Method for identifying and quantifying polysaccharides in complex carbohydrate compositions.

Abstract

There is a need for analytical methods to identify and quantify complex carbohydrate compositions, particularly methods using liquid chromatography-mass spectrometry ("LC-MS systems") to analyze the polysaccharide components of glycoproteins in samples. For example, there is a demand for the use of LC-MS systems in process control during the production of complex carbohydrates. [Solution] A method for analyzing the polysaccharide components of a glycoprotein in a sample, comprising: identification of the polysaccharide components; and absolute quantification of the polysaccharide components, the method comprising: (a) establishing a calibration curve for the polysaccharide components using an LC-MS system; (b) measuring the sample using the same LC-MS system; and (c) comparing the results of (a) and (b) thereby analyzing and comparing the polysaccharide components in the sample, wherein the glycoprotein is in its natural state.

Description

Technical Field

[0001] The present invention relates to an analytical method for identifying and quantifying a complex carbohydrate composition, which is a complex, and more particularly to the analysis of glycoproteins in a sample. The present invention further relates to the use of a liquid chromatography-mass spectrometry system ("LC-MS system") in such an analytical method, particularly in-process control during the production of complex carbohydrates, release control of the produced complex carbohydrates, stability control of the stored complex carbohydrates, and the use of the LC-MS system for optimizing the complex carbohydrate production process. For understanding, measuring, and controlling glycosylation in glycoprotein-based drugs, there is increasing pressure from regulatory authorities on biopharmaceutical companies to demonstrate satisfactory programs. However, the analysis of complex carbohydrate compositions, which are complexes, i.e., their identification and absolute quantification, is a difficult task. Such an analysis typically involves several assays and requires a complete analysis under GMP conditions for one week or more. For glycoprotein-based drugs, current state-of-the-art chemical assays in the art are performed on high-performance liquid chromatography systems (HPLC systems) equipped with various detectors: When identifying total polysaccharides (PS) and free PS, a pulsed amperometric detection detector is used, which requires sample preparation in a laboratory by hydrolyzing the sugar chains and detecting the monosaccharides released 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."

[0002] To show a satisfactory program for understanding, measuring, and controlling glycosylation in glycoprotein-based drugs, there is increasing pressure from regulatory authorities on biopharmaceutical companies. However, the analysis of complex carbohydrate compositions, which are complexes, i.e., their identification and absolute quantification, is a difficult task. Such an analysis typically involves several assays and requires a complete analysis under GMP conditions for one week or more. For glycoprotein-based drugs, current state-of-the-art chemical assays in the art are performed on high-performance liquid chromatography systems (HPLC systems) equipped with various detectors: When identifying total polysaccharides (PS) and free PS, a pulsed amperometric detection detector is used, which requires sample preparation in a laboratory by hydrolyzing the sugar chains and detecting the monosaccharides released 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." Such an analysis typically involves several assays and requires a complete analysis under GMP conditions for one week or more.

[0003] For glycoprotein-based drugs, current state-of-the-art chemical assays in the art are performed on high-performance liquid chromatography systems (HPLC systems) equipped with various detectors: When identifying total polysaccharides (PS) and free PS, a pulsed amperometric detection detector is used, which requires sample preparation in a laboratory by hydrolyzing the sugar chains and detecting the monosaccharides released 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." When identifying total polysaccharides (PS) and free PS, a pulsed amperometric detection detector is used, which requires sample preparation in a laboratory by hydrolyzing the sugar chains and detecting the monosaccharides released 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." As stated by ThermoFisher Scientific, the manufacturer of such HPLC systems, "Carbohydrates are difficult to analyze using common chromatographic and detection methods." These "electrochemical detections are optimized for carbohydrate analysis" (1). In conclusion, current Measurement, detection, and quantification can be considered the gold standard in carbohydrate quantification. However, one of the main drawbacks of this technique is that it requires monosaccharides, which can lead to problems with glycoproteins. Laboratory sample preparation and release assays for qualitative drugs are time-intensive and cost-intensive. .

[0004] Furthermore, all glycoprotein-based drugs must meet the purity and designation requirements set by the supervising government agency. Regarding the general release of Arsenal, further analysis of the release process It goes through a process (e.g., free glycans in the drug substance, glycan modification). These multiple different processes Depending on the release criteria, the release time for glycoprotein-based drugs will be delayed.

[0005] Mazsaroff et al. used (2) LC-MS systems and in-source fragmentation. This document describes the analysis of the carbohydrate structure of glycoproteins. This paper also describes the ratio of two peaks. The text describes an analytical method for relatively quantifying by identifying the relevant factors. It is clearly stated by the authors. As stated above, "the fragment ion peak area under optimal signal-to-noise ratio conditions is adjusted The absolute ratio of fragment ions present in the glycoprotein molecule being examined may not be reflected. However, the optimal conditions for comparing different batches of biopharmaceuticals to their baseline are based on the fragment ion ratio. It is important to understand that this provides the best measure of relative quantitative rate. The literature does not describe any absolute quantification of carbohydrate structure, and does not use a reference material calibration curve. It does not suggest that either.

[0006] Ivancic et al. (3) Develop methods for identifying and quantifying intact glycopeptides. As a model for this, we described the LC-MS analysis of complex polysaccharide-modified human A1 glycoprotein. This document describes how in-source fragmentation is used to fragment carbohydrates into glycopeptides. This document describes an analytical method that uses fragmented carbohydrates as markers for identification. This technique ultimately quantifies the "degree of glycation" of peptides in AAG proteins. The objective is to compare the peak intensity of glycated and identified peptides with that of non-glycated peptides. This means a comparison with the peak intensity of the ozone. Here again, this literature does not consider any absolute value of the carbohydrate structure. Neither the quantitative analysis nor the use of a reference material calibration curve is mentioned.

[0007] Carell et al. (U.S. Patent Application Publication No. 2020 / 041470) have found that (4) sugars The present invention discloses a derivatization reagent for polysaccharides as a protein compound. It is produced from a reference material. The use of this derivatization reagent and its isotopic substitutions in combination with the calibration curve is effective in collision-induced solutions. By using an LC-MS system capable of fragmenting ions selected by ion separation (CID), This enables the absolute quantification of polysaccharides such as [specific polysaccharides]. Derivatization of the sample is a necessary condition for quantification, This document describes any LC-MS method used to determine the state of glycoproteins in their native form. This has not been achieved. In such derivatization of polysaccharide components, glycoproteins in their natural state are not analyzed. Compared to the method, it is necessary to add a sample preparation step, and therefore such The method inevitably becomes more complicated. Furthermore, this method is specific to the fragmentation that arises from insource fragmentation. An advanced LC-MS system that can select and further fragment ions using CID. The use of such advanced equipment is not only more expensive in itself, but also more responsive. Verification of the analysis method also takes more time than methods without such requirements. Therefore, the use of a relatively simple LC-MS system and protocol with only minimal handling steps is considered particularly advantageous in the context of drug release assays.

[0008] Echeverria et al. describe an MS method for quantifying (5) 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. In the case of quantification, a mixture of isotope-labeled glycans is added to the sample as an internal standard, and the analysis is performed by matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) MS. Again, this document does not describe any analysis method for glycoproteins in their native state. Furthermore, this method does not use liquid chromatography to separate glycoproteins in the sample prior to MS analysis and only suggests the possibility of applying the reported mixture of isotope-labeled glycan standards to an LC-MS-based method. As discussed earlier, with the addition of sample preparation steps, such as the PNGase F-mediated release of the PS component from the carrier protein, the analysis method becomes more cumbersome and therefore throughput is reduced compared to methods that do not include such steps. Furthermore, PNGase F digestion is not applicable to all polysaccharide structures and therefore limits prior art methods to glycoproteins within the substrate specificity of

[0009] Similarly, Jeong et al. perform PNGase F-mediated release from (6) glycoproteins and then absolutely quantify the glycans after polymethylating the glycans by MALDI-TOF M describes the S method. Therefore, this document also does not describe a method for quantifying glycoproteins in their natural state. The methods described do not include LC-based separation of glycoproteins prior to MS analysis. As outlined above, the addition of sample preparation steps complicates the analysis method and reduces throughput. In this particular case, sample preparation involves not only PNGase F digestion (thereby necessarily imposing limitations on the method), but also a multiple methylation step, further complicating sample preparation. As a conclusion, there is a need for an improved analysis method for complex carbohydrates, particularly glycoprotein-based drugs. Therefore, an object of the present invention is to alleviate at least some of these drawbacks of the current state of the art. In particular, an object of the present invention is to provide an analysis method that bypasses sample preparation for glycoprotein-based drugs, thereby increasing analysis throughput and reducing release costs.

[0010] Furthermore, an object of the present invention is to combine multiple different release criteria into one assay, thereby providing an analysis method that further shortens the batch release time of glycoprotein-based drugs. In particular, an object of the present invention is to provide an analysis method that simultaneously provides information on the specific name and absolute quantification of glycoprotein-based drugs.

[0011] One or more of the above objects are achieved by the analysis method according to claim 1 and the LC- according to claim 12.

[0012]

[0013]

[0014]

[0015] One or more of the above objects are achieved by the analysis method according to claim 1 and the LC- according to claim 12. ​​​​​​​​​This is achieved by using an MS system. Further aspects of the present invention are described herein and independently claimed. This is disclosed in [the relevant document], and preferred embodiments are disclosed herein and in dependent claims.

