CONTROLLED RELEASE OF GLYCANS FROM GLYCOPROTEINS AND

ENVELOPED VIRUSES

The present invention relates to a method for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes. Further, the invention relates to a method for isolating glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes. Furthermore, the invention relates to kits for carrying out these methods.

BACKGROUND OF THE INVENTION

Glycostructures are composed of multiple glycosidically linked monosaccharides connected to form linear as well as complex, branched structures. Glycans are hydrophilic molecules that can vary in size from a single monosaccharide to extremely large polysaccharides. Typically, glycans are conjugated to proteins and lipids present on cell surfaces as well as other cellular compartments conjugated to proteins and lipids. Glycans encompassing N-linked or O-linked glycans are found on glycoproteins and proteoglycans, e.g. protein conjugates. Glycans are also coupled to lipids such as ceramide, e.g. glycolipids.

Protein-associated glycostructures influence efficacy and safety of therapeutically glycoproteins used on pharmacokinetic as well as pharmacodynamic and immunological level. As the quality, effectiveness, and safety of medicinal products has to be assured before said products are placed on the market, the quality of the active ingredients is monitored at the glycosylation level both during the production process and in the course of batch release.

Currently established methods for the determination of glycan structures are based on sample preparation with subsequent analytical separation of the individual differentiated glycans, e.g. High Performance Liquid Chromatography- (HPLC-) based separation methods such as Hydrophilic Interaction Liquid Chromatography (HILIC) and Reversed Phase High Performance Liquid Chromatography (RP-HPLC) and capillary electrophoresis. All these methods offer the advantage of immediate quantifiability of the glycans via the obtained calibrated peak area. The disadvantage is, however, the need for fluorescent labeling of the glycans which is essential in order to achieve the necessary sensitivity. Especially, the low sample volume for in-process controls is a limiting factor. In contrast thereto, mass spectrometric (MS) methods work label-free, but the evaluation of the MS spectra obtained is usually complex and complicated by adduct formation and the occurrence of multiple charged species with identical conformation and configuration. A reliable quantification of the glycans via the relative peak intensities is, therefore, only possible to a limited extent. The use of mass spectrometry in house is time-consuming and release analysis also requires high capital expenditures (CapEX) and operating expenditures (OPEX). This means that a device failure cannot be immediately remedied with internal resources. The use of less robust systems or external services in order to fulfil the Good Manufacturing Practice (GMP) is, thus, out of question.

A major disadvantage of HPLC and electrophoresis-based methods is the time expenditure for release and fluorescent labeling of the glycans. Enzymatic methods for glycan release often take several hours. Classical methods for fluorescent labeling via reductive amination with cyanoborohydride or picolinborane take several hours.

A further major problem of previous methods is the release and detection of proteinbound O-glycans. Previous standard procedures for the release of O-glycans rely on the process of beta-elimination/hydrazinolysis with free Hydra zinc, a highly toxic, unstable and carcinogenic substance (IARC Category 2A).

The introduction of the Quality-by-Design (QbD) initiative of the US Food and Drug Administration (FDA) in 2004 resulted in a paradigm shift with regard to the basic quality assurance measures which accompany the manufacturing process. While in the past, quality assurance was limited to the process-accompanying collection of measurement data on statically defined quality criteria and on a downstream error correction within the scope of corrective and preventive measures (CAPA), the focus of quality assurance measures is now shifted more strongly into the area of error prevention with a focus on more targeted error identification and more efficient error reduction. On the one hand, the quality by design (QbD) model is methodically supported by a knowledge-based, technical recording of the design space for the manufacturing process. On the other hand, it is supported by the timely and process- related collection of measurement data which allows in real-time immediate regulatory intervention in the process. The latter is summarized under the keyword “Process Analytical Technology (PAT)”. The QbD initiative, therefore, requires a knowledge-based approach for product-oriented quality control. The basis for this is firstly the scientific and technical justified definition of a target profile for the product to be manufactured (Quality Target Product Profile (QTPP)) and secondly the collection of data on Critical Material Parameters (CMPs) and Critical Process Parameters (CPPs) as well as their influence on the Critical Quality Attributes (CQAs). The drug-associated sugar structure characteristic is one of the most relevant CQAs, whose process-related and timely recording is essential for a better understanding of the relationship between influencing parameters (CMAs, CQAs) and quality attributes (CQAs) and finally to an improved understanding of product quality in terms of the QbD model.

Currently, the analysis of these glycans including sample preparation requires several hours. During this time, the process continues without any meaningful intervention. The CQA analysis for glycostructures is, therefore, not PAT-compatible. There is presently no chemical method available which allows the controlled and simultaneous removal of all kinds of glycostructures from glycoproteins or enveloped viruses. There is presently also no method available which allows the coupling to fluorescent markers to the glycostructures at the same time. Thus, there is a strong need for a cost-efficient and time-efficient controlled process allowing the removal of glycostructures from glycoproteins and/or enveloped viruses. In addition, the simultaneous labelling of said glycostructures would be advantageous. As the release and detection of O-glycans with the previously described methods requires the use of highly toxic and hazardous reagents that pose problems for occupational safety, the finding of a non-toxic alternative to the release of protein-bound O-Glycans would, therefore, also be beneficial.

The present invention discloses such a method. In particular, the inventors of the present invention provide a method which allows the removal of all types of glycostructures (N-, O-, and C-glycans) from glycoproteins in a time- and cost-effective manner as well as in a controllable way using a strong oxidizing reagent (= oxidizing redox potential/redox voltage, DIN 38404-6 of > +1 V under standard conditions) under alkaline conditions (e.g. pH of between 7.5 and 14). The strong oxidizing reagent is harmless and toxicologically safe. The glycostructures can optionally be labelled in situ at the same time with a tag, e.g. a fluorescent and charged compound. The oxidizing agents are preferably used in combination with a halide ion. The method of the present invention facilitates the concerted and accelerated release of protein-bound or virus-bound glycans and their simultaneous/controlled fluorescence and charge labelling for the purpose of an expedite detection. In addition, the method of the present invention enables an accelerated and sensitive analysis of the protein-bound or virus-bound sugar with robust standard methods. The release of glycostructures takes place with cheap chemicals instead of expensive enzymes. In addition, the glycostructures are exposed to the chemicals for a very short time (< 1 hour). In this way, the specific glycostructures - especially sialylations - are preserved. SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a method for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprising the steps of

(i) producing a mixture comprising an alkaline solution, preferably an alkaline aqueous solution, comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E°red) of > + 1.00 V, and

(ii) incubating the mixture produced in step (i), thereby releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

In a second aspect, the present invention relates to a method for isolating glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprising the steps of

(i) carrying out the method of the first aspect, thereby releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes, and

(ii) separating the glycostructures from the deglycanized proteins or enveloped viruses, thereby isolating the glycostructures from the glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

In a third aspect, the present invention relates to a composition comprising glycostructures obtainable by the method of the second aspect.

In a fourth aspect, the present invention relates to a composition comprising N-glycans (released from glycoproteins or enveloped viruses comprising glycostructures on their envelopes) coupled to a fluorescent compound comprising a primary amine functional group via an urea bond/linkage.

In a fifth aspect, the present invention relates to a composition comprising O-glycans (released from glycoproteins or enveloped viruses comprising glycostructures on their envelopes) coupled to a fluorescent compound comprising a primary amine functional group via a carboxamide ester bond/linkage.

In a sixth aspect, the present invention relates to the use of an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes. In a seventh aspect, the present invention relates to a kit for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprising

(i) an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V, and

(ii) optionally a reducing agent and/or a protein precipitant.

Preferably, the kit further comprises a fluorescent compound comprising a primary amine functional group and/or a halide ion.

In an eighth aspect, the present invention relates to a kit for isolating glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprising

(i) an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V,

(ii) a separation material, and

(iii) optionally a reducing agent and/or a protein precipitant.

Preferably, the kit further comprises a fluorescent compound comprising a primary amine functional group and/or a halide ion.

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

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. The term “oligopeptide”, as used herein, 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.

The term “glycan”, as used herein, refers to a molecule that is composed of multiple glycosidically linked monosaccharides connected to form linear as well as complex, branched structures. A glycan is a hydrophilic molecule that can vary in size from a single monosaccharide to extremely large polysaccharides. Typically, a glycan is present on cell surfaces as well as other cellular compartments conjugated to proteins and lipids. A glycan encompassing N-linked or O-linked structures is found on glycoproteins and proteoglycans, e.g. protein conjugates. A glycan is also coupled to lipids such as ceramide, e.g. glycolipids.

