Novel ligand for human tissue plasminogen activator

Abstract

The present invention relates to novel affinity ligands that bind to human tissue plasminogen activator (tPA). The novel affinity ligands of the present invention are powerful tools because they allow efficient purification methods for human tPA via affinity chromatography. In particular, the affinity ligands are powerful in the purification of recombinant human tPA that was produced in Chinese hamster ovary cells because the affinity ligands of the invention do not detectably bind to hamster tPA. Accordingly, the present invention also relates to a novel process of isolating and purifying tPA, specifically tPA from CHO cells.

Description

[0001] NOVEL LIGAND FOR HUMAN TISSUE PLASMINOGEN ACTIVATOR

[0002] TECHNICAL FIELD

[0003] The present invention relates to novel affinity ligands that bind to human tissue plasminogen activator (tPA). The novel affinity ligands of the present invention are powerful tools because they allow efficient purification methods for human tPA via affinity chromatography. In particular, the affinity ligands are powerful in the purification of recombinant human tPA that was produced in Chinese hamster ovary cells because the affinity ligands of the invention do not detectably bind to hamster tPA. Accordingly, the present invention also relates to a novel process of isolating and purifying tPA, specifically tPA from CHO cells.

[0004] BACKGROUND OF THE INVENTION

[0005] Human tissue plasminogen activator (t-PA) is a glycoprotein belonging to the superfamily of serine proteases. It activates plasminogen to plasmin in fibrinolysis. In particular, human tPA is a serine protease that converts the proenzyme plasminogen to plasmin, a fibrinolytic enzyme. Human tPA has 17 disulfide bridges and a molecular weight of about 65 kDa.

[0006] Human tPA facilitates the breakdown of blood clots. Thrombolytic therapy based on tPA is a strong clot-busting medication that dissolves intravascular blood clots and reconstitutes blood flow. Fibrinolytic tPA protein can be bioengineered from human tPA using recombinant biotechnology techniques. Known recombinant tPA (rtPA) proteins include alteplase, reteplase, and tenecteplase, which are used in clinical medicine to treat, inter alia, myocardial infarction and embolic or thrombotic stroke.

[0007] The manufacturing of recombinant glycoproteins is typically carried out by (fed) batch and perfusion cell culture bioreactor processes, and for purification of these proteins adsorption and ion exchange chromatography are commonly applied. Purification protocols for human tPA and its variants are described in the art, e.g., purification by immuno affinity chromatography (anti-tPA goat polyclonal antibody). WO 2011 / 015922 describes a purification process for tPA based on a series of ion exchange chromatography steps, immune affinity chromatography and ultrafiltration / diafiltration steps. WO 2012 / 066569 relates to a purification process focusing on the use of hydrophobic interaction chromatography.

[0008] The known methods of immune affinity chromatography have not always proven to be a suitable technique for commercial production of recombinant human tPA. There is thus a demand for providing efficient, scalable and robust purification solutions, which allow consistently producing recombinant human tPA, in particular at commercial scale, meeting all the required specifications. There is a particular need for an efficient purification of recombinant human tPA produced with Chinese hamster ovary cells. The present invention meets these needs by providing novel affinity ligand for human tPA. These novel affinity ligands are particularly advantageous because they allow a purification of human tPA via affinity chromatography, having specific binding affinity for human tPA but not to hamster tPA.

[0009] The above overview does not necessarily describe all problems solved by the present invention.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention provides a novel ligand for human tissue plasminogen activator (human tPA) and related applications, in particular in the purification of recombinant human tPA. The novel ligand exhibits a surprisingly high affinity for human tPA, and is particularly suitable for the affinity purification of recombinant human tPA, more specifically recombinant human tPA produced in Chinese hamster ovary (CHO) cells. Accordingly, the present invention also provides a novel process of isolating and purifying human tPA, more specifically human tPA from CHO cells. The novel affinity ligand for human tPA allows an industrially applicable, simple, cost effective, robust and highly efficient process of purification of recombinant human tPA.The present disclosure provides the following items 1 to 18, without being specifically limited thereto:

[0012] 1. An affinity ligand for recombinant human tissue plasminogen activator (human tPA), comprising the amino acid sequence of SEQ ID NO: 1 or a sequence with at least 85 % sequence identity thereto, wherein the human tPA affinity ligand comprises a tryptophan (W) at the position corresponding to position 18 of SEQ ID NO: 1.

[0013] 2. The affinity ligand for recombinant human tPA according to aspect 1, (further) comprising

[0014] (i) a basic amino acid at the position corresponding to position 13 of SEQ I D NO: 1 ;

[0015] (ii) an aromatic amino acid at the position corresponding to position 47 of SEQ ID NO: 1 ; and / or

[0016] (iii) a basic amino acid at the position corresponding to position 51 of SEQ ID NO: 1.

[0017] 3. The affinity ligand for recombinant human tPA according to aspect 2, wherein:

[0018] (i) the basic amino acid at the position corresponding to position 13 of SEQ ID NO: 1 is lysine (K);

[0019] (ii) the aromatic amino acid at the position corresponding to position 47 of SEQ I D NO: 1 is tyrosine (Y); and / or

[0020] (iii) the basic amino acid at the position corresponding to position 51 of SEQ ID NO: 1 is lysine (K).

[0021] 4. A multimeric affinity ligand for recombinant human tissue plasminogen activator (human tPA) comprising a first monomer and a second monomer, wherein:

[0022] (a) the first monomer comprises the amino acid sequence of SEQ ID NO: 1 or a sequence with at least 85 % sequence identity thereto, wherein the first monomer comprises a tryptophan (W) at the position corresponding to position 18 of SEQ ID NO: 1 ; and

[0023] (b) the second monomer comprises the amino acid sequence of SEQ ID NO: 2 or a sequence with at least 85 % sequence identity thereto.

[0024] 5. The multimeric affinity ligand for recombinant human tPA according to aspect 4, wherein the first monomer (further) comprises:

[0025] (i) a basic amino acid at the position corresponding to position 13 of SEQ ID NO: 1 ;

[0026] (ii) an aromatic amino acid at the position corresponding to position 47 of SEQ ID NO: 1 ; and / or

[0027] (iii) a basic amino acid at the position corresponding to position 51 of SEQ ID NO: 1 .

[0028] 6. The multimeric affinity ligand for recombinant human tPA according to aspect 5, wherein:

[0029] (i) the basic amino acid at the position corresponding to position 13 of SEQ ID NO: 1 is lysine (K);

[0030] (ii) the aromatic amino acid at the position corresponding to position 47 of SEQ I D NO: 1 is tyrosine (Y); and / or

[0031] (iii) the basic amino acid at the position corresponding to position 51 of SEQ ID NO: 1 is lysine (K).

[0032] 7. A multimer, preferably a dimer, of the affinity ligand for recombinant human tPA according to any one of aspects 1 to 3, wherein an affinity ligand for recombinant human tPA according to any one of aspects 1 to 3 is linked to the amino acid sequence of SEQ ID NO: 2 or a sequence with at least 85 % sequence identity thereto.

[0033] 8. The multimeric affinity ligand for recombinant human tPA according to any one of aspects 4 to 6, or the multimer according to aspect 7, comprising an amino acid sequence with at least 85 % sequence identity to any one of SEQ ID NOs: 3 to 11 .

[0034] 9. A multimer, preferably a dimer, of the affinity ligand for recombinant human tPA according to any one of aspects 1 to 3, wherein at least two affinity ligands for recombinant human tPA according to any one of aspects 1 to 3 are linked to each other.

[0035] 10. The affinity ligand for recombinant human tPA according to any one of aspects 1 to 3, or the multimeric affinity ligand for recombinant human tPA according to any one of aspects 4 to 6 and 8, or the multimer according to any one of aspects 7 to 9, which does not exhibit a detectable binding affinity to hamster tPA, preferably as determined by SPR.

[0036] 11. A fusion protein comprising:

[0037] (a) an affinity ligand for recombinant human tPA according to any one of aspects 1 to 3 and 10, or a multimeric affinity ligand for recombinant human tPA according to any one of aspects 4 to 6, 8 and 10, or a multimer according to any one of aspects 7 to 10; and (b) a protein having at least 85 % identity to SEQ ID NO: 12 which has no detectable binding affinity for human tPA, preferably as determined by SPR.

[0038] 12. The affinity ligand for recombinant human tPA according to any one of aspects 1 to 3 and 10, or the multimeric affinity ligand for recombinant human tPA according to any one of aspects 4 to 6, 8 and 10, or the multimer according to any one of aspects 7 to 10, or the fusion protein according to aspect 11 , which:

[0039] (a) is capable of binding to human tPA in a solution at neutral pH; and / or

[0040] (b) has a binding affinity for recombinant human tPA of less than 1 pM, as determined by SPR.

[0041] 13. A solid phase support, or an affinity separation matrix, comprising or having immobilized thereon, the affinity ligand for recombinant human tPA according to any one of aspects 1 to 3, 10 and 12, or the multimeric affinity ligand for recombinant human tPA according to any one of aspects 4 to 6, 8, 10 and 12, or the multimer according to any one of aspects 7 to 10 and 12, or the fusion protein according to aspect 11 or 12.

[0042] 14. Use of the affinity ligand for recombinant human tPA according to any one of aspects 1 to 3, 10 and 12, or the multimeric affinity ligand for recombinant human tPA according to any one of aspects 4 to 6, 8, 10 and 12, or the multimer according to any one of aspects 7 to 10 and 12, or the fusion protein according to aspect 11 or 12, or the solid phase support or affinity separation matrix according to aspect 13, in

[0043] (a) affinity purification of recombinant human tPA;

[0044] (b) sample analysis for the detection of recombinant human tPA; or

[0045] (c) detection methods for the detection of recombinant human tPA.

[0046] 15. A polynucleotide encoding the recombinant human tPA affinity ligand according to any one of aspects 1 to 3, 10 and 12, or the multimeric affinity ligand for recombinant human tPA according to any one of aspects 4 to 6, 8, 10 and 12, or the multimer according to any one of aspects 7 to 10 and 12, or the fusion protein according to aspect 11 or 12.

