WO2017144099A1

WO2017144099A1 - Affinity ligand for the purification of glycosylated biomolecules

Info

Publication number: WO2017144099A1
Application number: PCT/EP2016/053950
Authority: WO
Inventors: Hans Henning Von Horsten
Original assignee: Hochschule Für Technik Und Wirtschaft (Htw) Berlin
Priority date: 2016-02-25
Filing date: 2016-02-25
Publication date: 2017-08-31
Also published as: EP3419987A1

Abstract

The present invention relates to a compound of formula (I) wherein the substituents are as defined in the claims. Said compound may be used as a ligand in order to purify glycosylated bio molecules. The present invention further relates to a complex comprising said compound and a Lewis-acidic transition metal ion or to a complex comprising said compound and boric acid or diboronic acid. The present invention also relates to methods for producing an affinity matrix for the purification of glycosylated biomolecules. In addition, the present invention relates to an affinity matrix obtainable by said methods.

Description

AFFINITY LIGAND FOR THE PURIFICATION OF GLYCOSYLATED BIOMOLECULES

The present invention relates to a compound. Said compound may be used as a ligand in order to bind and purify glycosylated biomolecules. The present invention further relates to a complex comprising said compound and a Lewis-acidic transition metal ion or to a complex comprising said compound and boric acid or diboronic acid. The present invention also relates to methods for producing an affinity matrix for the purification of glycosylated biomolecules. In addition, the present invention relates to an affinity matrix obtainable by said methods. BACKGROUND OF THE INVENTION

Affinity media containing protein ligands have long been employed in the purification of biomolecules. One key limitation of current affinity media equipped with protein ligands is diffusive mass transfer. The principle factor restricting the overall rate of glycoprotein adsorption to an affinity column is the rate of diffusion of the targeted adsorbate from the particle surface to its adsorption sites within the porous structure of the particle. When it comes to the affinity capture of macromolecules such as therapeutic glycoproteins, it is important to note that the rates of intra- particle diffusion are reciprocally proportional to the adsorbate size. These limitations of diffusive mass transfer are especially enhanced when the ligand employed is itself a macro molecule such as protein A, an antibody or another protein molecule. Because of its size Protein A itself occupies a large amount of intrapore space in porous media and the presence of this large ligand within the porous structure of the matrix hinders diffusion of monoclonal antibody (mAb) adsorbate molecules to their binding sites. The dynamic binding capacities (DBCs) of current protein A resins and other affinity media with protein ligands are still too low to allow for sufficiently short enough processing times. Due to non-equilibrium mass transfer effects in the porous resin, dynamic binding capacity decreases with increasing concentration of the target glycoprotein at short residence times. Taken together, protein-based affinity ligands contribute to a limitation of binding capacity that is not present with small molecule affinity ligands.

Another major issue of multivalent protein-affinity ligands is the need for denaturing elution conditions. The strong multivalent binding between ligand and target protein typically requires harsh elution conditions such as a low pH shift where the protein ligands are denatured to perturb the steric fit and release the bound protein target. Depending on the intrinsic physicochemical stability of the target glycoprotein such denaturing affinity-elution steps can suffer from decreased product recovery. While limited product losses may be tolerated from a commercial point of view, the root cause for product losses in affinity/elution steps is a major issue of concern, namely the formation of undesirable product aggregates. Product aggregates in parenteral drug formulations compromise not only drug efficacy but also drug safety mainly by their potential to trigger immunogenic reactions with life-threatening consequences. The plain and simple adsorption of a glycoprotein at interfaces can induce physical instability and the formation of structurally altered protein and peptide molecules which may serve as nucleation precursors for the formation of aggregated seeds that can then serve as heterogeneous nuclei that foster protein aggregation. Rising protein concentration itself is yet another well-known trigger of aggregation. Target protein concentrations in a typical upstream harvest bulk range from 1 to 7 mg/ml. But once a protein has bound to a resin, local concentrations can rise to a level equivalent to resin capacity. Eluate fractions were reported to reach transient concentrations higher than 100 mg/1 and it might be expected that today's capacity-enhanced resins promote even higher transient protein concentrations. While this problem of shifting product concentration is inherent to the affinity chromatography technology, it becomes all the more critical in the context of denaturing elution conditions. Buffer conditions for binding and elution have a tremendous impact on whether or not undesirable product aggregation is triggered during affinity purification steps [1]. A buffer pH that is non-critical at typical product concentrations in the upstream harvest bulk may cause product aggregation at high concentrations on a resin surface [2]. In commercial manufacturing low pH elution steps are typically combined with a subsequent low pH hold step for dedicated virus depletion in line with the stipulations of ICHQ5A. Both low pH elution conditions and low pH hold for virus depletion can induce and promote conformational changes and concomitant unfolding and aggregation of target proteins. Interestingly, rate constants for aggregation after protein A chromatography were found to be considerably higher than those from low pH exposure alone [2]. Thus, the affinity/elution step at low pH is critical. In summary, the commonly applied elution of glycoprotein therapeutics at low pH is an underestimated problem that is not only responsible for product losses but also compromises drug safety.

Moreover, any protein-based affinity ligand is gradually leached from the affinity matrix. Trace levels of leached protein ligand that co-elute with the target protein from the affinity matrix must be removed at later steps in the purification process making sure that leached ligand is no longer detectable in the purified drug substance. Protein A leachables are particularly dangerous. The protein A affinity ligand is derived from a type I membrane protein of the human-pathogenic bacterium Staphylococcus aureus and thus is a potential danger to patient safety. mAbs and Fc fusion proteins are typically administered parenterally and thus any contamination with Staphylococcus aureus protein A, a known superantigenic virulence factor, may be expected to cause an immediate and powerful immune response towards protein A. Moreover, protein A has also been suspected of an intrinsic mitogenic activity. In light of this, EMA and FDA regulatory authorities require documented evidence of leached protein A clearance during bioprocessing of mAbs and Fc fusion proteins intended for human use. Biopharmaceuticals intended for clinical administration must be monitored precisely to the parts-per-million (ppm) level for leached protein A contamination during in-process controls and release analytics for bulk drug substance. This regulatory need for in-process and release analytics of column leachables impacts on manufacturing Cost of Goods Sold (COGS) by causing additional analytical monitoring effort. Moreover, any discovery of alarm level protein A leachables in an in-process control will result in re-work effort and concomitant product yield losses with every additional processing step implemented to bring down the leachable contamination to a level where no traces are detectable.

In light of the above named problems, there is a need for new highly selective affinity ligands of low molecular mass. Said ligands should offer the following advantages over the current state-of-the-art: (1) they should allow product capture and elution at constant pH so as to avoid triggering of process induced product aggregation, (2) they should enable higher ligand densities per matrix surface area and better diffusive mass transfer properties resulting in optimized breakthrough dynamics and increased dynamic binding capacities at high-flow rates, (3) they should be chemically and physically stable to last a maximum number of re-uses, and (4) they should be non-toxic and non-immunogenic when possibly co-eluted as leachables.

The inventor of the present invention provides such ligands (compounds). In particular, the ligands of the present invention allow product capture and elution at constant pH, i.e. without pH shift, and, thus, avoid the formation of undesirable product aggregation. The ligands according to the present invention are further of low molecular weight and size and are able to bind to glycostructures, e.g. N-glycans, with a selectivity greater than that of monovalent boric and boronic acids. They are, thus, very suitable for the purification of glycosylated biomolecules. In addition, the ligands according to the present invention are based on a partial structure common to natural human N-glycans and are, therefore, expected to be non-toxic and non-immunogenic. All these attributes render the ligands of the present invention as attractive candidates, especially for cost-effective large-scale purification of glycoproteins and enveloped viruses comprising glycostructures on their surface. SUMMARY OF THE INVENTION a first aspect, the present invention relates to a compound according to formula (I)

A is either absent or is selected from the group consisting of alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, arylalkyl, alkylaryl, heteroalkyl, heteroalkenyl, heterocycloalkyl, heterocycloalkenyl, heteroaryl, heteroarylalkyl, arylheteroalkyl, heteroarylheteroalkyl, heteroalkylaryl, alkylheteroaryl, heteroalkylheteroaryl, alkoxy, alkenoxy, cycloalkoxy, cycloalkenoxy, aryloxy, arylalkoxy, and alkylaryloxy;

X is a functional group that allows the covalent coupling of the compound to a solid support;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are independently from each other selected from the group consisting of H, -OH, (Ci-C6)alkoxy, and Rⁿ-C(0)-0-, wherein R¹¹ is H or (Ci-C₄)alkyl;

with the proviso that, when A is absent, then X is not -OH.