In the context of the present invention, the term “glycoprotein” refers to a protein that contains oligosaccharide chains (glycans) covalently attached to its polypeptide side-chains. In particular, the term “glycoprotein”, as used herein, refers to a protein that contains multiple glycosidically linked monosaccharides connected to form linear as well as complex, branched structures covalently attached to its polypeptide side-chains. The carbohydrate is attached to the protein in a process of co-translational or post-translational modification. This process is known as glycosylation such as N-glycosylation or O-glycosylation.

In one preferred embodiment, the glycoprotein is an antibody. 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 Vu-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.

In the context of the present invention, the term “glycostructure” refers to oligosaccharide chains (glycans) usually covalently attached to polypeptide side-chains of proteins or lipids. These proteins are designated as glycoproteins or glycolipids. 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 terms “glycan” and “glycostructure” are used interchangeably in the context of the present invention.

The term “N-glycosylation”, as used herein, means the addition of sugar chains to a nitrogen atom, e.g. the amide nitrogen on the side chain of asparagine (Asn), of a protein.

The term “O-glycosylation”, as used herein, means the addition of sugar chains to a hydroxyl oxygen on the side chain of hydroxylysine, hydroxyproline, serine, or threonine of a protein.

The term “N-glycan”, as used herein, means an N-linked polysaccharide or oligosaccharide. In particular, the term “N-glycan, as used herein, means a glycan that is or was attached to a protein via a glycosyl amide linkage where the glycan is attached to a nitrogen atom, e.g. the amide nitrogen of asparagine (Asn) residue, of a protein. Thus, the term “N- glycan”, as used herein, refers to the free, reducing glycan released by enzymatic processes, e.g. by N-glycosidases, such as Peptide-N-Glycosidases F or A (PNGase F or A), or by chemical processes from proteins. 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 MansGIcNAc2 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 “O-glycan”, as used herein, means an O-linked polysaccharide or oligosaccharide. In particular, the term “O-glycan”, as used herein, means a glycan that is or was attached to protein via a glycosidic linkage where the glycan is attached to an oxygen atom in an amino acid residue in a protein. Thus, the term “O-glycan”, as used herein, refers to the free, reducing glycan released by enzymatic processes, e.g. by O-glycosidases, such as Endo- a-N-Acetylgalactosaminidase, or by chemical processes from proteins. 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 “C-glycan”, as used herein, refers to a rare form of glycosylation where a sugar is added to a carbon on a tryptophan side chain.

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 cell membranes (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 “oxidizing agent (also designated as reactant)”, as used herein, refers to a compound that removes electrons from other reactants during a redox reaction. The oxidizing agent typically takes these electrons for itself, thus, gaining electrons and being reduced. An oxidizing agent is an electron acceptor.

The term „reducing agent (also designated as reductant)” as used herein, refers to a compound that loses an electron to an electron recipient in a redox chemical reaction. A reducing agent is, thus, oxidized when it loses electrons in the redox reaction. A reducing agent is an electron donator.

In one preferred embodiment, the reducing agent is selected from the group consisting of formic acid, ascorbic acid acetaldehyde, and formaldehyde.

The term “oxidizing agent comprising at least one aromatic residue and at least one halogen atom”, as used herein, refers to an aromatic chemical compound with an oxidizing redox potential that contains at least one halogen atom comprised within at least one substituent group on the aromatic ring. Such compounds include, but are not limited to, phenylsulfonylazanides, aromatic iodine (III) compounds such as iodosobenzoic acid and hypervalent iodine compounds such as (bis(trifluoroacetoxy)iodo) benzene.

In one preferred embodiment, the halogen atom is a covalently bound (non-radioactive) halogen atom selected from the group consisting of fluorine, chlorine, bromine, or iodine. In one more preferred embodiment, the oxidizing agent comprising at least one aromatic residue and at least one halogen atom is selected from the group consisting of a phenylsulfonylazanide, a hypervalent iodine compound, N,N-dichlorobenzensulfonamide, and a cyclic iodine (III) compound.

The term “redox potential (also known as reduction/oxidation potential)” is a measure of the tendency of a chemical species to acquire electrons from or lose electrons to an electrode and thereby be reduced or oxidized respectively. Redox potential is measured in volts (V) or milivolts (mV). Each species has its own intrinsic redox potential; for example, the more positive the reduction potential (reduction potential is more often used due to general formalism in electrochemistry), the greater the species' affinity for electrons and tendency to be reduced.

The term “standard reduction potential (E° _re d)”, as used herein, is the reduction potential of a molecule under specific, standard conditions. The standard reduction potential can be useful in determining the directionality of a reaction. The reduction potential of a given species can be considered to be the negative of the oxidation potential. The standard conditions for determining the standard reduction potential (E° _re d) are as follows: 25°C, a 1 activity for each ion participating in the reaction, a partial pressure of 101.325 kPa (absolute) (1 atm, 1.01325 bar) for each gas that is part of the reaction, and metals in their pure state. The standard reduction potential is defined relative to a standard hydrogen electrode (SHE) reference electrode, which is arbitrarily given a potential of 0.00 V.

The oxidizing agent used in the present invention has a standard reduction potential at 25°C (E°red) of > + 1.00 V, preferably of > + 2.12 V. In one preferred embodiment, the oxidizing agent having a standard reduction potential at 25°C (E°red) of > + 1.00 V is selected from the group consisting of an interhalogen compound, an interhalide compound, and a peroxo compound.

The term “interhalogen compound”, as used herein, refers to a molecule which contains two or more different halogen atoms (fluorine, chlorine, bromine, iodine, or astatine) and no atoms of elements from any other group. Most interhalogen compounds known are binary (composed of only two distinct elements). Their formulae are generally XY„, where n = 1, 3, 5 or 7, and X is the less electronegative of the two halogens. The value of “n” in interhalogens is always odd, because of the odd valence of halogens. They are all prone to hydrolysis and ionize to give rise to polyhalogen ions.

The term “peroxo compound”, as used herein, refers to an organic or inorganic molecule containing active oxygen. In particular, a peroxo compound is an organic or inorganic peroxo salt containing active oxygen such as e.g. peroxomonosulfate, peroxodi sulfate, perborate, peracetate, or percarbonate.

The term “halide ion”, as used herein, refers to a singlet halogen atom bearing a negative charge of -1. In the context of the present invention, the term “halide ion” encompasses nonradioactive halogen anions. Preferred halide ions are selected from the group consisting of fluoride (F-), chloride (C1-), bromide (Br-) and iodide (I-).

The term “aqueous solution”, as used herein, refers to a solution comprising water. The word aqueous (which comes from aqua) means pertaining to, related to, similar to, or dissolved in water. The aqueous solution of the present invention comprises glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

In one preferred embodiment, the aqueous solution is selected from the group consisting of a cell culture supernatant, a cell culture, a buffer solution, a body fluid sample, and water.

The term “cell culture supernatant (e.g. vertebrate cell culture supernatant)”, as used herein, refers to any cell culture supernatant comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes. It is usually obtained from a cell culture (e.g. vertebrate cell culture). Techniques to harvest the cell culture supernatant (e.g. vertebrate cell culture supernatant) from a cell culture (e.g. vertebrate cell culture) are known to the skilled person. This can be done, for example, by centrifugation, sedimentation and/or filtration techniques.

The term “body fluid sample”, as used herein, refers to any liquid sample derived from the body of an individual (e.g. mammal such as human) which comprises glycoproteins or enveloped viruses comprising glycostructures on their envelopes. Said body fluid sample may be a urine sample, blood sample, sputum sample, breast milk sample, cerebrospinal fluid (CSF) sample, cerumen (earwax) sample, gastric juice sample, mucus sample, lymph sample, endolymph fluid sample, perilymph fluid sample, peritoneal fluid sample, pleural fluid sample, saliva sample, sebum (skin oil) sample, semen sample, sweat sample, tears sample, cheek swab, vaginal secretion sample, liquid biopsy, or vomit sample including components or fractions thereof.

