Convective affinity adsorbents for purifying biologics

Abstract

The present disclosure provides materials and methods related to convective affinity adsorbents for the purification of a target biologic from a biological fluid or for the capture of process-related and product-related contaminants from a biological fluid.

Description

CONVECTIVE AFFINITY ADSORBENTS FOR PURIFYING BIOLOGICSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority from U.S. provisional application No. 63 / 710,304, filed October 22, 2024, the entire disclosure of which is incorporated herein by reference.FIELD

[0002] The present disclosure provides materials and methods related to convective affinity adsorbents for the purification of a target biologic from a biological fluid or for the capture of process-related and product-related contaminants from a biological fluid. In particular, the present disclosure provides compositions, and related methods, comprising membranes functionalized with affinity ligands for the purification of biological materials from biological fluids during the process of producing a biologic.INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 22, 2025, is named 000536-0014-W01-SL.xml and is 61,450 bytes in size.BACKGROUND

[0004] The high binding capacity characteristic of chromatography resins stems from the large surface area of their pores. However, the tortuous morphology and limited diameter of these pores reduce the transport of large biologies, such as large proteins, multiprotein constructs, viruses, virus-like particles, and extracellular vesicles, due to diffusion limitations. This prolongs the total time of the purification process, which may lead to a decreased product’s bioactivity and increased production costs. Thus, whilst resins functionalized with ligands for use in chromatography and purification applications are still widely utilized in the biopharmaceutical industry, there is general consensus on the need to transition to “convective” chromatographic adsorbents.

[0005] Convective adsorbents such as membranes, fiber mats, and monoliths offer an excellent alternative to resins as their open porosity eliminates or significantly reduces diffusive limitations and enables processing at significantly higher flow rates and lowerpressure drops. Affinity monoliths for biological separations have been demonstrated, but the large-scale manufacturing of monoliths is challenging and - to date - has not been fully accomplished. On the other hand, membranes and fiber mats can be easily produced at a large scale, but - to date - there is no affinity membrane or fiber mat that provide combined high binding capacity and high product yield to warrant industrial application for purifying high-value biologies, such as biotherapeutics and vaccines.

[0006] Thus, there remains a need for effective affinity membranes or affinity fiber mats for purifying biologies from biological fluids.SUMMARY

[0007] This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely an example of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise for purposes of example. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such features.

[0008] Provided in some embodiments are affinity adsorbents for purifying a biologic, the affinity adsorbent comprising: a convective porous substrate having a surface that can be functionalized; one or more branched polymers tethered to the surface of the convective porous substrate, wherein the one or more branched polymers comprise one or more reactive terminal groups; and a ligand conjugated to a terminus of the one or more branched polymers.

[0009] Provided in some aspects are methods of making an affinity adsorbent, the methods comprising providing a convective porous substrate having a surface that can be functionalized; tethering one or more branched polymers on a surface of the convective porous substrate, wherein the one or more branched polymers comprise one or more reactive terminal groups, wherein the one or more branched polymers comprise a preformed branched polymer; and conjugating a ligand to a terminus of the one or more branched polymers.

[0010] In some embodiments, provided are methods of purifying a target biologic from a biological fluid in flow-through mode, the method comprising: contacting the affinityadsorbent with a biological fluid comprising a target biologic; and collecting an effluent in flow-through mode, wherein the effluent comprises the target biologic; wherein the affinity adsorbent binds and retains at least one host cell protein (HCP), at least one high-risk HCP, at least one host cell nucleic acid, aggregates of the target biologic, and / or an impurity derived from the target biologic.

[0011] In some embodiments, provided are methods of purifying a target biologic from a biological fluid in bind-and-elute mode, the methods comprising: contacting the affinity adsorbent with a biological fluid comprising a target biologic; washing the affinity adsorbent to remove loosely bound contaminants; and releasing the target biologic using an appropriate elution buffer, thereby collecting a purified target biologic in the eluate.

[0012] These and other objects are achieved in whole or in part by the presently disclosed subject matter. Other objects and advantages of the presently disclosed subject matter will become apparent to those skilled in the art after a study of the following description, examples, and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0014] The presently disclosed subject matter can be better understood by referring to the following, example figure. The components in the figure are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the presently disclosed subject matter (often schematically). In the figure, like reference numerals designate corresponding parts throughout the different views. A further understanding of the presently disclosed subject matter can be obtained by reference to an embodiment set forth in the illustrations of the accompanying drawing. Although the illustrated embodiment is merely for purposes of example of systems for carrying out the presently disclosed subject matter, both the organization and method of operation of the presently disclosed subject matter, in general, together with further objectives and advantages thereof, may be more easily understood by reference to the drawings and the following description. The drawing is not intended to limit the scope of this presently disclosed subject matter, which is set forth with particularity in the claims as appended or as subsequently amended, but merely to clarify and provide examples of the presently disclosed subject matter.

[0015] Figure 1. Structure of functionalized membranes prepared by functionalizing the branched polymer grafted on the surface of the pores of the membrane.

[0016] Figures 2. Panels A- C show the results of experiments to test the efficacy of functionalized membranes in the removal of Chinese Hamster Ovary (CHO) host cell proteins (HCP) from cell culture harvests.DETAILED DESCRIPTION

[0017] The presently disclosed subject matter now will be described more fully hereinafter, in which some, but not all embodiments of the presently disclosed subject matter are described. Indeed, the presently disclosed subject matter can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.1. Definitions

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0019] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments“comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0020] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0021] “Correlated to” as used herein refers to compared to.

[0022] As used herein, “peptide” and “polypeptide,” unless otherwise specified, generally refer to polymer compounds of two or more amino acids joined through the main chain by peptide amide bonds (— C(0)NH— ). The term “peptide” typically refers to short amino acid polymers (e.g., chains having fewer than 25 amino acids), whereas the term “polypeptide” typically refers to longer amino acid polymers (e.g., chains having more than 25 amino acids).

[0023] As used herein, “sequence identity” generally refers to the degree two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits. The term “sequence similarity” refers to the degree with which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have similar polymer sequences. For example, similar amino acids are those that share the same biophysical characteristics and can be grouped into the families, e.g., acidic (e.g., aspartate, glutamate), basic (e.g., lysine, arginine, histidine), non-polar (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). The “percent sequence identity” (or “percent sequence similarity”) is calculated by: (1) comparing two optimally aligned sequences over a window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), (2) determining the number of positions containing identical (or similar) monomers (e.g., same amino acids occurs in both sequences, similar amino acid occurs in both sequences) to yield the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to yield the percent sequence identity or percent sequence similarity. For example, if peptides A and B are both 20 amino acids inlength and have identical amino acids at all but 1 position, then peptide A and peptide B have 95% sequence identity. If the amino acids at the non-identical position shared the same biophysical characteristics (e.g., both were acidic), then peptide A and peptide B would have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length and peptide D is 15 amino acids in length, and 14 out of 15 amino acids in peptide D are identical to those of a portion of peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity to an optimal comparison window of peptide C. For the purpose of calculating “percent sequence identity” (or “percent sequence similarity”) herein, any gaps in aligned sequences are treated as mismatches at that position.

[0024] As used herein, the term “target” or “target biologic” generally refers to a target protein, peptide, polypeptide, nucleic acid, ribonucleoprotein complex, nucleic acid construct, supramolecular construct, virus, viral construct, virus-like particle, cell, organelle, small molecule, and any combinations thereof, that may have a biological activity, such as an enzymatic activity, a gene-editing activity, an antigen-binding activity, a therapeutic activity, or a prophylactic activity. In some embodiments, the target or target biologic is an antibody or any antigen binding fragment / derivative thereof (e.g., monoclonal or polyclonal antibody). In some embodiments, the target biologic is a viral vector.

[0025] As used herein, the term “purified” or “to purify” refers to the isolation of a target biologic from a sample wherein the target biologic is present along with contaminants. Contaminants include process-related contaminants and product-related contaminants. Process-related contaminants result from the production process, and include host cell proteins (HCPs) and host cell nucleic acids (hcDNA and hcRNA), and fragments thereof, cell debris, cell metabolites, the inorganic and organic components of cell culture media (e.g., mono- and poly-saccharides, amino acids, vitamins, insulin, antioxidants, surfactants such as antifoams, iron, zinc, nickel, etc.), and other ingredients necessary for the production of the target biologic, such as plasmids and transfection reagents, or helper viruses. Product-related contaminants results from unwanted, yet unavoidable, alteration of the target biologic, and include aggregates of multiple target biologies, aggregates of one or more target biologic with process-related contaminants, fragments of the target biologic resulting from enzymatic or chemical processes, isoforms of the target biologic resulting from unwanted post-translational modifications (e.g., amidation,deamidation, phosphorylation, glycosylation, methylation, etc.), and the like. For example, antibodies are purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin that does not bind to the target molecule. The removal of non-immunoglobulin proteins and / or the removal of immunoglobulins that do not bind to the target molecule results in an increase in the percent of target-reactive immunoglobulins in the sample. In another example, recombinant polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample. The same applies to the purification of any biologies.

[0026] As used herein, the term “host cell protein” or “HCP” refers to any protein produced or encoded by the organism used to produce a recombinant polypeptide product and unrelated to the intended product. For example, HCPs are generally undesirable in a final drug substance.

[0027] As used herein, the term “host cell DNA” or “hcDNA” refers to any deoxyribonucleic acid produced by the organism used to produce a recombinant polypeptide product and unrelated to the intended product. For example, hcDNA are generally undesirable in a final drug substance.

[0028] As used herein, the term “chromatography” is a biophysical technology that enables the separation and purification of a target biologic from process-related and product-related contaminants by virtue of their different interactions with a stationary phase and a mobile phase. Examples of stationary phases include chromatographic resins, monoliths, and membranes. Examples of mobile phases include aqueous solutions with controlled composition and stable value of pH, known as buffers, such as equilibration buffers, binding buffers, washing buffers, elution buffers, and regeneration buffers, which are utilized during the various stages of the chromatographic process. Chromatographic techniques for the purification of biologies differ by the type or ligand utilized to interact with the target biologic and the process-related and product-related contaminants: (i) affinity chromatography utilize affinity ligands (vide infra , (ii) mixedmode or multimodal chromatography utilizes mixed-mode or multimodal ligands, which form multiple non-covalent interactions like affinity ligands, but feature a lower binding selectivity compared to affinity ligands; (Hi) ion-exchange chromatography and hydrophobic interaction chromatography utilize respectively ionic and hydrophobic ligands, which enable a single interaction mode and therefore feature non-selectivebinding; (iv) size-exclusion chromatography; (v) reverse-phase chromatography; etc. Additionally, chromatographic techniques for the purification of biologies differ by mode of operation: (a) bind-and-elute chromatography (BEC) is a method where the target biologic and, optionally, the process-related and product-related contaminants loaded in a binding buffer bind the stationary phase, and, following an optional washing step using a washing buffer, are separately released (eluted) from the stationary phase using an elution buffer; (b) flow-through chromatography (FTC) is a method whereby the process-related and product-related contaminants bind the stationary phase while the target biologic flows through unbound or without significant retention; and (c) weak-partitioning chromatography (WPC) is an isocratic method whereby both the target biologic and the contaminants initially bind to the stationary phase with different binding strength, thereby enabling the subsequent release of the target biologic before the release of the contaminants.

[0029] As used herein, the term “affinity ligand” or “affinity binder” refers to molecules that bind an assigned target with high selectivity and, by virtue of that, are applied in various biochemical and biomanufacturing processes, including but not limited to the purification of or the detection of a biological target from / in a complex medium. The affinity binding results from the combination of shape complementarity and multiple non-covalent interactions, including but not limited to ionic bonds, hydrogen bonding, polar interactions, hydrophobic interactions, Van der Waals interactions, it-it stacking, London forces, and the like. As a result, these ligands can recognize a target biological molecule in complex mixtures. The strength of ligand-target binding can be quantified by the dissociation constant (KD), with lower values of KD indicating higher affinity. Affinity ligands include proteins, especially antibodies and enzymes, aptamers, peptides, and other synthetic molecules, such as triazine scaffolds and the like.

[0030] As used herein, the term “viral vector” refers to an engineered, or modified, virus used for delivering a specific genetic payload into a target cell to enable various applications in genetic research, therapy, or vaccination. Viral vectors play a crucial role in various medical fields, including gene therapy, where they are used to correct or replace faulty genes responsible for genetic disorders, and platform vaccines, where they are used to deliver antigens or a nucleic acid sequence encoding for antigens. Viral vectors can be distinguished in enveloped and non-enveloped viral vectors. Enveloped viral vectors comprise a lipid membrane, known as “coat” or “envelope”, that surroundstheir protein coat, known as “capsid”, and allows the vector to fuse with the membrane of the target cell and initiate the process of delivering the genetic payload. Examples of enveloped viral vectors include retroviruses, such as lentiviruses, which are capable of infecting both dividing and non-dividing cells, and can integrate their genetic payload in the genome of the target cell; other examples of enveloped viral vectors include baculovirus and herpes virus. Non-enveloped viral vectors lack the membrane and utilize different mechanisms, such as endocytosis, to enter target cells. Examples of nonenveloped viral vectors include adenoviruses and adeno-associated viruses, which can deliver genes without integrating into the host genome, leading to transient expression, and are therefore often used in gene therapy.

[0031] As used herein, “branched polymers” are classes of polymers characterized by structure that includes branch points and side chains. Branched polymers are polymer chains that feature branch points, connecting three or more chain segments, and can exhibit either regular or irregular branching along the main chain. Examples of branched polymers include low-density polyethylene or branched polylactic acid, which consists of polymer chains with short branch chains. Branched polymers can have a highly branched three-dimensional structure, which is characterized by a large number of modified terminal functional groups, can be either regular or irregular, and presents with multiple terminal functional groups. These branched polymers are known for their excellent solubility and temperature resistance, which are attributed to their unique structure. Examples of such branched polymers include dendrimers and branched polyethylene glycol.

[0032] As used herein, a “reactive group” or “reactive moiety” is a specific atom or group of atoms that is capable of producing a chemical reaction with a second atom or a second group of atoms, optionally leading to the formation of a covalent bond with the second atom or a second group of atoms. Examples of reactive groups include hydroxyl, primary and secondary amines, thiols, carboxyl, alkyl halides, alkenes and alkynes, active esters, active imides, epoxides, etc. Reactive groups can be nucleophilic groups or electrophilic groups. Nucleophilic groups contain an electron-rich atom capable of donating a pair of electrons to form a covalent bond; examples of nucleophilic groups include hydroxyl (R-OH), primary amine (R-NH2), thiols (R-SH), etc. Electrophilic groups contain an electron-deficient atom capable of accepting a pair of electrons toform a covalent bond; examples of electrophilic groups include carboxyl (R-COOH), alkyl bromide (R-Br), etc.