[0016] The present invention will be described in more detail below. Various embodiments provided / disclosed herein It is understood that preferences and ranges can be combined as desired. Furthermore, in a particular embodiment, Accordingly, the selected definitions, embodiments, or scope may not apply.

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

[0018] When used herein, it is used in the context of the present invention (particularly in the context of the claims). "a," ​​"an," "the," and similar terms are used unless otherwise indicated herein. , or unless the context clearly contradicts it, it includes both singular and plural forms. It should be interpreted as such.

[0019] The terms "including" and "containing" as used herein "comprising)" and "comprising" are used in this specification to describe their opening Used in a free, non-restrictive sense. Various embodiments, preferences, and ranges can be combined as desired. It is understood that this is possible.

[0020] Throughout this specification, several abbreviations will be used, including the following: Area under the AUC curve CID collision-induced dissociation, MS technique EPA is a detoxified exotoxin A from Pseudomonas aeruginosa. ETD Electron transfer dissociation, MS technique HPLC (High-Performance Liquid Chromatography) IC-PAD Ion Chromatography - Pulse Current Measurement and Detection ISF Insource Fragmentation LC Liquid Chromatography LPS (Lipopolysaccharide) MS mass spectrometry PS polysaccharide RP inverse phase SEC size exclusion chromatography TIC total ion current, MS techniques

[0021] The abbreviations mentioned above and any further abbreviations used herein are common in this field.

[0022] The term "complex carbohydrate" is known in this field, and in particular refers to a compound shared by one or more polysaccharides. Describe the combined chemical substances. Such complex carbohydrates are involved in biological coupled reactions in living cells. (Can be obtained by "bio-conjugated reaction" or "biological conjugated reaction") or by the chemical reaction of polysaccharides It can be obtained by conjugation ("chemical" or "synthetic" complex carbohydrates). Suitable chemical substances include There are proteins / peptides and lipids, and the corresponding complex carbohydrates are glycoproteins (proteoglycans). These are glycolipids (including peptidoglycans and glycopeptides) and glycolipids.

[0023] The term glycoprotein includes "conventional glycoproteins" and "complex carbohydrate vaccines." In conventional glycoproteins, for example, the protein portion of antibodies or erythropoietin sugar is superimposed. The principle of "activity" is more prevalent in the protein portion, while glycans, for example, have a half-life. It plays a role in defining the period or other properties. Such conventional glycoproteins are used in formulations. It is widely used along the way. In complex carbohydrate vaccines, glycans are related antigens, therefore immunity Emphasis is placed on the glycan portion where a response is desired, while the protein portion simply represents the desired T cell memory. It functions as a carrier that triggers an immune response.

[0024] The term glycoproteins include "proteoglycans," "peptidoglycans," and "glycoproteins." It also contains "cydo." The term "proteoglycan" refers to a highly glycosylated tan. This refers to a protein. The basic proteoglycan unit is one or more covalently bonded glycosides. It consists of a core protein containing saminoglycan (GAG) chains. The attachment site is glycosamino A serine (Ser) residue that glycans join via tetrasaccharide crosslinking. The term "n" refers to the sugars that form a mesh-like layer outside the plasma membrane of most bacteria and which form the cell wall. This refers to polymers composed of proteins and amino acids. The term "glycopeptide" refers to the protein portion. This refers to glycoproteins containing up to 50 amino acids. Digestion of larger glycoproteins (e.g.) Such sugars can be made available through (for example, trypsin digestion), bacterial fermentation, or chemical synthesis. Petit Do.

[0025] The term "glycolipid" is known in this field, and in particular, refers to a lipid in which one or more sugars are aggregated. This describes substances linked by lycosidic (covalent) bonds. Therefore, it describes the characteristic features of glycolipids. A key characteristic is the presence of sugars (especially polysaccharides, as discussed herein) bound to the lipid portion. ru.

[0026] The term "polysaccharides" is well known in this field, and in particular, whether linear or branched, glycans This section describes polymerized carbohydrates composed of monosaccharide units linked together by cosidic bonds. Such polysaccharides are characterized by repeating units, and each repeating unit is described using the monosaccharide composition of its respective monosaccharide. The repeating units described above can also be chemically modified (e.g., amidation, sulfation, acetation). Contains one or more monosaccharides (e.g., chilled, phosphorylated). Typically found in the repeating units described above. Monosaccharides are cyclic or linear monosaccharides containing 3 to 7 carbon atoms. (The text then mentions a complex carbohydrate vaccine.) In certain cases, the complex polysaccharides originate from pathogenic species (e.g., E. coli), and the repeating units are associated with specific diseases. It is defined by the genetic properties of the pathogen. Therefore, repeat units are specific markers of pathogens. / Can be an identifier.

[0027] The term "polysaccharide component" therefore refers to one or more glycan chains of a complex carbohydrate. Glycans can be monomers or polymers of sugar residues, but typically few Both contain three sugars and can be linear or branched. Glycans are natural sugar residues ( For example, glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose (e.g., sucrose, mannose, fucose, arabinose, ribose, xylose, etc.) and / or modified sugars (For example, 2'-fluororibose, 2'-deoxybose, phosphomannose, 6'- It may contain (such as glycan N-acetylglucosamine). The term "glycan" refers to a sugar residue. This includes mopolymers and heteropolymers. The term "glycan" refers to complex carbohydrates (e.g., sugars). This term also includes the glycan components of proteins, peptides, glycolipids, etc. This term is a complex carbohydrate. This includes free glycans, including glycans that have been cleaved or otherwise released. ru.

[0028] The term "O-acetylated polysaccharide" as used herein refers to one of the repeating units. Alternatively, it refers to polysaccharides in which multiple monosaccharides are chemically modified by acetylation. The above monosaccharides exist Acetylated one or more of the hydroxyl groups present. Used in complex carbohydrate vaccines. In the case of repeat units derived from pathogens, O-acetylation of specific monosaccharides is an immunoassay of the pathogen. This can be extremely important for guiding the answer. Examples of polysaccharide components derived from pathogens are shown in Table 1. show.

[0029] The terms "glycan" and "glycan chain" are synonymous with "polysaccharide," as defined below. Accordingly, in the context of the present invention, "glycan" and the prefix "glyco-" refer to sugars. This also refers to the carbohydrate portion of complex carbohydrates such as proteins or glycolipids.

[0030] The term "serotype," as used herein, refers to different bacterial serotypes. This refers to a complex carbohydrate having a polysaccharide chain. Examples of cans are identified in Table 1 below.

[0031] The term "natural state" is known in this field and refers to the intact functional state of complex carbohydrates. Related to biomolecules such as the above. When referring to the natural state in this invention, the natural state is analyzed. The complex carbohydrate is derivatized during sample preparation using an enzyme such as PNGase F or other enzymes. This means that it is not modified by this method or does not undergo chemical reactions. Therefore, in its natural state Analysis of complex carbohydrates involves derivatization of complex carbohydrates or LC-MS step analysis of the sample. Before attaching to the sample, a sample preparation step is performed to release glycans from the carrier molecule, such as a carrier protein. It is different from any method including a smear. However, if the natural state is referred to herein, The term "natural state" refers to the conformation of a complex carbohydrate, i.e., secondary, tertiary, or quaternary structure. These are unrelated to the three-dimensional fold. Therefore, "analysis of glycoproteins in their natural state" " is an enzyme that removes polysaccharide components from carrier proteins using glycoproteins. (ii) Before the glycoprotein was introduced into the LC-MS system in each case, without any direct digestion. This is synonymous with "analysis of glycoproteins that do not undergo chemical reactions, such as derivatization."

[0032] This invention will be better understood by referring to the figures. [Brief explanation of the drawing]

[0033] [Figure 1] LC-MS spectra (TIC) coupled across the main chromatographic peaks of the analyzed glycoprotein Ec06A (a complex carbohydrate vaccine component containing polysaccharides from Escherichia coli (E. coli) serotype O6A that covalently binds to the EPA carrier protein; the structure of the PS repeat 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 binding to the EPA carrier protein; the structures of the repeat units of these polysaccharides are given in Table 1) were analyzed. MS function (x-axis - m / z; y-axis - relative intensity [%]), panel A shows in-source fragmentation and release of repeat unit-specific fragments of Escherichia coli (E. coli) serotype EcO6A (peak with a mass of 893.3262 g / mol), and as seen in panel B, release and selective assignment of glycan repeat units are 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 insource fragmentation occurs linearly based on the amount of serotype injected, as demonstrated here in a proof-of-concept using the glycoprotein EcO6A. Different injection amounts of protein (from 0.2 μg to 1.2 μg) lead to different signal responses of repeating units of EcO6A (fragment with molecular mass 893.32 g / mol; y-axis - response / [au]; x-axis - micrograms EcO6A). [Figure 3]The LC-MS spectra (with / without acetylation) are coupled across the main chromatographic peaks of glycoproteins EcO25B and EcO16 (TIC MS function, x-axis m / z; y-axis relative intensity [%]). The structures of the O-acetylated repeat units for each Escherichia coli (E. coli) O25B and O16 serotype are shown in Table 1. Panel A shows insource fragmentation of glycoprotein EcO25B with two distinct peaks: O-acetylated (862 g / mol) and O-acetylated (820 g / mol) repeat units. Panel B shows the same effect in glycoprotein EcO16 with two distinct peaks: O-acetylated and O-acetylated repeat units with masses of 716 g / mol and 674 g / mol.

