Method for identifying and quantifying polysaccharides in complex glycoconjugate compositions

Abstract

The present invention relates to analytical methods for identifying and quantifying complex glycoconjugate compositions, and in particular to the analysis of polysaccharide components of glycoproteins in a sample. The present invention further relates to the use of liquid chromatography-mass spectrometry systems ("LC-MS systems") in such analytical methods, for example, for process control during glycoconjugate production.

Description

[Technical Field]

[0001] The present invention relates to analytical methods for identifying and quantifying complex glycoconjugate compositions, particularly for the analysis of glycoproteins in a sample. The present invention further relates to the use of liquid chromatography-mass spectrometry systems ("LC-MS systems") in such analytical methods, particularly for in-process control during glycoconjugate production, release control of produced glycoconjugates, stability control of stored glycoconjugates, and glycoconjugate production process optimization.

[0002] Biopharmaceutical companies are under increasing regulatory pressure to demonstrate satisfactory programs for understanding, measuring, and controlling glycation in glycoprotein-based drugs. However, analysis of complex glycoconjugate compositions, i.e., their identification and absolute quantification, is a difficult task. Such analyses typically involve several assays and require a week or more for complete analysis under GMP conditions.

[0003] For glycoprotein-based drugs, state-of-the-art chemical assays are performed on high-performance liquid chromatography (HPLC) systems equipped with various detectors. For the determination of total polysaccharides (PS) and free PS, pulsed amperometric detectors are used, which require in-lab sample preparation by hydrolyzing glycans and detecting the released monosaccharides after ion chromatography separation. As stated by ThermoFisher Scientific, the manufacturer of such HPLC systems, "carbohydrates are difficult to analyze using common chromatographic and detection methods," but their "electrochemical detection has been optimized for carbohydrate analysis" (1). Consequently, amperometric detection and quantification can be considered the gold standard for carbohydrate quantification. However, due to one of the major drawbacks of this technique (the requirement of monosaccharides), in-lab sample preparation and release assays for glycoprotein-based drugs are time- and cost-intensive.

[0004] Furthermore, all glycoprotein-based drugs undergo additional analytical release testing (e.g., free glycans in drug substance, glycan modifications) beyond the general release arsenal for purity and specificity required by regulatory agencies. These multiple different release criteria result in slow release times for glycoprotein-based drugs.

[0005] Mazsaroff et al. (2) describe the analysis of carbohydrate structures of glycoproteins using an LC-MS system and in-source fragmentation. This paper describes an analytical method for relative quantification by determining the ratio of two peaks. As the authors clearly state, "Although the fragment ion peak area at optimal signal-to-noise conditions may not reflect the absolute ratio of fragment ions present in the glycoprotein molecule being investigated, it is important to understand that optimal conditions provide the best measure of relative quantification of fragment ion ratios when comparing different batches of biopharmaceuticals with their reference standards." In conclusion, this paper fails to describe any absolute quantification of carbohydrate structures, nor does it suggest the use of a reference material calibration curve.

[0006] Ivancic et al. (3) described LC-MS analysis of complex glycosylated human A1 acid glycoprotein as a model for developing identification and quantification methods for intact glycopeptide analysis. This paper describes an analytical method that uses in-source fragmentation to fragment carbohydrates and then uses the fragmented carbohydrates as markers for identifying glycopeptides. This technique ultimately aims to quantify the "degree of glycation" of peptides in the A1 acid glycoprotein. This involves comparing the peak intensity of peptides identified as glycated with that of non-glycosylated peptides. Again, this paper fails to describe any absolute quantification of carbohydrate structures, nor does it suggest the use of a reference material calibration curve.

[0007] Carell et al. (US Patent Application Publication No. 2020 / 041470) disclose a derivatization reagent for polysaccharides as glycoprotein compounds. The use of this derivatization reagent and its isotopic derivatives in combination with a calibration curve generated from a reference material allows absolute quantification of such polysaccharides using an LC-MS system capable of fragmenting selected ions by collision-induced dissociation (CID). Because analyte derivatization is a prerequisite for quantification, this document fails to describe any LC-MS method in which glycoproteins are in their native state. Such derivatization of polysaccharide components requires an additional sample preparation step compared to methods analyzing glycoproteins in their native state, making such methods necessarily more tedious. Furthermore, this method requires the use of an advanced LC-MS system capable of selecting and further fragmenting specific ions resulting from in-source fragmentation by CID. Not only is such advanced equipment itself more expensive, but validation of the corresponding analytical method also takes longer than methods without such requirements. Therefore, the use of relatively simple LC-MS systems and protocols that require minimal handling steps is considered advantageous, especially in the context of drug release assays.

[0008] Echeverria et al. (5) described an MS method for quantifying glycans that are part of monoclonal antibodies. Absolute quantification of polysaccharides is achieved after cleavage from the antibody by incubation with the enzyme PNGase F. For quantification, a mixture of isotopically labeled glycans is added to the sample as an internal standard, and analysis is performed by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) MS. Again, this publication fails to describe any analytical method for glycoproteins in their native state. Furthermore, this method does not use liquid chromatography to separate glycoproteins within the sample prior to MS analysis; it only suggests the possibility of applying the reported mixture of isotopically labeled glycan standards to LC-MS-based methods. As discussed previously, the addition of sample preparation steps, such as PNGase F-mediated release of PS components from the carrier protein, makes the analytical method more tedious and therefore reduces throughput compared to methods that do not include such steps. Furthermore, PNGase F digestion is not applicable to all polysaccharide structures, thus limiting prior art methods to glycoproteins within PNGase F's substrate specificity.

[0009] Similarly, Jeong et al. (6) described a MALDI-TOF MS method for absolute glycan quantification after PNGase F-mediated release from glycoproteins and subsequent polymethylation of the glycans. Therefore, this document also does not describe a method for quantifying glycoproteins in their native state. The described method also does not include LC-based separation of glycoproteins prior to MS analysis. As outlined above, adding sample preparation steps makes analytical methods more cumbersome and reduces throughput. In this particular case, sample preparation involves not only PNGase F digestion (which necessarily imposes limitations on the method) but also a polymethylation step, further complicating sample preparation.

[0010] In conclusion, there is a need for improved methods for the analysis of glycoconjugates, and in particular glycoprotein-based drugs.

[0011] It is therefore an object of the present invention to alleviate at least some of these drawbacks of the current state of the art.

[0012] In particular, it is an object of the present invention to provide an analytical method for glycoprotein-based drugs that bypasses sample preparation, thereby increasing analytical throughput and reducing release costs.

[0013] It is a further object of the present invention to provide an analytical method that combines multiple different release criteria into one assay, thereby further shortening batch release times for glycoprotein-based drugs.

[0014] In particular, it is an object of the present invention to provide an analytical method that simultaneously provides information on the identity and absolute quantification of glycoprotein-based drugs.

[0015] One or more of the above objects are achieved by the use of an analytical method according to claim 1 and an LC-MS system according to claim 12. Further aspects of the invention are disclosed herein and in the independent claims, and preferred embodiments are disclosed herein and in the dependent claims.

[0016] The present invention will be described in more detail below. It is understood that the various embodiments, preferences, and ranges provided / disclosed herein can be arbitrarily combined. Furthermore, depending on the specific embodiment, the selected definition, embodiment, or range may not be applicable.

[0017] Unless otherwise stated, the following definitions shall apply herein.

[0018] As used herein, "a," "an," "the," and similar terms as used in the context of the present invention (particularly in the context of the claims) are to be construed as encompassing both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0019] As used herein, the terms "including," "containing," and "comprising" are used herein in their open, non-limiting sense. It is understood that the various embodiments, preferences, and ranges are combinable.

[0020] Throughout this specification several abbreviations are used, including the following: Area under the AUC curve CID collision-induced dissociation, MS technique EPA Detoxified exotoxin A of Pseudomonas aeruginosa (P. aeruginosa) ETD electron transfer dissociation, MS technique HPLC High Performance Liquid Chromatography IC-PAD Ion Chromatography - Pulsed Amperometric Detection ISF In-source fragmentation LC liquid chromatography LPS lipopolysaccharide MS mass spectrometry PS polysaccharide RP reverse phase SEC Size Exclusion Chromatography TIC total ion current, MS technique

[0021] The above abbreviations and further abbreviations mentioned herein are common in the art.

[0022] The term "glycoconjugate" is known in the art and specifically describes a chemical entity covalently attached to one or more polysaccharides. Such glycoconjugates may be obtained by biological conjugation in living cells ("bioconjugation" or "bioconjugation") or by chemical conjugation of polysaccharides ("chemical" or "synthetic" glycoconjugates). Suitable chemical entities include proteins / peptides and lipids, with corresponding glycoconjugates being glycoproteins (including proteoglycans, peptidoglycans, and glycopeptides) and glycolipids.

[0023] The term glycoprotein includes "traditional glycoproteins" and "glycoconjugate vaccines." In traditional glycoproteins, the emphasis is on the protein portion, e.g., of an antibody or erythropoietin glycoprotein, where the "activity" principle resides more in the protein portion, and the glycans play a role in defining, e.g., half-life or other properties. Such traditional glycoproteins are widely used in pharmaceutical applications. In glycoconjugate vaccines, the emphasis is on the glycan portion, to which an immune response is desired, since the glycan is the relevant antigen, and the protein portion simply serves as a carrier to lead to the desired T cell memory immune response.

