Caustic-resistant affinity ligands for chromatography

Abstract

The present invention relates to the field of affinity purification. Disclosed are affitin ligand for separating a target structure from a liquid sample by affinity chromatography comprising or consisting of a polypeptide, wherein the polypeptide does not comprise an asparagine or glutamine residue flanked on both sides by glycine residues. Further disclosed are the use of said affitin ligands for affinity purification of a target structure as well as related methods.

Description

[0001] -347

[0002] - 1 -

[0003] CAUSTIC-RESISTANT AFFINITY LIGANDS FOR CHROMATOGRAPHY

[0004] FIELD OF THE INVENTION

[0005] 5

[0006] The present invention relates to the field of affinity chromatography, and more particularly to compounds and methods for purifying target structures and regenerating affinity matrices using affinity ligands that are resistant to degradation under caustic cleaning conditions.

[0007] BACKGROUND OF THE INVENTION

[0008] Affinity chromatography is a fundamental tool in bioprocessing, enabling selective isolation of biomolecules from complex mixtures. The purification of

[0009] 15 large, sensitive therapeutics such as viral vectors, virus-like particles, and other macromolecular assemblies presents significant challenges due to their structural fragility and the delicate nature of conventional affinity ligands.

[0010] A critical limitation in conventional affinity chromatography arises during

[0011] 20 cleaning-in-place (CIP) procedures that employ caustic reagents such as sodium hydroxide (NaOH). While NaOH is widely used for its strong sanitizing properties, it irreversibly denatures most protein- and peptide- based ligands, causing substantial loss of binding capacity, shortened matrix lifespan, and the accumulation of degraded fragments on the resin. These fragments can leach into the purified product during subsequent runs, contaminating the material, compromising purity, interfering with analytical characterisation, and posing immunogenic or safety risks. For fragile therapeutics like viral vectors, even minor ligand degradation can significantly reduce recovery and quality, while repeated caustic exposure

[0012] 30 accelerates these problems, making conventional ligands unsuitable for robust, repeated use. -347

[0013] - 2 -

[0014] Existing strategies, including chemically stabilized ligands, synthetic ligands, or disposable matrices, fail to adequately address the challenges posed by NaOH cleaning. Stabilized ligands often remain vulnerable to caustic degradation, synthetic ligands may not provide sufficient selectivity for large

[0015] 5 macromolecular targets, and disposable matrices increase costs and waste without eliminating the need for rigorous sanitation. Consequently, there is a pressing need for affinity matrices capable of withstanding caustic cleaning without losing ligand functionality or contaminating the product.

[0016] Conventional protein- and peptide-based affinity ligands are further limited by the intrinsic chemical instability of certain amino acid motifs when exposed to caustic reagents such as sodium hydroxide. Sequences containing asparagine followed by small or flexible residues (e.g., Asn-Gly, Asn-Ser, Asn-Thr) are highly prone to deamidation under alkaline conditions, resulting

[0017] 15 in structural alterations that impair ligand binding. Similarly, motifs containing glutamine adjacent to small residues (e.g., Gln-Gly, Gln-Ser) are susceptible to backbone hydrolysis at elevated pH, leading to fragmentation and loss of ligand integrity. The underlying reaction mechanism involves the main chain peptide nitrogen of the succeeding residue, which functions as the

[0018] 20 nucleophile and attacks the side chain carbonyl of for example the asparagine / glutamine. This cyclisation reaction results in a cyclic imide intermediate, which is then hydrolysed to an isomeration product, in this case aspartate / glutamate and isoaspartate / isoglutamate. Although these reactions occur spontaneously, the rate is severely increased at alkaline pH, which makes a cleavage of the peptide bond likely. (Geiger and Clark, 1987; Robinson and Robinson, 1991 ). Additional residues, including methionine and cysteine, are vulnerable to oxidation, while aromatic residues such as tryptophan and tyrosine can undergo alkaline degradation. Together, these sequence-specific vulnerabilities accelerate ligand deterioration during

[0019] 30 NaOH cleaning, increasing the risk of fragment leaching into the product stream and thereby compromising both process efficiency and product safety. - 3 -

[0020] The present invention addresses this need by providing affinity ligands, in the following called affitins or affitin ligands, that retain structural integrity and binding functionality even after exposure to harsh caustic conditions, including NaOH. By maintaining performance under such conditions, these

[0021] 5 ligands prevent leaching of fragments into the purified product, extend matrix lifespan, preserve high recovery and purity of target molecules, and enable efficient, repeated use of affinity matrices. This innovation allows rigorous cleaning-in-place with caustic agents while ensuring product safety, quality, and regulatory compliance, solving a critical limitation in the purification of complex biologies.

[0022] SUMMARY OF THE INVENTION

[0023] The present invention is directed to an affitin ligand comprising or consisting of a polypeptide, wherein the polypeptide does not comprise an asparagine

[0024] 15 or glutamine residue flanked on both sides by glycine residues. Preferably, the ligand is for separating a target structure from a liquid sample by affinity chromatography

[0025] In a preferred embodiment, the polypeptide comprises at least one

[0026] 20 asparagine-glycine (NG) dipeptide sequence.

[0027] In another preferred embodiment, the affitin ligand comprises at least one glutamine-glycine (QG) dipeptide sequence.

[0028] In a preferred embodiment the polypeptide of the affitin ligand follows formula II

[0029] VKVKFX1X2X3G X4EKEVDX5X6KI X7X8VX9RX10GX11X12V X13FX14YDDNGKX15 GXI6G X17VX18EX19X20A PKELLX21ML ARAEREK formula II, and - 4 -

[0030] X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, Xi 1 , X12, X13, X14, X15, Xi6, X17,

[0031] Xis, X19, X20, and X21 represents a single amino acid independently selected from the group consisting of naturally occurring amino acids.

[0032] The present invention is further directed to a chromatography matrix

[0033] 5 comprising a covalently bonded affitin ligands comprising or consisting of a polypeptide, wherein the polypeptide does not comprise an asparagine or glutamine residue flanked on both sides by glycine residues. In a preferred embodiment, the polypeptide comprises at least one asparagine-glycine (NG) dipeptide sequence.

[0034] In another preferred embodiment, the affitin ligand comprises at least one glutamine-glycine (QG) dipeptide sequence.

[0035] In another preferred embodiment, the chromatography matrix comprises a

[0036] 15 base material in form of a resin or a membrane.

[0037] In a preferred embodiment, the chromatography matrix has a ligand density between 4 mg / ml and 10 mg / ml.

[0038] In one embodiment the chromatography matrix according has less than a

[0039] 20 30% reduction in binding capacity for a target structure following an incubation in 0.1 M NaOH for at least 150 min.

[0040] In another embodiment the chromatography matrix has less than a 30% reduction in binding capacity following an incubation in 0.5 M NaOH for at least 30 min.

[0041] The present invention is further directed to the use of the chromatography matrix according to the present invention for affinity purification of a target structure.

[0042] 30 In a preferred embodiment, the affitin ligand retains binding capacity after exposure to sodium hydroxide during cleaning-in-place regeneration. - 5 -

[0043] In another embodiment, the affitin ligand retains at least 70% of its binding capacity after 10 or more regeneration cycles with sodium hydroxide at a concentration between 0.05 M and 0.5 M.

[0044] The present invention is also directed to a method for separating a target

[0045] 5 structure from a liquid sample comprising or consisting of the steps of:

[0046] (a) providing a liquid sample comprising a target structure, a chromatography matrix according to the present invention, at least one wash buffer, and at least one elution buffer,

[0047] (b) contacting said liquid sample with the chromatography matrix,

[0048] (c) optionally washing the chromatography matrix with at least one washing buffer,

[0049] 15 (d) eluting the target structure from the chromatography matrix with at least one elution buffer, and

[0050] (e) cleaning the chromatography matrix with a caustic cleaning liquid.

[0051] 20

[0052] In a preferred embodiment, the caustic cleaning liquid comprises sodium hydroxide at a concentration between 0.05 M and 0.5 M.

[0053] In a preferred embodiment, the chromatography matrix has a ligand density between 4 mg / ml and 10 mg / ml.

[0054] DETAILED DESCRIPTION OF THE INVENTION

[0055] DEFINITIONS

[0056] Before describing the present invention in detail, it is to be understood that

[0057] 30 this invention is not limited to specific compositions or process steps, as such may vary. -347

[0058] - 6 -

[0059] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0060] 5

[0061] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments; however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the

[0062] 15 description of the present application unless the context indicates otherwise.

[0063] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related. The following terms are defined for

[0064] 20 purposes of the invention as described herein.

[0065] The term "comprise" or variations such as "comprises" or "comprising" according to the present invention means the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The term "consisting essentially of’ according to the present invention means the inclusion of a stated integer or group of integers, while excluding modifications or other integers which would materially affect or alter the stated integer. The term "consisting of” or variations such as "consists of” according to the present invention means the inclusion of a

[0066] 30 stated integer or group of integers and the exclusion of any other integer or group of integers. Embodiments described herein as "comprising" one or - 7 - more features may also be considered as disclosure of the corresponding embodiments "consisting of" such features.

[0067] The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be

[0068] 5 construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Thus, for example, reference to "a ligand" includes a plurality of ligands and reference to "an antibody" includes a plurality of antibodies and the like.

[0069] “About” may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. In particular embodiments, the term “about” shall be understood herein as plus or minus

[0070] 15 (±) 5%, ± 4%, ± 3%, ± 2%, ± 1 %, ± 0.5%, ± 0.1 %, of the numerical value of the number with which it is being used.

[0071] Concentrations, amounts, volumes, percentages, and other numerical values may be presented herein in a range format. It is also to be understood that

[0072] 20 such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0073] The term “affitin” or “affitin ligand” refers to an artificial binding protein capable of selectively binding antigens or other target molecules. Affitins are structurally derived from the Sul7d protein family, a group of hyperthermostable, DNA-binding proteins such as Sac7d, Sso7d, and Aho7c, isolated from hyperthermophilic archaea of the Sulfolobus genus. By

[0074] 30 randomizing amino acid residues on the binding surface of Sul7d proteins and subjecting the resulting protein library to selection methods such as - 8 - ribosome display, binding specificity and affinity can be directed towards a wide variety of targets, including peptides, proteins, nucleic acids, viruses, bacteria, and other biological or synthetic molecules.

[0075] Affitins are antibody mimetics developed as alternatives to conventional

[0076] 5 antibodies for use in biotechnology, diagnostics, and therapeutic applications. They typically comprise 60 to 66 amino acids, have a molecular mass of approximately 7 kDa, and are markedly smaller than conventional antibodies (~130-150 kDa). Affitins exhibit exceptional thermal stability and durability through repeated freeze-thaw cycles. They can be produced in vitro in high yield and purity, often via recombinant bacterial expression systems, owing to their small size, high solubility, and low structural complexity. Their stability can be further enhanced through site-directed mutagenesis, grafting techniques, or other protein engineering strategies. The term “affitin” encompasses variants, derivatives, fusion constructs, chemically modified

[0077] 15 forms, and any engineered version retaining the structural scaffold and binding properties of the Sul7d family, preferably of wild-type Sac7d.

[0078] The term "wild-type Sac7d" as used herein refers to a naturally occurring Sac7d protein, such as the DNA-binding protein 7d derived from Sulfolobus

[0079] 20 acidocaldarius. In certain embodiments, the term "wild-type Sac7d" specifically refers to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 (MVKVKFKYKG EEKEVDTSKI KKVWRVGKMV SFTYDDNGKT GRGAVSEKDA PKELLDMLAR AEREKK, UniProt ID: P13123). In alternative embodiments, the term may encompass a variant of this polypeptide in which the N-terminal methionine (M) and the C-terminal lysine (K) residues present in SEQ ID NO: 1 have been removed, thereby yielding the polypeptide sequence set forth in SEQ ID NO: 2 (VKVKFKYKG E E KEVDTS KI KKVWRVGKMVS FTYD D N G KTG RGAVS E KDAP KE LLDM LA RAEREK). In alternative embodiments, the term may encompass a variant

[0080] 30 of this polypeptide with amino acid residues 2 to 59 of SEQ ID NO:1 (SEQ -347

[0081] - 9 -

[0082] ID NO: 3, VKVKFKYKGEEKEVDTSKIKKVWRVGKMVSFTYDDNGKTGRG AVSEKDAPKELLDML).

[0083] The term “specificity binding” refers to the ability of the affitin ligand of the present invention to detectably bind a target molecule (e.g., an epitope

[0084] 5 presented on an antigen) while exhibiting relatively little detectable reactivity with non-target molecules. Specificity may be determined, for example, by binding or competitive binding assays, such as those performed using Biacore® instruments, as described elsewhere herein. In certain embodiments, specificity is demonstrated by a ratio of affinity or avidity for the specific antigen versus nonspecific binding to irrelevant molecules of about 10:1 , about 20: 1 , about 50: 1 , about 100: 1 , about 10,000: 1 , or greater.

[0085] As used herein, the term “affinity” refers to the strength of binding between the affitin ligand of the present invention and a target molecule / structure (e.g.,

[0086] 15 an epitope on an antigen). The affinity of a binding protein may be, for instance, expressed as the dissociation constant (KD). The term "dissociation constant" or " KD" defines the specific binding affinity. As used herein, the term “KD" (usually measured in mol / L or abbreviated as M) refers to the dissociation equilibrium constant of an interaction between two molecules

[0087] 20 such as a first and a second protein or a protein and a small molecule. In the context of the present invention, the term KD is particularly used to describe the binding affinity between the affitin binding ligands and its target structure. Methods for determining the affinity of a binding protein are well known in the art and include, for example, those described in Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988). A preferred method for determining affinity is surface plasmon resonance analysis, such as with Biacore instruments or bio-layer interferometry (BLI) using Octet platforms.