[0034] In more general terms, in the first embodiment, the present invention relates to the polysaccharide composition of complex carbohydrates in a sample. This relates to a method for analyzing glycans. Such analysis is used for the identification of the above polysaccharide components and It is important to note that this refers to both the absolute quantification of the polysaccharide components mentioned above. The method involves (a) establishing a calibration curve for the polysaccharide component using an LC-MS system. (b) the step of measuring the sample with the same LC-MS system, and (c) (a) and (b) ) is a step of comparing the results with the above polysaccharide component in the above sample, thereby determining This includes the steps of analyzing and comparing. In one advantageous embodiment, the complex carbohydrate is a glycoprotein It's about quality.

[0035] In one preferred embodiment, the complex carbohydrate, preferably glycoprotein, is in its natural state. Therefore, the enzymatic release of PS components from carrier proteins, for example, PNGase F, is necessary. Since intercautionary release is not required, the method involves complex carbohydrates that are less susceptible to such enzymatic cleavage. For example, N-acetylglucosamine (Glc) that is not linked to alpha-1,3-fucose. PNGase F contains sugar motifs that are not substrate-specific, such as sugar motifs including NaAc. It can also be applied to the analysis of carbohydrates.

[0036] As will be outlined in more detail below, the method according to the present invention (i) Identification of polysaccharides in the substance, (ii) Absolute determination of polysaccharides bound to other chemical substances in complex carbohydrates such as proteins, (iii) Determination of free polysaccharides in the substance, (iv) Quantitative determination and identification of modifications such as acetylation in bound polysaccharides and free polysaccharides, Bini (v) Purity of samples containing complex carbohydrates, particularly glycoprotein-based formulation compositions Provide information on at least two, preferably three or more, and most preferably all of them. Provided. Further details regarding this aspect of the present invention, in particular the process steps and the terminology used. The following explanation will be provided.

[0037] Complex carbohydrates: This term has been discussed previously. The method of the present invention is widely applicable. Glycoproteins (including proteoglycans, peptidoglycans, and glycopeptides) and sugars It can be used for lipid analysis.

[0038] Glycoproteins: This term has been discussed previously. Specifically, glycoproteins are, This relates to a complex product in which sugars (i.e., glycans) are covalently bound to a carrier protein. It may be a biological complex that is a complex product prepared within the main cell, and the host cell mechanism involves glycans and It produces proteins and uses glycans as carrier proteins, for example, asparagine or arginine. They are linked via N-bonds. The complex consists of protein and glycan chains, for example, thiols. It can also be prepared by chemical bonding via alkylation. The method according to the present invention is for all Applicable to complex types. Insource fragmentation by MS without being constrained by theory. As long as this is the case, the way the sugar adheres is considered irrelevant. The reference material shows the same fragmentation. Therefore, it is irrelevant whether or not fragmentation occurs at the protein-glycan interface.

[0039] Particularly useful complex carbohydrates include carrier proteins to which one or more polysaccharides are attached. Such complex carbohydrates are used, for example, as the active ingredient in certain vaccines, and the polysaccharides of complex carbohydrates The aim is to induce a functional immune response against this species.

[0040] In embodiments of the present invention, the glycoprotein comprises one carrier protein and the carrier One or more polysaccharides that covalently bind to the protein, preferably the carrier protein mentioned above. It contains 1 to 4 polysaccharides that bind to it. In embodiments of the present invention, the glycoprotein is covalently bound to a carrier protein in Escherichia coli (E) It is a complex product containing the O antigen of *C. coli*. The term O antigen is known in this field. Yes, it is used in its usual context and should not be confused with O-binding. The term O antigen is one Generally, repeated glycan polymers contained within the LPS of bacteria such as Escherichia coli (E. coli) This refers to the O antigen of Escherichia coli (E. coli), which is an immunogenic repeat oligosaccharide (typically 1-4). It is a polymer of 0 repeating units and is typically used in serotyping and complex carbohydrate vaccine production. It is used.

[0041] Carrier protein: In embodiments of the present invention, the carrier protein is Pseudomonas aeruginosa (P. aer). Detoxified exotoxin A (EPA) of *E. coli*, flagellin of *E. coli* FliC), CRM197, maltose-binding protein (MBP), diphtheria toxovirus D, tetanus toxoid, detoxified hemolysin A for Staphylococcus aureus (S. aureus), Cran Ping factor A, clamping factor B, Escherichia coli (E. coli) heat-labile toxin, Escherichia coli (E. Cholera toxin B subunit (CTB), a detoxification variant of the heat-labile toxin of coli. Cholera toxin, cholera toxin detoxification variant, Escherichia coli (E. coli) SAT Protein, passenger domain of Escherichia coli (E. coli) SAT protein, pneumococcus Fungus (Streptococcus pneumoniae) pneumocysin, on the mussel. Mocyanin (KLH), Pseudomonas aeruginosa (P. aeruginosa) PcrV, Neococcus meningitidis (Ne) isseria meningitidis) outer membrane protein (OMPC) and unclassifiable From Haemophilus influenza It is selected from the group consisting of protein D.

[0042] In certain embodiments, the carrier protein is Pseudomonas aeruginosa. It is the detoxified exotoxin A (EPA) of uginosa. In such embodiments, E PA preferably has 1 to 20, preferably 1 to 10, and preferably 2 to 4 glycosylation sites. Includes.

[0043] In certain embodiments, EPA comprises four glycosylation sites. For example, EPA This explains an example of the biological coupling reaction of E. coli O antigen polysaccharides to somatic proteins. Please refer to International Publication No. 2017 / 035181.

[0044] Polysaccharides: This term has been discussed previously. Suitable polysaccharides are 1 to 100, for example, 1 to 50, 1-40, 1-30, 1-20, and 1-10, 3-50, 3-40, for example, a small At least 5, for example 5-40, for example 7-30, for example 7-25, for example 10-20, example For example, it contains 5 to 20 repeating units n. Such repeating units are (i) unmodified monosaccharides and / or (ii) Contains modified monosaccharides (i.e., contains or consists of them). The term "modified monosaccharides" is particularly relevant to chemically modified monosaccharides, and in non-limiting embodiments, the monosaccharides This includes N-acetylation, O-acetylation, amidation, and / or amination. Such monosaccharides This may include one or more modifications in the same monosaccharide, particularly one, two, or three of the above modifications. ru.

[0045] In embodiments of the present invention, suitable repeating units are mannose, rhamnose, and glucose. , fucose, galactose, modified mannose, modified rhamnose, modified glucose, modified fucose It contains monosaccharides selected from the group consisting of coase and modified galactose. Non-restrictive and exemplary structures of the E. coli (E. coli) O antigen polysaccharide are shown in Table 1 below. A single repeating unit of the E. coli (E. coli) O antigen polysaccharide is shown in this table. Each n is an independent integer from 1 to 100, for example, 1 to 50, 1 to 40, 1 to 30, 1 ~20, and 1~10, 3~50, 3~40, for example at least 5, for example 5~40, example For example, integers from 7 to 30, such as 7 to 25, such as 10 to 20, such as 5 to 20. However, in some cases it can be 1 or 2.

[0046] [Table 1]

[0047] [Table 2]

[0048] Sample: This term is known in this field. The sample is optionally diluted and then analyzed in the analytical system. This includes all materials that can be supplied. Such samples are particularly useful in (i) the production batches of complex carbohydrates. (i) multiple composites (including ongoing batches and released / stored manufacturing batches) This includes compositions containing carbohydrates, such as pharmaceutical compositions containing polyvalent vaccines.

[0049] Suitable samples include (i) a water-soluble matrix, in addition to complex carbohydrates, and (ii) optionally , containing polysaccharides (or free peptides in the case of glycopeptides, or free lipids in the case of glycolipids) (iii) Carrier proteins that do not have, (iii) optionally, carrier proteins (or in this specification) Peptides or lipids: polysaccharides not bound to "free PS", (iv) optionally non It contains related proteins (or peptides or lipids). The water-soluble matrix (i) is a batch Phosphates (e.g., phosphate buffers), inorganic salts (e.g., NaCl), sugar alcohols (e.g., D-sorbitol), one of the nonionic surfactants (e.g., polysorbate 80) Or may include multiple. Unrelated proteins (iv) may make up to 10%, up to 50%, or It may contain up to 90% process-related impurities (e.g., host cell proteins). The method is considered particularly beneficial if it is tolerant of a wide range of additional components in the sample. Therefore, without laboratory preparation and containing materials that interfere with standard analytical protocols... It is appropriate to analyze the sample without doing so.

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

[0051] In a further embodiment, the sample is a plurality of complex carbohydrates, for example 2 to 20, for example 4 to 10 It contains complex polysaccharides. Here again, the complex carbohydrates are preferably related to glycoproteins. The method according to the present invention involves, for example, a plurality of complex carbohydrates having glycans from a plurality of serotypes Analysis of complex samples including these is also possible. Therefore, when releasing preparations such as polyvalent conjugate vaccines... Even composite samples for analysis can be analyzed by the method according to the present invention. This method has advanced the manufacturing and production of formulations containing multiple serotypes, and the quality of such complex formulations. It is clear that quality and safety will be improved.

