molecular recognition elements, because the epitopes are manifold, therefore, a broad selection of molecular recognition elements is expected. Instead, if a monoclonal IgG is selected, a certain region of the target may be masked continuously and not accessible for an epitope-competitive binder. Such experiments can be conducted counterwise (poly- and monoclonal screens) to reveal the wider nature of different therapeutic agents in a disease context.

[0078] One advantage of this is that if a molecular recognition element library with different molecular recognition elements is exposed the endogenous patient’s body fluid sample, it may be possible to identify two or more molecular recognition elements which bind to the target particle. As each binding event can induce changes of the target particle, it may beP29176PC00

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[0080] possible that the one of the molecular recognition element induces a change, such as a conformational change of the target particle, which the other one of the molecular recognition element does not. Since both of them result in different target complexes with different molecular recognition elements [consisting of (magnetic bead)-(molecular recognition element)-(target particle)] they can both be screened against the same binder complex library thereafter. This allows to observe the formation of hit target pairs with one of them (for example the one with induced change of the target particle - e.g. by exposing a binding pocket of the target particle), while no such hit target pair is formed without this induced change.

[0081] In some embodiments, each molecular recognition element of the molecular recognition element library or the one molecular recognition element is bound to a magnetic bead (that is, before it is exposed to the endogenous patient’s body fluid sample). Alternatively, each molecular recognition element of the molecular recognition element library or the one molecular recognition element can be bound to a magnetic bead after it is exposed to the endogenous patient’s body fluid sample. In both cases an element or target complex is formed that consists of (magnetic bead)-(molecular recognition element)-(target particle). Thereby, it is in some embodiments possible to isolate the target particle from the rest of the endogenous patient’s body fluid sample by using the provided capillary.

[0082] For example, it is in some embodiments possible to isolate the formed target complex after exposing the molecular recognition element or the molecular recognition element library to the endogenous patient’s body fluid sample from the rest of the endogenous patient’s body fluid sample. This may in some embodiments include retaining the target inside the capillary under application of the magnetic field gradient and flushing the capillary. In some embodiments, the mixture comprising the target complex and the rest of the endogenous patient’s body fluid sample, may first be introduced into the capillary.

[0083] The target complex formed and optionally the isolated target complex or the mixture of target complex and the rest of the patient’s body fluid sample, can then subsequently be used in a screening method as discussed herein, which means that the target particleP29176PC00

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[0085] discussed herein is then part of the target complex, or, in other words, bound to the magnetic bead via the molecular recognition element. In some embodiments, the screening method is both a capturing, and optionally isolation, method for isolating an endogenous target, such as an endogenous target particle from a patient’s body fluid sample derived from a specific patient, and a screening method for identifying a binder which binds to the target particle. For example, in some general embodiments, the target complex formed (the target particle being bound to the magnetic bead via the molecular recognition element) may then be exposed to a binder complex library as discussed herein. The latter can be performed either in the presence of the rest of the patient’s body fluid or in its absence.

[0086] Therefore, in some embodiments, forming the at least one hit-target pair may comprise exposing the binder complex library to the formed and optionally isolated target complex, wherein the binder complex library comprises a plurality of different binder complexes, wherein each binder complex comprises a binder and a unique identifier, and wherein the at least one hit-target pair comprises the target complex (which comprises the target particle being bound to the magnetic bead) being bound to at least one binder complex of the binder complex library.

[0087] As noted above, the at least one hit-target pair may then in some embodiments be isolated inside the capillary by retaining the at least one hit-target pair inside the capillary under application of the magnetic field gradient and flushing the capillary. In some embodiments, then unique identifier of the binder complex of the isolated at least one hit-target pair may then be analyzed, e.g. as described herein.

[0088] Generally, during isolating the at least one hit-target pair inside the capillary, the at least one hit-target pair is retained inside the capillary, in particular in the magnetic trap section. The magnetic field gradient maintains the at least one hit-target pair in the magnetic trap section and immobilizes and / or concentrates it temporarily and spatially. The magnetic field gradient may move the beads, such as the beads of the target particles. For example, the magnetic field gradient may move the beads into the magnetic trap section, such as from areas outside the magnetic trap section (but inside the capillary) into the magnetic trapP29176PC00

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[0090] section. In general, the magnetic field gradient enables to temporarily immobilize the matter, e.g. particle or compound of interest, such as the hit-target pair. This can be exploited during flushing the capillary. Flushing may be performed with a washing solution. For example, the capillary may be introduced or dipped into a washing solution and a negative pressure can be applied. Thereby, the washing solution is sucked into and / or through the capillary. For example, the washing solution may flow through the magnetic trap section and / or pass the retained at least one hit-target pair. Thereby, the washing solution can isolate the hit-target pair by removing any residual elements, for example elements originating from the binder complex library, such as non-binding binder complexes. The washing solution may be also expelled again, either by switching between applying a negative pressure and a positive pressure to suck washing solution into the capillary (e.g. under applying a negative pressure) and to expel it (e.g. upon application of a positive pressure). It may also be possible to expel it through a second opening of the capillary, being different from first opening through which the washing solution is introduced, respectively sucked into the capillary. For flushing, typically a volume of 10 to 10000 nanoliters, e.g. of 10 to 1000 nanoliters, in particular 100 to 300 nanoliters may be introduced, e.g. sucked into, the capillary, e.g. per flushing cycle. These volume refer to the volume of washing solution for one flushing cycle (i.e. the volume being sucked into the capillary at once for flushing). It may be possible to repeat the flushing cycle, e.g. multiple times.

[0091] In some embodiments, the magnetic field gradient generator is temporarily (e.g. for a predefined time interval) switched off during flushing, before it is switched back on. The time interval may for example be 1 to 10 minutes or 1 to 5 minutes. It is understood that no magnetic field gradient is present inside the capillary when the magnetic field gradient generator is switched off. Such embodiments allow dispersion of the formed hit-target pair, respectively the target particle being bound to the magnetic bead and the at least one binder complex. After switching the magnetic field gradient generator back on, the formed hit-target pair, respectively the target particle being bound to the magnetic bead and the at least one binder complex, re-diffuse and re-accumulate in the magnetic trap section.P29176PC00

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[0093] In some embodiments, forming the at least one hit-target pair may comprise the step of providing at least one target complex. The at least one target complex may comprise the at least one target particle which is bound to a magnetic bead, for example by the molecular recognition element. In some embodiments, the at least one target complex may be comprised in an endogenous patient’s body fluid sample or derived therefrom. Forming the at least one hit-target pair may further comprise the step of exposing this provided at least one target complex to the binder complex library. This may be performed inside or outside the capillary. Thereby, at least one binder complex may be bound to the at least one target particle of the target complex to form the at least one hit-target pair. Thus, in such embodiments, the at least one target particle is already bound to the magnetic bead, before it is exposed to the library. Exposing the at least one target complex to the binder complex library may for example be performed in a vessel, such as a well. It is understood that binding occurs typically between the target particle and the binder of the corresponding binder complex. In some embodiments, the at least one target complex may be present in a patient’s body fluid sample or may be derived therefrom. It is understood that this may be achieved by obtaining or providing a body fluid sample from the patient which comprises the target particle (i.e. the target particle may be endogenous) and adding one or more magnetic beads being bound to molecular recognition elements upon which the target complex forms.

[0094] In some embodiments, providing the at least one target complex comprises combining a patient’s body fluid sample comprising the at least one target with one or more magnetic beads each being bound to a molecular recognition element and binding the target particle to at least one magnetic bead via the molecular recognition element to form the at least one target complex. This may for example be achieved by the embodiments of the method for obtaining a target complex and / or target particle as described herein and / or the methods according to the second aspect of the disclosure.

[0095] In some embodiments, providing at least one target complex may be performed outside the capillary by exposing at least one target particle to the magnetic beads being optionally each bound to a molecular recognition element outside the capillary, for example in aP29176PC00

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[0097] vessel, e.g. a well. It may in particular be possible that the at least one target particle is present in a patient’s body fluid sample (e.g. it may be endogenous). Thus, in such embodiments, providing at least one target complex may comprise exposing the patient’s body fluid sample comprising the at least one target particle to the magnetic beads being each bound to a molecular recognition element and binding the at least one target particle to at least one magnetic bead via the molecular recognition element. This step may be performed outside the capillary. Alternatively, it may be performed inside the capillary. In some embodiments, the thus formed target complex may be comprised in the patient’s body fluid sample to which the binder complex library is then exposed.

[0098] In some embodiments, exposing the at least one target complex comprising the at least one target particle bound to the magnetic bead via the molecular recognition element to the binder complex library and binding the at least one target particle to at least one binder complex to form the at least one hit-target pair is performed outside the capillary, such as for example in a vessel, e.g. a well. It may in some embodiments be possible to then introduce the formed at least one hit-target pair into the capillary. The hit-target pair may for example be comprised in a patient’s body fluid sample or in any other solvent. It may in some embodiments be possible that the at least one target complex is present in a patient’s body fluid sample. Thus, in such embodiments, exposing the at least one target complex comprising the at least one target particle bound to the magnetic bead to the binder complex library and binding the at least one target particle to at least one binder complex may comprise: exposing the patient’s body fluid sample comprising at least one target complex comprising the at least one target particle bound to the magnetic bead to the binder complex library and binding the at least one target particle to at least one binder complex. This step may be performed outside the capillary and optionally the formed hit-target pair being comprised in the patient’s body fluid sample may be introduced into the capillary. Alternatively, it may be performed inside the capillary.

[0099] In some embodiments the at least one target complex is provided by binding the magnetic bead to the at least one target particle, e.g. via a molecular recognition element. Binding the magnetic bead to the at least one target particle may either also be performed outsideP29176PC00

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[0101] the capillary, or it may be performed inside the capillary. In the latter case, one or more magnetic beads may be introduced into the capillary, e.g. as a suspension comprising the magnetic beads and a solvent, under application, respectively action, of the magnetic field gradient. The amount of the suspension being introduced into the capillary may be 10 to 10000 nanoliters, in particular 10 to 1000 nanoliters, in particular 100 to 300 nanoliters. Thereby, the introduced beads are immobilized in the capillary, for example in the magnetic trap section. Thereafter, the at least one target particle may be introduced into the capillary and transported to the magnetic beads in the capillary upon which binding, e.g. selective binding, of the magnetic beads to the at least one target particle occurs and the target complex forms. If binding the magnetic beads to the at least one target particle is performed outside the capillary, it may be performed in a separate vessel. For example, it may in such embodiments be possible to apply an external magnetic field gradient to pull down, immobilize and concentrate magnetic beads being bound to the target particle at a certain location of the separate vessel, such as at the bottom. Any free flowing liquid may be partly removed and reduced. This allows to obtain a volume which confines a higher concentration and density of the magnetic beads, respectively the target complex. In general, the target particle may be part of a patient’s body fluid sample.

[0102] In some embodiments, the capillary may be in fluidic communication with a pressure source, e.g. a pump or in particular a syringe pump. In some embodiments, the pressure source may have a picoliter sensitivity. That is, the pressure source can dispense a volume with an accuracy of 1 nL or less, in particular of 10 pL or less, in particular 1 pL or less, if desired. Such a pressure source can in general be used in embodiments of the disclosure to introduce elements, e.g. solutions or dispersions into the capillary. For example, the pressure source may be part of the screening apparatus mentioned herein. In some embodiments, the pressure source may be configured such that it can pump matter multiple times through the capillary. This is beneficial if for example the magnetic beads, the target complex or the hit-target pair are retained and / or immobilized inside the capillary. Then, a library such as the binder complex library, or the patient’s body fluid sample comprising the target particle can generally be exposed to the same magnetic bead comprising a molecular recognition element or to the target complex multiple times. This is particularlyP29176PC00

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[0104] advantageous for embodiments in which the endogenous target particle is captured from a patient’s body fluid sample, because the target particle may be scarce and therefore exposing it multiple times to the magnetic beads being bound to molecular recognition elements leads to a higher success rate.