[0033] As used herein, the “capping” of reactive group refers to the process of chemically modifying residual reactive groups to make them unreactive, thereby preventing further reactions or interactions. The capping of residual reactive groups is typically achieved using capping agents, namely reagents that specifically target the reactive groups to form with them a covalent bond that is chemically inert. Once a residual reactive group has been capped by a capping agent, the resulting covalent bond is not further modified within the scope of the functionalization and subsequent application.

[0034] As used herein, a “mixture” comprises a target biologic of interest (for which purification is desired) and one or more contaminant or impurity. In some embodiments, the mixture is produced from a host cell or organism that expresses the protein of interest (either naturally or recombinantly). Such mixtures include, for example, cell cultures, cell lysates, and clarified bulk (e.g., clarified cell culture supernatant).

[0035] As used herein, a “derivative” with respect to a peptide or polypeptide generally has the amino acid sequence of a reference peptide or variant, but additionally comprises a chemical modification of one or more of its amino acid side groups, a-carbon atoms, terminal amino group, or terminal carboxylic acid group. A chemical modification includes, but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties. Modifications at amino acid side groups include, without limitation, acylation of lysine s-amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine. Modifications of the terminal amino include, without limitation, the desamino, N-lower alkyl, N-di-lower alkyl, constrained alkyls (e.g., branched, cyclic, fused, adamantyl) and N-acyl modifications. Modifications of the terminal carboxy group include, without limitation, the amide, lower alkyl amide, constrained alkyls (e.g. branched, cyclic, fused, adamantyl) alkyl, dialkyl amide, and lower alkyl ester modifications. Lower alkyl is C1-C4 alkyl. Furthermore, one or more side groups, or terminal groups, may be protected by protective groups known to the ordinarily skilled peptide chemist. The a-carbon of an amino acid may be mono- or dimethylated.

[0036] Additionally, as used herein, a “variant” with respect to a peptide or polypeptide generally refers to a peptide or polypeptide whose base amino acid sequence was derivedfrom that of a reference peptide or polypeptide. A variant can include conservative and / or non-conservative amino acid substitutions (including non-natural amino acids and L and D forms).

[0037] As used herein, “hydrodynamic radius”, sometimes abbreviated R. / ,, is a parameter that describes the size of a particle or a molecule, such as a branched polymer, in a fluid, considering its shape, mass, and interactions with the surrounding fluid molecules. In some aspects it is defined as the radius of a hypothetical sphere that diffuses at the same rate as the branched polymer.2. Convective affinity adsorbents functionalized with affinity ligands for the purification of biological materials

[0038] The high binding capacity characteristic of chromatography resins stems from the large surface area of their pores. However, the tortuous morphology and limited diameter of these pores reduce the transport of large biologies, such as multi-protein constructs, viruses, virus-like particles, and extracellular vesicles, due to diffusion limitations. This prolongs the total time of the purification process, which may lead to a decreased product’s bioactivity and increased production costs. Thus, whilst resins functionalized with ligands for use in chromatography and purification applications are still widely utilized in the biopharmaceutical industry, there is general consensus on the need to transition to “convective” chromatographic adsorbents.

[0039] The limitations of chromatographic resins become evident in the purification of valuable complex biologies, including labile proteins such as enzymes and blood factors, engineered polynucleotides such as DNA plasmids and mRNA, engineered antibodies such as multi-specific monoclonal antibodies and their drug conjugates, viral vectors and virus-like particles, extracellular vesicles such as exosomes, and cells. Columns packed with affinity, mixed-mode, or ion-exchange chromatographic resins - either comprising natural stationary phases such as crosslinked agarose, or synthetic stationary phases such as polymethacrylate - provide good binding capacity and product purity. However, due to diffusional limitations and to limit pressure drops (or compaction of the packed bed), resin-packed columns (i) require operation at low flow rates, which prolongs the residence of the target biologic adsorbed on the stationary phase; (ii) produce large volumes of eluates at low product titer, which complicates the subsequent purification steps; and (ii) often provide limited yield of larger biologies whose size approaches thevalue of the pore diameter of the resin. Collectively, these phenomena limit the productivity, and consequently the availability and affordability of biotherapeutics, and pose concerns on the efficacy and safety of the biopharmaceutical formulation.

[0040] Convective adsorbents such as membranes, fiber mats, and monoliths offer an excellent alternative to resins as their open porosity eliminates or significantly reduces diffusive limitations and enables processing at significantly higher flow rates and lower pressure drops. Affinity monoliths for biological separations have been demonstrated, but the large-scale manufacturing of monoliths is challenging and - to date - has not been fully accomplished. On the other hand, membranes and fiber mats can be easily produced at a large scale, but - to date - there is no affinity membrane with sufficient binding capacity to warrant industrial application for purifying high-value biologies, such as biotherapeutics and vaccines.

[0041] Commonly employed materials for the production of chromatographic membranes and fiber mats are cellulose or regenerated cellulose, polyamides such as nylon, polyolefins such as polyethylene, polyesters such as polybutylene terephthalate, polysulfones and polyethersulfone, styrene maleic acid copolymers, etc.Chromatographic membranes are manufactured using methods such as phase inversion or by casting, with or without the addition of porogens. Chromatographic fiber mats are manufactured using methods such as wet-laying, electrospinning, melt blowing, melt electrospinning, etc. Both the base materials and the manufacturing techniques are inexpensive and yield large production volumes.

[0042] The limited binding capacity of chromatographic membranes and fiber mats stems from their lower specific surface (z.e., the area available for adsorbing the target biologic per unit of weight or unit of total volume of membrane) compared to porous resins. In order to increase the binding capacity, several approaches have been introduced including (i) impregnating the pore space of the membrane or fiber mat with (i.a) porous polymer or ceramic beads or (i.b) hydrogels in the form of either a continuous phase or microgels; and (ii) forming a permeable polymeric sheath coating on the surface of the membrane or the fibers in the mat. The porous beads, the continuous hydrogel or discrete microgels, and the polymeric sheath are functionalized with ligands that enable the chromatographic process to be conducted in various binding modes (z.e., affinity, mixed-mode, ion exchange, hydrophobic interaction, etc.) or operational modes (z.e., bind and elute, flow-through, or weak-partition). The design parameters of theporous beads, the hydrogel or microgels, and the polymeric sheath - namely, their molecular structure, microstructure (z.e., thickness, morphology), and ligand functionalization - determine the binding capacity of the convective adsorbent and the yield of target biologic, yet often in opposing trends: higher hydrogel contents or thicker sheaths typically increase binding capacity but decrease total permeability and product yield; similarly, higher ligand density increase binding capacity but decrease product yield.

[0043] As disclosed herein, in some embodiments provided are membranes functionalized with ligands for the chromatographic purification of biologies in bind-and-elute, flow-through, and weak-partitioning mode, including but not limited to the affinity purification of antibodies and viral vectors for gene and cell therapies. As discussed further herein, the disclosed functionalized membranes provide high binding capacity when operated at high flow rates and high product yield, thereby presenting an effective route to enhance the productivity of bioseparation processes.

[0044] Without being bound by any particular theory or mechanism of action, the optimal accessibility of the ligand on the functionalized membrane or fiber mat enables effective product binding and elution in a shorter timeframe compared to conventional resins or other convective adsorbents on the market. The ligand accessibility results from using a branched polymer that is (i) grafted on the surface of the membrane’s pores or the surface of the fibers in the fiber mat, and (ii) acts as a scaffold (or framework) for the conjugation at optimal density and display at optimal presentation of the ligands. The techniques for grafting of the branched polymer on the membrane or fiber mat, and for functionalizing the branched polymer with ligands are scalable.

[0045] In one aspect, an affinity convective adsorbent for purifying a biologic is provided. The convective adsorbent comprises a convective porous substrate having a surface that can be functionalized; branched polymers tethered to the surface of the convective porous substrate, wherein the one or more branched polymers comprise one or more reactive terminal groups; and ligands conjugated to a terminus of the one or more branched polymers.

[0046] In some aspects, the substrate of the convective adsorbent is a membrane, where the membrane can be cellulose membrane, a regenerated cellulose membrane, a polyester fiber mat, a nylon membrane, a polyamide fiber mat, a polyolefin membrane, a polyester membrane, combinations thereof, and the like.

[0047] In some aspects, the fiber mat comprises a cellulose fiber mat, a derivatized cellulose fiber mat, a polysulfone fiber mat, a nylon fiber mat, a polyimide fiber mat, a polyolefine fiber mat, a polyester fiber mat, combinations thereof, and the like.

[0048] In some aspects, the branched polymers or branched polymer grafted on the surface of the substrate include a branched polyester, a branched polyamide, a branched polyether, a branched polyamine, and / or a branched polyurethane. In some aspects, the branched polymer comprises a branched polyethylenimine (PEI), a poly(amidoamine) (PAMAM) dendrimer, a dendritic peptide, a peptide-PAMAM hybrid (DendriPep), a branched polyethylene glycol (PEG), a branched polylactic acid (PLA), and the like. As discussed herein, the branched polymers preferably comprise pre-formed branched polymers, i.e. formed prior to grafting to the surface of the substrate (membrane and / or fiber mat).

[0049] In contrast to existing chromatographic membranes and fiber mats whereby the grafted polymer is produced in situ on the surface of the substrate, for example by radical polymerization, or a continuous hydrogel phase or discrete microgels are randomly inserted in the pores of the substrate, in the present disclosure pre-formed branched polymer(s) are attached / grafted to the membrane / fiber mat. That is, the branched polymers are already formed and purified prior to their grafting onto the surface of the convective adsorbent, as opposed to forming the branched polymer in situ on the surface. By using preformed branched polymers, the present disclosure provides for the grafting of branched polymers with controlled and monodispersed hydrodynamic radius onto the pore surface of the substrate thereby achieving a thick uniform layer, whereby the thickness of the layer comprising the branched polymer is between about 2 nm and about 5 nm and forms between about 1 weight % and about 50 weight % of the final convective affinity adsorbent. More particularly, in some aspects, the hydrodynamic radius of the branched polymer is about 1 nm to about 20 nm, optionally about 2 nm to about 5 nm.

[0050] In some embodiments, the thickness of the layer comprising the branched polymer is about 2 nm, 3 nm, 4 nm, or 5 nm. That is, the uniform layer of pre-formed branched polymers grafted to the surface of the membrane / fiber mat extends about 2 nm to about 5 nm from the surface of the membrane / fiber mat. In some embodiments, the layer of branched polymer grafted to the surface of the membrane / fiber mat forms about 1 weight %, about 5 weight %, about 10 weight %, about 15 weight %, about 20 weight%, about 25 weight %, about 30 weight %, about 35 weight %, about 40 weight %, about 45 weight %, and / or about 50 weight % of the final convective affinity adsorbent. The layer of pre-formed branched polymers grafted onto the surface of the membrane, or the fiber mat allows for optimal density and spatial display of the ligand, and consequently optimal performance - namely, high binding capacity, high binding selectivity, and high elution yield - of the convective affinity adsorbent. In some aspects, the branched polymer grafted on the convective adsorbent is functionalized with an affinity ligand, whereby the grafting of the branched polymers to the convective adsorbent and the conjugation of the ligands to the branched polymer relies on reactive groups capable of forming covalent bonds. Without any restriction, the reactive groups present on the surface of the convective adsorbent for grafting the branched polymers and the reactive group available on the ligand for conjugation to the branched polymers are nucleophilic groups (e.g., hydroxyl, primary or secondary amine, or thiol, etc.), and optionally the same nucleophilic group, whereas the reactive groups displayed on the branched polymers are electrophilic groups (e.g., carboxyl, active ester, mal eimide, etc.).Alternatively, reactive groups present on the surface of the convective adsorbent for grafting the branched polymers, the reactive group available on the ligand for conjugation to the branched polymers, and the reactive groups displayed on the branched polymers are nucleophilic groups, and optionally the same group. In this second case, reagents (e.g., carbonyldiimidazole, iodoacetyl chloride, etc.) are utilized to modify the groups present on the surface of the convective adsorbent to enable the grafting the branched polymers and subsequently to modify the groups present on the branched polymers to enable the conjugation of the ligands to the branched polymers.

[0051] Thus, in some embodiments, the one or more branched polymers tethered to the surface of the convective porous substrate represent about 1% to about 50% of the weight of the convective porous substrate, optionally about 1% to about 20% of the weight of the convective porous substrate, optionally about 5% to about 10% of the weight of the convective porous substrate, optionally about 1% to about 5% of the weight of the convective porous substrate.

[0052] In some aspects, the ligand is conjugated to at least one of the one or more reactive terminal groups of the one or more branched polymers, optionally wherein the ligand is conjugated to at least one of the one or more reactive terminal groups other thanthe one or more reactive terminal groups tethering the one or more branched polymers to the surface of the convective porous substrate.

[0053] The affinity ligand used in the disclosed affinity membranes and substrates can be any suitable ligand with an affinity to the target molecule(s). For example, and without limitation, such a ligand can comprise a protein ligand, a peptide ligand, an aptamer ligand, a mixed-mode ligand, and / or a triazine ligand. A mixed-mode ligand comprises an alkylammonium group, and / or an aromatic group, and / or a carboxyl group, and / or an alkyl chain, and / or a thioether group.

[0054] Such peptide ligands comprise a target-binding peptide sequence and a peptide linker connecting the target-binding peptide sequence to the terminal group of the one or more branched polymers. The peptide sequence comprises about 4 to about 50 amino acids, optionally about 5 to about 20 amino acids, optionally about 10 to about 35 amino acids.

