TW201312115A - Improved glycosylation assay, glycoanalysis array and an assay system - Google Patents

Abstract

The present invention is to provide an improved glycosylation assay, glycoanalysis array and an assay system for performing glycosylation assays glycoanalysis array. The glycoanalysis array (102) comprise: a planar substrate (1); and a plurality of contact portions (10) present on a surface of said planar substrate (1) at a plurality of predetermined locations, a plurality of saccharide binding agents are provided in the plurality of contact portions (10), the plurality of saccharide binding agents act saccharide and glycan detectors and binding agents in predetermined locations on said planar substrate (1), each of said plurality of saccharide binding agents is present at a plurality of separate predetermined locations (10) on said surface, wherein said plurality of separate predetermined locations (10) are provided with a plurality of concentrations of said saccharide binding agent in a concentration curve; said planar substrate (1) is provided to contact with a sample comprising a glycoprotein, such that said glycoprotein binds specifically to at least one saccharide binding agent at the plurality of contact portions (10) and forms a detectable binding complex, such that a baseline for non-specific binding is determined according to said concentration curve.

Description

Improved glycosylation assay, sugar analysis array and assay system Field of invention

This application relates to an improved glycosylation assay (analysis).

Background of the invention

Oligosaccharides and polysaccharides are polymers composed of monosaccharide (sugar) units interconnected by glycosidic bonds. These polymers have a structure which can be described according to the linear sequence of monosaccharide subunits, which is called a two-dimensional structure of a polysaccharide. Polysaccharides can also be described in terms of structures formed in their three dimensions by their constituent monosaccharide subunits.

Like a strand of DNA or protein, a sugar chain has two different ends. In the case of sugar chains, these are reducing ends (equivalent to the aldehyde groups of linear sugar molecules) and non-reducing ends. However, unlike proteins and DNA, polysaccharides are generally branched, with substantially each saccharide unit in the polysaccharide being an optional branching point.

There are many proteins that bind to sugars. Many of these proteins specifically bind to specific short monosaccharide sequences or disaccharide sequences. Lectins are a large family of proteins that bind to sugars. Many plant lectins have been characterized and used in research. Many mammalian lectins are also characterized. An antibody is a protein that specifically recognizes a specific molecular structure. Like lectins, antibodies also recognize sugar structures. Glycosidases are enzymes that are capable of cleaving glycosidic bonds in sugar chains. Glycosidases also specifically recognize specific oligosaccharide sequences. Glycosyltransferases are enzymes that transfer sugar units to receptor molecules. In vivo, these receptor molecules are a growing glycan structure.

The structural determination of polysaccharides is very important for the development of glycobiology. Sugar creature Research involves subjects including bacterial cell walls, hemoglobin, and growth associated with viral diseases such as HIV infection, autoimmune diseases such as insulin-dependent diabetes mellitus and rheumatoid arthritis, and abnormal cell growth such as occurs in cancer. Factor and cell surface receptor structure.

The discovery of penicillin emphasizes the importance of sugar molecules, which are inhibitors of bacterial cell wall sugar molecule synthesis and may be the most successful antibiotics ever discovered.

Another example is the medical application of heparin, a mucopolysaccharide that inhibits blood coagulation and is currently widely used in medicine. Additional examples of medically important sugar molecules include: mucopolysaccharide (GAG), heparin sulfate, monoclonal antibodies, cytokines (eg, IL-8, TNF, and most successful EPO), chemokines (eg, Acidic fibroblast growth factor) and different growth factors. The above cytokines, chemokines and growth factors are also capable of binding to GAG and other polysaccharides and thus can also be considered as lectins.

The structural complexity of polysaccharides hinders their analysis. For example, sugars are believed to be synthesized by a non-template-dependent mechanism. In the absence of structural information, the researcher must therefore assume that the building block is selected from any sugar unit currently known. Furthermore, these units may be modified during the synthesis, for example by the addition of sulfate groups. Without the ability to measure this carbohydrate structure information, researchers were unable to determine the true, correct glycosylation pattern in cell populations such as tissues. Furthermore, these units may be modified during the synthesis, for example by the addition of sulfate groups, so that only knowing which types of sugars are added does not provide a full picture.

In addition, the connections between the sugar units are diverse. If the connected sugar list The element is a hexose, and the sugar may be attached to any of the C1, C2, C3, C4 or C6 atoms. Moreover, the linkage to the C1 atom may be either an alpha configuration or a beta configuration. In addition, the structural differences between many sugars are minor because the sugar unit may differ from the other (epimer) only by the position of the hydroxyl group.

In vivo, glycosylation is tissue dependent and can vary significantly with cell state. In vitro, glycosylation strongly depends on growth conditions: cell type, nutrient concentration, pH, cell density and age can affect the glycosylation pattern of glycoproteins. The amount of glycoforms and their relative abundance within the cell are influenced by the inherent structural properties of the individual proteins and the overall properties of the available glycosylation enzymes, including their type, concentration, kinetic characteristics, compartmentalization. This property has been shown to change upon changes in cellular state (eg, oncogenic transformation).

Summary of invention

The application provides an improved glycosylation assay. This improved assay can provide more accurate results. Examples of such improvements include, but are not limited to, assay optimization improvements for assay systems; K-fingerprint array formats involving array improvements; QC monitoring and correction associated with improvements in detection systems; and out of assay flags for arrays and systems Implementation.

According to at least some embodiments, a sugar analysis array is provided comprising a planar substrate and a plurality of sugar binding agents present at a plurality of predetermined locations on a surface of the substrate, each of the plurality of sugar binding agents being present on the surface a plurality of independent predetermined locations, wherein the plurality of independent predetermined locations relate to a plurality of concentrations of the sugar binder in the concentration curve at the location; The planar substrate is adapted to contact a sample comprising a glycoprotein such that the glycoprotein specifically binds to at least one sugar binding agent and forms a detectable binding complex to determine a baseline for non-specific binding based on the concentration profile.

According to at least some embodiments, a sugar analysis array is provided, comprising: a planar substrate; and a plurality of contacts, the plurality of contacts being present at a plurality of predetermined locations on a surface of the planar substrate, Providing a plurality of sugar binders in the plurality of contacts, the plurality of sugar binders being disposed in predetermined positions of the planar substrate; and each of the plurality of sugar binders present on the surface Separate predetermined locations, wherein the plurality of independent predetermined locations are provided with a plurality of concentrations of the sugar binder in a concentration curve; the planar substrate being configured to contact a sample comprising a glycoprotein such that The glycoprotein specifically binds to the at least one sugar binding agent at the plurality of contacts and forms a detectable binding complex to determine a baseline for non-specific binding based on the concentration profile.

Optionally, the contact portion is selected from the group consisting of a circular, elliptical, square groove, recess or hole. Alternatively, the detectable binding complex can be detected by a binding signal, and wherein in the concentration curve, the binding signal is linear in at least five of the total concentration of sugar binder. Optionally, the planar substrate has a recess or a hole. Optionally, the planar substrate comprises at least one of a film, glass or plastic. Optionally, the planar substrate is derivatized.