[0105] In some embodiments, exposing the at least one target complex to the binder complex library and binding at least one target particle to at least one binder complex may form a mixture. This mixture may comprise the at least one hit-target pair formed and optionally also non-binding binder complexes of the binder complex library. As the skilled person understands, non-binding binder complexes are complexes which cannot and / or do not bind to the target particle. Furthermore, the mixture may comprise other components, such as solvents and / or a buffer and / or a patient’s body fluid sample. In some embodiments, the formed mixture may be introduced into the capillary. The volume of the formed mixture comprising the at least one hit-target pair which is introduced into the capillary may for example be 10 to 10000 nanoliters, in particular 10 to 1000 nanoliters, in particular 100 to 300 nanoliters.

[0106] Introducing elements, such as the mixture mentioned above, washing solutions, hit-target pairs, target complexes, the binder complex library or the patient’s body fluid sample optionally comprising these elements, into the capillary may in general be performed by applying a negative pressure to the capillary. Thereby, the element to be introduced may be sucked into the capillary. The term “introducing” may also encompass that the inserted matter is flushed through the capillary, such as that it enters the capillary and then exits it again. Typically, the volume being introduced into the capillary is in the nanoliter range, such as below 10000, or below 1000 nanoliters, or below 500 nanoliters, or below 100 nanoliters, or below 50 nanoliters. In some embodiments, at least 1 nanoliter, in particular at least 10 nanoliter, is introduced into the capillary.

[0107] In some embodiments, the magnetic field gradient generator generates the magnetic field gradient inside the capillary during introducing the mixture into the capillary. Thereby, theP29176PC00

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[0109] magnetic beads, target complexes and / or hit-target pair may be moved into the magnetic trap section.

[0110] In some embodiments, the at least one hit-target pair is immobilized inside the capillary by the magnetic field gradient generated by the magnetic field gradient generator.

[0111] In some embodiments, the magnetic field gradient generator is temporarily (e.g. for a predefined time interval) switched off after initially immobilizing the at least one hit-target pair, before it is switched back on. The time interval may for example be 1 to 10 minutes or 1 to 5 minutes. It is understood that no magnetic field gradient is present inside the capillary when the magnetic field gradient generator is switched off. Such embodiments allow dispersion of the at least hit-target pair. After switching the magnetic field gradient generator back on, the at least one hit-target pair is immobilized again.

[0112] In some embodiments, the step of exposing the provided at least one target complex to the binder complex library and binding the at least one target particle to at least one binder complex of the binder complex library to form the at least one hit-target pair is performed outside the capillary. For example, it may be performed in a vessel, such as a well. Thereafter, at least one hit-target pair and optionally the rest of the formed mixture and / or non-binding binder complexes of the binder complex library may be introduced into the capillary, for example under application, respectively action of the magnetic field gradient generated by the magnetic field gradient generator. In some embodiments, the at least one target complex may be comprised in a patient’s body fluid sample, e.g. as described herein above.

[0113] In some embodiments, the provided at least one target complex is introduced into the capillary and immobilized inside the capillary by the magnetic field gradient generated by the magnetic field gradient generator. In some embodiments, the at least one target complex may be present, e.g. suspended, in a solvent, such as water or in a patient’s body fluid sample. The volume (e.g. the volume of the formed solution or suspension) being introduced into the capillary may for example be below 10000, or below 1000 nanoliters, orP29176PC00

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[0115] below 500 nanoliters, or below 100 nanoliters, or below 50 nanoliters, or it may in some embodiments be at least 1 nanoliter, in particular at least 10 nanoliters. For example, the at least one target complex may be introduced and immobilized in the magnetic trap section of the capillary. Thereafter, (that is, after the at least one target complex comprising the target particle bound to a magnetic bead has been introduced and immobilized in the capillary), the introduced and immobilized target complex is exposed to the binder complex library inside the capillary. This may for example be achieved by introducing the binder complex library into the capillary which contains already the immobilized at least one target complex. Therefore, in such embodiments, the target complex is first immobilized inside the capillary (without having yet been exposed to the binder complex library) and the exposure to the binder complex library occurs thereafter and directly inside the capillary.

[0116] In some embodiments, the magnetic field gradient generator is temporarily (e.g. for a predefined time interval) switched off after initially immobilizing the at least one target complex, before it is switched back on. The time interval may for example be 1 to 10 minutes or 1 to 5 minutes. It is understood that no magnetic field gradient is present inside the capillary when the magnetic field gradient generator is switched off. Such embodiments allow dispersion of the at least one target complex. After switching the magnetic field gradient generator back on, the at least one target particle is immobilized again.

[0117] In some embodiments, the at least one target complex is introduced such into the capillary that it is guided into the magnetic field gradient inside the capillary and thus into the magnetic trap section of the capillary, where it is immobilized by the action of the magnetic field gradient, e.g. the magnetic vector field. This may be achieved at least partially by the magnetic field gradient.

[0118] In some embodiments, the provided at least one target complex is introduced into the capillary under application of a negative pressure to the capillary. In general, the negative pressure may be provided by means of a suitable pump unit. The pump unit may for example be part of the screening apparatus.P29176PC00

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[0120] In some embodiments, exposing the provided at least one target complex to the binder complex library comprises introducing the binder complex library into the capillary, and in particular into the magnetic trap section, and guiding the binder complex library into the magnetic field gradient and to the immobilized at least one target complex. Thereby, binder complexes being able to bind to the at least one target particle of the target complex may bind thereto to form the at least one hit-target pair. Any binding complex that cannot and / or does not bind to the target complex, respectively the target particle, may remain free and thus not immobilized. Additionally, or alternatively, any binding complex that cannot and / or does not bind to the target complex, respectively the target particle, may be removed from the capillary. Removing such a binder complex from the capillary may for example be performed by flushing, e.g. with the washing solution or just by applying a positive pressure to the capillary such that these binder complexes are expelled from the capillary. In some embodiments, the binder complex library may be present in a solution. Such a solution may for example comprise a solvent, such as water. The volume of the solution comprising the binder complex library being introduced into the capillary may for example below 10000, or below 1000 nanoliters, or below 500 nanoliters, or below 100 nanoliters, or below 50 nanoliters, or it may in some embodiments be at least 1 nanoliter, in particular at least 10 nanoliters . In general, the binder complex library may comprise a solvent, such as water. The solvent is typically selected such that it preserves the structure of the target complex.

[0121] In some embodiments, the at least one target particle is obtained and / or isolated from a patient’s fluid sample, such as a patient’s body fluid sample. The body fluid sample may comprise the at least one target particle and optionally the patient’s body fluid. In some general embodiments, the body fluid may be blood, urine, saliva, cerebral fluid or spinal fluid. The body fluid sample may be considered a biological sample. In some general embodiments, the at least one target particle and / or the at least on target complex may be part of a patient’s body fluid sample. That is, in all embodiments described herein, the at least one target particle and / or the at least one target complex may be comprised in a patient’s body fluid sample, in particular during one or more of the steps selected from: forming the at least one hit-target pair, exposing the at least one target complex to the binder complex library and isolating the at least one hit-target pair.P29176PC00

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[0123] In some embodiments, the method may comprise the step of obtaining and / or isolating the at least one target particle from a patient’s body fluid sample. This step may comprise exposing one or more magnetic beads that each comprise a molecular recognition element to a patient’s body fluid sample.

[0124] The molecular recognition elements may in some embodiments be configured and / or selected such that they can bind, in particular selectively bind, to the at least one target particle being present in the patient’s body fluid sample. In some embodiments, the patient’s body fluid may be used as received from the patient, i.e. without further purification.

[0125] In some embodiments, different magnetic beads may comprise different molecular recognition elements, respectively may be bound to different molecular recognition elements. The molecular recognition elements may for example differ in the binding behavior to the target particle. The pairs of molecular recognition elements and magnetic beads may form a molecular recognition element library. Such as a molecular recognition element library as already described herein above. In some embodiments, such a molecular recognition element library of different molecular recognition elements being bound to different magnetic beads may be exposed to the patient’s body fluid sample. In this case, several different magnetic beads being bound to different molecular recognition elements may be exposed to the patient’s body fluid sample.

[0126] The step may further comprise binding the at least one target particle to the one or more magnetic beads via the molecular recognition elements. Thus, the molecular recognition elements may bind to the at least one target particle in the patient’s body fluid sample. Thereby, the target complex may be formed, which comprises, or consists of, the at least one target particle being bound to a magnetic bead via a molecular recognition element.

[0127] In some embodiments, a target particle of interest is defined before isolating the at least one target particle from a patient’s body fluid sample. That is, for example, a specific molecular recognition element may be designed to be able to bind and isolate the defined at least one target particle from the patient’s body fluid sample. It may be possible that aP29176PC00

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[0129] binding site and / or the target particle is unknown or not defined prior to the obtaining and / or capturing and / or isolating it from the patient’s body fluid sample.

[0130] In some embodiments providing the at least one target complex comprises immobilizing the magnetic beads inside the capillary under application of the magnetic field gradient and thereafter introducing the target particle, respectively the patient’s body fluid sample, into the capillary to bind the target particle to the magnetic beads via the molecular recognition elements to form the target complex. As noted above, the magnetic beads may typically comprise a molecular recognition element that is configured to bind to the at least one target particle. The molecular recognition element may in some embodiments be configured to bind, in particular selectively, to the at least one target particle.

[0131] Obtaining and / or isolating the at least one target particle from a patient’s body fluid sample may further comprise the step of isolating the at least one target particle being bound to the magnetic bead via the molecular recognition elements (i.e. the target complex) inside the capillary. This may be achieved by retaining the at least one target particle being bound to the magnetic bead (i.e. the target complex) under application, respectively action, of the magnetic field gradient. Furthermore, any remains of the patient’s body fluid sample which may be comprised in the patient’s body fluid sample provided may be removed from the capillary. This may be achieved by flushing the capillary with a washing solution, as for example described herein above, or by expelling the remains from the capillary upon application of a positive pressure. For example, besides the target particle, the patient’s body fluid sample may comprise cells, cellular components, proteins, antibodies, oligonucleotides, peptides, lipids, small molecules and the like.

[0132] As described herein above, obtaining and / or isolating the at least one target particle from a patient’s body fluid sample may comprise:

[0133] Providing the capillary and the magnetic field gradient generator being configured to generate a magnetic field gradient inside the capillary;P29176PC00

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[0135] Providing an endogenous patient’s body fluid sample comprising a target particle and optionally a plurality of different elements, e.g. biological elements (such as for example proteins, peptides, nucleic acids, hormones, cellular components, for example cell wall or fragments, lysosomes, etc., and also body fluid);

[0136] Forming at least one target complex by exposing a molecular recognition element or a molecular recognition element library (which library may comprise a plurality of different molecular recognition elements) to the endogenous patient’s body fluid sample and thus also to the at least one target particle being present therein, and wherein the at least one target complex comprises, or consists of, a target particle being bound to a magnetic bead via the molecular recognition element or via one molecular recognition element of the molecular recognition element library.

[0137] The last step mentioned above therefore allows to selectively bind a target particle which is initially present in an endogenous patient’s body fluid sample to a magnetic bead by means of the molecular recognition element. The molecular recognition element library may therefore be a library which comprises multiple different potential molecular recognition element candidates which may bind to the target particle. This molecular recognition element library is usually different from the binder complex libraries mentioned herein as it serves ultimately to immobilize the target particle on a magnetic bead via a suitable molecular recognition element being able to bind the target particle. For example, the molecular recognition element or the molecular recognition elements of the first binder library may bind to a first epitope of the target particle, while the binder complexes of the binder complex library mentioned herein may bind to a second epitope of the target particle being different from the first epitope. The molecular recognition element or the molecular recognition elements of the molecular recognition element library may therefore be seen as anchor elements to anchor the target particle to a magnetic bead and thereby immobilize them for a subsequent screening.