[0088] 30 The term "viral vector" or “viral particle” is widely used to refer to a viral particle that mediates nucleic acid transfer. Viral particles include various viral components and sometimes also host cell components in addition to -347

[0089] - 10 - nucleic acid(s). Illustrative viral vectors include, but are not limited to, retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, vaccinia virus vectors, and herpes simplex virus (HSV) vectors. Adeno-associated virus (AAV) has been characterized and

[0090] 5 developed as a potent viral vector to deliver genes in vitro in cultured cells and also in vivo. AAV is meanwhile a leading platform for in vivo delivery of gene therapies. AAV is a member of the Parvoviridae family. The AAV genome is composed of a linear single-stranded DNA molecule which contains approximately 4.7 kilobases (kb) and consists of two major open reading frames encoding the non-structural Rep (replication) and structural Cap (capsid) proteins. Flanking the AAV coding regions are two cis-acting inverted terminal repeat (ITR) sequences, approximately 145 nucleotides in length, with interrupted palindromic sequences that can fold into hairpin structures that function as primers during initiation of DNA replication. In

[0091] 15 addition to their role in DNA replication, the ITR sequences have been shown to be necessary for viral integration, rescue from the host genome, and encapsidation of viral nucleic acid into mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158:97-129). Multiple serotypes of AAV exist and offer varied tissue tropism. Known serotypes include, for example, AAV1 ,

[0092] 20 AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11 .

[0093] Vectors derived from AAV are particularly attractive for delivering genetic material because they are able to infect (transduce) a wide variety of nondividing and dividing cell types including muscle fibers and neurons and they are devoid of the virus structural genes, thereby eliminating the natural host cell responses to virus infection, e.g., interferon-mediated responses. In addition, wild-type viruses have never been associated with any pathology in humans.

[0094] According to the present invention scAAV are also within the group of AAVs.

[0095] 30 Self-complementary adeno-associated vectors (scAAV) are viral vectors engineered from the naturally occurring adeno-associated virus (AAV) for -347

[0096] - 11 - use in gene therapy. ScAAV is termed "self-complementary" because the coding region has been designed to form an intramolecular double-stranded DNA template.

[0097] Thus, in some embodiments, by an "AAV " is meant a vector or virus derived

[0098] 5 from an adeno-associated virus serotype, including without limitation, AAV- 1 , AAV-2, AAV-3, AAV-4, AAV-5, AAV- 6, AAV-7, AAV -8, AAV-9, AAV-10 and AAV-11 . AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, e.g., the rep and / or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences that provide for replication and packaging (e.g., functional ITRs) of the virus. The ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for

[0099] 15 functional rescue, replication and packaging. In one embodiment, the vector is an AAV-5 vector, with AAV-2 derived ITRs. Also, by an "AAV " is meant the protein shell or capsid, which provides an efficient vehicle for delivery of vector nucleic acid to the nucleus of target cells.

[0100] 20 The term "AAV" as used herein is intended to also encompass recombinant AAV as well as any AAV species deviating from the wild type, like engineered AAVs. Engineered AAVs encompass for example glycosylated, target fused, tagged or sequence modified AAVs. Adeno-associated viruses (AAVs) are herein also called viruses, viral particles, or viral vectors.

[0101] As used herein, the term “cell” or "cell line" refers to a single cell or to a population of cells capable of continuous or prolonged growth and division in vitro. In some embodiments, e.g., the terms "HEK293 cells", "293 cells" or their grammatical equivalents are used interchangeably here and refer to the

[0102] 30 host / packing cell line used in the methods disclosed herein. -347

[0103] - 12 -

[0104] Suitable cells and cell lines have been described for use in production of viral vectors like lentiviral vectors, AAVs and AdVs. The cells themselves may be selected from any biological organism, including prokaryotic (e.g., bacterial) cells and eukaryotic cells including insect cells, yeast cells and mammalian

[0105] 5 cells. Particularly desirable host cells are selected from among any mammalian species, including, without limitation, A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS 1 , COS 7, BSC 1 , BSC 40, BMT 10, VERO, WI38, HeLa, a HEK 293 cell, Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cells derived from mammals including human, monkey, mouse, rat, rabbit, and hamster.

[0106] During the manufacturing of especially AAVs, a percentage of capsids might not incorporate any of the transgenes and are referred to as empty capsids, empty AAVs or empty AAV particles. Additionally, capsids that contain fragments of the transgene or process related impurities are called partial

[0107] 15 capsids, partial AAVs or partial AAV particles. These undesired impurities are co-produced with the full capsids or full AAVs which contain the full length of the desired transgene.

[0108] The term “purification” as used herein, refers to increasing the degree of

[0109] 20 purity of a target structure, in this case e.g., viral particles like AAVs, for instance, by removing one or more impurities.

[0110] The term "impurity" or “contaminant” as used herein, refers to any foreign or objectionable molecules or species, including a biological macromolecule such as DNA, RNA, one or more host cell proteins, nucleic acids, endotoxins, lipids, impurities of synthetic origin like detergents, partial and / or empty viral particles as well as one or more additives which may be present in a sample containing the viral particles to be purified and thus to be separated from one or more of the impurities. Viral particle impurities include both, process-

[0111] 30 related impurities and / or product-related impurities. Exemplary process- related impurities include, but are not limited to, residual host-cell components (e.g., proteins, DNA - including extra-viral, chromatin- -347

[0112] - 13 - associated DNA - and / or RNA), residual viral production components (e.g., plasmid DNA, and / or helper viruses), residual cell culture components (e.g., antibiotics, supplements, inducers, and / or growth factors), and residual purification components (e.g. buffers, inorganic salts, enzymes, media,

[0113] 5 and / or detergents), as well as other contaminants. The aim of purifying viral particles is typically to generate viral particles that are substantially free of process related impurities like a host-cell protein, a host-cell DNA, a host-cell RNA, or a combination thereof. Exemplary product-related impurities can include, but are not limited to, empty capsids (where undesirable), aggregated viral particles, and degraded viral particles.

[0114] It is understood that a sample can be "substantially free" of one or more impurities but continue to have a small amount (e.g., undetectable level, or below an acceptable range) of one or more impurities, and that "substantially free" does not require complete removal of all impurities.

[0115] 15

[0116] As used herein, and unless stated otherwise, the term “sample” refers to any composition or mixture that contains target structures according to the invention. Samples may be derived from biological or other sources. Biological sources include eukaryotic and prokaryotic sources, such as plant

[0117] 20 and animal cells, tissues, and organs. The sample may also include diluents, buffers, detergents, and contaminating species, debris and the like that are found mixed with the target molecule. The sample may be "partially purified" (i.e., having been subjected to one or more purification steps, such as filtration steps) or may be obtained directly from a host cell producing the target structures, e.g., the sample may comprise harvested cell culture fluid.

[0118] The term "conjugate" refers to a molecule comprising or essentially consisting of at least a first molecule such as an affitin ligand of the invention attached, preferably chemically, to other substances such as to a second

[0119] 30 polypeptide or another moiety such as a functional group on the surface of a chromatography matrix. -347

[0120] - 14 -

[0121] The terms "polypeptide", "peptide", and "protein" relate to oligo- and polypeptides and refers to substances which comprise two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 10 or more consecutive amino acids linked to one

[0122] 5 another via peptide bonds. Amino acids are referred to herein using the name of the amino acid, the three-letter abbreviation, or the single letter abbreviation. Polypeptides may further comprise substances which contain not only amino acid components but also non-amino acid components such as sugars and phosphate structures, as well as substances containing bonds such as ester, thioether or disulfide bonds. According to the present invention, a nucleic acid such as RNA may encode a polypeptide or protein. Accordingly, a transcribable polynucleotide or a transcript thereof such as mRNAs may contain an open reading frame (ORF) encoding a polypeptide. Said polynucleotide may express the encoded polypeptide or protein. It is

[0123] 15 also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code. As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide”, “peptide” and / or the term “protein”.

[0124] Polypeptides as described herein may comprise amino acids with distinct

[0125] 20 biophysical properties, including but not limited to polarity, charge, and structural characteristics. In the context of the present application, and unless indicated otherwise, “X” can be any naturally occurring amino acid, in particular any of the standard amino acids (i.e., the 20 amino acids encoded in the standard genetic code). The term “amino acids with hydrophobic side chains” may refer to amino acids characterized by non-polar, hydrophobic side chains, as understood by those skilled in the art. Non-limiting examples include Alanine (Ala, A), Valine (Vai, V), Isoleucine (lie, I), Leucine (Leu, L), Methionine (Met, M), Phenylalanine (Phe, F), Tyrosine (Tyr, Y), and Tryptophan (Trp, W). The term “amino acids with hydrophilic side chains”

[0126] 30 refers to amino acids with side chains that exhibit a favourable interaction with aqueous environments, typically due to the presence of polar, charged, -347

[0127] - 15 - or hydrogen-bonding functional groups. These amino acids may be broadly classified as follows: Polar, uncharged amino acids, including Asparagine (Asn, N), Glutamine (Gin, Q), Serine (Ser, S), Threonine (Thr, T), Cysteine (Cys, C), Tyrosine (Tyr, Y); Positively charged (basic) amino acids, including

[0128] 5 Arginine (Arg, R), Lysine (Lys, K), Histidine (His, H); Negatively charged (acidic) amino acids, including Aspartic acid (Asp, D), Glutamic acid (Glu, E). The term “amino acids with aromatic side chains” refers to amino acids comprising aromatic structures, as recognized by those skilled in the art. These side chains confer unique chemical properties. Non-limiting examples include Phenylalanine (Phe, F), Tyrosine (Tyr, Y), and Tryptophan (Trp, W). Throughout this disclosure and in the appended claims, the conventional one-letter and three-letter codes for amino acid residues may be utilized, as defined in accordance with the nomenclature guidelines established by the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN).

[0129] 15

[0130] The terms “polynucleotide” and “nucleic acid” are used herein interchangeably. They refer to a polymeric form of nucleotides of any length: Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment,

[0131] 20 exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, synthetic polynucleotides, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified, such as by conjugation with a labelling component. If not stated otherwise, the term

[0132] 30 “nucleic acid” refers to any nucleic acid such as ribonucleic acid, deoxyribonucleic acid, xeno nucleic acid, single stranded or double stranded. -347

[0133] - 16 -

[0134] As used herein, the term "identity" when used in relation to nucleic acids or polypeptides, describes the degree of similarity between two or more nucleotide or polypeptide sequences. The percentage of "sequence identity" between two sequences can be determined by comparing two optimally

[0135] 5 aligned sequences over a comparison window, such that the portion of the sequence in the comparison window may comprise additions or deletions (gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. A sequence that is identical at every position in comparison to a reference

[0136] 15 sequence is said to be identical to the reference sequence and vice-versa. An alignment of two or more sequences may be performed using any suitable computer program. For example, a widely used and accepted computer program for performing sequence alignments is CLUSTALW vl .6 (Thompson, et al. (1994) Nucl. Acids Res., 22: 4673-4680).

[0137] 20

[0138] The “percent sequence identity” between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity may be calculated as the number of identical nucleotides I amino acids divided by the total number of nucleotides I amino acids, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimise alignment of two or more sequences. Sequence comparisons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled person.

[0139] 30 -347

[0140] - 17 -

[0141] As used herein, the term “binding conditions” refers to solution and process parameters under which a target molecule will selectively bind to a ligand immobilized on or associated with a stationary phase in an affinity chromatography system. Binding conditions may include, without limitation,

[0142] 5 buffer composition, pH, ionic strength, temperature, and the presence or absence of cofactors, chaotropic agents, or detergents, provided that such conditions permit the formation of a stable and selective interaction between the ligand and the target.

[0143] The term "matrix" or "chromatography matrix" are used interchangeably herein and refers to a solid phase through which the sample migrates in the course of a chromatographic separation. The matrix typically comprises a base material and ligands covalently bound to the base material by e.g., functional groups on the surface of the base material.

[0144] 15 A “ligand” is a functional group or molecule that is part of the chromatography matrix, typically it is attached to the base material of the matrix, and that determines the binding properties and interaction properties of the matrix. Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic

[0145] 20 interactions groups, metal affinity groups, affinity groups, bioaffinity groups, and mixed mode groups (combinations of the aforementioned). It is also possible that one ligand has more than one binding I interaction property.

[0146] As used herein, the term “reverse binding” refers to the ability of an affitin ligand to associate with, and subsequently dissociate from, a target structure under conditions that allow the ligand-target complex to be disrupted without permanent chemical modification of either the ligand or the target. Reversible binding is mediated predominantly by non-covalent interactions such as hydrogen bonding, ionic interactions, hydrophobic interactions, van der

[0147] 30 Waals forces, or metal ion coordination. In certain embodiments, dissociation may involve a transient alteration of the ligand’s conformation or tertiary / quaternary structure such that its structural integrity and / or binding -347

[0148] - 18 - activity is partially or wholly lost under non-binding conditions, but is restored upon re-establishment of binding conditions. Dissociation can be achieved by modifying one or more environmental parameters, including but not limited to pH, ionic strength, temperature, the presence of competing ligands,

[0149] 5 chaotropic agents, detergents, or changes in buffer composition.

[0150] The term "alkaline-stable" or "alkaline-resistant" or "caustic-stable" or "caustic-resistant" refers to the ability of the affitin ligands of the invention to withstand alkaline conditions without significantly losing the ability to bind to their respective target structure. The skilled person in this field can easily test caustic resistance by incubating (exposing for a set time) an affitin ligand of the invention (e.g., AAV-specific affitins) with sodium hydroxide solutions, e.g., as described in the Examples, and subsequent testing of the binding activity to target structures (e.g., respective AAVs) by routine experiments known to someone skilled in the art, for example, by chromatographic

[0151] 15 approaches.

[0152] The term “binding capacity” defines the amount of the target structure that can be bound by the chromatography matrix, thereby the dynamic binding capacity defines the amount of the target structure that can be bound by the

[0153] 20 chromatography matrix under flow conditions and the static binding capacity defines the amount of the target that can be bound by the chromatography matrix when it is equilibrated with the solution (liquid) comprising the target structure at a given concentration. Binding capacity can be expressed in terms of mass of target structure per volume of matrix (e.g., mg / mL).

[0154] To determine 10% dynamic binding capacity (10% DBC), a chromatography column is loaded at a controlled flow rate with a solution containing the target structure at a known concentration and the breakthrough of unbound target structure is monitored in the column's effluent using a detector, such as a UV

[0155] 30 detector. The concentration of the target structure in the effluent is plotted over the volume to generate a breakthrough curve. The volume at the point where the effluent concentration reaches 10% of the initial feed concentration -347

[0156] - 19 - corresponds to the 10% breakthrough point, and the amount of bound material at that point represents the 10% DBC.