Optionally, the array further includes predetermined locations of the plurality of indicia on the planar substrate to provide a high signal to support image analysis of the array after contact with the sample. Optionally, the array further comprises A plurality of sugar binders serve as calibration standards in predetermined positions of the planar substrate. Optionally, the planar substrate is divided into a plurality of pads, and each of the pads includes a plurality of independent calibration standards.

Alternatively, the sugar binding agent comprises a lectin or antibody, or a modified component or combination thereof.

Optionally, the concentration of each lectin on the substrate ranges from 0.01 mg/ml to 10 mg/ml.

Optionally, the concentration ranges from 0.001 mg/ml to 10 mg/ml.

Alternatively, the concentration ranges from 0.01 mg/ml to 5 mg/ml.

Optionally, each location has a spot size diameter, and wherein the spot size diameter is in the range of 0.05 mm to 75 mm.

Optionally, each location has a spot size diameter, and wherein the spot size diameter is in the range of 80 microns to 2500 microns.

Optionally, the glycoprotein comprises an IgG antibody or fragment.

Alternatively, the amount of IgG per position is in the range of 1.8-12 μg.

Alternatively, in a solution for contacting the IgG antibody or fragment with the sugar binding agent, the IgG concentration is in the range of 0.001 micromolar to 100 micromolar.

Optionally, the solution includes a detergent to unfold the IgG antibody or fragment and expose at least one glycan.

According to at least some embodiments of the present application, there is provided an assay system for performing a glycosylation assay using a plurality of arrays as described herein, comprising a kit for performing the glycosylation assay, by detecting a signal by binding a signal a detectable binding complex to obtain a binding data detector, and an assay a module for comparing reference data and the binding data obtained from the sample glycoprotein to measure the optimal lectin activity, measuring a lectin that only produces a signal with the sample glycoprotein, and by using a large number of samples based on The experimentally determined database of the type creation calculates the background value of the slide to compare the calculated baseline to determine the appropriate assay conditions.

According to at least some embodiments of the present application, there is provided an assay system for performing a glycosylation assay, comprising: - a plurality of arrays, - a measurement execution module, the plurality of arrays being coupled to the assay execution a module, or housed in the assay execution module; a detector coupled to the assay execution module, the detector detecting the binding of the sugar binder to the sample protein; - the assay data analyzer, Communicating with or connected to the detector; - measuring an external marker module; connecting to or communicating with the assay data analyzer; and - determining an optimization module, connected to the Measuring or communicating with the assay data analyzer; wherein each of the plurality of arrays comprises: - a planar substrate; and - a plurality of contacts, the plurality of contacts being present on the planar substrate At a plurality of predetermined positions on the surface, a plurality of sugar binders are disposed in the plurality of contacts, the plurality of sugar binders being disposed in predetermined positions of the planar substrate; Each of the sugar binding agents is present in the table a plurality of independent predetermined locations on the face, wherein the plurality of independent predetermined locations are provided with a plurality of concentrations of the sugar binder in a concentration curve; the planar substrate is configured to contact a sample comprising glycoprotein The glycoprotein is specifically bound to the at least one sugar binding agent at the plurality of contacts and forms a detectable binding complex to determine a baseline for non-specific binding based on the concentration profile.

Optionally, the contact portion is selected from the group consisting of a circular, elliptical, square groove, recess or hole.

Optionally, the array is inserted or provided to the assay execution module, optionally for assay optimization only once.

Optionally, the detector is combined with or separate from the assay execution module.

Optionally, the detector is combined with or separate from the assay data analyzer.

Optionally, the assay optimization module comprises a database comprising data relating to binding of the protein of interest under different experimental conditions.

Optionally, the assay optimization module receives an archive of optimized experimental results containing other specific types of proteins.

Optionally, the assay optimization module further determines suitable assay conditions according to one or more of the following parameters: a binding time for incubating the sample protein and the sugar binder, a temperature, and a pH of the buffer; sugar binding Agent signal, signal ratio, a set of specified sugar binders that react when exposure is not optimal, background values, and background values compared to the calibration standard background; The concentration of detergent in the rinse; and the protein concentration of the sample.

Optionally, the assay optimization module optimizes sample glycoprotein concentration, substrate format, and assay format.

Optionally, the sample glycoprotein comprises an IgG antibody, and wherein the assay optimization module determines whether the IgG sample antibody has Fab glycosylation or O-linked glycosylation in order to perform specific optimization conditions, and to issue such A warning about the presence of glycosylation.

Optionally, the assay optimization module determines an amount of exposure solution applied to the sample glycoprotein, wherein the exposure solution has a known percentage of detergent in the art for unfolding proteins selected from the group consisting of Acid salts, deoxycholate, C16TAB, LysoPC, CHAPS, amphoteric surfactants (both ionic detergents, Zwittergent), octyl glucoside, digitonin, rubour, C12E8, Triton X- A group consisting of 100 (Triton X-100), Noron P-40 SDS (sodium lauryl sulfate), and Tween-80.

Optionally, the assay optimization module determines a condition matrix involving one or more of the following: optimizing exposure solution concentration: 0.001 to 1%, optimizing temperature: 50-80 ° C, optimization of pretreatment incubation Time: 1 minute to 1 hour.

Optionally, the system further comprises at least one QC (Quality Control) monitoring selected from speckle evaluation by foreground uniformity, background uniformity, similarity between average median densities, saturation levels; Array verification of the quality of the entire planar substrate in comparison to spot, intra-array reproducibility; normalized evaluation between sample and corrected position; determination of the entire array signal.

Optionally, the system further comprises applying to the plurality of calibration samples A calibration protein comprising a predetermined position of the sugar binder is used to correct the reactivity of the sugar binder by the assay optimization module according to the gold fingerprint standard.

Optionally, the assay optimization module may determine an off-label according to a comparison of a binding signal of the sample glycoprotein to the calibration sample.

Optionally, the measurement optimization module issues a warning according to the measurement external flag.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference. These include, but are not limited to, WO 00/68688 and WO 01/84147 (US20060194269, US20070092915, US7056678 and US7132251), WO 02/37106 (US20040132131), and WO 02/44714 (US7079955 and US20040153252). In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Simple illustration

The present application is described herein by way of example only with reference to the accompanying drawings. In the present invention, the detailed description of the details of the present invention is to be considered as being Proposed in the description of the principles and conceptual aspects of the application. in In this regard, the details of the structure of the present application are not intended to be more detailed than the details necessary for the basic understanding of the present application, and the description of the accompanying drawings makes it possible to make several forms of the present application embodied in practice to those skilled in the art. Obvious.

In the drawings: FIG. 1 illustrates an exemplary array in accordance with at least some embodiments of the present application; FIG. 2 illustrates an exemplary system in accordance with at least some embodiments of the present application; FIG. 3 relates to Exemplary print concentrations of exemplary lectins of at least some embodiments of the application; FIG. 4 shows an exemplary QC and correction process for system operation of FIG. 2, and FIG. 5 shows test optimization experiment layout , sample and array types combined with different test sample exposure conditions, the experiment is to use an exemplary IgG antibody, Avastin, through the above system for an array type (a padded slide or multiple liners) Slides) for testing.