[0138] In some embodiments, each molecular recognition element of the molecular recognition element library or the one molecular recognition element is bound to a magnetic bead (that is, before it is exposed to the endogenous patient’s body fluid sample). Alternatively, eachP29176PC00

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[0140] molecular recognition element of the molecular recognition element library or the one molecular recognition element can be bound to a magnetic bead after it is exposed to the endogenous patient’s body fluid sample. In both cases an element or target complex is formed that consists of (magnetic bead)-(molecular recognition element)-(target particle). Thereby, it is in some embodiments possible to isolate the target particle from the rest of the endogenous patient’s body fluid sample by using the provided capillary.

[0141] For example, it is in some embodiments possible to isolate the formed target complex after exposing the molecular recognition element or the molecular recognition element library to the endogenous patient’s body fluid sample from the rest of the endogenous patient’s body fluid sample. This may in some embodiments include retaining the target inside the capillary under application of the magnetic field gradient and flushing the capillary. In some embodiments, the mixture comprising the target complex and the rest of the endogenous patient’s body fluid sample, may first be introduced into the capillary.

[0142] Obtaining and / or capturing and / or producing the at least one target particle from a patient’s body fluid sample, in particular such as according to the embodiments described herein above, may also represent a separate, second aspect of the disclosure. That is, in a second aspect, the present disclosure may relate to a method for obtaining at least one target particle from a patient’s body fluid sample.

[0143] The method according to an embodiment of the second aspect may in some embodiments comprise:

[0144] - Providing a capillary and a magnetic field gradient generator being configured to generate a magnetic field gradient inside the capillary;

[0145] - Providing a patient’s body fluid sample;

[0146] - Exposing one or more magnetic beads, which are each bound to a molecular recognition element being preferably configured to bind, in particular selectively, toP29176PC00

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[0148] the at least one target particle, to the patient’s body fluid sample and binding the at least one target particle to the magnetic beads via the molecular recognition element, optionally thereby forming a target complex;

[0149] - Isolating the at least one target particle bound to the magnetic beads inside the capillary by retaining it inside the capillary under application of the magnetic field gradient and removing any remains of the patient’s body fluid sample from the capillary.

[0150] Such a method may generally be considered as a method for obtaining a target complex and / or a target particle, in particular from an endogenous patient’s body fluid sample.

[0151] A target particle or target complex obtained by this method may be used the screening method according to any of the embodiments described with respect to the first aspect. It is further noted that the embodiments described with reference to obtaining the at least one target particle can apply to embodiments of the first and second aspect of the disclosure.

[0152] In some embodiments, the step of exposing one or more magnetic beads, which are each bound to a molecular recognition element being configured to bind to the patient’s body fluid sample and binding the at least one target particle to the magnetic beads may comprise or consist of exposing a library of different molecular recognition elements being bound to different magnetic beads to the patient’s body fluid sample and binding the at least one target particle to the magnetic beads via the molecular recognition element, optionally thereby forming a target complex to form at least one target complex.

[0153] In some embodiments, exposing the one or more magnetic beads to the patient’s body fluid sample may comprise a first alternative embodiment, namely introducing the one or more magnetic beads into the capillary and immobilizing them inside the capillary by the magnetic field gradient generated by the magnetic field gradient generator. In these embodiments, the magnetic beads being each bound to a molecular recognition element are typically introduced into the capillary as such, without having yet been exposed to other bindingP29176PC00

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[0155] partners or the patient’s body fluid sample. Only thereafter, the patient’s body fluid sample is introduced into the capillary upon which the at least one target particle being present in the patient’s body fluid sample binds to the magnetic beads being immobilized in the capillary via the molecular recognition element(s). The binding may therefore occur inside the capillary. Typically, the magnetic beads may be immobilized in the magnetic trap section of the capillary. They may, for example, be introduced into the capillary by suspending them in a solvent, such as water, and the formed suspension is then introduced into the capillary. In some embodiments, the volume of the suspension being introduced into the capillary may be 10 to 10000 nanoliters, in particular 10 to 1000 nanoliters, in particular 100 to 300 nanoliters. Thus, in this first alternative embodiment, the beads are first introduced and immobilized inside the capillary as such and alone and binding them to the at least one target particle being generally comprised in the patient’s body fluid sample may be performed thereafter.

[0156] In some embodiments, exposing the one or more magnetic beads to the patient’s body fluid sample may comprise a first alternative embodiment, namely exposing the one or more magnetic beads being each bound to a molecular recognition element to the patient’s body fluid sample and binding the at least one target particle being comprised in the patient’s body fluid sample, to the one or more magnetic beads via the molecular recognition element outside the capillary. That is, exposing and binding the magnetic beads to the patient’s body fluid sample is not performed in the capillary, but for example in a separate vessel. Thereafter, the at least one target particle which is now already bound to the one or more magnetic beads (and may therefore in some embodiments be considered as a target complex) may be introduced into the capillary and immobilized inside the capillary by the magnetic field gradient. As understood, the magnetic field gradient may be generated by the magnetic field gradient generator. The at least one target particle being bound to the one or more magnetic beads may for example be introduced into the capillary by suspending it in a solvent, such as water, and the formed suspension is then introduced into the capillary. In some embodiments, the volume of the suspension being introduced into the capillary may be 10 to 5000 nanoliters, in particular 100 to 300 nanoliters.P29176PC00

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[0158] Immobilizing the at least one target particle being bound to the one or more magnetic beads may be occur in the magnetic trap section of the capillary.

[0159] In some embodiments, forming the at least one hit-target pair comprises exposing the at least one target particle to the binder complex library and binding thereby at least one target particle to at least one binder complex. The at least one target particle may in such embodiments be used as such, e.g. not being bound (yet) to a magnetic bead. Only thereafter, i.e. after the at least one target particle has been bound to at least one binder complex, a magnetic bead may be bound thereto and the hit-target pair is formed. Thus, after exposure and binding, the magnetic bead may be bound to the at least one target particle being bound to the at least one binder complex. Thereby the at least one hit-target pair is formed. As noted above, the hit-target pair may comprise the target particle, which is bound to both the at least one binder complex and the magnetic bead. The magnetic bead may in particular be selectively bound to the at least one target particle. Thus, in such embodiments, the target particle is first bound to at least one of the binder complexes of the binder complex library and only thereafter, a magnetic bead is bound to the at least one target particle.

[0160] In some embodiments, exposing the at least one target particle to the binder complex library forms a mixture. This mixture may for example comprise the at least one target particle bound to the at least one binder complex. The mixture may optionally also contain other binder complexes of the binder complex library, which are not and / or cannot be bound to the at least one target particle. The mixture may also comprise the patient’s body fluid sample. Furthermore, the mixture may, for example, comprise a solvent, such as water. In some embodiments, the formed mixture may be introduced into the capillary after binding the magnetic bead to the at least one target particle, the latter being bound to the at least one binder complex. Thus, binding the magnetic bead to the at least one target particle may for example be performed outside the capillary. The volume of the formed mixture being introduced into the capillary may for example be 10 to 10000 nanoliters, in particular 10 to 1000 nanoliters, in particular 300 to 500 nanoliters. In specific embodiments, the at least one target particle may be present in a patient’s body fluid sample and the binder complexP29176PC00

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[0162] library may be added to the patient’s body fluid sample upon which at least one target particle being present in the patient’s body fluid sample binds to at least one binder complex. The whole patient’s body fluid sample may then be introduced into the capillary either after binding the magnetic bead to the at least one target particle being bound to the at least one binder complex thereby forming the at least one hit-target pair or before, in the latter case, the magnetic bead may either already be present in the capillary or may be introduced thereafter - in any case, the magnetic bead is bound to the at least one binder complex thereby forming the at least one hit-target pair. Thereafter, isolating the at least one hittarget pair may be performed.

[0163] In some embodiments, the magnetic field gradient generator may generate the magnetic field gradient inside the capillary during introducing the formed mixture into the capillary.

[0164] In some embodiments, exposing the at least one target particle to the binder complex library and binding the at least one target particle to at least one binder complex is performed outside the capillary, such as for example in a vessel, e.g. a well. It may in particular be possible that the at least one target particle is present in a patient’s body fluid sample. Thus, in such embodiments, exposing the at least one target particle to the binder complex library and binding the at least one target particle to at least one binder complex may comprise exposing the patient’s body fluid sample comprising the at least one target particle to the binder complex library and binding the at least one target particle to at least one binder complex. This step may be performed outside the capillary. Alternatively, it may be performed inside the capillary.

[0165] Binding the magnetic bead to the at least one target particle being bound to the at least one binder complex thereby forming the at least one hit-target pair, may either also be performed outside the capillary, or it may be performed inside the capillary.

[0166] In the latter case, one or more magnetic beads may be introduced into the capillary, e.g. as a suspension comprising the magnetic beads and a solvent, under application, respectively action, of the magnetic field gradient. This may for example be done by application of aP29176PC00

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[0168] negative pressure to the capillary. The amount of the suspension being introduced into the capillary may for example be 10 to 1000 nanoliters, in particular 100 to 300 nanoliters. Thereby, the introduced beads are immobilized in the capillary, for example in the magnetic trap section. Thereafter, the at least one target particle bound to the at least one binder complex may be introduced into the capillary and transported to the magnetic beads in the capillary upon which binding, e.g. selective binding, of the magnetic beads to the at least one target particle being bound to the at least one binder complex occurs and the hit-target pair forms. Alternatively, the formed hit-target pair (which may optionally be part of a mixture which may for example comprise the patient’s body fluid sample) may first be introduced into the capillary. Thereafter, one or more magnetic beads may be introduced into the capillary, e.g. as a suspension comprising the magnetic beads and a solvent, under application, respectively action, of the magnetic field gradient upon which binding, e.g. selective binding, of the magnetic beads to the at least one target particle being bound to the at least one binder complex occurs.

[0169] If binding of the magnetic beads to the at least one target particle being bound to the at least one binder complex is performed outside the capillary, it may be performed in a separate vessel. For example, it may in such embodiments be possible to apply an external magnetic field gradient to pull down, immobilize and concentrate magnetic beads being bound to the target particle at a certain location of the separate vessel, such as at the bottom. Any free flowing liquid may be partly removed and reduced. This allows to obtain a volume which confines a higher concentration and density of the magnetic, respectively the target complex.

[0170] In some general embodiments, each magnetic bead has at least about 800 - 5000, e.g.

[0171] 800 - 2500 molecular recognition element per 1 mm diameter of the magnetic bead. That is, a magnetic bead of 1 mm diameter may for example comprise 800 - 5000, e.g. 800 -2500, molecular recognition elements on its surface. In some embodiments, each magnetic bead may have a surface density of 250 - 2000 molecular recognition elements / mm2.P29176PC00

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[0173] In some general embodiments, the screening method comprises a negative control run. Such a negative control run may be performed identically to the screening method of any of the embodiments described herein with the exception that it is performed in the absence of the target particle. That is, the target complex used in the embodiments described herein differs from a negative control target complex in that it lacks, e.g. only lacks, the target particle. Performing such a negative control run allows to identify and rule out unselective binders or unselective binder complexes which bind for example not to the target particle but to other elements, such as the magnetic bead, a linker, a surface coating of the magnetic bead, etc.

