KR101743969B1 - Real time glycosylation monitoring method by measuring molecular weight of glycoproteins - Google Patents
Real time glycosylation monitoring method by measuring molecular weight of glycoproteins Download PDFInfo
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Abstract
Challenges to be solved: There needs to be a way to quickly screen for oligosaccharides in real time in the biopharmaceutical production process.
The present invention relates to a method and apparatus for analyzing the glycoprotein of the present invention by measuring the total molecular weight of the glycoprotein or the molecular weight of the protein after the removal of the glycoprotein and measuring the diversity including the glycoprotein composition and the glycoprotein variant and the content of each glycoprotein, ≪ / RTI >
Description
The present invention relates to a method for confirming the composition and diversity of oligosaccharides and the presence or absence of glycosylation at each sugar chain in real time through the measurement of the total molecular weight of the glycoprotein and the molecular weight of the protein after the desolvation.
Proteins are required to be analyzed in order to identify precisely these proteins because various mutants can be formed by the physicochemical changes of proteins depending on how post translational modification (PTM) is performed in the synthesis process and functions may be changed.
Glycosylation belongs to the posttranslational modification process (PTM) and is an important factor that determines the function of the protein. It is sensitive to the biochemical environment and is degraded by oligosaccharides and competes with synthetic enzymes. In addition, glycosylation represents the glycan heterogeneity in which various kinds of oligosaccharides are bonded to one sugar site.
Glycan is mainly divided into N-oligosaccharide and O-oligosaccharide. N-oligosaccharide is bound to asparagine residue and contains 2 GlcNAc (N-AcetyGlucosamine) and 3 mannose cores. And forms a typical structure based on these cores. O-glycans are attached to serine residues or threonine residues and, unlike N- oligosaccharides, have eight kinds of cores rather than one.
In the case of biopharmaceuticals, the pattern of oligosaccharides may vary depending on the production host and culture environment from the genetic engineering process to the mass production of the protein, and the inhomogeneous oligosaccharide may affect the quality and safety of the drug such as therapeutic efficacy, persistence in the body, A problem arises. Therefore, the International Conference on Harmonization (ICH) guidelines at the international level are required to identify the heterogeneity of oligosaccharides attached to glycoprotein drugs and to demonstrate that they are reproducible for each batch.
According to the guidelines for the quality, stability and efficacy of domestic recombinant drugs (Korea Food and Drug Administration, 2014.12), the primary structural characteristics of recombinant GMOs are determined by peptide mapping and confirmation of total molecular weight, To confirm that the sequence matches. In some cases, in-depth oligosaccharide analysis is required at the development stage and production stage of a biopharmaceutical, however, there is a need for an assay method capable of quickly and accurately confirming the oligosaccharide pattern in different culture conditions in a large amount. When analyzing by applying in-depth oligosaccharide analysis method, time, labor and cost are consumed. Also, it takes a lot of time to analyze the data, which can miss the crucial moment of controlling the culture conditions. Therefore, there is a need for an assay that allows fast and accurate identification of sugar chains. In fact, for the N-glycosylase analysis, it is subjected to the enzymatic treatment, purification and concentration, mass spectrometry and oligosaccharide labeling according to the use of PNGase F. In addition, O-oligosaccharide separation, purification and concentration, mass spectrometry and, in some cases, oligosaccharide labeling are necessary for the identification of O-oligosaccharides. Therefore, for the N-oligosaccharide and O-oligosaccharide analysis, the process from sample preparation to mass spectrometry and data interpretation is complex and time-consuming.
Further, in order to confirm whether or not the sugar chain is bound to the sugar chain, the sugar chain must be treated in the form of a sugar peptide, which requires more effort and time than sample analysis, mass analysis and data analysis.
Through the analysis of oligosaccharide and glycopeptide, it is possible to obtain accurate qualitative and quantitative results when confirming N-oligosaccharide and O-oligosaccharide profiling, oligosaccharide diversity and glycosylation share. However, real-time monitoring and production of actual glycoprotein biopharmaceuticals Since there are many difficulties in quality inspection, it is absolutely necessary to have a platform capable of monitoring a large amount of samples in a short time in real time.
In order to analyze the primary structural characteristics of glycoproteins according to the guidelines for quality, stability and efficacy of recombinant drugs, confirmation of total molecular weight is necessary. At present, the oligosaccharide is removed from the glycoprotein, followed by peptide mapping analysis to confirm whether the amino acid sequence conforms to the gene sequence of the desired product at the protein level. To confirm the total molecular weight, SDS-PAGE or western blotting However, according to these methods, various bands are identified due to the variability of the oligosaccharide. Recently, molecular mass measurement using MALDI-TOF MS has been used. However, according to this method, it is difficult to measure an accurate mass value due to nonuniformity of oligosaccharide bound to protein and ionization efficiency.
In the current guidelines, full molecular weight measurements using mass spectrometry are being used as additional confirmation factors for the primary structure. Furthermore, although the measurement of total molecular weight of glycoproteins is an important factor in the analysis of physicochemical properties, there is no analytical technique ensuring accuracy and reproducibility.
Accordingly, it is an object of the present invention to provide an assay method capable of overcoming the problems of the prior art and analyzing the total molecular weight, glycosylation according to sugar chain, and glycosylation diversity of glycoprotein with rapid, accurate and high reproducibility .