Detailed description of the preferred embodiment

This application is an improved glycosylation assay. In accordance with a preferred embodiment of the present application, such an assay may be optionally and preferably performed in accordance with U.S. Patent No. 7,056,678, the disclosure of which is incorporated herein by reference in its entirety in its entirety herein in its entirety in its entirety. For example, the patent describes a method for structural analysis of sugars comprising: setting a plurality of necessary sequences on a surface a specific and/or site-specific binding agent that brings the surface into contact with a mixture of sugars to be analyzed, such as an extract of sugar molecules from a particular compartment of a cell or tissue; washing or otherwise removing unbound sugar or a sugar fragment; a previously obtained essential sequence-specific and/or site-specific marker, or a mixture of essential sequence-specific and/or site-specific markers, is added to the surface; a marker that binds to the surface is obtained One or more images; and derived (derived) information related to the identification of sugars by image analysis.

In one aspect, the present application provides a sugar analysis array 102, comprising: a planar substrate 1; and a plurality of contacts 10 present on the surface of the planar substrate 1 At a predetermined position, a plurality of sugar binding agents are disposed in the plurality of contact portions 10 as detectors of glycan structures on the analysis sample in a predetermined position of the planar substrate 1; Each of the plurality of sugar binders is present at a plurality of independent predetermined locations 10 on the surface, wherein the plurality of independent predetermined locations 10 are provided with a plurality of concentrations of the sugar binder in a concentration curve The planar substrate 1 is disposed in contact with a sample comprising a glycoprotein such that the glycoprotein specifically binds to at least one sugar binding agent at the plurality of contacts 10 and forms a detectable binding complex such that A baseline for non-specific binding is determined based on the concentration curve.

In another aspect, an assay system 100 for performing a glycosylation assay is provided, comprising: - a plurality of arrays 102, each of the plurality of arrays 102 comprising: a planar substrate 1; Contact portions 10, the plurality of contact portions 10 are present at a plurality of predetermined positions on the surface of the planar substrate 1, at the plurality of contacts a plurality of sugar binders disposed in a predetermined position of the planar substrate 1 in the portion 10; each of the plurality of sugar binders having a plurality of independent reservations on the surface Position 10, wherein the plurality of independent predetermined positions 10 are provided with a plurality of concentrations of the sugar binder in a concentration curve; the planar substrate 1 is disposed in contact with a sample comprising a glycoprotein such that the sugar Protein specifically binds to at least one sugar binding agent at the plurality of contacts 10 and forms a detectable binding complex to determine a baseline for non-specific binding based on the concentration profile; - assay execution module 104 The plurality of arrays 102 are connected to the measurement execution module 104 or housed in the measurement execution module 104. The detector 105 is connected to the measurement execution module 104 through the detector 105. Detecting binding of the sugar binding agent to the sample protein; - measuring the data analyzer 106, communicating with or connecting to the detector 105; - measuring the external marker module 107; connecting to the measurement data analysis 106 or communications with the measurement data analyzer 106; and - measurement optimization module 108, connected to the measurement analyzer 106 or the communication data with the measurement data analyzer 106.

Optionally, the contact portion 10 is selected from the group consisting of a circular, elliptical, square groove, recess or hole.

Optionally, the array 102 is inserted or provided to the assay execution module 104.

Optionally, the detector 105 is combined with the measurement execution module 104 or separate from the measurement execution module 104.

Optionally, the detector 105 is combined with or separate from the assay data analyzer 106.

Optionally, the assay optimization module 108 includes a database containing data relating to binding of the protein of interest under different experimental conditions.

Optionally, the assay optimization module 108 receives an archive of optimized experimental results containing other specific types of proteins.

The surface on which the bonding agent is disposed may include, for example, a bead or an array, or a perforated plate (e.g., a 96-well plate), but preferably includes a planar substrate. The array on the planar substrate (preferably having depressions or holes) may optionally comprise any of a film, a glass or a plastic surface, and the like.

Optionally, the binding of the glycoconjugate marker can be detected by obtaining an image of the marker and generating a recognition site map of the polysaccharide to be analyzed to derive (derive) a partial structural distribution on the sample structure, thereby deriving at least a polysaccharide-related Partial sequence information.

The marker can optionally include a chromogen binder such that the image is provided by a combination of a binding agent, such as a composite, which is the color developed on the surface of the substrate. Alternatively, the marker may be a labeled binding agent such that an image of the marker is provided based on the signal from the marker. The image can be obtained, for example, by using a filter, a laser scanner, or by photographing and/or digitizing the image.

Additional methods and assays for determining the glycosylation pattern or "fingerprint" of a sample, such as a cell or human IgG, are also disclosed in U.S. Patent Application No. 20050186645, which is also incorporated herein by reference in its entirety herein This article is as fully explained in this article. This application describes a method for obtaining information on the carbohydrate content of a sugar molecule by adding a sugar molecule to a substrate to which one or more sugar binders (also referred to herein as first sugar binders) are attached. A first sugar binding agent that binds to the sugar molecule is identified and the resulting binding information is used to produce a fingerprint of the sugar molecule.

Essential sequence-specific and/or site-specific binding agents of the present application may include, for example, lectins (eg, colored lectins, fluorescent lectins, biotinylated lectins) or antibodies (eg, fluorescent antibodies, organisms) a labeled antibody, or an enzyme-labeled antibody). The method or assay can be performed using at least five lectins, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 lectins, although any number of lectins can optionally be used, for example From about 5 lectins to about 100 or more lectins.

For example, the method can be optionally performed using a set of 20-30 lectins printed on a film coated glass slide with 4-8 sets of repeats (or any other suitable repeat set), or alternatively A concentration range of dose response for each printed lectin is provided. A sample of intact glycoprotein is applied to the array and its binding pattern is detected by direct labeling using any fluorophore or by using a fluorophore-labeled probe directed against a protein moiety, such as an antibody, or a carbohydrate moiety-lectin. The resulting fingerprint is the glycosylation pattern of the highly specific sample. A large number of lectins, each with its specific recognition pattern, ensure high sensitivity of fingerprints to changes in glycosylation patterns. Many fluorescent labels can be used, such as FITC, Rhodamine, Cy3, Cy5, or any Alexa dye. These fluorescent and dye labels are collectively referred to herein as "chromogen labels." In addition, biotin-avidin systems and/or known in the art can be used. Or use any other suitable tag type for tagging. The sugar molecule can optionally be modified prior to analysis as described above.

The methods and assays of the present application can optionally be performed throughout the cell. Alternatively, the methods and assays can be performed in a cell preparation (non-intact cell material), such as a membrane protein extract, a cell homogenate, or a crude membrane mixture.