[0024] The term glycoprotein further includes "proteoglycans," "peptidoglycans," and "glycopeptides." The term "proteoglycan" refers to heavily glycosylated proteins. The basic proteoglycan unit consists of a core protein with one or more covalently attached glycosaminoglycan (GAG) chains. The attachment point is a serine (Ser) residue to which the glycosaminoglycan is joined via a tetrasaccharide bridge. The term "peptidoglycan" refers to the polymer of sugars and amino acids that forms a mesh-like layer on the outer plasma membrane of most bacteria and forms the cell wall. The term "glycopeptide" refers to a glycoprotein containing up to 50 amino acids in the protein portion. Such glycopeptides are available through digestion of larger glycoproteins (e.g., trypsin digestion), bacterial fermentation, or chemical synthesis.

[0025] The term "glycolipid" is known in the art and specifically describes a substance in which one or more sugars are linked to a lipid by a glycosidic (covalent) bond. Thus, a distinctive feature of a glycolipid is the presence of a sugar (particularly a polysaccharide, as discussed herein) attached to the lipid moiety.

[0026] The term "polysaccharide" is known in the art and specifically describes polymeric carbohydrates composed of monosaccharide units, whether linear or branched, linked together by glycosidic bonds. Such polysaccharides are characterized by repeating units, each of which is described by its respective monosaccharide composition. The repeating units contain one or more monosaccharides, which may be chemically modified (e.g., amidated, sulfated, acetylated, phosphorylated, etc.). Monosaccharides typically found in the repeating units are cyclic or linear monosaccharides containing 3 to 7 carbon atoms. In the specific case of glycoconjugate vaccines, the polysaccharide is derived from a pathogenic species (e.g., E. coli), and the repeating units are defined by the genetic makeup of the specific pathogen. Thus, the repeating units can be specific markers / identifiers for the pathogen.

[0027] The term "polysaccharide component" therefore refers to one or more glycan chains of a glycoconjugate. Glycans can be monomers or polymers of sugar residues, but typically contain at least three sugars and can be linear or branched. Glycans can contain natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyglucosamine, phosphomannose, 6'-sulfo-N-acetylglucosamine, etc.). The term "glycan" includes homopolymers and heteropolymers of sugar residues. The term "glycan" also encompasses glycan components of glycoconjugates (e.g., glycoproteins, isopeptides, glycolipids). This term also encompasses free glycans, including glycans cleaved or otherwise released from glycoconjugates.

[0028] The term "O-acetylated polysaccharide," as used herein, refers to a polysaccharide in which one or more monosaccharides of the repeating unit are chemically modified by acetylation. The monosaccharide has one or more of the hydroxyl groups present acetylated. In the case of pathogen-derived repeating units used in glycoconjugate vaccines, O-acetylation of specific monosaccharides can be crucial for the induction of an immune response to the pathogen. Examples of pathogen-derived polysaccharide components are shown in Table 1.

[0029] The terms "glycan" / "glycan chain" are synonymous with "polysaccharide" as defined below. Correspondingly, in the context of the present invention, "glycan" and the prefix "glyco-" also refer to the carbohydrate moiety of a glycoconjugate such as a glycoprotein or glycolipid.

[0030] The term "serotype," as used herein, refers to glycoconjugates having different polysaccharide chains derived from different bacterial serotypes. Examples of glycans from several E. coli serotypes are identified in Table 1 below.

[0031] The term "native state" is known in the art and refers to biomolecules, such as glycoconjugates, in an intact, functional state. When referring to the native state within the present invention, it means that the glycoconjugates being analyzed have not been derivatized or otherwise modified, for example with enzymes such as PNGase F, or subjected to chemical reactions during sample preparation. Analysis of glycoconjugates in their native state is therefore distinct from any method that involves derivatization of the glycoconjugates or a sample preparation step that releases glycans from a carrier molecule, e.g., a carrier protein, before subjecting the sample to the LC-MS step of analysis. However, when referring to the native state within this specification, the term native state does not relate to the conformation of the glycoconjugate, i.e., its three-dimensional fold, such as secondary, tertiary, or quaternary structure. Thus, "analysis of glycoproteins in their native state" is synonymous with "analysis of glycoproteins in which the glycoproteins have not (i) been subjected to enzymatic digestion with enzymes that remove polysaccharide components from the carrier protein, and (ii) have not been subjected to chemical reactions, e.g., derivatization, in each case prior to introducing the glycoprotein into the LC-MS system."

[0032] The invention may be better understood with reference to the drawings. [Brief explanation of the drawings]

[0033] [Figure 1] The coupled LC-MS spectra (TIC) across the main chromatographic peaks of the analyzed glycoprotein EcO6A (a glycoconjugate vaccine component containing a polysaccharide from Escherichia coli (E. coli) serotype O6A covalently bound to an EPA carrier protein; the structure of the PS repeating unit is given in Table 1) and a mixture of different glycoproteins EcO75, EcO2, EcO1A, EcO4, EcO25B, and EcO6A (polysaccharides from Escherichia coli (E. coli) serotypes O75, O2, O1A, O4, O25B, and O6A, each independently covalently bound to an EPA carrier protein; the structures of the repeating units of these polysaccharides are given in Table 1). MS features (x-axis - m / z; y-axis - relative intensity [%]). Panel A shows the in-source fragmentation of Escherichia coli (E. coli) serotype EcO6A and the release of repeat unit-specific fragments (peak with mass 893.3262 g / mol). As seen in panel B, the release of glycan repeat units and their selective assignment is also possible in complex protein samples, as seen in the hexavalent mixed mass with six different serotypes shown near the peak. [Figure 2] An exemplary calibration curve is shown, where in-source fragmentation occurs linearly based on the amount of serotype injected, here shown in a proof-of-concept using the glycoprotein EcO6A, with different injected amounts of protein (from 0.2 μg to 1.2 μg) leading to different signal responses of the repeating unit of EcO6A (fragment with a molecular mass of 893.32 g / mol; y-axis - response / [au]; x-axis - micrograms EcO6A). [Figure 3]Figure 1. Combined LC-MS spectra (with and without acetylation) across the main chromatographic peaks of glycoproteins EcO25B and EcO16 (TIC MS features, x-axis m / z; y-axis relative intensity [%]). The structures of the repeating units with and without O-acetylation for each E. coli (E. coli) O25B and O16 serotype are given in Table 1. Panel A shows the in-source fragmentation of glycoprotein EcO25B, which has two distinct peaks: repeating units with (862 g / mol) and without (820 g / mol) O-acetylation. Panel B shows the same effect on glycoprotein EcO16, which has two distinct peaks with masses of 716 g / mol and 674 g / mol for repeating units with and without O-acetylation.

[0034] In more general terms, in a first aspect, the present invention relates to a method for analyzing polysaccharide components (glycans) of glycoconjugates in a sample. It is important to note that such analysis refers to both the identification of said polysaccharide components and the absolute quantification of said polysaccharide components. The method according to the invention comprises the steps of (a) establishing a calibration curve for said polysaccharide components by an LC-MS system, (b) measuring a sample on the same LC-MS system, and (c) comparing the results of (a) and (b), thereby analyzing said polysaccharide components in said sample. In an advantageous embodiment, the glycoconjugate is a glycoprotein.

[0035] In a preferred embodiment, the glycoconjugates, preferably glycoproteins, are in their native state. Therefore, enzymatic release of PS components from the carrier protein, e.g., PNGase F-mediated release, is not required, and the method is also applicable to the analysis of glycoconjugates that are not susceptible to such enzymatic cleavage, e.g., glycoconjugates containing glycomotifs that are not within the substrate specificity of PNGase F, such as glycomotifs containing N-acetylglucosamine (GlcNAc) that are not linked to alpha-1,3-fucose.

[0036] As outlined in more detail below, the method according to the present invention comprises: (i) Identification of polysaccharides in (ii) absolute quantification of polysaccharides bound to other chemical entities in glycoconjugates such as proteins; (iii) Determination of free polysaccharides in (iv) Quantification and identification of modifications such as acetylation in bound and free polysaccharides; and (v) Purity of samples containing glycoconjugates, particularly glycoprotein-based pharmaceutical compositions This aspect of the invention, particularly the process steps and terminology used, will be explained in more detail below.

[0037] Glycoconjugates: This term is discussed above. The methods of the present invention are broadly applicable and may be used to analyze glycoproteins (including proteoglycans, peptidoglycans, and glycopeptides) and glycolipids.

[0038] Glycoprotein: This term has been discussed above. Specifically, glycoprotein refers to a conjugation product in which a polysaccharide (i.e., glycan) is covalently attached to a carrier protein. The conjugate may be a bioconjugate, a conjugation product prepared in a host cell, where the host cell machinery produces the glycan and protein and links the glycan to the carrier protein, for example, via an asparagine or arginine N-linkage. The conjugate may also be prepared by chemical coupling of the protein and glycan chain, for example, via thiol alkylation. The method according to the present invention is applicable to all types of conjugates. Without being bound by theory, it is believed that it does not matter how the sugar is attached, as long as it is in-source fragmented by MS. Whether fragmentation occurs at the protein-glycan interface is irrelevant, as the reference material will exhibit the same fragmentation.

[0039] Particularly useful glycoconjugates include carrier proteins to which one or more polysaccharides are attached, and such glycoconjugates are used, for example, as active ingredients in certain vaccines, with the aim of inducing a functional immune response against the polysaccharides of the glycoconjugate.