[0055] More particularly, example peptide ligands include, but are not limited to, Lentivirus-binding peptides of amino acid sequences EWKAAFIW (SEQ ID NO: 1), FEKISNAE (SEQ ID NO: 2), GEFENINW (SEQ ID NO: 3), GKEAAFAA (SEQ ID NO: 4), SIEINSSE (SEQ ID NO: 5), SKSAAEHE (SEQ ID NO: 6), SNEIEIAN (SEQ ID NO: 7), EHFEHWSE (SEQ ID NO: 8), FEKISNAE (SEQ ID NO: 2), C-cyclo[GSRAFVGDAD]C (SEQ ID NO: 9), SRQFVCGDSDRD (SEQ ID NO: 10), SRAFVGDADRD (SEQ ID NO: 11), and SFVRIGLSD (SEQ ID NO: 12). Furthermore, in some aspects, peptide ligands can include AdenoAssociated Virus-binding peptides, including for example those of amino acid sequences CYGHFSGYGNYGPC (SEQ ID NO: 13), CYGHFSPYGNYGPC (SEQ ID NO: 14), CYHFSYNYPC (SEQ ID NO: 15), CYHFSYNYPKSC (SEQ ID NO: 16), CYIHFSGYTNYNGSLKSC (SEQ ID NO: 17), CYIHFSGYTNYNPC (SEQ ID NO: 18), CYIHFSGYTNYNPSLKSC (SEQ ID NO: 19), CYIHFSPYTNYNPSLKSC (SEQ ID NO: 20), CYVHFSGYSNYSPSC (SEQ ID NO: 21), GCGQQYWIGPFTFGCG (SEQ ID NO: 22), GQQYWIGPFTFG (SEQ ID NO: 23), LETVKPGLYEPITHPRDYS (SEQ ID NO: 24), and SYDRPHTIPEYLGPKVTEL (SEQ ID NO: 25), AIVSPQFQEISLPTTSTVIDGSQSTDDDKIVQY (SEQ ID NO: 26), CDGSQSTDDDKIC (SEQ ID NO: 27), CDSQSTDDDKIC (SEQ ID NO: 28), CSGSTDDDKIC (SEQ ID NO: 29), CSGSTEQEKIC (SEQ ID NO: 30), CVIDGSQSTDDDKIC (SEQ ID NO: 31), CVIDGSQSTDDDKIVQYC (SEQ ID NO: 32), GCLITHPRDYS (SEQ ID NO: 33), GCLITHPRDYSGCG (SEQ ID NO: 34),GYIHFSGYTNYNPSLKS (SEQ ID NO: 35), GYWIGPFTGGGYIHFSGYT (SEQ ID NO: 36), GYWIGPFTGPGYIHFSGYT (SEQ ID NO: 37), GYWIGPFTPGPYIHFSGYT (SEQ ID NO: 38), LITHPRDYSPKLTPGLYEFG (SEQ ID NO: 39), and TVIDGSQSTDDDKIVQY (SEQ ID NO: 40). Still yet, the peptide ligands can be Host Cell Protein-binding peptides of amino acid sequences IYRIGR (SEQ ID NO: 41), YRFD (SEQ ID NO: 42), DRNI (SEQ ID NO: 43), HYFD (SEQ ID NO: 44), RYYYAI (SEQ ID NO: 45), DKSI (SEQ ID NO: 46), GSRYRY (SEQ ID NO: 47), AAHIYY (SEQ ID NO: 48), HSKIYK (SEQ ID NO: 49), HAIYPHRH (SEQ ID NO: 50), DICLPRWGCLW (SEQ ID NO: 51), EHIPA (SEQ ID NO: 52), DLCLRDWGCLW (SEQ ID NO: 53), and GPRPK (SEQ ID NO: 54).

[0056] The Lentivirus-binding capacity of the affinity adsorbent can be at least about 109transducing units per mL of adsorbent. Additionally, the yield of purified, celltransducing Lentivirus provided by the affinity adsorbent is at least 30%, and optionally at least 70%.

[0057] The Adeno Associated Virus-binding capacity of the affinity adsorbent is at least about 1013viral particles per mL of adsorbent. Moreover, the yield of purified, celltransducing Lentivirus provided by the affinity adsorbent is at least 50%, and optionally at least 90%.

[0058] The host cell protein-binding capacity of the affinity adsorbent is at least 10 mg of Host Cell Proteins per mL of adsorbent. The yield of purified target biologic provided by the affinity adsorbent is at least 80%, and optionally at least 90%.

[0059] For all of the disclosed affinity adsorbents, they can be configured to provide a reduction of Host Cell Proteins in an eluate of at least 100-fold, optionally at least about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 75-fold, about 99-fold or more.

[0060] In some embodiments, the peptide ligand comprises a target-binding peptide sequence and a peptide linker connecting the target-binding peptide sequence to the terminal group of the one or more branched polymers. The linking peptide segment can comprise 1 to 10 amino acids, optionally 1 to 2 amino acids, optionally 2 to 4 amino acids, optionally 3 to 5 amino acids. By way of example and not limitation, the linking peptide segment can be any peptide sequence C, GC, GSGC (SEQ ID NO: 55), GGGC (SEQ ID NO: 56), GSGSC (SEQ ID NO: 57), K, GK, GSGK (SEQ ID NO: 58), GGGK (SEQ ID NO: 59), and / or GSGSK (SEQ ID NO: 60).

[0061] In some embodiments, mixed-mode ligands, optionally an alkylamine or an alkylammonium group, are conjugated to the branched polymer alongside the peptide ligands.

[0062] In some embodiments, after ligand conjugation the residual reactive groups on the branched polymer are capped with appropriate capping agents.

[0063] To elaborate on the peptide ligands, in some embodiments the ligands can include a least one peptide ligand that is at least four amino acids in length and comprises: (i) at least one charged amino acid, and (ii) at least one amino acid comprising a side chain capable of hydrogen bond formation.

[0064] In some aspects, the pre-formed branched polymers are grafted to the convective substrate via a first end of the one or more branched polymers, and the ligands are conjugated to a second and opposing terminus of the branched polymers, where a distance from the first end of the branched polymer and the second and opposing end of the branched polymer is about 2 nm to about 5 nm. With such a configuration, the conjugated ligand is spaced apart from the surface of the membrane at a distance of about 2 nm to about 5 nm. With this design, the spacing of the conjugated ligand with respect to the surface of the membrane uniquely improves the binding capacity, the binding selectivity, the binding kinetics, the release kinetics, and the yield of release of the target biologic. In one example, the conjugation of branched PEI of average molecular weight of 25,000 grams per mole, followed by conversion of primary amine terminal groups of PEI to reactive groups iodoacetamide, followed by the conjugation of the ligand GKEAAFAAC (SEQ ID NO: 61) on the iodoacetamide-activated branched PEI at the density of about 0.01 to about 0.03 mmol of peptide per mL of convective adsorbent provides a lentivirus-binding capacity of about 109to 1010cell-transducing virions per mL of convective adsorbent, as compared to the lentivirus-binding capacity of less than 109cell-transducing virions per mL of convective adsorbent obtained by conjugating ligand GKEAAFAAC (SEQ ID NO: 61) to a hydrogel-filled membrane. In another example, the conjugation of branched PEI of average molecular weight of 25,000 grams per mole, followed by conversion of primary amine terminal groups of PEI to reactive groups iodoacetamide, followed by the conjugation of the ligands DRNIGC (SEQ ID NO: 62), YRFDGC (SEQ ID NO: 63), HSKIYKGC (SEQ ID NO: 64), GSRYRYGC (SEQ ID NO: 65), HAIYPHRHGC (SEQ ID NO: 66), GPRPKC (SEQ ID NO: 67) on the iodoacetamide-activated branched PEI at the density of about 0.03 toabout 0.05 mmol of peptide per mL of convective adsorbent provides a host cell proteinbinding capacity of about 10 to about 20 milligrams of protein per mL of convective adsorbent, as compared to the host cell protein-binding capacity of less than 5 milligrams of protein per mL of convective adsorbent obtained by conjugating ligands DRNIGC (SEQ ID NO: 62), YRFDGC (SEQ ID NO: 63), HSKIYKGC (SEQ ID NO: 64), GSRYRYGC (SEQ ID NO: 65), HAIYPHRHGC (SEQ ID NO: 66), GPRPK (SEQ ID NO: 67) directly to the surface of the membrane. In another example, increasing the grafted amount of branched PEI from 10 weight % to 100 weight % of the substrate followed by the conjugation of ligands DRNIGC (SEQ ID NO: 62), YRFDGC (SEQ ID NO: 63), HSKIYKGC (SEQ ID NO: 63), GSRYRYGC (SEQ ID NO: 65), HAIYPHRHGC (SEQ ID NO: 66), GPRPK (SEQ ID NO: 67) on the branched PEI at the density of about 0.1 mmol of peptide per mL of convective adsorbent decreased the host cell protein-binding capacity from about 20 to less than 5 milligrams of protein per mL of convective adsorbent.

[0065] In some aspects, multiple peptide and mixed-mode ligands can be conjugated to the branched polymer to further increase the binding capacity of the resulting convective affinity adsorbent. In one example, the conjugation of branched PEI of average molecular weight of 50,000 grams per mole, followed by conversion of primary amine terminal groups of PEI to reactive groups iodoacetamide, followed by the conjugation of the ligands DRNIGC (SEQ ID NO: 62), YRFDGC (SEQ ID NO: 63), HSKIYKGC (SEQ ID NO: 64), GSRYRYGC (SEQ ID NO: 65), HAIYPHRHGC (SEQ ID NO: 66), GPRPKC (SEQ ID NO: 67) on the iodoacetamide-activated branched PEI at the density of about 0.03 to about 0.05 mol of peptide per mL of convective adsorbent, further followed by the conjugation of ligand thiocholine chloride on the iodoacetamide-activated PEI at the density of about 0.05 to 0.1 mmol of thiocholine chloride per mL of convective adsorbent provides a host cell protein-binding capacity above 20 milligrams of protein per mL of convective adsorbent, as compared to the host cell protein-binding capacity of less than 20 milligrams of protein per mL of convective adsorbent obtained with the conjugation of ligands DRNIGC (SEQ ID NO: 62), YRFDGC (SEQ ID NO: 63), HSKIYKGC (SEQ ID NO: 64), GSRYRYGC(SEQ ID NO: 65), HAIYPHRHGC (SEQ ID NO: 66), GPRPKC (SEQ ID NO: 67) without the following conjugation of ligand thiocholine chloride. In one example, the conjugation of branched PEI of average molecular weight of 50,000 grams per mole, followed by conversion of primary amineterminal groups of PEI to reactive groups iodoacetamide, followed by the conjugation of the ligands DRNIGC (SEQ ID NO: 62), YRFDGC (SEQ ID NO: 63), HSKIYKGC (SEQ ID NO: 64), GSRYRYGC(SEQ ID NO: 65), HAIYPHRHGC (SEQ ID NO: 66), GPRPKC (SEQ ID NO: 67) on the iodoacetamide-activated branched PEI at the density of about 0.03 to about 0.05 mol of peptide per mL of convective adsorbent, further followed by the conjugation of ligand 2-(diethylamino)ethanethiol on the iodoacetamide-activated PEI at the density of about 0.05 to 0.1 mmol of thiocholine chloride per mL of convective adsorbent provides a host cell protein-binding capacity above 20 milligrams of protein per mL of convective adsorbent, as compared to the host cell protein-binding capacity of less than 20 milligrams of protein per mL of convective adsorbent obtained with the conjugation of ligands DRNIGC (SEQ ID NO: 62), YRFDGC (SEQ ID NO: 63), HSKIYKGC(SEQ ID NO: 64), GSRYRYGC(SEQ ID NO: 65), HAIYPHRHGC (SEQ ID NO: 66), GPRPKC (SEQ ID NO: 67) without the following conjugation of ligand 2-(diethylamino)ethanethiol.

[0066] As would be recognized by one of ordinary skill in the art based on the present disclosure, some amino acids are known to be electrically charged. In general, lysine (K), arginine (R) and histidine (H) are known to be amino acids having a positive charge (positively charged amino acids). Aspartic acid (D), glutamic acid (E), and such are known to be amino acids having a negative charge (negatively charged amino acids). In addition, alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), and the like are known to be amino acids that do not have a charge, or nonpolar amino acids. In some embodiments, and as described further herein, at least one charged amino acid that is present in the peptide ligands of the present disclosure includes arginine (R), histidine (H), lysine (K), and / or glutamate (E). In some embodiments, at least one charged amino acid that is not present in the peptide ligands of the present disclosure is aspartate (D).

[0067] As would be recognized by one of ordinary skill in the art based on the present disclosure, some amino acids comprising side chains that are capable of hydrogen bond formation (e.g., hydrogen bond donor and / or a hydrogen bond acceptor). These amino acids generally include methionine (M), serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), lysine (K), alanine (A), glycine (G), tyrosine (Y), and tryptophan (W). In someembodiments, at least one amino acid comprising a side chain capable of hydrogen bond formation that is present in the peptide ligands of the present disclosure includes tyrosine (Y), tryptophan (W), and / or glutamine (Q). In some embodiments, at least one amino acid comprising a side chain capable of hydrogen bond formation that is not present in the peptide ligands of the present disclosure is phenylalanine (F).

[0068] In some embodiments, and as described further herein, at least one peptide ligand comprises alanine (A) or valine (V). In some embodiments, the at least one peptide ligand does not comprise isoleucine (I), leucine (L), or proline (P). In some embodiments, an arginine (R) residue is flanked (i.e., directly adjacent to) by a tyrosine (Y) residue and / or a tryptophan (W) residue. In some embodiments, a valine (V) residue is flanked by a tyrosine (Y) residue and / or a tryptophan (W) residue. In some embodiments, an alanine (A) residue is flanked by a lysine (K) residue.

[0069] As would be recognized by one of ordinary skill in the art based on the present disclosure, some amino acids are hydrophilic or polar in nature. Generally, amino acids considered to be hydrophilic or polar amino acids include serine (S), threonine (T), cysteine (C), asparagine (N), glutamine (Q), and tyrosine (Y). In some embodiments, and as described further herein, the peptide ligands of the present disclosure can include one or more hydrophilic or polar amino acids. In some embodiments, two of the at least four amino acids in the peptide ligands of the present disclosure are hydrophilic amino acids. In some embodiments, three of the at least four amino acids in the peptide ligands of the present disclosure are hydrophilic amino acids. In some embodiments, four of the at least four amino acids in the peptide ligands of the present disclosure are hydrophilic amino acids.

[0070] As would be recognized by one of ordinary skill in the art based on the present disclosure, amino acids that are considered to be hydrophobic (i.e., have hydrophobic side chains) include glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), and tryptophan (W). In some embodiments, two of the at least four amino acids in the peptide ligands of the present disclosure are hydrophobic amino acids. In some embodiments, three of the at least four amino acids in the peptide ligands of the present disclosure are hydrophobic amino acids. In some embodiments, four of the at least four amino acids in the peptide ligands of the present disclosure are hydrophobic amino acids.