In embodiments including the use of intact cells, it is preferred to first immobilize the cells. For example, by adding a solution of 1% glutaraldehyde in Sorenson's buffer, pH 7.3 (Tousimis Research Corp., Rockville, Md), using Sorenssen after 24-48 hours After buffer washing, the cells can be immobilized in a suspension of RPMI medium (as described, for example, in Sanders et al, A high-yield technique for preparing cells fixed in suspension for scanning electron microscopy, The Journal of Cell Biology, Volume 67, 1975). , pages 476 480).

Alternatively, the cells can be fixed by immersing the cells in PBS/3.7% formaldehyde for 60 minutes at ambient temperature, followed by washing the cells with distilled water (as described, for example, in Nimrichter et al, Intact cell adhesion to glycan microarrays, Glycobiology, vol .14, no. 2; pp. 197-203, 2004).

Of course, any type of cell fixation process can optionally be performed to allow detection of binding of the carbohydrate binding agent to the cells.

The methods of the present application can be optionally and preferably performed in vitro.

The methods and assays of the present application can be optionally and preferably performed using the Qproteome Glycoprofiling kit (Qiagen, USA) or any GlycoScope type kit. Using carefully selected large amounts of data A set of glycoproteins, a glycoprotein of these proteins synthesized by a large group of enzymes, and a lectin used in such a kit are selected by analyzing a group of more than 80 lectins. Where possible, lectins on the array are grouped according to monosaccharide specificity; lectins in the group expressed as "complex" do not bind to monosaccharides, but can bind to complex N-linked glycans. The differences between the lectins in each group and each group are described in detail below.

Complex

The lectin in this group recognizes a branch of either of the two α-mannose residues located in the trimannosyl core of the complex N-linked complex glycan. Because some lectins in this group bind most of the glycan structure, they are sensitive to different antenna ends. Lectins expressed as complex (1) and complex (4) tend to 2,6-branched structures; lectin complexes (3) tend to 2,4-branched structures, and lectin complexes (2) ) Identify the two structures with similar affinities.

GlcNAc

The lectin in this group binds to N-acetylglucosamine (GlcNAc), and its β4-linked oligomer has an increased affinity for the subsequent chain length increase. There was no difference in carbohydrate specificity between the two lectins in this group, but differences in their binding patterns were observed and may be derived from the non-carbohydrate portion of the sample.

Glc/Man

The lectin group is a subgroup of mannose-binding lectins (see below), and since they bind to glucose in addition to binding to mannose, they are represented as Glc/Man-binding lectins. All lectins in this group bind to the dual antenna complex N-linked glycan with high affinity. Pro about its dual antenna structure In comparison with the force, lectin Glc/Man(1) and (2) bind to high mannose glycans with lower affinity, while lectin Glc/Man(3) binds to high mannose with higher affinity. Glycans.

Mannose

This group consists of lectins that specifically bind to mannose. These lectins can bind to the high mannose structure with lower affinity and can recognize the core mannose of the dual antenna composite structure.

End GlcNAc

The lectin specifically recognizes a terminal GlcNAc residue.

α-Gal

These lectins bind to the terminal α-galactose (α-Gal). The lectin α-Gal(1) binds to both α-galactose and α-GalNAc (α-N-acetylgalactosamine) and can bind to N and O-linked glycans. The lectin α-Gal (3) is mainly bound to a Galili antigen (Galal-3Gal) based on an N-linked antenna.

β-Gal

These lectins specifically bind to terminal (non-sialylated) β-galactose residues.

Gal/GalNAc

These lectins are specific for terminal galactose and N-acetylgalactosamine residues. The relative affinities of the different lectins in this group for galactose and N-acetylgalactosamine are different.

The lectins (2) and (5) from this group bind almost exclusively to Gal; the lectins (1), (3) and (4) bind almost exclusively to GalNAc. The relative affinities of the remaining lectins in the group to GalNAc/Gal are: (8) > (7) > (6).

Fucose

Lectins from this group bind to fucose residues in a variety of linkages.

The lectin fucose (6) preferentially binds to 1-2 linked fucose; the lectin fucose (8) preferentially binds to fucose linked to 1-3 and 1-6; lectin fucose ( 12) and (13) preferentially bind to Fuc1-4GlcNAc (Louis A antigen).

Due to steric hindrance, these lectins typically do not bind to the core fucose of the N-linked oligosaccharide on the intact glycoprotein.

Sialic acid

The sialic acid lectin reacts with a charged sialic acid residue. Members of this group were also observed to have intermediate specificity for other acidic groups such as sulfation. The lectin sialic acid (1) mainly recognizes 2-3 linked sialic acid; the lectin sialic acid (4) mainly recognizes 2-6 linked sialic acid.

It should be understood that these examples of methods and assays for detecting glycosylation are provided for purposes of discussion only and are not intended to limit the application in any way, and any other suitable methods and/or alternatives may be optionally employed in the present application. Determination.

The principles and operation of the present application can be better understood by referring to the appended claims and the accompanying drawings.

Example 1 Optimization and improvement of measurement system

This embodiment relates to an assay optimization improvement of the assay system. This assay system is typically used to determine glycosylation for many different types of proteins. However, specific improvements in this system for one type of protein have been discovered, which preferably involve antibodies, and preferably human IgG antibodies. It has been surprisingly found that glycan analysis of these proteins requires the exposure of glycans hidden in the protein structure, which can This is achieved by a specifically improved sugar analysis system. The system defines the optimal exposure conditions and leads the user through the optimization process, including determining parameters and analyzing parameters.

The system adjusts the array and associated array conditions for detection using the conditioned array, and includes optimal exposure temperature and exposure solution concentration in the sample buffer, optimization schemes and software. The system preferably verifies that the selected optimal exposure treatment enables accurate sugar analysis compared to the results obtained by conventional methods such as HPLC and/or by comparisons as described in more detail below. Although a complete analysis is preferably performed for the assay system described in Figure 2, the protein is optionally digested prior to analysis by HPLC or other assays. Protocols with or without an HPLC reference can be used to allow the user to perform internal calibration or to correct in a simple manner. The system preferably has assay software as well as related protocols and samples.

As shown in FIG. 1, the sugar analysis array 102 of the present application includes: a planar substrate 1; and a plurality of contact portions 10 present in a plurality of predetermined positions on the surface of the planar substrate 1. Wherein a plurality of sugar binding agents are disposed in the plurality of contact portions 10, the plurality of sugar binding agents acting as detectors for sugars and glycans in predetermined positions of the planar substrate 1 according to their binding specificities; Each of the plurality of sugar binders is present at a plurality of independent predetermined locations 10 on the surface, wherein the plurality of independent predetermined locations 10 are provided with a plurality of concentrations of the sugar binder in a concentration curve The planar substrate 1 is disposed in contact with a sample comprising a glycoprotein such that the glycoprotein specifically binds to at least one sugar binding agent at the plurality of contacts 10 and forms a detectable binding complex such that According to the concentration curve Determine the baseline for non-specific binding.