[0040] In an embodiment of the invention, the glycoprotein comprises a carrier protein and one or more polysaccharides covalently bound to the carrier protein, preferably 1 to 4 polysaccharides covalently bound to the carrier protein. In an embodiment of the invention, the glycoprotein is a conjugate product containing an E. coli O-antigen covalently linked to a carrier protein. The term O-antigen is known in the art and is used in its normal context and should not be confused with an O-linkage. The term O-antigen generally refers to the repetitive glycan polymer contained within the LPS of bacteria such as E. coli. E. coli O-antigens are polymers of immunogenic repeating oligosaccharides (typically 1-40 repeating units) and are typically used for serotyping and glycoconjugate vaccine production.

[0041] Carrier Protein: In an embodiment of the present invention, the carrier protein is detoxified exotoxin A (EPA) of Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli) flagellin (FliC), CRM197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus (S. aureus), clumping factor A, clumping factor B, Escherichia coli (E. coli) heat-labile toxin, detoxified variants of Escherichia coli (E. coli) heat-labile toxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, Escherichia coli (E. coli) SAT protein, passenger domain of Escherichia coli (E. coli) SAT protein, Streptococcus pneumoniae (Streptococcus pneumoniae pneumolysin, keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein (OMPC) of Neisseria meningitidis, and protein D from nontypeable Haemophilus influenzae.

[0042] In certain embodiments, the carrier protein is detoxified Pseudomonas aeruginosa exotoxin A (EPA). In such embodiments, EPA preferably comprises 1-20, preferably 1-10, preferably 2-4 glycosylation sites.

[0043] In certain embodiments, EPA comprises four glycosylation sites. See, e.g., WO 2017 / 035181 for a description of an example of bioconjugation of E. coli O-antigen polysaccharide to an EPA carrier protein.

[0044] Polysaccharide: This term has been discussed above. Suitable polysaccharides comprise repeating units n of 1-100, such as 1-50, 1-40, 1-30, 1-20, and 1-10, 3-50, 3-40, such as at least 5, such as 5-40, for example 7-30, for example 7-25, for example 10-20, for example 5-20. Such repeating units include (i.e., comprise or consist of) (i) unmodified monosaccharides and / or (ii) modified monosaccharides. The term "modified monosaccharide" particularly relates to chemically modified monosaccharides, and in non-limiting embodiments includes N-acetylation, O-acetylation, amidation, and / or amination of the monosaccharide. Such monosaccharides may comprise one or more modifications, particularly one, two, or three of the above modifications, in the same monosaccharide.

[0045] In an embodiment of the invention, suitable repeating units comprise monosaccharides selected from the group consisting of mannose, rhamnose, glucose, fucose, galactose, modified mannose, modified rhamnose, modified glucose, modified fucose, and modified galactose. Non-limiting exemplary structures of E. coli O-antigen polysaccharides are shown below in Table 1. A single repeating unit of each E. coli O-antigen polysaccharide is shown. In this table, each n is independently an integer from 1 to 100, such as 1 to 50, 1 to 40, 1 to 30, 1 to 20, and 1 to 10, 3 to 50, 3 to 40, such as at least 5, such as 5 to 40, such as 7 to 30, such as 7 to 25, such as 10 to 20, such as 5 to 20, but optionally can be 1 to 2.

[0046] [Table 1]

[0047] [Table 2]

[0048] Sample: This term is known in the art. A sample includes any material that can be fed, optionally after dilution, to an analytical system. Such samples include, inter alia, (i) production batches of glycoconjugate production (including ongoing batches and released / stored production batches), (ii) pharmaceutical compositions comprising multiple glycoconjugates, e.g., multivalent vaccines.

[0049] In addition to glycoconjugates, a suitable sample contains (i) a water-soluble matrix, (ii) optionally a carrier protein free of polysaccharides (or free peptides in the case of glycopeptides, or free lipids in the case of glycolipids), (iii) optionally polysaccharides not bound to the carrier protein (or peptides, or lipids, as used herein: "free PS"), and (iv) optionally unrelated proteins (or peptides, or lipids). The water-soluble matrix (i) may contain one or more of buffers (e.g., phosphate buffer), inorganic salts (e.g., NaCl), sugar alcohols (e.g., D-sorbitol), and non-ionic surfactants (e.g., polysorbate 80). The unrelated proteins (iv) may comprise up to 10%, up to 50%, or up to 90% process-related impurities (e.g., host cell proteins). It is considered particularly advantageous that the method according to the invention is tolerant to a wide range of additional components in the sample. Therefore, it is suitable to analyze samples without laboratory preparation and without materials that would interfere with standard analytical protocols.

[0050] In one embodiment, the sample contains a single glycoconjugate.

[0051] In a further embodiment, the sample contains multiple glycoconjugates, e.g., 2 to 20, e.g., 4 to 10, glycoconjugates. Again, the glycoconjugates are preferably associated with glycoproteins. Thus, the method according to the invention also allows for the analysis of complex samples containing multiple glycoconjugates, e.g., with glycans from multiple serotypes. Thus, even complex samples for the release of formulations, such as multivalent conjugate vaccines, can be analyzed by the method according to the invention. It is clear that the method according to the invention advances the manufacturing production of formulations containing multiple serotypes, improving the quality and safety of such complex formulations.

[0052] It is clear that the method according to the invention is also suitable for verifying the presence or absence of glycoconjugates, for example in blank samples, so that in certain embodiments the sample does not contain glycoconjugates.

[0053] In one embodiment, the sample contains one carrier protein or carrier peptide. In another embodiment, the sample contains two or more carrier proteins or carrier peptides. Thus, the method according to the invention is suitable for analyzing samples containing multiple different glycoproteins / glycopeptides, including not only the same carrier protein / peptide but also different carrier proteins / peptides with different glycosylation patterns. Thus, the term carrier protein / peptide refers to a single carrier protein / peptide as outlined above, but also to multiple carrier proteins / peptides present in a sample, e.g., 2-10, e.g., 2-5 different carrier proteins / peptides.

[0054] Analytical Method: The method of the present invention allows for the identification and absolute quantification of the total amount of polysaccharides in a sample. Furthermore, the method of the present invention is capable of distinguishing between the amount of bound and free polysaccharides in a sample. Thus, in one embodiment, the present invention provides a method for distinguishing and quantifying the bound and free polysaccharide content in a composition comprising at least one glycoconjugate. Furthermore, the method of the present invention is capable of confirming the presence of polysaccharide components (bound PS and / or free PS) in a sample.

[0055] As discussed above, the method of the present invention comprises three steps: (a) calibration, (b) measurement, and (c) identification and quantification of the polysaccharide components of glycoconjugates, preferably in their native state. These steps are described in more detail below and are further illustrated by examples in the figures provided.

[0056] Step (a): Establish a calibration curve for the polysaccharide components using an LC-MS system. To prepare the calibration curve, an aliquot of a reference material is provided to the LC-MS system. The reference material is a sample having known specific names and known absolute amounts of the polysaccharide components. Such a reference material can be obtained by subjecting a sample containing the polysaccharide components to a conventional analytical protocol.

[0057] In one embodiment of the present invention, step (a) comprises preparing a reference material and calibrating the LC-MS system using said reference material. Preparation of the reference material may be achieved according to steps a1 to a3, and optionally a4 and a5, as outlined below. Calibration of the reference material may be achieved according to step a6, as outlined below.

[0058] Step (a1): This step involves identifying the specific name of said glycoprotein in the reference material. Suitable analytical methods are known per se, examples being Western blot electrophoresis or MS.

[0059] Step (a2): The following step involves determining the total polysaccharide (PS) content of said glycoproteins in the reference material. Suitable analytical methods are known per se, for example IC-PAD after thrombus analysis.

[0060] Step (a3): The following step involves determining the free PS content in the reference material. Suitable analytical methods are known per se, for example IC-PAD after hydrolysis and separation of bound and unbound PS.

[0061] Step (a4): The following optional step involves determining the degree of modification of said glycoprotein (in a particular embodiment, the modification is O-acetylation). Suitable analytical methods are known per se, for example ion chromatography IC-CD after removal of the modifying group (e.g., release of the O-acetyl group by hydrolysis).

[0062] Step (a5): The following optional step involves determining the purity of said glycoprotein in the reference material. Suitable analytical methods are known per se, examples being RP-HPLC and / or SEC.

[0063] Following steps (a1...a3) and optionally (a4...a5), a reference material is obtained comprising the polysaccharide components of the glycoprotein, an aliquot of which is prepared for the following step (a6). Such an aliquot of reference material comprises a known concentration, i.e., a known absolute amount, of the polysaccharide components.

[0064] Step (a6): This step involves measuring an aliquot of the reference material using an LC-MS system. The acquired data (including retention time, peak identification, and AUC determination) allows for the establishment of a calibration curve. In this step, the reference material is first separated via LC, and the eluate undergoes in-source fragmentation (ISF) within the MS detector by adjusting the ionization voltage (cone voltage). It is understood that step a6 is performed after an aliquot of the reference material is acquired. Such a calibration curve is specific to the LC-MS system and parameters used.

[0065] As outlined above, the glycoprotein is preferably in its native state. However, in an alternative embodiment, it is possible to digest the glycoprotein with a peptidase, e.g., trypsin, before subjecting the sample to the LC-MS step of analysis. Such digestion with a peptidase, e.g., trypsin, only leads to the cleavage of peptide bonds, not glycopeptide bonds. In the case of a glycoprotein-containing sample, such digestion leads to the generation of glycopeptides that are subjected to the LC-MS step of analysis. In this case, the reference material is digested before performing step (a6), and the sample is digested before performing step (b1). The generated glycopeptides can be considered in each case still in their native state before introducing the sample into the LC-MS system, i.e., (i) not having undergone enzymatic digestion with an enzyme that removes polysaccharide components from the carrier protein, and (ii) not having undergone chemical reactions, e.g., derivatization. Digestion of glycoproteins using treatment with a peptidase, e.g., trypsin, is known to those skilled in the art.