[0052] It has become clear that the method according to the present invention is also suitable for verifying the presence or absence of complex carbohydrates, for example, in blank samples. Yes, and therefore in certain embodiments, the sample does not contain complex carbohydrates.

[0053] In one embodiment, the sample comprises one carrier protein or carrier peptide. Another embodiment Morphologically, the sample contains two or more carrier proteins or carrier peptides. Therefore, The method according to the present invention uses only the same carrier protein / peptide with different glycosylation patterns. Furthermore, it includes multiple different glycoproteins / glycopeptides, including different carrier proteins / peptides. Suitable for analyzing samples containing [specific components]. Therefore, the term carrier protein / peptide is appropriate. As outlined earlier, it refers to a single carrier protein / peptide, and also refers to the presence of a sample. Multiple carrier proteins / peptides, for example 2 to 10, for example 2 to 5 different carrier proteins It also refers to proteins / peptides.

[0054] Analytical method: The method according to the present invention enables the identification and absolute quantification of the total amount of polysaccharides in a sample. Furthermore, the method of the present invention makes it possible to distinguish between the amount of bound polysaccharides and the amount of free polysaccharides in the sample. It is possible. Therefore, in one embodiment, the present invention comprises at least one complex carbohydrate. This provides a method for identifying and quantifying the content of bound polysaccharides and free polysaccharides in a composition. Furthermore, the method of the present invention involves the polysaccharide components (bound PS and / or free PS) in the sample. It is possible to confirm its existence.

[0055] As discussed earlier, the method of the present invention consists of three steps: (a) calibration, polysaccharide composition of complex carbohydrates This includes (b) measurement, (c) identification and quantification of the compound carbohydrate, preferably in its natural state. These steps are explained in more detail below and further illustrated with examples in the provided diagrams. Let's assume that.

[0056] Step (a): Establish a calibration curve for polysaccharide components using an LC-MS system. To prepare the wire, aliquots of the reference material are provided to the LC-MS system. This refers to a sample having the above-mentioned polysaccharide component with a known specific name and a known absolute amount. The irradiated material was obtained by subjecting a sample containing polysaccharide components to a conventional analytical protocol. obtain.

[0057] In one embodiment of the present invention, step (a) prepares a reference material and the reference material This includes calibrating the LC-MS system using the following. The preparation of the reference material is outlined below. This can be achieved according to steps a1-a3 and optionally a4 and a5. Comparison of reference materials The correct answer can be achieved by following step a6, as outlined below.

[0058] Step (a1): This step involves identifying the specific name of the glycoprotein in the reference material. This includes the following. Suitable analytical methods are known in themselves, for example, Western blot electrolysis. Pneumophoresis or MS is available.

[0059] Step (a2): The following steps involve the polysaccharides (PS) of the glycoprotein in the reference material. This includes identifying the total content. Suitable analytical methods are known in themselves, for example, below There is an IC-PAD after the meeting.

[0060] Step (a3): The following steps involve identifying the free PS content in the reference material. Includes. Suitable analytical methods are known in themselves, for example, IC-PAD after hydrolysis and There is a separation between bound PS and unbound PS.

[0061] Step (a4): The following optional steps depend on the degree of modification of the glycoprotein ( This includes identifying that in certain embodiments the modification is O-acetylation. The analytical method itself is known, for example, removal of modifying groups (e.g., O-acetate by hydrolysis). There is an ion chromatography IC-CD after the release of the chill group.

[0062] Step (a5): The following optional steps involve the glycoprotein in the reference material. This includes determining the purity. Suitable analytical methods are known in themselves, for example, RP-H There is a PLC and / or SEC.

[0063] Steps (a1...a3) and optionally (a4...a5) are followed by glycoproteins A reference material containing a high-quality polysaccharide component is obtained. The aliquot is obtained in the following step (a6). It is prepared for this purpose. Such aliquots of reference material are known concentrations, i.e., known absolutes. Contains a large amount of polysaccharide components.

[0064] Step (a6): This step involves aliquoting the reference material using an LC-MS system. This includes measuring the acquired data (including dwell time, peak identification, and AUC determination). This allows for the establishment of a calibration curve. In this step, the reference material is first LC The eluate is separated via and the ionization voltage (cone voltage) is adjusted for MS detection. In-source fragmentation (ISF) occurs inside the extruder. After aliquots of the reference material are obtained. It is understood that step a6 will be performed. LC-MS system and the para used Such a calibration curve is specific to the meter.

[0065] As outlined earlier, glycoproteins are preferably in their natural state. However, alternatives are available. In one embodiment, before subjecting the sample to the LC-MS step of analysis, peptidase, for example, It is possible to digest glycoproteins using trypsin. Peptidases, for example, Such digestion using liposin leads only to the cleavage of peptide bonds, not glycopeptide bonds. No cleavage occurs. In the case of glycoprotein-containing samples, such digestion is not performed in the LC- This leads to the generation of glycopeptides that undergo the MS step. In that case, before step (a6) is executed. The reference material is digested, and the sample is digested before performing step (b1). The resulting sugar is... Before introducing the samples into the LC-MS system, Petido assumed that in each case the samples were still in their natural state. , that is, (i) enzymatic digestion using an enzyme that removes polysaccharide components from the carrier protein. (ii) It can be considered as not having undergone any chemical reaction, such as derivatization. It is possible. Digestion of glycoproteins using peptidases, such as trypsin, is a process that is carried out in our field. It is known to the person.

[0066] In a preferred embodiment of the present invention, the sample is digested using a peptidase enzyme. stomach.

[0067] Step (b): Measure the sample using the same LC-MS system. Measuring the sample is known in itself, and (b1) provide the sample to the LC-MS system. (b) including (b) measuring the sample with an LC-MS system.

[0068] The calibration in step (a) is performed by measuring a single sample in step (b) or step (b) It will be obvious to those skilled in the art that it can be used for measuring multiple samples in Steps (b1) and (2) can be repeated to measure multiple samples. For example, Two samples from the same serotype were prepared: the first sample (b1), and the first sample was measured (b2). ), then provide a second sample (b1'), and measure the second sample (b2') The same calibration curve is used for measurement. Therefore, measuring a sample is equivalent to measuring a single sample. This includes determining and measuring multiple samples.

[0069] Step (b1): The sample can be provided to the LC-MS system in any known format. Typical In particular, the injection of the sample from the vial into the system using an autosampler is chosen. The material can be obtained directly from the manufacturing batch. The sample is diluted to fit the concentration range of the calibration curve. To obtain. In the case of glycoprotein-containing samples, the samples are preferably in their natural state.

[0070] In an alternative embodiment, such a sample is eliminated using a peptidase such as trypsin. The resulting glycopeptides can be subjected to analysis. As discussed earlier, the generated glycopeptides are Before introducing the sample into the LC-MS system, assuming that each case is still in its natural state, that is, (i ) It has not undergone enzymatic digestion using enzymes that remove polysaccharide components from the carrier protein, (ii) It can be considered as not having undergone any chemical reaction, such as derivatization. Therefore, in the method according to the present invention, before subjecting the sample to the LC-MS step of analysis, glycopeptides are... The bond is not broken.

[0071] In that alternative embodiment, the reference material is also removed before performing step (a6). It will be transformed.

[0072] In a preferred embodiment of the method of the present invention, the sample is not digested even with peptidase. Furthermore, it cannot be digested by any enzyme. Methods that do not use enzymatic digestion involve analysis. It has the advantage of being simple to prepare.

[0073] Step (b2): According to the method of the present invention, the sample is the same as in step (a6) above. It is measured using parameters. Preferably, the sample is the same as the one used to prepare the calibration curve. The same LC-MS system is used for measurement. Preferably, the sample is measured after the calibration curve has been established. Measurements are taken within 8 hours. All these measurements improve data quality and are known to those skilled in the art. Yes, and the sensitivity of the LC-MS system to external factors should be considered.

[0074] In the first embodiment, the measurement of the sample is performed. • Identifying the specific name of the sample, • Identifying the PS content of glycoproteins, This includes the ability to determine the absolute amount of glycan chains.

[0075] In the second embodiment, measuring the sample is • Identifying the specific name of the sample, • Identifying the PS content of glycoproteins, • Identifying the acetylated PS content of glycoproteins, This includes the ability to further determine the degree of acetylation in the sample.

[0076] In a third embodiment, measuring the sample is • Identifying the specific name of the sample, • Identifying the PS content of glycoproteins, • To determine the purity of the sample, Includes.

[0077] In a fourth embodiment, measuring the sample is • Identifying the specific name of the sample, • Identifying the PS content of glycoproteins, • Identifying the acetylated PS content of glycoproteins, • To determine the purity of the sample, Includes.

[0078] In the fifth embodiment, measuring the sample is • Identifying the specific name of the sample, • Identifying the PS content of glycoproteins, • Identify the free PS content, This includes the ability to further specify the amount (relative or absolute) of unconjugated glycans. .