[0047] 16. A method for isolating or purifying recombinant human tissue plasminogen activator (human tPA) from a liquid, the method comprising:

[0048] (a) contacting the affinity ligand for recombinant human tPA according to any one of aspects 1 to 3, 10 and 12, or the multimeric affinity ligand for recombinant human tPA according to any one of aspects 4 to 6, 8, 10 and 12, or the multimer according to any one of aspects 7 to 10 and 12, or the fusion protein according to aspect 11 or 12, with a liquid containing recombinant human tPA;

[0049] (b) separating the recombinant human tPA bound in step (a) to the affinity ligand for recombinant human tPA, or to the multimeric affinity ligand for recombinant human tPA, or to the multimer, or to the fusion protein, respectively, from the liquid; (c) detaching or eluting said recombinant human tPA from the affinity ligand for recombinant human tPA, or the multimeric affinity ligand for recombinant human tPA, or the multimer, or the fusion protein, respectively.

[0050] 17. A method for isolating or purifying recombinant human tissue plasminogen activator (human tPA) from a liquid, the method comprising:

[0051] (a) contacting the solid phase support or affinity separation matrix according to aspect 13 with a liquid containing recombinant human tPA, thereby binding of recombinant human tPA in the liquid to the solid phase support or affinity separation matrix;

[0052] (b) detaching or eluting said recombinant human tPA from the solid phase support or affinity separation matrix, respectively.

[0053] 18. A method for determining the presence of recombinant human tissue plasminogen activator (human tPA) in a liquid, the method comprising:

[0054] (a) contacting the affinity ligand for recombinant human tPA according to any one of aspects 1 to 3, 10 and 12, or the multimeric affinity ligand for recombinant human tPA according to any one of aspects 4 to 6, 8, 10 and 12, or the multimer according to any one of aspects 7 to 10 and 12, or the fusion protein according to aspect 11 or 12, or the solid phase support or affinity separation matrix according to aspect 13, with a liquid containing recombinant human tPA;

[0055] (b) determining the recombinant human tPA bound in step (a) to the affinity ligand for recombinant human tPA, or the multimeric affinity ligand for recombinant human tPA, or the multimer, or the fusion protein, or the solid phase support or affinity separation matrix, respectively;

[0056] (c) optionally detaching or eluting said recombinant human tPA from the affinity ligand for recombinant human tPA, or the multimeric affinity ligand for recombinant human tPA, or the multimer, or the fusion protein, or the solid phase support or affinity separation matrix, respectively, prior to determining the recombinant human tPA.

[0057] This summary of the invention is not limiting, and other aspects and embodiments of the invention will become evident from the following description, examples and drawings.

[0058] DETAILED DESCRIPTION OF THE INVENTION

[0059] The present invention provides a novel ligand for human tissue plasminogen activator (human tPA) and related applications, in particular in the purification of recombinant human tPA. The novel ligand exhibits a surprisingly high affinity for human tPA and is particularly suitable for the affinity purification of recombinant human tPA, more specifically recombinant human tPA produced in Chinese hamster ovary (CHO) cells. Accordingly, the present invention also provides a novel process of isolating and purifying human tPA, more specifically human tPA from CHO cells. The novel affinity ligand for human tPA allows an industrially applicable, simple, cost effective, robust and highly efficient process of purification of recombinant human tPA.

[0060] The present invention allows the (production and) purification of biologically active recombinant human tPA. Without wishing to be bound by theory, the present invention allows the (production and) purification of recombinant human tPA with a level of purity sufficient for technical applications and / or therapeutic use.

[0061] The novel proteins of the present invention have specific binding affinity for recombinant human tPA. The novel proteins of the present invention are particularly advantageous because as affinity ligands for human tPA, they allow precise purification of human tPA, for example in affinity chromatography. In particular, the ligands of the present invention have a specific binding affinity of less than 1 pM to human tPA, but do not bind to hamster tPA or bind only with very low affinity to hamster tPA. This is of particular importance because recombinant human tPA that is expressed in CHO cells might contain a contamination of hamster tPA that is removed by affinity chromatography using the specific ligands of the present invention. The affinity ligands for human tissue plasminogen activator (human tPA) provided by the present invention are characterized by the amino acid sequence of SEQ ID NO: 1 or a sequence with at least 85 % sequence identity thereto, wherein the recombinant human tPA affinity ligand comprises a tryptophan (W) at the position corresponding to position 18 of SEQ ID NO: 1.

[0062] Before the present invention is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects and embodiments only and is not intended to limit the scope of the present invention, which is reflected by the appended items. 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 belongs. This includes a skilled person working in the field of protein engineering and purification, but also including a skilled person working in the field of binding molecules for human tPA and their use as affinity ligands in the purification of human tPA in affinity chromatography.

[0063] Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0064] Throughout this specification and the items, which follow, unless the context requires otherwise, the word “comprise”, and variants such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. The term “comprise(s)” or “comprising” may encompass a limitation to “consists of’ or “consisting of’, should such a limitation be necessary for any reason and to any extent. Several documents (for example: patents, patent applications, scientific publications, manufacturer’s specifications, instructions, UniProt Accession Number, etc.) may be cited throughout the present specification. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein may be characterized as being “incorporated by reference". In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

[0065] All sequences referred to herein are disclosed in the attached sequence listing that, with its whole content and disclosure, forms part of the disclosure content of the present specification.

[0066] General Definitions of Important terms used in the Application

[0067] The terms “human tPA” or “human tissue plasminogen activator” may be used interchangeably herein and describe a human tissue plasminogen activator (serine protease). More specifically, human tissue plasminogen activator (human tPA) is a glycoprotein belonging to the superfamily of serine proteases. It activates plasminogen to plasmin in fibrinolysis. In particular, human tPA is a serine protease that converts the proenzyme plasminogen to plasmin, a fibrinolytic enzyme (which itself is also a serine protease).

[0068] The terms “human tPA” or “recombinant human tPA” as used herein includes recombinant human tissue plasminogen activator. The terms „human tPA” or “recombinant human tPA” comprises all polypeptides which show an amino acid sequence identity of at least 80 %, 85 %, 90 %, 95 %, 96 % or 97 % or more, or 100 % to SEQ ID NO: 21. In some embodiments, the terms “human tPA” or “recombinant human tPA” refer to an amino acid sequence with at least 90 % identity to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the term “human tPA” refers to an amino acid sequence with at least 95 % identity to the amino acid sequence of SEQ ID NO: 20. In preferred embodiments, the terms „human tPA” or “recombinant human tPA” refer to an amino acid sequence with at least 98 % identity to the amino acid sequence of SEQ ID NO: 21. Preferred embodiments relate to recombinant human tissue plasminogen activator with at least 98 % identity to amino acids 36-562 of the amino acid sequence of Uni Prot P00750 (SEQ ID NO: 21). In various embodiments of the present invention, a variant of the tPA of SEQ ID NO: 21 as described herein comprises a substitution of threonine (T) at position 103 with asparagine (N), a substitution of asparagine (N) at position 117 with glutamine (Q) (both within the kringle 1 domain), and the substitution of lysine (K), histidine (H) and two arginine (two R) with tetra-alanine (tetra-A) amino acids at positions 296- 299 in the protease domain of tPA. Accordingly, in preferred embodiments of the present invention, the term „human tPA” comprises polypeptides, which have an amino acid sequence identity of at least 80 %, 85 %, 90 %, 95 %, 96 % or 97 % or more, or 100 % to SEQ ID NO: 21 , and which comprise:

[0069] (i) an asparagine (N) at the position corresponding to position 103 of SEQ ID NO: 21 ;

[0070] (ii) a glutamine (Q) at the position corresponding to position 117 of SEQ ID NO: 21 ; and

[0071] (iii) a tetra-alanine (tetra-A) at the positions corresponding to positions 296-299 of SEQ ID NO: 21.

[0072] In preferred embodiments, the term “human tPA” refers to an amino acid sequence with at least 98 % identity to the amino acid sequence of SEQ ID NO: 21 and comprising the aforementioned substitutions at the positions corresponding to positions 103, 117, and 296-299 of SEQ ID NO: 21.

[0073] In various embodiments of the present invention, a variant of the tPA of SEQ ID NO: 21 as described herein exhibits the same biological activity, in particular serine protease activity, of native human tPA, in particular human tPA having the amino acid sequence of SEQ ID NO: 21. In preferred embodiments, a recombinant human tPA according to the present invention exhibits the same biological activity, in particular serine protease activity, as Tenecteplase. Tenecteplase (International non-proprietary name, INN) is an EMEA- and FDA-approved recombinant thrombolytic agent used to manage intravascular clots. Tenecteplase binds to the fibrin component of a blood clot (thrombus). It acts within the endogenous fibrinolytic coagulation cascade to selectively catalyze the cleavage of plasminogen to plasmin.

[0074] Accordingly, the present invention allows the (production and) purification of biologically active recombinant human tPA. Without wishing to be bound by theory, the present invention allows the (production and) purification of recombinant human tPA while maintaining the biological activity, in particular serine protease activity, of native human tPA. Specifically, without wishing to be bound by theory, the present invention allows the (production and) purification of recombinant human tPA while maintaining the same higher fibrin specificity, increased resistance to inactivation by endogenous inhibitor plasminogen activator inhibitor, and / or a longer half-life of recombinant human tPA, as compared to native tPA.

[0075] Human tPA may be obtained from natural sources or recombinantly produced by cell culture techniques. Human tPA may be produced by transfecting eucaryotic cells such as for example Chinese hamster ovary (CHO) cells. In preferred embodiments of the present invention, recombinant human tPA refers to human tPA produced in CHO cells.

[0076] The term “fusion protein” relates to a protein comprising at least a first protein joined genetically to at least a second protein. A fusion protein is created through joining of two or more genes that originally coded for separate proteins. Thus, a fusion protein may comprise a multimer of identical or different proteins which are expressed as a single, linear polypeptide.