[0174] In some embodiments, the negative control run may comprise:

[0175] - Providing a capillary and a magnetic field gradient generator being configured to generate a magnetic field gradient inside the capillary;

[0176] - Exposing the binder complex library to the negative control target complex (e.g. in the absence of the target particle), wherein the binder complex library comprises a plurality of different binder complexes, wherein each binder complex comprises a binder and a unique identifier and optionally forming one or more unselective binder complexes. The one or more unselective binder complexes may for example bind to elements used in the negative control run, such as magnetic beads of the negative control target complex;

[0177] - Isolating the formed one or more unselective binder complexes inside the capillary by retaining the formed one or more unselective binder complexes inside the capillary under application of the magnetic field gradient and flushing the capillary;

[0178] - Analyzing the unique identifier of the isolated formed one or more unselective binder complexes and optionally thereby identifying the unselective binders of the one or more unselective binder complexes.P29176PC00

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[0180] This has the advantage that the negative control run identifies any unselective binders of the binder complex library. Thus, when the screening method is performed in the presence of the target particle and binder complexes with binders are identified that also have been identified in the negative control run, it is clear that they are unselective binders, which may be disregarded. In some embodiments, binders which have been identified in the control run are subtracted from the binders identified in the screening method, i.e. the at least one other run which is not a negative control run.

[0181] In some embodiments, the binder and the unique identifier of each binder complex of the binder complex library form together a linked genotype-phenotype pair. In this genotypephenotype pair, the binder of each binder complex may form the phenotype and the unique identifier of each binder complex may form the genotype. The genotype may typically be in the form of a nucleic acid, such as DNA, RNA or PNA (peptide nucleic acid). The phenotype may for example be in the form of a small molecule, a peptide, a aptamer or a protein.

[0182] In some embodiments, the binder and the unique identifier of each binder complex are comprised in a phage. The binder may for example be expressed on the surface of the phage, in particular such that it is accessible for the target particle. The unique identifier may be present inside the phage. In such embodiments, each binder complex of the library may be a phage. Since at least every identifier is unique, also every phage of the binder complex library may be unique. The binder complex library may for such embodiments be considered as a phage library. The screening, in particular forming the at least one hit-target pair and / or isolating the at least one hit-target pair, may be performed at least partially inside the capillary. Forming the at least one hit-target pair and isolating the at least one hit-target pair and optionally analyzing the unique identifier may be a phage display. In some embodiments, the phage comprising the binder of the isolated hit-target pair is isolated and used to infect bacteria to amplify the phage. Analyzing may comprise sequencing and / or reading the unique identifier, which may generally be present as DNA inside the phage.

[0183] In some embodiments, the binder and the unique identifier of each binder complex are comprised in a ribosome complex. The binder may for example be expressed by a ribosomeP29176PC00

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[0185] of the ribosome complex, in particular such that it is accessible for the target particle. The unique identifier may be linked to the ribosome. In such embodiments, each binder complex of the library may be a ribosome complex. Since at least every identifier is unique, also every ribosome complex of the binder complex library may be unique. The binder complex library may for such embodiments be considered as a ribosome library. The screening, in particular forming the at least one hit-target pair and / or isolating the at least one hit-target pair may be performed inside the capillary. Forming the at least one hit-target pair and isolating the at least one hit-target pair and optionally analyzing the unique identifier may be a ribosome display.

[0186] In some embodiments, the binder and the unique identifier of each binder complex are comprised in an mRNA-peptide complex. The binder may for example be present as a peptide. The unique identifier may be linked to the peptide and may be an mRNA. A puromycin moiety may be arranged between the peptide and the mRNA. In such embodiments, each binder complex of the library may be an mRNA-peptide complex. Since at least every identifier is unique, also every mRNA-peptide complex of the binder complex library may be unique. The binder complex library may for such embodiments be considered as a mRNA-peptide library. The screening, in particular forming the at least one hit-target pair and / or isolating the at least one hit-target pair may be performed inside the capillary. Forming the at least one hit-target pair and isolating the at least one hit-target pair and optionally analyzing the unique identifier may be a mRNA display.

[0187] In some embodiments, the binder and the unique identifier of each binder complex are comprised in a yeast cell. The binder may for example be expressed by the yeast cell on its surface, in particular such that it is accessible for the target particle. Commonly, the binder may be fused to a Aga2p protein on the yeast cell surface. The unique identifier may be arranged inside the yeast cell. In such embodiments, each binder complex of the binder complex library may be a yeast cell. Since at least every identifier is unique, also every yeast cell of the library may be unique. The binder complex library may for such embodiments be considered as a yeast cell library. The screening, in particular forming the at least one hit-target pair and / or isolating the at least one hit-target pair may be performedP29176PC00

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[0189] inside the capillary. Forming the at least one hit-target pair and isolating the at least one hittarget pair and optionally analyzing the unique identifier may be a yeast display.

[0190] In some embodiments it may also be possible that each unique identifier is an oligonucleotide. For example, an oligonucleotide consisting of 5 to 25 nucleotides, in particular 8 to 20 nucleotides, more particular 10 to 15 nucleotides. In some embodiments, the oligonucleotide may be selected from DNA, RNA and a peptide nucleic acid. It may also be possible that the unique identifier is an oligopeptide. Although it is possible that the unique identifier is an oligonucleotide encoding for the binder of the corresponding binder complex (i.e. binder and unique identifier are a phenotype-genotype pair), this does not necessarily have to be the case. It may also be possible that the unique identifier serves as a unique barcode. The information which unique identifier is bound to which binder may for example be stored in a database. By comparing the identified unique identifier, the binder which binds to the target particle can be determined and identified. For example, it is possible that the screening method comprises or is a DECL screening (DNA encoded chemical library). In a DECL screening, the binders may be chemical molecules, such as organic chemical molecules, e.g. small molecules, which are bound to a unique DNA strand. The DNA strand serves as the unique identifier. The DNA strand may act as a barcode which can be used to identify a particular binder, e.g. by associating the DNA strand sequence with a database that associates the DNA strand sequence with the binder it is bound to.

[0191] In some embodiments, a database may be provided which associates for each binder complex its unique identifier to the binder of the corresponding binder complex. In other words, the database may associate each unique identifier with a binder. In some embodiments, analyzing the unique identifier may therefore include identifying the unique identifier, for example by determining its sequence. After identifying the unique identifier, the binder of the binder complex which comprises the identified unique identifier can be identified by associating the identified unique identifier to its binder using the database. The database may be stored on a circuit, such as a server, or on a cloud. In some embodiments,P29176PC00

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[0193] identifying the binder of the binder complex which comprises the identified unique identifier can be computer-implemented.

[0194] In some embodiments, analyzing the unique identifier of the binder complex of the isolated at least one hit-target pair comprises sequencing to determine the oligonucleotide sequence of the unique identifier of the corresponding binder. Sequencing may for example be performed as NGS (next generation sequencing).

[0195] In some embodiments, analyzing the unique identifier of the binder complex of the isolated at least one hit-target pair may comprise amplifying the unique identifier to produce multiple copies of the corresponding unique identifier of the at least one hit-target pair. In some embodiments, amplifying may be performed by PCR amplification of the unique identifier. In such embodiments, the unique identifier is typically an oligonucleotide. Amplifying the unique identifier may be performed before sequencing.

[0196] In some embodiments, the binder complex or at least its unique identifier of the at least one hit-target pair may be isolated after flushing the capillary. Isolation may for example comprise, or consist of, detaching the binder complex from the target particle and / or detaching the target particle from the magnetic bead. Furthermore, the target particle and / or the binder complex may be removed from the capillary. While it may in some embodiments be possible that detaching the binder complex from the target particle and / or the target particle from the magnetic bead may be performed inside the capillary, it may also be possible to do this outside the capillary. The first case may typically be done under application of the magnetic field gradient, such that the magnetic bead being optionally bound to the target particle remains inside the capillary, even after detaching the binder complex from the hit-target pair and / or the target particle from the magnetic bead. After detaching, the binder complex may be expelled from the capillary. In the second case, application of the magnetic field gradient is ceased, and the hit-target pair is expelled from the capillary. Detaching the binder complex and / or the target particle may be performed thereafter, e.g. outside the capillary. The analysis step may typically be conducted after detaching the binder complex and / or the target particle.P29176PC00

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[0198] Detaching the binder complex from the target particle and / or the target particle from the magnetic bead may be achieved by exposing the hit-target pair to conditions which degrade the target particle, but particularly maintain the at least one identifier of the binder complex, respectively leave it unaltered. For example, detaching may be achieved by exposing the hit-target pair to irradiation, such as UV irradiation, or by increasing the temperature beyond a denaturation temperature at which denaturation of the target particle occurs. For example, the temperature may be raised to 50 °C or more, in particular 70 °C or more, in particular 80 °C or more. In some embodiments, the temperature may be raised to 50 ° to 90 °C, in particular 70 °C to 80 °C. Detaching the binder complex from the target particle and / or the target particle form the magnetic bead may also comprise exposing the hit-target pair to a detaching solution comprising agents which are configured to release the binder complex from the hit-target pair, such as protein denaturation agents. It may also be possible that a cleavable unit may be arranged between the magnetic bead and the target particle. In particular, the cleavable unit may be bound to both the magnetic bead and the target particle. The cleavable unit may for example be configured such that it can be cleaved upon activation by an external stimulus, such as UV light, thermal activation, enzymatic digestion or chemical cleavage by a cleaving agent. Cleaving the clearable unit may allow to detach the hit-target pair from the magnetic bead. As an example, Stul can be used as a cleavable unit.

[0199] In some embodiments, the capillary may be a nano capillary. A nano capillary is understood as a capillary having a total inner volume of less than 1000 nanoliters. The term “total inner volume” refers to the volume which can be filled with a fluid when the fluid is filled into the capillary. In some embodiments, the total inner volume of the capillary is 500 nanoliters or less, in particular 100 nanoliters or less, more particular 50 nanoliters or less.

[0200] In some embodiments, the capillary may have a circular cross-section with an inner diameter of 10 to 300 pm, in particular 50 to 150 pm. The “inner diameter” refers to the diameter of the cylindric inner volume which can be filled with a fluid. Thus, it excludes the capillary walls. In some embodiments, the capillary may have a volume of 2000 nanoliters or less, e.g. 1000 nanoliters or less. e.g. 500 nanoliters or less.P29176PC00

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[0202] The magnetic field gradient generator may comprise, or consist of, an electromagnet or a permanent magnet. In some embodiments, the magnetic field gradient generator may comprise, or consist of, one, two, or more electromagnets. The two electromagnets may face each other and define a gap between them. The capillary may be inserted into this gap, in particular such that it is sandwiched between the two electromagnets. Each electromagnet may comprise a cone shape with a cone tip. The cone tip of each electromagnet may direct towards the capillary. In some embodiments, the cone tips of the two electromagnets may be facing each other and may particularly be aligned with each other, in particular such that they are on the same level along the longitudinal capillary axis. Also, other geometries of the electromagnets are possible, which are configured to generate a magnetic field gradient inside the capillary.

[0203] The magnetic trap section as described herein may have a length, i.e. extension along the longitudinal capillary axis, of 0.1 to 1 mm, or 0.5 to 5 mm, or 0.2 to 0.3 , or 1 to 2 mm.

[0204] The capillary may in some embodiments have a thickness (i.e. extension perpendicular to the longitudinal capillary axis) of 50 to 500 pm, in particular 100 to 250 pm. In some embodiments, the capillary may have a length (i.e. extension along the longitudinal capillary axis) of 0.5 to 10 cm , in particular 2 to 5 cm.

[0205] The screening apparatus, which typically comprises the magnetic field gradient generator and the capillary, may in some embodiments comprise an irradiation source and / or a heating source. The irradiation source may be configured to provide irradiation inside the capillary, such as inside the magnetic trap section. The heating source may be configured to heat the inside of the capillary, such as the magnetic trap section, to a specific temperature.