As shown in Figure 2, assay system 100 is provided for glycosylation assays. The assay system 100 has a plurality of arrays 102, each comprising a solid (preferably planar) substrate on which a plurality of sequence or site-specific sugar binding agents are provided in a predetermined order (unlimited for the format of the sequence) For an example, see Example 2) below. Such binding agents include, but are not limited to, antibodies, lectins, and the like. Non-limiting examples of such lectins have been previously described. The solid substrate may optionally comprise a porous membrane, such as nitrocellulose, or a non-porous substrate, such as glass (as is known in the art, it is preferred to derivatize the latter type of substrate to allow the protein to bind firmly thereto). The array 102 can alternatively be prepared in accordance with the K fingerprint array format described in Example 2 below.

Array 102 is preferably processed by components of the kits specified in the assay protocol and assay optimization scheme, for example, including one or more washing and/or blocking buffers as described herein. The array 102 can optionally be inserted (or otherwise provided) to the assay execution module 104 for automated preparation and execution of assay protocols as described herein. In assay execution module 104, sample proteins are applied to each array 102 followed by one or more washing steps. Optionally, one or more blocking, equilibration and/or washing steps are also performed prior to application of the sample protein. "Closed" refers to the application of a buffer to block non-specific interactions. Such blocking may also optionally be performed after the sample protein is applied to the array 102. The array 102 is also referred to below as a "slide."

Binding of the sugar binding agent to the sample protein is detected by detector 105 after application of the sample protein and optionally after one or more washing steps. Note Preferably, the sample protein is first bound to the array 102 without a sugar binder, and then the sugar binder is applied, but in any case, the complex between the sugar binder and the sample protein is passed through the detector 105. The formation of the substance is detected. Detector 105 can optionally be combined with, or alternatively separable from, detection execution module 104.

Detector 105 preferably receives the raw signal that directly indicates the combination; for example, if a colorimetric indicator is used to detect the formation of the binding complex, image analysis is optionally performed with detector 105 to obtain the original binding signal. Optionally and preferably, the data is then analyzed by assay data analyzer 106 to determine if binding does occur. The detector 105 is in communication with the assay data analyzer 106, but may alternatively be combined with or alternatively separated from the assay data analyzer 106.

Binding is measured by calculating a glycoconjugate signal, such as a lectin signal, and by calculating the lectin signal ratio. For example, as described in certain embodiments below, an optional correction result or QC (Quality Control) factor can be considered to determine if a bond has occurred. Optionally, as described in embodiment 4, the outer sign module 107 is determined to flag any anomalous or "outlier" results, and also to detect problems associated with the assay; further as described in more detail below, by further consideration These results may optionally be excluded.

The assay optimization module 108 then receives the combined results. The assay optimization module 108 preferably includes a database containing data relating to binding of the protein of interest under different experimental conditions, and in this embodiment the protein of interest is an IgG antibody. The assay optimization module 108 preferably receives an archive of optimized experimental results containing other specific types of proteins, such as IgG antibodies. The file preferably includes data relating to:

Calibration standard

Required samples in different exposure conditions (different exposure solution concentrations and different exposure temperatures)

Reference sample

A non-limiting example of an experimental layout for such an experiment (see, for example, Figure 5) is: Figure 5 shows an example of an optimization/exposure correction experiment result. This experiment is preferably performed once to determine the optimal conditions for future experiments using the sample. The experimental layout (required slides and samples with relevant test exposure conditions) is shown below. When the experiment is performed, as shown in Figure 5, a set of results/sugar analysis is generated by a system with additional information such as username, date, and the like. The report shown in Figure 5 is loaded (uploaded) into the assay optimization software module for analysis, and the system produces a report indicating the best conditions for running future measurements.

Each pad or slide is related to the type of material and/or the combination of material and specific sample exposure conditions, and thus to the experimental set that sets the experimental conditions. For example, for pad slides (eg, the number of slides is 2), there is one material on each slide under specific exposure conditions (temperature, exposure solution concentration, and exposure time). Provide a common calibration mark between slides To achieve correction data.

Multi-pad slides have a substrate, such as a planar substrate, but with multiple sub-regions. For such slides, the first liner is typically a calibration standard. Alternatively, the individual liners can have samples, reference standards, and controls. More than one probe having a different detection agent (e.g., a colorimetric agent having a different color) can be provided in the same slide or pad/sub-region.

Moreover, if available, assay optimization module 108 can optionally receive comparative HPLC (high performance liquid chromatography) data for the same protein sample and at least one glycoconjugate protein present in array 102; optionally One or more methods known in the art are used to obtain the data.

The assay optimization module 108 then compares all available reference data to actual data obtained from the sample protein to calculate the optimal lectin activity, measure the lectin that only produces signals when the sample is exposed, and pass and use The experimentally determined database created by a large number of sample types is compared to the calculated baseline calculated to calculate the background value of the slide to determine the most appropriate assay conditions.

Non-limiting examples of such parameters include binding time, temperature, and exposure solution concentration values for buffers for incubation of sample proteins and carbohydrate binders; lectin (sugar binder) signals, signal ratios, when exposure is not the most a specified set of lectins, background values, and background values compared to a calibration standard background; ES (exposed solution) concentration in sample buffer (eg, 0.01% of sample buffer volume); and sample protein concentration. The assay optimization module 108 can also specifically determine whether the IgG sample antibody protein has Fab glycosylation or O-linked glycosylation; if so, specific optimization conditions are implemented. Send to the user A warning about the presence of this glycosylation, the presence of this glycosylation may have a negative impact on the accuracy of the results. Optionally, optimize one or more of the following other parameters:

- Sample concentration

- Slide format

- Measurement format

- Interpreting algorithms (scripts)

Optimization was performed to tailor the assay to a specific mAb (antibody) and to allow for maximal exposure of Fc (immune molecule)-associated glycans. The optimization includes correction of at least three parameters: exposure solution concentration, temperature, and incubation time of the exposure pretreatment to expose the glycan to the array lectin.

The exposure solution can optionally include any component suitable for exposing the unexposed glycan structure, which can be otherwise blocked. Optionally and preferably, the solution contains a detergent; more preferably, the detergent is selected from the group consisting of cholate, deoxycholate, C16TAB, LysoPC, CHAPS, amphoteric surfactant (facultative ion detergent, Zwittergent, SDS, octyl glycoside, digitonin, Rebourrol, C12E8, Triton X-100, Trinex P-40, and Tween-80, all in proportion It is a percentage known in the art for unfolding proteins.

Since differences in protein sequences may affect the biochemical and structural properties of the antibody, exposure conditions may vary slightly depending on the monoclonal antibody.

The condition matrix to be controlled may optionally include: optimizing the exposure solution concentration: 0.001% to 1%

Optimized temperature: 50-80 ° C

Optimization time for pretreatment incubation: 1 minute to 1 hour

The selection of optimal conditions is automatically performed by a software method as described herein.

Example 2 Multi-concentration array format

Multi-concentration array formats involve improvements to their physical arrays to achieve more optimized binding results. This new grid format uses a concentration curve printed on each array for each lectin (or antibody-sugar binder) compared to a single concentration in previous array types.