In a second aspect, the present invention relates to a complex comprising:

the compound according to the first aspect; and

a Lewis-acidic transition metal ion.

In a third aspect, the present invention relates to a complex comprising: (a) the compound according to the first aspect; and

(b) a compound selected from the group consisting of boric acid and diboronic acids.

In a fourth aspect, the present invention relates to a method of producing an affinity matrix for the purification of glycosylated biomolecules, comprising the steps:

(i) providing a solid support; and

(ii) covalently coupling the compound according to the first aspect to said solid support via functional group X, thereby producing a functionalized solid support; and

(iii) optionally contacting the functionalized solid support produced in step (ii)

either with a Lewis-acidic transition metal ion

- or with a compound selected from the group consisting of boric acid and diboronic acids,

thereby producing said affinity matrix.

In a fifth aspect, the present invention relates to a method of producing an affinity matrix for the purification of glycosylated biomolecules, comprising the steps:

(i) providing a solid support; and

(ii) covalently coupling the complex according to the second or third aspect to said solid support via functional group X, thereby producing said affinity matrix.

In a sixth aspect, the present invention relates to an affinity matrix obtainable by the method according to the fourth or fifth aspect.

In a seventh aspect, the present invention relates to the use of the compound according to the first aspect, the complex according to the second or third aspect, or the affinity matrix according to the sixth aspect for the purification of glycosylated biomolecules.

In an eighth aspect, the present invention relates to a method for purifying glycosylated biomolecules from a cell culture supernatant comprising the steps of:

(i) providing a cell culture supernatant comprising glycosylated biomolecules, and

(ii) applying the cell culture supernatant to the affinity matrix according to the sixth aspect.

In a ninth aspect, the present invention relates to a method for purifying glycosylated biomolecules from a cell culture supernatant comprising the steps of:

(ii) precipitating the glycosylated biomolecules from the cell culture supernatant with the compound according to the first aspect, or with the complex according to the second or third aspect.

This summary of the invention does not necessarily describe all features of the present invention. Other embodiments will become apparent from a review of the ensuing detailed description. DETAILED DESCRIPTION OF THE INVENTION

Definitions

Before the present invention is described in 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 embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

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

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations 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 integer or step.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank Accession Number sequence submissions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. 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.

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 stated integer or group of integers and the exclusion of any other integer or group of integers. The terms "polypeptide" and "protein" are used interchangeably in the context of the present invention and refer to a long peptide-linked chain of amino acids, e.g. one that is typically 50 amino acids long or longer than 50 amino acids.

In the context of the present invention, the term "oligopeptide" refers to a short peptide- linked chain of amino acids, e.g. one that is typically less than about 50 amino acids long and more typically less than about 30 amino acids long. Said short peptide-linked chain of amino acids is typically more than about 2 amino acids long.

In the context of the present invention, the term "fusion protein" refers to a polypeptide comprising a polypeptide or polypeptide fragment coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins.

The terms "antibody", "immunoglobulin", "Ig" and "Ig molecule" are used interchangeably in the context of the present invention. The CH2 domain of each heavy chain contains a single site for N-linked glycosylation at an asparagine residue linking an N-glycan to the antibody molecule, usually at residue Asn-297 (Kabat et al, Sequence of proteins of immunological interest, Fifth Ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Included within the scope of the term are classes of Igs, namely, IgG, IgA, IgE, IgM, and IgD. Also included within the scope of the terms are the subtypes of IgGs, namely, IgGl, IgG2, IgG3 and IgG4. The terms are used in their broadest sense and include monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, single chain antibodies, and multispecific antibodies (e.g. bispecific antibodies).

The term "antibody fragment", as used herein, refers to a fragment of an antibody that contains at least the portion of the CH2 domain of the heavy chain immunoglobulin constant region which comprises an N-linked glycosylation site of the CH2 domain and is capable of specific binding to an antigen, i.e. chains of at least one VL and/or VH-chain or binding part thereof.

The terms "Fc domain" and "Fc region", as used herein, refer to a C-terminal portion of an antibody heavy chain that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system.

The term "glycosylated bio molecules", as used herein, refers to molecules including large macro molecules such as proteins or lipids that contain oligosaccharide chains (glycans).

The term "glycoproteins", as used herein, refers to proteins that contain oligosaccharide chains (glycans) covalently attached to their polypeptide side-chains. The carbohydrate is attached to the protein in a co-translational or posttranslational modification. This process is known as glycosylation such as N-glycosylation or O-glycosylation. The term "glyco lipids", as used herein, refers to carbohydrate-attached lipids. They occur where a carbohydrate chain is associated with phospholipids on the exoplasmic surface of the cell membrane. The carbohydrates are found on the outer surface of all eukaryotic cell membranes. The carbohydrate structure of the glycolipid is controlled by the glycosyltransferases that add the lipids and glycosylhydrolases that modify the glycan after addition. Glyco lipids also occur on the surface of enveloped viruses including those used as attenuated life vaccines.

In the context of the present invention, the term "glycostructure" encompasses glycoproteins and/or glycooligopeptides that contain oligosaccharide chains (glycans) covalently attached to their polypeptide/oligopeptide side-chains. The carbohydrate is attached to the structure in a co-translational or posttranslational modification. As mentioned above, this process is known as glycosylation such as N-glycosylation or O-glycosylation.

The term "N-glycosylation", as used herein, means the addition of sugar chains which to the amide nitrogen on the side chain of asparagine. The term "O-glycosylation", as used herein, means the addition of sugar chains on the hydroxyl oxygen on the side chain of hydroxylysine, hydroxyproline, serine, or threonine.

The term "N-glycan", as used herein, means an N-linked polysaccharide or oligosaccharide. An N-linked oligosaccharide is for example one that is or was attached by an N- acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in a protein. The predominant sugars found on N-glycoproteins are glucose, galactose, mannose, fucose, N- acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialic acid (e.g., N-acetyl- neuraminic acid (NANA)). The processing of the sugar groups occurs co-translationally in the lumen of the ER and continues in the Golgi apparatus for N-linked glycoproteins. N-glycans have a common pentasaccharide core of Man₃GlcNAc₂ ("Man" refers to mannose; "Glc" refers to glucose; and "NAc" refers to N-acetyl; GlcNAc refers to N-acetylglucosamine). N-glycans differ with respect to the number of branches (antennae) comprising peripheral sugars (e.g., GlcNAc, galactose, fucose and sialic acid) that are added to the Man3GlcNAc₂ core structure which is also referred to as the "trimannose core", the "pentasaccharide core" or the "paucimannose core". N- glycans are classified according to their branched constituents (e.g., high mannose, complex or hybrid).

The term "high mannose type N-glycan", as used herein, means an N-linked polysaccharide or oligosaccharide which has five mannose residues (Man₅), or more mannose residues (e.g. Mane, Man₇, or Mans).

The term "complex type N-glycan", as used herein, means a N-linked polysaccharide or oligosaccharide which typically has at least one GlcNAc attached to the 1 ,3 mannose arm and at least one GlcNAc attached to the 1,6 mannose arm of a "trimannose" core. Complex N-glycans may also have galactose ("Gal") or N-acetylgalactosamine ("GalNAc") residues that are optionally modified with sialic acid or derivatives (e.g., "NANA" or "NeuAc", where "Neu" refers to neuraminic acid and "Ac" refers to acetyl). Complex N-glycans may also have intrachain substitutions comprising "bisecting" GlcNAc and core fucose ("Fuc"). Complex type N-glycans in the context of the present invention may contain zero (GO), one (Gl), or two (G2) galactoses as well as one fucose attached to the first GlcNAc on the reducing end (denoted as GOF, GIF, G2F, respectively).

