Method for analyzing glycan structure
|発明の名称 （英語）||Method for analyzing glycan structure|
In order to provide an analysis method capable of determining glycan structures with an excellent detection sensitivity, the method of the present invention comprises a process of conducting three-step quadrupole mass spectrometric analysis while changing CID energy values; a process of drawing an energy decomposition profile including a yield curve showing the relation between the CID energy and the measured amount at each specific product ion; a process of preparing a reference profile; and a process of analyzing the glycan structure of a sample by comparing the obtained energy decomposition profile and the reference profile.
COPYRIGHT KIPO 2014
[Reference numerals] (AA) Fab glycosylation (potential); (BB) Fc glycosylation; (CC) Peptide / Saccharide peptide mixture; (DD) Signal intensity; (EE) Energy decomposition yield curve; (FF) Electrode potential; (GG) Oxonium ion; (HH) Framgment; (II) Detection; (Q1) Complete mass filtering; (Q2) Collision-induced organic dissociation; (Q3) Fragment ion filtering
300 Billion dollars in the world can be done through the sale of patents in the expiration of the duration of the therapeutic antibody in preparation for, if the surrogate is how their biological similar can be applied in the FDA interest to determine whether it have been raised. In this regard, for the evaluation of the guidance of the biological analogue of said initial draft is, February 2012 in the United States food and drug administration (FDA) was issued from. Among them which has been conventionally is highlighted, effectiveness and safety of biopharmaceuticals are strongly influenced by a slight structure which may have effects (particularly of a sugar addition pattern) for evaluating a difference of the importance, and, in order to understand the deviation between lots, using a plurality of lots to structural analysis by the conclusion that should be performed.
With regard to therapeutic antibodies, in the conserved regions of the heavy chain (Fc) Asn297 N- consisting in a common sequence by the presence of an glycosylation, glycosylation N- certain structures are in terms of effectiveness and safety of the therapeutic antibody exerts an influence on a known to be zero. For example, the core structure of the fucose addition in the case that there is no, antibody dependent cellular cytotoxicity appeared 10 times is becomes greater. In addition, according to recent studies, glycolilyluramine acid (Neu5Gc) - α 1,3- galactose and galactose (α field Gal epitope) of the like nonhuman oligosaccharide motif are immunogenic, and a cell expressing a particular IgE in Phil laxi anaphylatoxin to a patient causes it became clear that the invention may be practiced. Even more advanced according to a study, α field Gal the immunogenicity of the epitope, mainly, an antigen binding region (Fab) which occurs by extra N- glycans it became clear that is as defined above. Is made in view of such facts, in the antibody sugar adding heterogeneity in their biological and influence one may need to be extensively illuminated.
Here, for explanation of glycoprotein sugar chain, the sugar chains of glycoproteins, sugar chain asparagine residues N- glycosidic bond is attached is attached to the sugar chain (N type sugar chain), and serine and threonine and the like O- glycosidic bond that binds to the sugar chain (O type sugar chain) of roughly divided into 2 species. N type sugar chain is represented by the following structural formula showing a common of and with the core structure, which binds to the reducing end terminus is asparagine, referred to as a surface on the opposite end is non reducing end and others.
N type sugar chain include, at a nonreducing terminal mannose of the core structure that binds the plurality of high-mannoos type, the nonreducing end N- acetylglucosamine (or less, hereinafter referred to as GlcNAc) 1 has a branch of the one or a plurality, each branch also galactose, sialic acid and fucose and the like having a structure in which the binding of the complex type, and high-mannoos type and complex type having the soft magnetic thin film has a hybrid of both of the bootie. For two or more complex type and hybrid to your brother, a diverging point of the core structure which can be on mannoose GlcNAc is bonded to the (biphenyl GlcNAc sector), and GlcNAc fucose on the reducing end capable of binding to the (core fucose) and the like are well known.
Diversity is such a structure, even a single glycoprotein have been observed to, Glycoform Heterogeneity called the like. By way of example, N type sugar chain binding sites having 1 points in the sugar chain structure of human serum immunoglobulin G from the interpretation of, the sugar chain structure is of the order of 34 kind to be present in the sample has been reported (non Patent literature 1).
The structural difference between the glycoprotein sugar chain in the functionality thereof to exert a large influence on, as described above has been found in recent years (for example, non Patent document 2), which has a variety of quantitatively analyzed by the sugar chain structure, which a certain structure is included any ratio of high efficiency with a method in which the profile has been demanded.
As a method for analyzing the structure of the sugar chain, glycoprotein chemical or enzymatically N from type to release the sugar chain, or a chemically modified (labeled) was carried out to atmosphere after the purification, and the like HPLC MALDI - TOF MS of mass spectrometry method that embodies a method of detecting the combination to be used therein. The method includes, after reverse phase or normal phase HPLC label linked sugar chains are separated from each other by the structure by since it is easier for, by purification of the removed contaminants can be measured with high sensitivity or the like which is an advantage of the other hand, processing becomes complicated when the prior point, glycoprotein sugar chain modification site in the case where there is a plurality of, information for each sugar chain modification site may not be obtained disadvantage of the like.