[0077] The present invention encompasses a fusion protein comprising an affinity ligand for human tPA as described above fused to at least one further protein. The terms “affinity ligand” or ..binding protein" may be used interchangeably herein and describe a protein that is capable to bind to recombinant human tPA. As described herein, a binding protein refers to a protein with detectable interaction with human tPA, as determined by suitable methods such as for example SPR (surface plasmon resonance) analysis or BLI (bio-layer interferometry) or any other appropriate technology known to a person skilled in the art.

[0078] The term “binding affinity” refers to the ability of the proteins of the invention to bind to human tPA.

[0079] The term “amino acid sequence identity” refers to a quantitative comparison of the identity (or differences) of the amino acid sequences of two or more proteins. “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. To determine the sequence identity, the sequence of a query protein is aligned to the sequence of a reference protein or polypeptide, for example, to the amino acid sequence of the polypeptide of SEQ ID NO: 1. Methods for sequence alignment are well known in the art. For example, the NCBI BLAST similarity program is preferably employed (Camacho et al. (2009, BMC Bioinformatics 10: 421). For multiple alignment analysis, ClustalL is preferably used (Sievers & Higgins, 2021 , Methods Mol Biol. 2231 : 3-16). Thus, the percentage of an amino acid sequence identity may be calculated using a published or commercially available software with an algorithm which conducts comparison using a base sequence (e.g., SEQ ID NO: 1 in the present invention) as a reference sequence. For example, BLAST, FASTA, or GENETYX (manufactured by Software Development Co., Ltd.) may be used, and these may be run with default parameters. Each amino acid of the query sequence that differs from the aligned reference amino acid sequence at a given position is counted as one difference. An insertion or deletion in the query sequence is also counted as one difference. The sum of differences is then related to the length of the aligned reference sequence to yield a percentage of non-identity or identity, respectively.

[0080] The terms “protein” and “polypeptide” refer to any chain of two or more amino acids linked by peptide bonds and does not refer to a specific length of the product. Thus, “peptides”, “protein”, “amino acid chain”, or any other term used to refer to a chain of two or more amino acids, are included within the definition of “polypeptide”, and the term “polypeptide” may be used instead of, or interchangeably with, any of these terms. The term “polypeptide” is also intended to refer to the products of post-translational modifications of the polypeptide like, e.g., glycosylation, which are well known in the art.

[0081] A “linker” as used herein is preferably an amino acid sequence that joins at least two moieties, e.g., two affinity ligands of the present invention, or two monomers of a multimeric affinity ligand of the present invention. The linker as used herein is a peptide linker of up to 30 amino acids. Two or more moieties may be linked via a peptide linker of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In preferred embodiments, a linker comprises two amino acids, in particular isoleucine (I) and alanine (A). In the fusion proteins of the present invention, a recombinant human tPA affinity ligand and a protein having at least 85 % sequence identity to SEQ ID NO: 12 as described elsewhere herein, may also be linked by a linker, in particular a peptide linker.

[0082] The term “chromatography” refers to separation technologies, which employ a mobile phase and a stationary phase to separate one type of molecules (e.g., recombinant human tPA) from other molecules (e.g., contaminants) in a liquid or sample. The (liquid) mobile phase contains a mixture of molecules and transports these across or through a stationary phase (such as a solid matrix). Due to the differential interaction of the different molecules in the mobile phase with the stationary phase, molecules in the mobile phase can be separated.

[0083] The term “affinity chromatography” refers to a specific mode of chromatography in which a ligand (e.g., affinity ligand of the invention) coupled to a stationary phase (e.g. solid support) interacts with a molecule (i.e. , recombinant human tPA) in the mobile phase (the liquid sample), i.e., the ligand has a specific binding affinity for the molecule to be captured. As understood in the context of the invention, affinity chromatography involves the addition of a liquid or (liquid) sample containing recombinant human tPA to a stationary phase which comprises a chromatography ligand, such as the affinity ligand of the invention. The terms “solid support” or “solid matrix” are used interchangeably for the stationary phase.

[0084] The terms "affinity matrix" or "affinity separation matrix", as used interchangeably herein, refer to a matrix, e.g., a chromatographic matrix, onto which an affinity ligand of the invention is attached. The attached affinity ligand is capable of specific binding to a protein of interest (e.g., recombinant human tPA) which is to be purified from a liquid or a (liquid) sample.

[0085] The term “affinity purification” as used herein refers to a method of purifying human tPA from a liquid or (liquid) sample by binding human tPA to a ligand of the invention that is immobilized to a matrix. Thereby, human tPA is removed from the liquid or (liquid) sample and thereby purified.

[0086] Detailed description of the embodiments of the invention

[0087] The present invention will now be further described. In the following passages different aspects of the invention are defined in more detail. Each aspect defined below may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0088] The novel affinity ligands of the present invention exhibit a specific binding affinity for human tPA. In particular, the invention provides affinity ligands for human tissue plasminogen activator (human tPA), comprising the amino acid sequence of SEQ ID NO: 1 or a sequence with at least 85 % sequence identity thereto, wherein the human tPA affinity ligand comprises a tryptophan (W) at the position corresponding to position 18 of SEQ ID NO: 1.

[0089] Structural characteristics of the affinity ligand. In some embodiments, the ligand (or affinity ligand) for human tPA comprises the amino acid sequence of SEQ ID NO: 1 wherein the human tPA affinity ligand comprises a tryptophan (W) at the position corresponding to position 18 of SEQ ID NO: 1. In some embodiments, the ligand (or affinity ligand) for human tPA comprises an amino acid with at least 85 % sequence identity to SEQ ID NO: 1 wherein the human tPA affinity ligand comprises a tryptophan (W) at the position corresponding to position 18 of SEQ ID NO: 1.

[0090] In some embodiments, the affinity ligand for human tPA has at least 85 %, 86 %, 87 %, 88 %, 89%, 90 %, 91 %, 92 %, 93 %, 94 %, 95%, 96 %, 97 %, 98 %, 99 %, or 100 % sequence identity to SEQ ID NO: 1 wherein the human tPA affinity ligand comprises a tryptophan (W) at the position corresponding to position 18 of SEQ ID NO: 1.

[0091] In preferred embodiments, the affinity ligand for human tPA further comprises (i) a basic amino acid at the position corresponding to position 13 of SEQ ID NO: 1 ; (ii) an aromatic amino acid at the position corresponding to position 47 of SEQ ID NO: 1 ; and / or (iii) a basic amino acid at the position corresponding to position 51 of SEQ ID NO: 1. As described herein, the terms “basic amino acid” and “alkaline amino acid” may be used interchangeably herein.

[0092] In more preferred embodiments of the affinity ligand for human tPA (i) the basic amino acid at the position corresponding to position 13 of SEQ ID NO: 1 is lysine (K); (ii) the aromatic amino acid at the position corresponding to position 47 of SEQ ID NO: 1 is tyrosine (Y); and / or (iii) the basic amino acid at the position corresponding to position 51 of SEQ ID NO: 1 is lysine (K).

[0093] In particularly preferred embodiments, a variant of the human tPA affinity ligand according to the present invention comprises a tryptophan (W) at the position corresponding to position 18 of SEQ ID NO: 1 , a lysine (K) at the position corresponding to position 13 of SEQ ID NO: 1 ; a tyrosine (Y) at the position corresponding to position 47 of SEQ ID NO: 1 , and a lysine (K) at the position corresponding to position 51 of SEQ ID NO: 1.

[0094] Structural characteristics: Multimers. Some embodiments refer to an affinity ligand for a human tPA, wherein the affinity ligand comprises the amino acid sequence of SEQ ID NO: 1 , or an amino acid sequence with at least 85 % sequence identity thereto as described elsewhere herein, wherein the affinity ligand is a multimer.

[0095] Some embodiments refer to a dimer of the human tPA affinity ligand, wherein the affinity ligand comprises the amino acid sequence of SEQ ID NO: 1 , or an amino acid sequence with at least 85 % sequence identity thereto as described elsewhere herein, wherein at least two human tPA affinity ligands as described above are linked to each other. In the present invention, two human tPA affinity ligands comprising the amino acid sequence of SEQ ID NO: 1 , or an amino acid sequence with at least 85 % sequence identity thereto as described elsewhere herein, may be directly fused to each other, or may be linked by a peptide linker. In preferred embodiments, the peptide linker comprises or consists of two amino acids, in particular isoleucine (I) and alanine (A).

[0096] Some embodiments refer to a multimeric ligand, preferably a dimeric ligand, for human tissue plasminogen activator (human tPA) comprising a first monomer and a second monomer, wherein:

[0097] (a) the first monomer comprises the amino acid sequence of SEQ ID NO: 1 or a sequence with at least 85 % sequence identity thereto, wherein the first monomer comprises a tryptophan (W) at the position corresponding to position 18 of SEQ ID NO: 1 ; and

[0098] (b) the second monomer comprises the amino acid sequence of SEQ ID NO: 2 or a sequence with at least 85 % sequence identity thereto.

[0099] Some embodiments refer to the multimeric human tPA affinity ligand, preferably a dimeric human tPA affinity ligand, as described above, wherein the first monomer (further) comprises (i) a basic amino acid at the position corresponding to position 13 of SEQ ID NO: 1 ; (ii) an aromatic amino acid at the position corresponding to position 47 of SEQ ID NO: 1 ; and / or (iii) a basic amino acid at the position corresponding to position 51 of SEQ ID NO: 1. As described herein, the terms “basic amino acid” and “alkaline amino acid” may be used interchangeably.