The term "hybrid type N-glycan", as used herein, means a N-linked polysaccharide or oligosaccharide which has at least one GlcNAc on the terminal of the 1,3 mannose arm of the trimannose core and zero or more mannoses on the 1,6 mannose arm of the trimannose core.

The term "paucimannose type N-glycan" as used herein, means small oligomannose N- glycans as they are commonly found in plants and invertebrates and having the composition Man₃- ₄GlcNAc2.

The term "O-glycan", as used herein, means an O-linked polysaccharide or oligosaccharide. O-linked glycans are usually attached to the peptide chain through serine or threonine residues. O-linked glycosylation is a true post-translational event which occurs in the Golgi apparatus and which does not require a consensus sequence and no oligosaccharide precursor is required for protein transfer. The most common type of O-linked glycans contain an initial GalNAc residue (or Tn epitope), these are commonly referred to as mucin-type glycans. Other O-linked glycans include glucosamine, xylose, galactose, fucose, or mannose as the initial sugar bound to the Ser/Thr residues. O-Linked glycoproteins are usually large proteins (> 200 kDa) that are commonly biantennary with comparatively less branching than N-glycans.

The term "enveloped viruses comprising glycostructures on their envelopes", as used herein, refers to viruses having viral envelopes covering their protective protein capsids. The envelopes typically are derived from portions of the host (phospholipids and proteins), but include some (viral) glycostructures such as glycoproteins and/or glycooligopeptides. Functionally, viral envelopes help viruses to enter host cells and may help them to avoid the host immune system. (Viral) glycostructures such as glycoproteins and/or glycooligopeptides on the surface of the envelopes serve to identify and bind to receptor sites on the host's membrane. The viral envelope then fuses with the host's membrane, allowing the capsid and viral genome to enter and infect the host. The influenza virus and many animal viruses are enveloped viruses.

The term "functional group", as used herein, means any specific group of atoms and bonds within a larger molecule that is responsible for the characteristic chemical reactions of this molecule. In this particular context, the term "functional group that allows the covalent coupling of the compound", as used herein, means a specific group of atoms and bonds within a larger molecule that renders the larger molecule reactable with another specific chemical or functional group so that a covalent bond is formed between these two molecules. Suitable functional groups include halide (CI, Br, F or I), hydroxyl, alkoxy (i.e.— OR where e.g. R=alkyl C1-C30), carbonyl, carboxyl, anhydride, methacryl, epoxide, vinyl, nitrile, nitro, sulfate, sulfonyl, mercapto, sulfide, amino, amine, imine, amide and imide. Coupling reactions include all bioconjugation techniques known to someone skilled in the art including but not limited to N-hydroxysuccinimide-amino coupling, isocyanate-amino coupling, isothiocyanate-amino coupling, maleimide-sulfhydryl- coupling, azide-mediated Staudinger ligation, Huisgen Cyclization of Azides, Huisgen Sharpless Meldal azide-alkyne click chemistry, Suzuki coupling, Buchwald-Hartwig reaction, etc.

The term "alkyl", as used herein, refers to a saturated straight or branched carbon chain.

Preferably, the chain comprises from 1 to 30 carbon atoms, i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, e.g. methyl, ethyl propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, or dodecyl. The alkyl groups are optionally substituted.

The term "heteroalkyl", as used herein, refers to a saturated straight or branched carbon chain. Preferably, the chain comprises from 1 to 30 carbon atoms, i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30, e.g. methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, or dodecyl which is interrupted one or more times, e.g. 1 , 2, 3, 4, 5, with the same or different heteroatoms. Preferably, the heteroatoms are selected from O, S, and N, e.g. -(CH₂)_n-X- (CH₂)_mCH₃, with n = 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30, m = 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30, and X = S, O or NR* with R' = H or hydrocarbon (e.g. Ci to C₆ alkyl). In particular, "heteroalkyl" refers to -0-CH₃, -OC₂H₅, -CH₂-0-CH₃, -CH₂-0-C₂H₅, -CH₂-0-C₃H₇, -CH2-O-C4H9, -CH₂-0-C₅Hi i, -C₂H₄-0-CH₃, -C₂H4-0-C₂H₅, -C₂H₄-0-C₃H₇, - C2H4-O-C4H9 etc. Heteroalkyl groups are optionally substituted.

The terms "cycloalkyl" and "heterocycloalkyl", as used herein, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl", respectively, with preferably 3, 4, 5, 6, 7, or 8 carbon atoms forming a ring, e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl etc. The terms "cycloalkyl" and "heterocycloalkyl" are also meant to include bicyclic, tricyclic and polycyclic versions thereof. If bicyclic, tricyclic or polycyclic rings are formed, it is preferred that the respective rings are connected to each other at two adjacent carbon atoms, however, alternatively the two rings are connected via the same carbon atom, i.e. they form a spiro ring system or they form "bridged" ring systems. The term "heterocycloalkyl" preferably refers to a saturated ring having five members of which at least one member is an N, O or S atom and which optionally contains one additional O or one additional N; a saturated ring having six members of which at least one member is an N, O or S atom and which optionally contains one additional O or one additional N or two additional N atoms; or a saturated bicyclic ring having nine or ten members of which at least one member is an N, O or S atom and which optionally contains one, two or three additional N atoms. "Cycloalkyl" and "heterocycloalkyl" groups are optionally substituted. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, spiro[3,3]heptyl, spiro[3,4]octyl, spiro[4,3]octyl, spiro[3,5]nonyl, spiro[5,3]nonyl, spiro[3,6]decyl, spiro[6,3]decyl, spiro[4,5]decyl, spiro[5,4]decyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, adamantyl, and the like. Examples of heterocycloalkyl include l-(l,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2- piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, l,8-diazo-spiro[4,5]decyl, 1,7-diazo- spiro[4,5]decyl, l,6-diazo-spiro[4,5]decyl, 2,8-diazo-spiro[4,5]decyl, 2,7-diazo-spiro[4,5]decyl, 2,6-diazo-spiro[4,5]decyl, l,8-diazo-spiro[5,4]decyl, 1,7 diazo-spiro[5,4]decyl, 2,8-diazo- spiro[5,4]decyl, 2,7-diazo-spiro[5,4]decyl, 3,8-diazo-spiro[5,4]decyl, 3,7-diazo-spiro[5,4]decyl, 1 ,4-diazabicyclo[2.2.2]oct-2-yl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

The term "aryl", as used herein, preferably refers to an aromatic monocyclic ring containing 6 carbon atoms, an aromatic bicyclic ring system containing 10 carbon atoms or an aromatic tricyclic ring system containing 14 carbon atoms. Examples are phenyl, naphthyl, anthracenyl, or phenanthryl. The aryl group is optionally substituted.

The term "arylalkyl", as used herein, refers to an alkyl moiety, which is substituted by aryl, wherein alkyl and aryl have the meaning as outlined above. An example is the benzyl radical. Preferably, in this context the alkyl chain comprises from 1 to 24 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24, e.g. methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl. The arylalkyl group is optionally substituted at the alkyl and/or aryl part of the group. Preferably the aryl attached to the alkyl has the meaning phenyl, naphthyl, anthracenyl, or phenanthryl. The term "arylalkyl" can also be abbreviated to "aralkyl".