[0206] In certain specific embodiments, the screening method may comprise: Providing a capillary and a magnetic field gradient generator being configured to generate a magnetic field gradient inside the capillary; Providing at least one target complex comprising a target particle bound to a magnetic bead; Exposing the at least one target complex to a binderP29176PC00

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[0208] complex library, the binder complex library comprising a plurality of different binder complexes, wherein each binder complex comprises a binder and a unique identifier, and binding the at least one target particle to at least one binder to form at least one hit-target pair; Isolating the at least one hit-target pair inside the capillary by retaining the at least one hit-target pair inside the capillary under application of the magnetic field gradient and flushing the capillary; Analyzing the unique identifier of the binder complex of the isolated at least one hit-target pair.

[0209] In certain specific embodiments, the screening method may comprise: Providing a capillary and a magnetic field gradient generator being configured to generate a magnetic field gradient inside the capillary; Providing at least one target complex comprising a target particle bound to a magnetic bead; Exposing the at least one target complex to a binder complex library, the binder complex library comprising a plurality of different binder complexes, wherein each binder complex comprises a binder and a unique identifier, and binding at least one target particle to at least one binder to form a mixture comprising at least one hit-target pair; Introducing the formed mixture into the capillary; Isolating the at least one hit-target pair inside the capillary by retaining the at least one hit-target pair inside the capillary under application of the magnetic field gradient and flushing the capillary; Analyzing the unique identifier of the binder complex of the isolated at least one hit-target pair.

[0210] In certain specific embodiments, the screening method may comprise: Providing a capillary and a magnetic field gradient generator being configured to generate a magnetic field gradient inside the capillary; Providing at least one target complex comprising a target particle bound to a magnetic bead; Introducing the at least one target complex into the capillary and immobilizing it inside the capillary by the magnetic field gradient generated by the magnetic field gradient generator; Exposing the immobilized at least one target complex to a binder complex library, the binder complex library comprising a plurality of different binder complexes, wherein each binder complex comprises a binder and a unique identifier, and binding at least one target particle to at least one binder to form at least one hit-target pair; Isolating the at least one hit-target pair inside the capillary by retaining the at least oneP29176PC00

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[0212] hit-target pair inside the capillary under application of the magnetic field gradient and flushing the capillary; Analyzing the unique identifier of the binder complex of the isolated at least one hit-target pair.

[0213] In certain specific embodiments, the screening method may comprise: Providing a capillary and a magnetic field gradient generator being configured to generate a magnetic field gradient inside the capillary; Exposing at least one target particle to a binder complex library, the binder complex library comprising a plurality of different binder complexes, wherein each binder complex comprises a binder and a unique identifier, and binding the at least one target particle to at least one binder; Binding a magnetic bead to the at least one target particle being bound to the at least one binder, thereby forming at least one hittarget pair; Isolating the at least one hit-target pair inside the capillary by retaining the at least one hit-target pair inside the capillary under application of the magnetic field gradient and flushing the capillary; Analyzing the unique identifier of the binder complex of the isolated at least one hit-target pair.

[0214] In some embodiments, the screening method comprises: Providing a capillary and a magnetic field gradient generator being configured to generate a magnetic field gradient inside the capillary; Forming at least one hit-target pair by exposing a binder complex library to at least one target particle being present in a patient’s body fluid sample, wherein the binder complex library comprises a plurality of different binder complexes, wherein each binder complex comprises a binder and a unique identifier, and wherein the at least one hittarget pair comprises a target particle being bound to a magnetic bead and being bound to at least one binder complex of the binder complex library; Isolating the at least one hit-target pair inside the capillary by retaining the at least one hit-target pair inside the capillary under application of the magnetic field gradient and flushing the capillary; Analyzing the unique identifier of the binder complex of the isolated at least one hit-target pair.

[0215] In some embodiments, forming at least one hit-target pair comprises: Providing a patient’s body fluid sample which comprises at least one target complex comprising the at least one target particle bound to a magnetic bead; and exposing the provided the patient’s body fluidP29176PC00

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[0217] sample comprising the at least one target complex to the binder complex library and binding the at least one target particle to at least one binder complex to form the at least one hittarget pair.

[0218] In some embodiments forming at least one hit-target pair comprises: Exposing a patient’s body fluid sample, comprising the at least one target particle to the binder complex library and binding the at least one target particle to at least one binder complex; and binding the magnetic bead to the at least one target particle being bound to the at least one binder complex thereby forming the at least one hit-target pair.

[0219] A third aspect of the present disclosure relates to a method for capturing and optionally for isolating a target particle, such as an endogenous target particle, from a patient’s body fluid sample and for screening for a binder which binds to the target particle. Such a method may comprise the method for obtaining and / or capturing and optionally isolating at least one target particle according to any of the embodiments described herein, such as for example the embodiments of the second aspect. Subsequently, the method may comprise a screening method, such as a screening method as described with respect to any of the embodiment herein. The screening method may comprise step i. Forming at least one hittarget pair by exposing a binder complex library to the at isolated at least one target particle, which may be part of a target complex, wherein the binder complex library comprises a plurality of different binder complexes, wherein each binder complex comprises a binder and a unique identifier, and wherein the at least one hit-target pair comprises a target particle being bound to a magnetic bead and being bound to at least one binder complex of the binder complex library. The method may further comprise step ii. Isolating the at least one hit-target pair inside the capillary by retaining the at least one hit-target pair inside the capillary under application of the magnetic field gradient and flushing the capillary. The method may further comprise step iii. Analyzing the unique identifier of the binder complex of the isolated at least one hit-target pair.

[0220] The following non-limiting numbered examples form also part of the present disclosure. They may be combined with any embodiment described herein:P29176PC00

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[0222] A 1. example relates to a screening method comprising:

[0223] Providing a magnetic field gradient generator being configured to generate a magnetic field gradient, e.g. at a specific position inside a vessel, optionally such a vessel may also be provided;

[0224] Forming at least one hit-target pair by exposing a binder complex library to at least one target particle, wherein the binder complex library comprises a plurality of different binder complexes, wherein each binder complex comprises a binder and a unique identifier, and wherein the at least one hit-target pair comprises a target particle being bound to a magnetic bead, e.g. via a molecular recognition element, and being bound to at least one binder complex of the binder complex library;

[0225] Isolating the at least one hit-target pair by retaining the at least one hit-target pair under application of the magnetic field gradient, such as in a magnetic trap section, and flushing the retained at least one hit-target pair;

[0226] - Analyzing the unique identifier of the binder complex of the isolated at least one hittarget pair.

[0227] A 2. example relates to a screening method comprising:

[0228] Providing a capillary and a magnetic field gradient generator being configured to generate a magnetic field gradient inside the capillary;

[0229] Forming at least one hit-target pair by exposing a binder complex library to at least one target particle, wherein the binder complex library comprises a plurality of different binder complexes, wherein each binder complex comprises a binder and a unique identifier, and wherein the at least one hit-target pair comprises a target particle being bound to a magnetic bead, e.g. via a molecular recognition element, and being bound to at least one binder complex of the binder complex library;P29176PC00

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[0231] Isolating the at least one hit-target pair inside the capillary by retaining the at least one hit-target pair inside the capillary under application of the magnetic field gradient and flushing the capillary;

[0232] - Analyzing the unique identifier of the binder complex of the isolated at least one hittarget pair.

[0233] A 3. example relates to the screening method according to the 1. or 2. example, wherein forming the at least one hit-target pair comprises:

[0234] Providing at least one target complex comprising the at least one target particle bound to a magnetic bead;

[0235] Exposing the provided at least one target complex to the binder complex library and binding the at least one target particle to at least one binder complex to form the at least one hit-target pair.

[0236] A 4. example relates to the screening method according to the 3. example, wherein the at least one target complex is present in a patient’s body fluid sample and wherein exposing comprises combining the binder complex library with the patient’s body fluid sample, in particular outside the capillary.

[0237] A. 5. example relates to the screening method according to the 3. or 4. example, wherein providing at least one target complex comprises combining a patient’s body fluid sample comprising the at least one target particle with one or more magnetic beads being optionally each bound to a molecular recognition element and binding the target particle to at least one magnetic bead to form the at least one target complex, wherein optionally exposing the at least one target complex to the binder complex library is performed in the presence of the patient’s body fluid sample, respectively the rest of the patient’s body fluid sample.

[0238] A 6. example relates to the screening method according to any of the 3. to 5. examples wherein exposing the provided at least one target complex to the binder complex libraryP29176PC00

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[0240] and binding at least one target particle to at least one binder complex forms a mixture comprising the at least one hit-target pair; and wherein the formed mixture is introduced into the capillary.

[0241] A 7. example relates to the screening method according to the 6. example wherein during introducing the formed mixture into the capillary, the magnetic field gradient generator generates the magnetic field gradient inside the capillary.

[0242] An 8. example relates to the screening method according to any of the 3. to 6. examples, wherein exposing the provided at least one target complex to the binder complex library and binding at least one target particle to at least one binder complex is performed outside the capillary.

[0243] A 9. example relates to the screening method according to any of the 3. to 5. examples, wherein the provided at least one target complex is introduced into the capillary and immobilized inside the capillary by the magnetic field gradient generated by the magnetic field gradient generator before the at least one target complex is exposed to the binder complex library and wherein exposing the provided at least one target complex to the binder complex library is performed inside the capillary.

[0244] A 10. example relates to the screening method according to the 9. example, wherein the provided at least one target complex is introduced into the capillary such that it is guided into the magnetic field gradient inside the capillary.

[0245] An 11. example relates to the screening method according to the 9. or 10. example, wherein the provided at least one target complex is introduced into the capillary under application of a negative pressure to the capillary.

[0246] A 12. example relates to the screening method according to any of the 9. to 11. examples, wherein exposing the provided at least one target complex to the binder complex library comprises introducing the binder complex library into the capillary and guiding the binderP29176PC00

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[0248] complex library into the magnetic field gradient and to the immobilized at least one target complex.

[0249] A 13. example relates to the screening method according to the 12. example, wherein any binding complex that does not bind to the target particle passes the immobilized at least one target complex and / or the magnetic field gradient and / or is removed from the capillary.

[0250] A 14. example relates to the screening method according to any of the 3. to 5. examples, wherein providing the at least one target complex comprises immobilizing the magnetic bead inside the capillary under application of the magnetic field gradient and thereafter introducing the target particle into the capillary to bind the target particle to the magnetic bead to form the at least one target complex.

[0251] A 15. example relates to the screening method according to the 14. example, wherein after forming the at least one target complex, the at least one target complex is exposed to the binder complex library, wherein optionally exposing the provided at least one target complex to the binder complex library comprises introducing the binder complex library into the capillary and guiding the binder complex library into the magnetic field gradient and to the immobilized at least one target complex.

[0252] A 16. example relates to the screening method according to any of the previous examples, wherein the at least one target particle is obtained from a patient’s body fluid sample comprising the at least one target particle and / or wherein the at least one target particle and / or the at least one target complex is comprised in a patient’s body fluid sample.

[0253] A 17. example relates to the screening method according to the 16. example, wherein obtaining the at least one target particle comprises:

[0254] Exposing one or more magnetic beads, which comprise a molecular recognition element being configured to bind to the at least one target particle, to the patient’sP29176PC00

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[0256] body fluid sample and binding the at least one target particle to the one or more magnetic beads via the molecular recognition element;

[0257] Optionally, isolating the at least one target particle bound to the one or more magnetic beads inside the capillary by retaining it inside the capillary under application of the magnetic field gradient and removing any remains of the patient’s body fluid sample from the capillary.

[0258] An 18. example relates to the screening method according to the 17. example, wherein exposing the one or more magnetic beads to the patient’s body fluid sample comprises (a) either introducing the one or more magnetic beads into the capillary and immobilizing them inside the capillary by the magnetic field gradient generated by the magnetic field gradient generator, followed by introducing the patient’s body fluid sample into the capillary and binding the at least one target particle to the one or more magnetic beads; or (b) exposing the one or more magnetic beads to the patient’s body fluid sample and binding the at least one target particle to the one or more magnetic beads outside the capillary, followed by introducing the at least one target particle being bound to the one or more magnetic beads into the capillary and immobilizing it inside the capillary by the magnetic field gradient generated by the magnetic field gradient generator.