[0100] Some embodiments refer to the multimeric human tPA affinity ligand, preferably a dimeric human tPA affinity ligand, as described above, wherein (i) the basic amino acid at the position corresponding to position 13 of SEQ ID NO: 1 is lysine (K); (ii) the aromatic amino acid at the position corresponding to position 47 of SEQ ID NO: 1 is tyrosine (Y); and / or (iii) the basic amino acid at the position corresponding to position 51 of SEQ ID NO: 1 is lysine (K). In particularly preferred embodiments, a multimeric human tPA affinity ligand, preferably a dimeric human tPA affinity ligand, of the present invention comprises a variant of the amino acid sequence of SEQ ID NO: 1, which has a tryptophan (W) at the position corresponding to position 18 of SEQ ID NO: 1 , a lysine (K) at the position corresponding to position 13 of SEQ ID NO: 1 ; a tyrosine (Y) at the position corresponding to position 47 of SEQ ID NO: 1 , and a lysine (K) at the position corresponding to position 51 of SEQ ID NO: 1.

[0101] In preferred embodiments of the multimeric human tPA affinity ligand, preferably a dimeric human tPA affinity ligand, the first monomer comprising the amino acid sequence of SEQ ID NO: 1 or a sequence with at least 85 % sequence identity thereto as described elsewhere herein, is located C-terminally relative to the second monomer comprising the amino acid sequence of SEQ ID NO: 2 or a sequence with at least 85 % sequence identity thereto as described elsewhere herein. Accordingly, in preferred embodiments of a dimeric human tPA affinity ligand of the present invention, the second monomer comprising the amino acid sequence of SEQ ID NO: 2 or a sequence with at least 85 % sequence identity thereto as described elsewhere herein, is the N- terminal monomer, and the first monomer comprising the amino acid sequence of SEQ ID NO: 1 or a sequence with at least 85 % sequence identity thereto as described elsewhere herein, is the C-terminal monomer. Some embodiments refer to the multimer, preferably a dimer, of a human tPA affinity ligand as described above, wherein a human tPA affinity ligand comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof as described above, is linked to the amino acid sequence of SEQ ID NO: 2 or a sequence with at least 85 % sequence identity thereto. Particularly preferred embodiments of the present invention refer to a dimer of a human tPA affinity ligand comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof as described above, wherein a human tPA affinity ligand as described is linked to the amino acid sequence of SEQ ID NO: 2 or a sequence with at least 85 % sequence identity thereto. In such a multimer, preferably dimer, of the present invention, the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence with at least 85 % sequence identity thereto as described elsewhere herein, and the amino acid sequence of SEQ ID NO: 2 or a sequence with at least 85 % sequence identity thereto, may be directly fused to each other, or may be linked by a peptide linker. In preferred embodiments, the peptide linker comprises or consists of two amino acids, in particular isoleucine (I) and alanine (A).

[0102] In preferred embodiments, the amino acid sequence with at least 85 % sequence identity to SEQ ID NO: 2 is located at the N-terminus, and the amino acid sequence with at least 85 % sequence identity to SEQ ID NO: 1 with tryptophan at position corresponding to position 18 is located at the C-terminus of the dimer.

[0103] Some embodiments refer to the multimer of a human tPA affinity ligand as described above, comprising an amino acid sequence with at least 85 % sequence identity to any one of SEQ ID NOs: 3 to 11. A multimeric human tPA affinity ligand comprising an amino acid sequence with at least 85 % sequence identity to any one of SEQ ID NOs: 3 to 11 is preferably a dimeric human tPA affinity ligand. This is in accordance with SEQ ID NOs: 3 to 11 , all of which are composed of the monomer units according to SEQ ID NOs: 1 and 2 as described elsewhere herein. In all of SEQ ID NOs: 3-11 , the (second) monomer comprising the amino acid sequence of SEQ ID NO: 2 or a sequence with at least 85 % sequence identity thereto as described elsewhere herein, is located at the N-terminus, and the (first) monomer comprising the amino acid sequence of SEQ ID NO: 1 or a sequence with at least 85 % sequence identity thereto as described elsewhere herein, is located at the C-terminus.

[0104] Some embodiments refer to a human tPA affinity ligand comprising the amino acid sequence of any one of SEQ ID NOs: 3 to 11 , or a sequence with at least 85 % sequence identity thereto, wherein the human tPA affinity ligand comprises a tryptophan (W) at the position corresponding to position 18 of SEQ ID NO: 1.

[0105] Some embodiments refer to a human tPA affinity ligand comprising the amino acid sequence of any one of SEQ ID NOs: 3 to 11 , or a sequence with at least 85 % sequence identity thereto, wherein the human tPA affinity ligand comprises a tryptophan (W) at the position corresponding to position 18 of SEQ ID NO: 1 , and wherein the human tPA affinity ligand comprises a basic [alkaline] amino acid at the position corresponding to position 13 of SEQ ID NO: 1; an aromatic amino acid at the position corresponding to position 47 of SEQ ID NO: 1 ; and / or a basic [alkaline] amino acid at the position corresponding to position 51 of SEQ ID NO: 1.

[0106] Some embodiments refer to a human tPA affinity ligand comprising the amino acid sequence of any one of SEQ ID NOs: 3 to 11 , or a sequence with at least 85 % sequence identity thereto, wherein the human tPA affinity ligand comprises a tryptophan (W) at the position corresponding to position 18 of SEQ ID NO: 1 , and wherein (i) the basic [alkaline] amino acid at the position corresponding to position 13 of SEQ ID NO: 1 is lysine (K); (ii) the aromatic amino acid at the position corresponding to position 47 of SEQ ID NO: 1 is tyrosine (Y); and / or (iii) the basic [alkaline] amino acid at the position corresponding to position 51 of SEQ ID NO: 1 is lysine (K). Accordingly, a multimeric, preferably dimeric, human tPA affinity ligand of the present invention comprising an amino acid sequence with at least 85 % sequence identity to any one of SEQ ID NOs: 3 to 11 comprises a tryptophan (W) at the position corresponding to position 74 of any of SEQ ID NOs: 3, 4, 5, 6, and 11. In preferred embodiments, a multimeric, preferably a dimeric, human tPA affinity ligand of the present invention comprising an amino acid sequence with at least 85 % sequence identity to any one of SEQ ID NOs: 3 to 11 further comprises (i) a basic amino acid at the position corresponding to position 69 of any of SEQ ID NOs: 3, 4, 5, 6, and 11 ; (ii) an aromatic amino acid at the position corresponding to position 103 of any of SEQ ID NOs: 3, 4, 5, 6, and 11 ; and / or (iii) a basic amino acid at the position corresponding to position 107 of of any of SEQ ID NOs: 3, 4, 5, 6, and 11. Preferably, the basic amino acid at the position corresponding to position 69 of any of SEQ ID NOs: 3, 4, 5, 6, and 11 is lysine (K); the an aromatic amino acid at the position corresponding to position 103 of any of SEQ ID NOs: 3, 4, 5, 6, and 11 is tyrosine (Y); and / or the basic amino acid at the position corresponding to position 107 of any of SEQ ID NOs: 3, 4, 5, 6, and 11 is lysine (K).

[0107] According to further embodiments, a multimeric, preferably dimeric, human tPA affinity ligand of the present invention comprising an amino acid sequence with at least 85 % sequence identity to any one of SEQ ID NOs: 3 to 11 comprises a tryptophan (W) at the position corresponding to position 76 of any of SEQ ID NOs: 7, 8, 9, and 10. In preferred embodiments, such a multimeric, preferably dimeric, human tPA affinity ligand of the present invention comprising an amino acid sequence with at least 85 % sequence identity to any one of SEQ ID NOs: 3 to 11 further comprises (i) a basic amino acid at the position corresponding to position 71 of any of SEQ ID NOs: 7, 8, 9, and 10; (ii) an aromatic amino acid at the position corresponding to position 105 of any of SEQ ID NOs: 7, 8, 9, and 10; and / or (iii) a basic amino acid at the position corresponding to position 109 of any of SEQ ID NOs: 7, 8, 9, and 10. Preferably, the basic amino acid at the position corresponding to position 71 of any of SEQ ID NOs: 7, 8, 9, and 10 is lysine (K); the an aromatic amino acid at the position corresponding to position 105 of any of SEQ ID NOs: 7, 8, 9, and 10 is tyrosine (Y); and / or the basic amino acid at the position corresponding to position 109 of any of SEQ ID NOs: 7, 8, 9, and 10 is lysine (K).

[0108] Functional characteristics of the affinity ligand. One of the advantages of the herein disclosed affinity ligands is the specific binding to human tPA. In specific embodiments, the human tPA affinity ligand comprising the amino acid sequence of any one of SEQ ID NO: 1 , or a sequence with at least 85 % sequence identity thereto, wherein the human tPA affinity ligand comprises a tryptophan (W) at the position corresponding to position 18 of SEQ ID NO: 1 as described elsewhere herein, has a binding affinity of less than 1 pM for recombinant human tPA as disclosed herein. In some embodiments, the affinity ligand has a binding affinity of less than 500 nM for recombinant human tPA as disclosed herein. In some embodiments, the affinity ligand has a binding affinity of less than 100 nM for recombinant human tPA as disclosed herein. In preferred embodiments, the affinity ligand has a binding affinity of less than 50 nM for recombinant human tPA as disclosed herein. In preferred embodiments, the affinity ligand has a binding affinity of less than 20 nM for recombinant human tPA as disclosed herein. Binding affinity is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank the strength of bimolecular interactions. The binding affinity and dissociation constants can be measured quantitatively. Methods for determining binding affinities are well known to the skilled person and can be selected, for instance, from the following methods that are well established in the art: surface plasmon resonance (SPR), Bio-layer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), kinetic exclusion analysis (KinExA assay), flow cytometry, fluorescence spectroscopy techniques, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA), and enhanced chemiluminescence (ECL). Typically, the dissociation constant KD is determined at temperatures in the range of 20°C and 30°C. If not specifically indicated otherwise, KD values recited herein are determined at 25°C, more specifically at 25°C by SPR. Even more specifically, the binding affinity (KD) is determined by SPR at 25°C at about neutral pH, in particular at pH 7.3ln various embodiments of the present invention, the binding affinity for human tPA may be determined by the Sierra SPR-32 system (Bruker).