[0157] Yield” defines how much target structure is obtained after each process step compared to the amount of loaded target structure. For example - "elution

[0158] 5 yield” of an affinity chromatography step defines how much target structure that was bound could be eluted and is typically calculated as (Total Amount Eluted / lnitial Amount Bound)x 0. Recovery defines how much target structure is obtained overall. Recovery is typically calculated as (Total Amount Obtained / lnitial Amount Loaded )X100.

[0159] CAUSTIC-RESISTANT AFFINITY LIGANDS

[0160] A first aspect of the present invention relates to caustic-resistant affinity ligands. In particular, disclosed herein are affitin ligands useful for the

[0161] 15 separation of a target structure from a sample, such as a liquid sample, by affinity chromatography.

[0162] Surprisingly, it has been found that affitin ligands, not only provide an ideal chromatographic medium for the selective separation of target structures bound by said affitin ligands from complex fluid samples when immobilised

[0163] 20 on a chromatography support such as resins or membranes but also exhibit substantial resistance to caustic reagents such as NaOH used to wash, sanitise the chromatographic medium, and / or elute target structures during chromatography.

[0164] Affitins comprise a peptide of approximately 7 kDa and typically about 60-66 amino acids. The small molecular size and favourable steric properties of these peptide-based ligands enable efficient and versatile functionalisation onto chromatography matrixes, such as resins. Affitins can be recombinantly expressed in host systems such as E. coli and produced at scale, while

[0165] 30 allowing sequence adaptation for selected targets and desired biophysical characteristics. -347

[0166] - 20 -

[0167] While a majority of proteins do not withstand drastic changes of pH during elution / washing steps during affinity chromatography, it has been surprisingly found that the affitin ligands of the invention can withstand such conditions despite harbouring peptide sequences typically believed to be sensitive to

[0168] 5 caustic degradation such as asparagine-glycine (NG) dipeptide and / or glutamine-glycine (QG) dipeptide sequences. Interestingly, affitin ligands of the invention do show remarkably resistance to caustic reagents during e.g. cleaning in place (CIP) procedures during affinity chromatography allowing reusability as well as efficient and regulatory-compliant separation / isolation of target structures.

[0169] Without wishing to be bound by theory, it is hypothesised that the formation of alpha-helices by the affitins with a hydrophobic inner core may stabilise the polypeptide against unfolding and gives protection from exposure to harsh reactive chemical surroundings, such as alkalinity.

[0170] 15

[0171] While it was surprisingly shown by the applicant that affitin ligands of the invention harbouring at least one asparagine-glycine (NG) dipeptide and / or at least one glutamine-glycine (QG) dipeptide, which are commonly associated with caustic-sensitivity, tolerate caustic conditions without

[0172] 20 substantial loss of binding capacity, the presence of asparagine or glutamine residues flanked on both sides by glycine (e.g., GNG or GQG tripeptide sequences) is not tolerated leading to rapid loss of binding capacity following cleaning procedures.

[0173] Accordingly, the present invention refers in particular to an affitin ligand for separating a target structure from a liquid sample by affinity chromatography comprising or consisting of a polypeptide, wherein the polypeptide does not comprise an asparagine or glutamine residue flanked on both sides by glycine residues. In some embodiments, the polypeptide does not comprise

[0174] 30 an asparagine residue flanked on both sides by glycine residues. In some embodiments, the polypeptide does not comprise a glutamine residue flanked on both sides by glycine residues. In some embodiments, the -347

[0175] - 21 - polypeptide does not comprise an asparagine residue flanked on both sides by glycine residues and a glutamine residue flanked on both sides by glycine residues.

[0176] In certain embodiments, the polypeptide of the affitin ligand of the invention

[0177] 5 comprises at least 1 , 2, 3, 4, or 5 asparagine-glycine (NG) dipeptides within the sequence of the polypeptide. In a preferred embodiment, the polypeptide comprises between 1 and 3 asparagine-glycine (NG) dipeptides. In a preferred embodiment, the polypeptide comprises at least one glutamine- asparagine-glycine (NG) dipeptide.

[0178] In certain embodiments, the polypeptide of the affitin ligand of the invention comprises at least 1 , 2, 3, 4, or 5 glutamine-glycine (QG) dipeptides within the sequence of the polypeptide. In a preferred embodiment, the polypeptide comprises between 1 and 3 glutamine-glycine (QG) dipeptides. In a preferred

[0179] 15 embodiment, the polypeptide comprises at least one glutamine-glycine (QG) dipeptide.

[0180] In some embodiments, the polypeptide of the affitin ligand of the invention comprises at least one asparagine-glycine (NG) dipeptide and at least one

[0181] 20 glutamine-glycine (QG) dipeptide.

[0182] In some embodiments, the disclosed affitin ligands of the invention exhibit stability in alkaline conditions meaning that the ligands retain a certain binding capacity to their target structure compared to ligands before alkaline treatment. For example, in certain embodiments, the affitin ligands have less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% reduction in binding capacity following an incubation in about 0.01 M, 0.05 M, 0.1 M, 0.25 M, 0.5 M, 1.0 M, or 2.0 M NaOH or KOH for at least about 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 240 minutes, 360 minutes, 480 minutes,

[0183] 30 or 24 hours. In some embodiments, other caustic reagents commonly used for cleaning or sanitizing chromatography matrices, such as LiOH, CsOH, or -347

[0184] - 22 - mixtures thereof, may also be employed, and the affitin ligands have less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% reduction in binding capacity under such conditions.

[0185] 5 The term “incubating in caustic reagents” refers to maintaining the affitin ligand, or a matrix comprising the affitin ligand, in contact with a caustic solution for a defined duration, typically under controlled conditions of concentration, temperature, and time, such that the ligand is subjected to continuous treatment. “Incubating” generally implies a static, semi-static or non-static treatment where the ligand remains immersed in the caustic solution for a specified period (e.g., at least 5 minutes, 30 minutes, 150 minutes, or longer).

[0186] Unless otherwise specified, incubation in caustic reagents may be carried out under standard conditions. Such conditions may include maintaining the

[0187] 15 affitin ligand, or a matrix comprising the affitin ligand, in contact with a caustic solution at a temperature of about 20 °C to 25 °C (room temperature), at a pH determined by the caustic solution (typically greater than about 12), and under static or gently agitated conditions or with active flow-through. In some embodiments, incubation may be performed for at least about 15 minutes, at

[0188] 20 least about 30 m inutes, at least about 60 m inutes, at least about 120 m inutes, at least about 150 minutes, or at least about 180 minutes. In certain embodiments, the concentration of the caustic reagent during incubation may be between about 0.05 M and about 0.5 M sodium hydroxide, although higher or lower concentrations, such as about 0.01 M or about 2.0 M, may also be used. The preferred standard condition for comparing caustic stability is at 20 °C under static conditions for a defined period of time and with a defined concentration of the caustic reagent.

[0189] In some embodiments, the affitin ligands have less than about 30%, 25%,

[0190] 30 20%, 15%, 10%, or 5% reduction in binding capacity under these alkaline washing conditions. -347

[0191] - 23 -

[0192] In certain preferred embodiments, the affitin ligands have less than about 30% reduction in binding capacity following an incubation in 0.1 M NaOH for at least 150 minutes. In further preferred embodiments, the reduction in binding capacity of the affitin ligand following an incubation in 0.5 M NaOH

[0193] 5 for at least 30 minutes is less than about 30%.

[0194] In certain embodiments, the affitin ligands retain at least about 95%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% of their binding capacity following an incubation in about 0.01 M, 0.05 M, 0.1 M, 0.25 M, 0.5 M, 1.0 M, or 2.0 M NaOH or KOH for at least about 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 240 minutes, 360 minutes, 480 minutes, or 24 hours. In some embodiments, other caustic reagents commonly used for cleaning or sanitizing chromatography matrices, such as LiOH, CsOH, or mixtures

[0195] 15 thereof, may also be employed, and the affitin ligands retain at least about 95%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% of their binding capacity under such conditions.

[0196] In certain preferred embodiments, the affitin ligands retain at least about 70%, preferably 80% of their binding capacity following an incubation in 0.1

[0197] 20 M NaOH for at least 150 minutes. In further preferred embodiments, the affitin ligands retain at least about 70%, preferably 80% of their binding capacity following an incubation in 0.5 M NaOH for at least 30 minutes.

[0198] AFFITIN LIGANDS

[0199] In certain embodiments, the affitin ligands of the invention comprise or consists of a variant of a member of the Sac7d, Sso7d, or Aho7c family, preferably of the Sac7d family. Affitins are small artificial binding proteins that can be adapted to bind selected target structures. Due to their small size, well-defined structure, and chemical stability, affitin ligands can be designed

[0200] 30 to offer high-affinity interactions with a wide range of target molecules. Surprisingly it was found that, when immobilized on a chromatography matrix -347

[0201] - 24 - such as resins, affitin ligands offer the benefit of specific binding to target structures and enhanced resistance to chemical degradation e.g., during cleaning in place (CIP) with caustic reagents.

[0202] Affitin ligands according to the invention may be selected from affitins

[0203] 5 belonging to the Sac7d family. In certain embodiments, the affitin ligands of the present invention comprise or consist of a polypeptide of the variant of a member of the Sac7d family according to formula I (SEQ ID NO: 4), II (SEQ ID NO: 5), or III (SEQ ID NO: 6):

[0204] MVKVKFX1X2X3G X4EKEVDX5X6KI X7X8VX9RX10GX11X12V X13FX14YDDNGKX15 GXI6G X17VX18EX19X20A PKELLX21ML ARAEREKK formula I,

[0205] VKVKFX1X2X3G X4EKEVDX5X6KI X7X8VX9RX10GX11X12V Xi3FXi4YDDNGKXi5

[0206] 15 GXI6G X17VX18EX19X20A PKELLX21ML ARAEREK formula II,

[0207] VKVKFX1X2X3G X4EKEVDX5X6KI X7X8VX9RX10GX11X12V Xi3FXi4YDDNGKXi5 GXI6G X17VX18EX19X20A PKELLX21ML

[0208] 20 formula III wherein,

[0209] Xi , X2, X3, X4, X5, X6, X7, X8, X9, X10, X11 , X12, X13, X14, X15, X16, X17, X18, X19, X20, and X21 represents a single amino acid independently selected from the group consisting of naturally occurring amino acids.

[0210] In embodiments, at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids of Xi, X2, X3, X4, X5, X6, X7, X8, X9, Xio, Xu , X12, Xi3, X14, X15, X , X17, X , Xi9, X20, and X21 do not correspond to an

[0211] 30 amino acid of a wild-type member of the Sac7d family according to SEQ ID NO: 1 -3. -347

[0212] - 25 -

[0213] In some embodiments, the affitin ligand comprises a polypeptide, wherein the polypeptide refers to a polypeptide with between 1-25 amino acids, between 1-20 amino acids, between 1-15 amino acids, between 5-20 amino acids, between 10-20 amino acids, or between 12-16 amino acids e.g., about

[0214] 5 12, 13, 14, 15, or 16 amino acids, which are mutated, deleted, or inserted, as compared to a wild-type member of the Sac7d family according to SEQ ID NO: 1 -3. In a preferred embodiment, said between 1 -25 between 1 -20, between 1 -15 amino acids, between 5-20 amino acids, between 10-20 amino acids, or between 12-16 amino acids e.g., about 12, 13, 14, 15, or 16 amino acids, are mutated as compared to a wild-type member of the Sac7d family according to SEQ ID NO: 1 -3.

[0215] In embodiments, mutations, deletions, or insertions may occur at amino acid positions other than Xi , X2, X3, X4, X5, Xe, X7, Xs, X9, X10, X11, X12, X13, X14, X15, X , X17, X , X19, X20, and X21 of the polypeptide with an amino acid

[0216] 15 sequence according to formula 1 (SEQ ID NO: 4), formula II (SEQ ID NO: 5) or formula III (SEQ ID NO: 6).

[0217] In some embodiments, the affitin ligand comprises a polypeptide, wherein the polypeptide comprises a N-terminal M. In some embodiments, said

[0218] 20 polypeptide comprises a C-terminal cysteine. Preferably, said polypeptide comprises a N-terminal methionine and a C-terminal cysteine. Preferably, the affitin ligand comprises only a single C-terminal cysteine, while being devoid of further cysteine residues in its amino acid sequence.

[0219] In some embodiments, the affitin ligand comprises a polypeptide, wherein the polypeptide comprises an N-terminal affinity tag, such as a histidine tag (His-tag), preferably a polyhistidine tag, more preferably an RGS- Hiss tag (RGSHHHHHH, SEQ ID NO: 7), even more preferably an RGS- Hiss tag comprising an N-terminal methionine (MRGSHHHHHH, SEQ ID NO: 8), and

[0220] 30 most preferably an N-terminal RGS-Hise tag comprising an N-terminal methionine and a GS linker (MRGSHHHHHHGS, SEQ ID NO: 9). -347

[0221] - 26 -

[0222] In some embodiments, the affitin ligand comprises a polypeptide, wherein the polypeptide comprises amino acids 1 - 54, 1 - 55, 1 -56, 1 - 57, 1 - 58, 1

[0223] - 59, 1 - 60, 1 - 61 , 1 - 62, 1 - 63, 1 - 64, or 1 - 65 according to SEQ ID NOs: 4-6. In some embodiments, the polypeptide comprises amino acids 2 - 54, 2

[0224] 5 - 55, 2 - 56, 2 - 57, 2 - 58, 2 - 59, 2 - 60, 2 - 61 , 2 - 62, 2 - 63, 2 - 64, 2 - 65, or 2 - 66 according to SEQ ID NOs: 4-6, 19-46.

[0225] In certain embodiments, the affitin ligand comprises a polypeptide, wherein the polypeptide comprises or consists of a polypeptide with an amino acid sequence according to SEQ ID NOs: 1-6, 19-46 or a polypeptide with at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of an amino acid sequence according to SEQ ID

[0226] 15 NOs: 1 -6, 19-46.

[0227] Surprisingly, it has been found that certain mutations within the polypeptide of the variant of a member of the Sac7d family of the affitin ligand of the invention may not significantly affect the binding affinity of the affitin ligand to

[0228] 20 its target structure. Said tolerated mutations corresponds to positions within the sequence of the polypeptide, which show low conservation within the sequence of the Sac7d-family. In some embodiments, the affitin ligand comprises a polypeptide, wherein the polypeptide comprises at least one, at least two, at least tree, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or at least eleven tolerated mutations at positions selected from the group according to table 1 within the amino acid sequence according to SEQ ID NO: 1 or 4.