The solution used in the context of the present invention is an alkaline solution. The term “alkaline solution”, as used herein, refers to any solution having a pH of between 7.5 and 14. Said solution comprises glycoproteins or enveloped viruses comprising glycostructures on their envelopes. In particular, alkaline solutions are solutions of metal hydroxides such as sodium hydroxide or potassium hydroxide. These metal hydroxides belong to alkali hydroxides. In a preferred embodiment, the alkaline solution is an alkaline aqueous solution.

The term “tag” as used herein, broadly encompasses a variety of types of molecules which are detectable through spectral properties (e.g. fluorescent makers or “fluorophores” and coloured markers (“chromophores”)).

The terms “fluorescent compound” or “fluorophore”, as used herein, refer to a compound which absorbs light and can be detected by measuring absorbance at an appropriate wavelength.

The term “fluorescent compound comprising a primary amine functional group”, as used herein, refers to any fluorescent compound bearing a primary amine functional group that may undergo chemical bonding via the nitrogen atom while the overall molecule retains its fluorescence. Specifically, these molecules retain very good detectability features such as a sufficiently broad stokes shift, i.e. a good separation between the excitation and emission maxima, a sufficiently high quantum yield, molar extinction coefficient, photostability and excited-state-lifetime as well as low tendency for quenching and photobleaching when coupled to oligosaccharides via the primary amine functional group.

In one preferred embodiment, the fluorescent compound comprising a primary amine functional group is selected from the group consisting of 2-amino-benzamide (2-AB), 2-aminobenzoic acid, procainamide, 8-aminonaphthalene-l,3,6-trisulfonic acid (ANTS), 8-aminonaphthalene disulfonic acid (ANDS), aminopyrenetrisulfonic acid (APTS), and aminoacridone.

The term “protein precipitant”, as used herein, refers to a molecule which helps to separate the protein from any extra component that may be mixed with it. It is an important part of downstream processing and can be done with a variety of different techniques. While there are a number of different methods of precipitation, the two most popular ones are Salt Induced Precipitation (“Salting Out”), e.g. with ammonium sulfate, isoelectric precipitation, or precipitation with trichloroacetic acid (in particular 2,2,2-trichoroacetic acid). In the context of the present invention, the protein precipitant is used to precipitate the deglycanized proteins and enveloped viruses. In this way, the separation of the released glycostructures from the deglycanized proteins or enveloped viruses is possible.

In one preferred embodiment, the protein precipitant is selected from the group consisting of trichloroacetic acid (in particular 2,2,2-trichoroacetic acid), ammonium sulfate, methanol, acetone, and pyrogallol red/molybdate reagent. In an alternative preferred embodiment, proteins are co-precipitated with functionalized resin bead slurry, such as a slurry of hydroxylated silica particles or silica-resin beads functionalized with butyl-, Octyl- or Octadecyl-side chains.

In the context of the present invention, the term “kit of parts (in short: kit)” is understood to be any combination of at least some of the components identified herein, which are combined, coexisting spatially, to a functional unit, and which can contain further components.

Embodiments of the invention

The inventors of the present invention provide a method which allows the removal of all types of glycostructures (N-, O-, and C-glycans) from glycoproteins in a time- and cost- effective manner as well as in a controllable way using a strong oxidizing reagent (= oxidizing redox potential/redox voltage, DIN 38404-6 of > +1 V under standard conditions) under alkaline conditions (e.g. pH of between 7.5 and 14). The strong oxidizing reagent is harmless and toxicologically safe. The glycostructures can optionally be labelled in situ at the same time with a tag, e.g. a fluorescent and charged compound. The oxidizing agents are preferably used in combination with a halide ion. The method of the present invention facilitates the concerted and accelerated release of protein-bound or virus-bound glycans and their simultaneous/controlled fluorescence and charge labelling for the purpose of an expediate detection. In addition, the method of the present invention enables an accelerated and sensitive analysis of the protein-bound or virus-bound sugar with robust standard methods. The release of glycostructures takes place with cheap chemicals instead of expensive enzymes. In addition, the glycostructures are exposed to the chemicals for a very short time (< 1 hour). In this way, the specific glycostructures - especially sialylations - are preserved.

Thus, in a first aspect, the present invention relates to a (an) (in vitro) method for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprising the steps of: (i) producing a mixture comprising an alkaline solution, preferably an alkaline aqueous solution, comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E°red) of > + 1.00 V, preferably of > + 2.12 V, and

(ii) incubating the mixture produced in step (i), thereby releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes. Alternatively, the mixture can be provided in step (i) of the above method.

The present inventors found that the use of an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V allows under alkaline conditions the rapid, non-specific, and non-toxic release of glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes. The released glycostructures can immediately/directly be further processed for analytical purposes e.g. identifying HPLC, mass spectroscopy or capillary electrophoretic profiles of samples. In addition, the glycostructures are released in order to overcome the difficulties in obtaining sufficient quantities of glycans for studies or in order to establish a glycome (the entire complement of sugars, whether free or present in more complex molecules of an organisms, organ, tissue, or cell, e.g. recombinant cell).

Preferably, the oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1,00 V is comprised in the mixture in a concentration of between 0.5 % (w/v) and 30 % (w/v), more preferably of between 0.5 % (w/v) and 20 % (w/v), even more preferably of between 0.5 % (w/v) and 10 % (w/v), and most preferably of between 0.5 % (w/v) and 4 % (w/v), e.g. of 0.5, 1, 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 % (w/v).

The mixture can be produced by any method known to the skilled person which allows the blending of the different components, e.g. by stirring or shaking the different components. In particular, the mixture can be produced by adding an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V to an alkaline solution, such as an alkaline aqueous solution, comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes. Alternatively, the mixture can be produced by adding an alkaline solution, such as an alkaline aqueous solution, comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes to an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V.

In one preferred embodiment, the mixture is produced by adding an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V to an alkaline solution, preferably an alkaline aqueous solution, comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

Preferably, the oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V is added to the alkaline solution, in particular alkaline aqueous solution, comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes in a concentration of between 0.5 % (w/v) and 30 % (w/v), more preferably of between 1 % (w/v) and 10 % (w/v), even more preferably of between 1 % (w/v) and 8 % (w/v), and most preferably of between 1 % (w/v) and 5 % (w/v), e.g. of 0.5, 1, 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 % (w/v).

In one further preferred embodiment, the at least one halogen atom is a covalently bound (non-radioactive) halogen atom selected from the group consisting of fluorine, chlorine, bromine or iodine.

In one (additional or alternative) preferred embodiment, the oxidizing agent comprising at least one aromatic residue and at least one halogen atom is selected from the group consisting of a phenylsulfonylazanide, a hypervalent iodine compound, N,N-dichlorobenzensulfonamide, and a iodine (III) compound. Said oxidizing agents comprise at least one covalently bound (nonradioactive) halogen atom selected from the group consisting of fluorine, chlorine, bromine or iodine. It should be noted that the oxidizing agent is not limited to these compounds.

In one more preferred embodiment, the phenylsulfonylazanide is selected from the group consisting of sodium;chloro-(4- methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro- (4-Methylbenzene)sulfonamide, Chloramine T) and sodium;benzenesulfonyl(chloro)azanide (also designated as Chloramine B), the hypervalent iodine compound is selected from the group consisting of X3-iodane (iodine (III), iodine oxidation number +3), preferably phenyl{bis[(trifluoroacetyl)oxy]}-Z3-iodane (also designated as Bis(trifluoroacetoxy)iodo)benzene), and X5-iodane (iodine (V), iodine oxidation number +5), the N,N-dichlorobenzensulfonamide is selected from the group consisting of N,N-dichloro-4- methylbenzenesulfonamide (Dichloramine-T), N,N-di chlorobenzenesulfonamide (Dichloramine-B), or the iodine (III) compound is selected from the group consisting of iodoxybenzoic acid, iodosobenzoic acid, and Dess Martin Periodinane.

It should be noted that the oxidizing agent is not limited to these compounds.

The use of sodium;chloro-(4-methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro-(4-Methylbenzene)sulfonamide, Chloramine T) is particularly preferred.

In one alternative preferred embodiment, the oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V, preferably of > + 2.12 V, is selected from the group consisting of an interhalogen compound, a peroxo compound, and a (strongly oxidizing) heavy metal containing compound. The oxidizing agent can further be any other organic compound with the above standard reduction potential.]