[0109] In some embodiments, the human tPA affinity ligand as described above, or the multimeric human tPA affinity ligand as described above, or the multimer as described above, does not exhibit a detectable binding affinity or very low binding affinity to hamster tPA, for example, as determined by SPR.

[0110] Fusion proteins. In some embodiments, the invention refers to a fusion protein comprising: (a) a human tPA affinity ligand of the present invention as described herein, or a multimeric human tPA affinity ligand of the present invention as described herein, or a multimer ligand of the present invention as described herein; and (b) a protein with at least 85 % amino acid sequence identity to the sequence of SEQ ID NO: 12, which has no detectable binding affinity for human tPA, for example, as determined by SPR.

[0111] Further, the protein with at least 85 % amino acid sequence identity to the sequence of SEQ ID NO: 12 is a protein having no detectable binding affinity for the Fc domain of immunoglobulin, for example, as determined by SPR.

[0112] In preferred embodiments, the protein with at least 85 % amino acid sequence identity to the sequence of SEQ ID NO: 12 is a protein comprising an acidic amino acid selected from aspartic acid (D) and glutamic acid (E) at the position corresponding to position 11 of SEQ ID NO: 12, and a basic amino acid selected from any one of arginine (R), lysine (K), and histidine (H) at the position corresponding to position 29 of SEQ ID NO: 12, wherein the polypeptide has no detectable binding affinity for the Fc domain of immunoglobulin, for example, as determined by SPR.

[0113] In some embodiments, the invention refers to a fusion protein comprising:

[0114] (a) a human tPA affinity ligand of the present invention as described herein, or a multimeric human tPA affinity ligand of the present invention as described herein, or a multimer ligand of the present invention as described herein; and

[0115] (b) a protein of SEQ ID NO: 12, which has no detectable binding affinity for human tPA, for example, as determined by SPR.

[0116] In some embodiments, the invention refers to a human tPA affinity ligand of the present invention as described herein, or a multimeric human tPA affinity ligand of the present invention as described herein, or a multimer of the present invention as described herein, or a fusion protein of the present invention as described herein, which:

[0117] (a) is capable of binding to human tPA in a solution at neutral pH, in particular pH 6.7 to pH 7.3, preferably about pH 7.0, or which is capable of isolating human tPA from a solution at neutral pH, in particular about pH 7; and / or

[0118] (b) has a binding affinity for human tPA of less than 1 pM, as determined by SPR.

[0119] In some embodiments, the invention refers to a human tPA affinity ligand of the present invention as described herein, or a multimeric human tPA affinity ligand of the present invention as described herein, or a multimer of the present invention as described herein, or a fusion protein of the present invention as described herein, which is capable of binding to human tPA in a solution at pH 6.5 to pH 7.5, preferably pH 6.7 to pH 7.3, or which is capable of isolating human tPA from a solution at pH 6.5 to pH 7.5, preferably pH 6.7 to pH 7.3, and / or (b) has a binding affinity for human tPA of less than 1 pM, as determined by SPR.

[0120] In some embodiments, the invention refers to a fusion protein of the present invention, which has a binding affinity of less than 1 pM for recombinant human tPA as described herein. In some embodiments, the fusion protein of the present invention has a binding affinity of less than 500 nM for recombinant human tPA as described herein. In some embodiments, the fusion protein of the present invention has a binding affinity of less than 100 nM for recombinant human tPA as described herein. In preferred embodiments, the fusion protein of the present invention has a binding affinity of less than 50 nM for recombinant human tPA as described herein. In preferred embodiments, the fusion protein of the present invention has a binding affinity of less than 20 nM for recombinant human tPA as described herein.

[0121] Molecules for purification. In one embodiment, additional amino acid sequences include one or more peptide sequences that confer an affinity to certain chromatography column materials. The affinity ligand for human tPA may include specific attachment sites for the attachment to solid supports, preferably at the C-terminal end, such as cysteine or lysine.

[0122] Affinity separation matrix. One embodiment of the invention refers to an affinity separation matrix comprising an affinity ligand for the human tPA of the present invention as described herein, or a multimeric human tPA affinity ligand of the present invention as described herein, or a multimer of the present invention as described herein, or a fusion protein of the present invention as described herein. One particular embodiment refers to such an affinity separation matrix wherein the human tPA affinity ligand comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence with at least 85 % sequence identity thereto as described elsewhere herein. In preferred embodiments, the affinity separation matrix is a solid support. The affinity separation matrix comprises at least one affinity ligand for human tPA of the present invention as described herein.

[0123] Solid support matrices for affinity chromatography are known in the art and include, e.g., without being limited thereto, agarose and stabilized derivatives of agarose, cellulose or derivatives of cellulose, controlled pore glass, monolith, silica, zirconium oxide, titanium oxide, or synthetic polymers, and hydrogels of various compositions and combinations of the above.

[0124] The formats for solid support matrices can be of any suitable well-known kind. Such solid support matrix for coupling a novel affinity ligand of the present invention might comprise, e.g., one of the following, without being limited thereto: columns, capillaries, particles, membranes, filters, monoliths, fibers, pads, gels, slides, plates, cassettes, or any other format commonly used in chromatography and known to someone skilled in the art.

[0125] In one embodiment, the matrix is comprised of substantially spherical beads, for example Sepharose or Agarose beads. Matrices in particle form can be used as a packed bed or in a suspended form including expanded beds. In other embodiments of the invention, the solid support matrix is a membrane, for example a hydrogel membrane. In some embodiments, the affinity purification may involve a membrane as a matrix to which the affinity ligand for human tPA as described herein is covalently bound. The solid support can also be in the form of a membrane in a cartridge.

[0126] Use in affinity purification. Some embodiments relate to the use of the novel affinity ligand for recombinant human tPA of the present invention as disclosed herein, or a multimeric human tPA affinity ligand of the present invention as described herein, or a multimer of the present invention as described herein, or a fusion protein of the present invention as described herein, or a solid phase support or affinity separation matrix of the present invention as described herein, for the affinity purification of human tPA, sample analysis for the detection of human tPA, or detection methods for the detection of human tPA.

[0127] Some embodiments relate to the use of the affinity separation matrix of the present invention comprising the novel affinity ligand for human tPA of the present invention as disclosed herein, for the affinity purification of human tPAs. Accordingly, the affinity separation matrix of the present invention comprising a tPA affinity ligand of the present invention as disclosed herein is encompassed for use in the affinity purification of a human tPA.

[0128] Method for affinity purification of human tPA. As described herein, affinity chromatography (also called affinity purification) makes use of specific binding interactions between molecules. Methods for immobilization of protein and methods for affinity chromatography are well-known in the field of protein purification and can be easily performed by a skilled person in this field using standard techniques and equipment.

[0129] The present invention provides a method for purification of human tPA, the method comprising: (i) contacting a liquid containing human tPA with the affinity ligand of the present invention as described herein, or an affinity separation matrix of the present invention as described herein; (ii) purifying the human tPA; and (iii) obtaining the human tPA.

[0130] The present invention provides a method for purification of human tPA, the method comprising:

[0131] (i) providing a liquid that contains human tPA; (ii) providing an affinity separation matrix of the present invention comprising at least one affinity ligand of the present invention as described herein coupled to said affinity separation matrix; (iii) contacting said affinity separation matrix of

[0132] (ii) with the liquid of (i); (iv) purifying said human tPA from said affinity separation matrix, and (v) obtaining the human tPA.

[0133] Some embodiments refer to a method of purification as described above, the method comprising eluting the human tPA from the affinity separation matrix at pH 4.0 or higher. By eluting the fusion protein at mild acidic to neutral pH (in particular pH 6.7 to pH 7.3, such as pH 7.0), aggregation of the fusion protein is prevented.

[0134] In various embodiments, the method of affinity purification of the present invention may further comprise one or more washing steps after elution of human tPA from the affinity separation matrix, by using a suitable solution such as sodium hydroxide.

[0135] In various embodiments, the method of affinity purification of the present invention may further comprise one or more steps for isolating the purified human tPA via methods that are known to a person skilled in the art.

[0136] In some embodiments, the method of purification of the present invention involves a chromatography column containing a solid support (i.e. affinity separation matrix) to which the affinity ligand of the present invention as described herein is covalently bound. The affinity ligand of the present invention as described herein may be attached to a suitable solid support via conventional coupling techniques. Methods for immobilization of protein ligands to solid supports are well-known in the field of protein engineering and purification and can easily be performed by a skilled person in this field using standard techniques and equipment.

[0137] In various embodiments, the affinity ligands of the present invention as disclosed herein generated or obtained by any of the methods as described below are conjugated to a solid support. In some embodiments, the affinity ligand comprises an attachment site for site-specific covalent coupling of an affinity ligand of the present invention to a solid support. Specific attachment sites comprise without being limited thereto, natural amino acids, such as cysteine or lysine, which enable specific chemical reactions with a reactive group of the solid phase, or a linker between the solid phase and the protein, i.e., a tPA affinity ligand of the present invention. Polynucleotides, vectors, host cells. Some embodiments refer to a polynucleotide encoding the human tPA affinity ligand of the present invention as described herein, or the multimeric human tPA affinity ligand of the present invention as described herein, or the multimer of the present invention as described herein, or the fusion protein of the present invention as described herein.

[0138] The present invention encompasses an isolated polynucleotide or nucleic acid molecule encoding an affinity ligand for human tPA affinity ligand of the present invention as described herein. A further embodiment also encompasses proteins encoded by the polynucleotides.

[0139] Further provided is a vector, in particular an expression vector, comprising the isolated polynucleotide or nucleic acid molecule of the present invention encoding the affinity ligand of the present invention as described herein, as well as a host cell comprising the isolated polynucleotide or the expression vector of the present invention. For example, one or more polynucleotides, which encode the affinity ligand of the present invention as disclosed herein, may be expressed in a suitable host (cell) and the produced protein can be isolated. A vector means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) that can be used for transfer of protein-encoding information into a host cell. Suitable vectors that may be applied are known in the art.