[0259] A 19. example relates to the screening method according to the 1. or 2. example and optionally any of the 16. to 18. example, wherein forming the at least one hit-target pair comprises:

[0260] Exposing the at least one target particle to the binder complex library and binding the at least one target particle to at least one binder complex;

[0261] Binding the magnetic bead to the at least one target particle being bound to the at least one binder complex thereby forming the at least one hit-target pair.P29176PC00

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[0263] A 20. example relates to the screening method according to the 19. example, wherein the at least one target particle is present in a patient’s body fluid sample and wherein exposing the at least one target particle to the binder complex library comprises combining the binder complex library with the patient’s body fluid sample, in particular outside the capillary.

[0264] A 21. example relates to the screening method according to the 20. example, wherein exposing the at least one target particle to the binder complex library forms a mixture comprising the at least one target particle being bound to the at least one binder complex; and wherein the formed mixture is introduced into the capillary after binding the magnetic bead to the at least one target particle being bound to the at least one binder complex.

[0265] A 22. example relates to the screening method according to the 21. example, wherein during introducing the formed mixture into the capillary, the magnetic field gradient generator generates the magnetic field gradient inside the capillary.

[0266] A 23. example relates to the screening method according to any of the 19. to 22. examples, wherein exposing the at least one target particle to the binder complex library and binding the at least one target particle to at least one binder complex and binding the magnetic bead to the at least one target particle being bound to the at least one binder complex is performed outside the capillary.

[0267] A 24. example relates to the screening method according to any of the previous examples, wherein the binder and unique identifier of each binder complex of the binder complex library form a linked genotype-phenotype pair in which the binder forms the phenotype and the unique identifier the genotype.

[0268] A 25. example relates to the screening method according to the 24. example, wherein the binder and unique identifier of each binder complex are comprised in a phage, a ribosome complex or a yeast cell.P29176PC00

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[0270] A 26. example relates to the screening method according to any of the previous examples, wherein each unique identifier is an oligonucleotide such as DNA, RNA or peptide nucleic acid, or an oligopeptide.

[0271] A 27. example relates to the screening method according to the 26. example, wherein analyzing the unique identifier of the binder complex of the isolated at least one hit-target pair comprises sequencing to determine the oligonucleotide sequence of the unique identifier of the binder complex of the isolated at least one hit-target pair.

[0272] A 28. example relates to the screening method according to the 27. example, wherein analyzing the unique identifier of the binder complex of the isolated at least one hit-target pair comprises PCR amplification of the oligonucleotide before sequencing.

[0273] A 29. example relates to the screening method according to any of the previous examples, wherein after flushing the capillary, the binder complex of the at least one hit-target pair is isolated, in particular by detaching it from the target particle, and removing it from the capillary.

[0274] A 30. example relates to the screening method according to the 29. example, wherein detaching the binder complex comprises exposing the hit-target pair to irradiation, such as UV irradiation, and / or increasing the temperature, or to a detaching solution comprising agents which are configured to release the binder complex.

[0275] A 31. example relates to the screening method according to any of the previous examples, wherein the capillary has a total inner volume of 500 nanoliters or less, in particular 100 nanoliters or less, in particular 50 nanoliters or less.

[0276] A 32. example relates to the screening method according to any of the previous examples, wherein the at least one target particle is an endogenous protein having been obtained from a patient.P29176PC00

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[0278] A 33. example relates to a method for capturing, and optionally isolating, the at least one target particle and / or at least one target complex from a patient’s body fluid sample comprising the at least one target particle, the method comprising:

[0279] Exposing one or more magnetic beads each being bound to a molecular recognition element, which comprise a molecular recognition element being configured to bind to the at least one target particle, to the patient’s body fluid sample and binding the at least one target particle to the one or more magnetic beads via the molecular recognition element;

[0280] Optionally isolating the at least one target particle bound to the one or more magnetic beads inside a capillary by retaining it inside the capillary under application of a magnetic field gradient generated by a magnetic field gradient generator and removing any remains of the patient’s body fluid sample from the capillary.

[0281] A 34. example relates to the method according to the 33. example, wherein exposing the one or more magnetic beads each being bound to a molecular recognition element to the patient’s body fluid sample comprises: (a) either introducing the one or more magnetic beads each being bound to a molecular recognition element into the capillary and immobilizing them inside the capillary by the magnetic field gradient generated by the magnetic field gradient generator, followed by introducing the patient’s body fluid sample into the capillary and binding the at least one target particle to the one or more magnetic beads via the molecular recognition element; or (b) exposing the one or more magnetic beads each being bound to a molecular recognition element to the patient’s body fluid sample and binding the at least one target particle to the one or more magnetic beads via the molecular recognition element outside the capillary, followed by introducing the at least one target particle being bound to the one or more magnetic beads into the capillary and immobilizing it inside the capillary by the magnetic field gradient generated by the magnetic field gradient generator.P29176PC00

[0282] 53 / 72

[0283] A 35. example relates to the method according to the 33. or 34. example, wherein the step of exposing one or more magnetic beads each being bound to a molecular recognition element to the patient’s body fluid sample comprises or consists of exposing a library of different molecular recognition elements being bound to magnetic beads to the patient’s body fluid sample to form at least one target complex.

[0284] A 36. example relates to a method for capturing, and optionally isolating, a target particle, such as an endogenous target particle, from a patient’s body fluid sample and additionally for screening for a binder which binds to the target particle, the method comprising: (a) The method for capturing, and optionally isolating, at least one target particle according to any of examples 33 to 35; and subsequently (b) a screening method, such as screening method as described in any of the 1. to 31. example, which comprises: (i) Forming at least one hittarget pair by exposing a binder complex library to the at least one target particle, optionally in the presence of the rest of patient’s body fluid sample, wherein the binder complex library comprises a plurality of different binder complexes, wherein each binder complex comprises a binder and a unique identifier, and wherein the at least one hit-target pair comprises a target particle being bound to a magnetic bead and being bound to at least one binder complex of the binder complex library; (ii) Isolating the at least one hit-target pair inside the capillary by retaining the at least one hit-target pair inside the capillary under application of the magnetic field gradient and flushing the capillary; (iii) Analyzing the unique identifier of the binder complex of the isolated at least one hit-target pair.

[0285] A 37. example relates to a method for capturing, and optionally isolating, a target particle, such as an endogenous target particle, from a patient’s body fluid sample, the method comprising: (a) Providing a capillary and a magnetic field gradient generator being configured to generate a magnetic field gradient inside the capillary; (b) Providing an endogenous patient’s body fluid sample comprising a target particle and optionally a plurality of different elements, e.g. biological elements (such as for example proteins, peptides, nucleic acids, hormones, cellular components, for example cell wall or fragments, lysosomes, etc., and also body fluid); (c) Forming at least one target complex by exposing a molecular recognition element or a molecular recognition element library (which libraryP29176PC00

[0286] 54 / 72

[0287] may comprise a plurality of different molecular recognition elements) to the endogenous patient’s body fluid sample and thus also to the at least one target particle being present therein, wherein the molecular recognition element or each molecular recognition element of the molecular recognition element library is bound to a magnetic bead, and wherein the at least one target complex comprises, or consists of, a target particle being bound to a magnetic bead via the molecular recognition element or via one molecular recognition element of the molecular recognition element library.

[0288] A 38. example relates to a method for capturing, and optionally isolating, a target particle from a patient’s body fluid sample and additionally for screening for a binder which binds to the target particle, the method comprising the method of the 37. example and further comprising: Forming at least one hit-target pair by exposing a binder complex library to the target complex, wherein the binder complex library comprises a plurality of different binder complexes, wherein each binder complex comprises a binder and a unique identifier, and wherein the at least one hit-target pair comprises the target complex being bound to at least one binder complex of the binder complex library.

[0289] A 39. example relates to the method of the 38. example, wherein the binder complex library is exposed to the target complex in the presence of the endogenous patient’s body fluid sample (respectively any remains of it).

[0290] A 40. example relates to the method of the 38. example, wherein the binder complex library is exposed to the target complex in the absence of the endogenous patient’s body fluid sample (respectively any remains of it).

[0291] A 41. example relates to the method of the 40 example, wherein the target complex is isolated before the binder complex library is exposed to the target complex.

[0292] A 42. example relates to a method for capturing, and optionally isolating, the at least one target particle and / or at least one target complex from a patient’s body fluid sample comprising the at least one target particle, the method comprising:P29176PC00

[0293] 55 / 72

[0294] Exposing one or more magnetic beads each being bound to a molecular recognition element, which comprise a molecular recognition element being configured to bind to the at least one target particle, to the patient’s body fluid sample and binding the at least one target particle to the one or more magnetic beads via the molecular recognition element;

[0295] Optionally isolating the at least one target particle bound to the one or more magnetic beads by retaining it under application of a magnetic field gradient generated by a magnetic field gradient generator, such as in a magnetic trap section, and removing any remains of the patient’s body fluid sample.

[0296] A 43. example relates to the method according to the 42. example, wherein the step of exposing one or more magnetic beads each being bound to a molecular recognition element to the patient’s body fluid sample comprises or consists of exposing a library of different molecular recognition elements being bound to magnetic beads to the patient’s body fluid sample to form at least one target complex.

[0297] A 44. example relates to a method for capturing, and optionally isolating, a target particle, such as an endogenous target particle, from a patient’s body fluid sample and additionally for screening for a binder which binds to the target particle, the method comprising: (a) The method for capturing, and optionally isolating, at least one target particle according to any of examples 42 or 43; and subsequently (b) a screening method, such as screening method as described in any of the 1. to 32. example, which comprises: (i) Forming at least one hittarget pair by exposing a binder complex library to the at least one target particle, optionally in the presence of the rest of patient’s body fluid sample, wherein the binder complex library comprises a plurality of different binder complexes, wherein each binder complex comprises a binder and a unique identifier, and wherein the at least one hit-target pair comprises a target particle being bound to a magnetic bead and being bound to at least one binder complex of the binder complex library; (ii) Isolating the at least one hit-target pair by retaining the at least one hit-target under application of the magnetic field gradient, such as in aP29176PC00

[0298] 56 / 72

[0299] magnetic trap section and flushing retained at least one hit-target pair; (iii) Analyzing the unique identifier of the binder complex of the isolated at least one hit-target pair.

[0300] A 45. example relates to a method for capturing, and optionally isolating, a target particle, such as an endogenous target particle, from a patient’s body fluid sample, the method comprising: (a) Providing a magnetic field gradient generator being configured to generate a magnetic field gradient inside a provided vessel; (b) Providing an endogenous patient’s body fluid sample comprising a target particle and optionally a plurality of different elements, e.g. biological elements (such as for example proteins, peptides, nucleic acids, hormones, cellular components, for example cell wall or fragments, lysosomes, etc., and also body fluid); (c) Forming at least one target complex by exposing a molecular recognition element or a molecular recognition element library (which library may comprise a plurality of different molecular recognition elements) to the endogenous patient’s body fluid sample and thus also to the at least one target particle being present therein, wherein the molecular recognition element or each molecular recognition element of the molecular recognition element library is bound to a magnetic bead, and wherein the at least one target complex comprises, or consists of, a target particle being bound to a magnetic bead via the molecular recognition element or via one molecular recognition element of the molecular recognition element library.

[0301] A 46. example relates to a method for capturing, and optionally isolating, a target particle from a patient’s body fluid sample and additionally for screening for a binder which binds to the target particle, the method comprising the method of the 45. example and further comprising: Forming at least one hit-target pair by exposing a binder complex library to the target complex, wherein the binder complex library comprises a plurality of different binder complexes, wherein each binder complex comprises a binder and a unique identifier, and wherein the at least one hit-target pair comprises the target complex being bound to at least one binder complex of the binder complex library.P29176PC00

[0302] 57 / 72

[0303] A 47. example relates to the method of the 46. example, wherein the binder complex library is exposed to the target complex in the presence of the endogenous patient’s body fluid sample (respectively any remains of it).