[0229] Table 1 : Tolerated Mutations of SEQ ID NO: 1 and 4

[0230] 30 -347

[0231] - 27 -

[0232] 5

[0233] MODIFICATION OF C-TERMINUS

[0234] In certain embodiments, the C-terminus of the affitin ligand comprises a

[0235] 15 polypeptide, wherein the polypeptide comprises a short amino acid sequence that is configured to modify structural characteristics and / or to facilitate functionalisation of the polypeptide, for example, to enable immobilisation on solid supports, such as chromatography matrixes, preferably chromatography resins.

[0236] 20

[0237] Such C-terminal extensions may support site-specific coupling, increase surface accessibility, or enhance electrostatic interactions with negatively charged surfaces. For example, positively charged sequences such as polylysine tags (e.g., K, KK, KKK, KKKK (SEQ ID NO: 10), KKKKK (SEQ ID NO: 11 ), and KKKKKK (SEQ ID NO: 12)) may enhance binding to negatively charged materials via ionic interactions. In other cases, terminal cysteine residues may be used to enable covalent coupling via thiol-specific chemistries.

[0238] Accordingly, the C-terminus of the polypeptide of the affitins according to the

[0239] 30 invention may comprise one or more amino acid sequences selected from the group consisting of C, GKK, EKSGKK (SEQ ID NO: 13), EKQKK (SEQ ID NO: 14), ARAEREKK (SEQ ID NO: 15), ARAEREK (SEQ ID NO: 16), -347

[0240] - 28 -

[0241] ARAEKKK (SEQ ID NO: 17), EKQKK (SEQ ID NO: 18), EKK, K, KK, KKK, KKKK (SEQ ID NO: 10), KKKKK (SEQ ID NO: 11 ), and KKKKKK (SEQ ID NO: 12), or combinations thereof.

[0242] In a further embodiment, the polypeptide comprises a C-terminal cysteine

[0243] 5 residue, either alone or in combination with one or more of the aforementioned sequences.

[0244] MULTIMERS

[0245] In some embodiments, affitin ligands comprising multimeric polypeptides may be used as affinity ligands according to the invention. As used herein, a multimeric polypeptide refers to a polypeptide containing two or more domains. In embodiments, the domains are polypeptides described herein. In some embodiments, the multimeric polypeptide comprises one or more

[0246] 15 domains having an amino acid sequence according to any one of SEQ ID NOs 4-6, 19-46. In some embodiments, the multimeric polypeptide comprises two or more domains having an amino acid sequence independently selected from any one of SEQ ID NOs 4-6, 19-46, wherein the two domains are the same. In some embodiments, the multimeric

[0247] 20 polypeptide comprises two or more domains having an amino acid sequence independently selected from any one of SEQ ID NOs 4-6, 19-46, wherein the two domains are different. In some embodiments, the two or more domains are connected by a linker. In some embodiments, said linker is selected from the group of polypeptides, polynucleotide, nucleosides, chemically modified nucleosides, photo-labile linkers, fatty acid chains, natural and artificial amino-acids at the likes.

[0248] MODIFICATIONS

[0249] It was shown by the applicant that the N- or C-terminus of the polypeptide

[0250] 30 comprising the affitin ligand according to the invention may be modified. Surprisingly, N- or C-terminal modification of the polypeptide does not significantly affect affinity properties of the ligand coupled to a -347

[0251] - 29 - chromatographic matrix thus enabling the attachment of moieties useful within the scope of the invention. For instance, in certain embodiments, the polypeptide of the affitin ligands according to the invention may be N- and / or C-terminally modified with e.g., peptide-tags, small molecules, antibodies or

[0252] 5 other modifications known by the skilled person in the art such as, for instance, useful for the production of the ligand, purification of the ligand, increasing the stability of the ligand, enabling coupling to a chromatography matrix, preferably resins, and the likes.

[0253] In some embodiments, the affitin ligand according to the invention is conjugated to at least one agent operably linked to said ligand. In a preferred embodiment, the ligand is selected from the group of polypeptides, DNA, RNA, small molecules, antibodies, nanobodies, single chain variable domains, and immunoglobulin fragments.

[0254] 15 In some embodiments, the polypeptides described herein comprise a protein tag, preferably a polyhistidine tag. In a preferred embodiment, the polyhistidine tag is located at the N-terminus of the polypeptide sequence. In embodiments, the polyhistidine tag is located at the C-terminus of the polypeptide sequence. In embodiments, the polypeptides are biotinylated. In

[0255] 20 embodiments, the polypeptides described are extended at the N- or C- terminus. In embodiments, the extension comprises a polypeptide linker.

[0256] As known by the skilled person in the art, any method or approach for the modification of the polypeptide of the invention may be utilized. For instance, when the modification relates to a second polypeptide, said second polypeptide may be encoded in frame on the same vector as the first polypeptide. Small molecules and the likes may be coupled, for instance, directly to the polypeptide by means of chemical synthesis approaches, such as click chemistry.

[0257] 30

[0258] TARGET STRUCTURES -347

[0259] - 30 -

[0260] Affitin ligands according to the invention may be adapted for selected target structures by e.g., randomizing amino acid residues on the binding surface of the wildtype Sac7d family proteins and subjecting the resulting protein library to selection methods such as ribosome display. Thereby, binding

[0261] 5 specificity and affinity can be directed towards a wide variety of targets, including peptides, proteins, nucleic acids, viruses, bacteria, and other biological or synthetic molecules (Kalichuk et al., Methods Mol Biol. 2020;2070:19-41. doi: 10.1007 / 978-1 -4939-9853-1 _2. PMID: 31625088).

[0262] In some embodiments, affitin ligand according the invention specifically and / or selectively binds the target structure. In certain embodiments, the affitin ligands of the invention selectively bind a target structure selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, lipids, viruses including adeno-associated viruses, adenoviruses, lentiviruses, herpesviruses, vesicular stomatitis virus, orthomyxoviruses,

[0263] 15 flaviviruses, coronaviruses, paramyxoviruses, filoviruses, retroviruses, papillomaviruses, polyomaviruses, poxviruses, bacteriophages, and viruslike particles, bacteria including Gram-positive bacteria, Gram-negative bacteria, spore-forming bacteria, and bacterial cell fragments, fungi, yeast, protozoa, parasites including Plasmodium spp., Trypanosoma spp., and

[0264] 20 Leishmania spp., rickettsiae, mycoplasmas, chlamydiae, prions, other microorganisms, extracellular vesicles including exosomes, microvesicles, and apoptotic bodies, cells including mammalian cells, plant cells, insect cells, and stem cells, cell membranes, organelles, toxins including protein toxins, bacterial exotoxins, and mycotoxins, small molecules including metabolites, pharmaceutical agents, cofactors, natural products, and synthetic organic compounds, nanomaterials including nanoparticles, quantum dots, polymer particles, and dendrimers, and hybrid biological- synthetic complexes including bioconjugates, drug-protein conjugates, antibody-drug conjugates, and nucleic acid-lipid complexes. -347

[0265] - 31 -

[0266] In a preferred embodiment, the affitin ligands of the invention selectively bind a target structure selected from the group consisting of proteins, nucleic acids, carbohydrates, lipids, small molecules, vesicles, viruses, microorganisms, cells, organelles, and cell fragments.

[0267] 5

[0268] In a preferred embodiment, the target structure specifically bound by the affitin ligands is a virus particle or capsid or a variant of a virus particle or capsid, preferably selected from the group of adeno-associated viruses, adenoviruses, lentiviruses, herpesviruses, vesicular stomatitis virus, orthomyxoviruses, flaviviruses, coronaviruses, paramyxoviruses, filoviruses, retroviruses, papillomaviruses, polyomaviruses, poxviruses, bacteriophages, and virus-like particles.

[0269] AAV-BINDING AFFITIN LIGANDS

[0270] 15 Adeno-associated viruses are in high demand for medical applications such as gene therapy. However, large-scale production at acceptable quality is currently a serious industry bottleneck holding wide-spread adoption of these next-generation therapies back. In particular, repeated reusing of affinity matrixes specific for AAVs, such as affinity matrixes with protein ligand tags,

[0271] 20 is currently severely limited due to the sensitivity of protein-based ligands to cleaning / sanitation / elution conditions with caustic reagents. Necessary treatment of affinity matrixes with protein-based ligands with caustic reagents can not only result in decreased binding capacity of the ligands in subsequent runs limiting purification efficiency but also in significant leaching of ligand fragments following degradation into the purified sample. Impurities are especially problematic for purified target structures used in medical applications such as AAVs for gene therapy. Surprisingly, the affitin ligands of the application are particularly efficient in separating AAV particles or capsids or a variant of an AAV particle and capsid from fluid samples and

[0272] 30 allow for stringent washing / elution / sanitisation during affinity chromatography thus allowing repeated use of the matrixes for subsequent runs. -347

[0273] - 32 -

[0274] In some embodiments, the affitin ligands of the invention selectively bind an adeno-associated virus (AAV) particle or capsid or a variant of a AAV virus particle or capsid.

[0275] It has surprisingly been found that affitins ligands comprising or consisting of

[0276] 5 a polypeptide according to SEQ ID NOs: 19-46 not only bind efficiently to an adeno-associated virus (AAV) particle or capsid or a variant of a AAV virus particle or capsid but also show low reduction of binding capacity when incubated with caustic reagents for a prolonged time.

[0277] Accordingly, in certain embodiments, the affitin ligands selectively bind an adeno-associated virus (AAV) particle or capsid or a variant of a AAV virus particle or capsid. Preferably, said affitins comprise a polypeptide of table 2 selected from the group of SEQ ID NOs: 19-46.

[0278] Table 2: Affitin ligands selectively binding AAV serotypes

[0279] 30 -347

[0280] -33-

[0281] 5

[0282] 30 - 34 -

[0283] In a preferred embodiment, the target structures specifically bound by the affitin ligands of the invention are AAV particles or capsids or a variant of an AAV particle and capsid, preferably, AAV serotype 5, 8, or 9.

[0284] VECTORS

[0285] 5

[0286] The sequences of the polypeptides comprising the affitin ligand of the invention can be cloned in any appropriate vector by any molecular genetic methods known in the art. Accordingly, a further aspect of the present invention relates to a nucleic acid molecule coding for the polypeptide of the affitin ligand of the invention.

[0287] These recombinant DNA constructs comprising a nucleotide sequence, coding for a polypeptide comprising a variant as described above, are used in connection with a vector, such as a plasmid, phagemid, phage or viral

[0288] 15 vector.

[0289] These recombinant acid molecules can be produced by techniques described in Sambrook et al. ,1989 (Sambrook J, Fritsch EF and Maniatis T (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, New York). Alternatively, the DNA sequences may be

[0290] 20 chemically synthesized using, for example, synthesizers.

[0291] Recombinant constructs of the invention comprise the expression vectors that can express the RNA and thus lead to production of proteins from the above genetic sequences. The vector may thus further comprise regulatory sequences, including a suitable promoter operably linked to the open reading frame (ORF) of the genetic sequences herein disclosed. The vector may further comprise a selectable marker sequence such as an antibiotic resistance gene. Specific initiation and bacterial secretory signals also may be required for efficient translation of the coding sequences when bacteria

[0292] 30 as used as the expression host.

[0293] PRODUCTION OF AFFITINS - 35 -

[0294] A further aspect of the present invention relates to a method for producing the affitin ligands of the invention, comprising the steps of a) culturing cells, wherein the cells have been transformed by a nucleic acid molecule coding for the affitins of the invention, and

[0295] 5 b) recovering the affitins.

[0296] Cells are transfected or transformed with vectors containing the sequences coding for the affitins comprising the polypeptides as disclosed above. The cells are then cultured in such conditions as to have the polypeptides expressed and favourably secreted. The conditions of culture of the cells are the conditions generally used for recombinant antibody production and are known in the art. Such conditions that are known in the art can also be optimized by the person skilled in the art if needed. Kunert and Reinhart (Appl

[0297] 15 Microbiol Biotechnol. 2016; 100: 3451- 3461 ) review such methods and provide ample references thereto. One can use bacterial, phage (Shukra et al, Eur J Microbiol Immunol (Bp). 2014; 4(2): 91 -98) or eukaryotic systems of production. One shall prefer to use eukaryotic cells in order to obtain proper post- translational modifications such as glycosylation. One can use CHO

[0298] 20 (Chinese Hamster Ovary) cells, PER.C6 cells (human cell line, Pau et al, Vaccine. 2001 21 ; 19(17-19):2716-21 ), HEK 293b cells (Human embryonic kidney cells 293), NS0 cells (cell line derived from the non-secreting murine myeloma) or EB66 cells (a duck cell line Valneva, Lyons, France).

[0299] Also provided by the present disclosure are host cells containing at least one of the DNAs constructs coding for an affitin ligand as disclosed herein. The host cell can be any cell for which expression vectors are available. As indicated above, it may be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or a prokaryotic cell, such as a bacterial cell.

[0300] 30

[0301] Introduction of the recombinant construct into the host cell is performed by any method known in the art (such as calcium phosphate transfection, -347

[0302] - 36 - lipofection, DEAE, dextran mediated transfection, electroporation or phage infection). The vectors can be inserted within the genome of the host cell or be maintained as an extragenomic vector (such as a Bacterial Artificial Chromosome or a Yeast Artificial Chromosome). When introduced within the

[0303] 5 cell genome, such introduction may be random or targeted using methods known in the art (homologous recombination or the like).

[0304] BACTERIAL HOSTS AND EXPRESSION

[0305] Useful expression vectors for bacterial use are constructed by inserting the recombinant DNA sequence together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desirable, to provide amplification within the host.

[0306] 15

[0307] Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0308] EUKARYOTIC HOSTS AND EXPRESSION

[0309] 20

[0310] Examples of the eukaryotic host cells include vertebrate cells, insect cells, and yeast cells. In particular, one can use the cells mentioned above.

[0311] The transformed or transfected cells are cultured according to methods known in the art and the affitins are recovered from intracellular or extracellular fractions (depending on whether it is secreted or not).