The term "heteroaryl", as used herein, preferably refers to a five or six-membered aromatic monocyclic ring wherein at least one of the carbon atoms is replaced by 1, 2, 3, or 4 (for the five membered ring) or 1 , 2, 3, 4, or 5 (for the six membered ring) of the same or different heteroatoms, preferably selected from O, N and S; an aromatic bicyclic ring system with 8 to 12 members wherein 1, 2, 3, 4, 5, or 6 carbon atoms of the 8, 9, 10, 11 or 12 carbon atoms have been replaced with the same or different heteroatoms, preferably selected from O, N and S; or an aromatic tricyclic ring system with 13 to 16 members wherein 1, 2, 3, 4, 5, or 6 carbon atoms of the 13, 14, 15, or 16 carbon atoms have been replaced with the same or different heteroatoms, preferably selected from O, N and S. Examples are furanyl, thiophenyl, oxazolyl, isoxazolyl, 1,2,5- oxadiazolyl, 1,2,3-oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, thiazolyl, isothiazolyl, 1,2,3-thiadiazolyl, 1,2,5-thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, 1,2,3- triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1-benzo furanyl, 2-benzofuranyl, indoyl, isoindoyl, benzothiophenyl, 2-benzothiophenyl, lH-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, 2,1-benzosoxazoyl, benzothiazolyl, 1,2-benzisothiazolyl, 2,1-benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, 2,3-benzodiazinyl, quinoxalinyl, quinazolinyl, quinolinyl, 1,2,3- benzotriazinyl, or 1,2,4-benzotriazinyl.

The terms "alkenyl" and "cycloalkenyl", as used herein, refer to olefmic unsaturated carbon atoms containing chains or rings with one or more double bonds. Examples are propenyl and cyclohexenyl. Preferably, the alkenyl chain comprises from 2 to 30 carbon atoms, i.e. 2, 3, 4, 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, e.g. ethenyl, 1 -propenyl, 2-propenyl, iso-propenyl, 1 -butenyl, 2-butenyl, 3-butenyl, iso-butenyl, sec- butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, hexenyl, heptenyl, octenyl. Preferably the cycloalkenyl ring comprises from 3 to 8 carbon atoms, i.e. 3, 4, 5, 6, 7, or 8, e.g. 1-cyclopropenyl, 2-cyclopropenyl, 1-cyclo butenyl, 2-cyclo butenyl, 1-cyclopentenyl, 2-cyclopentenyl, 3- cyclopentenyl, 1 -cyclohexenyl, 2-cyclohexenyl, 3-cyclo hexenyl, cyclo heptenyl, cyclooctenyl.

The terms "heteroalkenyl" and "heterocycloalkenyl", as used herein, refer to unsaturated versions of "heteroalkyl" and "heterocycloalkyl", respectively. Thus, the term "heteroalkenyl" refers to an unsaturated straight or branched carbon chain. Preferably, the chain comprises from 2 to 30 carbon atoms, i.e. 2, 3, 4, 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, which is interrupted one or more times, e.g. 1, 2, 3, 4, 5, with the same or different heteroatoms. Preferably, the heteroatoms are selected from O, S, and N. In case that one or more of the interrupting heteroatoms is N, the N may be present as an -NR'- moiety, wherein R' is hydrogen or hydrocarbon (e.g. Ci to C₆ alkyl), or it may be present as an =N- or -N= group, i.e. the nitrogen atom can form a double bond to an adjacent C atom or to an adjacent, further N atom. "Heteroalkenyl" groups are optionally substituted. The term "heterocycloalkenyl" represents a cyclic version of "heteroalkenyl" with preferably 3, 4, 5, 6, 7, or 8 atoms forming a ring. The term "heterocycloalkenyl" is also meant to include bicyclic, tricyclic and polycyclic versions thereof. If bicyclic, tricyclic or polycyclic rings are formed, it is preferred that the respective rings are connected to each other at two adjacent atoms. These two adjacent atoms can both be carbon atoms; or one atom can be a carbon atom and the other one can be a heteroatom; or the two adjacent atoms can both be heteroatoms. However, alternatively the two rings are connected via the same carbon atom, i.e. they form a spiro ring system or they form "bridged" ring systems. The term "heterocycloalkenyl", as used herein, preferably refers to an unsaturated ring having five members of which at least one member is an N, O or S atom and which optionally contains one additional O or one additional N; an unsaturated ring having six members of which at least one member is an N, O or S atom and which optionally contains one additional O or one additional N or two additional N atoms; or an unsaturated bicyclic ring having nine or ten members of which at least one member is an N, O or S atom and which optionally contains one, two or three additional N atoms. "Heterocycloalkenyl" groups are optionally substituted. Additionally, for heteroalkenyl and heterocycloalkenyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule.

The term "alkylaryl", as used herein, refers to an aryl moiety that is substituted by alkyl, wherein aryl and alkyl have the meaning as outlined above.

The term "heteroarylalkyl", as used herein, refers to an alkyl moiety that is substituted by heteroaryl, wherein alkyl and heteroaryl have the meaning as outlined above.

The term "arylheteroalkyl", as used herein, refers to a heteroalkyl moiety that is substituted by aryl, wherein heteroalkyl and aryl have the meaning as outlined above.

The term "heteroarylheteroalkyl", as used herein, refers to a heteroalkyl moiety that is substituted by heteroaryl, wherein heteroalkyl and heteroaryl have the meaning as outlined above.

The term "heteroalkylaryl", as used herein, refers to an aryl moiety that is substituted by heteroalkyl, wherein aryl and heteroalkyl have the meaning as outlined above.

The term "alkylheteroaryl", as used herein, refers to a heteroaryl moiety that is substituted by alkyl, wherein heteroaryl and alkyl have the meaning as outlined above.

The term "heteroalkylheteroaryl", as used herein, refers to a heteroaryl moiety that is substituted by heteroalkyl, wherein heteroaryl and heteroalkyl have the meaning as outlined above.

The term "alkoxy", as used herein, refers to an alkyl-O- moiety, wherein alkyl has the meaning as outlined above.

The term "alkenoxy", as used herein, refers to an alkenyl-O- moiety, wherein alkenyl has the meaning as outlined above.

The term "cycloalkoxy", as used herein, refers to a cycloalkyl-O- moiety, wherein cycloalkyl has the meaning as outlined above.

The term "cycloalkenoxy", as used herein, refers to a cycloalkenyl-O- moiety, wherein cycloalkenyl has the meaning as outlined above.

The term "aryloxy", as used herein, refers to an aryl-O- moiety, wherein aryl has the meaning as outlined above. The term "arylalkoxy", as used herein, refers to an arylalkyl-O- moiety, wherein arylalkyl has the meaning as outlined above.

The term "alkylaryloxy", as used herein, refers to an alkylaryl-O- moiety, wherein alkylaryl has the meaning as outlined above.

Embodiments of the invention

The present invention will now be further described. In the following passages different aspects of the invention are defined in more detail. Each aspect so defined 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, unless clearly indicated to the contrary.

The inventor of the present invention developed new ligands (compounds) based on specific coupling enabled derivatives of the branched oligosaccharide 3,6-Trimannose. These ligands are functionalized at the reducing end of the branching mannose with a functional group that can be used for position specific coupling of the branched 3,6-Trimannosyl-ligand to an affinity matrix.

In a first aspect, the present invention relates to a compound (ligand) according to formula

(I):

wherein

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are independently from each other selected from the group consisting of H, -OH, (Ci-C6)alkoxy, and Rⁿ-C(0)-0-, wherein R^{1 1} is H or (Ci-C4)alkyl, preferably R^{1 1} is methyl;

with the proviso that, when A is absent, then X is not -OH, or in other words, with the proviso that -A-X together is not -OH.

The inventor of the present invention found that the above compound (ligand) allows product capture and elution at constant pH, i.e. without pH shift, and, thus, avoids the formation of undesirable product aggregation. The above compound is further of low molecular weight and size and is able to bind to glycostructures, e.g. N-glycans, with a selectivity greater than that of monovalent boric and boronic acids. It is, thus, very suitable for the purification of glycosylated biomolecules. In addition, the above compound is based on a partial structure common to natural human N-glycans and is, therefore, expected to be non-toxic and non-immunogenic. The above compound is, thus, an attractive candidate, especially for cost-effective large-scale purification of glycoproteins and enveloped viruses comprising glycostructures on their surface.