[0079] In the sixth embodiment, measuring the sample is • Identifying the specific name of the sample, • Identifying the PS content of glycoproteins, • Identifying the acetylated PS content of glycoproteins, • Identify the free PS content, This includes further specifying the amount (relative or absolute) of unconjugated glycans, and The degree of combined acetylated PS can also be considered.

[0080] In the seventh embodiment, measuring the sample is • Identifying the specific name of the sample, • Identifying the PS content of glycoproteins, • Identifying the acetylated PS content of glycoproteins, • Identify the free PS content, • Identify the content of free acetylated PS. This includes the following, which allows for further determination of the amount of free acetylated PS.

[0081] In the eighth embodiment, measuring the sample is • Identifying the specific name of the sample, • Identifying the PS content of glycoproteins, • Identify the free PS content of the sample, • To determine the purity of the sample, Includes.

[0082] In the ninth embodiment, measuring the sample is • Identifying the specific name of the sample, • Identifying the PS content of glycoproteins, • Identifying the acetylated PS content of glycoproteins, • Identify the free PS content of the sample, • To determine the purity of the sample, Includes.

[0083] In the tenth embodiment, measuring the sample is • Identifying the specific name of the sample, • Identifying the PS content of glycoproteins, • Identifying the acetylated PS content of glycoproteins, • Identify the free PS content of the sample, • Identify the free acetylated PS content of the sample, • To determine the purity of the sample, Includes.

[0084] Therefore, in embodiments of the present invention, the simultaneous use of a sample and one or more of the following specific names Identification is achieved: PS content of the sample, acetylated PS content, free PS content, free A Cetylated PS content and purity. In particular, simultaneous identification of the sample and the following parameters is required. Typically, the sample preparation step involves releasing the glycan from a carrier molecule, such as a carrier protein. This is not possible in methods that involve the derivatization of glycoproteins (introduction of further impurities): The free PS content, free acetylated PS content, and purity of the sample.

[0085] In other embodiments, the sugars in the sample relating to the embodiments outlined in the first to tenth embodiments The method for analyzing the polysaccharide components of protein is (a) using an LC-MS system to analyze the above polysaccharide components. The step (a) is to establish a calibration curve for minutes, and this step is to prepare reference material. (Steps a1-a3 and optionally a4, a5) and using the above reference material, LC-M This includes calibrating the S system (step a6), and the calibration curve is obtained via LC as shown above. By separating aliquots of the material and adjusting the ionization voltage, the LC eluate is detected by MS. (b) The establishment step, which is established by subjecting the internal fragmentation of the vessel to insource fragmentation, and Using the same LC-MS system and the same parameters as in step (a6), The sample is measured using an MS system, and the results of (c)(a) and (b) are compared to this. This further includes analyzing the polysaccharide components in the above-mentioned sample.

[0086] In particular, further fragmentation using, for example, CID or ETD is not necessary. Therefore, one solid In the application method, the present invention as described herein is obtained after insource fragmentation. This provides a method that does not require additional fragmentation of the network.

[0087] In one embodiment, the present invention is described herein in which no internal standard is added to the sample. This provides a method for generating separate calibration curves to provide reliable results. It turned out that this was sufficient. Omitting the internal standard resulted in the handling step being a source of wisdom. Therefore, the simplification of the method means that there is no need to use internal standards, which is considered a benefit. It will be done.

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

[0089] Step (c): Compare the results of (a) and (b) to determine the polysaccharide components in the sample. Analyzing: Until now, LC-MS systems have been used to compare polysaccharides in complex carbohydrates with calibration curves. It was not used for the absolute determination of related components. Therefore, step (c) is performed in (c1) sample. (c) Identify the characteristic peaks of each PS and (c) the area under the curve of such peaks (A This includes comparing the UC with the calibration curve. The measurement is repeated for multiple samples (e.g., containing the same serotype or the same glycoprotein). In the embodiment described above, step (c) is similarly performed on each sample using the same calibration curve. It is obvious to those skilled in the art that this can be repeated.

[0090] In a second aspect, the present invention relates to a liquid for analyzing the polysaccharide component of complex carbohydrates in a sample. Regarding the use of chromatography-mass spectrometry systems ("LC-MS" systems). In a particular embodiment, the glycoprotein is in its natural state. "Sample", "complex carbohydrate", The above definitions of "polysaccharides," "carrier proteins," and "natural state" are as defined in this second part of the present invention. The same applies to the following characteristics. In particular, the term "complex carbohydrate" means "glycoprotein". Including the meaning of taste, more specifically, it means "a carrier protein to which one or more polysaccharides are attached." This includes. This aspect of the present invention will be described in further detail below.

[0091] Analysis: Here too, the above analysis is necessary for the identification of the polysaccharide components and the decomposition of the polysaccharide components. It is important to note that this includes both quantitative and comparative analysis. The identification of PS components is the PS component described above. This includes confirming the presence and absence of [something]. Therefore, for example, if the sample is from a manufacturing batch... Therefore, if a specific PS component is expected to be present in the sample, the identification of the above PS component is performed. This is to confirm its presence. Similarly, it can be expected that a specific PS component will not be present in the sample. This can also be confirmed in the same way. The absolute quantification of the above PS component can be done, for example, by μg[PS- This includes specifying the concentration measured in units of [component] / mL[sample].

[0092] In embodiments, the present invention relates to in-process control in the production of complex carbohydrates, particularly complex carbohydrates This also relates to the use of LC-MS systems for in-process control in the manufacturing of quality vaccines.

[0093] In embodiments, the present invention provides release control of manufactured complex carbohydrates, particularly complex carbohydrate frames. This also relates to the use of LC-MS systems for release control in the manufacturing of chin. The LC-MS system can be used as a release assay for drug substances or formulations. The above-mentioned drug substance or preparation is one or more complex carbohydrates, in particular one or more complex carbohydrate frameworks Contains chin.

[0094] In embodiments, the present invention relates to the control of the stability of stored complex carbohydrates, particularly stored This also concerns the use of LC-MS systems for stability control of complex carbohydrate vaccines. Therefore, the LC-MS system is used as an assay for the shelf life of drug substances or formulations. The above-mentioned drug substance or preparation contains one or more complex carbohydrates, particularly one or more complex carbohydrates. Includes quality vaccines.

[0095] In embodiments, the present invention relates to the development of complex carbohydrate production, particularly complex carbohydrate vaccine production. This also relates to the use of LC-MS systems for process optimization, as described herein. The use of the C-MS system greatly simplifies the identification of critical parameters, and therefore This allows for faster development and shorter timelines during scaling up.

[0096] LC-MS method: An HPLC system connected to a mass spectrometry detector is generally an LC-MS system. These are called stems. These detectors identify the molecular mass of a specific sample present in the sample. It is possible to do so. Their precision and mass accuracy often allow for the determination of the molecular mass of a molecule. It is called the gold standard for that purpose.

[0097] Known side effects of analyzing complex carbohydrates of glycoprotein sugars using an LC-MS system are... This is the fragmentation of glycans when the substance is injected into the detector. This process is called "in-source fragmentation." It is called ISF and relates to the voltage applied during injection (the so-called "cone voltage"). SF can lead to unknown / unidentifiable ion fragmentation in composite samples, therefore good performance It is not ideal for MS detectors with high performance. Therefore, suppliers recommend low performance during system performance quantification. Test on ISF (<2%)

[0098] In the MS field, ISF is generally known and is derived from the rest of the conjugate by glycan modification (e.g.) For example, the peptide chain can be completely removed from the glycopeptide, thus reducing the glycan occupancy rate. This is undesirable for the stoker. On the other hand, sugar fragmentation is performed, and the reporter ion is simple Further identification and characterization of the sugar composition. However, this fragmentation is usually much higher. It requires energy, and therefore requires different MS techniques (CID, ETD).

[0099] The inventors were surprised to find that the complex carbohydrates subjected to LC-MS analysis showed a difference in the corresponding mass. The effect of ISF in separating glycan repeat units, which are clearly visible as individual peaks (Figure 1A), is evident. We found that after adjusting the cone voltage, this effect was observed when injecting a single complex carbohydrate. Furthermore, this phenomenon can also be observed in more complex mixtures, such as compositions containing multiple complex carbohydrates (Figure 1B). This effect occurs with all polysaccharides from different serotypes, and each serotype-specific polysaccharide is distinct. Because it has a certain mass, it can be used to identify a specific serotype in the sample being analyzed. This can be achieved (through its polysaccharides).

[0100] In one embodiment, the LC-MS system is used to obtain solutions from liquid chromatography. This is an LC-MS system that uses in-source fragmentation of the effluent. Such a system is, for example, For example, Waters Corporation, Sciex, ThermoFisher It is commercially available from Scientific.

[0101] In one embodiment, the LC-MS system further includes a switching valve in front of the MS detector. Such a valve allows only a portion of the eluate to be deflected to the MS detector. Here again, Such systems are commercially available.

[0102] In the embodiment, the glycoprotein is as defined above, and in particular one carrier It comprises a protein and one or more polysaccharides covalently bonded to the protein. In this embodiment, the sample consists of two components, each carrying one or more covalently bonded polysaccharides. It contains the above-mentioned carrier proteins.