[0140] Suitable host cells include prokaryotes or eukaryotes, for example a bacterial host cell, a yeast host cell or a non-human host cell carrying a vector. A suitable host may be a microbial expression host that is capable to express the protein of the invention. Suitable bacterial expression host cells or systems are known in the art. Various mammalian or insect cell culture systems as known in the art can also be employed to express recombinant proteins.

[0141] Furthermore, an isolated cell comprising a polynucleotide or nucleic acid of the present invention, or a vector of the present invention, as disclosed herein, is provided. Method for producing human tPA. The present invention provides a method for producing recombinant human tPA, the method comprising: (i) providing a liquid containing recombinant human tPA; (ii) contacting the liquid of (i) with an affinity ligand of the present invention as described herein, or contacting the liquid of (i) with the affinity separation matrix of the present invention as described herein; and (iii) purifying the recombinant human tPA.

[0142] In various embodiments of the method for producing recombinant human tPA, the method comprises eluting the recombinant human tPA from the affinity separation matrix at pH 4.0 or higher. By eluting the fusion protein at mild acidic (e.g., pH 4.0 to pH 4.3) aggregation of the human tPA is prevented.

[0143] In various embodiments, the method of producing a human tPA may further comprise one or more washing steps.

[0144] Isolating or purifying human tPA. The present invention provides a method for isolating or purifying human tPA from a liquid or liquid sample, the method comprising:

[0145] (a) contacting a human tPA affinity ligand of the present invention as described herein, or a multimeric human tPA affinity ligand of the present invention as described heren, or a multimer of the present invention as described herein, or a fusion protein of the present invention as described herein, with a liquid or a liquid sample containing human tPA;

[0146] (b) separating the human tPA bound in step (a) to the human tPA affinity ligand, or to the multimeric human tPA affinity ligand, or to the multimer, or to the fusion protein, respectively, from the liquid or the liquid sample;

[0147] (c) detaching or eluting said human tPA from the human tPA affinity ligand, or the multimeric human tPA affinity ligand, or the multimer, or the fusion protein, respectively.

[0148] The present invention further provides a method for isolating or purifying tissue plasminogen activator (human tPA) from a liquid or a liquid sample, the method comprising:

[0149] (a) contacting a solid phase support or affinity separation matrix of the present invention as described herein with a liquid or a liquid sample containing human tPA, thereby binding of human tPA in the liquid or the liquid sample to the solid phase support or affinity separation matrix;

[0150] (b) detaching or eluting said human tPA from the solid phase support or affinity separation matrix, respectively.

[0151] Methods to analyze the presence of human tPA. The present invention further provides the use of an affinity ligand for human tPA of the present invention as described herein, or an affinity matrix of the present invention as described herein in methods to determine or analyze the presence of human tPA.

[0152] One embodiment refers to a method for analyzing the presence of tissue plasminogen activator (human tPA) in a liquid or a liquid sample, the method comprising:

[0153] (a) contacting a human tPA affinity ligand of the present invention as described herein, or a multimeric human tPA affinity ligand of the present invention as described herein, or a multimer of the present invention as described herein, or a fusion protein of the present invention as described herein, or a solid phase support or affinity separation matrix of the present invention as described herein, with a liquid or a liquid sample containing human tPA;

[0154] (b) determining the human tPA bound in step (a) to the human tPA affinity ligand, or the multimeric human tPA affinity ligand, or the multimer, or the fusion protein, or the solid phase support or affinity separation matrix, respectively;

[0155] (c) optionally detaching or eluting said human tPA from the human tPA affinity ligand, or the multimeric human tPA affinity ligand, or the multimer, or the fusion protein, or the solid phase support or affinity separation matrix, respectively, prior to determining the human tPA.

[0156] In some embodiments of the method of analyzing the presence of human tPA of the present invention in a liquid or a liquid sample, the method comprises in step (c) the elution of human tPA at pH 4.0 or higher. The elution may be performed under conditions of mild acidic pH (e.g., pH 4.0 to pH 4.3).

[0157] Method of quantification of human tPA. The present invention further provides a method of quantification of a human tPA, the method comprising: (i) providing a liquid or a liquid sample that contains human tPA; (ii) providing a matrix to which the affinity ligand of the present invention as described herein is covalently coupled; (iii) contacting said affinity separation matrix with the liquid under conditions that permit binding; (iv) eluting said human tPA, preferably at pH 4.0 or higher; and (v) quantitating the amount of eluted human tPA. The elution may be performed under conditions of mild acidic pH (e.g., pH 4.0 to pH 4.3). Methods to determine the presence of human tPA in liquids or in liquid samples might be quantitative or qualitative. Such methods are well known to the skilled person and can be selected, for instance but limited to, from the following methods that are well established in the art: enzyme-linked immunosorbent assay (ELISA), enzymatic reactions, surface plasmon resonance (SPR), or chromatography.

[0158] Method of producing the affinity ligand of the invention. The present invention further provides a method for the production of the affinity ligand of the present invention as described herein, the method comprising the step(s): (i) culturing a (suitable) host cell under conditions suitable for the expression of the affinity ligand so as to obtain said affinity ligand; and (ii) optionally isolating said affinity ligand. Suitable conditions for culturing a prokaryotic or eukaryotic host are well known to a person skilled in the art.

[0159] The affinity ligand of the present invention as described herein may be prepared by any conventional and well-known techniques such as plain organic synthetic strategies, solid phase- assisted synthesis techniques, or by commercially available automated synthesizers. They may also be prepared by conventional recombinant techniques, alone or in combination with conventional synthetic techniques.

[0160] The present invention provides a method for the preparation of the affinity ligand for human tPA, as detailed above, said method comprising the steps: (i) providing a nucleic acid molecule encoding the affinity ligand of the present invention as described herein; (ii) introducing said nucleic acid molecule into an expression vector; (iii) introducing said expression vector into a host cell; (iv) culturing the host cell in a culture medium; (v) subjecting the host cell to culturing conditions suitable for expression thereby producing the affinity ligand; optionally (vi) isolating the polypeptide produced in step (v); and (vii) optionally conjugating the affinity ligand to a solid matrix as described above. In various embodiments of the present invention the production of the affinity ligand for human tPA of the present invention is performed by cell-free in vitro transcription and translation.

[0161] EXAMPLES

[0162] The following Examples are provided for further illustration of the invention. The invention, however, is not limited thereto, and the following Examples merely show the practicability of the invention on the basis of the above description. For a complete disclosure of the invention reference is made also to the literature cited in the application which is incorporated completely into the application by reference.

[0163] EXAMPLE 1. Purification of affinity ligands

[0164] The untagged proteins were purified by using an AKTA avant system (Cytiva). Initial small-scale purifications were performed via IgG Sepharose (Cytiva) followed by size exclusion chromatography (Cytiva: Superdex200 26 / 600 or Sephacryl S200) in 20 mM citric acid, 150 mM NaCI, 1 mM EDTA, 5mM TCEP pH 6.5. The initial capturing step of the scaled-up process was performed by using a SP65 cation exchange chromatography column (Purolite; binding buffer: 20 mM BisTris, 1 mM EDTA, 100 mM acetic acid pH 4; elution buffer: 40 mM Tris, 1 mM EDTA, 1 mM DTT pH 8). Afterwards the pooled elution fractions were polished using an anion exchange chromatography (Q65, Purolite; binding buffer: 20 mM Tris, 1 mM EDTA, 1 mM DTT pH 8; elution buffer: 20 mM Tris, 1 mM EDTA, 1 M NaCI, 1 mM DTT pH 8). Finally, the protein was buffer exchanged and concentrated using a SartoFlow Smart TFF system (Sartorius). Protein concentrations were determined by absorbance measurement at 280 nm using the molar absorbent coefficient. Further analysis included RP-HPLC and SE-HPLC. Reversed phase chromatography (RP-HPLC) has been performed using an Agilent 1290 Infinity II System and a PLRP-S (5 pm, 300 A) column (Agilent). The resulting purity of all proteins was >95 %. Analytic size exclusion chromatography (SE-HPLC) has been performed using an Agilent 1290 Infinity II System and a Superdex75 increase 5 / 150 GL or Superdex200 increase 5 / 150 GL (both Cytiva). No aggregation or oligomers were obtained.

[0165] EXAMPLE 2. Binding analysis of affinity ligands by SPR The purified proteins were immobilized on a High Capacity Amine sensor chip (Bruker) using PDEA after NHS / EDC activation with Sierra SPR-32 system (Bruker). The chip was equilibrated with SPR running buffer (PBS 0.05 % Tween, 500 mM NaCI pH 7.3). Upon binding, target analyte was accumulated on the surface increasing the refractive index. This change in the refractive index was measured in real time and plotted as response or resonance units versus time. The analyte (recombinant human tPA) was applied to the chip in serial dilutions with a flow rate of 30 pl / min. The association was performed for 180 seconds and the dissociation for 240 seconds. After each run, the chip surface was regenerated with 30 pl regeneration buffer (10 mM glycine pH 2.0) and equilibrated with running buffer. Binding studies were carried out using Sierra SPR- 32 system (Bruker); data evaluation was operated via Sierra Analyser software, provided by the manufacturer. For comparative analysis of different ligands a report point fit at the steady state was used (Table 1). The final analysis for lead candidates was done using the Langmuir 1 :1 model (RMin=0). Evaluated dissociation constants (KD) were standardized against the immobilized protein and indicated. Shown is the change in refractive index measured in real time and plotted as response or resonance unit [RU] versus time [sec]. Ligands bind to human recombinant human tPA with a KD below 1 pM.

[0166] The multimer SEQ ID NO: 3 comprises from N- to C-terminal SEQ ID NO: 2 and SEQ ID NO: 1. Multimers of SEQ ID NO: 5 and SEQ ID NO: 6 comprise SEQ ID NO: 2. All ligands comprise at least 85 % identical amino acid sequences to SEQ ID NO: 1 and at least 85 % identical amino acid sequences to SEQ ID NO: 2.