[0304] A 48. example relates to the method of the 46. example, wherein the binder complex library is exposed to the target complex in the absence of the endogenous patient’s body fluid sample (respectively any remains of it).

[0305] A 49. example relates to the method of the 48. example, wherein the target complex is isolated before the binder complex library is exposed to the target complex.

[0306] BRIEF DESCRIPTION OF THE FIGURES

[0307] The herein described disclosure will be more fully understood from the detailed description given herein below and the accompanying figures which should not be considered limiting to the disclosure described in the appended claims. The figures are showing:

[0308] Fig. 1 a schematic view of a capillary and a magnetic field gradient generator as they can be used in some embodiments of the present disclosure;

[0309] Fig. 2 a schematic representation of different steps of the method according to an embodiment of the present disclosure;

[0310] Fig. 3 a schematic representation of different steps of the method according to another embodiment of the present disclosure;

[0311] Fig. 4 a schematic representation of a method of obtaining a target particle from a patient sample;

[0312] Fig. 5 a diagram showing isolated and analyzed binders in a first screening experiment;P29176PC00

[0313] 58 / 72

[0314] Fig. 6 a diagram showing isolated and analyzed binders in a second screening experiment with CAIX immobilized with an anti-CAIX mAb.

[0315] DETAILED DESCRIPTION OF FIGURES

[0316] Fig. 1 shows a capillary 1 being mounted to capillary support 13. Capillary 1 has a length L (extension along the longitudinal capillary axis) and a thickness T (extension perpendicular to the longitudinal capillary axis). Capillary 1 is sandwiched between two magnets 2a and 2b, which may for example be electromagnets, being part of magnetic field gradient generator 2. As can be seen, magnets 2a and 2b may for example have a conical shape and the tips of each cone face towards capillary 1 and / or towards each other. Upon activation of the magnetic field gradient generator 2, a magnetic field gradient can be generated between magnets 2a and 2b which is present inside the capillary 2.

[0317] Fig. 2 shows different steps of a screening method according to an embodiment of the disclosure: (a) A capillary 1 and a magnetic field gradient generator 2 are provided. The magnetic field gradient generator 2 generates a magnetic field gradient inside capillary 1. Various target complexes 11 are arranged inside capillary 1 and within the magnetic field gradient generated. Each target complex 11 comprises a target particle 6 being bound to a magnetic bead 10, e.g. via a molecular recognition element 12. The molecular bead may in general comprise the molecular recognition element, e.g. the molecular recognition element may be part of it, or it can be a separate element of target complex 11. The target complexes are immobilized inside capillary 1 by the action of magnetic field gradient 3. (b) A binder complex library 5 comprising a plurality of different binder complexes 7 is provided. Each binder complex 7 comprises binder 8 being bound to a unique identifier 9. In this case, the unique identifier 9 is a DNA. The different shapes of the binders of the binder complexes represent different binders. The binder complex library 5 is then introduced into capillary 1 and is directed into the magnetic field gradient 3 and towards the immobilized target complexes 11. (c) Only binder complexes whose binders are capable of binding to target particle 6 bind to an immobilized target complex 11. Thereby hit-target pairs 4 are formed.P29176PC00

[0318] 59 / 72

[0319] The formed hit-target pairs may in this, or any other embodiment described herein, be immobilized inside capillary 1 due to the action of magnetic field gradient 3. The other, nonbinding binder complexes being present in the introduced binder complex library are present in the capillary, but they may in this, or in any other embodiment as described herein, not be immobilized, e.g. not be able to follow the magnetic field gradient gradient. This is because they do not bind to the immobilized target complexes 11. (d) Capillary 1 is then flushed, such as with a washing solution. Flushing can comprise applying a positive pressure to the capillary. It may also be possible that flushing comprises one or more cycles of applying a negative pressure to the capillary to introduce a volume of the washing solution, followed by applying a positive pressure to the capillary to expel the introduced washing solution (optionally together with any non-immobilized elements, such as nonbinding binder complexes of the library). As can be seen, flushing may remove the nonbinding binder complexes from the capillary, while the formed hit-target pairs 4 remain immobilized inside the capillary, (e) The hit-target pairs are then detached. This may for example be achieved by irradiation or heating (implied by the dotted lines). This may lead to detachment of the binder complex from the corresponding target particle and / or its detachment from the magnetic bead (either together with the target particles or under denaturation of the target particles. The detached binder complexes 7 can then be expelled from the capillary. In a next step, the unique identifiers of the isolated binder complexes can be analyzed and optionally the binding binders identified. This may for example comprise an optional amplification step of the unique identifier DNA and PCR.

[0320] Fig. 3 shows different steps on a screening method according to another embodiment of the disclosure: (a) a vessel is provided which contains a plurality of target particles 6. Target particles 6 may for example be present in a solution, such as an aqueous solution or in a patient’s body fluid sample. Then, binder complex library 5 is added to the vessel, which includes a plurality of binder complexes (of. Fig. 2). (b) Only binder complexes 7 whose binder is capable of binding to target particle 6 bind to target particles 6. Thereby target particle 6 is bound to specific binder complexes 7 of the binder complex library, while other non-binding binder complexes are present in the vessel but do not bind to the target particles 6. Thereafter, magnetic beads 10 which may comprise a molecular recognitionP29176PC00

[0321] 60 / 72

[0322] element being configured to selectively bind to target particles 6 are added. It is understood that it is generally also possible to first add the beads 10 to the target particle 6 to form target complexes and only thereafter add the binder complex library, (c) Upon binding of the magnetic beads to the target particles which are bond to binder complexes 7, hit-target pairs 4 are formed. They may be part of a mixture which also comprises the non-binding binder complexes of binder complex library 5. The formed mixture, which may for example comprise a patient’s body fluid sample, is then inserted into the capillary 1, e.g. by applying a negative pressure to capillary 1 upon which the mixture is sucked into the capillary, (d) Under action of the magnetic field gradient 3 generated by magnetic field gradient generator 2, the hit-target pairs are retained and / or immobilized inside capillary 1, while other elements of the mixture (such as the solvent of the mixture or other non-binding binder complexes) are not immobilized, (e) Capillary 1 is then flushed, such as with a washing solution. Flushing may comprise expelling fluid, such as the solvent from the mixture, from the capillary, e.g. by applying a positive pressure to the capillary. Flushing can generally comprise applying a positive pressure to the capillary. It may also be possible that flushing comprises one or more cycles of applying a negative pressure to the capillary to introduce a volume of the washing solution, followed by applying a positive pressure to the capillary to expel the introduced washing solution (optionally together with any non-immobilized elements, such as non-binding binder complexes of the library). As can be seen, flushing may remove the non-binding binder complexes from the capillary, while the formed hittarget pairs 4 remain immobilized inside the capillary, (f) The immobilized hit-target pairs 4 are retained inside the capillary due to the action of the magnetic field gradient 3. (g) Application of the magnetic field gradient is ceased and the hit-target pairs 4 are expelled from capillary 1. They may be collected again in vessel 14. Thereafter, the binder complex may be detached and the unique identifier be analyzed.

[0323] Fig. 4 shows certain steps of a method of obtaining, respectively isolating, at least one target particle 6 from a patient’s body fluid sample 15. (a) The body fluid sample 15 may for example be provided in vessel 14, e.g. a well. The patient’s body fluid sample 15 may comprise the target particle 6 of interest, but also other components (represented for example by the triangle, circle, hexagon and circular sections). Patient body fluid sampleP29176PC00

[0324] 61 / 72

[0325] 15 may be exposed to magnetic beads 10 which may be introduced into the vessel and added to the patient’s body fluid sample as shown, (b) Since magnetic beads 10 comprise molecular recognition elements which are configured to selectively bind to target particle 6, the magnetic beads will bind only to target particle 6 and not to other components of the body fluid. Thereby, target complex 11 is formed which comprises the magnetic bead 10 being bound to target particle 6. (c) As a next step, the formed mixture is sucked into capillary 1. (d) Under action of magnetic field gradient 3 being generated by magnetic field gradient generator 2, the formed target complexes 11 are immobilized inside capillary 1, while all other components of the patient’s body fluid 15 are not immobilized, (e) The immobilized target complex 11, respectively the target particle 6 can then be isolated, for example by retaining it inside capillary 1 under action of magnetic field gradient 3, while any remains, such as the other components mentioned herein above, are removed from capillary 1. The immobilized and isolated target complex 11 may now be directly used for screening, such as described in step (a) of Fig. 2. Alternatively, the application of the magnetic field gradient may be ceased and the target complexes 11 be removed from capillary 1. For screening they may then be exposed to a binder complex library outside capillary 1, as for example described in a similar manner for step (a) of Fig. 3 (with the difference the instead of target particles 6, the target complexes 11 would be used; as noted above, steps (a) and (b) shown in Fig. 3 may generally also be performed in reversed order, i.e.to first expose target particles 6 to magnetic beads 10 to form the target complexes followed by exposing the target complexes to a binder complex library to form a mixture, followed by introducing the mixture into capillary 1).

[0326] Fig. 5 shows the application of the method according to an embodiment of the disclosure for screening a binder complex library (see Example 1. Every grey dot represents a specific binder of a binder complex. It can be seen that 7 biotinylated binders were identified from a large DELT library (ER = enrichment ratio; LIB = Library).

[0327] Fig. 6 shows the enrichment of specific compounds in an application of the method according to an embodiment of the disclosure for screening a large library with over 109binder complexes against carbonic anhydrase (CAIX) being immobilized on magnetic beadsP29176PC00

[0328] 62 / 72

[0329] by means of an anti-CAIX monoclonal antibody (see Example 2). It can be seen that the method allows to selectively identify different binder complexes. Of those, 9 are sulfonamides (labeled by “SA” followed by a number) and 2 are coumarins (labeled by "CM” followed by a number). Both coumarins and sulfonamides are known binders for CAIX (see for example Nat. Rev. 2008, 7, 168). From a pool of over 109potential binder complexes, the method allows to efficiently identify a small number of suitable binders in one experiment.

[0330] EXAMPLE 1

[0331] Materials: 2 pL cereblon protein sample; 1 pL binder complex library; 400 nL magnetic bead suspension; 1 mL buffer; 10 pL PCR mix.

[0332] 1.0 pL protein sample were mixed with 1.0 pL of the binder complex library. The binder complex library contains around 20- 109binder complexes comprising small molecule binders being bound to unique DNA identifiers. The mixture was incubated for 20 min, before an aqueous suspension of 200 nL of magnetic beads was added and the formed suspension as incubated again for 20 min. Thereafter, the mixture was introduced into the capillary under application of a negative pressure and under application of the magnetic field gradient for immobilization. The capillary was flushed 8 x with 1 pL of an aqueous selection buffer. Fresh buffer was used for every flushing cycle. Thereafter, the system was heated to 72 °C for 5 to 10 min in a volume of 250 nL and washed with water. The magnetic beads were separated and the remaining solution was lyophilized outside the capillary. Thereafter, the lyophilizate was resuspended in 1 pL water, 1 pL protein sample 200 nL of the aqueous magnetic bead suspension. Thereafter, the mixture was introduced into the capillary under application of the magnetic field gradient for immobilization. The capillary was flushed 4 x with 1 pL of an aqueous selection buffer. Fresh buffer was used for every flushing cycle. Thereafter, the system was heated to 72 °C for 5 to 10 min in a volume of 250 nL and washed with water. The magnetic beads were separated and the remaining solution was dispensed into 10 pL of the PCR mix.P29176PC00

[0333] 63 / 72

[0334] PCR was performed using a thermal cycler. Cycling was performed 40x between 40 °C and 70 °C and the mixture was purified and the DNA identifiers were sequenced.