[0103] In the embodiment, the carrier protein is as defined above. For example, the carrier protein The protein contains Pseudomonas aeruginosa's detoxified exotoxin A (EPA), and Escherichia coli. (E. coli) Flagellin (FliC), CRM197, maltose-binding protein (MBP), diphtheria toxoid, tetanus toxoid, Staphylococcus aureus (S. aureus) Detoxified hemolysin A, clamping factor A, clamping factor B, Escherichia coli (E. coli) li) Heat-labile toxin, detoxified variant of Escherichia coli (E. coli) heat-labile toxin, cholera toxin Syn B subunit (CTB), cholera toxin, detoxified variant of cholera toxin, E. coli SAT protein, E. coli SAT protein Passenger domain, Streptococcus pneumoniae e) Pneumocyanin, hemocyanin (KLH), Pseudomonas aeruginosa sa) PcrV, outer membrane protein of Neisseria meningitidis White matter (OMPC) and unclassifiable type Haemophilus influenzae (Haemophilus Selected from a group consisting of protein D from influenza. Specific implementation In this state, the carrier protein is Pseudomonas aeruginosa It is the detoxified exotoxin A (EPA) of ).

[0104] In the embodiment, the polysaccharide is as defined above. For example, the polysaccharide is 1~ It contains 100, for example, 5 to 20 repeating units. An example structure is shown in Table 1 above.

[0105] In the embodiment, the sample is a water-soluble matrix; optionally, it does not contain polysaccharides. Carrier protein; optionally bound to a carrier protein (or lipid or peptide) No polysaccharides; optionally contains unrelated proteins.

[0106] The list of references provides additional information in the context of this invention. These references are references. It is used in conjunction with [the source]. (1)thermofisher.com / ch / en / home / industria l / chromatography / chromatography-learning -center / ion-chromatography-information / i on-chromatography-analysis-analyte / analy zing-carbohydrates-hpae-pad-ion-chromato graphy.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] To further illustrate the present invention, the following examples are provided. These examples do not limit the scope of the present invention. It is provided without the intention of being used.

[0108] I. Preparation of Reference Materials Larger quantities of glycoprotein samples are obtained directly from the manufacturing batch, aliquoted, and then processed. Store properly (e.g., -80°C). Differentially adjust the thawed aliquots of the glycoprotein sample. A characterization step is then performed.

[0109] 1) Identify the correct specific name of the reference material. Regarding the specificity of the target glycoprotein to the polysaccharide chain... Using specific antibodies selected and tested, follow the standard Western blot protocol. u. Selectively, the correct specific name of the carrier protein is determined by an antibody specific to the carrier protein. It is identified by use.

[0110] 2) Identify the total polysaccharide content in the reference material. Total acid hydrolysis to monosaccharides is performed, and Then, according to the following instructions, ion chromatography and pulse current measurement detection (IC-PAD) Analysis continues using the following method. Aliquots are taken from the reference material and trifluoric acid is used to a final concentration of 1.8 M. Hydrolyze using (TFA) at 120°C for 2 hours. Optimal hydrolysis conditions (temperature, TF) A (concentration and time) can vary depending on the starting concentration of the polysaccharide. The optimal conditions are absolute. Quantitative release of all monosaccharides from polysaccharide chains without further degradation of the target monosaccharide molecules. You must demonstrate that you will do it.

[0111] After hydrolysis, the sample is cooled to room temperature, and then SpeedVac is typically heated at 30°C. Dry by using it overnight. Completely resuspend the dried sample in H2O (milli-Q grade). Transfer the sample to an HPLC vial. Use a commercially available monosaccharide (e.g., mannose) as a calibration standard. Prepare the set. The monosaccharide that is the target of quantification undergoes modification during the hydrolysis step described above. (For example, when N-acetylglucosamine becomes glucosamine), use the appropriate monosaccharide. Alternatively, the hydrolysis procedure described above may be performed similarly on the set of calibration standards.

[0112] A pulse current measuring device with disposable gold electrodes attached to polytetrafluoroethylene (PTFE) Ion chromatography systems equipped with a constant detector (PAD) (e.g., Dionex) Prepare the ICS-5000. Dionex CarboPac PA1 analytical column ( 4 x 250 mm) and optionally Dionex CarboPac PA1 Guard Color Use a 4x50mm sample. For sample elution, use eluent A and 16mM NaOH. For column cleaning, equilibrate the system using eluent B 500 mM NaOH. To convert it. Then, use the following instrument method / gradient profile to select the sample and calibration standard set. Inject the toxin; -0~24 minutes, 100% eluent A, 1 mL / min flow (elution), -25~32 minutes, 100% eluent B, 1 mL / minimum flow rate (washing), -33 to 60 minutes, 100% eluent A, 1 mL / minimum flow rate (re-equilibration).

[0113] These gradients may need to be optimized depending on the targeted monosaccharide.

[0114] Use the area under the curve from the measured calibration set to obtain a calibration curve and then quantify the unknown sample. Optionally, then use the amount of the targeted monosaccharide to back-calculate the absolute amount of repeating units / polysaccharide in μg / mL in the glycoprotein sample.

[0115] 3) Determine the free polysaccharide content in the reference material. Polysaccharides bound to the carrier protein are removed by a C4 cartridge and then fully acid hydrolyzed 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 cartridge) can be selected for complete retention of the carrier protein. Since the sample preparation step is laborious, it is recommended to use an additional monosaccharide (e.g., galactose) not present in the reference material as an internal standard to compensate for potential sample loss during preparation.

[0116] Take two aliquots of the reference material and add the same amount of internal standard. Equilibrate a C4 cartridge (e.g., Chromafix C4-SPE from Macherey-Nagel) according to the manufacturer's manual: e.g., 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 free polysaccharides: Apply one of the two aliquots to the cartridge and subsequently 2 column volumes of 5% acetonitrile <N ​​​​​​​​​​​​​ Apply it. Measure the volume of the collected flow-through and add an equal volume of 5% acetonitrile to the other aliquot not applied to Cartridge C. Use a SpeedVac to dry both prepared samples typically overnight at 30 °C. Resuspend both dried samples completely in H2O (Milli-Q grade) and then perform total acid hydrolysis to monosaccharides according to the procedure described above: Add trifluoroacetic acid (TFA) to both samples to a final concentration of 1.8 M and hydrolyze at 120 °C for 2 hours (the optimal hydrolysis conditions can vary and should be optimized). After hydrolysis, cool both samples to room temperature and then dry typically overnight at 30 °C using a SpeedVac. Resuspend both dried samples completely in H2O (Milli-Q grade) and transfer to HPLC vials. Prepare an ion chromatography system (e.g., Dionex ICS-5000) equipped with a pulsed amperometric detector (PAD) with a disposable gold electrode installed on polytetrafluoroethylene (PTFE). Use a Dionex CarboPac PA1 analytical column (4 × 250 mm) and optionally a Dionex CarboPac PA1 guard column (4 × 50 mm). For sample elution, use eluent A, 16 mM NaOH, and for column cleaning, use eluent B, 500 mM NaOH to equilibrate the system. Subsequently, inject both prepared samples using the following instrument method / gradient profile:

[0117] - 0 - 24 minutes, 100% eluent A, 1 mL / min flow rate (elution), - 25 - 32 minutes, 100% eluent B, 1 mL / min flow rate (washing), - 33 - 60 minutes, 100% eluent A, 1 mL / min flow rate (re-equilibration).

[0118] Depending on the target monosaccharide, it may be necessary to optimize these gradients.

[0119] Area under the curve from the target monosaccharide (e.g., mannose) and the applied internal standard ( Normalize the measured area using, for example, galactose. Freeze of the analyzed sample. The monosaccharide content allows for a relative comparison of the normalized areas of the two measured aliquots. It is identified by the following. Aliquotes processed with the C4 cartridge are available polysaccharides. Free polysaccharides in the sample compared to an untreated aliquot of the C4 cartridge, representing 100% of the type. This represents the proportion. Using the obtained free polysaccharide value, from the total polysaccharide content identified above, Calculate the amount of "bound polysaccharides".

[0120] 4) Determine the degree of modification of the polysaccharide chain in the reference material. This optional step is weakly a Lucali hydrolysis was used, followed by analysis by ion chromatography and conductivity detection. The identification of the O-acetylated sugar portion follows.

[0121] Take an aliquot of the reference material and perform the desalination step according to the manufacturer's manual. Remove free acetate molecules from the sample (e.g., Zeba Spin column, PD-10 column) (Mu) Add the internal standard to the desalted sample (e.g., 20 μg / mL of propionic acid), and the final Hydrolysis is performed at 37°C for 2 hours using NaOH at a concentration of 10 mM. Optimal hydrolysis conditions ( The NaOH concentration, temperature, and time may vary depending on the starting concentration of the polysaccharide. The conditions are to quantitatively extract all O-acetyl groups from the polysaccharide chain without further degradation of the acetate in the sample. We must indicate that we will release it.