It is preferred that A is selected from the group consisting of (Ci-C3o)alkyl, (C₂-C₃o)alkenyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkenyl, (C₆-Ci₄)aryl, (C₆-Ci4)aryl(Ci-C₂₄)alkyl, (Ci- C₂₄)alkyl(C6-Ci₄)aryl, (Ci-C₃o)heteroalkyl, (C₂-C₃o)heteroalkenyl, (C₃-C8)heterocycloalkyl, (C₃- C8)heterocycloalkenyl, (C₃-Ci₃)heteroaryl, (C₃-Ci₃)heteroaryl(Ci-C₂₄)alkyl, (C6-Ci₄)aryl(Ci- C₂₄)heteroalkyl, (C₃-Ci₃)heteroaryl(Ci-C₂₄)heteroalkyl, (Ci-C₂₄)heteroalkyl(C6-Ci₄)aryl, (Ci- C₂₄)alkyl(C₃-Ci₃)heteroaryl, (Ci-C₂₄)heteroalkyl(C₃-Ci₃)heteroaryl, (Ci-C₃o)alkoxy, (C₂- C₃o)alkenoxy, (C₃-C₈)cycloalkoxy, (C₃-C8)cycloalkenoxy, (C6-Ci₄)aryloxy, (C6-Ci₄)aryl(Ci- C₂₄)alkoxy, and (Ci-C₂₄)alkyl(C6-Ci₄)aryloxy. A may be, for example, poly ethylengly col (e.g. PEG 12) or polysorbate.

It is preferred that X is selected from the group consisting of -OH, -N0₂, -NH₂, -COOH,

-NCO, azlactone-, N-hydroxysuccinimide-, maleinimide, carboxamido-, alkynyl, -N₃, and -SH. These functional groups are able to support stable coupling to a solid support by undergoing covalent chemical bonding.

It is more preferred that -A-X together is selected from the group consisting of -C₆H₄-X, - CioH₆-X,-Ci4H₈-X, -0-C₆H₄-X, -O-CioHe-X, and -0-Ci₄H₈-X. Preferably, X is selected from the group consisting of -OH, -NO2, -NH₂, -COOH, -NCO, azlactone-, N-hydroxysuccinimide-, maleinimide, carboxamido-, alkynyl, -N3, and -SH.

It is even more preferred that -A-X together is a substituent according to formula III:

It is further preferred that R¹ and R² are independently from each other selected from the group consisting of H, -OH, and -(Ci-Ce)alkoxy and/or that R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are -OH.

In another embodiment, the compound has a structure according to formula (II):

wherein

L is selected from the group consisting of alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, arylalkyl, alkylaryl, heteroalkyl, heteroalkenyl, heterocycloalkyl, heterocycloalkenyl, heteroaryl, heteroarylalkyl, arylheteroalkyl, heteroarylheteroalkyl, heteroalkylaryl, alkylheteroaryl, and heteroalkylheteroaryl;

X is a functional group that allows the covalent coupling of the compound to a solid support; and

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are independently from each other selected from the group consisting of H, -OH, (Ci-C6)alkoxy, and Rⁿ-C(0)-0-, wherein R^{1 1} is H or (Ci-C4)alkyl, preferably R^{1 1} is methyl. It is preferred that L is selected from the group consisting of (Ci-C3o)alkyl, (C₂-C₃o)alkenyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkenyl, (C₆-Ci₄)aryl, (C₆-Ci₄)aryl(Ci-C₂₄)alkyl, (Ci- C₂₄)alkyl(C₆-Ci₄)aryl, (Ci-C₃o)heteroalkyl, (C₂-C₃o)heteroalkenyl, (C₃-C8)heterocycloalkyl, (C₃- C8)heterocycloalkenyl, (C₃-Ci₃)heteroaryl, (C₃-Ci₃)heteroaryl(Ci-C₂₄)alkyl, (C₆-Ci₄)aryl(Ci- C₂₄)heteroalkyl, (C₃-Ci₃)heteroaryl(Ci-C₂₄)heteroalkyl, (Ci-C₂₄)heteroalkyl(C6-Ci₄)aryl, (Ci- C₂₄)alkyl(C₃-Ci₃)heteroaryl, and (Ci-C₂₄)heteroalkyl(C₃-Ci₃)heteroaryl. L may be, for example, poly ethylengly col (e.g. PEG 12) or polysorbate.

It is preferred that X is selected from the group consisting of -OH, -N0₂, -NH₂, -COOH, -NCO, azlactone-, N-hydroxysuccinimide-, maleinimide, carboxamido-, alkynyl, -N₃, and -SH. These functional groups are able to support stable coupling to a solid support by undergoing covalent chemical bonding.

It is further preferred that R¹ and R² are independently from each other selected from the group consisting H, -OH, and -(Ci-Ce)alkoxy and/or that R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are - OH.

If it is intended to form a complex between the compound and a Lewis-acidic transition metal ion, it is particularly preferred that R³, R⁴ and R⁷, R⁸ are -OH. Alternatively, R⁴, R⁵ and R⁸, R⁹ are -OH.

If it is intended to form a complex between the compound and boric acid or diboronic acids, it is particularly preferred that R³, R⁴ and R⁷, R⁸ are -OH. Alternatively, R⁵, R⁶ and R⁹, R¹⁰ are - OH.

It is most preferred that the compound (ligand) is 4-Aminophenyl-3,6-Trimannose. This compound (ligand) is shown in Figure 3.

The compound according to the first aspect is capable of binding to the glycosylated biomolecules, e.g. glycoproteins, particularly to the glycostructures of the glycoproteins, gly co lipids, or glycostructures on the envelopes of enveloped viruses. In particular, the compound according to the first aspect is capable of binding to the pyranoid-diol functionality of said glycoproteins or glycostructures, preferably to the 1 ,2 cis-diol and/or 1 ,3 trans-diol functionality of said glycoproteins or glycostructures. The compound, thus, allows the purification of glycosylated biomolecules from cell culture supernatants.

In a second aspect, the present invention relates to a complex comprising:

(a) the compound according to the first aspect; and

(b) a Lewis-acidic transition metal ion.

Preferably, the Lewis-acidic transition metal ion is selected from the group consisting of Cu(II), Mn(II), Fe(II), Fe(III), and Co(II).

Preferably, the diboronic acid is selected from the group consisting of 1,4- phenylendiboronic acid (synm. 1,4-benzenediboronic acid), 2-Nitrobenzene-l,4-diboronic acid (synm. 2-nitro-l,4-phenylenediboronic acid), 2,2'-Bithiophene-5,5'-diboronic acid, 4-4'- biphenyldiboronic acid, 2,5-thiophenediboronic acid, stilbene-4,4-diboronic acid, 4,4'-oxybis(l,4- benzene)diboronic acid, dibenzofuran-4,6-diboronic acid and derivatives thereof.

The complexes according to the second or third aspect are capable of binding to the glycosylated biomolecules, e.g. glycoproteins, particularly to the glycostructures of the glycoproteins, glyco lipids, or glycostructures on the envelopes of enveloped viruses. In particular, the complexes according to the second or third aspect are capable of binding to the pyranoid-diol functionality of said glycoproteins or glycostructures, preferably to the 1,2 cis-diol and/or 1,3 trans-diol functionality of said glycoproteins or glycostructures. The complexes, thus, allow the purification of glycosylated biomolecules from cell culture supernatants.

(i) providing a solid support; and

either with a Lewis-acidic transition metal ion

or with a compound selected from the group consisting of boric acid and diboronic acids,

thereby producing said affinity matrix.

Preferably, the Lewis-acidic transition metal ion is selected from the group consisting of

Cu(II), Mn(II), Fe(II), Fe(III), and Co(II).