[0071] 3. Methods of making convective affinity adsorbents functionalized with affinity ligands for the purification of biological materials

[0072] Methods of making affinity membranes are also provided. Such methods include providing a convective porous substrate having a surface that can be functionalized, tethering one or more branched polymers on a surface of the convective porous substrate, wherein the one or more branched polymers comprise one or more reactive terminal groups, wherein the one or more branched polymers comprise a pre-formed branched polymer, and conjugating a ligand to a terminus of the one or more branched polymers.

[0073] In some aspects, the convective porous substrate comprises a membrane and / or a fiber mat. The method may also include where the membrane includes a cellulose membrane, a derivatized cellulose membrane, a polysulfone membrane, a nylon membrane, a polyimide membrane, a polyolefine membrane, and / or a polyester membrane. The fiber mat can comprise a cellulose fiber mat, a derivatized cellulose fiber mat, a polysulfone fiber mat, a nylon fiber mat, a polyimide fiber mat, a polyolefine fiber mat, and / or a polyester fiber mat.

[0074] In some aspects of the methods of making, the branched polymers or branched polymer grafted on the surface of the substrate include a branched polyester, a branched polyamide, a branched polyether, a branched polyamine, and / or a branched polyurethane. In some aspects, the branched polymer comprises a branched polyethylenimine (PEI), a poly(amidoamine) (PAMAM) dendrimer, a dendritic peptide, a peptide-PAMAM hybrid (DendriPep), a branched polyethylene glycol (PEG), a branched polylactic acid (PLA), and the like. As discussed herein, the branched polymers preferably comprise pre-formed branched polymers, i.e. formed prior to grafting to the surface of the substrate (membrane and / or fiber mat).

[0075] In contrast to existing chromatographic membranes and fiber mats whereby the grafted polymer is produced in situ on the surface of the substrate, for example by radical polymerization, or a continuous hydrogel phase or discrete microgels are randomly inserted in the pores of the substrate, in the present methods of making affinity membranes pre-formed branched polymer(s) are attached / grafted to the membrane / fiber mat. That is, the branched polymers are already formed prior to engraftment onto a surface of the membrane, as opposed to forming the branched polymer in situ on the surface. By using preformed branched polymers, the present disclosure provides for the grafting of branched polymers with controlled and monodispersed hydrodynamic radiusonto the pore surface of the substrate thereby achieving a thick uniform layer, whereby the thickness of the layer comprising the branched polymer is between about 2 nm and about 5 nm and forms between about 1 weight % and about 50 weight % of the final convective affinity adsorbent. More particularly, in some aspects, the hydrodynamic radius of the branched polymer is about 1 nm to about 20 nm, optionally about 2 nm to about 5 nm.

[0076] In some embodiments, the thickness of the layer comprising the branched polymer is about 2 nm, 3 nm, 4 nm, or 5 nm. That is, the uniform layer of pre-formed branched polymers grafted to the surface of the membrane / fiber mat extends about 2 nm to about 5 nm from the surface of the membrane / fiber mat. In some embodiments, the layer of branched polymer grafted to the surface of the membrane / fiber mat forms about 1 weight %, about 5 weight %, about 10 weight %, about 15 weight %, about 20 weight %, about 25 weight %, about 30 weight %, about 35 weight %, about 40 weight %, about 45 weight %, and / or about 50 weight % of the final convective affinity adsorbent. The layer of pre-formed branched polymers grafted onto the surface of the membrane, or the fiber mat allows for optimal density and spatial display of the ligand, and consequently optimal performance - namely, high binding capacity, high binding selectivity, and high elution yield - of the convective affinity adsorbent. In some aspects, the branched polymer grafted on the convective adsorbent is functionalized with an affinity ligand, whereby the grafting of the branched polymers to the convective adsorbent and the conjugation of the ligands to the branched polymer relies on reactive groups capable of forming covalent bonds. Without any restriction, the reactive groups present on the surface of the convective adsorbent for grafting the branched polymers and the reactive group available on the ligand for conjugation to the branched polymers are nucleophilic groups (e.g., hydroxyl, primary or secondary amine, or thiol, etc.), and optionally the same nucleophilic group, whereas the reactive groups displayed on the branched polymers are electrophilic groups (e.g., carboxyl, active ester, mal eimide, etc.).Alternatively, reactive groups present on the surface of the convective adsorbent for grafting the branched polymers, the reactive group available on the ligand for conjugation to the branched polymers, and the reactive groups displayed on the branched polymers are nucleophilic groups, and optionally the same group. In this second case, reagents (e.g., carbonyldiimidazole, iodoacetyl chloride, etc.) are utilized to modify the groups present on the surface of the convective adsorbent to enable the grafting thebranched polymers and subsequently to modify the groups present on the branched polymers to enable the conjugation of the ligands to the branched polymers.

[0077] Thus, in some embodiments, the one or more branched polymers tethered to the surface of the convective porous substrate represent about 1% to about 50% of the weight of the convective porous substrate, optionally about 1% to about 20% of the weight of the convective porous substrate, optionally about 5% to about 10% of the weight of the convective porous substrate, optionally about 1% to about 5% of the weight of the convective porous substrate.

[0078] In some aspects of these methods, the ligand is conjugated to at least one of the one or more reactive terminal groups of the one or more branched polymers, optionally wherein the ligand is conjugated to at least one of the one or more reactive terminal groups other than the one or more reactive terminal groups tethering the one or more branched polymers to the surface of the convective porous substrate.

[0079] The affinity ligand used in the disclosed methods of making affinity membranes and substrates can be any suitable ligand with an affinity to the target molecule(s). For example, and without limitation, such a ligand can comprise a protein ligand, a peptide ligand, an aptamer ligand, a mixed-mode ligand, and / or a triazine ligand. A mixed-mode ligand comprises an alkylammonium group, and / or an aromatic group, and / or a carboxyl group, and / or an alkyl chain, and / or a thioether group.

[0080] Such peptide ligands comprise a target-binding peptide sequence and a peptide linker connecting the target-binding peptide sequence to the terminal group of the one or more branched polymers. The peptide sequence comprises about 4 to about 50 amino acids, optionally about 5 to about 20 amino acids, optionally about 10 to about 35 amino acids.

[0081] More particularly, example peptide ligands in these methods include, but are not limited to, Lentivirus-binding peptides of amino acid sequences EWKAAFIW (SEQ ID NO: 1), FEKISNAE (SEQ ID NO: 2), GEFENINW (SEQ ID NO: 3), GKEAAFAA (SEQ ID NO: 4), SIEINSSE (SEQ ID NO: 5), SKSAAEHE (SEQ ID NO: 6), SNEIEIAN (SEQ ID NO: 7), EHFEHWSE (SEQ ID NO: 8), FEKISNAE (SEQ ID NO: 2), C-cyclo[GSRAFVGDAD]C (SEQ ID NO: 9), SRQFVCGDSDRD (SEQ ID NO: 10), SRAFVGDADRD (SEQ ID NO: 11), and SFVRIGLSD(SEQ ID NO: 12). Furthermore, in some aspects, peptide ligands can include AdenoAssociated Virus-binding peptides, including for example those of amino acid sequences CYGHFSGYGNYGPC (SEQ IDNO: 13), CYGHFSPYGNYGPC (SEQ ID NO: 14), CYHFSYNYPC (SEQ ID NO: 15), CYHFSYNYPKSC (SEQ ID NO: 16), CYIHFSGYTNYNGSLKSC (SEQ ID NO: 17), CYIHFSGYTNYNPC (SEQ ID NO: 18), CYIHFSGYTNYNPSLKSC (SEQ ID NO: 19), CYIHFSPYTNYNPSLKSC (SEQ ID NO: 20), CYVHFSGYSNYSPSC (SEQ ID NO: 21), GCGQQYWIGPFTFGCG (SEQ ID NO: 22), GQQYWIGPFTFG (SEQ ID NO: 23), LETVKPGLYEPITHPRDYS (SEQ ID NO: 24), and SYDRPHTIPEYLGPKVTEL (SEQ ID NO: 25), AIVSPQFQEISLPTTSTVIDGSQSTDDDKIVQY (SEQ ID NO: 26), CDGSQSTDDDKIC (SEQ ID NO: 27), CDSQSTDDDKIC (SEQ ID NO: 28), CSGSTDDDKIC (SEQ ID NO: 29), CSGSTEQEKIC (SEQ ID NO: 30), CVIDGSQSTDDDKIC (SEQ ID NO: 31), CVIDGSQSTDDDKIVQYC (SEQ ID NO: 32), GCLITHPRDYS (SEQ ID NO: 33), GCLITHPRDYSGCG (SEQ ID NO: 34), GYIHFSGYTNYNPSLKS (SEQ ID NO: 35), GYWIGPFTGGGYIHFSGYT (SEQ ID NO: 36), GYWIGPFTGPGYIHFSGYT (SEQ ID NO: 37), GYWIGPFTPGPYIHFSGYT (SEQ ID NO: 38), LITHPRDYSPKLTPGLYEFG (SEQ ID NO: 39), and TVIDGSQSTDDDKIVQY (SEQ ID NO: 40). Still yet, the peptide ligands can be Host Cell Protein-binding peptides of amino acid sequences IYRIGR (SEQ ID NO: 41), YRFD(SEQ ID NO: 42), DRNI (SEQ ID NO: 43), HYFD (SEQ ID NO: 44), RYYYAI (SEQ ID NO: 45), DKSI (SEQ ID NO: 46), GSRYRY (SEQ ID NO: 47), AAHIYY (SEQ ID NO: 48), HSKIYK (SEQ ID NO: 49), HAIYPHRH (SEQ ID NO: 50), DICLPRWGCLW (SEQ ID NO: 51), EHIPA (SEQ ID NO: 52), DLCLRDWGCLW (SEQ ID NO: 53), and GPRPK (SEQ ID NO: 54).

[0082] In some embodiments of these methods of making, the peptide ligand comprises a target-binding peptide sequence and a peptide linker connecting the target-binding peptide sequence to the terminal group of the one or more branched polymers. The linking peptide segment can comprise 1 to 10 amino acids, optionally 1 to 2 amino acids, optionally 2 to 4 amino acids, optionally 3 to 5 amino acids. By way of example and not limitation, the linking peptide segment can be any peptide sequence C, GC, GSGC (SEQ ID NO: 55), GGGC (SEQ ID NO: 56), GSGSC (SEQ ID NO: 57), K, GK, GSGK (SEQ ID NO: 58), GGGK (SEQ ID NO: 59), and / or GSGSK (SEQ ID NO: 60). In some aspects, methods of making the affinity adsorbents further include tethering the one or more branched polymers on the surface of the convective porous substrate by means of an amide bond, or a thioether bond, or an ether bond, or a secondary amine bond, or a tertiary amine bond, or a carbamate bond, or a carbonate bond, or an ester bond.Additionally, such methods can further comprise capping residual reactive groups of the one or more branched polymers after conjugating the ligand to the terminus of the one or more branched polymers. Moreover, conjugating the ligand to the terminus of the one or more branched polymers can, in some applications, further comprise the use of one or more coupling buffers, optionally wherein the one or more coupling buffers comprises Tris buffer, EDTA-Na, TCEP and / or a combination thereof.

[0083] To elaborate further, the methods of making affinity adsorbents, e.g. functionalized membranes, may also include where grafting the one or more branched polymers on a surface of the membrane includes activating the membrane to provide a succinimidyl ester-activated membrane. Such activation can include, for example, immersing the membrane in a solution of 4-dimethylaminopyridine (DMAP) and N,N'-disuccinimidylcarbonate (DSC) followed by rinsing.

[0084] The methods of making the functionalized membranes disclosed herein can be performed in a way to achieve the ligand spacing as described herein. For example, the methods of making may include where the one or more branched polymers is grafted to the surface of the membrane via a first end of the one or more branched polymers, where the ligand is conjugated to a terminus of the one or more branched polymers at a second and opposing end of the one or more branched polymers, where a distance from the first end of the one or more branched polymers and the second and opposing end of the one or more branched polymers is about 2 nm to about 5 nm. The method may also include whereby the conjugated ligand is spaced apart from the surface of the membrane at a distance of about 2 nm to about 5 nm.

[0085] In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD that is at least about 10'3M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD that is at least about 10'4M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD that is at least about 10'5M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD that is at least about 10'6M for the HCP, the host cell nucleic acid, and / or the target protein,and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD that is at least about 10'7M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD that is at least about 10'8M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD that is at least about 10'9M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD that is at least about IO'10M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle.

[0086] In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about IO'10M to about 10'3M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'9M to about 10'3M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'8M to about 10'3M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'7M to about 10'3M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'6M to about 10'3M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'5M to about 10'3M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'4M to about 10'3M for the HCP, the host cell nucleic acid,and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD that is lower than about 10'5M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about IO'10M to about 10'4M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'9M to about 10'3M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'9M to about 10'4M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'9M to about 10'5M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'8M to about 10'3M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'8M to about 10'4M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'8M to about 10'5M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'7M to about 10'3M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'7M to about 10'4M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'7M to about 10'5M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellularvesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'6M to about 10'3M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'6M to about 10'4M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle. In some embodiments, at least one peptide ligand of the present disclosure exhibits a KD from about 10'6M to about 10'5M for the HCP, the host cell nucleic acid, and / or the target protein, and / or the target viral vector, and / or the target extracellular vesicle.

[0087] In some embodiments, where the membranes are functionalized with peptide ligands, the ligands conjugated to the branched polymers have the same amino acid sequence. In other embodiments, peptide ligands with different sequences can be conjugated to the same branched polymers to form an affinity membrane.

[0088] In some embodiments, the at least one peptide ligand is no more than 40 amino acids in length. In some embodiments, the at least one peptide ligand is no more than 35 amino acids in length. In some embodiments, the at least one peptide ligand is no more than 30 amino acids in length. In some embodiments, the at least one peptide ligand is no more than 25 amino acids in length. In some embodiments, the at least one peptide ligand is no more than 20 amino acids in length. In some embodiments, the at least one peptide ligand is no more than 16 amino acids in length. In some embodiments, the at least one peptide ligand is no more than 12 amino acids in length. In some embodiments, the at least one peptide ligand is no more than 8 amino acids in length. In some embodiments, the at least one peptide ligand is no more than 4 amino acids in length. In some embodiments, the at least one peptide ligand is from about 4 to about 20 amino acids in length. In some embodiments, the at least one peptide ligand is from about 4 to about 16 amino acids in length. In some embodiments, the at least one peptide ligand is from about 4 to about 12 amino acids in length. In some embodiments, the at least one peptide ligand is from about 4 to about 8 amino acids in length. In some embodiments, the at least one peptide ligand is from about 8 to about 16 amino acids in length. In some embodiments, the at least one peptide ligand is from about 12 to about 20 amino acids in length.