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

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

[0124] Prepare an ion chromatography system equipped with a conductivity detector and suppressor installed ( e.g., Dionex ICS-5000). Use a Dionex IonPac A S11-HC analytical column (4 × 250 mm) and optionally a Dionex IonPa c AS11-HC guard column (4 × 50 mm). For sample elution, use eluent A, 1 mM NaOH, and for column cleaning, use eluent B 100 mM Na OH to equilibrate the system. Subsequently, inject the sample and calibration standard set using the following instrument method / gradient profile: - 0 - 15 minutes, 100% eluent A, 1.5 mL / minimum flow rate, suppressor 4 mA (elution), - 16 - 21 minutes, 100% eluent B, 1.5 mL / minimum flow rate, suppressor 372 mA (cleaning), - 22 - 25 minutes, 100% eluent A, 1.5 mL / minimum flow rate, suppressor 4 mA (re-equilibration). - 22 - 25 minutes, 100% eluent A, 1.5 mL / minimum flow rate, suppressor 4 mA (re-equilibration ).

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

[0126] ​​​ Using an internal standard, the area under the curve is normalized for both the measurement calibration set and the unknown sample. The process is then performed. The resulting calibration curve is then used to quantify the acetate concentration in the unknown sample. The degree of O-acetylated monosaccharides is determined by the amount of acetic acid per specified polysaccharide content (as described above). It can be calculated by specifying the ratio of salt.

[0127] 5) Optionally, determine the relative purity of the reference material. This is done using size exclusion chromatography. Achieved by chromatography and / or reverse-phase chromatography. Appropriate for the target glycoprotein. Select a column (for example, for size exclusion, use Supelco TSKgel G3000S WXL (Cosmosil 5C4-AR-300 in reverse phase), according to the manufacturer's manual. Therefore, the chromatography step is performed. For example, absorption measured at 215 nm The relative absorption of glycoproteins at 215 nm was compared using luminosity. The purity can be determined according to the HPLC software manual for glycoproteins. Determine the relative purity (in percentage).

[0128] II. Analysis using an LC-MS system The glycoprotein samples to be analyzed are obtained directly from the manufacturing batch. The polysaccharide content of the above samples. To identify and quantify the quantity, we use the same glycoprotein species, for example, the same carrier protein. A pre-prepared reference material having the same glycan structure as described above is used.

[0129] The sample to be analyzed is the reference material used in the protocol described below, and its composition and glycan composition are as follows: If identical in structure, it may contain multiple different glycoproteins. Unless otherwise specified, prepare the eluate using MS-grade chemical substances. Note: It is not necessary to derivatize the glycoproteins to be analyzed (e.g., via isotopic labeling). Furthermore, no enzymatic treatment is required, and therefore, lengthy sample preparation before analysis is unnecessary. Proteins can be considered to be analyzed in their natural state, as described above. Furthermore, No internal standard for quantity is added to the sample; instead, the generation of a separate calibration curve is sufficient.

[0130] 1) Optionally, perform buffer exchange between the sample buffer and the calibration reference standard material. This step is desirable to prevent certain matrix components from entering the MS detector. However, buffer exchange also removes free polysaccharide fragments, thereby, This may hinder the accurate determination of the free polysaccharide content in the above sample. Instead, it is recommended to use a switching valve between the LC system and the MS system. The valve is designed to divert undesirable components into water after elution from liquid chromatography. It can be measured in grams. If performed, centrifugal force with an appropriate molecular weight cutoff is required. Using a luta, the buffer was added to eluate A (H2O milli-Q grade, 0.1% v / v formic acid). Replace at least three times. The amount of sample (e.g., 30 μg) depends on the glycoprotein being targeted. It must be preserved and optimized.

[0131] 2) Prepare HPLC vials using the sample to be analyzed and the reference material. Use the optimal concentration. It depends on the LC-MS system used. Different preparations are made to create the calibration curve. By injecting equal volumes at the same concentration, or by injecting different volumes at the same concentration, the reference material Different calibration points can be used. This affects the linearity and fraction of the LC system's injector. Depending on the sample being analyzed (e.g., potential interference due to absorption over time into the vial), a specific sample It must be optimized for that purpose.

[0132] 3) Prepare the LC-MS system. As an example system, use A from Waters. CQUITY-Synapt G2 HDMS uses NaI to provide manufacturer manuals It is prepared by calibrating the MS detector according to the following procedure. , a reversed-phase column suitable for the user's system (e.g., ACQUITY BEH300 C4, Use a 1.7 μm, 2.1 × 150 mm (Waters) sample. Hold the sample appropriately and elute it. To do this, depending on the size of the glycoprotein to be analyzed, different carbon polymer lengths of reversed phase materials are used. You can choose between the following: Elution A (H2O milli-Q grade, 0.1% v / v formic acid) and Elution B (Acetyl acetate). Equilibrate the system with tonitrile (0.1% v / v formic acid).

[0133] 4) Next, inject the calibration points for the sample and reference material. The following instrument method / gradient profile is used. Suitable: - First, 95% eluate A, 5% eluate 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] Depending on the target glycoprotein and the system / column used, these gradients and The injection volume can be adjusted.

[0135] 5) Analyze the eluted proteins using the attached detector. Optionally, Obtain a UV absorption signal at the desired wavelength. If a UV absorption signal is desired, use a larger amount of sample. It may be necessary to inject, and the injection concentration must be optimized. Optionally, split A switch / 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 in conjunction with electrospray ionization in positive resolution mode. Acquire data under optimized conditions. The following parameters are used for TOF Synapt G2 HD. Used with MS spectrometer: -1 second scan time, -3kV capillary voltage, -37V sampling cone voltage, -120℃ source voltage, -450℃ desolvation temperature, -20L / H cone gas flow, -550 L / H solvent removal gas flow, -MS function 500~2000 Da, 4 eV trap present, no transition collision energy. -Optionally: MSMS function at target mass 100-1000 Da, with 4 eV trap. A ramp transition collision energy of 18-25 eV is obtained. - Obtain a spray of Leucine Enkephalin. It locks every 30 seconds.

[0136] The critical parameter is the sampling that causes insource fragmentation of the glycan chain. This is the cone voltage. Depending on the glycoprotein being analyzed, it is necessary to optimize this cone voltage. could be.

[0137] 6) Processing of acquired data. Data processing is handled by the software package provided by the manufacturer. This is done by using a cage (e.g., MassLynx, Waters). Furthermore, the main chromatographic peak representing the eluted glycoprotein (TIC MS function) The spectra are combined. If the LC-MS acquisition parameters are optimized as appropriate, the insole - Fragmentation produces high specific mass peaks derived from the polysaccharide chains of the glycoprotein being analyzed (Figure) (See 1A). Results for samples containing six different EPA complex carbohydrates were similar to those described above (Figure 1). (See B) Processing is possible. Depending on the glycan structure and optimized parameters, many The mass associated with the repeating units of sugars can be observed. Using the specific mass peak, the sugars being analyzed can be analyzed. The specific name of the protein can be identified.

[0138] Secondly, in the next step, we use the extracted chromatogram of the identified specific mass peaks: The area under the curve at the main chromatographic peak (representing eluted glycoproteins) is shown in the reference diagram. Corresponds to the amount of polysaccharides injected and bound to the material. Calibration curve (amount of polysaccharides vs. area under the curve) (See Figure 2) Using this method, the polysaccharide content (in the main chromatographic peak) in the unknown sample was determined. It is possible to identify a quantifiable absolute quantity.

[0139] Optionally, the extracted chromatogram of the identified specific mass peaks is further used to analyze free mass The relative amount of sugars can be determined: all curves below the main chromatographic peak. The sum of the products is compared to the area under the curve of the principal chromatographic peak (representing eluted glycoproteins). Compare. Identifying free polysaccharides in a sample involves different fragments of polysaccharide chains (e.g., monosaccharides, disaccharide fragments). Extracting chromatograms of modified saccharides, polysaccharide-specific oxonium ions, etc. This can be further optimized.

[0140] Selectively, the chromatographic main peak (representing eluted glycoproteins) is analyzed. The degree of modification of polysaccharides is determined using a composite spectrum. In the case of O-acetylated polysaccharide chains, The identified specific mass peak has a mass difference of 42 Da, representing the deacetylated form of the glycan. It may have neighboring peaks (see Figure 3). By comparing the areas under the curves of these two peaks... This allows us to determine the degree of modification.

[0141] Optionally, use the acquired UV chromatogram and / or TIC chromatogram. This allows for the determination of the relative purity of the analyzed sample. The sum of the areas under all curves outside the curve represents the principal chromatographic peak (representing eluted glycoproteins). Compare this with the area under the curve.

[0142] III. Conclusion Polyvalent complex carbohydrate vaccines (currently, for example, each independently covalently binds to the EPA carrier protein) The combined E. coli O-antigen polysaccharides, i.e., 10 drug substances Regarding the development of a decavalent E. coli (ExPEC) vaccine that includes [specific type of vaccine] Prior to the present invention, each release of individual drug substances was typically performed using a PS content assay. Several different assays, including (same as step I.2 above), followed by (i.e., PS-containing) Depending on the results of the quantitative assay, a free PS content assay (same as in step I.3 above) and Depending on the serotype, an assay to identify O-acetylation (same as step I.4 above) is required. Please note that this required (Mr. / Ms.). This series dependence of the two other assays is a quality control issue. This leads to low throughput during quality control (QC) release. In addition, specific name and purity assays. (Similar to steps I.1 and I.5 above) Typically, this also involves the release of each drug substance. It should be done for that purpose.