[0066] In a preferred embodiment of the invention, the sample is not digested with a peptidase sugar enzyme.

[0067] Step (b): Measuring the sample on the same LC-MS system. Measuring a sample on an LC-MS system is known per se and involves (b1) providing a sample to an LC-MS system and (b2) measuring the sample on the LC-MS system.

[0068] It will be apparent to those skilled in the art that the calibration in step (a) can be used to measure a single sample in step (b) or to measure multiple samples in step (b). Thus, steps (b1) and (2) can be performed repeatedly to measure multiple samples. For example, two samples from the same serotype are measured using the same calibration curve by providing a first sample (b1), measuring the first sample (b2), subsequently providing a second sample (b1'), and measuring the second sample (b2'). Thus, measuring a sample includes measuring a single sample and measuring multiple samples.

[0069] Step (b1): The sample can be provided to the LC-MS system in any known manner. Typically, an autosampler is used to inject the sample-containing vial into the system. The sample can be obtained directly from the production batch. The sample can be diluted to fit the concentration range of the calibration curve. In the case of glycoprotein-containing samples, the sample is preferably in its native state.

[0070] In an alternative embodiment, such samples may be digested with a peptidase, such as trypsin, to release glycopeptides for analysis. As discussed above, the glycopeptides produced can be considered in each case still in their native state, i.e., (i) not having been subjected to enzymatic digestion with an enzyme that removes polysaccharide components from the carrier protein, and (ii) not having been subjected to chemical reactions, e.g., derivatization, prior to introducing the sample into the LC-MS system. Thus, in the methods according to the invention, glycopeptide bonds are not cleaved prior to subjecting the sample to the LC-MS step of analysis.

[0071] In that alternative embodiment, the reference material is also digested before carrying out step (a6).

[0072] In a preferred embodiment of the method of the present invention, the sample is not digested with peptidases or with any enzymes. Methods that do not use enzymatic digestion have the advantage that the analysis is much simpler.

[0073] Step (b2): According to the method of the present invention, the sample is measured using the same parameters as in step (a6) above. Preferably, the sample is measured on the same LC-MS system used to prepare the calibration curve. Preferably, the sample is measured within 8 hours after the calibration curve is established. All these measurements improve data quality and take into account the sensitivity of the LC-MS system to external factors, as known to those skilled in the art.

[0074] In a first embodiment, the measurement of the sample is Identifying the identity of the sample; - To identify the PS content of glycoproteins and This allows the absolute amount of glycan chains to be determined.

[0075] In a second embodiment, measuring the sample comprises: Identifying the identity of the sample; - To identify the PS content of glycoproteins and To identify the acetylated PS content of glycoproteins and This allows the degree of acetylation in the sample to be further identified.

[0076] In a third embodiment, measuring the sample comprises: Identifying the identity of the sample; - To identify the PS content of glycoproteins and Identifying sample purity; Includes.

[0077] In a fourth embodiment, measuring the sample comprises: Identifying the identity of the sample; - To identify the PS content of glycoproteins and To identify the acetylated PS content of glycoproteins and Identifying sample purity; Includes.

[0078] In a fifth embodiment, measuring the sample comprises: Identifying the identity of the sample; - To identify the PS content of glycoproteins and - To determine the free PS content and This allows the amount of unconjugated glycans (relative or absolute) to be further determined.

[0079] In a sixth embodiment, measuring the sample comprises: Identifying the identity of the sample; - To identify the PS content of glycoproteins and To identify the acetylated PS content of glycoproteins and - To determine the free PS content and This allows for further determination of the amount of unconjugated glycans (relative or absolute) and also takes into account the degree of bound acetylated PS.

[0080] In a seventh embodiment, measuring the sample comprises: Identifying the identity of the sample; - To identify the PS content of glycoproteins and To identify the acetylated PS content of glycoproteins and - To determine the free PS content and To identify the free acetylated PS content and This allows the amount of free acetylated PS to be further specified.

[0081] In an eighth embodiment, measuring the sample comprises: Identifying the identity of the sample; - To identify the PS content of glycoproteins and - Determining the free PS content of the samples; Identifying the purity of the sample; Includes.

[0082] In a ninth embodiment, measuring the sample comprises: Identifying the identity of the sample; - To identify the PS content of glycoproteins and To identify the acetylated PS content of glycoproteins and - Determining the free PS content of the samples; Identifying the purity of the sample; Includes.

[0083] In a tenth embodiment, measuring the sample comprises: Identifying the identity of the sample; - To identify the PS content of glycoproteins and To identify the acetylated PS content of glycoproteins and - Determining the free PS content of the samples; - Determining the free acetylated PS content of the sample; Identifying the purity of the sample; Includes.

[0084] Thus, in an embodiment of the present invention, simultaneous identification of a sample and one or more of the following parameters is achieved: PS content, acetylated PS content, free PS content, free acetylated PS content, and purity of the sample. In particular, simultaneous identification of a sample and one or more of the following parameters is not usually possible in the case of methods that include a sample preparation step that releases glycans from carrier molecules, e.g., carrier proteins, or derivatization of glycoproteins (introducing further impurities): free PS content, free acetylated PS content, and purity of the sample.

[0085] In another embodiment, a method for analyzing polysaccharide components of glycoproteins in a sample according to aspects outlined in the first to tenth embodiments includes: (a) establishing a calibration curve for the polysaccharide components by an LC-MS system, where step (a) comprises providing a reference material (steps a1-a3 and optionally a4, a5) and calibrating the LC-MS system with the reference material (step a6), the calibration curve being established by separating an aliquot of the reference material via LC and subjecting the LC eluate to in-source fragmentation within an MS detector by adjusting the ionization voltage; (b) measuring a sample on the same LC-MS system using the same parameters as in step (a6); and (c) comparing the results of (a) and (b), thereby analyzing the polysaccharide components in the sample.

[0086] In particular, no further fragmentation, for example using CID or ETD, is required. Thus, in one embodiment, the invention described herein provides a method in which no additional fragmentation of ions obtained after in-source fragmentation is required.

[0087] In one embodiment, the present invention provides the method described herein, wherein no internal standard is added to the sample.It has been found that generating a separate calibration curve is sufficient to provide reliable results.The omission of an internal standard is considered to be a benefit, since it reduces handling steps and therefore simplifies the method.

[0088] The purity of the sample can be determined in known manner, for example, via a refractive index detector, a UV detector in the LC portion of an LC-MS system. Alternatively or additionally, the purity can be determined by MS. This can be done by looking at the total ion current.

[0089] Step (c): Comparing the results of (a) and (b), thereby analyzing the polysaccharide components in the sample: Until now, LC-MS systems have not been used for absolute quantification of polysaccharide components in glycoconjugates against a calibration curve. Thus, step (c) involves (c1) identifying a characteristic peak for each PS in the sample, and (c2) comparing the area under the curve (AUC) of such peak with the calibration curve. It will be apparent to those skilled in the art that in embodiments where the sample measurement in step (b) is repeated for multiple samples (e.g., of the same serotype or containing the same glycoprotein), step (c) can likewise be repeated for each sample using the same calibration curve.

[0090] In a second aspect, the present invention relates to the use of a liquid chromatography-mass spectrometry system ("LC-MS" system) for analyzing the polysaccharide components of glycoconjugates in a sample. In a preferred embodiment, the glycoproteins are in their native state. The above definitions of "sample," "glycoconjugate," "polysaccharide," "carrier protein," and "native state" apply equally to this second aspect of the invention. In particular, the term "glycoconjugate" encompasses the meaning of "glycoprotein," and more specifically, "carrier protein to which one or more polysaccharides are attached." This aspect of the invention will be explained in more detail below.

[0091] Analysis: Again, it is important to note that analyzing includes both identifying the polysaccharide component and absolute quantification of the polysaccharide component. Identification of a PS component includes confirmation of its presence and confirmation of its absence. Thus, if a particular PS component is expected to be present in a sample, for example because the sample is from a manufacturing batch, identification of the PS component is confirmation of its presence. Similarly, the absence of a particular PS component may be expected in a sample, and that can be confirmed as well. Absolute quantification of the PS component includes determination of its concentration, measured, for example, in μg [PS-component] / mL [sample].

[0092] In an embodiment, the present invention also relates to the use of an LC-MS system for in-process control in the production of glycoconjugates, in particular in the production of glycoconjugate vaccines.

[0093] In an embodiment, the present invention also relates to the use of an LC-MS system for the controlled release of manufactured glycoconjugates, in particular in the production of glycoconjugate vaccines. Thus, the LC-MS system can be used as a release assay for drug substances or formulations, said drug substances or formulations comprising one or more glycoconjugates, in particular one or more glycoconjugate vaccines.

[0094] In an embodiment, the present invention also relates to the use of an LC-MS system for stability control of stored glycoconjugates, in particular for stability control of stored glycoconjugate vaccines. Thus, the LC-MS system can be used as an assay for the shelf life of drug substances or formulations, said drug substances or formulations comprising one or more glycoconjugates, in particular one or more glycoconjugate vaccines.