[0089] In some embodiments, and as described further herein, the affinity membranes functionalized with peptide ligands of the present disclosure can be used to purify a target biologic from a biological fluid in bind-and-elute mode, or in flow-through mode, or in weak-partitioning mode. In some embodiments, the biological fluid is a cell culture fluid that comprises a cell culture supernatant, and / or a cellular lysate. In some embodiments, the cell culture fluid comprises a pH from about 3.0 to about 9.0. In some embodiments, the cell culture fluid comprises a pH from about 4.0 to about 9.0. In some embodiments, the cell culture fluid comprises a pH from about 5.0 to about 9.0. In some embodiments, the cell culture fluid comprises a pH from about 6.0 to about 9.0. In some embodiments, the cell culture fluid comprises a pH from about 7.0 to about 9.0. In some embodiments, the cell culture fluid comprises a pH from about 3.0 to about 8.0. In some embodiments, the cell culture fluid comprises a pH from about 3.0 to about 7.0. In some embodiments, the cell culture fluid comprises a pH from about 3.0 to about 6.0. In some embodiments, the cell culture fluid comprises a pH from about 4.0 to about 8.0. In some embodiments, the cell culture fluid comprises a pH from about 5.0 to about 7.0.

[0090] In some embodiments, the cell culture fluid comprises a conductivity of about 1 to about 50 mS / cm. In some embodiments, the cell culture fluid comprises a conductivity of about 5 to about 50 mS / cm. In some embodiments, the cell culture fluid comprises a conductivity of about 10 to about 50 mS / cm. In some embodiments, the cell culture fluid comprises a conductivity of about 15 to about 50 mS / cm. In some embodiments, the cell culture fluid comprises a conductivity of about 20 to about 50 mS / cm. In some embodiments, the cell culture fluid comprises a conductivity of about 30 to about 50 mS / cm. In some embodiments, the cell culture fluid comprises a conductivity of about 40 to about 50 mS / cm. In some embodiments, the cell culture fluid comprises a conductivity of about 1 to about 40 mS / cm. In some embodiments, the cell culture fluid comprises a conductivity of about 1 to about 30 mS / cm. In some embodiments, the cell culture fluid comprises a conductivity of about 1 to about 20 mS / cm. In some embodiments, the cell culture fluid comprises a conductivity of about 1 to about 15 mS / cm. In some embodiments, the cell culture fluid comprises a conductivity of about 10 to about 40 mS / cm. In some embodiments, the cell culture fluid comprises a conductivity of about 20 to about 30 mS / cm.4. Methods of Using Membranes Functionalized with Ligands in Flow-Through or Weak-Partitioning Modes for the Purification of Target BiologiesAs described further herein, the present disclosure also provides improved methods for purifying a target biologic from a biological fluid comprising and one or more product-and / or process-related contaminants or contaminants, as compared to currently used methods. In some embodiments, the method includes contacting an affinity adsorbent disclosed herein with a biological fluid (e.g., a cell culture fluid or cell lysate) comprising the target biologic, and collecting the effluent in flow-through mode, with the effluent comprising the target biologic. In some aspects, the affinity adsorbent binds and retains at least one host cell protein (HCP), at least one high-risk HCP, at least one host cell nucleic acid, aggregates of the target biologic, and / or an impurity derived from the target biologic.

[0091] Furthermore, in some aspects, such methods can include contacting a functionalized membrane as disclosed herein with a biological fluid that includes a target biologic, and collecting an effluent in flow-through mode, where the effluent includes the target biologic, where the functionalized membrane binds and retains at least one host cell protein (HCP), at least one high-risk HCP, at least one host cell nucleic acid, aggregates of the target biologic, and / or an impurity derived from the target biologic. Such purification methods may also include performing affinity chromatography on the biological fluid that contains the target biologic before or after contacting the functionalized membrane.

[0092] In some embodiments of the methods of making affinity adsorbents the method is performed under static binding conditions. In some aspects the method is performed under dynamic binding conditions.

[0093] The methods can result in a yield of the target biologic of at least about 50%, or at least about 75%, or more. Such methods can result in the affinity adsorbent reducing Host Cell Proteins in the effluent of at least 100-fold, optionally at least 10-fold, optionally at least 5-fold.

[0094] Continuing further, the present disclosure also provides methods of purifying a target biologic from a biological fluid in bind-and-elute mode, the method comprising contacting the affinity adsorbent with a biological fluid comprising a target biologic; washing the affinity adsorbent to remove loosely bound contaminants; and releasing the target biologic using an appropriate elution buffer, thereby collecting a purified target biologic in the eluate. Such methods can further comprise performing chromatographyon the biological fluid comprising the target biologic before or after contacting the functionalized membrane.

[0095] Such methods can be performed under static binding conditions, or under dynamic binding conditions.

[0096] Such methods can result in a yield of the target biologic of at least about 50%, or of at least about 75%.

[0097] Such methods can result in the affinity adsorbent reducing Host Cell Proteins in the effluent of at least 100-fold, optionally at least 10-fold, optionally at least 5-fold.

[0098] Purification by such methods may result in a yield of the target biologic of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and preferably at least about 50% or more. The methods may also result in a purity of the target biologic of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and preferably at least about 75% or more.

[0099] The methods of the present disclosure can further comprise an isocratic washing the functionalized membrane to remove one or more unbound target biologies into a supernatant or mobile phase; and then collecting the supernatant or mobile phase containing the one or more unbound target biologies. In an embodiment, the washing step can also occur after the contacting step and after the collection of the supernatant or mobile phase.

[0100] In some embodiments, the method can be performed under any binding conditions suitable for use with the composition or adsorbent, including both static binding conditions and dynamic binding conditions. In some embodiments the unbound target biologies are collected into a supernatant when the methods are performed under static binding conditions. In some embodiments the unbound target biologies are collected into a mobile phase when the methods are performed under dynamic binding conditions. The methods of the present disclosure can optionally include flow-through chromatography and weak-partitioning chromatography.

[0101] The binding affinity of the functionalized membrane for the host cell proteins and host cell nucleic acids, as compared to the one or more target molecules, can be altered by changes in the following: properties and concentration of the one or more target proteins; the properties and concentration of the host cell proteins; the composition, concentration, and pH of the mixture; and / or the loading conditions and residence time of the contacting and washing steps. Any of these variables can be changed to variables which are suitable according to the methods of the present disclosure and result in increased or decreased binding affinity as required for the present disclosure.

[0102] In some embodiments, the contacting step comprises a high ionic strength binding buffer or low ionic strength binding buffer. A low ionic strength binding buffer comprises a buffer of between 1-50 mM NaCl. In one embodiment the low ionic strength binding buffer comprises 20mM NaCl. A high ionic strength binding buffer comprises a buffer of between 100-500mM NaCl. In one embodiment the low ionic strength binding buffer comprises 150mM NaCl.

[0103] In some embodiments, the contacting step can comprise a low pH buffer of between pH 5-9. In some embodiments, the contacting step can comprise a low pH buffer of between pH 5-8. In some embodiments, the contacting step can comprise a low pH buffer of between pH 5-7. In some embodiments, the contacting step can comprise a low pH buffer of between pH 6-9. In some embodiments, the contacting step can comprise a low pH buffer of between pH 6-8. In some embodiments, the contacting step can comprise a low pH buffer of between pH 6-7. In some embodiments, the contacting step can comprise a low pH buffer of between pH 7-9. In some embodiments, the contacting step can comprise a low pH buffer of between pH 7-8.

[0104] As would be appreciated by one of ordinary skill in the art based on the present disclosure, the methods described herein can be used before or after any purification method typically used to purify and / or isolate a given target molecule. For example, the methods disclosed herein can be used before or after ion exchange chromatography (e.g., cation exchange chromatography, anion exchange chromatography, and / or mixed mode chromatography), before or after affinity chromatography (e.g., Protein A affinity chromatography), and / or before or after size exclusion chromatography or other filtration treatment. In some embodiments, the methods disclosed herein are used after a cell culture fluid has been clarified, but prior to performing a chromatography step (e.g., Protein A affinity chromatography).

[0105] In some embodiments, the methods of the present disclosure are particularly suitable for use in the manufacturing of therapeutic antibodies, which can greatly benefit from the adoption of the compositions of peptides and adsorbents of the present disclosure owing to their potential of transforming the downstream process from a pipeline of “batch” chromatographic steps operated in “bind-and-elute” mode to a pipeline of continuous and connected chromatographic train operated in flow-through” mode. The methods described herein are also applicable to the purification of other target biologies, such as gene therapy products. These include, for example, viruses for in vivo (e.g., adenovirus and adeno-associated virus) and in vitro (e.g., lentivirus and baculovirus) gene therapy. Unlike proteins, viruses are much larger in size (> 20 nm), yet much lower in titer (108- 1013vg / mL, corresponding to levels ranging from ng / mL pg / mL, much lower than the typical mg / mL titer of proteins in cell culture harvests) and often in biochemical stability (e.g., all viruses quickly lose infectivity when exposed to the typical elution conditions (low pH) currently utilized for their purification; specific adeno-associated virus serotypes are very prone to irreversible adhesion and aggregation; lentiviruses are very sensitive to pH variations outside of the physiological range). As a result, affinity-based purification in capture-and-elute mode fails to provide the product yield and quality requested by clinics and biotech companies worldwide. The compositions and methods of the present disclosure circumvent these issues by enabling flow-through purification of viruses. The key benefits of this approach include, but are not limited to, (i) flowing the cell culture fluid from the bioreactor to capture HCPs and host cell nucleic acids while excluding the viruses, thereby improving product recovery; (ii) rapid clearance of HCPs at minimal residence time (upon adjusting the particle diameter), thereby improving product stability; (iii) operating in flow-through mode avoids virus adsorption on the resin and exposure to variations in conductivity and pH (associated to washing / elution buffers in current bind-and-elute affinity purification), thereby reducing product aggregation and preserving its transduction activity.

[0106] Additional applications of the compositions and methods of the present disclosure include the detection of low-abundance proteins in biological fluids, such as cell culture harvests, plant / tissue extracts, bodily fluids (e.g., blood, serum, plasma, sweat, urine, saliva). In this context, the prevalence of mass spectrometry (MS)-based analytical techniques for process monitoring and diagnostic applications has posed an accent on the need to enrich and / or isolate low-abundance proteins that are often key markers ofproduct quality of disease. MS-based analysis relies on the ionization of the analyte species in the sample: abundant analytes, due to their higher titer, capture most of the electrons, at the expense of low-titer analytes, which become undetected. The compositions and methods of the present disclosure can overcome these limitations by concentrating the HCPs and releasing them in a controlled fashion: (i) all HCPs are initially captured on the functionalized membrane; (ii) the HCPs are “eluted” using a linear or a stepwise gradient, which progressively releases cohorts of HCPs from the adsorbent and directly into the analytical equipment. The low-abundance proteins are present in the eluted stream at a much higher concentration and are more likely to be detected.In accordance with these embodiments, the compositions and methods of the present disclosure can be used in the production of any biologic, including but not limited to, biologic molecules such as antibodies (monoclonal and polyclonal) and antibody fragments (e.g., single-chain variable fragments (scFv), single-chain antibodies (scAb), fragment antigen binding molecules (Fab fragments), diabodies, glycoengineered antibodies, bi-specific antibodies, antibody-drug conjugates, as well as any combinations, derivatives, variants, and fusions thereof. For example, the peptide compositions and methods of the present disclosure can be used to purify any of the currently available therapeutic antibodies, including but not limited to, abciximab (Reopro), adalimumab (Humira, Amj evita), alefacept (Amevive), alemtuzumab (Campath), basiliximab (Simulect), belimumab (Benlysta), bezlotoxumab (Zinplava), canakinumab (Haris), certolizumab pegol (Cimzia), cetuximab (Erbitux), daclizumab (Zenapax, Zinbryta), denosumab (Prolia, Xgeva), efalizumab (Raptiva), golimumab (Simponi, Simponi Aria), inflectra (Remicade), ipilimumab (Yervoy), ixekizumab (Taltz), natalizumab (Tysabri), nivolumab (Opdivo), olaratumab (Lartruvo), omalizumab (Xolair), palivizumab (Synagis), panitumumab (Vectibix), pembrolizumab (Keytruda), rituximab (Rituxan), tocilizumab (Actemra), trastuzumab (Herceptin), secukinumab (Cosentyx), ustekinumab (Stelara), infliximab, and bevacizumab. In some embodiments, the present disclosure includes a method for purifying any of the aboverecited therapeutic antibodies by combining a cell culture fluid comprising one of the above-recited antibodies with a composition comprising any of the functionalized membranes disclosed herein. Furthermore, the compositions and methods of the present disclosure can be used in the production of viral vectors for gene and cell therapies suchas AAV -based therapies Hemgenix for hemophilia B, Zolgensma for muscular atrophy, Luxturna for congenital amaurosis, Roctavian for hemophilia A, Elevidys for Duchenne muscular dystrophy; and LVVs for the production of Abecma and Carvykti for multiple myeloma, Breyanzi for B-cell lymphoma, Skysona for cerebral adrenoleukodystrophy, and Zynteglo for P-thalassemia.5. Methods of Using Membranes Functionalized with Ligands in Bind-and-Elute Mode for the Purification of Target Biologies

[0107] The disclosed functionalized membranes and methods of using the same are directed to purifying biologies. As would be understood by one of ordinary skill in the art based on the present disclosure, the functionalized membranes of the present disclosure can be used to purify any biological target from a biological fluid. For example, the target biologic can be a polypeptide, a protein, an oligonucleotide, a polynucleotide, a virus or a viral capsid, a portion of the viral capsid, a virus-like particle, a cell or a cell organelle, and extracellular vesicle or an exosome, or a small molecule. In some embodiments, the target biologic is a protein, such as an antibody, an antibody fragment, an antibody-drug conjugate, a drug-antibody fragment conjugate, a Fc-fusion protein, a hormone, an anticoagulant, a blood coagulation factor, a growth factor, a morphogenic protein, a therapeutic enzyme, an engineered protein scaffold, an interferon, an interleukin, or a cytokine. As would be understood by one of ordinary skill in the art based on the present disclosure, the target biologic can be any protein, peptide, or polypeptide produced in a cell, including any endogenous, exogenous, or recombinant proteins produced by a cell, and the methods and compositions described herein can facilitate their purification from process-related contaminants and product-related contaminants. In some embodiments, at least one peptide ligand of the present disclosure binds the target biologic without binding or with minimal binding or process-related contaminants and product-related contaminants. In some embodiments, the least one peptide ligand of the present disclosure retains the target biologic during one or multiple washing steps aimed at removing any process-related contaminants and product-related contaminants present in the adsorbent after the binding step. In some embodiments, the least one peptide ligand of the present disclosure releases the target biologic during the elution step, whereby the purified target biologic is released from the stationary phase into the mobile phase.