In one more preferred embodiment, the interhalogen compound is iodine monochloride, the peroxo compound is selected from the group consisting of hydrogen peroxide, peracetate, percarbonate, perborate, peroxomonosulfate, and peroxodi sulfate, or the (strongly oxidizing) heavy metal containing compound is lead tetraacetate.

The use of ammoniumperoxodi sulfate (APS), potassium peroxodisulfate, or sodium peroxodisulfate is particularly preferred.

Preferably, the alkaline solution, in particular alkaline aqueous solution, has a pH of between 7.5 and 14, more preferably of between 11 and 14, and even more preferably of between 12 and 14, e.g. a pH of 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, or 14.

Preferably, the aqueous solution is selected from the group consisting of a cell culture supernatant, a cell culture, a buffer solution, a body fluid sample, and water.

More preferably, the aqueous solution is a cell culture supernatant or a body fluid sample. The cell culture supernatant is usually obtained from a cell culture (e.g. vertebrate cell culture). Techniques to harvest the cell culture supernatant (e.g. vertebrate cell culture supernatant) from a cell culture (e.g. vertebrate 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, a fish, an amphibian, a reptilian, or an avian cell culture supernatant. In particular, the vertebrate cell culture supernatant is a mammalian cell culture supernatant. It is particularly preferred that (i) the mammalian cell culture supernatant is a human, hamster, canine, or monkey cell culture supernatant, e.g. 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 (IgH-2) cell culture supernatant, or (iv) the avian cell culture supernatant is a duck cell culture supernatant.

The body fluid sample may be a urine sample, blood sample, sputum sample, breast milk sample, cerebrospinal fluid (CSF) sample, cerumen (earwax) sample, gastric juice sample, mucus sample, lymph sample, endolymph fluid sample, perilymph fluid sample, peritoneal fluid sample, pleural fluid sample, saliva sample, sebum (skin oil) sample, semen sample, sweat sample, tears sample, cheek swab, vaginal secretion sample, liquid biopsy, or vomit sample including components or fractions thereof.

The glycostructures released from the glycoproteins and enveloped viruses retain their free, reducing end and can further be tagged specifically for structural analysis, separation purposes e.g. chromatographic separation, introduction of functional groups for subsequent chemical modifications, e.g. covalent attachment to solid phases, or addition of other tags. The tag may be any molecule which is detectable through spectral properties (e.g. fluorescent makers or “fluorophores” and coloured markers (“chromophores”)). For the labelling, the tag may be added to the alkaline solution, such as alkaline aqueous solution. Thus, in one even more preferred embodiment, the mixture further comprises a fluorescent compound comprising a primary amine functional group. In this case, the (in vitro) method for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprises the steps of

(i) producing a mixture comprising an alkaline solution, preferably an alkaline aqueous solution, comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes, an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E°red) of > + 1.00 V, and a fluorescent compound comprising a primary amine functional group, and

(ii) incubating the mixture produced in step (i), thereby releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

The mixture can be produced by adding an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of> + 1.00 V to an alkaline solution, in particular an alkaline aqueous solution, comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and a fluorescent compound comprising a primary amine functional group. Preferably, the fluorescent compound comprising a primary amine functional group is comprised in the mixture in a concentration of between 1 mM and 1000 mM, more preferably of between 50 mM and 800 mM, even more preferably of between 100 mM and 500 mM, and most preferably of between 100 mM and 300 mM, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mM.

Alternatively, the mixture can be produced by adding a fluorescent compound comprising a primary amine functional group to an alkaline solution, in particular an alkaline aqueous solution, comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V.

Preferably, the fluorescent compound comprising a primary amine functional group is added to the alkaline solution, in particular alkaline aqueous solution, comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1,00 V in a concentration of between 0.05 M and 8 M, more preferably of between 0.1 M and 0.5 M, even more preferably of between 0.1 M and 0.3 M, and most preferably of between 0.2 M and 0.3 M, e.g. 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, or 8 M.

In one still even more preferred embodiment, the fluorescent compound comprising a primary amine functional group is selected from the group consisting of 2-amino-benzamide (2-AB), 2-aminobenzoic acid, procainamide, 8-aminonaphthalene-l,3,6-trisulfonic acid (ANTS), 8-aminonaphthalene disulfonic acid (ANDS), aminopyrenetri sulfonic acid (APTS), and aminoacridone. It should be noted that the fluorescent compound comprising a primary amine functional group is not limited to these compounds.

The present inventors found that the presence of a halide ion in the mixture further improves the release of glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes. Thus, in one even more preferred embodiment, the mixture further comprises a halide ion. In this case, the (in vitro) method for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprises the steps of: (i) producing a mixture comprising an alkaline solution, preferably an alkaline aqueous solution, comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes, an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E°red) of > + 1.00 V, and a halide ion, and

(ii) incubating the mixture produced in step (i), thereby releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

The mixture can be produced by adding an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of> + 1.00 V to an alkaline solution, in particular an alkaline aqueous solution, comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and a halide ion.

Preferably, the halide ion is comprised in the mixture in a concentration of between 20 mM and 500 mM, more preferably of between 30 mM and 250 mM, even more preferably of between 50 mM and 100 mM, and most preferably of between 80 mM and 100 mM, e.g. 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mM.

Alternatively, the mixture can be produced by adding a halide ion to an alkaline solution, in particular an alkaline aqueous solution, comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V.

Preferably, the halide ion is added to the alkaline solution, in particular alkaline aqueous solution, comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1,00 V in a concentration of between 20 mM and 500 mM, more preferably of between 30 mM and 250 mM, even more preferably of between 50 mM and 100 mM, and most preferably of between 80 mM and 100 mM, e.g. 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mM.

In one still even more preferred embodiment, the halide ion is selected from the group consisting of fluoride (F-), chloride (C1-), bromide (Br-), and iodide (I-).

In one most preferred embodiment, the mixture further comprises a fluorescent compound comprising a primary amine functional group and a halide ion. In this case, the (in vitro) method for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprises the steps of:

(i) producing a mixture comprising an alkaline solution, preferably an alkaline aqueous solution, comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes, an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E°red) of > + 1.00 V, a fluorescent compound comprising a primary amine functional group, and a halide ion, and

(ii) incubating the mixture produced in step (i), thereby releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

The method is carried out under conditions sufficient to release glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes. Preferably, the incubation step is carried out over a time period of between 1 and 30 minutes, more preferably of between 5 and 20 minutes, and even more preferably of between 10 and 20 minutes, e.g. over a period of 1, 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 minutes. In the examples the mixture was incubated over a time period of 15 minutes.

Exemplarily, the method for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprises the steps of:

(i) producing a mixture comprising an alkaline aqueous solution having a pH of between 7.5 and 14 comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and sodium peroxodi sulfate, potassium peroxodi sulfate, ammoniumperoxodi sulfate (APS) or sodium;chloro-(4-methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro-(4- Methylbenzene)sulfonamide, Chloramine T), and

(ii) incubating the mixture produced in step (i), thereby releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

In another example the method for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprises the steps of:

(i) producing a mixture comprising an alkaline aqueous solution having a pH of between 7.5 and 14 comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and sodium peroxodi sulfate, potassium peroxodi sulfate, ammoniumperoxodi sulfate (APS) or sodium;chloro-(4-methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro-(4- Methylbenzene)sulfonamide, Chloramine T) and 2-amino-benzamide (2-AB), and

(ii) incubating the mixture produced in step (i), thereby releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes. This method also allows the fluorescent labelling of the released glycostructures.

In another example, the method for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprises the steps of:

(i) producing a mixture comprising an alkaline aqueous solution having a pH of between 7.5 and 14 comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and sodium peroxodi sulfate, potassium peroxodi sulfate, ammoniumperoxodi sulfate (APS) or sodium;chloro-(4-methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro-(4- Methylbenzene)sulfonamide, Chloramine T) and potassium iodide, and

(ii) incubating the mixture produced in step (i), thereby releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

In another example, the method for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprises the steps of:

(i) producing a mixture comprising an alkaline aqueous solution having a pH of between 7.5 and 14 comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and sodium peroxodi sulfate, potassium peroxodi sulfate, ammoniumperoxodi sulfate (APS) or sodium;chloro-(4-methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro-(4- Methylbenzene)sulfonamide, Chloramine T), 2-amino-benzamide (2-AB), and potassium iodide, and

(ii) incubating the mixture produced in step (i), thereby releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

In one still most preferred embodiment, the method further comprises the step (iii) of adding a reducing agent and/or a protein precipitant to the mixture incubated in step (ii). The inventors of the present patent application found that the subsequent addition of a reducing agent efficiently stops the release of the glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes. It neutralizes the alkaline solution, preferably an alkaline aqueous solution. Alternatively or additionally, a protein precipitant is added. The inventors of the present patent application further found that the subsequent addition of a protein precipitant helps in a later isolation step to separate the released glycostructures from the deglycanized proteins or enveloped viruses.