[0335] EXAMPLE 2

[0336] Materials: 200 nL CAIX (42 kDa 10 pM), 10 pL binder complex library, Anti-Carbonic Anhydrase IX [M75], Mouse lgG1 , Kappa, PC Biotin-NHS ester (CCT-1225), Beads: 19.64 pL MagSi-STA 1.0, 1 mL selection buffer, 10 pL PCR mix, mPEG-biotin, MW 2kDa, 20mg / mL, DPBS (Gibco), 25 mM Tris and 150 mM NaCI, pH 7.5 (CAIX).

[0337] 1 pL mAbs was mixed with 19.64 pL beads for ideal spacing (2.6 mg / mL, 40% labeling efficiency). Incubation was performed for 30 min at RT (room temperature) in overhead shaker. Then, the mixture was saturated with Biotin-PEG 2kDa and washed 4x with 100 p DPBS. 4 pL protein sample were mixed with 400 nL magnetic bead mixture (approx.. 5x more protein than bead capacity) and the resulting mixture was incubated for 80 min at RT in an overhead shaker. 100 nL bead mixture (Take 1.1 pL of the mixture and supernatant wash 1x with 100 pL PBST and 2x with 100 pL PBS) were used and taken up and immobilized by the magnetic field gradient inside the capillary. Next, 10 pL of the binder complex library were taken up into the capillary and flushed over the magnetic beads (2x in and out, flowrate = 2 pL / min). Thereafter, a washing step has been performed by washing the plug of magnetic beads with 20 pL buffer (Flowrate 2 pL / min) and the washing fractions were collected (5x 4 pL). Separation from the magnetic beads was performed under UV irradiation for 15 min at 2% intensity to cleave proteins and binders. Then, the elution fraction was collected (total 4 pL; Flowrate 2 pL / min) and the resulting compounds were amplified by PCR and sequenced.

[0338] LIST OF DESIGNATIONS

[0339] 1 capillary

[0340] 2 magnetic field gradient generatorP29176PC00

[0341] 64 / 72 3 magnetic field gradient

[0342] 4 hit-target pair

[0343] 5 binder complex library

[0344] 6 target particle

[0345] 7 binder complex

[0346] 8 binder

[0347] 9 unique identifier

[0348] 10 magnetic bead

[0349] 11 target complex

[0350] 12 molecular recognition element 13 capillary support

[0351] 14 vessel

[0352] 15 body fluid sample of a patient

Claims

P29176PC0065 / 72Claims1. Screening method comprising:Providing a capillary (1) and a magnetic field gradient generator (2) being configured to generate a magnetic field gradient (3) inside the capillary (1);Forming at least one hit-target pair (4) by exposing a binder complex library (5) to at least one target particle (6), wherein the binder complex library (5) comprises a plurality of different binder complexes (7), wherein each binder complex (7) comprises a binder (8) and a unique identifier (9), and wherein the at least one hit-target pair (4) comprises a target particle (6) being bound to a magnetic bead (10) and being bound to at least one binder complex (7) of the binder complex library (5);Isolating the at least one hit-target pair (4) inside the capillary (1) by retaining the at least one hit-target pair (4) inside the capillary (1) under application of the magnetic field gradient (3) and flushing the capillary (1);- Analyzing the unique identifier (9) of the binder complex (7) of the isolated at least one hit-target pair (4).

2. The screening method according to claim 1 , wherein forming the at least one hit-target pair (4) comprises:Providing at least one target complex (11) comprising the at least one target particle (6) bound to a magnetic bead (10);Exposing the provided at least one target complex (11) to the binder complex library (5) and binding the at least one target particle (6) to at least one binder complex (7) to form the at least one hit-target pair (4).P29176PC0066 / 723. The screening method according to claim 2, wherein exposing the provided at least one target complex (11) to the binder complex library (5) and binding at least one target particle (6) to at least one binder complex (7) forms a mixture comprising the at least one hit-target pair (4); and wherein the formed mixture is introduced into the capillary (1), wherein optionally during introducing the formed mixture into the capillary (1), the magnetic field gradient generator (2) generates the magnetic field gradient (3) inside the capillary (1).

4. The screening method according to claim 2, wherein the provided at least one target complex (11) is introduced into the capillary (1) and immobilized inside the capillary (I) by the magnetic field gradient (3) generated by the magnetic field gradient generator (2) before the at least one target complex (11) is exposed to the binder complex library (5) and wherein exposing the provided at least one target complex (II) to the binder complex library (5) is performed inside the capillary (1), wherein optionally the provided at least one target complex (11) is introduced into the capillary (1) such that it is guided into the magnetic field gradient (3) inside the capillary (1).

5. The screening method according to claim 3 or 4 wherein exposing the provided at least one target complex (11) to the binder complex library (5) comprises introducing the binder complex library (5) into the capillary (1) and guiding the binder complex library (5) into the magnetic field gradient (3) and to the immobilized at least one target complex (11), wherein optionally any binding complex (7) that does not bind to the target particle (6) passes the immobilized at least one target complex (11) and / or the magnetic field gradient (3) and / or is removed from the capillary (1).

6. The screening method according to claim 2, wherein providing the at least one target complex (11) comprises immobilizing the magnetic bead (10) inside the capillary (1) under application of the magnetic field gradient and thereafter introducing the target particle (6) into the capillary (1) to bind the target particle (6) to the magnetic beadP29176PC0067 / 727. The screening method according to claim 6, wherein after forming the at least one target complex (11), the at least one target complex (11) is exposed to the binder complex library (5), wherein optionally exposing the provided at least one target complex (11) to the binder complex library (5) comprises introducing the binder complex library (5) into the capillary (1) and guiding the binder complex library (5) into the magnetic field gradient (3) and to the immobilized at least one target complex (11).

8. The screening method according to any of the previous claims, wherein the at least one target particle (6) is obtained from a patient’s body fluid sample comprising the at least one target particle (6) and / or wherein the at least one target particle (6) and / or the at least one target complex is comprised in a patient’s body fluid sample.

9. The screening method according to claim 8, wherein obtaining the at least one target particle (6) comprises:Exposing one or more magnetic beads (10), which comprise a molecular recognition element (12) being configured to bind to the at least one target particle (6), to the patient’s body fluid sample and binding the at least one target particle (6) to the one or more magnetic beads (10) via the molecular recognition element (12);Optionally isolating the at least one target particle (6) bound to the one or more magnetic beads (10) inside the capillary (1) by retaining it inside the capillary (1) under application of the magnetic field gradient (3) and removing any remains of the patient’s body fluid sample from the capillary (1).

10. The screening method according to claim 9, wherein exposing the one or more magnetic beads (10) to the patient’s body fluid sample comprises:a. either introducing the one or more magnetic beads (10) into the capillary (1) and immobilizing them inside the capillary (1) by the magnetic field gradientP29176PC0068 / 72(3) generated by the magnetic field gradient generator (2), followed by introducing the patient’s body fluid sample into the capillary (1) and binding the at least one target particle (6) to the one or more magnetic beads (10); orb. exposing the one or more magnetic beads (10) to the patient’s body fluid sample and binding the at least one target particle (6) to the one or more magnetic beads (10) outside the capillary (1), followed by introducing the at least one target particle (6) being bound to the one or more magnetic beads (10) into the capillary (1) and immobilizing it inside the capillary (1) by the magnetic field gradient (3) generated by the magnetic field gradient generator (2).

11. The screening method according to claim 1 and optionally any of claims 8 to 10, wherein forming the at least one hit-target pair (4) comprises:Exposing the at least one target particle (6) to the binder complex library (5) and binding the at least one target particle (6) to at least one binder complex (7);Binding the magnetic bead (10) to the at least one target particle (6) being bound to the at least one binder complex (7) thereby forming the at least one hit-target pair (4).

12. The method according to claim 11, wherein exposing the at least one target particle (6) to the binder complex library (5) forms a mixture comprising the at least one target particle (6) being bound to the at least one binder complex (7); and wherein the formed mixture is introduced into the capillary (1) after binding the magnetic bead (10) to the at least one target particle (6) being bound to the at least one binder complex (7).

13. The screening method according to any of the previous claims, wherein the binder (8) and unique identifier (9) of each binder complex (7) of the binder complex library formP29176PC0069 / 72a linked genotype-phenotype pair in which the binder (8) forms the phenotype and the unique identifier (9) the genotype, wherein optionally the binder (8) and unique identifier (9) of each binder complex (7) are comprised in a phage, a ribosome complex or a yeast cell.

14. The screening method according to any of the previous claims, wherein each unique identifier (9) is an oligonucleotide such as DNA, RNA or peptide nucleic acid, or an oligopeptide and wherein analyzing the unique identifier (9) of the binder complex (7) of the isolated at least one hit-target pair (4) comprises sequencing to determine the oligonucleotide sequence of the unique identifier (9) of the binder complex (7) of the isolated at least one hit-target pair (4).

15. Method for capturing, and optionally isolating, at least one target particle (6) and / or at least one target complex (11) from a patient’s body fluid sample comprising the at least one target particle (6), the method comprising:a. Exposing one or more magnetic beads (10), which are each bound to a molecular recognition element (12) to the patient’s body fluid sample and binding the at least one target particle (6) to at least some of the magnetic beads (10) via the molecular recognition element (12) to form at least one target complex;b. Optionally, isolating the at least one target particle (6) bound to at least some of the magnetic beads (10) inside a capillary (1) by retaining it inside the capillary (1) under application of a magnetic field gradient (3) generated by a magnetic field gradient generator (2) and removing any remains of the patient’s body fluid sample from the capillary (1).

16. The method according to claim 15, wherein exposing the one or more magnetic beads (10) to the patient’s body fluid sample comprises:P29176PC0070 / 72i. either introducing the one or more magnetic beads (10) each being bound to a molecular recognition element (12) into the capillary (1) and immobilizing them inside the capillary (1) by the magnetic field gradient (3) generated by the magnetic field gradient generator (2), followed by introducing the patient’s body fluid sample into the capillary (1) and binding the at least one target particle (6) to the one or more magnetic beads (10) via the molecular recognition element (12); orii. exposing the one or more magnetic beads (10) each being bound to a molecular recognition element (12) to the patient’s body fluid sample and binding the at least one target particle (6) to the one or more magnetic beads (10) via the molecular recognition element (12) outside the capillary (1), followed by introducing the at least one target particle (6) being bound to the one or more magnetic beads (10) into the capillary (1) and immobilizing it inside the capillary (1) by the magnetic field gradient (3) generated by the magnetic field gradient generator (2).

17. The method according to claim 15 or 16, wherein in step a. a library of different molecular recognition elements (12) being bound to magnetic beads is exposed to the patient’s body fluid sample to form at least one target complex.

18. A method for capturing, and optionally isolating, a target particle, such as an endogenous target particle, from a patient body fluid sample and for screening for a binder which binds to the target particle, the method comprising:a. The method for capturing, and optionally isolating, at least one target particle (6) according to any of claims 15 to 17; and subsequentlyb. a screening method comprising:P29176PC0071 / 72i. Forming at least one hit-target pair (4) by exposing a binder complex library (5) to the at isolated at least one target particle (6), wherein the binder complex library (5) comprises a plurality of different binder complexes (7), wherein each binder complex (7) comprises a binder (8) and a unique identifier (9), and wherein the at least one hit-target pair (4) comprises a target particle (6) being bound to a magnetic bead (10) and being bound to at least one binder complex (7) of the binder complex library (5);ii. Isolating the at least one hit-target pair (4) inside the capillary (1) by retaining the at least one hit-target pair (4) inside the capillary (1) under application of the magnetic field gradient (3) and flushing the capillary (1);iii. Analyzing the unique identifier (9) of the binder complex (7) of the isolated at least one hit-target pair (4).

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