[0108] In some embodiments, the target biologic can be a virus, viral capsid, or viral vector propagated in a cell. In some embodiments, such viruses, viral capsids, or viral vectors are engineered to deliver genetic material into cells for gene therapy, oncolytic applications, or vaccination; therefore, the various embodiments of the present disclosure can be used to purify the target biologic viruses, viral capsids, or viral vectors before they are administered to a cell or a subject. For example, the target biologic can be a retrovirus (RV), an adenovirus (AV), an adeno-associated virus (AAV), a lentivirus (LV), a baculovirus, or a herpes simplex virus (HSV). As would be understood by one of ordinary skill in the art based on the present disclosure, the target biologic can be any viral vector produced in a cell, and the methods and compositions described herein can facilitate their purification from HCPs.

[0109] In some embodiments, the target biologic can be a cell in a stem cell, a progenitor cell, or an immune effector cell. In some embodiments, the immune effector cell includes, but is not limited to, a T cell or a Natural Killer (NK) cell, including immune effector cells engineered to include a chimeric antigen receptor (CAR), such as CAR-T cells and CAR-NK cells. In some embodiments, the target biologic can be an extracellular vesicle or an exosome. In accordance with these embodiments, the affinity adsorbent or functionalized membranes of the present disclosure can be used in the production of any biologic, including but not limited to, biologic molecules such as antibodies and antibody fragments (e.g., single-chain variable fragments (scFv), singlechain antibodies (scAb), and fragment antigen binding molecules (Fab fragments), diabodies, glycoengineered antibodies, bi-specific antibodies, antibody-drug conjugates, as well as any combinations, derivatives, variants, and fusions thereof. For example, the affinity membranes of the present disclosure can be used to purify any of the currently available therapeutic antibodies, including but not limited to, abciximab (Reopro), adalimumab (Humira, Amj evita), alefacept (Amevive), alemtuzumab (Campath), basiliximab (Simulect), belimumab (Benlysta), bezlotoxumab (Zinplava), canakinumab (Haris), certolizumab pegol (Cimzia), cetuximab (Erbitux),daclizumab (Zenapax, Zinbryta), denosumab (Prolia, Xgeva), efalizumab (Raptiva), golimumab (Simponi, Simponi Aria), inflectra (Remicade), ipilimumab (Yervoy), ixekizumab (Taltz), natalizumab (Tysabri), nivolumab (Opdivo), olaratumab (Lartruvo), omalizumab (Xolair), palivizumab (Synagis), panitumumab (Vectibix), pembrolizumab (Keytruda), rituximab (Rituxan), tocilizumab (Actemra), trastuzumab (Herceptin),secukinumab (Cosentyx), ustekinumab (Stelara), infliximab, and bevacizumab.Furthermore, the compositions and methods of the present disclosure can be used in the production of viral vectors for gene and cell therapies such as AAV-based therapies Hemgenix for hemophilia B, Zolgensma for muscular atrophy, Luxturna for congenital amaurosis, Roctavian for hemophilia A, Elevidys for Duchenne muscular dystrophy; and LVVs for the production of Abecma and Carvykti for multiple myeloma, Breyanzi for B-cell lymphoma, Skysona for cerebral adrenoleukodystrophy, and Zynteglo for P-thalassemia. In some embodiments, the present disclosure includes a composition comprising any of the peptide ligands disclosed herein, and one of the above-recited therapeutic antibodies.6. Examples

[0110] The following examples are included to further illustrate various embodiments of the presently disclosed subject matter. However, those of ordinary skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the presently disclosed subject matter.

[0111] Example 1

[0112] Preparation of polyethyleneimine (PEI)-cellulose membranes. Regenerated cellulose membranes were initially rinsed with N,N'-Dimethylformamide (DMF), and air dried. A total membrane area of 100 cm2was immersed in a solution of 1.80 g of 4-dimethylaminopyridine (DMAP) and 2.24 g of N,N'-disuccinimidylcarbonate (DSC) in 35 mL of DMF for 3 hours at room temperature and gentle agitation. The membranes were then rinsed with DMF, dimethyl sulfoxide (DMSO), and isopropyl alcohol (IP A). The succinimidyl ester-activated cellulose membrane sheets were air dried and immersed in a solution of 10 g of branched polyethylenimine (PEI; molecular weight: 25 kDa or 70 kDa; MilliporeSigma, Burlington, MA) in 90 mL of MilliQ water for 3 hrs at room temperature under gentle agitation. The resulting PEI-cellulose membrane sheets were washed with water, ethanol, and acetonitrile (ACN), and air dried at room temperature. The resulting dry PEI-cellulose membrane sheet was cut into disks (thickness: 70 pm; diameter: 50 mm; volume: 0.14 mL).

[0113] Example 2

[0114] Preparation of GKEAAFAA-cellulose membranes. A set of 5 PEI-cellulose disks (see Example 1) were placed in a solution of 0.448 mL of iodoacetyl chloride(IAC) and 0.695 mL of triethyl amine (TEA) in 50 mL of ACN at room temperature under gentle mixing and in dark. After 3 hours, the disks were washed with ACN, acetone, and DMF. The unreacted primary amines of PEI were acetylated by incubating the disks in a solution of 5.7 mL of acetic anhydride and 10.2 mL of diisopropylethylamine (DIPEA) in 30 mL of N-methyl-2-pyrrolidone (NMP) for 3 hours at room temperature under mild agitation. The completion of the reaction was confirmed via Kaiser test. The iodoacetyl -activated cellulose disks were incubated in a solution of 100 mg of a test ligand, here GKEAAFAAC (SEQ ID NO: 61), in 10 mL of 50 mM Tris buffer with 5 mM EDTA-Na and 25 mM TCEP at pH 8.5 (coupling buffer) for 12 hrs at room temperature, under dark, and under end-to-end mixing. The GKEAAFAA-cellulose disks were rinsed in coupling buffer and incubated in 10 mL of a 25 mM solution of P-mercaptoethanol in coupling buffer for 2 hrs at room temperature, under dark, and under end-to-end mixing. The GKEAAFAA-cellulose disks were washed with 1 M sodium chloride and water, and finally stored in 20% v / v ethanol at 4°C.

[0115] Example 3

[0116] Preparation of peptide-functionalized (LG6 / TC and LG6 / MEDA) cellulose membranes. A first set of 5 PELcellulose disks were placed in a solution of 0.448 mL of iodoacetyl chloride (IAC) and 0.695 mL of triethyl amine (TEA) in 50 mL of ACN at room temperature under gentle mixing and in dark. After 3 hours, the resin was washed with ACN, acetone, and DMF. For each set of five iodoacetyl-activated cellulose disks were incubated in a solution of 0.064 mg of LG6-DRNIC, 0.073 mg of LG6-YRFDC, 0.090 mg of LG6-HSKIYKC, 0.094 mg of LG6-GSRYRYC, 0.112 mg of LG6-HAIYPHRHC, and 0.068 mg of LG6-GPRPKC in 10 mL of ethanol and 40 mL of 50 mM Tris buffer with 5 mM EDTA-Na and 0.2 mM NaCl at pH 8.5 (coupling buffer) for 18 hrs at room temperature, under dark, and end-to-end mixing. The resulting LG6-cellulose disks were rinsed in coupling buffer and incubated in 50 mL of a 25 mM solution of thiocholine chloride (TC) in coupling buffer for 2 hrs at room temperature, under dark, and under end-to-end mixing. The resulting LG6 / TC-cellulose disks were washed with 1 M NaCl (aq.) and MilliQ water, and finally stored in 20% v / v ethanol at 4°C.

[0117] A second set of 5 PELcellulose disks were placed in a solution of 0.448 mL of iodoacetyl chloride (IAC) and 0.695 mL of tri ethyl amine (TEA) in 50 mL of ACN at room temperature under gentle mixing and in dark. After 3 hours, the resin was washedwith ACN, acetone, and DMF. For each set of five iodoacetyl-activated cellulose disks were incubated in a solution of 0.064 mg of LG6-DRNIC, 0.073 mg of LG6-YRFDC, 0.090 mg of LG6-HSKIYKC, 0.094 mg of LG6-GSRYRYC, 0.112 mg of LG6-HAIYPHRHC, and 0.068 mg of LG6-GPRPKC in 10 mL of ethanol and 40 mL of 50 mM Tris buffer with 5 mM EDTA-Na and 0.2 mM NaCl at pH 8.5 (coupling buffer) for 18 hrs at room temperature, under dark, and end-to-end mixing. The resulting LG6-cellulose disks were rinsed in coupling buffer and incubated in 50 mL of a 25 mM solution of (2-mercaptoethyl)dimethylammonium chloride (MED A) in coupling buffer for 2 hrs at room temperature, under dark, and under end-to-end mixing. The resulting LG6 / MEDA-cellulose disks were washed with 1 M NaCl (aq.) and MilliQ water, and finally stored in 20% v / v ethanol at 4°C.

[0118] Example 4

[0119] Production and harvest of Lentivirus (LVV) particles loaded with a Green Fluorescence Protein (GFP) transgene. Viral production cells (ThermoFisher Scientific, Waltham, MA) were initially cultured in BalanCD LV-MAX media at 8% CO2 and 37°C to reach a density of 3.5 - 5.5 - 106cells / mL for at least four passages. The cells were diluted to 1.5 IO6cells / mL at 24 hrs before transfection and adjusted to 2.5- 106cells / mL immediately before transfection. The TransIT-VirusGEN™ transfection reagent (Minis Bio™, Madison, WI) and the plasmids pALD-LentiEGFP-K, pALD-Rev-K, pALD-VSV-G-K, and pALD-GagPol-K (Aldevron, Fargo, ND) were dissolved at the mass ratio of 2.4:1 (LVMax transfection reagent:DNA) in a volume of OptiMEM™ I complex-forming solution (ThermoFisher, Waltham, MA) equal to 10% of the cell culture volume and at the ratio of 0.625 pg of total DNA per 106cells, mixed, and incubated for 15 min at room temperature before being added to the HEK293F cell suspension at 4- 106cells / mL. After transfection, the cells were incubated for 48-55 hrs at 37°C. The cell culture supernatant (~ 106cells per mL at 85% viability) was clarified via depth filtration followed by microfiltration: briefly, a volume of 1 L of fluid was loaded on a Millistak+® CE25 pod depth filter followed by a Millistak+® CE50 pod depth filter at the flux of 150 liters per square meter per hour, treated with 50 U / mL of benzonase and 2 mM MgCL for 30 min at 37°C, and finally filtered using a Polysep™ II cartridge filters (1.0 / 0.5 pm). The filters were flushed with 25 mM PIPES, 100 mM NaCl, pH 7.4 to increase LVV recovery. Unless immediately used, all samples were stored at -80°C.

[0120] Example 5

[0121] Purification of Lentivirus (LVV) using GKEAAFAA-cellulose membranes.Two GKEAAFAA-cellulose membrane layers were housed in a 25 mm Whatman filter holder and equilibrated with 50 membrane volumes (MVs) of 25 mM PIPES buffer with 100 mM NaCl at pH 7.4 (binding buffer) at the flow rate of 10 MV / min. A volume of 5 mL of clarified feed (LVV particle titer: 2 - 1010vp / mL; genome titer: 3.9 - 108vg / mL; functional titer: 7 - 107TU / mL; HEK293 HCP titer: 0.05 mg / mL) was loaded on the holder in down-flow at 3 MV / min. After washing the membranes with 50 MVs of binding buffer, the LVV elution was conducted in up-flow using 50 MVs of 25 mM PIPES buffer with 650 mM NaCl at pH 7.4 at 10 MV / min. Finally, the membranes were regenerated with 50 MVs of 0.1 M glycine with 2 M NaCl at pH 2.0 and cleaning-in-place (CIP) was conducted with 50 MVs of 0.5 M NaOH (aq). In parallel, LVV purification using MustangQ devices (MV: 0.86 mL; Cytiva, Uppsala, Sweden) was conducted as follows: the membranes were initially equilibrated with 50 MVs of 10 mM histidine buffer with 150 mM NaCl at pH 7.0, loaded with 150 MVs of clarified clarified feed, and washed with 60 MVs of binding buffer. The LVV elution was performed in three steps using 20 MVs of 10 mM histidine buffer at pH 7.0 added with NaCl at the concentration of 0.4, 1.0, and 1.5 M, at 10 MV / min. All chromatographic steps were conducted at 4°C while continuously monitoring the conductivity, pH, and UV absorbance of the column effluents at 254, 260, and 280 nm.

[0122] The titer of p24 protein and HEK293 HCPs in the resulting chromatographic fractions were respectively measured using HIV ELISA (Abeam, Waltham, MA) and HEK293 HCP ELISA (Cygnus, Southpoint, NC) kits following the manufacturer’s instructions. From the values of HCP titer, the reduction values (RVs) and logarithmic reduction values (LRV) of HCPs were derived using Equation 1:

[0123] Equation 1 RV =CmE / c"^ LRV = log10(RV)CHCP,L / CAAV,L

[0124] Where CHCP.E and CHCP.L are the HCP titers in the elutates and corresponding loads; CLW.E and CLW.L are the LVV titers in the elutates and corresponding loads.

