Method for analyzing glycan structure
|発明の名称 （英語）||Method for analyzing glycan structure|
|発明の概要（英語）||In order to provide an analysis method that is capable of determining a glycan structure with high detection sensitivity, a method of the present invention includes the steps of: carrying out triple quadrupole mass spectrometry at various values of CID energy; creating an energy-resolved profile including yield curves representing relationships between (i) a value of the CID energy and (ii) measured amounts of specific types of product ions; preparing a reference profile, and identifying a glycan structure of a test material by comparing the energy-resolved profile with the reference profile.|
Foreseeing the patent expiry of therapeutic antibodies that are selling more than $30 billion worldwide, there is growing interest over how biosimilar substitutes could win FDA-approval in the near future. The first draft guidance on the evaluation of biosimilarity was published by FDA in February 2012, in which emphasis was placed on the importance of evaluating minor structural differences that can significantly affect the potency and safety of biopharmaceuticals, with specific reference to glycosylation patterns, and that such structural characterization be conducted on multiple lots to understand the lot-to-lot variability.
With regard to therapeutic antibodies, which have a common N-glycosylation consensus sequence at Asn297 in the conserved (Fc) region of heavy chain, some specific features of N-glycosylation have been characterized to affect potency and safety. For example, absence of core fucosylation was demonstrated to enhance antibody-dependent cellular cytotoxicity by 10-fold. Moreover, recent studies revealed that non-human oligosaccharide motifs such as glycolylneuraminic acid (Neu5Gc) and galactose-α-1,3-galactose (α-Gal epitope) are immunogenic and can cause anaphylaxis in patients expressing specific IgE. Further characterization elucidated more specifically that immunogenicity of α-Gal epitope was primarily attributed to an extra N-glycan occurring within the antigen-binding (Fab) region. These findings have raised the issue of antibody glycosylation to the level that global picture of its heterogeneity and biological impact is urgently needed.
Here, glycans of glycoproteins are explained. The glycans of glycoproteins are largely classified into two types of glycans, i.e., (i) N-glycoside-linked glycans (N-glycans) linked to an asparagine residue and (ii) O-glycoside-linked glycans (O-glycans) linked to serine, threonine, or the like. The N-glycans have a common core structure (see the following structural formula) whose terminal linked to asparagine is referred to as a reducing terminal and whose terminal opposite to the reducing terminal is referred to as a nonreducing terminal.
The N-glycans are classified into (i) high-mannose type having more than one mannose linked to the nonreducing terminal of the core structure, (ii) complex type having, at the nonreducing terminal, one or more N-acetylglucosamine (hereinafter referred to as GlcNAc) branches to each of which galactose, sialic acid, fucose, and the like are linked, and (iii) hybrid type having both a high-mannose type branch and a complex type branch. It is well known that, for example, the complex type and the hybrid type can have GlcNAc linked to mannose at a branching point of the core structure (bisecting GlcNAc) and can have fucose linked to GlcNAc at the reducing terminal (core fucose).
Such a structural diversity is observed in a single glycoprotein, and is called, for example, Glycoform Heterogeneity. For example, one paper reports that a glycan structure analysis of human serum immunoglobulin G having a single N-glycan binding site revealed that the human serum immunoglobulin G had 34 types of glycan structures (Non Patent Literature 1).
As described above, recent studies revealed that differences in glycan structure significantly affect functions of glycoproteins (see, for example, Non Patent Literature 2). Accordingly, there are demands for a method for a quantitative analysis of glycan structures having diversity and highly efficient profiling as to types and proportions of the glycan structures.
One example of the method for analyzing a glycan structure is a method of (i) chemically or enzymatically isolating an N-glycan from a glycoprotein, (ii) chemically modifying (labeling) and purifying the N-glycan, and then (iii) detecting the N-glycan by a combination of HPLC and mass spectrometry such as MALDI-TOF MS. This method has advantages such as (i) being capable of easily separating labeled glycans according to structure by reversed-phase or normal-phase HPLC and (ii) being capable of removing impurities through the purification and thereby allowing highly sensitive measurement. On the other hand, this method has disadvantages such as (i) requiring complicated pretreatment and (ii) being incapable of obtaining information of each glycosylation site in a case where the glycoprotein has more than one glycosylation sites.