On the other hand, glycoprotein of fragmentation by an enzyme such as trypsin and, fragmented peptide measured in a state that a glycopeptide linked sugar chain is linked to the layer in such a method of providing the surface (patent document 1). At this time utilized for the measurement is principally a nano ion with mass won HPLC field ESI and analysis, by integrating the signal derived from glycopeptides in addition to quantify, MSn as determined by the determination of an estimate of the sugar chain structure of a sugar chain modification site and are possible as well. In addition, 3 step 4 by using 중극형의 mass spectrometric system, every glycopeptides specific cleavage ion multiplexes (MRM) and quantified by monitoring the user's reaction with good method to measure the Fig. agents have been reported (non Patent literature 3).
1. In a sample having a sugar chain in a method for analyzing a sugar chain structure, while varying the value of MS/MS CID energy measurement as the step of carrying out, wherein the sample ions of a particular product, CID at each value of the energy measuring step, in each of the specific product ion CID showing the relationship between the energy quantity curve measured quantity and the energy profile generating and producing step of decomposition, having a structure in a sample of a known reference sugar chain, wherein the particular product ion of the same species as the measured amounts of product ions and, CID energy including a curve showing the relationship between the quantity of preparing a reference profile preparation step, preparing the obtained by the processes of the energy degradation profiles by comparing the reference profile, for identifying the sugar chain structure of the sample comprising the step of identifying, the specific product ion is, monosaccharide or disaccharide is protonated derived from 2 and at least one of product ions, wherein the measuring step, a low-mass cutoff (Low field mass cutoff) is not formed by using a mass analysis apparatus MS/MS measurement was performed, wherein the sample is, 당펩티드인 characterized in that, a method for analyzing a sugar chain structure.
2. Method according to claim 1, wherein the particular product ion is, m/z and 470 are each 163, 168, 186, 204, 274, 290, 292, 308, 366, 454 is selected from the group consisting of product ion species of at least 2 of said product ions which, a method for analyzing a sugar chain structure.
3. Method according to claim 2, wherein the particular product ion is, m/z is 163, 204, 274 and 366 are each product ions characterized in that it comprises, a method for analyzing a sugar chain structure.
4. Method according to any one of claims 1-3, wherein the particular product ions, phosphorus ions is 138 m/z further comprising a product, in the generation step, wherein m/z is 138 by using the measured amounts of each product ion by standardizing quantity curve, wherein the energy to create a dissolution profile of the, a method for analyzing a sugar chain structure.
5. Method according to any one of claims 1-3, the sample means, having a concentration of a sugar chain is known and the standard sample is added, the specific product ions, phosphorus ions is 138 m/z further comprising a product, wherein the ion in the sample 138 and m/z is the measured value of the product, wherein the m/z is in the standard sample 138 by comparison of the measured amounts of product ions, wherein the sample further comprising the step of quantitative quantify characterized in that, a method for analyzing a sugar chain structure.
6. Method according to claim 5, wherein the quantifying step, wherein m/z 138 is a maximum of the product ions in the energy CID quantity, each sample and the standard sample, m/z 138 is based on the product ion having a measured quantity, the sample characterized in that the quantification of the perform, a method for analyzing a sugar chain structure.
7. Method according to claim 6, wherein the value of the maximum energy CID, having a plurality of sugar chain with respect to the samples, m/z 138 is the value of the product ions having energy while changing CID MS/MS is measured in advance by the measurement, the maximum value of the measured quantity and CID energy, of a value of m/z of the sample using a precursor ion from the linear regression analysis from a calibration curve created by, of the sample of the precursor ions of m/z object is to be interpreted based on the value of a value estimated by the, a method for analyzing a sugar chain structure.
8. Method according to any one of claims 1-3, wherein the measurement step, by using a quadrupole mass spectrometer 3 step 4 MS/MS characterized by a measurement, a method for analyzing a sugar chain structure.
9. AMEND STATUS: Delete
10. Method according to any one of claims 1-3, are the gram, N 당펩티드인 having the sugar chains which, a method for analyzing a sugar chain structure.
11. Method according to any one of claims 1-3, are the gram, O 당펩티드인 having the sugar chains in which, a method for analyzing a sugar chain structure.
|発明の概要||抗体医薬品を含むタンパク質医薬品の薬効と安全性を左右する糖鎖構造プロファイルを、10分間で精密定量化する質量分析システム、Energy-Resolved Oxonium Ion Monitoring (Erexim) 法を開発しました。|
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