Preferably, the diboronic acid is selected from the group consisting of ,4- phenylendiboronic acid (synm. 1,4-benzenediboronic acid), 2-Nitrobenzene-l,4-diboronic acid

(synm. 2-nitro-l,4-phenylenediboronic acid), 2,2'-Bithiophene-5,5'-diboronic acid, 4-4'- biphenyldiboronic acid, 2,5-thiophenediboronic acid, stilbene-4,4-diboronic acid, 4,4'-oxybis(l,4- benzene)diboronic acid, dibenzofuran-4,6-diboronic acid and derivatives thereof.

The solid support may be a membrane or a solid phase chromatographic support such as a resin or a monolith. The solid support may be in the format of a chromatography column or a membrane adsorber. The solid support may also be a bead, particularly a set of beads. In a fifth aspect, the present invention relates to a method of producing an affinity matrix for the purification of glycosylated biomolecules, comprising the steps:

(i) providing a solid support; and

The solid support may be a membrane or a solid phase chromatographic support such as a resin or a monolith. The solid support may be in the format of a chromatography column or a membrane adsorber. The solid support may also be a bead, particularly a set of beads.

Covalent coupling as described in the above methods allows the formation of a very active and stable complex. For example: the complex is permanently bound and will not desorb/leach over time, the elimination of "crosstalk" between the complexes permits multiplexed assays, the complexes are favorably presented on the surface of the bead such that binding moieties are available for interaction with the glycosylated biomolecules, and binding kinetics can approach those of solution-based binding.

The affinity matrix according to the sixth aspect is capable of binding to the glycosylated biomolecules, e.g. glycoproteins, particularly to the glycostructures of the glycoproteins, glycolipids, or glycostructures on the envelopes of enveloped viruses. In particular, the affinity matrix according to the sixth aspect is capable of binding to the pyranoid-diol functionality of said glycoproteins or glycostructures, preferably to the 1,2 cis-diol and/or 1,3 trans-diol functionality of said glycoproteins or glycostructures. The affinity matrix, thus, allows the purification of glycosylated biomolecules from cell culture supernatants.

In a seventh aspect, the present invention relates to the use of

(i) the compound according to the first aspect; or

(ii) the complex according to the second or third aspect; or

(iii) the affinity matrix according to the sixth aspect

for the purification of glycosylated biomolecules.

It is preferred that the glycosylated biomolecules are selected from the group consisting of (a) glycoproteins,

(b) glycolipids, and

(c) enveloped viruses comprising glycostructures on their envelopes.

The glycoproteins may be antibodies, antibody fragments, fusion proteins, virus proteins, virus protein fragments, antigens, cytokines, clotting factors, growth factors, enzymes, or hormones. Preferably, the antibody or the antibody fragment is selected from the group consisting of IgG, preferably IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE. Preferably, the fusion protein comprises the Fc region of an antibody, e.g. the Fc region of IgG, such as the Fc region of IgGl , IgG2, IgG3, IgG4, IgM, IgA, IgD or IgE. Preferably, the cytokine is interferon or interleukin. Preferably, the clotting factor is factor VII, factor IX, or factor VIII, in particular full-length and B-domain deleted versions thereof. Preferably, the enzyme is a glucocerebrosidase, an iduronidase, arylsulfatase A and B, alpha-glucosidase, or iduronat-2-sulfatase. Preferably, the hormone is a follicle stimulating hormone (FSH), luteinizing hormone (LH), chorionic gonadotropin (HCG), or a tyroid stimulating hormone (TSH). The virus proteins or virus protein fragments may be comprised in the envelope membrane of an enveloped virus. Said enveloped virus may be an influenza virus, a rabies virus, chikungunya virus, vaccinia virus including modified vaccinia ankara (MVA) virus, mumps virus, measles virus, canine distemper virus, or a peste des petits ruminants virus (PPRV).

The glycolipids may be glyceroglyco lipids, e.g. galactolipids, sulfolipids (SQDG), or glycosphingo lipids, e.g. cerebrosides, gangliosides, globosides, sulfatides or glycophosphosphingo lipids. The glycolipids may be comprised in the envelope membranes of enveloped viruses. Glycosphingolipids (GSL) are particularly preferred. Glycosphingo lipids contain a hydrophobic ceramide anchor N-acylsphingosine and a hydrophilic head-group composed of saccharides. They are normally found at the outer surface of cell membranes. The composition of the saccharide-moiety is cell type specific and depends on the developmental stage of the organism or can change with the oncogenic state of a cell.

Said enveloped viruses may be influenza viruses, rabies viruses, chikungunya viruses, vaccinia viruses including modified vaccinia ankara (MVA) viruses, mumps viruses, measles viruses, canine distemper viruses, or peste des petits ruminants viruses (PPRV).

It is more preferred that the glycosylated biomolecules carry N-glycans. It is even more preferred that the N-glycans are selected from the group consisting of high-mannose type N- glycans, complex type N-glycans, hybrid type N-glycans, and paucimannose type N-glycans. The N-glycans may be biantennary N-glycans of the hybrid type or biantennary N-glycans of the complex type.

The cell culture supernatant comprising glycosylated biomolecules is obtained from a cell culture. Techniques to harvest the cell culture supernatant from a cell culture are known to the skilled person. This can be done, for example, by centrifugation, sedimentation and/or filtration techniques.

The cell culture supernatant may be a vertebrate cell culture supernatant, in particular a mammalian, a fish, an amphibian, a reptilian, or an avian cell culture supernatant. It is particularly preferred that (i) the mammalian cell culture supernatant is a human, hamster, canine, or monkey cell culture supernatant, preferably a Chinese hamster ovary (CHO) cell culture supernatant, (ii) the fish cell culture supernatant is an Ictalurus punctatus (channel catfish) ovary (CCO) cell culture supernatant, (iii) the amphibian cell culture supernatant is a Xenopus laevis kidney cell culture supernatant, the reptilian cell culture supernatant is an Iguana iguana heart cell culture supernatant, or (iv) the avian cell culture supernatant is a duck cell culture supernatant.

Preferably, the method further comprises the step(s) of:

(iii) washing the affinity matrix with a washing buffer, and/or

(iv) releasing the glycosylated bio molecules from the affinity matrix with an elution buffer containing a competitive eluent.

The washing buffer may be any buffer which allows the removal of unspecific binding partners from the affinity matrix. As washing buffer, standard phosphate buffered saline (PBS) may be used. The elution buffer may be any buffer which allows the release of the glycosylated biomolecules from the affinity matrix. As elution buffer, standard PBS supplemented with fructose may be used. The competitive eluent may be any eluent which allows the displacement of the glycosylated biomolecules from the affinity matrix. The competitive eluent may be selected from the group consisting of furanoid cis diol sugars or glycosides, pyranoid 1,2,3 triol sugars or glycosides, sugar alcohols, e.g. sorbitol, adonitol, mannitol, xylitol, dulcitol, pentaerythritol, or erythritol, alpha hydroxy carbonic acids, e.g. glycolate, lactate, malate, tartrate, citrate, alpha- hydroxy caprylate, salicylate or glucuronate, and hydroxylamino compounds, e.g. triethanolamin or Tris-hydroxymethyl-aminomethan (TRIS).

The cell culture supernatant comprising glycosylated biomolecules is obtained from a cell culture. Techniques to harvest the cell culture supernatant from a cell culture are known to the skilled person. This can be done, for example, by centrifugation, sedimentation and/or filtration techniques. The cell culture supernatant may be a vertebrate cell culture supernatant, in particular a mammalian, a fish, an amphibian, a reptilian, or an avian cell culture supernatant. It is particularly preferred that (i) the mammalian cell culture supernatant is a human, hamster, canine, or monkey cell culture supernatant, preferably a Chinese hamster ovary (CHO) cell culture supernatant, (ii) the fish cell culture supernatant is an Ictalurus punctatus (channel catfish) ovary (CCO) cell culture supernatant, (iii) the amphibian cell culture supernatant is a Xenopus laevis kidney cell culture supernatant, the reptilian cell culture supernatant is an Iguana iguana heart cell culture supernatant, or (iv) the avian cell culture supernatant is a duck cell culture supernatant.