Preferably, the reducing agent is added in a concentration of between 0.1 M and 1 M, e.g. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 M, and/or the protein precipitant is added in a concentration of between 0.5 M and 4 M, e.g. 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, or 4 M .

More preferably, the reducing agent is selected from the group consisting of formic acid, ascorbic acid acetaldehyde, and formaldehyde, and/or the protein precipitant is selected from the group consisting of trichloroacetic acid, methanol, acetone, and pyrogallol red/molybdate reagent.

Exemplarily, the method for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprises the steps of:

(i) producing a mixture comprising an alkaline aqueous solution having a pH of between 7.5 and 14 comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and sodium peroxodi sulfate, potassium peroxodi sulfate, ammoniumperoxodi sulfate (APS) or sodium;chloro-(4-methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro-(4- Methylbenzene)sulfonamide, Chloramine T), and

(ii) incubating the mixture produced in step (i), thereby releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

After step (ii), formic acid and/or trichloroacetic acid is (are) added.

In another example, the method for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprises the steps of:

(i) producing a mixture comprising an alkaline aqueous solution having a pH of between 7.5 and 14 comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and sodium peroxodi sulfate, potassium peroxodi sulfate, ammoniumperoxodi sulfate (APS) or sodium;chloro-(4-methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro-(4- Methylbenzene)sulfonamide, Chloramine T) and 2-amino-benzamide (2-AB), and

(ii) incubating the mixture produced in step (i), thereby releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

After step (ii), formic acid and/or trichloroacetic acid is (are) added.

In another example, the method for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprises the steps of: (i) producing a mixture comprising an alkaline aqueous solution having a pH of between 7.5 and 14 comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and sodium peroxodi sulfate, potassium peroxodi sulfate, ammoniumperoxodi sulfate (APS) or sodium;chloro-(4-methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro-(4- Methylbenzene)sulfonamide, Chloramine T) and potassium iodide, and

(ii) incubating the mixture produced in step (i), thereby releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

After step (ii), formic acid and/or trichloroacetic acid is (are) added.

In another example, the method for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprises the steps of:

(i) producing a mixture comprising an alkaline aqueous solution having a pH of between 7.5 and 14 comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and sodium peroxodi sulfate, potassium peroxodi sulfate, ammoniumperoxodi sulfate (APS) or sodium;chloro-(4-methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro-(4- Methylbenzene)sulfonamide, Chloramine T), 2-amino-benzamide (2-AB), and potassium iodide, and

(ii) incubating the mixture produced in step (i), thereby releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

After step (ii), formic acid and/or trichloroacetic acid is (are) added.

Preferably, the method is carried out at a temperature of between 20°C and 90°C, more preferably at a temperature of between 20°C and 70°C, and even more preferably at a temperature of between 50°C and 70°C, e.g. at room temperature (RT) or at 70°C. In the examples the mixture was incubated over 15 minutes at room temperature (RT).

The method allows the release of glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes. Preferably, at least 70% of the glycostructures attached to the glycoproteins or enveloped viruses comprised in the alkaline solution, such as alkaline aqueous solution, are released with the method of the present invention. More preferably, at least 80% of the glycostructures attached to the glycoproteins or enveloped viruses comprised in the alkaline solution, such as alkaline aqueous solution, are released with the method of the present invention. Even more preferably at least 90% of the glycostructures attached to the glycoproteins or enveloped viruses comprised in the alkaline solution, such as alkaline aqueous solution, are released with the method of the present invention. Still even more preferably, at least 95 % of the glycostructures attached to the glycoproteins or enveloped viruses comprised in the alkaline solution, such as alkaline aqueous solution, are released with the method of the present invention. Most preferably, at least 99% or even 100% of the glycostructures attached to the glycoproteins or enveloped viruses comprised in the alkaline solution, such as alkaline aqueous solution, are released with the method of the present invention. For example, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the glycostructures attached to the glycoproteins or enveloped viruses comprised in the alkaline solution, such as alkaline aqueous solution, are released with the method of the present invention.

The method allows the release of any glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes. The glycostructures which are released with the method of the present invention are preferably selected from the group consisting of N-glycans, O-glycans, N- and O-glycans, C-glycans, and lipid-linked glycans, or mixtures thereof.

In a second aspect, the present invention relates to a (an) (in vitro) method for isolating glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprising the steps of:

(i) carrying out the method of the first aspect, thereby releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes, and

(ii) separating the glycostructures from the deglycanized proteins or enveloped viruses, thereby isolating the glycostructures from the glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

In particular, the glycostructures are isolated from the deglycanized proteins or enveloped viruses.

Thus, the present invention relates to a method for isolating glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprising the steps of:

(i) producing a mixture of an alkaline solution, preferably an alkaline aqueous solution, comprising glycoproteins or enveloped viruses comprising glycostructures on their envelopes and an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E°red) of > + 1.00 V, preferably of > + 2.12 V,

(ii) incubating the mixture produced in step (i), thereby releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes, and (iii) separating the glycostructures from the deglycanized proteins or enveloped viruses, thereby isolating the glycostructures from the glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

In particular, the glycostructures are isolated from the deglycanized proteins or enveloped viruses.

With regard to the specific embodiments of steps (i) and (ii), it is referred to the first aspect of the present invention.

In one preferred embodiment, the separation in step (iii) is carried out by a technique selected from the group consisting of chromatography, filtration, electrophoresis, and centrifugation.

In one more preferred embodiment, the chromatography is selected from the group consisting of hydrophilic interaction chromatography (HILIC) and reversed-phase chromatography (RP-HPLC), or the electrophoresis is selected from the group consisting of capillary electrophoresis, capillary gel electrophoresis, and gel electrophoresis.

In one even more preferred embodiment, the RP-HPLC is carried out with a Cl 8 column, the HILIC is carried out with a ZIC-HILIC column (zwitterionic hydrophilic interaction liquid chromatography column), or the gel electrophoresis is a horizontal flatbed gel electrophoresis.

As mentioned above, the separation of the glycostructures from the deglycanized proteins or enveloped viruses is done in an even more preferred embodiment with a Cl 8 column. The long hydrophobic Cl 8 chains bind the deglycanized proteins or enveloped viruses reversibly. The glycostructures remain in the mobile phase.

Alternatively, the separation of the glycostructures from the deglycanized proteins or enveloped viruses is done in an even more preferred embodiment with a ZIC-HILIC column (zwitterionic hydrophilic interaction liquid chromatography column). The separation takes place by hydrophilic and weak electrostatic interactions of the zwitterionic phase with the glycostructures. The deglycanized proteins or enveloped viruses remain in the mobile phase.]

Subsequently, the purity and/or identify of the released glycostructures can be determined. The skilled person is aware of techniques how to determine the purity and/or identity of glycostructures. The purity and/or identity of the glycostructures is preferably measured/determined using (i) spectrometry, e.g. mass spectrometry (MS), (ii) chromatography, e.g. liquid chromatography (LC) such as high-performance liquid chromatography (HPLC), (iii) gel electrophoresis, e.g. horizontal flatbed gel electrophoresis or SDS gel electrophoresis, (iv) Western blot/Immunoblot analysis, or (v) combinations thereof.

In a third aspect, the present invention relates to a composition comprising glycostructures obtainable by the method of the second aspect.

The glycostructures which are released with the method of the present invention are preferably selected from the group consisting of N-glycans, O-glycans, N- and O-glycans, C-glycans, and lipid-linked glycans, or mixtures thereof. More preferably, the composition comprises N- glycans and/or O-glycans.