[0312] ISOLATION OF THE MOLECULES

[0313] The recombinant protein produced can be separated and purified by any of various known separation methods utilizing the physical or chemical property

[0314] 30 of the protein, from the intracellular or extracellular fraction. -347

[0315] - 37 -

[0316] In particular, one can use methods such as precipitation, ultrafiltration, various types of liquid chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, dialysis, and a combination

[0317] 5 thereof.

[0318] In general, any method known and used to purify recombinant polypeptides is adapted for the purification of the molecules herein disclosed.

[0319] If a tag has been introduced within the recombinant sequence (such as a poly- Histidine tag), one can purify the molecules using this tag. However, in certain embodiments, it is preferred to use affinity to purify the molecules.

[0320] One can, in particular, use the fact that the molecule herein produced binds to a specific target and use any affinity method (affinity column, FACS,

[0321] 15 beads) to isolate such molecules.

[0322] One particular advantage of the molecules herein disclosed is that they don’t need to be glycosylated to be active and can thus be produced in any type of cells, and not necessarily eukaryotic cells. They are particularly well produced in bacterial cells.

[0323] 20

[0324] TARGET PRODUCTION

[0325] A preferred target structure according to the invention are viral vectors, particularly AAVs. Viral vector production is known to the person skilled in the art. In the following, the production of AAV vectors is described in more detail as an example.

[0326] Suitable compositions of the liquid sample comprising AAV vectors, the washing buffers and the elution buffers, as well as the general conditions for performing the separation are well known in the art of affinity

[0327] 30 chromatography. For instance, AAV vectors can be produced in various cell lines in adherent or suspension cell culture formats using transient transfection or co-infection methods. Preferably, AAV vectors are produced -347

[0328] - 38 - in suspension cell lines. Depending on specific serotypes and production times, viral particles including full, partial and empty species can be secreted out of cells into culture medium or contained inside cells at various ratios.

[0329] Initially, stable AAV producer cells were generated by transfection and

[0330] 5 selection of human-derived cells, like HeLa or HEK293 cells, with an rAAV transfer vector containing the ITR cassette and a packaging construct containing Rep and Cap. Production of recombinant AAV vectors (rAAV) was then achieved by infection with auxiliary viruses such as adenoviruses (AdV) that provide the helper function. After identification of AdV genes required for AAV vector packaging, a helper virus-free method was established using a duo or triple transfection protocol consisting of two or three plasmids including a constructed helper plasmid instead of an auxiliary virus. This system is widely used in research and drug development. In addition, development of baculovirus expression vectors provides another method to

[0331] 15 produce rAAV viruses in insect Sf9 cells. These different technologies are shown to be able to produce sufficient quantities of rAAV viruses for use in laboratories and clinical trials.

[0332] A cell lysis step is generally required at harvest to release viral particles into

[0333] 20 the supernatant if the viral particles are not secreted by the cells. Suitable methods are known to the experts. For chemical lysis, typical cell lysis reagents in form of detergents such as Triton® X-100, Tween® 20, Deviron® 13-S9; Deviron® C16 or mixtures thereof can be used, optionally in combination with NaCI.

[0334] After cell lysis or secretion by the cells, the AAVs need to be purified. Typical AAV purification processes include clarification, concentration and diafiltration using tangential flow filtration, chromatography purification by using affinity chromatography and ion exchange chromatography. In some

[0335] 30 processes, ultracentrifugation and gradient ultracentrifugation are used instead of chromatography or in addition to chromatography. Final steps in -347

[0336] - 39 -

[0337] AAV purification typically involve concentration and diafiltration into suitable excipient buffer composition and sterile filtration.

[0338] In the following an exemplary process is described in more detail.

[0339] 5 Typically, the selected cells are expanded in suitable culture media in a bioreactor under suitable conditions. Typically, the cells are grown as suspension culture. For example, in suspension culture of HEK293 cells suitable seeding numbers before transfection are 0.5 to 1 .1 10A6 viable cells per ml.

[0340] Suitable methods for the transduction are known in the art. In one embodiment, cells can be transduced in vitro by combining a rAAV with the cells, e.g., in appropriate media, and screening for those cells harboring the DNA of interest using conventional techniques such as Southern blots and / or

[0341] 15 PCR, or by using selectable markers.

[0342] Transfection can be performed using any of the techniques known in the art, including but not limited to electroporation, lipofection, e.g. with a lipofectamine, cationic polymers and cationic lipids. Any suitable transfection media may be used. In one embodiment of the transfection process,

[0343] 20 adherent or suspension human embryonic kidney (HEK293) cells are transfected with a dual or triple DNA plasmid polyethylenimine (PEI) coprecipitation.

[0344] After a suitable virus production period post transfection or infection, the cells are optionally lysed and the viral particles, like AAVs or any other viral particles that have been produced, are harvested. In some embodiments, the cells are dissociated from the bioreactor before the cell lysis process is initiated. In some embodiments, the cells are lysed in situ.

[0345] The released viral particles can then be isolated and / or purified, whereby

[0346] 30 preferably a chromatographic purification on an affinity matrix is included. -347

[0347] - 40 -

[0348] Preferably, the mixture obtained from the cell culture or after lysis is first filtered or centrifuged.

[0349] In one embodiment the mixture is filtered through a filter that removes large molecule contaminants and cellular debris but that permits viral particles to

[0350] 5 pass therethrough.

[0351] In a preferred embodiment, the released viral particles can be separated and purified from the cell culture medium using clarification. Clarification is a process, which includes at least one filtration step. It might involve one or more steps selected from centrifugation, tangential flow filtration, depth filtration, and sterile filtration. Clarification is a process in which relatively larger components such as cells, lysed cells, cellular debris, protein aggregates and other bulky mainly process related impurities are removed from a solution, typically the solution resulting from culturing cells and

[0352] 15 optionally lysing them. Clarification filters include depth filtration, charged depth filtration and similar microfiltration techniques. The resulting sample is a clarified sample.

[0353] Tangential flow filtration can be used to concentrate the mixture of purified

[0354] 20 viral particles and to remove salts and process related contaminants like proteins or nucleic acids. Tangential flow filtration (TFF) refers to a generally rapid and efficient method for filtration or purification of a solution containing target product and / or impurities during which a solution or liquid stream flows parallel to a filtering membrane.

[0355] Centrifugation can, for example, be a low-speed centrifugation to remove larger particles like cellular debris. This can be for example done at 10000 to 12000 g for 10 to 30 minutes. The released viral particles can be found in the supernatant.

[0356] 30 In some embodiments, a nuclease, typically an endonuclease, is added, e.g. to reduce the amount of host cell DNA. It can be added directly to the mixture in the bioreactor before, while or after lysis. The nuclease may be one that -347

[0357] - 41 - degrades both DNA and RNA. In one embodiment, the endonuclease is a genetically engineered endonuclease from Serratia marcescens that is sold under the name Benzonase(R) (EMD Millipore, US). If the activity of the nuclease depends on the presence of certain divalent cations it might be

[0358] 5 favourable to do the nuclease treatment separately, e.g. prior to the addition of a chelator.

[0359] AFFINITY PURIFICATION

[0360] The affitin ligands of the invention are, particularly useful for the isolation and / or purification of a target structure by e.g., affinity chromatography from e.g., clarified cell culture solutions or other natural sources such as biological samples (e.g., serum). In purification based on affinity chromatography, a target of interest (e.g. protein or molecule) is selectively isolated according to its ability to specifically and reversibly bind to a ligand that has typically

[0361] 15 been covalently coupled to a chromatography matrix.

[0362] It has surprisingly been found that the affitin ligands according to the invention conjugated to e.g., a chromatography support / matrix allow the selective binding as well as subsequent elution of target structures while

[0363] 20 tolerating treatment with caustic reagents or treatment under caustic conditions useful for e.g., elution of the target structure and / or washing / sanitising the chromatography matrix.

[0364] Accordingly, a further aspect of the present invention refers to an affitin ligand of the invention conjugated to a chromatography matrix useful for separating a target structure from a liquid sample by affinity chromatography.

[0365] In some embodiment, the chromatography matrix is a chromatographic resin. In some embodiment, the chromatography matrix is a chromatographic membrane. In certain embodiments, the chromatography matrix is a

[0366] 30 hydrophilic polymer. -347

[0367] - 42 -

[0368] A further aspect relates to an affinity separation matrix comprising the affitin ligand according to invention.

[0369] The terms "purifying," "separating," or "isolating," are used interchangeably herein and refer to increasing the degree of purity of the target viral particles

[0370] 5 from a composition or sample comprising the target structure and one or more impurities.

[0371] The term "chromatography" refers to any kind of technique which separates an analyte of interest (e.g. a target viral particle) from other molecules present in a sample. Usually, the target structure is separated from other molecules as a result of differences in rates at which the individual molecules of the mixture bind to and / or migrate through a chromatography matrix under the influence of a moving liquid phase, also called mobile phase.

[0372] 15 The term "matrix", "chromatography matrix", or “chromatography support” are used interchangeably herein and refers to a solid phase through which the sample migrates in the course of a chromatographic separation. The matrix typically comprises a base material and ligands covalently bound to the base material. The matrix of the present invention may comprise or

[0373] 20 consists e.g. of resin particles, particles, a membrane or a monolith. Suitable base materials are described herein or otherwise known in the art.

[0374] Preferably, the affinity chromatography matrix of the invention comprises or consists of:

[0375] (a) a porous base material having a surface comprising a plurality of functional groups, and

[0376] (b) a plurality of affitin ligands bound to said functional groups of the base material.

[0377] 30 Preferably, the affitin ligands are covalently bound to the porous base material, that means covalent bonds are formed between the affitin ligands and the functional groups on the e.g. membrane, resin particle or monolith. -347

[0378] - 43 -

[0379] Coupling may be realized via one or more functional groups on the affitin ligand, like functional groups at the N-terminus, the C-terminus or at any other position within the peptide sequence, preferably coupling is realized via one or more functional groups at the C-terminus.

[0380] 5

[0381] The affitin ligands according to the invention may be attached to the base material of the matrix by any type of covalent attachment. Methods of covalently coupling polypeptides to a surface are known by those of skill in the art, and peptide tags that can be used to attach a ligand to a matrix are known to those of skill in the art. Further, ligands can be attached (i.e., coupled, linked, or adhered) to a solid surface using any reagents or techniques known in the art. A detailed overview of techniques as well as protocols suitable to attach ligands on base materials can be found in Greg T. Hermanson, Bioconjugate Techniques, Third Edition, Academic Press, 2013, especially in Chapter 15 (Greg T. Hermanson, Chapter 15 -

[0382] 15 Immobilization of Ligands on Chromatography Supports, Editor(s): Greg T. Hermanson, Bioconjugate Techniques (Third Edition), Academic Press, 2013, Pages 589-740, ISBN 9780123822390, https: / / doi.org / 10.1016 / B978- 0-12-382239-0.00015-7). Covalent attachment can for example be performed by directly bonding the functional groups of the affitin ligand to

[0383] 20 suitable functional groups on the base material like OH, NH2, carboxyl, phenol, anhydride, aldehyde, epoxide, vinyl, halogen or thiol etc. It is also possible to attach the ligands via suitable linkers. Most preferred are base materials that are functionalized with hydroxy, amino, carboxyl, aldehyde or epoxy groups. The affitin ligand may comprise any functional group or plurality of functional groups suitable to attach the ligand by covalent bonding to the functional groups on the matrix, preferably via a single point attachment. The functional group may be located at any position within the ligand. Preferably it is located close to (one of the last 5 amino acids) or at the N- and / or C-terminus of the ligand, most preferred it is located close to

[0384] 30 or at the C-terminus of the ligand. Preferably, the attachment of the affitin ligands to the base material is performed by reacting affitin ligands -347

[0385] - 44 - comprising at least one thiol- or at least one amino-group with a base material that comprises epoxy groups.

[0386] In one embodiment the chromatography matrix comprises affitin ligands that are attached to the base material via reductive amination generating a

[0387] 5 secondary amine bond between the ligand and the base matrix whereby an amino group that is preferably located at the C-terminus of the affitin ligand is reacted with an aldehyde group on the base material. In another embodiment, the chromatography matrix comprises affitin ligands that are attached to the base material via reacting thiol group that is preferably located at the C-terminus of the affitin ligand is reacted with an aldehyde group on the base material. If the affitin ligand comprises more than one functional group suitable for attaching it to the base material, typically the resulting chromatography matrix will comprise ligands that are attached via functional groups that are located at different positions within the affitin

[0388] 15 ligand. The presence of more than one suitable functional group at the C- terminus will increase the percentage of ligands attached via the C-terminus. The skilled person can adjust the modification of the C-terminus to achieve the most preferred way of ligand attachment.

[0389] 20 It is also possible to generate the matrix by polymerizing monomers comprising the affitin ligands and a polymerizable moiety. Examples of matrices generated by polymerization of suitable monomers are polystyrene, polymethacrylamide or polyacrylamide-based matrices generated by polymerizing suitable styrole or acryloyl monomers.

[0390] In another embodiment the chromatography matrix can be generated by grafting the affitin ligands onto the base material or from the base material. For grafting from processes with controlled free-radical polymerisation, such as, for example, the method of atom-transfer free-radical polymerisation

[0391] 30 (ATRP), are suitable. A very preferred one-step grafting from polymerisation reaction of acrylamides, methacrylates, acrylates, methacrylates etc. which are functionalized e.g. with ionic, hydrophilic or hydrophobic groups can be -347

[0392] - 45 - initiated by cerium (IV) on a hydroxyl-containing support, without the support having to be activated.

[0393] Particulate base materials, also called resins, can be prepared, for example, from organic polymers. Organic polymers of this type can be

[0394] 5 polysaccharides, such as agarose, dextranes, starch, cellulose, etc., or synthetic polymers, such as poly(acrylamides), poly(methacrylamides), poly(acrylates), poly(methacrylates), hydrophilically substituted poly(alkyl allyl ethers), hydrophilically substituted poly(alkyl vinyl ethers), poly(vinyl alcohols), poly(styrenes) and copolymers of the corresponding monomers. These organic polymers can preferably also be employed in the form of a crosslinked hydrophilic network. This also includes polymers made from styrene and divinylbenzene, which can preferably be employed, like other hydrophobic polymers, in a hydrophilized form.