Preferably, the method further comprises the step(s) of:

(iii) washing the precipitate with a washing buffer, and/or

(iv) releasing the glycosylated bio molecules from the precipitate with an elution buffer

(dissociating buffer) containing a competitive eluent (competitive dissociating reactant).

The washing buffer may be any buffer which allows the removal of unspecific binding partners from the precipitate. As washing buffer, phosphate buffered saline (PBS) may be used.

The elution buffer may be any buffer which allows the dissociation of the precipitate and, thus, the release of the glycosylated biomolecules from the precipitate. Thus, it may also be designated as dissociation buffer. As elution buffer (dissociation buffer), phosphate buffered saline supplemented with fructose may be used.

The competitive eluent may be any eluent which allows the displacement of the glycosylated biomolecules from the precipitate. It is further capable of dissociating the precipitate. Thus, it may also be designated as competitive dissociating reactant. The competitive eluent (competitive dissociating reactant) may be selected from the group consisting of furanoid cis diol sugars or glycosides, pyranoid 1,2,3 triol sugars or glycosides, sugar alcohols, e.g. sorbitol, adonitol, mannitol, xylitol, dulcitol, pentaerythritol, or erythritol, alpha hydroxy carbonic acids, e.g. glycolate, lactate, malate, tartrate, citrate, alpha-hydroxy caprylate, salicylate or glucuronate, and hydroxylamino compounds, e.g. triethanolamin or Tris-hydroxymethyl-aminomethan (TRIS).

Preferably, steps (ii), (iii), and/or (iv) of the methods of the eighth or ninth aspect are conducted at a pH between 6 and 8, more preferably at a pH between 6.5 and 7.5, even more preferably at a pH between 6.8 and 7.2, e.g. at a neutral pH (pH 7). This means that it is preferred that the washing and/or elution (dissociation) buffer has such a pH. This also means that the binding of the glycosylated biomolecules to the affinity matrix is performed at such a pH or that the precipitation of the glycosylated biomolecules from the cell culture supernatant is performed at such a pH.

The skilled person is aware of techniques how to determine the purity of the glycosylated biomolecules such as glycoproteins or enveloped viruses. The purity of the glycoproteins is preferably measured using (i) spectrometry, preferably mass spectrometry (MS), (ii) chromatography, preferably liquid chromatography (LC), more preferably high performance liquid chromatography (HPLC), (iii) gel electrophoresis, preferably SDS gel electrophoresis, (iv) Westemblot/Immunoblot, or (v) combinations thereof. It is preferred that the chromatography, preferably liquid chromatography (LC), more preferably high performance liquid chromatography (HPLC), is combined with spectrometry, preferably mass spectrometry (MS). Accordingly, the purity of the purified glycoprotein is preferably measured using liquid chromatography-mass spectrometry (LC-MS) and is more preferably measured using high performance liquid chromatography-mass spectrometry (HPLC-MS).

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Schematic representation of a biantennary fucosylated G2F N-glycan, a complex glycan structure commonly found on therapeutic IgGl-type antibodies. Characteristic partial structures of complex N-glycan are highlighted in the dotted circles. The combination of fucosyl- chitobiose and 3,6-trimannose is termed "N-glycan core" structure.

Figure 2: Schematic representation of the synthesis of the compound (ligand) 4- Aminophenyl-3,6-Trimannose. PhC (OMe)₃ = Trimethylorthobenzoate, p-TsOH = para- Toluenesulfonic acid, TFA = Trifluoroacetic acid, BF₃ OEt₂ = Boron tri- fluoride diethyl etherate, OTCA= Trichloroacetimidate-moiety, Ac = acetyl, Bz = Benzoyl, MeOH = Methanol, NaOMe= Sodium methoxide, and K₂C0₃= Potassium carbonate.

Figure 3: Schematic representation of the compound (ligand) 4-Aminophenyl-3,6- Trimannose.

Figure 4: Schematic representation of hypothetical bivalent ligand binding: The borate charged 4-Aminophenyl-trimannosyl-ligand forms a tetradentate diester with the juxtaposed biantennary N-glycan of the target glycoprotein.

Figure 5: Schematic representation of hypothetical bivalent ligand binding: The proposed 4-Aminophenyl-trimannosyl ligand forms a bivalent metal coordination complex with the juxtaposed N-glycan of the target glycoprotein. Here, coordination complex formation at C3/C4 hydroxyl groups is shown illustratively. The other remaining hydroxyl groups at C2 and C6 may as well support formation of coordination complexes with transition metal ions. M(II,III) symbolize Lewis-acidic transition metal ions. EXAMPLES

Example 1 : Synthesis of 4-Aminophenyl-3,6-Trimannose

4-Nitrophenyl mannoside 1 is treated with trimethylorthobenzoate and p-TsOH in acetonitrile to yield a bisorthoester, which is subsequently hydro lysed by exposure to aqueous trifluoroacetic acid to give the 2,4-di-O-benzoate 2 (Figure 2). Bisglycosylation of diol 2 by treatment with boron tri- fluoride diethyl etherate and an excess of tetraacetyl-a-D-mannopyranoside trichloroacetimidate donor 3 then yields trisaccharide 4 in good yield. Conversion of the nitro-group to an amino group is achieved by catalytic hydrogeno lysis with the Palladium on Carbon method. This gives compound 5. To give the complete ligand 4-Aminophenyl-3,6-Trimannose 6, compound 5 is subjected to Zemplen Deacetylation, i.e. treatment of compound 5 in methanol with a catalytic amount of sodium methoxide at room temperature, and to O-benzoate deprotection with potassium carbonate in methanol for 2 hours at room temperature (Figure 2). Example 2: Production of an affinity matrix with 4-Aminophenyl-3,6-Trimannose

20μΜ Aminophenyl-3,6-Trimannose are reacted with 1 ml VERY HIGH Density GLYOXAL 6BC pre-activated agarose beads (ABT Agarose bead Technologies Ltd. /20μΜ equivalents of aldhyde groups per ml of beads), pre-equilibrated in 0.2 M disodium phosphate. The reaction is allowed to proceed for at least 2 hours. The beads are filtered and resuspended in 2 bead volumes of 2M sodiumcyanoborohydride solution. Unreacted glyoxal sites are blocked with 20 μΐ of ethanolamine per every ml of glyoxal beads with 5 ml of water and the resultant solution is added to the ligand-coupled bead slurry and the reaction is allowed to proceed for 1 hour. Then the beads are washed on a filter funnel with at least 5 volumes of water or buffer (but not coupling buffer). The ligand-coupled glyoxal beads are stored in a preservative-containing phosphate buffer.

Example 3: Purification of glycosylated bio molecules with the affinity matrix

The following experiment illustrates the process of the present invention and in no way is intended to limit the disclosure. An affinity column prepared with ligand functionalized resin from example 2 is equilibrated with loading buffer (lOOmM Phosphate, 200mM 1,4-phenylendiboronate pH 8,0). A sample containing N-glycosylated ovalbumin and conalbumin and non-glycosylated lysozyme in lOOmM Phosphate buffer, pH 8.0 is loaded onto the column. After loading the material onto the column unbound material is washed out for about 8 column volumes (CV) by loading buffer (s. above). Bound ovalbumin and conalbumin are eluted by one step rise to 100% of elution buffer (about 5 CV) containing 200mM TRIS, lOOmM Sorbitol pH 8. The step is performed at room temperature. REFERENCES

[1] Shukla AA, Gupta P, Han X, Protein aggregation kinetics during Protein A chromatography: Case study for an Fc fusion protein Journal of Chromatography A, Volume 1171, Issues 1-2, 9 November 2007, Pages 22-28

[2] Mazzer AR, Perraud X, Halley J, O'Hara J, Bracewell DJ, Protein A chromatography increases monoclonal antibody aggregation rate during subsequent low pH virus inactivation hold Journal of Chromatography A, Volume 1415, 9 October 2015, Pages 83-90

Claims

A compound according to formula (I):

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are independently from each other selected from the group consisting of H, -OH, (Ci-C₆)alkoxy, and Rⁿ-C(0)-0-, wherein R¹¹ is H or (Ci-C₄)alkyl;

with the proviso that, when A is absent, then X is not -OH.