In a fourth aspect, the present invention relates to a composition comprising N-glycans (released from glycoproteins or enveloped viruses comprising glycostructures on their envelopes) coupled to a fluorescent compound comprising a primary amine functional group via a urea bond/linkage. The N-glycans comprised in this composition are released from glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

In a fifth aspect, the present invention relates to a composition comprising O-glycans (released from glycoproteins or enveloped viruses comprising glycostructures on their envelopes) coupled to a fluorescent compound comprising a primary amine functional group via a carboxamide ester bond/linkage. The O-glycans comprised in this composition are released from glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

In a sixth aspect, the present invention relates to the (in vitro) use of an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V, preferably of > + 2.12 V, for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

In one preferred embodiment, the oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V is used at an alkaline pH. Preferably, the oxidizing agent is used at a pH of between 7.5 and 14, more preferably of between 11 and 14, and even more preferably of between 12 and 14, e.g. at a pH of 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, or 14.

In one (alternative or additional) preferred embodiment, the oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V is used in a concentration of between 0.5 % (w/v) and 30 % (w/v), more preferably of between 1 % (w/v) and 10 % (w/v), even more preferably of between 1 % (w/v) and 8 % (w/v), and most preferably of between 1 % (w/v) and 5 % (w/v), e.g. of 0.5, 1, 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 % (w/v).

In one more preferred embodiment, the at least one halogen atom is a covalently bound (non-radioactive) halogen atom selected from the group consisting of fluorine, chlorine, bromine or iodine.

In one (additional or alternative) more preferred embodiment, the oxidizing agent comprising at least one aromatic residue and at least one halogen atom is selected from the group consisting of a phenylsulfonylazanide, a hypervalent iodine compound, N,N- dichlorobenzensulfonamide, and a cyclic iodine (III) compound. Said oxidizing agents comprise at least one covalently bound (non-radioactive) halogen atom selected from the group consisting of fluorine, chlorine, bromine or iodine. It should be noted that the oxidizing agent is not limited to these compounds.

In one even more preferred embodiment, the phenylsulfonylazanide is selected from the group consisting of sodium;chloro-(4- methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro- (4-Methylbenzene)sulfonamide, Chloramine T) and sodium;benzenesulfonyl(chloro)azanide (also designated as Chloramine B), the hypervalent iodine compound is selected from the group consisting of X3-iodane (iodine (III), iodine oxidation number +3), preferably phenyl{bis[(trifluoroacetyl)oxy]}-X3-iodane (also designated as Bis(trifluoroacetoxy)iodo)benzene), and X5-iodane (iodine (V), iodine oxidation number +5), the N,N-dichlorobenzensulfonamide is selected from the group consisting of N,N-dichloro-4- methylbenzenesulfonamide (Dichloramine-T) and N,N-dichlorobenzenesulfonamide (Dichloramine-B), or the cyclic iodine (III) compound is selected from the group consisting of iodoxybenzoic acid, iodosobenzoic acid, and Dess Martin Periodinane.

It should be noted that the oxidizing agent is not limited to these compounds.

The use of sodium;chloro-(4-methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, Natrium-N-chloro-(4-Methylbenzene)sulfonamide, Chloramine T) is particularly preferred.

In one alternative more preferred embodiment, the oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V, preferably of > + 2.12 V, is selected from the group consisting of an interhalogen compound, a peroxo compound, and a (strongly oxidizing) heavy metal containing compound. The oxidizing agent can further be any other organic compound with the above standard reduction potential.

In one even more preferred embodiment, the interhalogen compound is iodine monochloride, the peroxo compound is selected from the group consisting of hydrogen peroxide, peracetate, percarbonate, perborate, peroxomonosulfate, and peroxodi sulfate, or the (strongly oxidizing) heavy metal containing compound is lead tetraacetate.

The use of sodium peroxodisulfate, potassium peroxodi sulfate, or ammoniumperoxodisulfate (APS) is particularly preferred.

In a seventh aspect, the present invention relates to (the (in vitro) use of) a kit for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprising

(i) an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V, preferably of > + 2.12 V, and

(ii) optionally a reducing agent and/or a protein precipitant.

In a preferred embodiment, the kit further comprises a tag, in particular a fluorescent compound comprising a primary amine functional group. In an alternative preferred embodiment, the kit further comprises a halide ion. In a more preferred embodiment, the kit further comprises a tag, in particular a fluorescent compound comprising a primary amine functional group, and a halide ion.

The present inventors found that the use of an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V allows under alkaline conditions the rapid, non-specific, and non-toxic release of glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes. Thus, the above-mentioned kit comprising an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V allows the release/removal of glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes in a rapid, non-specific, and non-toxic way.

Preferably, the at least one halogen atom is a covalently bound (non-radioactive) halogen atom selected from the group consisting of fluorine, chlorine, bromine or iodine.

In one preferred embodiment, the oxidizing agent comprising at least one aromatic residue and at least one halogen atom is selected from the group consisting of a phenylsulfonylazanide, a hypervalent iodine compound, N,N-dichlorobenzensulfonamide, and a cyclic iodine (III) compound. Said oxidizing agents comprise at least one covalently bound (non-radioactive) halogen atom selected from the group consisting of fluorine, chlorine, bromine or iodine. It should be noted that the oxidizing agent is not limited to these compounds.

In one more preferred embodiment, the phenylsulfonylazanide is selected from the group consisting of sodium;chloro-(4- methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro- (4-Methylbenezne)sulfonamide, Chloramine T) and sodium;benzenesulfonyl(chloro)azanide (also designated as Chloramine B), the hypervalent iodine compound is selected from the group consisting of X3-iodane (iodine (III), iodine oxidation number +3), preferably phenyl{bis[(trifluoroacetyl)oxy]}-X3-iodane (also designated as Bis(trifluoroacetoxy)iodo)benzene), and X5-iodane (iodine (V), iodine oxidation number +5), the N,N-dichlorobenzensulfonamide is selected from the group consisting of ,N-dichloro-4- methylbenzenesulfonamide (Dichloramine-T) and N,N-dichlorobenzenesulfonamide (Dichloramine-B), or the cyclic iodine (III) compound is selected from the group consisting of iodoxybenzoic acid, iodosobenzoic acid, and Dess Martin Periodinane.

It should be noted that the oxidizing agent is not limited to these compounds.

The use of sodium;chloro-(4-methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro-(4-Methylbenezne)sulfonamide, Chloramine T) is particularly preferred.

In one alternative preferred embodiment, the oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V, preferably of > + 2.12 V, is selected from the group consisting of an interhalogen compound, a peroxo compound, and a (strongly oxidizing) heavy metal containing compound. The oxidizing agent can further be any other organic compound with the above standard reduction potential.]

In one more preferred embodiment, the interhalogen compound is iodine monochloride, the peroxo compound is selected from the group consisting of hydrogen peroxide, peracetate, percarbonate, perborate, peroxomonosulfate, and peroxodi sulfate, or the (strongly oxidizing) heavy metal containing compound is lead tetraacetate.

The use of sodium peroxodisulfate, potassium peroxodi sulfate, or ammoniumperoxodisulfate (APS) is particularly preferred. The glycostructures released from the glycoproteins and enveloped viruses retain their free, reducing end and can further be tagged specifically for structural analysis, separation purposes e.g. chromatographic separation, introduction of functional groups for subsequent chemical modifications, e.g. covalent attachment to solid phases, or addition of other tags. The tag may be any molecule which is detectable through spectral properties (e.g. fluorescent makers or “fluorophores” and coloured markers (“chromophores”)).

In particular, the tag is a fluorescent compound comprising a primary amine functional group. In one even more preferred embodiment, the fluorescent compound comprising a primary amine functional group is selected from the group consisting of 2-amino-benzamide (2-AB), 2-aminobenzoic acid, procainamide, 8-aminonaphthalene-l,3,6-trisulfonic acid (ANTS), 8-aminonaphthalene disulfonic acid (ANDS), aminopyrenetri sulfonic acid (APTS), and aminoacridone. It should be noted that the fluorescent compound comprising a primary amine functional group is not limited to these compounds.

The halide ion is preferably selected from the group consisting of fluoride (F-), chloride (Cl-), bromide (Br-), and iodide (I-).