Develop multi-concentration formats to increase system reproducibility and generate more accurate signals. Not wishing to be limited to closed lists, the multi-concentration array format provides at least the following improvements: higher reproducibility of fingerprints and interpretation levels

Improved and more reliable way to define baselines in the case of certain non-specific bindings

Multi-concentration formats can solve a variety of problems, such as "lectin zero", reproducibility and sample concentration, which can be very important for high background or low signal. The solution to this problem is to reduce the amount of sugar binder printed on the array.

The multi-concentration format defines multiple spots for each binder, including a concentration profile for each sugar binder, such as a lectin, and thus requires more spots per array. To achieve this goal, the array format is modified and each lectin is included with additional spots. The board format was also changed to include the concentration and other marking spots required for all improved automated image analysis. Adding more spots may reduce the automatic image analysis software to accurately detect the array The ability to spot and position the virtual measurement grid (set the spot value and calculate the fingerprint). In order to improve image analysis capabilities, it is possible to provide additional marker spots, including pre-labeled proteins that provide high signal during the processed array scan.

As noted above, the multi-concentration format defines a concentration profile for each sugar binding agent, such as a lectin. In this non-limiting embodiment, a concentration curve is given for the amount of lectin on a solid substrate of an array printed over a plurality of spots. Preferably, for each lectin (sugar binding agent) on the array substrate, there are a plurality of different spots that are associated with different protein concentrations. Alternatively, the concentration ranges from 0.01 mg/ml to 10 mg/ml; preferably, the range is from 0.038 mg/ml to 3.5 mg/ml, and more preferably from 0.05 mg/ml to 1 mg/ml (as per The milligram amount of lectin in the milliliter sample solution is given). For example, the concentration of lectin ConA is given in Figure 3, with 7 different concentrations (thus there are 7 different spots on the physical substrate, the amount of each containing lectin is given in the table).

The multi-concentration print format was used to calculate the signal curve for each lectin on the slide. As noted above, system 100 preferably automatically determines which concentrations are acceptable; if there is a problem with the signal and linearity, up to 2 concentration points (within the total number of concentrations) in the curve can optionally be ignored. The calculated curve and signal quality determine the error value of the submitted fingerprint. The binding signal is expected to be linear in at least five of the total number of lectin concentrations; this parameter is one of the parameters determined by the assay optimization module 108 and is one of the criteria for selecting a particular set of experimental conditions as described in Example 1.

Multi-concentration formats are optionally prepared on the array by contact or non-contact printing, preferably planar substrates as previously described. Contact name as indicated by its name Brushing involves contacting a solid device, such as a needle, coated with a solution containing a sugar binder, with the surface of a planar substrate. Non-contact printing may, for example, optionally involve other means of spraying or a solution containing a sugar binder to deposit the sugar binder on the planar substrate.

For contact printing, the spot size diameter preferably varies from 0.05 mm to 75 mm. For non-contact printing, the spot size diameter preferably varies from 80 microns to 2500 microns.

"Diameter" refers to the longest axis (size) of the spot; the spots do not need to be circular or symmetrical about their axis.

The recommended amount of IgG for the IgG protein is in the range of 1.8-12 μg, depending on the amount of sample protein per spot. The recommended IgG concentration is in the range of 0.001 micromolar to 100 micromolar; particularly preferably 0.1 μM.

Example 3 QC monitoring and correction

QC (Quality Control) monitoring and calibration involves improvements in the assay system.

The QC monitoring identifies technical issues in the wet (slide treatment) and dry (scan) phases of the workflow of the system 100. The main problems detected were the quality of the slides, the quality of the scans, the correlation between the copies (if any) and the problems during the image analysis steps. The numerical scores of the slides and samples in the experiment are provided to the user (for technical quality, the grade is 0-1, and for the image analysis grid correction quality, the grade is -100 to -200). More detailed information about the internal components of each score is provided in the report providing each sample.

Calculation phase:

The calculation phase of the algorithm begins with testing each spot on the slide; Each spot receives a score based on its uniformity and background level. Some spots can be excluded during this process. The entire slide is then scored based on its average spot quality, correlation between two identical sub-blocks, and other technical parameters. The next stage is to test the correlation between the replica slides (two sample slides, or two control slides). If the copy is poorly correlated, the system automatically selects a better slide as the basis for the calculation.

The following table summarizes the main monitoring used to calculate slide scores; it should be noted that the following table is an example for one type of slide and may be different for other slide formats and/or surface types.

A non-limiting list of QC monitoring is provided as follows: Spot assessment - In this process, the spots are evaluated by the uniformity of the foreground, the uniformity of the background, the similarity between the mean median densities, and the saturation level. The low quality spots between the copies are excluded during the normalization phase.

Slide Verification - In this procedure, the entire slides were evaluated and each slide received a score based on its technical quality (based on slide background, control spots, intra-slide reproducibility). Low quality slides are defined as slides where >=50% of the spots are of low quality, or there is low intra-slide reproducibility or low inter-slide reproducibility. Eliminate low quality slides.

Evaluation of the adaptive algorithm - The "adaptation" algorithm is used for normalization between the sample and the control slide. In this process, the quality of the adaptive algorithm is evaluated (based on the variation of the slope coefficient, the signal for the adapted lectin versus zero, signal %>0).

Evaluation of samples and controls - In this process, the combined signals of the experiments were tested. (Determining that the combined signal of the lectin not used for fitting is higher than zero, producing a lectin %>0 of the combined signal).

Figure 4 shows a flow chart for QC monitoring within system 100 for implementing Figure 2. As shown in Figure 4, which relates to a solid array surface as a "slide," as a non-limiting example only, the initial portion of the process involves "raw data." In a portion of the process, as shown, it was repeated for all slides, and all experimental and control spots (positions on the array) were evaluated for their signals. Control spots included spot monitoring (controls involving specific spots) and block monitoring (controls involving sections (parts) of slides or slides). After estimating these spots, the quality inside the slide (reproducibility in the slide) was estimated. Each slide is then scored; the slide is considered acceptable (ie, has sufficiently high quality data) or failed. Estimation is also made if the replicated slide is available.

Next, in the section dealing with the "calculated data", "gold monitoring" (previously determined data with desired results) is estimated and scored. If their scores are sufficiently high, the experimental data results can be analyzed accordingly as described herein. Otherwise, the experimental data is unacceptable because the underlying measurements were determined to be unreliable.

Correction standard algorithm

In this non-limiting example, a calibration protein is added to the assay as a standard. The standard protein was calibrated as a calibration tool for analysis. For each assay, a specific calibration standard protein should be selected (some of the broader assay types such as common IgG assays may include common calibration standards). The calibration standard protein is a variant type of glycoprotein to which an assay is established (for example, as in Example 1, the standard protein may be a known IgG antibody having known glycosylation properties). Calibration standard proteins were included in each experiment and were used to correct lectin reactivity (corrected for the expected fingerprint of the standard protein or the binding profile of the carbohydrate binder) according to the "gold standard". The calculation is based on the normalization between the real fingerprint generated in the experiment and the "golden" fingerprint, and corrects the outlier response produced by the experimental conditions. This program enables quantitative and accurate sugar analysis.