[0395] 15 Alternatively, inorganic materials, such as silica, zirconium oxide, titanium dioxide, aluminium oxide, etc., can be employed as particulate base materials. It is equally possible to employ composite materials, i.e., for example, particles which can themselves be magnetised by copolymerisation of magnetisable particles or of a magnetisable core. It is also possible to use

[0396] 20 core shell materials whereby the shell, i.e. at least the surface or a coating, has OH groups.

[0397] However, preference is given to the use of hydrophilic base materials which are stable to hydrolysis or can only be hydrolysed with difficulty since the chromatography matrixes according to the invention should preferably withstand alkaline cleaning or regeneration at e.g. basic pH over an extended use duration.

[0398] The base matrix may consist of irregularly shaped or spherical particles, whose particle size can be between 2 and 1000 pm. Preference is given to

[0399] 30 average particle sizes between 3 and 300 pm, in a most preferred embodiment the average particle size is between 20 - 63 pm. -347

[0400] - 46 -

[0401] The particulate base material may, in particular, be in the form of non-porous or preferably porous particles. The average pore sizes can be between 2 and 300 nm. Preference is given to pore sizes between 5 and 200 nm, most preferred average pore size is between 40 - 110 nm.

[0402] 5

[0403] In a very preferred embodiment, the particulate base material is formed by copolymerisation of a hydrophilically substituted alkyl vinyl ether selected from the group of 1 ,4-butanediol monovinyl ether, 1 ,5-pentanediol monovinyl ether, diethylene glycol monovinyl ether or cyclo-'hexane-'dimethanol monovinyl ether and divinylethyleneurea (1 ,3-divinylimidazolin-2-one) as crosslinking agent.

[0404] An example of a suitable commercially available vinylether based base material is Eshmuno®, Merck KGaA, Germany.

[0405] 15 A membrane or membrane matrix according to the invention can be distinguished from particle-based chromatography systems by the fact that the interaction between a fluid sample, e.g. the target structure and / or contaminants, and the matrix does not take place in the pores of a particle, but mainly in the throughpores of the membrane. Accordingly, in certain

[0406] 20 embodiments, the affitin membrane comprises or consists of a porous membrane matrix having a surface comprising a plurality of functional groups. Exemplary types of membranes according to the invention are flat sheet systems, stacks of membranes, microporous polymer sheets with incorporated cellulose, polystyrene or silica-based membranes as well as radial flow cartridges, hollow fiber modules and hydrogel membranes. Membranes may also be produced by 3D printing processes.

[0407] In certain embodiments, the porous membrane matrix comprises a polymeric support impregnated with a (crosslinked) hydrogel network, also known as

[0408] 30 hydrogel membranes. In a preferred embodiment, the porous membrane matrix comprises or consists of a macroporous polymeric support - 47 - impregnated with a crosslinked hydrogel network and the plurality of functional groups is present on the surface of the hydrogel network.

[0409] Such membranes thus comprise a membrane polymeric support and a

[0410] 5 hydrogel formed within the pores of said support. The membrane polymeric support provides mechanical strength to the hydrogel. The hydrogel determines the properties of the final product, like pore size and binding chemistry. The membrane polymeric support can consist of any porous membrane like polymeric membranes, ceramic based membranes and woven or non-woven fibrous material. Suitable polymeric materials for membrane polymeric supports are cellulose or cellulose derivatives as well as other preferably inert polymers like polyethylene, polypropylene, polybutylenterephthalate or polyvinylidene-difluoride.

[0411] The hydrogels can be formed through in-situ reaction of one or more

[0412] 15 polymerizable monomers with one or more crosslinkers and / or one or more cross-linkable polymers to form a cross-linked gel that has preferably macropores. Suitable polymerizable monomers include monomers containing vinyl or acryl groups. Preferred are monomers comprising an additional functional group that either directly forms the ligand of the matrix

[0413] 20 or is suitable for attaching the ligands. Suitable crosslinkers are compounds containing at least two vinyl or acryl groups. In a preferred embodiment, the hydrogel network comprises or consists of a polyacrylamide or copolymers of acrylamide formed in situ within the support.

[0414] Further details about suitable polymeric supports, monomers, crosslinkers etc. as well as suitable production conditions can be found in WO04073843 and WO2010 / 027955. WO2014134147 discloses membranes mixed mode membranes that optionally comprise more than one group like hydrophobic groups, hydrophilic groups or ion exchange groups. Especially preferred are

[0415] 30 membranes made of an inert, flexible fiber web support comprising assembly within and around the fiber web support a porous polyacrylamide hydrogel - 48 - comprising hydroxyl groups like Natrix® type membranes, Merck KGaA, Germany.

[0416] Examples of suitable membranes of the present invention are

[0417] 5

[0418] - Membranes with a polyethersulfone (PES)-based support and a crosslinked polymeric coating, functionalized with suitable ligands, like Mustang® type membranes, Pall.

[0419] - Membranes made of stabilized reinforced cellulose, functionalized with suitable ligands, like Sartobind® type membranes, Sartorius.

[0420] - Membranes made of stabilized reinforced cellulose, comprising a hydrogel with suitable ligands, like Sartobind® Jumbo Membranes, Sartorius, made of stabilized reinforced cellulose

[0421] 15

[0422] - Membranes made of a fine fiber non-woven scaffold comprising a hydrogel with suitable ligands, like 3MTMEmphaze™ Hybrid Purifier type membranes, 3M.

[0423] 20 - Membranes made of an inert, flexible fiber web support comprising within and around the fiber web support a porous polyacrylamide hydrogel with suitable cation exchange and hydrophobic ligands , like Natrix® type Chromatography membranes, Merck KGaA, Germany.

[0424] The membranes to be used in the present invention preferably have mean flow pore diameters of 0.3 to 2 pm, more preferably, between 0.6 and 1.2 pm. Mean Flow Pore (MFP) Diameter refers to the average diameter of the pores in a membrane through which fluid can flow. MFP diameter is determined by capillary flow porometry which is used to construct a pore size

[0425] 30 distribution curve that can then be used to determine the mean flow pore diameter. -347

[0426] - 49 -

[0427] The membranes may have different sizes and formats depending on the application or the equipment that is used. Typically, the thickness of the membranes is between 10 and 2000 pm, preferably between 50 and 500 pm, most preferred between 150 and 250 pm.

[0428] 5

[0429] A monolith or a monolithic sorbent, similar to a membrane, has throughpores, like interconnected channels, so that liquid can flow from one side of the monolith, through the monolith, to the other side of the monolith.

[0430] Since the mobile phase is flowing through these throughpores, molecules to be separated are transported by convection rather than by diffusion. Due to their structure monolithic sorbents show flow rate independent separation efficiency and dynamic capacity.

[0431] The monolith is typically formed in situ from reactant solutions and can have

[0432] 15 any shape or confined geometry, typically with frit-free construction, which guarantees convenience of operation. Preferably, monolithic materials have a binary porous structure, mesopores and macropores. The micron-sized macropores are the throughpores and ensure fast dynamic transport and low backpressure in applications; mesopores contribute to sufficient surface area

[0433] 20 and thus high loading capacity.

[0434] The monoliths can be made of organic, inorganic or organic / inorganic hybrid materials. Preferred are organic polymer-based monoliths.

[0435] The synthesis of organic polymer monoliths is typically done by a one-step polymerization providing a tunable porous structure with tailored functional groups. Generally, a pre-polymerization mixture consisting of the monomers, crosslinkers, porogenic solvents, and initiators in an appropriate ratio is polymerized in a suitable container, also called mould, determining the format

[0436] 30 of the monolith. Polymerization is typically initiated by heating, use of UV radiation, microwave or y-ray radiation in the presence of initiators. After reaction for the prescribed time at an appropriate temperature, the resulting -347

[0437] - 50 - material is typically washed with solvents to remove unreacted components and porogenic solvents.

[0438] Suitable organic polymers are polymethacrylates, polyacrylamides,

[0439] 5 polystyrenes, polyurethanes, etc., like Poly(methacrylic acid-ethylene dimethacrylate), Poly(glycidyl methacrylate-ethylene dimethacrylate) or Poly(acrylamide-vinylpyridine-N,N'-methylene bisacrylamide).

[0440] Inorganic monoliths can be made of silica or other inorganic oxides. Preferably they are made of silica. Silica monoliths are normally prepared via a sol-gel method with phase separation. This mainly includes hydrolysis, condensation, and polycondensation of silica precursors. Typically, tetraethoxysilane (TEOS) or tetramethylorthosilicate (TMOS) is distributed in a suitable solvent in the presence of a porogen (e.g. polyethylene glycol) (PEG)), followed by the addition of a catalyst, acid or base, or a binary

[0441] 15 catalyst, acid and base in sequence. After reaction for a prescribed time, the resulting gel-like product is washed with solvents to remove unreacted precursor, porogen, and catalyst, followed by the proper post treatment, typically a heat treatment.

[0442] 20

[0443] The monoliths can be modified with suitable functional groups, preferably at least ion exchange groups, to generate the targeted interaction with the sample comprising the target molecule and thus the targeted separation.

[0444] Membranes and monoliths can also be produced by 3D printing processes.

[0445] When the chromatography matrix is used in a chromatographic separation it is typically used in a separation device, also called column or housing, as a means for holding the matrix. Suitable columns or housings are known to the skilled person.

[0446] 30 The affitin ligand conjugated to the matrix may be packed in columns of various sizes and operated at various linear velocities or immobilized affitin -347

[0447] - 51 - ligand can be contacted with a solution under conditions favourable to form a complex between the ligand and the specific target structure. Non-binding materials can be washed away. Suitable wash conditions and buffers can readily be determined by one of skill in the art.

[0448] 5

[0449] According to the present invention the term “buffer” or “solvent” is used for any liquid composition that is used to load, wash, elute, re-equilibrate, strip and / or sanitize a chromatography matrix.

[0450] A "buffer" is a solution that resists changes in pH by the action of its acidbase conjugate components. Various buffers which can be employed depending, for example, on the desired pH of the buffer are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975). Non- limiting examples of buffers include MES, MOPS, MOPSO, Tris, HEPES, phosphate,

[0451] 15 acetate, citrate, succinate, and ammonium buffers, as well as combinations of these.

[0452] It has surprisingly been found that higher ligand densities on a chromatographic matrix, preferably a resin or a membrane, can result in

[0453] 20 improved caustic stability of affitin ligands. As used herein, the term “ligand density” refers to the amount of affitin ligand immobilized on the chromatography matrix, normalised to the volume of the chromatography matrix. In case of resin particles, it is normalized to the settled resin bed volume, in case of membranes it is normalized to the membrane volume and typically expressed as milligrams of ligand per millilitre of settled resin or membrane volume (mg / ml).

[0454] In some embodiments, the ligand density of the chromatography matrix of the present invention, preferably resin or membrane, is at least about 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9

[0455] 30 mg / ml, 10 mg / ml, 12 mg / ml, 15 mg / ml, or 20 mg / ml. Ligand densities between about 4 mg / ml and 10 mg / ml are preferred. In further embodiments, -347

[0456] - 52 - the matrix comprising the ligands of the present invention exhibits improved caustic stability relative to lower ligand densities, as measured by retention of a higher binding capacity compared to matrices with lower ligand density and / or other ligand structure following incubation or regeneration with caustic

[0457] 5 cleaning solutions such as sodium hydroxide or potassium hydroxide under the conditions described herein. Preferably the matrices of the present invention retain at least about 70%, 80%, 90%, or 95% of binding capacity following incubation or regeneration with caustic cleaning solutions such as sodium hydroxide or potassium hydroxide under the conditions described herein.

[0458] USE OF AFFITIN LIGANDS

[0459] A further aspect of the invention refers to the use of the affitin ligands according to the invention for affinity purification of a target structure.

[0460] 15

[0461] In certain embodiments, the invention refers to the use of the affitin ligands for affinity purification of a target structure, wherein the affitin ligands retain binding functionality / capacity after repeated exposure and / or incubation to caustic reagents, such as sodium hydroxide (NaOH) or potassium hydroxide

[0462] 20 (KOH), during cleaning-in-place (CIP) or regeneration processes typically employed in the maintenance of chromatography matrices.

[0463] As used herein, the term “regeneration” or “regeneration cycle” refers to a cleaning procedure in which the chromatography matrix or solid support to which an affitin ligand is coupled is contacted with a caustic solution (e.g., NaOH or KOH) under conditions sufficient to remove bound impurities, sanitize the matrix, and restore binding performance for subsequent cycles of use. A regeneration cycle typically comprises (i) incubation of the ligandcontaining matrix with the caustic solution for a defined time (e.g., 5 to 60

[0464] 30 minutes), followed by (ii) rinsing or equilibration of the matrix to restore appropriate operating conditions. -347

[0465] - 53 -

[0466] As used herein, the term “exposing to caustic reagents” refers to bringing the affitin ligand, or a matrix comprising the affitin ligand, into contact with a caustic solution under conditions effective to achieve a desired purpose, such as regeneration, cleaning, sanitization, or elution of a bound target

[0467] 5 molecule. Caustic solutions are solutions of e.g. sodium hydroxide, potassium hydroxide, lithium hydroxide, caesium hydroxide, or mixtures thereof in water, buffers or aqueous solutions optionally comprising one or more organic solvents like ethanol or isopropanol. Such caustic solutions typically have a pH ranging from 12 to 14 and comprise between 0.01 M and 2 M of sodium hydroxide and / or other suitable bases. “Exposing” encompasses both brief and prolonged contact and does not require maintenance of constant conditions (e.g., temperature, concentration, or mixing) throughout the exposure period. Thus, in some embodiments, “exposing” may comprise a wash, rinse, or flow-through treatment in which

[0468] 15 the caustic reagent is applied dynamically.

[0469] Accordingly, “exposing” may be used to describe operational conditions in processes such as column regeneration, sanitization, or elution where flow- through or cycling may occur, while “incubating” may be used to describe

[0470] 20 laboratory or test conditions designed to measure the chemical stability of the affitin ligands under prolonged caustic challenge.