[0167] SEQ ID NOs: 3-11 bind with only very low affinity to hamster tPA. The binding to hamster tPA is neglectable (at least 10 x less binding affinity compared to the binding to human tPA).

[0168] SEQ ID NO: 12 and SEQ ID NO: 22 do not bind to recombinant human tPA.

[0169] Mutants of the dimers 229389 (SEQ ID NO: 7), 229381 (SEQ ID NO: 9), 229393 (SEQ ID NO: 10), 229390 (SEQ ID NO: 8) having W74Y or W74F showed no binding to recombinant human tPA. The tryptophan in position 74 of the dimeric human tPA ligands seems to be essential for binding to recombinant human tPA. Position 74 of the dimeric human tPA ligand corresponds to position 18 of SEQ I D NO: 1 .

[0170] Table 1. Steady state binding affinities of ligands to recombinant human tPA

[0171] EXAMPLE 3. Purification of recombinant human tPA via affinity ligands

[0172] High coupling density to resin. Affinity ligands were purified to homogeneity and immobilized at 20 mg per mL on activated Praesto Epoxy Jetted 50 or Praesto Epoxy Jetted 80 (Purolite) according to the manufacturer’s instructions, coupling conditions: 45 °C for 3 h, pH 9.5 per mL resin. All ligands were successfully coupled to the epoxy activated resins. The achieved coupling density was about 15-20 mg / ml for coupled ligands.

[0173] DBC10%. Coupled resin was packed into super compact 5 / 50 column (Gotec GmbH) or omnifit 6.6 / 100 column (Diba). Human tPA was used as load (cone. 0.2 mg / ml; in 1x PBS, 500 mM NaCI, 0.04% Tween20, pH 7.3). The sample was applied to the matrix comprising immobilized affinity ligand until 10 % target breakthrough at 6 min residence time. Unbound sample was washed off with 1xPBS, pH 7.3. The loaded sample was quantified and calculated as dynamic binding capacity DBC10 %. Results: The binding capacity (DBC10) for affinity ligand for 230645 29 mg / ml, for 229381 29 mg / ml, 230669 was 26 mg / ml, for 229393 23 mg / ml, and for 230389 20 mg / ml. Caustic stability reflects the remaining binding capacity (in %) compared to the binding capacity at 0 h.

[0174] Caustic stability. Columns were incubated with 0.1 M NaOH for 25 h at room temperature (22 °C + / - 3 °C); 2 sets were measured. DBC10 was measured after incubation with 0.1 M NaOH and compared to DBC10 values without NaOH incubation. Results: After 25 h in 0.1 M NaOH, 230669 showed >90 %, 230645 has 89 % remaining target binding capacity, 230670 has 82 % remaining binding capacity, 230679 has 81 % remaining binding capacity (DBC10 in %).

[0175] Elution of human tPA at pH 4.2. Resin with coupled affinity ligand 229389 (SEQ ID NO: 7), or 230669 (SEQ ID NO: 3), or 230645 (SEQ ID NO: 4) was loaded with recombinant human tPA (cone. 0.2 mg / ml; in 1x PBS, 500 mM NaCI, 0.04% Tween20, pH 7.3) with a contact time of 6 minutes a wash step with 1xPBS, 500 mM NaCI + 0,04% Tween20, pH 7.3 was included to remove unbound sample followed by a second wash step with 50 mM acetic acid pH 4.8 to pH 5.0. The bound sample was eluted with 100 mM acetic acid at pH 4.2. >91 % of recombinant human tPA could be eluted at pH 4.2 via resin with coupled 229389 (SEQ ID NO: 7). 89 % of recombinant human tPA could be eluted at pH 4.2 via resin with coupled 230645 (SEQ ID NO: 4). >90 % of recombinant human tPA could be purified at pH 4.2 via resin with coupled 230669 (SEQ ID NO: 3). Eluted human tPA showed high purity on SDS-PAGE.

[0176] No variation was observed for two glycoforms of human tPA, Type I human tPA (glycosylated at Asn-219) and Type II human tPA (not glycosylated at Asn-219) ratios between load and elution fractions. EXAMPLE 4. Validation of purified human tPA

[0177] Human tPA was analyzed for homogeneity using size exclusion chromatography (SE-HPLC). Analysis has been performed on Agilent 1290 Infinity II HPLC system and a Superdex200 increase 5 / 150 GL (Cytiva). No aggregation of eluted fusion protein was detected.

[0178] EXAMPLE 5. Ligand detection in Protein A ELISA (leaching assay)

[0179] To determine low levels of leached variants in affinity chromatography is important for obtaining reliable results. Protein A ELISA Kits for the detection of native and recombinant Protein A (Repligen, Cat. No. 9000-1) were used for leaching assays according to manufacturer’s instructions, except using 0.1 % PBST as dilution buffer. Samples: eluted fraction of Praesto Jetted Epoxy50_230669 (SEQ ID NO: 3 immobilized to Praesto Jetted Epoxy50). The leaching of the affinity ligand was <25 ng per mg human tPA and showed good detectability in PBST buffer, comparable to rProtein A standard.

[0180] SEQUENCES

[0181] SEQ ID NO: 1: Affinity ligand for human tPA

[0182] AKFDKAQQAAFYKILHLWNLTEEQRNAFIQSLRDDPSVSYEVLAEAYKLNKAQAPK

[0183] SEQ ID NO: 2: Affinity ligand for human tPA

[0184] AKFDEAQQAAFYEILHLPNLTEKQRNQFIQYLRDDPSVSLEVLAEAQKLNDSQAPK

[0185] SEQ ID NO: 3: Affinity ligand for human tPA (dimer 230669; dimer of SEQ ID NO: 2 and SEQ ID NO: 1)

[0186] AKFDEAQQAAFYEILHLPNLTEKQRNQFIQYLRDDPSVSLEVLAEAQKLNDSQAPKAKFDKAQQ

[0187] AAFYKILHLWNLTEEQRNAFIQSLRDDPSVSYEVLAEAYKLNKAQAPK

[0188] SEQ ID NO: 4: Affinity ligand for human tPA (dimer 230645)

[0189] AKFDEAQQAAFYEILHLPNLTEKQRNQFIQYLRDDPSVSLKVLAEAQKLNDSQAPKAKFDEAQQ

[0190] AAFYKILHLWNLTEEQRNAFIQSLRDDPSVSYEVLAEAYKLNKSQAPK

[0191] SEQ ID NO: 5: Affinity ligand for human tPA (dimer 230671)

[0192] AKFDEAQQAAFYEILHLPNLTEKQRNQFIQYLRDDPSVSLEVLAEAQKLNDSQAPKAKFDEAQQ

[0193] AAFYKILHLWNLTEEQRNAFIQSLRDDPSVSYEVLAEAYKLDKSQAPK

[0194] SEQ ID NO: 6: Affinity ligand for human tPA (dimer 230670)

[0195] AKFDEAQQAAFYEILHLPNLTEKQRNQFIQYLRDDPSVSLEVLAEAQKLNDSQAPKAQFDEIQQ

[0196] AAFYKILHLWNLTEEQRNAFIQSLRDDPSVSYEVLAEAYKLNKSQAPK

[0197] SEQ ID NO: 7: Affinity ligand for human tPA (dimer 229389)

[0198] AKFDEAQQAAFYEILHLPNLTEKQRNQFIQYLRDDPSVSLKVLGEAQKLNDSQAPKIAAKFDEA

[0199] QQAAFYKILHLWNLTEEQRNAFIQSLRDDPSVSYEVLGEAYKLNKSQAPK

[0200] SEQ ID NO: 8: Affinity ligand for human tPA (dimer 229390)

[0201] AKFDEAQQAAFYEILHLPNLTEKQRNLFIQLLRDDPSVSLHVLGVAQTLNDSQAPKIAAKFDEAQ

[0202] QAAFYKILHLWNLTEEQRNAFIQSLRDDPSVSREVLGEAYKLNKSQAPK

[0203] SEQ ID NO: 9: Affinity ligand for human tPA (dimer 229381)

[0204] AKFDEAQQAAFYEILHLPNLTEQQRNEFIQELRDDPSVSLQVLGVAQALNDSQAPKIAAKFDEA

[0205] QQAAFYKILHLWNLTEEQRNAFIQSLRDDPSVSREVLGEAYKLNKSQAPK SEQ ID NO: 10: Affinity ligand for human tPA (dimer 229393)

[0206] AKFDEAQQAAFYEILHLPNLTEQQRNRFIQVLRDDPSVSLTVLGAAQLLNDSQAPKIAAKFDEA

[0207] QQAAFYKILHLWNLTEEQRNAFIQSLRDDPSVSREVLGEAYKLNKSQAPK

[0208] SEQ ID NO: 11: Affinity ligand for human tPA (dimer 230679)

[0209] AQFDKIQQAAFYEILHLPNLTEKQRNQFIQYLRDDPSVSKKILAEAKKLNDAQAPKAQFDEIQQA

[0210] AFYKI LH LWN LTEKQRHQFIQYLRDDPSVSYEI LAEAYKLN KAQAPK

[0211] SEQ ID NO: 12: non tPA binding protein

[0212] AQH DKIQQAADKEI LH LPN LTEEQRN KFRQSLRDDPSVSAEI LAEAKKLN DAQAPK

[0213] SEQ ID NO: 13: Affinity ligand for human tPA (fusion protein 230669)

[0214] AKFDEAQQAAFYEILHLPNLTEKQRNQFIQYLRDDPSVSLEVLAEAQKLNDSQAPKAKFDKAQQ

[0215] AAFYKILHLWNLTEEQRNAFIQSLRDDPSVSYEVLAEAYKLNKAQAPKAQHDKIQQAADKEILHL

[0216] PNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKAQHDKIQQAADKEILHLPNLTEEQR NKFRQSLRDDPSVSAEILAEAKKLNDAQAPK

[0217] SEQ ID NO: 14: Affinity ligand for human tPA (fusion protein 230645)