Another example is a method of (i) breaking a glycoprotein into glycopeptides, which are peptides to which a glycan is linked, by an enzyme such as trypsin and then (ii) measuring the glycopeptides thus obtained (Patent Literature 1). This measurement is carried out mostly by use of a mass spectrometer using nano HPLC-ESI as an ion source. This mass spectrometer makes it possible to not only accumulate glycopeptide-derived signals and quantify glycopeptide but also determine a glycosylation site and estimate a glycan structure through MSn measurement. Another paper reports a method of measuring a fragment ion specific to each glycopeptide with good quantitativity with the use of a triple quadrupole mass spectrometer by a multiple reaction monitoring (MRM) method (Non Patent Literature 3).
1. A method for analyzing a glycan structure of a test material having a glycan, comprising the steps of:
(a) measuring specific types of product ions produced from the test material at various values of CID energy by MS/MS;
(b) creating an energy-resolved profile including yield curves representing relationships between (i) the values of the CID energy and (ii) measured amounts of the respective specific types of product ions;
(c) preparing a reference profile including yield curves representing relationships between (i) the values of the CID energy and (ii) measured amounts of respective same types of product ions produced from a reference test material as the specific types of product ions, the reference test material being a test material having a glycan and whose structure is known; and
(d) identifying the glycan structure of the test material by comparing the energy-resolved profile obtained in the step (b) with the reference profile,
the specific types of product ions including at least two types of product ions derived from a protonated monosaccharide or disaccharide, and
in the step (a), the measurement by MS/MS being carried out by use of a mass spectrometer which causes no Low-mass cutoff.
2. The method according to claim 1, wherein the specific types of product ions include at least two types of product ions selected from the group consisting of product ions having m/z of 163, 168, 186, 204, 274, 290, 292, 308, 366, 454, and 470, respectively.
3. The method according to claim 2, wherein the specific types of product ions include product ions having m/z of 163, 204, 274, and 366, respectively.
4. The method according to claim 1, wherein:
the specific types of product ions further include a product ion having m/z of 138; and
in the step (b), the energy-resolved profile is created by normalizing the yield curves with use of a measured amount of the product ion having m/z of 138.
5. The method according to claim 1, wherein:
in the step (a), the measuring is performed by using a sample in which a standard material which has a glycan and whose concentration is known is added in addition to the test material;
the specific types of product ions include a product ion having m/z of 138; and
the method further comprises the step of (e) quantifying the test material by comparing (i) a measured amount of the product ion having m/z of 138 produced from the test material and (ii) a measured amount of the product ion having m/z of 138 produced from the standard material.
6. The method according to claim 5, wherein in the step (e), the test material is quantified on basis of (i) the measured amount of the product ion having m/z of 138 produced from the test material and (ii) the measured amount of the product ion having m/z of 138 produced from the standard material, each of which measured amounts are obtained at a value of the CID energy at which the product ion having m/z of 138 becomes maximum.
7. The method according to claim 6, wherein the value of the CID energy at which the product ion having m/z of 138 becomes maximum is a value estimated based on a calibration curve and a value of m/z of a precursor ion of the test material to be analyzed, the calibration curve being created by (I) measuring, at various values of the CID energy, in advance the product ion having m/z of 138 in a plurality of test materials each having a glycan by MS/MS and then (II) carrying out linear regression analysis with use of (i) values of the CID energy at which measured amounts of the product ion having m/z of 138 in the plurality of test materials become maximum and (ii) values of m/z of precursor ions of the plurality of test materials.
8. The method according to claim 1, wherein in the step (a), the measurement by MS/MS is carried out by use of a triple quadrupole mass spectrometer.
9. The method according to claim 1, wherein the test material is a glycopeptide.
10. The method according to claim 9, wherein the glycopeptide is a glycopeptide having an N-glycan.
11. The method according to claim 9, wherein the glycopeptide is a glycopeptide having an O-glycan.
|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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