[0095] In embodiments, the present invention also relates to the use of LC-MS systems for process optimization in glycoconjugate manufacturing, particularly in the development of glycoconjugate vaccine manufacturing. The use of LC-MS systems as described herein greatly simplifies the identification of critical parameters, thus enabling faster development and shorter timelines during scale-up.

[0096] LC-MS Method: HPLC systems coupled to mass spectrometry detectors are commonly referred to as LC-MS systems. These detectors are capable of identifying the molecular mass of specific analytes present in a sample. Their precision and mass accuracy are often referred to as the gold standard for identifying the molecular mass of a molecule.

[0097] A known side effect of analyzing glycoconjugates on glycoproteins with LC-MS systems is the fragmentation of glycans when samples are injected into the detector. This effect is called "in-source fragmentation" (ISF) and is related to the voltage applied during injection (the so-called "cone voltage"). ISF is not ideal for a well-performing MS detector, as it can lead to unknown / unidentifiable ion fragmentation in complex samples. Therefore, suppliers test for low ISF (<2%) during system performance quantification.

[0098] In the MS field, ISF is generally known and can completely remove glycan modifications (e.g., peptide chains from glycopeptides) from the rest of the conjugate, making it undesirable for glycan occupancy testing. On the other hand, glycofragmentation is performed to further identify and characterize the monosaccharide composition by reporter ions. However, this fragmentation usually requires much higher energy and therefore requires different MS techniques (CID, ETD).

[0099] We surprisingly found that glycoconjugates subjected to LC-MS analysis exhibited the effect of ISF in separating glycan repeating units, which were clearly seen as distinct peaks of corresponding masses (Figure 1A). After adjusting the cone voltage, this effect was observed not only when injecting a single glycoconjugate, but also in more complex mixtures, such as compositions containing multiple glycoconjugates (Figure 1B). This effect occurred for all polysaccharides from different serotypes, and because each serotype-specific polysaccharide has a distinct mass, it could be used to identify a specific serotype (via its polysaccharide) in the analyzed sample.

[0100] In one embodiment, the LC-MS system is an LC-MS system that uses in-source fragmentation of the eluate obtained from liquid chromatography. Such systems are commercially available from, for example, Waters Corporation, Sciex, and ThermoFisher Scientific.

[0101] In one embodiment, the LC-MS system further comprises a switching valve before the MS detector. Such a valve allows only a portion of the eluate to be diverted to the MS detector. Again, such systems are commercially available.

[0102] In embodiments, the glycoprotein is as defined above, in particular comprising one carrier protein and one or more polysaccharides covalently bound to said protein, hi certain embodiments, the sample comprises two or more carrier proteins, each carrying one or more covalently bound polysaccharides.

[0103] In an embodiment, the carrier protein is as defined above. For example, the carrier protein may be detoxified P. aeruginosa exotoxin A (EPA), E. coli flagellin (FliC), CRM197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified S. aureus hemolysin A, clumping factor A, clumping factor B, E. coli heat-labile toxin, detoxified variants of E. coli heat-labile toxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, E. coli SAT protein, passenger domain of E. coli SAT protein, Streptococcus pneumoniae (Streptococcus pneumoniae) In certain embodiments, the carrier protein is selected from the group consisting of P. pneumoniae pneumolysin, keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein (OMPC) of Neisseria meningitidis, and protein D from Haemophilus influenza nontypeable. In certain embodiments, the carrier protein is detoxified exotoxin A (EPA) of Pseudomonas aeruginosa.

[0104] In embodiments, the polysaccharide is as defined above. For example, the polysaccharide comprises 1 to 100, e.g., 5 to 20, repeating units. Exemplary structures are shown in Table 1 above.

[0105] In embodiments, the sample comprises a water-soluble matrix; optionally, a carrier protein that does not contain polysaccharides; optionally, polysaccharides that are not bound to a carrier protein (or lipid or peptide); and optionally, unassociated proteins.

[0106] The list of cited documents provides additional information in the context of the present invention and is incorporated by reference. (1)thermofisher.com / ch / en / home / industrial / chromatography / chromatography-learning-center / ion-chromatography-information / ion-chromatography-analysis-analyte / analyzing-carbohydrates-hpae-pad-ion-chromatography.html (2) Mazsaroff et al., Anal.Chem., 1997 (3) Ivancic et al., Analytical Biochemistry, 2010 (4) U.S. Patent Application Publication No. 2020 / 041470 (5) Echeverria et al., Analytical Chemistry, 2015 (6) Jeong et al., Analytical Chemistry, 2012

[0107] The following examples are provided to further illustrate the present invention, and are not intended to limit the scope of the invention.

[0108] I. Preparation of Reference Materials Larger quantities of glycoprotein samples are taken directly from the manufacturing batch, aliquoted and stored appropriately (e.g., at -80° C.) Different characterization steps are performed on thawed aliquots of the glycoprotein samples.

[0109] 1) Identifying the correct identity of the reference material. Standard Western blot protocols are followed using specific antibodies selected and tested for specificity to the polysaccharide chains of the glycoprotein of interest. Optionally, the correct identity of the carrier protein is identified using an antibody specific for the carrier protein.

[0110] 2) Identify the total polysaccharide content in the reference material. Total acid hydrolysis to monosaccharides is performed, followed by analysis by ion chromatography with pulsed amperometric detection (IC-PAD) according to the following instructions: An aliquot of the reference material is hydrolyzed at 120°C for 2 hours using trifluoroacetic acid (TFA) at a final concentration of 1.8 M. Optimal hydrolysis conditions (temperature, TFA concentration, and time) may vary depending on the starting polysaccharide concentration. Optimal conditions should demonstrate quantitative release of all monosaccharides from the polysaccharide chain without further degradation of the monosaccharide molecules targeted for absolute quantification.

[0111] After hydrolysis, the samples are cooled to room temperature and then dried using a SpeedVac, typically overnight at 30°C. The dried samples are thoroughly resuspended in HO (Milli-Q grade) and the samples are transferred to HPLC vials. A set of calibration standards is prepared using commercially available monosaccharides (e.g., mannose). If the monosaccharide targeted for quantitation undergoes modification during the hydrolysis step above (e.g., N-acetylglucosamine becomes glucosamine), the appropriate monosaccharide is used, or alternatively, the hydrolysis procedure described above is also performed on a set of calibration standards.

[0112] Prepare an ion chromatography system (e.g., Dionex ICS-5000) equipped with a pulsed amperometric detector (PAD) with disposable gold electrodes mounted on polytetrafluoroethylene (PTFE). Use a Dionex CarboPac PA1 analytical column (4 x 250 mm) and, optionally, a Dionex CarboPac PA1 guard column (4 x 50 mm). Equilibrate the system with eluent A, 16 mM NaOH, for sample elution and eluent B, 500 mM NaOH, for column cleaning. Subsequently, inject the sample and calibration standard sets using the following instrument method / gradient profile: -0~24 minutes, 100% eluent A, 1 mL / min flow (elution), -25-32 min, 100% eluent B, 1 mL / min flow rate (wash), -33~60 min, 100% eluent A, 1 mL / min flow (re-equilibration).

[0113] Depending on the monosaccharide being targeted, these gradients may need to be optimized.

[0114] The area under the curve from the measured calibration set is used to obtain a calibration curve, which is then used to quantify the unknown samples. Optionally, the amount of targeted monosaccharide is then used to back-calculate the absolute amount of repeating unit / polysaccharide in μg / mL in the glycoprotein sample.

[0115] 3) Identify the free polysaccharide content in the reference material. Polysaccharides bound to carrier proteins are removed by a C4 cartridge, followed by total acid hydrolysis to monosaccharides, followed by ion chromatography and pulsed amperometric detection. Depending on the size of the glycoprotein, cartridge materials with different carbon polymer lengths (e.g., C8 cartridges) can be selected to fully retain the carrier protein. Because the sample preparation step is laborious, it is recommended to use additional monosaccharides (e.g., galactose) not present in the reference material as an internal standard to compensate for potential sample losses during preparation.

[0116] Two aliquots of the reference material are taken and an equal amount of internal standard is added. Equilibrate a C4 cartridge (e.g., Chromafix C4-SPE from Macherey-Nagel) according to the manufacturer's instructions: for example, 5 column volumes of 100% methanol, then 5 column volumes of 100% acetonitrile, then 5 column volumes of 5% v / v acetonitrile. During the next step, collect the flow-through containing the free polysaccharides: apply one of the two aliquots to the cartridge, followed by 2 column volumes of 5% acetonitrile. Measure the volume of the collected flow-through and add an equal amount of 5% acetonitrile to the other aliquot that was not applied to the C4 cartridge. Dry both prepared samples using a SpeedVac, typically overnight at 30°C. Both dried samples are thoroughly resuspended in HO (Milli-Q grade) and then subjected to total acid hydrolysis to monosaccharides according to the procedure described above: trifluoroacetic acid (TFA) is added to both samples to a final concentration of 1.8 M and hydrolyzed at 120°C for 2 hours (optimal hydrolysis conditions may vary and should be optimized). After hydrolysis, both samples are cooled to room temperature and then dried using a SpeedVac, typically at 30°C overnight. Both dried samples are thoroughly resuspended in HO (Milli-Q grade) and transferred to HPLC vials.