[0471] In some embodiments, the affitin ligands retain at least about 95%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% of their binding capacity after exposure to one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty-five, thirty, forty, fifty, seventy-five, one hundred, one hundred fifty, two hundred, three hundred, five hundred, seven hundred, or one thousand regeneration cycles with caustic reagents such as sodium hydroxide (NaOH) or potassium hydroxide (KOH) at concentrations of about 0.01 M, 0.05 M, 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M, 0.5 M, 0.75 M, 1.0

[0472] 30 M, 1 .25 M, 1 .5 M, 1 .75 M, or 2.0 M. In certain embodiments, the affitin ligands retain at least about 90%, 80%, 70%, 60%, 50%, or 40% of their binding -347

[0473] - 54 - capacity after one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty-five, thirty, forty, fifty, seventy-five, or one hundred regeneration cycles with sodium hydroxide at concentrations of about 0.05 M, 0.1 M, 0.2 M, 0.25 M, 0.3 M, 0.4 M, 0.5 M, 0.75 M, or 1.0 M.

[0474] 5

[0475] In further embodiments, the affitin ligands retain their functionality when exposed to other caustic cleaning agents commonly used in column regeneration and sanitation procedures, including LiOH, CsOH, or mixtures thereof, under similar concentration ranges and regeneration cycles as described herein.

[0476] In certain preferred embodiments, the affitin ligands retain at least 70%, preferably at least 80% of their binding capacity after 10 or more regeneration cycles with sodium hydroxide at a concentration between 0.05 M and 0.5 M, most preferred at a concentration of 0.5 M.

[0477] 15

[0478] In some embodiments, treatment with caustic reagents may also be employed for elution of the bound target molecule from the affitin ligand. For example, after affinity capture of a target protein, nucleic acid, or other biomolecule, the matrix comprising the affitin ligand may be contacted with a

[0479] 20 solution comprising sodium hydroxide, potassium hydroxide, or other caustic reagents under conditions sufficient to disrupt ligand-target interactions and thereby release the bound target. In certain embodiments, elution may be performed using caustic solutions at concentrations between about 0.01 M and 0.5 M, optionally in combination with other salts or buffers. In some embodiments, elution with caustic reagents may be combined with or followed by regeneration of the matrix under similar conditions. Advantageously, the affitin ligands of the invention retain at least about 95%, 90%, 80%, 70%, 60%, or 50% of their binding capacity following such caustic elution steps, thereby enabling repeated cycles of binding, elution, and

[0480] 30 regeneration without substantial loss of functionality.

[0481] METHOD FOR AFFINITY CHROMATOGRAPHY -347

[0482] - 55 -

[0483] Another aspect of the present invention relates to an affinity chromatography method for separating / isolating a target structure, wherein an affitin ligand conjugated to a chromatography matrix as disclosed above is utilized.

[0484] In certain embodiments, the method comprises, consists, or essentially

[0485] 5 consists of the steps of:

[0486] (a) providing a liquid sample comprising a target structure, an affitin ligand conjugated to a chromatography matrix according to the invention, at least one wash buffer, and at least one elution buffer,

[0487] (b) contacting said liquid sample with the chromatography matrix,

[0488] (c) optionally but preferably washing the chromatography matrix with at least one washing buffer,

[0489] 15 (d) eluting the target structure from the chromatography matrix with at least one elution buffer, and

[0490] (e) optionally but preferably cleaning the chromatography matrix with a caustic solution, in this case a caustic cleaning liquid.

[0491] 20 According to the present invention the term “buffer” or “solvent” is used for any liquid composition that is used to load, wash, elute, re-equilibrate, strip, clean and / or sanitize a chromatography matrix.

[0492] As used herein, the term “caustic cleaning liquid” refers to a caustic solution comprising one or more caustic reagents suitable for cleaning, sanitising, or regenerating the chromatography matrix following use. In some embodiments, the caustic cleaning liquid comprises sodium hydroxide (NaOH), potassium hydroxide (KOH), lithium hydroxide (LiOH), cesium hydroxide (CsOH), or mixtures thereof. Most preferred it comprises NaOH. The caustic cleaning liquid may be applied at concentrations ranging from

[0493] 30 about 0.01 M to about 2.0 M of the one or more bases, and may be used under conditions sufficient to remove bound material, reduce microbial -347

[0494] - 56 - contamination, or restore binding capacity of the matrix. In certain embodiments, cleaning with the caustic liquid may be conducted for a brief exposure (e.g., seconds to minutes) or for longer incubation periods (e.g., 30 minutes to several hours), under static or gently agitated conditions at a

[0495] 5 temperature of about 20 °C to 25 °C, unless otherwise specified. In some embodiments, the caustic cleaning liquid may additionally be employed as part of an elution step to release the bound target structure from the matrix.

[0496] The affinity chromatography method for isolating / separating a target structure according to the invention may be performed in bind-elute mode. When doing affinity chromatography, the target structure is bound to the affinity chromatography matrix while the impurities do not bind and flow through. After optional washing of the matrix-bound target structures, the structures are eluted from the matrix with a suitable elution buffer.

[0497] 15 In some embodiments, the method for isolation of the target structures may include one or more of the following process steps: clarification filtration

[0498] 20 dialysis / diafiltration tangential flow filtration treatment with nuclease, e.g. RNase and / or DNase treatment with chloroform ion exchange chromatography affinity chromatography

[0499] 30 multimodal chromatography hydrophobic interaction chromatography -347

[0500] - 57 - centrifugation ultracentrifugation

[0501] (PEG) precipitation

[0502] 5 flocculation

[0503] In a preferred embodiment, the ffinity chromatography method according to the present invention may be performed by:

[0504] (a) providing an affitin ligand of the invention conjugated to a chromatography matrix targeted against a desired target structure.

[0505] (b) loading the (liquid) sample comprising the target structure, if present a chelator and / or process related impurities, onto the affitin chromatography matrix of step a) under conditions that allow binding

[0506] 15 between the target structure and the affinity chromatography matrix;

[0507] (c) undertaking at least one wash step; and

[0508] (d) eluting the target structure from the affinity chromatography matrix.

[0509] 20 Preferably, the affinity chromatography further comprises step (e): cleaning the chromatography matrix with a caustic cleaning liquid.

[0510] Suitable conditions and buffers for affinity chromatography are known to the skilled person. They might vary depending on the matrix that is used and the target structure. It is aware to the skilled person that one or more buffers, typically 1 to 4 buffers, can be used for equilibration, washing and elution.

[0511] Preferably, in step a), prior to loading the target structure to the affinity chromatography matrix, the matrix is conditioned (equilibrated) with a

[0512] 30 suitable starting buffer to facilitate binding between the target structure and the affinity chromatography matrix. Typically, the starting buffer has a pH -347

[0513] - 58 - between 6 and 9. In one embodiment the starting buffer, also called equilibration buffer, is the same buffer as the loading buffer.

[0514] The loading of the (liquid) sample comprising the target structure onto the

[0515] 5 affinity matrix is performed at a pH, which is preferably in the range between 6 and 9. PBS pH 7.0 to 7.5 is a preferred loading buffer. However, other standard neutral pH buffers such as 10-50 mM sodium phosphate or Tris can also be used. Adding 0.1 -0.5 M NaCI or KCI may prevent nonspecific adsorption due to protein / protein interactions.

[0516] Suitable conditions and buffers to undertake one or more wash steps, preferably 1 to 3 wash steps, might vary between the pH 5 and 9. PBS pH 7.0 to 7.5 is a preferred wash buffer. However, other standard neutral pH buffers such as 10-50 mM sodium phosphate or Tris can be used. Adding 0.05 to 0.5 M NaCI and / or KCI and / or chelator and / or detergents / surfactants

[0517] 15 and / or amino acids may prevent nonspecific adsorption due to protein / protein interactions and improve impurity removal.

[0518] Conditions and buffers to elute the target structure from the affinity chromatographic matrix may vary. A preferred elution buffer typically

[0519] 20 comprises a pH in the range between 2 and 3. Glycine buffer at a pH of 2.5 is a preferred elution buffer. However, other buffers in a pH range of 2 to 3, such as 50-100 mM citrate or phosphate can also be used. Adding 0.1 -0.5 M NaCI or KCI and / or chelator and / or detergents / surfactants and / or amino acids may enhance elution efficiency of the target.

[0520] Suitable flow rates for all process steps for pumping the respective buffers through the matrix are between 1 MV or CV / min and 20 MV or CV / min, preferred are flow rates of 8-10 MV or CV / min.

[0521] The term “column volume” (CV) refers to the total volume of the porous

[0522] 30 structure of the matrix inside of a packed column or generally packed housing through which the mobile phase and target structures can flow and interact. -347

[0523] - 59 -

[0524] If the matrix is a membrane the column volume is typically called the membrane volume (MV).

[0525] In one embodiment, for equilibration, washing and elution, 5 to 50

[0526] 5 column / membrane volumes (CV / MV) of the respective buffers may be applied.

[0527] In another embodiment, another chromatographic method is used in combination with affinity chromatography. Suitable chromatographic methods are for example ion exchange chromatography or multimodal chromatography. In one embodiment, only a cation- or only an anion exchange or only a multimodal chromatography is performed prior or after affinity chromatography. In another embodiment first an affinity chromatography and then at least one other chromatography selected from

[0528] 15 anion exchange, cation exchange or multimodal chromatography is performed.

[0529] BRIEF DESCRIPTION OF THE DRAWINGS

[0530] 20

[0531] Figure 1 shows UV signal curve tracked at 280nm of chromatography run for affitin ligand NF03 specifically binding AAV5.

[0532] Figure 2 shows UV signal curve tracked at 280nm of chromatography run for affitin ligand NF36 specifically binding AAV5.

[0533] Figure 3 shows chromatogram of NF13 barring prototype P1 prior and after incubation in 0.5M NaOH for 150 minutes

[0534] Figure 4 shows chromatogram of NF38 barring prototype P2 prior and after incubation in 0.5M NaOH for 150 minutes.

[0535] 30

[0536] Figure 5 shows chromatogram of AAV9 from clarified lysate with affinity ligand conjugated on membrane base material after caustic sanitization; AAV -347

[0537] - 60 - capture run after initial membrane sanitization with 0.1 M NaOH for 10 min shown in solid line and after 0.5 M NaOH for 30 min shown in dotted line

[0538] 5

[0539] 15

[0540] 20

[0541] 30 -347

[0542] - 61 -

[0543] EXAMPLES

[0544] Example 1 : Dynamic binding (association) of AAV5 to immobilised affinity ligand to chromatographic stationary phase

[0545] 5

[0546] The performance of covalently immobilized affinity ligands (NF03: SEQ ID NO: 33 and NF36: SEQ ID NO: 31 ) was demonstrated in a dynamic purification process. Therefore, epoxy activated Eshmuno® resin was modified with the purified affinity ligand. The modification of epoxy activated Eshmuno® resin was carried out in phosphate buffer with addition of salts at pH 9.0 at 40°C temperature for 6 hours in the ratio of 8mg affinity ligand quantity for the 1 ml of Eshmuno® resin after reducing the dimeric polypeptides with DTT for at least 30 minutes (referring to the initial coupling protocol).

[0547] 15 After modification, the obtained affinity ligand baring Eshmuno® resin is washed to remove non-covalently bound ligand and stored in 20% ethanol and 150mM NaCI containing solution. The affinity ligand density was calculated to be 5.09 mg / ml for NF03 Eshmuno® resin and 6.91 mg / mL for NF36 Eshmuno® resin. The Eshmuno® NF03 and Eshmuno® NF36 affinity

[0548] 20 ligand baring resins were used to manufacture two 0.196 mL column prototype with 0.5cm x 1 cm column dimensions. The dynamic binding performance was each measured by loading 40 mL of clarified HEK293T cell culture containing 5.0 x 10A12vp / mL AAV5 capsids concentration to the resin prototype. The AAV5 capsids present in the clarified HEK293T cell culture could be bound within neutral pH conditions (PBS, pH 7.4) to the equilibrated resin, followed by an additional wash step (PBS, pH 7.4). The pH induced elution took place using 50mM Glycine, 150mM NaCI, 0.001 % Pluronic, pH 2.5. The NF03 resin was able to bind and elute 8.30 x 10A14vp / mL with a respecting 98% elution step recovery. The NF36 resin was able to bind and

[0549] 30 elute 9.01 x 10A14vp / mL with a respecting 104% elution step recovery. The dynamic binding capacities at 10% breakthrough were 4.96 x 10A14vp / mL -347

[0550] - 62 - for the NF03 resin and 5.36 x 10A14vp / mL for the NF36 resin. The overall recovery of all AAV5 particles was calculated to be 96% (NF03) and 106% (NF36). Further, the resins have been sanitized and cleaned using 25 column volumes Strip buffer (100mM Tris, 2M NaCI) followed by 25 column volumes

[0551] 5 CIP buffer (0.1 M NaOH).

[0552] To determine if the binding capacity of the affinity ligand baring Eshmuno® resins is affected by harsh cleaning conditions, the resins were incubated with 0.1 M NaOH for 150 min without flow. Afterwards, the dynamic binding performance was measured again using 40 mL of clarified HEK293T cell culture containing 5.0 x 10A14vp / mL AAV5 capsids concentration to the resin prototype using the same protocol as described. After the 0.1 M NaOH treatment, the NF03 resin was able to bind and elute 7.93 x 10A14vp / mL with a respecting 90% elution step recovery. The NF36 resin was able to bind and elute 8.76 x 10A14vp / mL with a respecting 108% elution step recovery. The

[0553] 15 dynamic binding capacities at 10% breakthrough were 4.89 x 10A14vp / mL for the NF03 resin and 3.92 x 10A14vp / mL for the NF36 resin. The overall recovery of all AAV5 particles was calculated to be 107% (NF03) and 108% (NF36). The remaining binding capacity at 10% breakthrough, compared to before the treatment with 0.1 M NaOH, were at 98.6% for the NF03 resin and

[0554] 20 73.1 % for the NF36 resin.

[0555] The belonging chromatography runs are demonstrated using the UV signal curve tracked at 280nm in Figure 1 (NF03, zoomed in), Figure 2 (NF36, zoomed in), whereas the AAV5 purification run protocol is summarized in Table 3 for all described runs and the yield values are summarized in Table 4.