For example, assay data analyzer 106 of system 100 can optionally compare the actual combined results to the expected combined results based on the type of protein. For example, a "golden" standard or binding fingerprint can optionally be assayed for IgG antibodies or other types of proteins and then used to compare actual binding results obtained from a particular corrected protein. Preferably, however, a gold standard fingerprint for the same corrected protein printed on the solid substrate of array 102 is obtained. A non-limiting exemplary method is described as follows: Fingerprint comparison: Compare the fingerprint of the CS (corrected sample) sample with the gold fingerprint (which is the "gold" standard result set): corrected to the average gold standard and calibration standard sample

Perform a T test, discard the outliers, and calculate the regression of non-exclusive lectins

(sugar binder)

If in the T test, 1/3 or more lectin-CS is discarded

If the resulting slope <X or >Y-CS is unqualified

If the resulting R ² <x-CS is unqualified

Further, alternatively, the following correction factor for each glycoconjugate protein is obtained from the above-described correction method: CS value/gold fingerprint value. This correction factor is preferably applied to the binding data of the actual sample protein by, for example, the assay data analyzer 106.

Example 4 Out-of-assay markers for arrays and systems

Development of an external marker to reduce errors and to detect problems in the assay by marking "outliers" or abnormal results. As for the sample variant in which the glycosylation pattern greatly deviates from the range of data generated during the assay development process, as described above, the assay external marker module 107 of the second graph system generates information that warns the user of a problem. During the development of a specific assay for a given glycoprotein sample, a set of glycosylation variants that may be present in the sample was created. Develop, optimize, and use the set of tests for the explain script. High accuracy interpretation of the glycosylation pattern in the region defined by these variants can be expected. This guarantee cannot be provided for variants in which the glycosylation pattern deviates significantly from the extent of the region. However, the system will identify the glycosylation pattern of the sample and the original sample and The derivatives are significantly different; warning information ("assay external markers") is provided for this result and may, for example, be optionally displayed to the user or sent to the experimental log file, or otherwise recorded.

Some non-limiting examples of situations in which it is not desired to be bound by a closed list that may trigger the determination of an out-of-label include:

1. Does not reflect the appearance of new variants of the actual expected fingerprint (for example, detecting unanticipated sugars or polysaccharides in a particular glycoprotein):

a. Predictable variants that cannot be prepared by biochemicals (eg, different distributions between dual antennas and three antennas)

b. Unpredictable variants containing abnormal glycosylation structures (abnormal in the context of a particular sample)

2. Abnormal fingerprint of the tested sample protein

3. User error, such as using the wrong sample

4. Determine the fault itself; for example, from a low signal to no signal.

The “out of measure” warning is based on some simple knowledge-based rules. These rules are the result of studying the combination of specific sample variant behavior and lectin knowledge accumulation. In most assays, the basic rule for defining a fingerprint as "out of measure" is:

1. Significant changes in the ratio between the two antennas and the three-four antenna structure and the expected ratio (the rule is based on the complex and the mannose lectin binding motif (motif))

2. The emergence of new units (sugar/polysaccharide motifs). In most cases, the unit detects yes/no and, after detection, is ready for an “out of measure” warning (high mannose structure and terminal GalNAc unit are the most common, because in many cases, it is not desirable Combined results appear)

3. Based on, for example, the type or type of sample protein, or previous results of the same sample protein, unexpectedly, a low signal of lectin that is expected to react with the minimum is expected.

Test by running a script on the sample baseline. The result is that in most cases the fingerprint is within the limits of the assay and has very few "out of measure" fingerprints. In most cases, this warning is reasonable.

Example 5 - IgG experiment

The above system was tested for an array type (a pad slide) using an exemplary IgG antibody, Avastin. The experimental results are shown in Figure 5. In short, Avastin and many different lectins as sugar binders (as shown, complex (1), complex (3), complex (4), GlcNAc (1) and GlcNAc (2)) and the background sample and the different sample exposure conditions parameters were incubated together to determine the optimal conditions for sample determination (as shown in Figure 5). The different sample names under "Sample Name" relate to standards (samples 78 and 85) and Avastin itself (samples 79-84; different samples are given to indicate changes in conditions, etc.). After the "Background" column, the five columns to the right refer to different lectins, where different lectin names are given.

The optimal conditions for this protein are as follows (given as the best Avastin exposure conditions in the lowermost right corner): IgG concentration (micromolar): 0.1; exposure solution concentration - 0.01% (involving detergent solution in incubation solution) The amount, as previously described, was used to expose the specific sugar/polysaccharide motif; the exposure temperature was 71 ° C; and the optimum time for exposure was found to be 15 minutes. It should be noted that this is merely a non-limiting example; different conditions may be required to use different arrays and/or assay types.

Although this application has been described in a limited number of implementations It is to be understood that many variations, modifications, and other applications are possible in the application.

1‧‧‧Flat substrate

10‧‧‧Flat substrate/contact/predetermined position

100‧‧‧Measurement system

102‧‧‧Array

104‧‧‧Execution module

105‧‧‧Detector

106‧‧‧Measurement Data Analyzer

107‧‧‧Measurement of external marking module

108‧‧‧Measurement optimization module

1 shows an exemplary array in accordance with at least some embodiments of the present application; FIG. 2 illustrates an exemplary system in accordance with at least some embodiments of the present application; and FIG. 3 relates to at least some in accordance with the present application. Exemplary print concentrations of exemplary lectins of an embodiment; FIG. 4 shows an exemplary QC and calibration process for system operation of FIG. 2, and FIG. 5 shows test optimized experimental layout, samples, and arrays The result of combining the type with different test sample exposure conditions, using an exemplary IgG antibody, Avastin, for one array type (one padded slide or multiple padded slides) by the above system experimental.

1‧‧‧Flat substrate

10‧‧‧Flat substrate/contact/predetermined position

102‧‧‧Array

Claims (39)