[0143] One of the advantages of the present invention is that these five different assays are all performed in one step. The analysis procedure can be summarized in Section II above, and sample preparation only requires a dilution step. This reduces the time required for QC testing chemical analysis from approximately two weeks to approximately two days. This allows for a reduction in the time, materials, and operators required to obtain the same related information. Therefore, this method significantly reduces batch release time at a lower cost. To make someone do it.

[0144] Furthermore, the assay can be performed with individual drug substances, but if the individual drug substances are mixed together... It can be performed even after the formulation has become more complex, for example, the final polyvalent compound It can even be applied to carbohydrate vaccine compositions.

[0145] Furthermore, due to the high specificity of the spectrometer, the sample purity is generally much lower during ongoing testing. The quantity can be precisely measured. This reduces the cost and time during process optimization, and therefore... This will reduce the overall development costs of commercially manufactured glycoprotein-based vaccines.

Claims

1. A method for analyzing the polysaccharide components of glycoproteins in a sample, thereby, The above analysis is, Identification of the aforementioned polysaccharide components, - Absolute determination of the polysaccharide component, Includes, The aforementioned method, (a) A step of establishing a calibration curve for the polysaccharide component using an LC-MS system, (b) The step of measuring the sample using the same LC-MS system, (c) A step of comparing the results of (a) and (b), thereby determining the amount of the sample The steps include analyzing and comparing the aforementioned polysaccharide components, A method comprising the glycoprotein being in its natural state.

2. The glycoprotein comprises one carrier protein and one covalently bonded to the carrier protein. The method according to claim 1, comprising one or more polysaccharides.

3. The aforementioned carrier protein is the detoxified exotoxin A (EP) of Pseudomonas aeruginosa. A) Escherichia coli (E. coli) flagellin (FliC), CRM197, maltose knot Composite protein (MBP), diphtheria toxoid, tetanus toxoid, Staphylococcus aureus ( Detoxified hemolysin A, clamping factor A, clamping factor B, colon (S. aureus) Heat-labile toxins of E. coli, detoxified variants of heat-labile toxins of E. coli. cholera toxin B subunit (CTB), cholera toxin, detoxification of cholera toxin Variant, Escherichia coli (E. coli) SAT protein, Escherichia coli (E. coli) SAT The passenger domain of the protein, Streptococcus pneumoniae Pseudomonas aeruginosa (P. ae) pneumocyanin, hemocyanin (KLH), Pseudomonas aeruginosa (P. ae) ruginosa) PcrV, Neisseria meningitidi s) outer membrane proteins (OMPC), and unclassifiable type Haemophilus influenza (Haem Selected from the group consisting of protein D from Ophilus influenza, Preferably, the detoxified exotoxin A of Pseudomonas aeruginosa. The method according to claim 1 or 2.

4. The polysaccharide comprises 1 to 100, preferably 5 to 20, repeating units. The repeating unit includes unmodified monosaccharides and / or modified monosaccharides, according to any one of claims 1 to 3. Methods used.

5. The aforementioned sample is • A water-soluble matrix, wherein the matrix may optionally contain buffers, inorganic materials, etc. A water-soluble matrix containing one or more salts, sugar alcohols, and nonionic surfactants. ks, - Selectively, carrier proteins that do not contain polysaccharides, - Selectively, polysaccharides not bound to carrier proteins ("free PS") - Selectively, unrelated proteins The method according to any one of claims 1 to 4, further comprising:

6. The sample contains a number of different glycoproteins, preferably 2 to 20, for example, 4 to 10 glycoproteins. The protein contains the polysaccharide component and / or the carrier protein. The method according to any one of claims 1 to 5, wherein the method is different.

7. Step (a) is to prepare reference material (steps a1 to a3 and optionally a4, a5) and calibrating the LC-MS system using the reference material (step a6): (a1) Identify the specific name of the glycoprotein, (a2) Identify the total polysaccharide (PS) content of the glycoprotein in the reference material. (a3) Identify the free PS content in the reference material. (a4) Optionally, the degree of modification of the glycoprotein in the reference material, particularly O-acetate To determine the degree of chilling, (a5) By optionally specifying the purity of the glycoprotein in the reference material hand, This allows for obtaining a reference material containing the polysaccharide component of the glycoprotein and determining its purity. What happened afterwards, (a6) Measuring the aliquot of the reference material using an LC-MS system. 、 This allows for the establishment and measurement of the aforementioned calibration curve. The method according to any one of claims 1 to 6, including the method described in any one of claims 1 to 6.

8. - In step (a1), the specific name of the glycoprotein is determined by Western blot. Identified by 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 hydrolyzed and bonded PS is debonded. After separation from the combined PS, it is identified by the IC-PAD and / or - In step (a4), the degree of modification, particularly the degree of O-acetylation, is determined by the modifying group After the release, especially after the release of the O-acetyl group by hydrolysis, ion chromatography Identified by IC-CD and / or - In step (a5), the purity of the glycoprotein is determined by RP-HPLC and / or Identified by the SEC and / or - In step (a6), the calibration curve separates the aliquots via LC. By adjusting the ionization voltage, the eluate from the LC is infused into the MS detector. The method according to claim 7, which is established by subjecting it to fragmentation.

9. Step (b) above is: (b1) To provide a sample, (b2) Using the same LC-MS system and the same parameters as in (a6), the sample Measuring and The method according to claim 7 or 8, including the method described in claim 7 or 8.

10. The measurement (b2) described above involves identifying the specific name of the sample and, as follows: - Identification of the PS content of the glycoprotein, or - Identification of the PS content and acetylated PS content of the glycoprotein, or - Identification of the PS content of the glycoprotein and identification of the sample purity, or - Identification of the PS content and acetylated PS content of the glycoprotein, and identification of the sample purity. , or - Identification of the PS content of the glycoprotein and identification of the free PS content, or - Identification of the PS content and acetylated PS content of the glycoprotein, and the free PS content Identification of, or - Identification of the PS content and acetylated PS content of the glycoprotein, and the free PS content and identification of the free acetylated PS content, - Identification of the PS content of the glycoprotein and identification of the free PS content and purity of the sample. , or - Identification of the PS content and acetylated PS content of the glycoprotein, and release of the sample. Identification of PS content and purity, or - Identification of the PS content and acetylated PS content of the glycoprotein, and release of the sample. Identification of PS content, free acetylated PS content, and purity. The method according to claim 9, comprising one of the following.

11. Step (c) is, (c1) Identifying the characteristic peaks of each PS in the sample, (c2) Comparing the AUC of such peaks with the calibration curve, The method according to any one of claims 1 to 10, including the method described in any one of claims 1 to 10.

12. Liquid chromatography-mass spectrometry for analyzing the polysaccharide components of glycoproteins in a sample. The use of the system ("LC-MS system"), and the analysis described above, - Identifying the aforementioned polysaccharide components, - To absolutely quantify the aforementioned polysaccharide components, Includes, The aforementioned glycoprotein is used in its natural state.

13. The LC-MS system is used to analyze the insoles obtained from the liquid chromatography. The use according to claim 12, which is an LC-MS system that uses fragmentation.

14. The LC-MS system further comprises a switching valve in front of the MS detector, claim 12 or Use as described in 13.

15. The glycoprotein comprises one carrier protein and one covalently bonded to the carrier protein. The use according to any one of claims 12 to 14, comprising one or more polysaccharides.

16. The glycoprotein comprises a carrier protein, and the carrier protein is derived from Pseudomonas aeruginosa (P. a). Detoxified exotoxin A (EPA) of E. coli (E. coli) flagellate (FliC), CRM197, maltose-binding protein (MBP), diphtheria toxin Toxoid, tetanus toxoid, detoxifying hemolysin A for Staphylococcus aureus (S. aureus), Lamping factor A, clamping factor B, Escherichia coli (E. coli) heat-labile toxin, Escherichia coli ( E. coli) Detoxification variant of the thermolabile toxin, cholera toxin B subunit (CTB) ), cholera toxin, cholera toxin detoxification variant, Escherichia coli (E. coli) SA T protein, passenger domain of Escherichia coli (E. coli) SAT protein, lung Streptococcus pneumoniae, pneumocysin, sukashi Shellfish hemocyanin (KLH), Pseudomonas aeruginosa PLCrV, Neisseria meningitidis ( Outer membrane proteins (OMPCs) of Neisseria meningitidis, and classification Is it a non-functional form of Haemophilus influenzae? Selected from the group consisting of protein D, preferably Pseudomonas aeruginosa It is the detoxified exotoxin A of Aeruginosa, and / or The polysaccharide comprises 1 to 100, preferably 5 to 20, repeating units, and the repeating units are unmodified. The use according to any one of claims 12 to 15, comprising monosaccharides and / or modified monosaccharides.

17. The aforementioned sample is • A water-soluble matrix, wherein the matrix may optionally contain buffers, inorganic materials, etc. A water-soluble matrix containing salts, sugar alcohols, and nonionic surfactants, - Selectively, carrier proteins that do not contain polysaccharides, - Selectively, polysaccharides not bound to carrier proteins, and - Selectively, unrelated proteins The use according to any one of claims 12 to 16, further comprising:

18. - In-process control in the production of complex carbohydrates, - Control of the release of produced complex carbohydrates, - Stability control of stored complex carbohydrates, and / or - Process optimization in the production of complex carbohydrates Use according to any one of claims 12 to 17 for the purpose of

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