[0117] Prepare an ion chromatography system (e.g., Dionex ICS-5000) equipped with a pulsed amperometric detector (PAD) with disposable gold electrodes mounted on polytetrafluoroethylene (PTFE). Use a Dionex CarboPac PA1 analytical column (4 x 250 mm) and, optionally, a Dionex CarboPac PA1 guard column (4 x 50 mm). Equilibrate the system with eluent A, 16 mM NaOH, for sample elution and eluent B, 500 mM NaOH, for column cleaning. Subsequently, inject both prepared samples using the following instrument method / gradient profile: -0~24 minutes, 100% eluent A, 1 mL / min flow (elution), -25-32 min, 100% eluent B, 1 mL / min flow rate (wash), -33~60 min, 100% eluent A, 1 mL / min flow (re-equilibration).

[0118] Depending on the monosaccharide being targeted, these gradients may need to be optimized.

[0119] The area under the curve from the targeted monosaccharide (e.g., mannose) and the applied internal standard (e.g., galactose) is used to normalize the measured area. The free monosaccharide content of the analyzed sample is determined by relative comparison of the normalized areas of the two measured aliquots. The aliquot processed by the C4 cartridge represents the percentage of free polysaccharides in the sample compared to the aliquot not processed by the C4 cartridge, which represents 100% of the available polysaccharides. The resulting free polysaccharide value is used to calculate the amount of "bound polysaccharide" from the total polysaccharide content determined above.

[0120] 4) Identifying the degree of modification of polysaccharide chains in reference materials: This optional step describes the identification of O-acetylated sugar moieties using mild alkaline hydrolysis followed by analysis by ion chromatography and conductivity detection.

[0121] An aliquot of the reference material is taken and a desalting step is performed according to the manufacturer's instructions to remove free acetate molecules in the sample (e.g., Zeba Spin Column, PD-10 Column). An internal standard is added to the desalted sample (e.g., 20 μg / mL propionic acid) and hydrolyzed using NaOH at a final concentration of 10 mM for 2 h at 37 °C. Optimal hydrolysis conditions (NaOH concentration, temperature, and time) may vary depending on the starting polysaccharide concentration. Optimal conditions should quantitatively release all O-acetyl groups from the polysaccharide chains without further degradation of acetate in the sample.

[0122] After hydrolysis, allow the sample to cool to room temperature and then filter it (e.g., centrifugal filter, PES 3 kDa) to remove residual proteins that may interfere with HPLC analysis. Prepare the filter according to the manufacturer's instructions and collect the filtrate containing the released acetate molecules.

[0123] Prepare a set of calibration standards using commercially available acetate standards for ion chromatography. The calibration standard set must contain the same amount of internal standard (e.g., 20 µg / mL of propionic acid) as previously applied.

[0124] Prepare an ion chromatography system (e.g., Dionex ICS-5000) equipped with an installed conductivity detector and suppressor. Use a Dionex IonPac AS11-HC analytical column (4 x 250 mm) and, optionally, a Dionex IonPac AS11-HC guard column (4 x 50 mm). Equilibrate the system with eluent A, 1 mM NaOH, for sample elution and eluent B, 100 mM NaOH, for column cleaning. Subsequently, inject the sample and calibration standard sets using the following instrument method / gradient profile: - 0 to 15 min, 100% eluent A, 1.5 mL / min flow rate, suppressor 4 mA (elution), -16 to 21 minutes, 100% eluent B, 1.5 mL / min flow rate, suppressor 372 mA (washing), -22~25 min, 100% eluent A, 1.5 mL / min flow, suppressor 4 mA (re-equilibration).

[0125] Depending on the ion chromatography system, the suppressor settings can vary.

[0126] The internal standard is used to normalize the area under the curve for both the calibration set and the unknown samples. The resulting calibration curve is then used to quantify the acetate concentration in the unknown samples. The degree of O-acetylation of monosaccharides can be calculated by determining the ratio of acetate per specified polysaccharide content (as described above).

[0127] 5) Optionally, determine the relative purity of the reference material. This is achieved by size exclusion chromatography and / or reversed-phase chromatography. Select a column appropriate for the target glycoprotein (e.g., Supelco TSKgel G3000SWXL for size exclusion, Cosmosil 5C4-AR-300 for reversed-phase) and perform the chromatography step according to the manufacturer's instructions. For example, absorbance measured at 215 nm can be used to determine the relative purity of the glycoprotein compared to other compounds that absorb at 215 nm. Determine the relative purity of the glycoprotein (in %) according to the HPLC software instructions.

[0128] II. Analysis using LC-MS system The glycoprotein samples to be analyzed are obtained directly from the production batch, and to identify and quantify the polysaccharide content of said samples, a reference material, previously prepared as described above, reflecting the same glycoprotein species, e.g., having the same glycan structure on the same carrier protein, is used.

[0129] The sample to be analyzed can also contain several different glycoproteins, provided that the reference materials used in the protocol described below are identical in terms of composition and glycan structure. Unless otherwise stated, MS grade chemicals are used to prepare eluents. NOTE: The glycoproteins to be analyzed do not need to be derivatized (e.g., via isotope labeling). Furthermore, no enzymatic treatment is required, and therefore no lengthy sample preparation before analysis is necessary. Glycoproteins can be considered to be analyzed in their native state, as described above. Furthermore, no internal standard for quantification is added to the sample; instead, the generation of a separate calibration curve is sufficient.

[0130] 1) Optionally, perform a buffer exchange of the sample buffer and the calibration reference standard material. This step may be desirable to prevent certain matrix components from entering the MS detector. However, buffer exchange may also remove free polysaccharide fragments, thereby preventing accurate determination of the free polysaccharide content in the sample. Instead, it is recommended to use a switching valve between the LC and MS systems, which can be programmed to divert undesired components into water after elution from the liquid chromatography. If performed, exchange the buffer into eluent A (HO Milli-Q grade, 0.1% v / v formic acid) at least three times using a centrifugal filter with an appropriate molecular weight cutoff. The amount of sample (e.g., 30 µg) depends on the glycoprotein of interest and must be optimized.

[0131] 2) Prepare HPLC vials with the sample to be analyzed and the reference material. The optimal concentration depends on the LC-MS system used. To generate a calibration curve, different calibration points of the reference material can be used by injecting equal amounts at different prepared concentrations or by injecting different volumes at the same concentration. This depends on the injector linearity of the LC system and the sample to be analyzed (e.g., potential interference due to absorption into the vial over time), and must be optimized for the specific sample.

[0132] 3) Prepare the LC-MS system. As an example, an ACQUITY-Synapt G2 HDMS from Waters is prepared by calibrating the MS detector using NaI according to the manufacturer's manual. A reversed-phase column suitable for the user's system (e.g., ACQUITY BEH300 C4, 1.7 μm, 2.1 × 150 mm, Waters) capable of eluting the protein to be analyzed is used. Different carbon polymer lengths of reversed-phase materials can be selected depending on the size of the glycoprotein to be analyzed to appropriately retain and elute the sample. Equilibrate the system with eluent A (H2O Milli-Q grade, 0.1% v / v formic acid) and eluent B (acetonitrile, 0.1% v / v formic acid).

[0133] 4) Subsequently inject the calibration points of the sample and reference material. The following instrument method / gradient profile is suitable: -First, 95% eluent A, 5% eluent B, 0.4 mL / minimum flow rate, -1 min, 95% eluent A, 5% eluent B, 0.4 mL / min flow; -9 minutes, 60% eluent A, 40% eluent B, 0.4 mL / min flow; -11 minutes, 5% eluent A, 95% eluent B, 0.4 mL / min flow; -12 minutes, 5% eluent A, 95% eluent B, 0.4 mL / min flow; -12.5 minutes, 95% eluent A, 5% eluent B, 0.4 mL / min flow; -15 minutes, 95% eluent A, 5% eluent B, 0.4 mL / min flow.

[0134] These gradients and injection volumes can be adapted depending on the target glycoprotein and the system / column used.

[0135] 5) Analyze the eluted proteins using an attached detector. Optionally, acquire a UV absorption signal at the desired wavelength. If a UV absorption signal is desired, a larger sample volume may need to be injected and the injection concentration must be optimized. Optionally, a split / switch valve is used between the LC system and the MS system to prevent oversaturation of the MS detector. The system's MS detector (e.g., quadrupole TOF Synapt G2 HDMS spectrometer, Waters) is used with electrospray ionization in positive resolution mode. Acquire data under instrument optimized conditions. The following parameters were used for the TOF Synapt G2 HDMS spectrometer: -1 second scan time, -3kV capillary voltage, -37V sampling cone voltage, -120℃ source voltage, -450℃ desolvation temperature, -20L / H cone gas flow, -550L / H desolvation gas flow, -MS function 500-2000 Da, with 4 eV trap, no transfer collision energy Optionally: MSMS function with target mass 100-1000 Da, 4 eV trap, and ramp transfer collision energy 18-25 eV. -Lock Leucine Enkephalin spray acquisition at 30 second intervals.

[0136] A critical parameter is the sampling cone voltage, which results in in-source fragmentation of the glycan chains. Depending on the glycoprotein being analyzed, this cone voltage may need to be optimized.

[0137] 6) Processing of acquired data. Data processing is performed using the manufacturer's software package (e.g., MassLynx, Waters). First, spectra across the main chromatographic peak (TIC MS feature) representing the eluted glycoprotein are combined. If the LC-MS acquisition parameters are appropriately optimized, in-source fragmentation produces high specific mass peaks derived from the polysaccharide chains of the analyzed glycoprotein (see Figure 1A). The results of a sample containing six different EPA glycoconjugates can be processed similarly to the above (see Figure 1B). Depending on the glycan structure and optimized parameters, masses associated with the repeating units of the polysaccharide can be observed. The specific mass peaks can be used to confirm the identity of the analyzed glycoprotein.