A sugar analysis array comprising a planar substrate and a plurality of sugar binders present at a plurality of predetermined locations on a surface of the substrate, each of the plurality of sugar binders being present at a plurality of independent predetermined locations on the surface Where the plurality of independent predetermined locations relate to a plurality of concentrations of the sugar binder in the concentration curve at the location; the planar substrate being adapted to contact a sample comprising a glycoprotein such that the glycoprotein is at least one The carbohydrate binding agent specifically binds to and forms a detectable binding complex to determine a baseline for non-specific binding based on the concentration profile. A sugar analysis array, comprising: a planar substrate; and a plurality of contact portions present at a plurality of predetermined positions on a surface of the planar substrate, in the plurality of contact portions Provided with a plurality of sugar binders disposed in predetermined positions of the planar substrate; each of the plurality of sugar binders being present at a plurality of independent predetermined locations on the surface, wherein Said plurality of independent predetermined positions are provided with a plurality of concentrations of said sugar-binding agent in a concentration curve; said planar substrate being arranged to be in contact with a sample comprising a glycoprotein such that said glycoprotein is in said plurality of contacts The portion specifically binds to at least one sugar binding agent and forms a detectable binding complex to determine a baseline for non-specific binding based on the concentration curve. The array of claim 1 or 2, wherein the detectable binding complex is detectable by a binding signal, and wherein the binding signal is at a sugar binder concentration in the concentration curve total At least five of the numbers are linear. The array of claim 1 or 2, wherein the planar substrate has a recess or a hole. The array of claim 4, wherein the planar substrate comprises at least one of a film, glass or plastic. The array of claim 5, wherein the planar substrate is derivatized. The array of claim 3, wherein the array further comprises predetermined locations of the plurality of indicia on the planar substrate to provide a high signal to support the array after contact with the sample Image analysis. The array of claim 7, wherein the array further comprises a plurality of sugar binders at a predetermined position of the planar substrate as a calibration standard. The array of claim 8 wherein the planar substrate is divided into a plurality of pads, and wherein each of the pads comprises a plurality of independent calibration standards. The array of claim 1 or 2, wherein the saccharide-binding agent comprises a lectin or an antibody, or a modified component or combination thereof. The array of claim 10, wherein the concentration of each lectin on the substrate ranges from 0.01 mg/ml to 10 mg/ml. The array of claim 11, wherein the concentration ranges from 0.001 mg/ml to 10 mg/ml. The array according to claim 12, wherein the concentration The range is from 0.01 mg/ml to 5 mg/ml. The array of claim 11, wherein each location has a spot size diameter, and wherein the spot size diameter is in the range of 0.05 mm to 75 mm. The array of claim 11, wherein each location has a spot size diameter, and wherein the spot size diameter is in the range of 80 microns to 2500 microns. The array of claim 14 or 15, wherein the glycoprotein comprises an IgG antibody or fragment. The array according to claim 16, wherein the amount of the IgG per position is in the range of 1.8 to 12 μg. The array according to claim 17, wherein in the solution for contacting the IgG antibody or fragment with the sugar binding agent, the IgG concentration is in the range of 0.001 μmol to 100 μmol. . The array of claim 18, wherein the solution comprises a detergent for unfolding the IgG antibody or fragment and exposing at least one glycan. The array of claim 3, wherein the planar substrate has a recess or a hole. The array of claim 3, wherein the saccharide-binding agent comprises a lectin or an antibody, or a modified component or combination thereof. An assay system for performing a glycosylation assay using a plurality of arrays according to any one of claims 1 to 21, comprising a kit for performing the glycosylation assay, by detecting a signal by binding a signal Detectable a binding complex to obtain a binding data detector, and an assay optimization module for comparing reference data and the binding data obtained from the sample glycoprotein to calculate optimal lectin activity, The slide assay background values were calculated by comparing only the lectin that produced the signal to the sample glycoprotein, and the calculated baseline defined by a database based on an experiment created using a large number of sample types to determine appropriate assay conditions. An assay system for performing a glycosylation assay, comprising: a plurality of arrays, a measurement execution module, the plurality of arrays being coupled to the assay execution module, or being housed in the assay execution module a detector coupled to the assay execution module, the binding of the sugar binding agent to the sample protein is detected by the detector; a data analyzer is measured, in communication with the detector or connected to the detector; Determining an external sign module; connecting to the measurement data analyzer or communicating with the measurement data analyzer; and measuring an optimization module, connecting to the measurement data analyzer or communicating with the measurement data analyzer; wherein Each of the plurality of arrays includes: a planar substrate; and a plurality of contacts present at a plurality of predetermined locations on a surface of the planar substrate, in the plurality of contacts Provided with a plurality of sugar binders, the plurality of sugar binders are provided In a predetermined position of the planar substrate; each of the plurality of sugar binders is present at a plurality of independent predetermined locations on the surface, wherein the plurality of independent predetermined locations are disposed in a concentration curve a plurality of concentrations of the sugar binding agent; the planar substrate being disposed in contact with a sample comprising a glycoprotein such that the glycoprotein specifically binds to at least one sugar binding agent at the plurality of contacts and forms The bound complex is detected to determine a baseline for non-specific binding based on the concentration curve. The assay system of claim 22 or 23, wherein the assay optimization module further determines suitable assay conditions based on one or more of the following parameters: for incubating the sample protein and the carbohydrate binder Binding time, temperature, and pH of the buffer; sugar binder signal, signal ratio, a set of specified sugar binders that react when exposure is not optimal, background values, and background values compared to the calibration standard background; The concentration of detergent in the buffer; and the protein concentration of the sample. The assay system of claim 24, wherein the assay optimization module optimizes sample glycoprotein concentration, substrate format, and assay format. The assay system of claim 25, wherein the sample glycoprotein comprises an IgG antibody, and wherein the assay optimization module determines whether the IgG sample antibody has Fab glycosylation or O-linked glycosylation, Specific optimization conditions are implemented and warnings about the presence of such glycosylation are issued. The assay system of claim 26, wherein the An assay optimization module determines an amount of an exposure solution to be applied to the sample glycoprotein, wherein the exposure solution has a detergent known in the art for spreading proteins, the detergent being selected from the group consisting of SDS (Ten) Sodium dialkyl sulphate, cholate, deoxycholate, C16TAB, LysoPC, CHAPS, amphoteric surfactant, octyl glucoside, digitonin, Rebourrol, C12E8, Triton X-100 , a group consisting of Novo P-40, and Tween-80. The assay system of claim 27, wherein the assay optimization module determines a condition matrix relating to one or more of the following: optimizing exposure solution concentration: 0.001% to 1%, optimized temperature: 50- Optimization time for pretreatment incubation at 80 ° C: 1 minute to 1 hour. The assay system of any of claims 22 or 23 or 25-28, further comprising at least one QC (Quality Control) monitor selected from the group consisting of foreground uniformity, background uniformity, and mean median density Similarity, saturation level spot evaluation; array verification of the quality of the entire planar substrate based on the background of the array, control spots, intra-array reproducibility; normalized evaluation between sample and corrected position; The entire array of signals is determined by the group of components. The assay system according to any one of claims 22 or 23 or 25-28, further comprising as a calibration sample applied to a plurality of correction proteins comprising a predetermined position of the sugar binder to be based on a gold fingerprint Standard, the reactivity of the sugar binder is corrected by the assay optimization module. The assay system of claim 30, wherein the assay optimization module is based on the sample glycoprotein and the calibration sample The comparison of the signals is used to determine the off-label. The measurement system according to claim 31, wherein the measurement optimization module issues a warning according to the measurement external flag. The array of claim 2, wherein the contact portion is selected from the group consisting of a circular, elliptical, square groove, recess or hole. The assay system of claim 23, wherein the contact portion is selected from the group consisting of a circular, elliptical, square groove, recess or hole. The assay system of claim 23, wherein the array is inserted or provided to the assay execution module. The measuring system according to claim 23, wherein the detector is combined with the measurement execution module or separate from the measurement execution module. The assay system of claim 23, wherein the detector is combined with or separate from the assay data analyzer. The assay system of claim 23, wherein the assay optimization module comprises a database comprising data relating to binding of the protein of interest under different experimental conditions. The assay system of claim 23, wherein the assay optimization module receives an archive of optimized experimental results containing other specific types of proteins.