[0125] The titer of encapsidated GFP transgenes in the chromatographic fractions was measured by real-time quantitative PCR (RT-qPCR). Briefly, 10 pL of each sample were incubated with 1 pL of TurboDNAse (ThermoFisher, Waltham, MA) for 60 min at 37°C followed by enzyme inactivation for 10 min at 95°C. The DNAse-treated samples were purified using a Purelink Viral RNA / DNA Kit (ThermoFisher Scientific, Waltham, MA)to isolate the encapsidated RNA. The samples were then combined with TaqMan fast virus, custom TaqMan probe, and the primers listed in Table 1, and analyzed using a CFX Duet Real-Time qPCR System (Bio Rad, Hercules, CA). Plasmid pALD-LentiEGFP-K was used as a standard.

[0126] Table 1. Primer and probe sequences for LW quantification via real time quantitative pCR (RT-qPCR).Primer DNA sequenceForward primer CCCAGTTCCGCCCATTCTC (SEQ ID NO: 68)Reverse primer GCCTCGGCCTCTGCATAAATAAA (SEQ ID NO: 69)Probe ATGGCTGACTAATTTTT (SEQ ID NO: 70)

[0127] The titer of cell-transducing LVVs in the chromatographic fractions was measured by fluorescence flow cytometry using HT1080 cells. The cells were initially cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% v / v fetal bovine serum (FBS) at 5% CO2 and 37°C until 80-90% confluence was reached. The cells were released from the culture flasks using trypsin, counted using a hemocytometer and trypan blue for cell viability, and plated in a 96-well plate at 7,000 cells / mL. Plates were centrifuged at 900g for 5 min and kept in an incubator for 4 hrs. At the onset of the transduction assay, the culture media in the plates were replaced with equal volumes of samples prepared via serial dilution (lOx) of the fractions containing LVVs in DMEM media supplemented with 8 pg / mL of polybrene. After 12 hrs, the spent medium was replaced with fresh DMEM medium supplemented with 10% v / v FBS, and the cells were incubated for 60 hrs. The fractions of cells expressing GFP were measured using a CytoFLEX flow Cytomer (Beckman, Brea, CA) and the values of transduction units (TU) per mL were calculated using Equation 2. Only dilutions that yielded %GFP+cells between 1% to 25% were considered for LVV transduction concentration. rmoo-117 r i / TU \ number of cells x (% )

[0128] Equation 2 Transduction [ - )= - - - - -\ mL / volume (mL) x dilution factor

[0129] Where the transductions units (TU) per mL were determined based on the number of cells at the time of transduction, the number of HT1080 cells expressing GFP, the total volume of sample per well, and the dilution factor.

[0130] Example 6

[0131] Removal of Chinese Hamster Ovary (CHO) host cell proteins (HCP) from cell culture harvests using LG6 / TC- and LG6 / MEDA-cellulose membranes. Six LG6 / TC-cellulose membranes or six LG6 / MEDA-cellulose membranes were housed in a 25 mm Whatman filter holder and equilibrated with 50 MVs of 10 mM BisTris buffer at pH 7.4 (binding buffer) at the flow rate of 10 MV / min. A volume of 16 mL of clarified CHO cell culture harvest containing an IgGi-type monoclonal antibody (IgGi titer: 8.23 mg / mL; CHO HCP titer: 0.226 mg / mL) was loaded on the holder at 10 MV / min while continuously monitoring the conductivity, pH, and UV absorbance of the effluents at 280 nm. The effluent was apportioned in 0.4-mL fractions.

[0132] The titer of CHO HCPs in the chromatographic fractions were respectively measured using G.3 CHO HCP ELISA (Cygnus, Southpoint, NC) kits following the manufacturer’s instructions.

[0133] The flow-through fractions were analyzed for using a BioResolve SEC, 200A size exclusion chromatographic (SEC) column with PBS at pH 7.4 as mobile phase. A sample volume of 10 pL was injected at the flowrate of 0.5 mL / min and the UV absorbance of the effluent was continuously monitored at 280 nm.

[0134] Example 7

[0135] Affinity purification of Lentivirus (LVV) using GKEAAFAA-cellulose membranes

[0136] The high binding capacity characteristic of chromatography resins stems from the large surface area of their pores. However, the tortuous morphology and limited diameter of these pores reduce the transport of large biologies, such as viruses, due to diffusion limitations. Membranes offer an excellent alternative to resins as their open porosity eliminates diffusive limitations and enables processing at significantly higher flow rates and lower pressure drops. To date, however, most of the commercial affinity membranes are dedicated to protein purification, whereas only a pseudo-affinity sulfated cellulose membrane is used for purifying influenza A virions. Developing an affinity membrane is particularly beneficial for LVV purification as it would safeguard the transductionactivity of the purified virions by reducing their contact time with the chromatographic matrix.

[0137] Addressing these needs, we conjugated LVV-binding peptide ligand GKEAAFAA (SEQ ID NO: 4) on cellulose membranes. To this end, we grafted a branched polyamine (PEI) on the surface of cellulose membranes and used the resulting tentacular grafted layer for conjugating GKEAAFAA (SEQ ID NO: 4) ligands at a comparable density (Figure 1). We evaluated the resulting adsorbents by measuring their dynamic binding capacity at 10% breakthrough (DBCio%) at the residence time (RT) of 0.5 min, along with LVV recovery and HCP clearance (Table 2).

[0138] Table 2. LW purification performance of GKEAAFAA-cellulose membranes and Mustang Q membranes.P RT DBCio% Recovery of roductiHCPMembrane (m (TU / mL of Transducting vityLRVin) resin) LVVs (%) (TU / mL•min)GKEAAFAA- (1.23 ± 2.22 ± 0.5 74.0 ± 2.0 % 5.9 108Cellulose 0.41) IO90.121.49 ± Mustang Q 0.5 85.0 ± 0.5 %0.05

[0139] Example 8

[0140] Removal of Chinese Hamster Ovary (CHO) host cell proteins (HCP) from cell culture harvests using LG6 / TC- and LG6 / MEDA-cellulose membranes

[0141] The results shown in Figure 2 (Panels A-C) show that LG6 / TC- and LG6 / MEDA-cellulose membranes possess an excellent binding capacity for host cell proteins, either monomeric (process-related contaminants), or aggregated to the product (product-related contaminants), as well as host cell nucleic acids in the forms of host cell DNA, chromatin, and product-chromatin aggregates (product-related contaminants). The LG6 / TC- and LG6 / MEDA-cellulose membranes were continuously loaded with clarifiedCHO cell culture harvests containing therapeutic monoclonal antibodies and the effluent collected and apportioned in fractions that were analyzed via analytical size-exclusion chromatography (SEC-HPLC). At lower values of loading the LG6 / TC- and, albeit to a lesser extent, the LG6 / MEDA-cellulose remove aggregates ( / .< ., species featuring a retention time lower than the retention time of the product) and other process-related impurities (z.e., species featuring a higher time lower than the retention time of the product), while letting the antibody product flow through unbound.It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

1. CLAIMS2.What is claimed is:

1. An affinity adsorbent for purifying a biologic, the affinity adsorbent comprising: a convective porous substrate having a surface that can be functionalized;4.one or more branched polymers tethered to the surface of the convective porous substrate, wherein the one or more branched polymers comprise one or more reactive terminal groups; and5.a ligand conjugated to a terminus of the one or more branched polymers.

2. The affinity adsorbent of claim 1, wherein the convective porous substrate comprises a membrane.

3. The affinity adsorbent of claim 1, wherein the convective porous substrate comprises a fiber mat.

4. The affinity adsorbent of claim 2, wherein the membrane comprises a cellulose membrane, a derivatized cellulose membrane, a polysulfone membrane, a nylon membrane, a polyimide membrane, a polyolefine membrane, and / or a polyester membrane.

5. The affinity adsorbent of claim 3, wherein the fiber mat comprises a cellulose fiber mat, a derivatized cellulose fiber mat, a polysulfone fiber mat, a nylon fiber mat, a polyimide fiber mat, a polyolefine fiber mat, and / or a polyester fiber mat.

6. The affinity adsorbent of any one of claims 1 to 5, wherein the one or more branched polymers comprises a branched polyester, a branched polyamide, a branched polyether, a branched polyamine, and / or a branched polyurethane, optionally wherein the one or more branched polymers comprise a pre-formed branched polymer.

7. The affinity adsorbent of claim 6, wherein a hydrodynamic radius of the branched polymer is about 1 nm to about 20 nm.

8. The affinity adsorbent of claim 6 or 7, wherein a hydrodynamic radius of the branched polymer is about 2 nm to about 5 nm.

9. The affinity adsorbent of any one of claims 6 to 8, wherein the branched polyamide is a poly(amidoamine) (PAMAM) dendrimer, a branched polypeptide, or a polypeptide-PAMAM hybrid.

10. The affinity adsorbent of any one of claims 6 to 8, wherein the branched polyamine is a polyethylenimine (PEI).

11. The affinity adsorbent of any one of claims 6 to 8, wherein the branched polyether is a polyethylene glycole (PEG).

12. The affinity adsorbent of any one of claims 6 to 8, wherein the branched polyester is a branched polylactic acid (PLA).

13. The affinity adsorbent of any one of claims 1 to 12, wherein the one or more reactive terminal groups of the branched polymer comprise a hydroxyl, an amine, a thiol, a carboxyl, a nitrile, an activated ester, an aldehyde, an acyl halide, a malehymide, an iodoacetamide, an epoxide, an alkyl halide, an alkene, an alkine, and / or an azide.

14. The affinity adsorbent of any one of claims 1 to 13, wherein the one or more branched polymers is tethered to the surface of the convective porous substrate via a reactive terminal group of the one or more branched polymers.

15. The affinity adsorbent of claim 14, whereby the one or more branched polymers tethered to the surface of the convective porous substrate represent about 1% to about 50% of the weight of the convective porous substrate.

16. The affinity adsorbent of claim 14, whereby the one or more branched polymers tethered to the surface of the convective porous substrate represent about 1% to about 20% of the weight of the convective porous substrate.

17. The affinity adsorbent of claim 14, whereby the one or more branched polymers tethered to the surface of the convective porous substrate represent about 5% to about 10% of the weight of the convective porous substrate.

18. The affinity adsorbent of claim 14, whereby the one or more branched polymers tethered to the surface of the convective porous substrate represent about 1% to about 5% of the weight of the convective porous substrate.

19. The affinity adsorbent of any one of claims 1 to 18, wherein the ligand is conjugated to at least one of the one or more reactive terminal groups of the one or more branched polymers, optionally wherein the ligand is conjugated to at least one of the one or more reactive terminal groups other than the one or more reactive terminal groups tethering the one or more branched polymers to the surface of the convective porous substrate.

20. The affinity adsorbent of any one of claims 1 to 19, wherein the ligand comprises a protein ligand, a peptide ligand, an aptamer ligand, a mixed-mode ligand, and / or a triazine ligand.

21. The affinity adsorbent of claim 20, wherein the mixed-mode ligand comprises an alkylammonium group, and / or an aromatic group, and / or a carboxyl group, and / or an alkyl chain, and / or a thioether group.

22. The affinity adsorbent of claim 20, wherein the peptide ligand comprises a targetbinding peptide sequence and a peptide linker connecting the target-binding peptide sequence to the terminal group of the one or more branched polymers.

23. The affinity adsorbent of claim 22, wherein the peptide sequence comprises about 4 to about 50 amino acids, optionally about 5 to about 20 amino acids, optionally about 10 to about 35 amino acids.

24. The affinity adsorbent of claim 22 or 23, wherein the peptide is selected from the group consisting of Lentivirus-binding peptides of amino acid sequences EWKAAFIW (SEQ ID NO: 1), FEKISNAE (SEQ ID NO: 2), GEFENINW (SEQ ID NO: 3), GKEAAFAA (SEQ ID NO: 4), SIEINSSE (SEQ ID NO: 5), SKSAAEHE (SEQ ID NO: 6), SNEIEIAN (SEQ ID NO: 7), EHFEHWSE (SEQ ID NO: 8), FEKISNAE (SEQ ID NO: 2), C-cyclo[GSRAFVGDAD]C (SEQ ID NO: 9), SRQFVCGDSDRD (SEQ ID NO: 10), SRAFVGDADRD (SEQ ID NO: 11), and SFVRIGLSD (SEQ ID NO: 12).

25. The affinity adsorbent of claim 22 or 23, wherein the peptide is selected from the group consisting of AdenoAssociated Virus-binding peptides of amino acid sequences CYGHFSGYGNYGPC (SEQ ID NO: 13), CYGHFSPYGNYGPC (SEQ ID NO: 14), CYHFSYNYPC (SEQ ID NO: 15), CYHFSYNYPKSC (SEQ ID NO: 16), CYIHFSGYTNYNGSLKSC (SEQ ID NO: 17), CYIHFSGYTNYNPC (SEQ ID NO: 18), CYIHFSGYTNYNPSLKSC (SEQ ID NO: 19), CYIHFSPYTNYNPSLKSC (SEQ ID NO: 20), CYVHFSGYSNYSPSC (SEQ ID NO: 21), GCGQQYWIGPFTFGCG (SEQ ID NO: 22), GQQYWIGPFTFG (SEQ ID NO: 23), LETVKPGLYEPITHPRDYS(SEQ ID NO: 24), and SYDRPHTIPEYLGPKVTEL (SEQ ID NO: 25), AIVSPQFQEISLPTTSTVIDGSQSTDDDKIVQY (SEQ ID NO: 26), CDGSQSTDDDKIC (SEQ ID NO: 27), CDSQSTDDDKIC (SEQ ID NO: 28), CSGSTDDDKIC (SEQ ID NO: 29), CSGSTEQEKIC (SEQ ID NO: 30), CVIDGSQSTDDDKIC (SEQ ID NO: 31), CVIDGSQSTDDDKIVQYC (SEQ ID NO: 32), GCLITHPRDYS (SEQ ID NO: 33), GCLITHPRDYSGCG (SEQ ID NO: 34), GYIHFSGYTNYNPSLKS (SEQ ID NO: 35), GYWIGPFTGGGYIHFSGYT (SEQ ID NO: 36), GYWIGPFTGPGYIHFSGYT (SEQ ID NO: 37), GYWIGPFTPGPYIHFSGYT(SEQ ID NO: 38), LITHPRDYSPKLTPGLYEFG (SEQ ID NO: 39), and TVIDGSQSTDDDKIVQY (SEQ ID NO: 40).