The compound of claim 1 , wherein A is selected from the group consisting of (Ci-C3o)alkyl, (C₂-C3o)alkenyl, (C3-Cs)cycloalkyl, (C3-C8)cycloalkenyl, (C₆-Ci₄)aryl, (C₆-Ci₄)aryl(Ci- C₂₄)alkyl, (Ci-C₂₄)alkyl(C₆-Ci₄)aryl, (Ci-C₃o)heteroalkyl, (C₂-C₃o)heteroalkenyl, (C₃- C8)heterocycloalkyl, (C3-C8)heterocycloalkenyl, (C3-Ci3)heteroaryl, (C3- Ci3)heteroaryl(Ci-C24)alkyl, (C6-Ci4)aryl(Ci-C24)heteroalkyl, (C3-Ci3)heteroaryl(Ci- C24)heteroalkyl, (Ci-C24)heteroalkyl(C6-Ci4)aryl, (Ci-C24)alkyl(C3-Ci3)heteroaryl, (Ci- C24)heteroalkyl(C3-Ci3)heteroaryl, (Ci-C3o)alkoxy, (C2-C3o)alkenoxy, (C3- Cs)cycloalkoxy, (C3-C8)cycloalkenoxy, (C6-Ci4)aryloxy, (C6-Ci4)aryl(Ci-C24)alkoxy, and (Ci-C24)alkyl(C6-Ci4)aryloxy.

The compound of claims 1 or 2, wherein the compound has a structure according to formula

(II

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are independently from each other selected from the group consisting of H, -OH, (Ci-C6)alkoxy, and Rⁿ-C(0)-0-, wherein R¹¹ is H or (Ci-C₄)alkyl.

4. The compound of claim 3, wherein L is selected from the group consisting of (Ci-C3o)alkyl, (C₂-C3o)alkenyl, (C3-Cs)cycloalkyl, (C3-C8)cycloalkenyl, (C₆-Ci4)aryl, (C₆-Ci4)aryl(Ci- C₂4)alkyl, (Ci-C₂4)alkyl(C₆-Ci₄)aryl, (Ci-C₃o)heteroalkyl, (C₂-C₃o)heteroalkenyl, (C₃- C8)heterocycloalkyl, (C3-C8)heterocycloalkenyl, (C3-Ci3)heteroaryl, (C3- Ci3)heteroaryl(Ci-C₂4)alkyl, (C6-Ci4)aryl(Ci-C₂4)heteroalkyl, (C3-Ci3)heteroaryl(Ci-

C₂4)heteroalkyl, (Ci-C₂4)heteroalkyl(C6-Ci4)aryl, (Ci-C₂4)alkyl(C3-Ci3)heteroaryl, and (C 1 -C₂4)heteroalkyl(C3-C 13)heteroaryl.

5. The compound of claims 1 or 2, wherein -A-X together is selected from the group consisting of-C₆H₄-X, -CioH₆-X,-Ci₄H8-X, -0-C₆H₄-X, -O-CioHe-X, and -0-Ci₄H₈-X.

6. The compound of any one of claims 1 to 5, wherein X is selected from the group consisting of -OH, -NO2, -NH₂, -COOH, -NCO, azlactone-, N-hydroxysuccinimide-, maleinimide, carboxamido-, alkynyl, -N3, and -SH.

7. The compound of claim 1 , wherein -A-X together is a substituent according to formula III:

8. The compound of any one of claims 1 to 7, wherein R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are -OH.

9. The compound of any one of claims 1 to 7, wherein R¹ and R² are independently from each other selected from the group consisting of H, -OH, and -(Ci-C6)alkoxy. 10. A complex comprising:

(a) the compound according to any one of claims 1 to 9; and

(b) a Lewis-acidic transition metal ion.

11. The complex of claim 10, wherein the Lewis-acidic transition metal ion is selected from the group consisting of Cu(II), Mn(II), Fe(II), Fe(III), and Co(II).

12. A complex comprising:

(a) the compound according to any one of claims 1 to 9; and

13. The complex of claim 12 wherein the diboronic acid is selected from the group consisting of 1 ,4-phenylendiboronic acid (synm. 1,4-benzenediboronic acid), 2 -Nitrobenzene- 1,4- diboronic acid (synm 2-nitro-l,4-phenylenediboronic acid), 2,2'-Bithiophene-5,5'- diboronic acid, 4-4'-biphenyldiboronic acid, 2,5-thiophenediboronic acid, stilbene-4,4- diboronic acid, 4,4'-oxybis(l,4-benzene)diboronic acid, dibenzofuran-4,6-diboronic acid and derivatives thereof.

14. A method of producing an affinity matrix for the purification of glycosylated biomolecules, comprising the steps:

(i) providing a solid support; and

(ii) covalently coupling the compound according to any one of claims 1 to 9 to said solid support via functional group X, thereby producing a functionalized solid support; and

either with a Lewis-acidic transition metal ion

thereby producing said affinity matrix.

15. The method of claim 14, wherein the Lewis-acidic transition metal ion is selected from the group consisting of Cu(II), Mn(II), Fe(II), Fe(III), and Co(II).

16. The method of claim 14 wherein the diboronic acid is selected from the group consisting of ,4-phenylendiboronic acid (synm. 1,4-benzenediboronic acid), 2 -Nitrobenzene- 1,4- diboronic acid (synm 2-nitro-l,4-phenylenediboronic acid), 2,2'-Bithiophene-5,5'- diboronic acid, 4-4'-biphenyldiboronic acid, 2,5-thiophenediboronic acid, stilbene-4,4- diboronic acid, 4,4'-oxybis(l,4-benzene)diboronic acid, dibenzofuran-4,6-diboronic acid and derivatives thereof.

17. A method of producing an affinity matrix for the purification of glycosylated biomolecules, comprising the steps:

(i) providing a solid support; and

(ii) covalently coupling the complex according to any one of claims 10 to 13 to said solid support via functional group X, thereby producing said affinity matrix.

18. An affinity matrix obtainable by the method of any one of claims 14 to 17.

Use of

(i) the compound according to any one of claims 1 to 9;

(ii) the complex according to any one of claims 10 to 13; or

(iii) the affinity matrix according to claim 18

for the purification of glycosylated biomolecules. 20. The use of claim 19, wherein the glycosylated biomolecules are selected from the group consisting of

(a) glycoproteins,

(b) gly co lipids, and

(c) enveloped viruses comprising glycostructures on their envelopes.

21. The use of claims 19 or 20, wherein the glycosylated biomolecules carry N-glycans.

22. The use of claim 21, wherein the N-glycans are selected from the group consisting of high- mannose type N-glycans, complex type N-glycans, hybrid type N-glycans, and paucimannose type N-glycans.

23. A method for purifying glycosylated biomolecules from a cell culture supernatant comprising the steps of:

(i) providing a cell culture supernatant comprising glycosylated biomolecules, and (ii) applying the cell culture supernatant to the affinity matrix according to claim 18.

24. The method of claim 23, wherein the method further comprises the step(s) of:

(iii) washing the affinity matrix with a washing buffer, and/or

(iv) releasing the glycosylated biomolecules from the affinity matrix with an elution buffer containing a competitive eluent.

25. A method for purifying glycosylated bio molecules from a cell culture supernatant comprising the steps of:

(i) providing a cell culture supernatant comprising glycosylated bio molecules, and

(ii) precipitating the glycosylated bio molecules from the cell culture supernatant with the compound according to any one of claims 1 to 9, or with the complex according to any one of claims 10 to 13.

26. The method of claim 25, wherein the method further comprises the step(s) of:

(iii) washing the precipitate with a washing buffer, and/or

(iv) releasing the glycosylated bio molecules from the precipitate with an elution buffer containing a competitive eluent.