[0556] Table 3: AAV5 dynamic purification protocol

[0557] 30 -347

[0558] -63-

[0559] 5

[0560] Table 4: AAV5 dynamic purification run summary

[0561] 30 -347

[0562] - 64 -

[0563] Example 2: Identification of caustic-stable affinity ligands for AAV8 binding

[0564] The affinity ligands

[0565] 5 Dynamic binding (association) of AAV8 to immobilized and 0.5 M NaOH treated affinity ligands

[0566] The caustic stability of covalently immobilized affinity ligands specifically binding AAV8, NF13 (SEQ ID NO: 36) and NF38 (SEQ ID NO: 37), was demonstrated in a dynamic purification process. Therefore, epoxy activated Eshmuno® resin was modified with purified affinity ligand as described earlier.

[0567] The affinity ligand density was calculated to be 4.28 mg / mL (NF13, prototype P1 ) and 4.79 mg / mL (NF38, prototype P2). The Eshmuno® affinity ligand

[0568] 15 baring resins were used to manufacture a 0.196 mL column prototype with 0.5cm x 1 cm column dimensions.

[0569] The dynamic binding performance was measured and calculated results are described earlier. The dynamic binding capacity at 10% breakthrough was calculated from flow through fractions to be 7.44x10A14 vp / mL for P1 and

[0570] 20 4.75x10A14 vp / mL for P2.

[0571] To determine if the dynamic binding capacity of the affinity ligand baring Eshmuno® resins is affected by harsh cleaning conditions, the resins were incubated with 0.5 M NaOH for 150 min without flow. Afterwards, the dynamic binding performance was measured again using 70 mL of clarified HEK293T cell lysate containing 4.5-4.7x10A12 vp / mL particle concentration to the resin prototype using the same protocol as described. After 0.5 M NaOH treatment, the NF13 resin was able to bind and elute 1.2 x10A15 vp / mL resin with 85% yield. The NF38 baring resin was able to bind and elute 1 x10A15 vp / mL resin with a respecting 80% yield, both similar results as measured before NaOH

[0572] 30 treatment. The overall recovery of all AAV8 particles was calculated to be 89% (NF13) and 85% (NF38). The extrapolated remaining dynamic binding -347

[0573] - 65 - capacity at 10% breakthrough, compared to before the treatment with 0.5 M NaOH, were at 85% for the NF13 resin and 91 % for the NF38 resin.

[0574] The belonging chromatography runs are demonstrated using the UV signal curve tracked at 280nm in Figure 3 (NF13) and Figure 4 (NF38), whereas the

[0575] 5 AAV8 purification run is summarized in Table 5 for NF13 related values and in Table 6 for NF38 related values. The overall comparison result is described in Table 7. Both ligand variants that were treated and used for dynamic purification were proofed to be stable and can keep the functionality to bind AAV8 after incubation in 0.5M NaOH after 150 minutes.

[0576] Table 5: Process conditions for AAV8 purification run after NaOH treatment using NF13 barring prototype P1. Run summary data was calculated over

[0577] ELISA.

[0578] 30 -347

[0579] 5

[0580] Table 6: Process conditions for AAV8 purification run after NaOH treatment using NF38 barring prototype P2. Run summary data was calculated over

[0581] ELISA.

[0582] 30 -347

[0583] Table 7: AAV8 dynamic purification run summary

[0584] *after NaOH incubation

[0585] 5

[0586] 10

[0587] 20

[0588] 30 -347

[0589] - 68 -

[0590] Example 3: Influence of NaOH on static binding (association) of AAV9 to immobilized affinity ligand to chromatographic stationary phase (resin)

[0591] The purpose of this experiment was to determine whether the resin treatment

[0592] 5 with various sodium hydroxide concentrations affects resin AAV9-binding capacity. Two Eshmuno® NF08 (SEQ ID NO: 20) affinity ligand baring resins having different affinity ligand densities were compared to two commercially available AAV9 chromatography resins.

[0593] To test the performance of covalently immobilized affinity ligand (NF08), epoxy activated Eshmuno® resin was modified with purified affinity ligand. The modification of epoxy activated Eshmuno® resin was then caried out in phosphate buffer with addition of salts at pH 9.0 at 40°C temperature for 6 hours in the ratio of 8mg affinity ligand (polypeptide comprising ligand ID

[0594] 15 NF08 with C-terminal Cysteine) for the 1 ml of Eshmuno® resin after reducing the dimeric polypeptides with DTT for at least 30 minutes.

[0595] After modification the obtained affinity ligand baring Eshmuno® resin is washed to remove non-covalently bound ligand and stored in 20% ethanol

[0596] 20 and 150mM NaCI containing solution.

[0597] Before the performance test for static binding capacity, the loose resin was soaked in sodium hydroxide solutions of either 0.1 , 0.5, or 1 .0mol / l for 2 hours at room temperature. A reference experiment was conducted using resin without sodium hydroxide treatment. After caustic treatment, the resin was thoroughly washed with buffer.

[0598] The static binding performance was measured by subjecting the resins to purified AAV9 capsids solution following an incubation for four hours. The resins were able to bind AAV9 capsids present in the solution, and upon pH

[0599] 30 induced elution (50mM Glycine, 150mM NaCI, 0.001 % Pluronic, pH 3.0), different amounts of AAV9 capsids of the bond capsids were eluted -347

[0600] - 69 - depending on the caustic resin treatment beforehand. Static AAV9 binding capacity was determined based on elution pool fraction.

[0601] As shown in Table 8, as the concentration of sodium hydroxide increased, the static binding capacity decreases. Some resins lose up to 100% of their

[0602] 5 initial functionality.

[0603] Table 8: Static binding capacity summary table for caustic resin treatment

[0604] 30 -347

[0605] - 70 -

[0606] 5 Given the two different affinity ligand densities in Table 8 for the Eshmuno® NF08 affinity ligand baring resins, it was shown that a higher ligand density results in a higher AAV9 static binding capacity as well as an increased caustic stability at different sodium concentrations. Compared to competitor resin (Resin A and Resin B, Table 8) the Eshmuno® NF08 affinity ligand baring resins showed >66% remaining AAV9 capacity post 1.0 mol / l sodium hydroxide treatment for two hours.

[0607] Example 4: Influence of NaOH on dynamic binding (association) of

[0608] 15 AAV9 to immobilized affinity ligand to chromatographic stationary phase

[0609] Resin-based matrix: Eshmuno® AAV9

[0610] The performance of covalently immobilized affinity ligand (NF08) was

[0611] 20 demonstrated in a dynamic purification process, thereby the affinity ligand first was prepared by C-terminal fusion of C-tag to the polypeptide.

[0612] The modification of epoxy activated Eshmuno® resin was then caried out in 0.55M phosphate buffer pH 9.0 at 40°C temperature for 6 hours in the ratio of 8mg affinity ligand quantity for the 1 ml of Eshmuno® resin after reducing the dimeric polypeptides with DTT for at least 30 minutes.

[0613] After modification the obtained affinity ligand baring Eshmuno® resin is washed to remove non-covalently bound ligand and stored in 20% ethanol and 150mM NaCI containing solution.

[0614] The obtained affinity ligand density was 7.85mg / ml resin.

[0615] 30

[0616] The Eshmuno® NF08 affinity ligand baring resin was used to manufacture a 0.196 mL column prototype with 0.5cm x 1cm column dimensions. The -347

[0617] - 71 - dynamic binding performance was measured by loading 125 mL of clarified HEK293T cell culture containing 1.2 x 10A12vp / mL AAV9 capsids concentration to the resin prototype. The AAV9 capsids present in the clarified HEK293T cell culture could be bound within neutral pH conditions

[0618] 5 (PBS, pH 7.4) to the in PBS, followed by an additional wash step (PBS, pH 7.4). The pH induced elution took place using 50mM Glycine, 150mM NaCI, 0.001% Pluronic, pH 3.0. After elution, the resin was subjected to dynamic cleaning using 25 column volumes of Strip buffer (100mM Tris, 2M NaCI) followed by 10 column volumes of 0.5M NaOH solution. All steps were carried out at 1 minute residence time for 1 column volume.

[0619] The described experiment was repeated five times with the same column, thereby taking the samples of the loaded viral particles and eluted viral particles summarized in Table 9.

[0620] Table 9. AAV9 dynamic purification run summary

[0621] The resin was able to bind > 4.00 x 10A14 vp / ml CV and elute > 90% amount of the AAV9 viral particles. Regardless the caustic cleaning with 0.5M NaOH for 10 minutes at each cycle for 5 cycles, the resin maintained > 4.00 x 10A14 vp / ml CV dynamic binding capacity and > 90% elution rate compared to the particles bound.

[0622] 30

[0623] Example 5 : Membrane-based matrix: Natrix® AAV9: -347

[0624] - 72 -

[0625] The performance of covalently immobilized affinity ligand (NF08) was demonstrated in a dynamic purification process. The ligand was conjugated to a Natrix® membrane.

[0626] 5

[0627] Table 10: Summary of Results

[0628] Table 11 : Buffer compositions

[0629] 30

[0630] The buffers used for the AAV affinity capture chromatography pre-run (initial sanitization and cleaning) and the main run were prepared according to the -347

[0631] - 73 - buffer table provided above (table 11 ). All buffers were filtered through a 0.2 pm bottle-top filter before use.

[0632] The membrane chromatography device was attached to a chromatography system, such as the Akta™ avant or Akta™ pure system.

[0633] 5

[0634] Before the main run, the affinity membrane was sanitized with sodium hydroxide (NaOH) using the pre-run procedure. First, the membrane was equilibrated with Buffer A at a flow rate of 10 membrane volumes per minute (MV / min). Next, the membrane was flushed with Buffer B at a flow rate of 10 MV / min. Following the flush, a STRIP step was performed at a flow rate of 10 MV / min, which was succeeded by a CIP step at a flow rate of 4 MV / min. Two different CIP steps were compared, caustic sanitization with 0.1 M NaOH for 10 min and 0.5 M NaOH for 30 min. After completing the CIP step, the membrane was re-equilibrated with Buffer A2 at a flow rate of 10 MV / min

[0635] 15 and finally equilibrated again with Buffer A at 10 MV / min.

[0636] After the successful pre-run, the AAV-containing feed was sterile filtered and connected to the chromatography system. The main run (Figure 5) was initiated with an equilibration step at a flow rate of 8 MV / min, followed by the

[0637] 20 sample application at the same flow rate of 8 MV / min. The flow-through during the sample application was collected to determine the dynamic binding capacity of the membrane. The application of feed onto the membrane was stopped after loading 1 E+16 viral particles per mL membrane volume (VP / mL MV). The sample application step was followed by a wash step using Buffer A at 8 MV / min. Elution of the viral particles was performed using a step elution with 100% Buffer B at 8 MV / min. Following elution, a STRIP step and a CIP step were conducted at flow rates of 4 MV / min and 2 MV / min, respectively. The membrane was then preequilibrated using Buffer A2 at 2 MV / min, followed by an equilibration step

[0638] 30 using Buffer A at 4 MV / min. -347

[0639] - 74 -

[0640] Samples from the different steps (flow-through fractions of the sample application, wash, elution, STRIP, and CIP) were analyzed externally using ELISA-based methods for quantification of viral capsids. The 10% dynamic binding capacity (10% DBC) was calculated after analyzing the flow-through

[0641] 5 fractions and compared regarding the different membrane sanitization protocols. High dynamic binding capacities (>5E+15 viral particles per mL membrane volume) were determined independent from the initial caustic sanitization (see table 10) demonstrating the caustic stability of the affinity ligand conjugated on the chromatography membrane.

[0642] 15

[0643] 20

[0644] 30

Claims

1. -347- 75 -CLAIMS1 . An affitin ligand for separating a target structure from a liquid sample by5 affinity chromatography comprising or consisting of a polypeptide, wherein the polypeptide does not comprise an asparagine or glutamine residue flanked on both sides by glycine residues.

2. The affitin ligand of claim 1 , wherein the polypeptide comprises at least one asparagine-glycine (NG) dipeptide sequence.

3. The affitin ligand of claim 1 or 2, wherein the polypeptide comprises at least one glutamine-glycine (QG) dipeptide sequence.

4. The affitin ligand of claim 1 , wherein the polypeptide follows formula II15 VKVKFX1X2X3G X4EKEVDX5X6KI X7X8VX9RX10GX11X12V X13FX14YDDNGKX15 GXI6G X17VX18EX19X20A PKELLX21ML ARAEREK formula II, andXi, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17,20 X , X19, X20, and X21 represents a single amino acid independently selected from the group consisting of naturally occurring amino acids.

5. The affitin ligand of claim 4, wherein the polypeptide follows a sequence according to SEQ ID NOs: 19-466. A chromatography matrix comprising a covalently bonded affitin ligand of any of claims 1 to 5.

7. The chromatography matrix according to claim 6 comprising a base material in form of a resin or a membrane.-347- 76 -8. The chromatography matrix according to claims 6 and 7, wherein the matrix has less than a 30% reduction in binding capacity following an incubation in 0.1 M NaOH for at least 150 min.

9. The chromatography matrix according to one or more of of claims 6 to5 8, wherein the matrix has less than a 30% reduction in binding capacity following an incubation in 0.5 M NaOH for at least 30 min.

10. The chromatography matrix according to one or more of claims 6 to 9, wherein the affitin ligand specifically binds a target structure selected from the group of proteins, nucleic acids, carbohydrates, lipids, small molecules, vesicles, viruses, microorganisms, cells, organelles, and cell fragments.11 . The chromatography matrix according to one or more of claims 6 to 10,15 whereby the chromatography matrix has a ligand density between 3 mg / ml and 10 mg / ml.

12. Use of the chromatography matrix of any of claims 6 to 11 for affinity purification of a target structure.20 13. The use of claim 12, wherein the chromatography matrix retains at least 70% of its binding capacity after 10 or more regeneration cycles with sodium hydroxide at a concentration between 0.05 M and 0.5 M.

14. A method for separating a target structure from a liquid sample comprising or consisting of the steps of:(f) providing a liquid sample comprising a target structure, a chromatography matrix according to one or more of claims 6 to 10, at least one wash buffer, and at least one elution buffer,(g) contacting said liquid sample with the chromatography matrix,30- 77 -(h) optionally washing the chromatography matrix with at least one washing buffer,(i) eluting the target structure from the chromatography matrix with at least one elution buffer, and5(j) cleaning chromatography matrix with a caustic cleaning liquid.

15. The method of claim 14, wherein the caustic cleaning liquid comprises sodium hydroxide at a concentration between 0.05 M and 0.5 M.152030