26. The affinity adsorbent of claim 22 or 23, wherein the peptide is selected from the group consisting of Host Cell Protein-binding peptides of amino acid sequences IYRIGR (SEQ ID NO: 41), YRFD (SEQ ID NO: 42), DRNI (SEQ ID NO: 43), HYFD (SEQ ID NO: 44), RYYYAI (SEQ ID NO: 45), DKSI (SEQ ID NO: 46), GSRYRY(SEQ ID NO: 47), AAHIYY (SEQ ID NO: 48), HSKIYK (SEQ ID NO: 49), HAIYPHRH (SEQ ID NO: 50), DICLPRWGCLW (SEQ ID NO: 51), EHIPA (SEQ ID NO: 52), DLCLRDWGCLW (SEQ ID NO: 53), and GPRPK(SEQ ID NO: 54).

27. The affinity adsorbent of claim 22, wherein the peptide linker comprises 1 to 10 amino acids, optionally 1 to 2 amino acids, optionally 2 to 4 amino acids, optionally 3 to 5 amino acids.

28. The affinity adsorbent of claim 27, wherein the linker is C, GC, GSGC (SEQ ID NO: 55), GGGC (SEQ ID NO: 56), and / or GSGSC (SEQ ID NO: 57).

29. The affinity adsorbent of claim 27, wherein the linker is K, GK, GSGK (SEQ ID NO: 58), GGGK (SEQ ID NO: 59), and / or GSGSK (SEQ ID NO: 60).

30. The affinity adsorbent of claim 24, wherein the Lentivirus-binding capacity of the affinity adsorbent is at least 109transducing units per mL of adsorbent.

31. The affinity adsorbent of claim 24, wherein the yield of purified, cell-transducing Lentivirus provided by the affinity adsorbent is at least 30%, and optionally at least 70%.

32. The affinity adsorbent of claim 25, wherein the Adeno Associated Virus-binding capacity of the affinity adsorbent is at least 1013viral particles per mL of adsorbent.

33. The affinity adsorbent of claim 25, wherein the yield of purified, cell-transducing Lentivirus provided by the affinity adsorbent is at least 50%, and optionally at least 90%.

34. The affinity adsorbent of claim 26, wherein the host cell protein-binding capacity of the affinity adsorbent is at least 10 mg of Host Cell Proteins per mL of adsorbent.

35. The affinity adsorbent of claim 26, wherein the yield of purified target biologic provided by the affinity adsorbent is at least 80%, and optionally at least 90%.

36. The affinity adsorbent of any one of claims 1 to 35, wherein the affinity adsorbent is configured to provide a reduction of Host Cell Proteins in an eluate of at least 100-fold.

37. The affinity adsorbent of any one of claims 1 to 35, wherein the affinity adsorbent is configured to provide a reduction of Host Cell Proteins in the effluent of at least 10-fold.

38. The affinity adsorbent of any one of claims 1 to 35, wherein the affinity adsorbent is configured to provide a reduction of aggregates in the effluent of at least 5-fold.

39. A method of making an affinity adsorbent, the method comprising:39.providing a convective porous substrate having a surface that can be functionalized; tethering one or more branched polymers on a surface of the convective porous substrate, wherein the one or more branched polymers comprise one or more reactive terminal groups, wherein the one or more branched polymers comprise a pre-formed branched polymer; and40.conjugating a ligand to a terminus of the one or more branched polymers.

40. The method of claim 39, wherein the convective porous substrate comprises a membrane.

41. The method of claim 39, wherein the convective porous substrate comprises a fiber mat.

42. The method of claim 40, wherein the membrane comprises a cellulose membrane, a derivatized cellulose membrane, a polysulfone membrane, a nylon membrane, a polyimide membrane, a polyolefine membrane, and / or a polyester membrane.

43. The method of claim 41, wherein the fiber mat comprises a cellulose fiber mat, a derivatized cellulose fiber mat, a polysulfone fiber mat, a nylon fiber mat, a polyimide fiber mat, a polyolefine fiber mat, and / or a polyester fiber mat.

44. The method of any one of claims 39 to 43, wherein the one or more branched polymers comprises a branched polyester, a branched polyamide, a branched polyether, a branched polyamine, and / or a branched polyurethane.

45. The method of claim 44, wherein a hydrodynamic radius of the branched polymer is about 1 nm to about 20 nm.

46. The method of claim 44 or 45, wherein a hydrodynamic radius of the branched polymer is about 1 nm to about 5 nm.

47. The method of any one of claims 44 to 46, wherein the branched polyamide is a poly(amidoamine) (PAMAM) dendrimer, a branched polypeptide, or a polypeptide-PAMAM hybrid.

48. The method of any one of claims 44 to 46, wherein the branched polyamine is a polyethylenimine (PEI).

49. The method of any one of claims 44 to 46, wherein the branched polyether is a polyethylene glycole (PEG).

50. The method of any one of claims 44 to 46, wherein the branched polyester is a branched polylactic acid (PLA).

51. The method of any one of claims 39 to 50, wherein the one or more reactive terminal groups of the branched polymer comprise a hydroxyl, an amine, a thiol, a carboxyl, a nitrile, an activated ester, an aldehyde, an acyl halide, a malehymide, an iodoacetamide, an epoxide, an alkyl halide, an alkene, an alkyne, and / or an azide.

52. The method of any one of claims 39 to 51, wherein the one or more branched polymers is tethered to the surface of the convective porous substrate via a reactive terminal group of the one or more branched polymers.

53. The method of claim 52, whereby the one or more branched polymers tethered to the surface of the convective porous substrate represent about 1% to about 50% of the weight of the convective porous substrate.

54. The method of claim 52, whereby the one or more branched polymers tethered to the surface of the convective porous substrate represent about 1% to about 20% of the weight of the convective porous substrate.

55. The method of claim 52, whereby the one or more branched polymers tethered to the surface of the convective porous substrate represent about 5% to about 10% of the weight of the convective porous substrate.

56. The method of claim 52, whereby the one or more branched polymers tethered to the surface of the convective porous substrate represent about 1% to about 5% of the weight of the convective porous substrate.

57. The method of any one of claims 39 to 56, wherein the ligand is conjugated to at least one of the one or more reactive terminal groups of the one or more branched polymers, optionally wherein the ligand is conjugated to at least one of the one or more reactive terminal groups other than the one or more reactive terminal groups tethering the one or more branched polymers to the surface of the convective porous substrate.

58. The method of any one of claims 39 to 57, wherein the ligand comprises a protein ligand, a peptide ligand, an aptamer ligand, a mixed-mode ligand, and / or a triazine ligand.

59. The affinity adsorbent of claim 58, wherein the mixed-mode ligand comprises an alkylammonium group, and / or an aromatic group, and / or a carboxyl group, and / or an alkyl chain, and / or a thioether group.

60. The method of claim 58, wherein the peptide ligand comprises a target-binding peptide sequence and a peptide linker connecting the target-binding peptide sequence to the terminus of the one or more branched polymers.

61. The method of claim 60, wherein the peptide sequence comprises about 4 to about 50 amino acids, optionally about 5 to about 20 amino acids, optionally about 10 to about 35 amino acids.

62. The method of claim 60 or 61, wherein the peptide is selected from the group consisting of Lentivirus-binding peptides of amino acid sequences EWKAAFIW (SEQ ID NO: 1), FEKISNAE (SEQ ID NO: 2), GEFENINW (SEQ ID NO: 3), GKEAAFAA (SEQ ID NO: 4), SIEINSSE (SEQ ID NO: 5), SKSAAEHE (SEQ ID NO: 6), SNEIEIAN (SEQ ID NO: 7), EHFEHWSE (SEQ ID NO: 8), FEKISNAE (SEQ ID NO: 2), C-cyclo[GSRAFVGDAD]C (SEQ ID NO: 9), SRQFVCGDSDRD (SEQ ID NO: 10), SRAFVGDADRD (SEQ ID NO: 11), and SFVRIGLSD (SEQ ID NO: 12).

63. The method of claim 60 or 61, wherein the peptide is selected from the group consisting of AdenoAssociated Virus-binding peptides of amino acid sequences CYGHFSGYGNYGPC (SEQ ID NO: 13), CYGHFSPYGNYGPC (SEQ ID NO: 14), CYHFSYNYPC (SEQ ID NO: 15), CYHFSYNYPKSC (SEQ ID NO: 16), CYIHFSGYTNYNGSLKSC (SEQ ID NO: 17), CYIHFSGYTNYNPC (SEQ ID NO: 18), CYIHFSGYTNYNPSLKSC (SEQ ID NO: 19), CYIHFSPYTNYNPSLKSC (SEQ ID NO: 20), CYVHFSGYSNYSPSC (SEQ ID NO: 21), GCGQQYWIGPFTFGCG (SEQ ID NO: 22), GQQYWIGPFTFG (SEQ ID NO: 23), LETVKPGLYEPITHPRDYS (SEQ ID NO: 24), and SYDRPHTIPEYLGPKVTEL (SEQ ID NO: 25), AIVSPQFQEISLPTTSTVIDGSQSTDDDKIVQY (SEQ ID NO: 26), CDGSQSTDDDKIC (SEQ ID NO: 27), CDSQSTDDDKIC (SEQ ID NO: 28), CSGSTDDDKIC (SEQ ID NO: 29), CSGSTEQEKIC (SEQ ID NO: 30), CVIDGSQSTDDDKIC (SEQ ID NO: 31), CVIDGSQSTDDDKIVQYC (SEQ ID NO: 32), GCLITHPRDYS (SEQ ID NO: 33), GCLITHPRDYSGCG (SEQ ID NO: 34), GYIHFSGYTNYNPSLKS (SEQ ID NO: 35), GYWIGPFTGGGYIHFSGYT (SEQ ID NO: 36), GYWIGPFTGPGYIHFSGYT (SEQ ID NO: 37), GYWIGPFTPGPYIHFSGYT(SEQ ID NO: 38), LITHPRDYSPKLTPGLYEFG (SEQ ID NO: 39), and TVIDGSQSTDDDKIVQY (SEQ ID NO: 40).

64. The method of claim 60 or 61, wherein the peptide is selected from the group consisting of Host Cell Protein-binding peptides of amino acid sequences IYRIGR (SEQ ID NO: 41), YRFD (SEQ ID NO: 42), DRNI (SEQ ID NO: 43), HYFD (SEQ ID NO: 44), RYYYAI (SEQ ID NO: 45), DKSI (SEQ ID NO: 46), GSRYRY (SEQ ID NO: 47), AAHIYY(SEQ ID NO: 48), HSKIYK (SEQ ID NO: 49), HAIYPHRH (SEQ ID NO: 50), DICLPRWGCLW (SEQ ID NO: 51), EHIPA (SEQ ID NO: 52), DLCLRDWGCLW (SEQ ID NO: 53), and GPRPK (SEQ ID NO: 54).

65. The method of claim 60, wherein the peptide linker comprises 1 to 10 amino acids, optionally 1 to 2 amino acids, optionally 2 to 4 amino acids, optionally 3 to 5 amino acids.

66. The method of claim 65, wherein the linker is C, GC, GSGC (SEQ ID NO: 55), GGGC (SEQ ID NO: 56), and / or GSGSC (SEQ ID NO: 57).

67. The method of claim 65, wherein the linker is K, GK, GSGK (SEQ ID NO: 58), GGGK (SEQ ID NO: 59), and / or GSGSK (SEQ ID NO: 60).

68. The method of any one of claims 39 to 67, wherein tethering the one or more branched polymers on the surface of the convective porous substrate by means of an amide bond, or a thioether bond, or an ether bond, or a secondary amine bond, or a tertiary amine bond, or a carbamate bond, or a carbonate bond, or an ester bond.

69. The method of any one of claims 39 to 68, further comprising capping residual reactive groups of the one or more branched polymers after conjugating the ligand to the terminus of the one or more branched polymers.

70. The method of any one of claims 39 to 69, wherein conjugating the ligand to the terminus of the one or more branched polymers further comprises the use of one or more coupling buffers, optionally wherein the one or more coupling buffers comprises Tris buffer, EDTA-Na, TCEP and / or a combination thereof.

71. A method of purifying a target biologic from a biological fluid in flow-through mode, the method comprising:70.contacting the affinity adsorbent of any one of claims 1 to 38 with a biological fluid comprising a target biologic; and collecting an effluent in flow-through mode, wherein the effluent comprises the target biologic;71.wherein the affinity adsorbent binds and retains at least one host cell protein (HCP), at least one high-risk HCP, at least one host cell nucleic acid, aggregates of the target biologic, and / or an impurity derived from the target biologic.

72. The method of claim 71, wherein the method further comprises performing chromatography on the biological fluid comprising the target biologic before or after contacting the functionalized membrane.

73. The method of claim 71 or 72, wherein the method is performed under static binding conditions.

74. The method of claim 71 or 72, wherein the method is performed under dynamic binding conditions.

75. The method of any one of claims 71 to 74, wherein the method results in a yield of the target biologic of at least about 50%.

76. The method of any one of claims 71 to 74, wherein the method results in a purity of the target biologic of at least about 75%.

77. The method of any one of claims 71 to 76, wherein the affinity adsorbent reduces Host Cell Proteins in the effluent of at least 100-fold.

78. The method of any one of claims 71 to 76, wherein the affinity adsorbent reduces Host Cell Proteins in the effluent of at least 10-fold.

79. The method of any one of claims 71 to 76, wherein the affinity adsorbent reduces Host Cell Proteins in the effluent of at least 5 -fold.

80. A method of purifying a target biologic from a biological fluid in bind-and-elute mode, the method comprising:81.contacting the affinity adsorbent of any one of claims 1 to 38 with a biological fluid comprising a target biologic;82.washing the affinity adsorbent to remove loosely bound contaminants; and releasing the target biologic using an appropriate elution buffer, thereby collecting a purified target biologic in the eluate.

81. The method of claim 80, wherein the method further comprises performing chromatography on the biological fluid comprising the target biologic before or after contacting the functionalized membrane.

82. The method of claim 80 or 81, wherein the method is performed under static binding conditions.

83. The method of claim 80 or 81, wherein the method is performed under dynamic binding conditions.

84. The method of any one of claims 80 or 81, wherein the method results in a yield of the target biologic of at least about 50%.

85. The method of any one of claims 80 or 81, wherein the method results in a purity of the target biologic of at least about 75%.

86. The method of any one of claims 80 to 85, wherein the affinity adsorbent reduces Host Cell Proteins in an eluate of at least 100-fold.

87. The method of any one of claims 80 to 85, wherein the affinity adsorbent reduces Host Cell Proteins in an eluate of at least 10-fold.

88. The method of any one of claims 80 to 85, wherein the affinity adsorbent reduces Host Cell Proteins in an eluate of at least 5 -fold.

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