[0138] Second, in the next step, the extracted chromatogram of the identified specific mass peaks is used: the area under the curve in the main chromatographic peak (representing the eluted glycoprotein) corresponds to the amount of injected and bound polysaccharide in the reference material. A calibration curve (polysaccharide amount vs. area under the curve; see Figure 2) can be used to identify the absolute amount of polysaccharide content (in the main chromatographic peak) in an unknown sample.

[0139] Optionally, extracted chromatograms of the identified specific mass peaks can be further used to identify the relative amounts of free polysaccharides: the sum of the areas under the curve of all but the main chromatographic peak is compared to the area under the curve of the main chromatographic peak (representing the eluted glycoprotein). The identification of free polysaccharides in a sample can be further optimized by extracting chromatograms of different fragments of the polysaccharide chain (e.g., monosaccharides, disaccharide fragments, modified saccharides, polysaccharide-specific oxonium ions, etc.).

[0140] Optionally, the combined spectrum across the main chromatographic peak (representing the eluted glycoprotein) is used to identify the degree of polysaccharide modification. In the example of an O-acetylated polysaccharide chain, the identified specific mass peak may have a neighboring peak with a mass difference of 42 Da, representing the deacetylated form of the glycan (see Figure 3). By comparing the areas under the curve of these two peaks, the degree of modification can be identified.

[0141] Optionally, the acquired UV and / or TIC chromatograms can be used to determine the relative purity of the analyzed sample: the sum of the areas under the curves of all peaks other than the main chromatographic peak is compared to the area under the curve of the main chromatographic peak (representing the eluted glycoprotein).

[0142] III. Conclusion It should be noted that for multivalent glycoconjugate vaccines (e.g., a 10-valent E. coli (ExPEC) vaccine is currently under development, containing 10 different E. coli O-antigen polysaccharides, each independently covalently coupled to an EPA carrier protein, i.e., 10 drug substances), prior to the present invention, release of each individual drug substance typically required several different assays, including a PS content assay (similar to step I.2 above), followed by (i.e., depending on the results of the PS content assay) a free PS content assay (similar to step I.3 above) and, depending on the serotype, an assay to identify O-acetylation (similar to step I.4 above). This sequential dependency of the two other assays leads to low throughput during quality control ("QC") release. In addition, identity and purity assays (similar to steps I.1 and I.5 above, respectively) should also typically be performed for each drug substance release.

[0143] One advantage of the method of the present invention is that all five of these different assays can now be combined into one analytical procedure (Section II above), with only a dilution step in sample preparation, thereby reducing the time required for QC testing chemistry from approximately two weeks to approximately two days, resulting in a reduction in the time, materials, and operators required to obtain the same relevant information. Thus, the method significantly reduces batch release time at a lower cost.

[0144] Furthermore, assays can be performed on the individual drug substances, but also after the individual drug substances have been combined into more complex formulations, for example even on the final multivalent glycoconjugate vaccine composition.

[0145] Furthermore, the high specificity of spectroscopic detectors allows for in-process sample quantification, where sample purity is typically much lower, reducing costs and time during process optimization and therefore the overall development cost of commercially produced glycoprotein-based vaccines. The present invention includes the following aspects. [1] 1. A method for analyzing polysaccharide components of glycoproteins in a sample, whereby The analysis Identification of said polysaccharide components; Absolute quantification of said polysaccharide components; Including, The method comprises: (a) establishing a calibration curve of the polysaccharide components by an LC-MS system; (b) measuring said samples on the same LC-MS system; (c) comparing the results of (a) and (b), thereby analyzing the polysaccharide components in the sample; wherein the glycoprotein is in its native state. [2] The method according to [1], wherein the glycoprotein comprises one carrier protein and one or more polysaccharides covalently bound to the carrier protein. [3] The carrier protein may be detoxified exotoxin A (EPA) of Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli) flagellin (FliC), CRM197, maltose-binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus (S. aureus), clumping factor A, clumping factor B, Escherichia coli (E. coli) heat-labile toxin, detoxified variants of Escherichia coli (E. coli) heat-labile toxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, Escherichia coli (E. coli) SAT protein, passenger domain of Escherichia coli (E. coli) SAT protein, Streptococcus pneumoniae (Streptococcus The method according to [1] or [2], wherein the exotoxin is selected from the group consisting of Pseudomonas aeruginosa pneumolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, Neisseria meningitidis outer membrane protein (OMPC), and protein D from nontypeable Haemophilus influenzae, and is preferably detoxified exotoxin A from Pseudomonas aeruginosa. [4] the polysaccharide comprises 1 to 100, preferably 5 to 20 repeating units; The method according to any one of [1] to [3], wherein the repeating unit comprises an unmodified monosaccharide and / or a modified monosaccharide. [5] The sample is a water-soluble matrix, said matrix optionally comprising one or more of buffers, inorganic salts, sugar alcohols, and non-ionic surfactants; Optionally, a polysaccharide-free carrier protein; Optionally, polysaccharides that are not bound to carrier proteins ("free PS"); Optionally, unrelated proteins The method according to any one of [1] to [4], further comprising: [6] The method according to any one of [1] to [5], wherein the sample contains a number of different glycoproteins, preferably 2 to 20, for example 4 to 10 glycoproteins, and the glycoproteins differ in the polysaccharide component and / or the carrier protein. [7] The step (a) includes preparing a reference material (steps a1 to a3 and optionally a4, a5) and calibrating the LC-MS system using the reference material (step a6): (a1) identifying the specific name of the glycoprotein; (a2) determining the total polysaccharide (PS) content of the glycoprotein in the reference material; (a3) ​​determining the free PS content in the reference material; (a4) optionally identifying the degree of modification, in particular the degree of O-acetylation, of said glycoprotein in said reference material; (a5) optionally, determining the purity of the glycoprotein in the reference material, Thereby, obtaining a reference material containing said polysaccharide component of the glycoprotein, determining its purity, and then (a6) measuring an aliquot of the reference material by an LC-MS system, thereby establishing said calibration curve. The method according to any one of [1] to [6], comprising: [8] In step (a1), the specific name of the glycoprotein is identified by Western blot electrophoresis or MS, and / or In step (a2), the total PS content is determined by IC-PAD after hydrolysis, and / or In step (a3), the free PS content is determined by IC-PAD after hydrolysis and separation of bound and unbound PS, and / or In step (a4), the degree of modification, in particular the degree of O-acetylation, is determined by ion chromatography IC-CD after release of the modifying group, in particular after release of the O-acetyl group by hydrolysis, and / or In step (a5), the purity of the glycoprotein is determined by RP-HPLC and / or SEC, and / or The method according to [7], wherein in step (a6), the calibration curve is established by separating the aliquot via LC and subjecting the LC eluate to in-source fragmentation within an MS detector by adjusting the ionization voltage. [9] The step (b) (b1) providing a sample; (b2) measuring the sample using the same LC-MS system and the same parameters as in (a6); The method according to [7] or [8], comprising:

[10] The measuring (b2) includes identifying the specific name of the sample and: Determining the PS content of said glycoprotein, or - determining the PS content and acetylated PS content of said glycoprotein, or Identifying the PS content of said glycoproteins and determining the purity of the sample, or Determining the PS content and acetylated PS content of said glycoproteins and determining the purity of the sample; or Determining the PS content of said glycoprotein and determining the free PS content, or - Determining the PS content and acetylated PS content of said glycoproteins and determining the free PS content, or - Determining the PS content and acetylated PS content of said glycoproteins, as well as the free PS content and free acetylated PS content, Determining the PS content of said glycoproteins and determining the free PS content and purity of said sample, or Determining the PS content and acetylated PS content of said glycoproteins and determining the free PS content and purity of said sample, or Identifying the PS content and acetylated PS content of said glycoprotein and identifying the free PS content and free acetylated PS content and purity of said sample. The method according to [9], comprising one of the following:

[11] The step (c) (c1) identifying a characteristic peak of each PS in the sample; (c2) comparing the AUC of such peaks with the calibration curve; The method according to any one of [1] to

[10] , comprising:

[12] 1. Use of a liquid chromatography-mass spectrometry system ("LC-MS system") to analyze polysaccharide components of glycoproteins in a sample, said analyzing comprising: identifying said polysaccharide components; absolute quantification of said polysaccharide components; Including, The glycoprotein is in its native state.

[13] The use according to

[12] , wherein the LC-MS system is an LC-MS system that uses in-source fragmentation of the eluate obtained from the liquid chromatography.

[14] The use according to

[12] or

[13] , wherein the LC-MS system further comprises a switching valve in front of the MS detector.

[15] The use according to any one of

[12] to

[14] , wherein the glycoprotein comprises one carrier protein and one or more polysaccharides covalently bound to the carrier protein.

[16] The glycoprotein includes a carrier protein, and the carrier protein may be detoxified exotoxin A (EPA) of Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli) flagellin (FliC), CRM197, maltose-binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus (S. aureus), clumping factor A, clumping factor B, Escherichia coli (E. coli) heat-labile toxin, a detoxified variant of Escherichia coli (E. coli) heat-labile toxin, cholera toxin B subunit (CTB), cholera toxin, a detoxified variant of cholera toxin, Escherichia coli (E. coli) SAT protein, a passenger domain of Escherichia coli (E. coli) SAT protein, a Streptococcus pneumoniae (Streptococcus pneumoniae pneumolysin, keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, Neisseria meningitidis outer membrane protein (OMPC), and protein D from nontypeable Haemophilus influenzae, preferably Pseudomonas aeruginosa aeruginosa exotoxin A, and / or The use according to any one of

[12] to

[15] , wherein the polysaccharide comprises 1 to 100, preferably 5 to 20, repeating units, and the repeating units comprise unmodified monosaccharides and / or modified monosaccharides.