[0218] AKFDEAQQAAFYEILHLPNLTEKQRNQFIQYLRDDPSVSLKVLAEAQKLNDSQAPKAKFDEAQQ

[0219] AAFYKILHLWNLTEEQRNAFIQSLRDDPSVSYEVLAEAYKLNKSQAPKAKFDEAQQAAFYEILHL PNLTEKQRNQFIQYLRDDPSVSLKVLAEAQKLNDSQAPKAKFDEAQQAAFYKILHLWNLTEEQ RNAFIQSLRDDPSVSYEVLAEAYKLNKSQAPK

[0220] SEQ ID NO: 15: Affinity ligand for human tPA (fusion protein 230671)

[0221] AKFDEAQQAAFYEILHLPNLTEKQRNQFIQYLRDDPSVSLEVLAEAQKLNDSQAPKAKFDEAQQ

[0222] AAFYKILHLWNLTEEQRNAFIQSLRDDPSVSYEVLAEAYKLDKSQAPKAQHDKIQQAADKEILHL

[0223] PNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKAQHDKIQQAADKEILHLPNLTEEQR NKFRQSLRDDPSVSAEILAEAKKLNDAQAPKC

[0224] SEQ ID NO: 16: Affinity ligand for human tPA (fusion protein 230670)

[0225] AKFDEAQQAAFYEILHLPNLTEKQRNQFIQYLRDDPSVSLEVLAEAQKLNDSQAPKAQFDEIQQ

[0226] AAFYKILHLWNLTEEQRNAFIQSLRDDPSVSYEVLAEAYKLNKSQAPKAQHDKIQQAADKEILHL

[0227] PNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKAQHDKIQQAADKEILHLPNLTEEQR NKFRQSLRDDPSVSAEILAEAKKLNDAQAPKC

[0228] SEQ ID NO: 17: Affinity ligand for human tPA (fusion protein 229389) AKFDEAQQAAFYEILHLPNLTEKQRNQFIQYLRDDPSVSLKVLGEAQKLNDSQAPKIAAKFDEA QQAAFYKILHLWNLTEEQRNAFIQSLRDDPSVSYEVLGEAYKLNKSQAPKIAAQHDKIQQAADK

[0229] EILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKIAAQHDKIQQAADKEILHLPNL TEEQRN KFRQSLRDDPSVSAEI LAEAKKLN DAQAPKC

[0230] SEQ ID NO: 18: Affinity ligand for human tPA (fusion protein 229381)

[0231] AKFDEAQQAAFYEILHLPNLTEQQRNEFIQELRDDPSVSLQVLGVAQALNDSQAPKIAAKFDEA QQAAFYKILHLWNLTEEQRNAFIQSLRDDPSVSREVLGEAYKLNKSQAPKIAAQHDKIQQAADK

[0232] EILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKIAAQHDKIQQAADKEILHLPNL TEEQRN KFRQSLRDDPSVSAEI LAEAKKLN DAQAPKC

[0233] SEQ ID NO: 19: Affinity ligand for human tPA (fusion protein 230679)

[0234] AQFDKIQQAAFYEILHLPNLTEKQRNQFIQYLRDDPSVSKKILAEAKKLNDAQAPKAQFDEIQQA AFYKILHLWNLTEKQRHQFIQYLRDDPSVSYEILAEAYKLNKAQAPKAQHDKIQQAADKEILHLP NLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKAQHDKIQQAADKEILHLPNLTEEQRN

[0235] KFRQSLRDDPSVSAEI LAEAKKLN DAQAPKC

[0236] SEQ ID NO: 20: Affinity ligand for human tPA (fusion protein 229373)

[0237] AKFDEAQQAAFYEILHLPNLTEQQRNRFIQVLRDDPSVSLTVLGAAQLLNDSQAPKIAAKFDEA QQAAFYKILHLWNLTEEQRNAFIQSLRDDPSVSREVLGEAYKLNKSQAPKIAAQHDKIQQAADK

[0238] EILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKIAAQHDKIQQAADKEILHLPNL TEEQRN KFRQSLRDDPSVSAEI LAEAKKLN DAQAPKC

[0239] SEQ ID NO: 21 : human tPA (amino acids 36-562 of the amino acid sequence of UniProt P00750) SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGT CQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQ KPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGN

[0240] GSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVL KNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCG

[0241] GILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIAL LQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHVRLYP

[0242] SSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWG LGCGQKDVPGVYTKVTNYLDWIRDNMRP

[0243] SEQ ID NO: 22 Non-tPA binding protein

[0244] IAAKFDEAQQAAFYEILHLPNLTEEQRNAFIQSLRDDPSVSLEVLGEAQKLNDSQAPK

[0245] ***

Claims

CLAIMS1. An affinity ligand for tissue plasminogen activator (tPA), comprising the amino acid sequence of SEQ ID NO: 1 or a sequence with at least 85 % sequence identity thereto, wherein the tPA affinity ligand comprises(i) a tryptophan (W) at the position corresponding to position 18 of SEQ ID NO: 1 ;(ii) a basic amino acid at the position corresponding to position 13 of SEQ ID NO: 1 ;(iii) an aromatic amino acid at the position corresponding to position 47 of SEQ ID NO: 1 ; and(iv) a basic amino acid at the position corresponding to position 51 of SEQ ID NO: 1.

2. The affinity ligand for tPA according to claim 1 , wherein:(i) the basic amino acid at the position corresponding to position 13 of SEQ ID NO: 1 is lysine (K);(ii) the aromatic amino acid at the position corresponding to position 47 of SEQ ID NO: 1 is tyrosine (Y); and / or(iii) the basic amino acid at the position corresponding to position 51 of SEQ ID NO: 1 is lysine (K).

3. A multimeric affinity ligand for tissue plasminogen activator (tPA) comprising a first monomer and a second monomer, wherein:(a) the first monomer comprises the amino acid sequence of SEQ ID NO: 1 or a sequence with at least 85 % sequence identity thereto, wherein the first monomer comprises (i) a tryptophan (W) at the position corresponding to position 18 of SEQ ID NO: 1 , (ii) a basic amino acid at the position corresponding to position 13 of SEQ ID NO: 1 , (iii) an aromatic amino acid at the position corresponding to position 47 of SEQ ID NO: 1 , and (iv) a basic amino acid at the position corresponding to position 51 of SEQ ID NO: 1 ; and(b) the second monomer comprises the amino acid sequence of SEQ ID NO: 2 or a sequence with at least 85 % sequence identity thereto.

4. The multimeric tPA affinity ligand according to claim 3, wherein:(i) the basic amino acid at the position corresponding to position 13 of SEQ I D NO: 1 is lysine (K);(ii) the aromatic amino acid at the position corresponding to position 47 of SEQ ID NO: 1 is tyrosine (Y); and / or(iii) the basic amino acid at the position corresponding to position 51 of SEQ I D NO: 1 is lysine (K).

5. The multimeric tPA affinity ligand according to claim 3 or 4, which is a dimer.

6. The multimeric tPA affinity ligand according to any one of claims 3 to 5, comprising an amino acid sequence with at least 85 % sequence identity to any one of SEQ ID NOs: 3 to 11.

7. The tPA affinity ligand according to claims 1 or 2, or the multimeric tPA affinity ligand according to any one of claims 3 to 6, which does not exhibit a detectable binding affinity to hamster tPA.

8. A fusion protein comprising:(a) tPA affinity ligand according to any one of claims 1 , 2, and 7, or the multimeric tPA affinity ligand according to any one of claims 3 to 7; and(b) a protein with at least 85 % identity to SEQ ID NO: 12, which has no detectable binding affinity for tPA.

9. A solid phase support, or an affinity separation matrix, comprising the tPA affinity ligand according to any one of claims 1 , 2, and 7, or the multimeric tPA affinity ligand according to any one of claims 3 to 7, or the fusion protein according to claim 8.

10. Use of the tPA affinity ligand according to any one of claims 1 , 2, and 7, or the multimeric tPA affinity ligand according to any one of claims 3 to 7, or the fusion protein according to claim 8, or the solid phase support or affinity separation matrix according to claim 9, in(a) affinity purification of tPA;(b) sample analysis for the detection of tPA; or(c) detection methods for the detection of tPA.

11. A polynucleotide encoding the tPA affinity ligand according to any one of claims 1 , 2, and 7, or the multimeric tPA affinity ligand according to any one of claims 3 to 7, or the fusion protein according to claim 8.

12. A method for isolating or purifying tissue plasminogen activator (tPA), the method comprising:(a) contacting the tPA affinity ligand according to any one of claims 1 , 2, and 7, or the multimeric tPA affinity ligand according to any one of claims 3 to 7, or the fusion protein according to claim 8, with a sample containing tPA;(b) separating the tPA bound in step (a) to the tPA affinity ligand, or the multimeric tPA affinity ligand, or the fusion protein, respectively, from the sample;(c) detaching or eluting said tPA from the tPA affinity ligand, or the multimeric tPA affinity ligand, or the fusion protein, respectively.

13. A method for isolating or purifying tissue plasminogen activator (tPA), the method comprising:(a) contacting the solid phase support or affinity separation matrix according to claim 9 with a sample containing tPA, thereby binding of tPA in the sample to the solid phase support or affinity separation matrix;(b) detaching or eluting said tPA from the solid phase support or affinity separation matrix, respectively.

14. A method for determining the presence of tissue plasminogen activator (tPA) in a liquid sample, the method comprising:(a) contacting the tPA affinity ligand according to any one of claims 1 , 2 , and 7, or the multimeric tPA affinity ligand according to any one of claims 3 to 7, or the fusion protein according to claim 8, or the solid phase support or affinity separation matrix according to claim 9, with a liquid sample containing tPA;(b) determining the tPA bound in step (a) to the tPA affinity ligand, or the multimeric tPA affinity ligand, or the fusion protein, or the solid phase support or affinity separation matrix, respectively.(c) optionally detaching or eluting said tPA from the tPA affinity ligand, or the multimeric tPA affinity ligand, or the fusion protein, or the solid phase support or affinity separation matrix, respectively, prior to determining the tPA.

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