The inventors of the present patent application found that the use of a reducing agent efficiently stops the release of the glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes. It has neutralizing functions. Alternatively or additionally, a protein precipitant is used. The inventors of the present patent application further found that the use of a protein precipitant helps in a later isolation step to separate the released glycostructures from the deglycanized proteins or enveloped viruses.

Thus, optionally, a reducing agent and/or a protein precipitant is (are) part of the kit. The reducing agent is preferably selected from the group consisting of formic acid, ascorbic acid acetaldehyde, and formaldehyde, and/or protein precipitant is preferably selected from the group consisting of trichloroacetic acid, methanol, and acetone.

In a preferred example, the kit for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprises

(i) sodium peroxodisulfate, potassium peroxodisulfate, ammoniumperoxodi sulfate (APS) or sodium;chloro-(4-methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro-(4-Methylbenzene)sulfonamide,

Chloramine T),

(ii) 2-amino-benzamide (2-AB),

(iii) potassium iodide, and (iv) optionally formic acid and/or trichloroacetic acid.

The kit is preferably used to carry out the method of the first aspect. It preferably comprises instructions on how to carry out the method of the first aspect.

In an eighth aspect, the present invention relates to (the (in vitro) use of) a kit for isolating glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprising

(i) an oxidizing agent comprising at least one aromatic residue and at least one halogen atom or an oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V, preferably of > + 2.12 V,

(ii) a separation material, and

(iii) optionally a reducing agent and/or a protein precipitant.

In a preferred embodiment, the kit further comprises a tag, in particular a fluorescent compound comprising a primary amine functional group. In an alternative preferred embodiment, the kit further comprises a halide ion. In a more preferred embodiment, the kit further comprises a tag, in particular a fluorescent compound comprising a primary amine functional group, and a halide ion.

The above-mentioned kit allows the release of all glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes. It also allows the separation of the released glycostructures from the deglycanized proteins or enveloped viruses. In this way, the glycostructures are isolated from the glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

The released glycostructures can immediately/directly be further processed for analytical purposes e.g. identifying HPLC, mass spectroscopy or capillary electrophoretic profiles of samples.

Preferably, the at least one halogen atom is a covalently bound (non-radioactive) halogen atom selected from the group consisting of fluorine, chlorine, bromine or iodine.

In one preferred embodiment, the oxidizing agent comprising at least one aromatic residue and at least one halogen atom is selected from the group consisting of a phenylsulfonylazanide, a hypervalent iodine compound, N,N-dichlorobenzensulfonamide, and a cyclic iodine (III) compound. Said oxidizing agents comprise at least one covalently bound (non-radioactive) halogen atom selected from the group consisting of fluorine, chlorine, bromine or iodine. It should be noted that the oxidizing agent is not limited to these compounds.

In one more preferred embodiment, the phenylsulfonylazanide is selected from the group consisting of sodium;chloro-(4- methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro- (4-Methylbenzne)sulfonamide, Chloramine T) and sodium;benzenesulfonyl(chloro)azanide (also designated as Chloramine B), the hypervalent iodine compound is selected from the group consisting of X3-iodane (iodine (III), iodine oxidation number +3), preferably phenyl{bis[(trifluoroacetyl)oxy]}-X3-iodane (also designated as Bis(trifluoroacetoxy)iodo)benzene), and X5-iodane (iodine (V), iodine oxidation number +5), the N,N-dichlorobenzensulfonamide is selected from the group consisting of ,N-dichloro-4- methylbenzenesulfonamide (Dichloramine-T) and N,N-dichlorobenzenesulfonamide (Dichloramine-B), or the cyclic iodine (III) compound is selected from the group consisting of iodoxybenzoic acid, iodosobenzoic acid, and Dess Martin Periodinane.

It should be noted that the oxidizing agent is not limited to these compounds.

The use of sodium;chloro-(4-methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro-(4-Methylbenzene)sulfonamide, Chloramine T) is particularly preferred.

In one alternative preferred embodiment, the oxidizing agent having a standard reduction potential at 25°C (E° _re d) of > + 1.00 V, preferably of > + 2.12 V, is selected from the group consisting of an interhalogen compound, a peroxo compound, and a (strongly oxidizing) heavy metal containing compound. The oxidizing agent can further be any other organic compound with the above standard reduction potential.]

In one more preferred embodiment, the interhalogen compound is iodine monochloride, the peroxo compound is selected from the group consisting of hydrogen peroxide, peracetate, percarbonate, perborate, peroxomonosulfate, and peroxodi sulfate, or the (strongly oxidizing) heavy metal containing compound is lead tetraacetate.

The use of sodium peroxodisulfate, potassium peroxodi sulfate, or ammoniumperoxodisulfate (APS) is particularly preferred.

In particular, the tag is a fluorescent compound comprising a primary amine functional group. In one even more preferred embodiment, the fluorescent compound comprising a primary amine functional group is selected from the group consisting of 2-amino-benzamide (2-AB), 2-aminobenzoic acid, procainamide, 8-aminonaphthalene-l,3,6-trisulfonic acid (ANTS), 8-aminonaphthalene disulfonic acid (ANDS), aminopyrenetri sulfonic acid (APTS), and aminoacridone. It should be noted that the fluorescent compound comprising a primary amine functional group is not limited to these compounds.

The halide ion is preferably selected from the group consisting of fluoride (F-), chloride (Cl-), bromide (Br-), and iodide (I-).

Optionally, a reducing agent and/or a protein precipitant is (are) part of the kit. The reducing agent is preferably selected from the group consisting of formic acid, ascorbic acid acetaldehyde, and formaldehyde, and/or protein precipitant is preferably selected from the group consisting of trichloroacetic acid, methanol, and acetone.

The separation material is any material allowing the separation of the released glycostructures from the deglycanized proteins or enveloped viruses. In this way, the glycostructures are isolated from the glycoproteins or enveloped viruses comprising glycostructures on their envelopes.

In one even more preferred embodiment, the separation material is selected from the group consisting of a chromatography material, filtration material, and electrophoresis material.

In still one even more preferred embodiment, the chromatography material is selected from a hydrophilic interaction chromatography (HILIC) column and a reversed-phase chromatography (RP-HPLC) column, or the electrophoresis material is selected from the group consisting of capillary electrophoresis material, capillary gel electrophoresis material, and gel electrophoresis material.

In one most preferred embodiment, the RP-HPLC is carried out with a Cl 8 column, the HILIC is carried out with a ZIC-HILIC column (zwitterionic hydrophilic interaction liquid chromatography column), or the gel electrophoresis is a horizontal flatbed gel electrophoresis.

In a preferred example, the kit for releasing glycostructures from glycoproteins or enveloped viruses comprising glycostructures on their envelopes comprises

(i) sodium peroxodisulfate, potassium peroxodisulfate, ammoniumperoxodi sulfate (APS) or sodium;chloro-(4-methylphenyl)sulfonylazanide (also designated as Tosylchloramide-sodium, sodium-N-chloro-(4-Methylbenzene)sulfonamide,

Chloramine T),

(ii) 2-amino-benzamide (2-AB),

(iii) potassium iodide, (iv) gel electrophoreses material, in particular horizontal flatbed gel electrophoresis material, and

(v) optionally formic acid and/or trichloroacetic acid.

The kit is preferably used to carry out the method of the second aspect. It preferably comprises instructions on how to carry out the method of the second aspect.

In a further aspect, the present invention relates to the use of glycostructures of the third, fourth, fifth aspect of the present invention for enzymatic transglycosylation. It is preferred that the glycostructures are modified glycostructures (modified to oxazolone).

Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope of invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art in the relevant fields are intended to be covered by the present invention.

The following figures and examples are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Controlled release of glycans from chicken ovalbumin. A and B: Glycans released from chicken ovalbumin - a model protein carrying protein-bound N-glycan structures were separated by Thin Layer Chromatography on LuxPlate® Silica gel 60 F254 glass plates and detected as spots migrating closely behind the solvent front (A and B). Untreated ovalbumin control samples did not yield these spots. B: Orcinol staining identified these spots as saccharides. A: TLC Plates under UV 254 nm illumination. B: Orcinol stain of the same plates . CP/I: Ovalbumin samples treated with Chloramine-T/potassium iodide as described in the example section, KPS/I: Ovalbumin samples treated with potassium peroxodisulfate/potassium iodide as described in the example section. C: Binding of the released glycans from chicken ovalbumin to porous graphitic carbon (PGC). Glycans released from chicken ovalbumin by CP/I and KPS/I were bound to PGC particles. Bound glycans eluted from these PGC particles were spotted onto TLC silica plates and stained with orcinol. Orcinol staining detected saccharides in both PGC-eluates, indicating that the released glycan oligosaccharides consisted of at least three glycosidically linked hexose units. PGC is known not to bind Mono- and a majority of disaccharides.