[17] The sample is a water-soluble matrix, said matrix optionally comprising buffers, inorganic salts, sugar alcohols, and non-ionic surfactants; Optionally, a polysaccharide-free carrier protein; Optionally, a polysaccharide that is not conjugated to a carrier protein, and Optionally, unrelated proteins The use according to any one of

[12] to

[16] , further comprising:

[18] In-process control in the production of complex carbohydrates, Controlling the release of produced complex carbohydrates, Controlling the stability of stored glycoconjugates, and / or ·Process optimization in the production of complex carbohydrates The use according to any one of

[12] to

[17] for the treatment of

Claims

1. 1. A method for analyzing polysaccharide components of glycoproteins in a sample, comprising: the glycoprotein comprises a carrier protein and one or more polysaccharides covalently bound to the carrier protein; The analysis - Identifying the polysaccharide components; - absolute quantification of the polysaccharide components; Including, The method comprises: (a) establishing a calibration curve for the polysaccharide components, comprising introducing a reference material comprising the glycoprotein into an LC-MS system; (b) measuring said samples on the same LC-MS system; (c) comparing the results of (a) and (b), thereby analyzing the polysaccharide components in the sample; wherein steps a and b comprise injecting glycoproteins or glycopeptides derived therefrom into said LC-MS system and subjecting them to in-source fragmentation in a mass spectrometer at a cone voltage that separates the glycan repeat units.

2. 2. The method of claim 1, wherein the glycoprotein is not subjected to enzymatic digestion with an enzyme that removes the polysaccharide components from the carrier protein prior to introducing the glycoprotein into the LC-MS system, and the glycoprotein is not subjected to a chemical reaction prior to introducing the glycoprotein into the LC-MS system.

3. The carrier protein may be a detoxified exotoxin A (EPA) of Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli) flagellin (FliC), CRM197, maltose-binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus (S. aureus), clumping factor A, clumping factor B, Escherichia coli (E. coli) heat-labile toxin, a detoxified variant of Escherichia coli (E. coli) heat-labile toxin, cholera toxin B subunit (CTB), cholera toxin, a detoxified variant of cholera toxin, Escherichia coli (E. coli) SAT protein, a passenger domain of Escherichia coli (E. coli) SAT protein, a Streptococcus pneumoniae (Streptococcus pneumoniae) 3. The method of claim 1 or 2, wherein the exotoxin is selected from the group consisting of Pseudomonas pneumoniae pneumolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, outer membrane protein (OMPC) of Neisseria meningitidis, and protein D from nontypeable Haemophilus influenza, preferably detoxified exotoxin A of Pseudomonas aeruginosa.

4. the polysaccharide comprises 1 to 100, preferably 5 to 20 repeating units; The method of any one of claims 1 to 3, wherein the repeating units comprise unmodified monosaccharides and / or modified monosaccharides.

5. The sample is a water-soluble matrix, said matrix optionally comprising one or more of buffers, inorganic salts, sugar alcohols, and non-ionic surfactants; - optionally a polysaccharide-free carrier protein; Optionally, polysaccharides that are not bound to a carrier protein ("free PS"); Optionally, an unrelated protein The method of any one of claims 1 to 4, further comprising:

6. 6. The method according to any one of claims 1 to 5, wherein the sample comprises a number of different glycoproteins, preferably 2 to 20, such as 4 to 10 glycoproteins, which glycoproteins differ in their polysaccharide components and / or in their carrier proteins.

7. The step (a) includes preparing a reference material (steps a1 to a3 and optionally a4, a5) and calibrating an LC-MS system using the reference material (step a6): (a1) identifying the specific name of the glycoprotein; (a2) determining the total polysaccharide (PS) content of the glycoprotein in the reference material; (a3) ​​determining the free PS content in the reference material; (a4) optionally identifying the degree of modification, in particular the degree of O-acetylation, of said glycoprotein in said reference material; (a5) optionally, determining the purity of the glycoprotein in the reference material, Thereby, obtaining a reference material containing said polysaccharide component of the glycoprotein, determining its purity, and then (a6) measuring an aliquot of said reference material by an LC-MS system; thereby establishing said calibration curve. The method according to any one of claims 1 to 6, comprising:

8. In step (a1), the specific name of the glycoprotein is identified by Western blot electrophoresis or MS, and / or In step (a2), the total PS content is determined by IC-PAD after hydrolysis, and / or In step (a3), the free PS content is determined by IC-PAD after hydrolysis and separation of bound and unbound PS, and / or in step (a4), the degree of modification, in particular the degree of O-acetylation, is determined by ion chromatography IC-CD after release of the modifying group, in particular after release of the O-acetyl group by hydrolysis, and / or In step (a5), the purity of the glycoprotein is determined by RP-HPLC and / or SEC, and / or 8. The method of claim 7, wherein in step (a6), the calibration curve is established by separating the aliquots via LC and subjecting the LC eluate to in-source fragmentation inside an MS detector by adjusting the ionization voltage.

9. The step (b) (b1) providing a sample; (b2) measuring the sample using the same LC-MS system and the same parameters as in (a6); 9. The method of claim 7 or 8, comprising:

10. The measuring (b2) includes identifying the specific name of the sample and: - Identifying the PS content of said glycoprotein, or - Determining the PS content and acetylated PS content of said glycoprotein, or - Identifying the PS content of the glycoprotein and determining the purity of the sample, or - Identifying the PS content and acetylated PS content of the glycoprotein and determining the purity of the sample, or - Determining the PS content and free PS content of the glycoprotein, or - Determining the PS content and acetylated PS content of the glycoprotein and determining the free PS content, or - Identifying the PS content and acetylated PS content of the glycoprotein and identifying the free PS content and free acetylated PS content; - Determining the PS content of the glycoprotein and determining the free PS content and purity of the sample, or - Determining the PS content and acetylated PS content of the glycoprotein and determining the free PS content and purity of the sample, or Identifying the PS content and acetylated PS content of the glycoprotein and identifying the free PS content and free acetylated PS content and purity of the sample and one of:

11. The step (c) (c1) identifying a characteristic peak of each PS in the sample; (c2) comparing the AUC of such peaks with said calibration curve; The method according to any one of claims 1 to 10, comprising:

12. Use of a liquid chromatography-mass spectrometry system ("LC-MS system") to analyze polysaccharide components of glycoproteins in a sample by injecting an eluate containing a sample obtained from liquid chromatography of the LC-MS system into a mass spectrometer of the LC-MS system and subjecting the sample to in-source fragmentation, wherein the glycoprotein comprises a carrier protein and one or more polysaccharides covalently bound to the carrier protein; The analyzing step comprises: Injecting glycoproteins into the LC-MS system and subjecting them to in-source fragmentation in a mass spectrometer at a cone voltage that separates the glycan repeat units; establishing a calibration curve of the polysaccharide components by the LC-MS system using a reference material comprising the glycoprotein; - identifying the polysaccharide component; - absolute quantification of said polysaccharide components; Including, use.

13. 13. The use according to claim 12, wherein the glycoprotein is not subjected to enzymatic digestion with an enzyme that removes the polysaccharide components from the carrier protein prior to introducing the glycoprotein into the LC-MS system, and the glycoprotein is not subjected to a chemical reaction prior to introducing the glycoprotein into the LC-MS system.

14. The use according to claim 12 or 13, wherein the LC-MS system further comprises a switching valve before the MS detector.

15. The carrier protein may be a detoxified exotoxin A (EPA) of Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli) flagellin (FliC), CRM197, maltose-binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus (S. aureus), clumping factor A, clumping factor B, Escherichia coli (E. coli) heat-labile toxin, a detoxified variant of Escherichia coli (E. coli) heat-labile toxin, cholera toxin B subunit (CTB), cholera toxin, a detoxified variant of cholera toxin, Escherichia coli (E. coli) SAT protein, a passenger domain of Escherichia coli (E. coli) SAT protein, a Streptococcus pneumoniae (Streptococcus pneumoniae) pneumoniae pneumolysin, keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein (OMPC) of Neisseria meningitidis, and protein D from nontypeable Haemophilus influenza, preferably detoxified exotoxin A of Pseudomonas aeruginosa; and / or 15. The use according to any one of claims 12 to 14, wherein the polysaccharide comprises 1 to 100, preferably 5 to 20, repeating units, the repeating units comprising unmodified and / or modified monosaccharides.

16. The sample is a water-soluble matrix, said matrix optionally comprising buffers, inorganic salts, sugar alcohols, and non-ionic surfactants; - optionally a polysaccharide-free carrier protein; - optionally a polysaccharide that is not conjugated to a carrier protein, and Optionally, an unrelated protein The use according to any one of claims 12 to 15, further comprising:

17. - In-process control in the production of complex carbohydrates, - Controlling the release of produced glycoconjugates, - controlling the stability of stored glycoconjugates, and / or - Process optimization in the production of complex carbohydrates Use according to any one of claims 12 to 16 for

18. A method described in any one of claims 1 to 11, wherein the cone voltage is 37V.

19. The use described in any one of claims 12 to 17, wherein the cone voltage is 37V.

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