Figure 2: HILIC-FDL profile of IgGl-Fc-Glycans simultaneously released and labelled with 2-aminobenzamide (2-AB). A sample containing human IgGl was subjected to treatment with an alkaline solution of ammonium peroxodisulfate and 2-aminobenzamide (2-AB). After removal of excess 2-AB the sample was separated by HILIC-HPLC on an OTU Amino- HPLC column. 2-AB-Labelled glycans consecutively eluting from the column were recorded by fluorescence detection. The retention times of the 2-AB- fluorescent peaks eluting after 20 min. from the Amino-HILIC HPLC column correspond to 2-AB-oligosaccharide sizes matching GOF-, GIF- and G2F -N-glycan structures, which are expected to be dominant in IgGl mAb. These data indicate that is is possible to simultaneously release N-Glycans from ovalbumin and label them with 2-AB in a single step.

Figure 3: (A) ESLMS profile and (B) identification of the 2-AB-labelled glycans of the > 20 min fraction of the HILIC-FLD (Hydrophilic Interaction Liquid Chromatography- with Fluorescence Detection). 2-AB-fluorescent peaks eluting from HILIC-FLD after 20 min. (see Figure 2) were collected and then analyzed by ESLMS (Electrospray-Mass Spectrometry). Figure 3A shows the ESLMS spectra obtained from the collected HILIC-FLD peaks. Figure 3B shows that the dominant peaks in the ESLMS spectra could all be matched with the expected masses of the dominant IgGl N-glycans labelled with 2-AB via a urea bond. EXAMPLES

Experiment 1: Controlled release of protein-bound glycans

Thin Layer Chromatography of released glycans

100 .l Ovalbumin (20mg/ml) were treated with 1 O .1 NaOH (saturated solution), 100 .l sodium tetraborate (saturated aquous solution) and

(1) lOO .1 sodium tosylchloramide (10%) aqueous solution and 2yl of potassium iodide (saturated aquous solution) or

(2) lOOpl potassium peroxodisulfate (saturated aqueous solution) and 2pl of potassium iodide (saturated aquous solution) or

(3) lOOpl ammonium peroxodisulfate (saturated aqueous solution)

All mixtures (1) to (3) were incubated at 37°C for 15 minutes. Following the incubation, the reaction was terminated by adding 40pl pure formic acid and 50 pl trichloroacetic acid (saturated aqueous solution). Samples containing potassium iodide were treated with 400pl of Ethylacetate and then all samples were vigorously vortexted. Then all samples were centrifuged at top speed in a tabletop centrifuge for 3 minutes. 4pl of each aqueous phase supernatant from mixtures (1) to (3) were spotted onto LuxPlate® Silica gel 60 F254 glass plates (2,5 x 7,5cm) (Supelco) and developed in a saturated TLC mini chamber equipped with a 3:3:2 solvent mix of 2-propanol, glacial acetic acid and water. After the solvent front had reached a point 1 cm below the top of the TLC plate, the plates were removed from the developing chamber and dryed at RT for 10 minutes. Then the plates were inspected under 254nm UV light and photographed (Figure 1A). The dry plates were then sprayed with Orcinol-Sulfuric Acid-TLC- Spray- Reagent (180mg Orcinol dissolved in 5ml H2O, then 75ml ice cold ethanol were added and the mix placed in an ice bath; Finally, 10ml concentrated sulfuric acid (95-98%) were slowly added to the cold mixture under stirring, reagent is stored in the dark at 4°C) and then developed by placing the orcinol-spayed plates on a hot plate at 120°C for 10 minutes. Presence of carbohydrates is indicated by a brown greyish purple spot color (Figure IB). The spots migrating closely behind the solvent from that were already visible under 254nm UV light (Figure 1A) were positively identified as (glycan-oligo-)saccharides by oricinol staining (Figure IB). Glycans liberated and released from the ovalbumin proteins migrate closely behind the solvent front whereas the intact ovalbumin glycoprotein is retained at the starting line (Figures 1A and B).

Porous Graphitic Carbon Extraction and Orcinol Detection of released glycans lOOpl Ovalbumin (20mg/ml) were treated with 1 Opl NaOH (saturated solution), and

(1) 38 pl sodium tetraborate (saturated aquous solution), 3 Opl water and 20pl sodium tosyl chi orami de (10%) aquous solution and 2pl of potassium iodide (saturated aquous solution) or

(2) 38pl sodium tetraborate (saturated aquous solution) and 50pl potassium peroxodisulfate (4% (w/v) aquous solution) and 2pl of potassium iodide (saturated aquous solution)

All mixtures (1) to (2) were incubated at 37°C for 15 minutes. Following the incubation, the reaction was terminated by adding 50pl trichloroacetic acid (saturated aquous solution) and brief vortexing. All samples were centrifuged at 21100 x g in a Hereaus Pico21 tabletop centrifuge for 3 minutes. Each supernatant was then transferred into a fresh 5ml conical shaped polypropylen microfuge tube (Sarstedt AG&Co, Numbrecht, Germany, Cat. -No. 72.701) equipped with lOmg porous graphitic carbon (PGC) powder (removed from a Supelclean™ ENVI-Carb™ SPE Tube, Sigma Aldrich). 2ml pure distilled water were added to each tube and the PGC powder in each tube was resuspended and wetted with the samples. Then an additional 2,5ml pure distilled water was added to each tube and each tube was then vortexted. Tubes were then placed on a rotating wheel and incubated for 5 min at RT. Tubes were then placed in a storage rack until the PGC powder in each tube had settled. Supernatants were removed and discarded. The PGC powder in each tube was then washed with 3 ml water. Again the PGC powder was allowed to settle and the supernatant discarded. Then the PGC powder in each tube was finally washed with 1ml water in an identical procedure. The settled and washed PGC powder in each tube was eluted with 80pl of 80% (v/v) acetonitrile. 6pl (3x2pl) of each eluate were spotted onto a TLC plate and allowed to dry. The dry plates were then sprayed with Orcinol-Reagent (as described above) and then developed by placing the orcinol-spayed plates on a hot plate at 120°C for 10 minutes. Presence of carbohydrates is indicated by a brown greyish purple spot color. PGC -bound oligosaccharides were identified for both the HOC1- treated sample as well as the K2S2O8/KI -treated samples (Figure 1C). This results indicates that the bound and released carbohydrate species are likely to consist of oligosaccharides composed of ^3 hexose units, since the graphitic carbon clean-up step has been reported to not to retain monosaccharides and a majority of disaccharides (Jensen PH, Karlsson NG, Kolarich D and Packer NH, Structural Analysis of N- and O-Glycans Released from Glycoproteins, Nat.

Protoc, 2012, 7, 1299-1310).

Experiment 2: Simultaneous release and fluorescent labelling of glycans of an IgGl- format therapeutic antibody (from CHO cell culture)

500 pl cell culture supernatant with 2 mg/ml IgGl mAb was treated with 1 mg 2- aminobenzamide (2-AB) and 0.5 ml sodium hydroxide solution (1 N). Then, 3% (w/v) ammonium peroxodi sulfate was added and the mixture was incubated for 15 min at RT. Afterwards, the reaction mixture was neutralized with formic acid and purified using a C18- SPE column. Here, unreacted 2-AB was separated. The 2-AB labelled glycans (fraction 2 of the C18-SPE elution) were subsequently analysed with HILIC-FLD (OTU Amino-HILIC, Applichrom, Oranienburg) (Jasco 1520 fluorescence detector). Labelled glycans elute at retention times >20 min (see Figures 2 and 3). The glycans eluted after 20 min from the HILIC which correspond to the dextran standard glucose units for the GOF, GIF and G2F structures, which are expected to be dominant in IgGl mAb. These peaks were collected, concentrated in the Speedvac and analyzed in the ES1-MS (positive ion mode). The masses found could also be clearly assigned to the expected IgGl-type mAb glycostructures, namely GOF, GIF and G2F.