In order to solve the above problems, the inventors of the present invention invented an LC / MS-based glycoprotein analysis method capable of confirming the sugar chain aspect and the sugar chain occupancy rate in real time through intact total molecular weight measurement of the glycoprotein, Respectively. The present invention relates to an assay method capable of real-time monitoring in which time, cost, and labor are significantly reduced from sample preparation, analysis and data interpretation compared with an assay method for confirming the sugar chain aspect and the sugar content through conventional separation and purification of sugar chains and sugar chains to be.
The present invention
a) measuring the total molecular weight of the protein using a mass spectrometer;
b) As a result of step a), when the mass value shows a single peak, it is judged that the sugar is not bound protein. The mass value is represented by 162.14 (hexose), 203.19 (HexNAc) Judging the protein to be a glycoprotein when it comprises a difference of one or more of 146.14 (fucose), 291.25 (NeuAc) and 307.25 (NeuGc);
c) if the protein is determined to be a glycoprotein in the step b), removing the N-glycoconjugate through an enzyme treatment and measuring the molecular weight of the N-glycosylated protein; And
d) If the mass value in the step c) shows a single peak, it is determined that the N-glycoprotein is the bound glycoprotein. If the total molecular weight measured in the step a) and the molecular weight of the N-oligosaccharide- And determining the composition of the N-glycoside by using the difference.
In addition,
A) The molecular weight of the protein from which the N-linked oligosaccharide of step c) has been removed shows a number of peaks and the difference in the value of each peak is 162.14 (Hexa), 203.19 (HexNAc), 146.14 (Fucose), 291.25 ) And 307.25 (NeuGc), and determining the O-sugar chain-bound glycoprotein when it is the same as the total molecular weight measurement result in the step a); And
B) determining the composition of the O-oligosaccharide by using the difference in the mass value of each peak based on the molecular weight measurement result of the step a) or c) ≪ / RTI >
In addition,
As a result of measuring the molecular weight of the protein from which the N-sugar chain was removed in the step c), a plurality of peaks were found and the difference in the value of each peak was 162.14 (Hexa), 203.19 (HexNAc), 146.14 (Fucose), 291.25 ) And 307.25 (NeuGc), and when there is a difference from the total molecular weight measurement result of step (a), judging the oligosaccharide to be a glycoprotein having an N-oligosaccharide and an O-oligosaccharide bonded thereto;
(C) determining the N-glycosylation using the total molecular weight of the glycoprotein and the difference in mass value of the glycoprotein from which the N-sugar chain is removed in the step (c); And
C) determining the composition of the O-sugar chain using the difference in mass value of each peak based on the result of measurement of the molecular weight of the glycoprotein from which the N-oligosaccharide has been removed in the step c) And more particularly to a real-time carbohydrate monitoring method using the same.
In the present invention, the content of oligosaccharide relative to the total molecular weight is determined using the formula (total molecular weight of glycoprotein - molecular weight measured after removing N-glycoprotein) / total molecular weight of glycoprotein * 100 (% And a method of monitoring a sugar chain protein molecular weight in real time.
In the present invention, after step (b), the amount of oligosaccharide to total molecular weight (total molecular weight - total molecular weight of glycoprotein - O-oligosaccharide composition is restricted molecular weight} / total molecular weight of glycoprotein * 100 The method comprising the steps of: (a) measuring the molecular weight of the glycoprotein;
In the present invention, after step (c), the total molecular weight of the glycoprotein - the mass value of the O-sugar composition and the mass value of the N- And determining the amount of the sugar chain relative to the total molecular weight using the method of the present invention.
The present invention also provides a method for determining the N-glycoside composition of d), wherein the difference in mass value between the detection peaks of step a) or step c) is 42.01 Da (O-acetylation), 79.95 Da (sulfate), 79.96 Da The present invention relates to a method for real-time sugar chain monitoring using a molecular weight measurement of a glycoprotein, which is characterized in that there is an N-glycosylation mutation.
Also, the present invention relates to a method for determining the amount of O-glycosylation of an oligosaccharide when the difference in mass value between the detection peaks of step a) or step c) in step b) The present invention relates to a method for real-time carbohydrate monitoring using measurement of molecular weight of glycoprotein.
Further, the present invention is characterized in that there is an O-oligosaccharide mutation when the difference in mass value between the detection peaks of step c) in the step of determining the O-sugar composition of the above c) represents a multiple of 1 or more of 42.01 Da (O-acetylation) The present invention relates to a real-time sugar chain monitoring method using the measurement of glycoprotein molecular weight.
In addition,
a) measuring the total molecular weight of a glycoprotein known to have a theoretical amino acid composition and molecular weight using a mass spectrometer; And
b) determining the sugar chain composition from the difference between the known theoretical molecular weight and the measured total molecular weight peak as a result of the step a), and a real time sugar chain monitoring method using the glycoprotein molecular weight measurement.
In addition,
c) comparing the number of N-glycosides and the number of fucoses from the composition of the oligosaccharide determined in the above step b) to determine the number of the N-glycosides in the theoretically predictable N- And determining the content of the glycoprotein by measuring the molecular weight of the glycoprotein.
In addition,
and d) determining O-sugar chain composition from the sugar chain composition determined in step b) and the N-saccharide content determined in step c).
In addition,
Wherein the saccharide content of the glycoprotein is calculated using the formula (total molecular weight of glycoprotein - theoretical protein molecular weight using protein amino acid composition) / total molecular weight of glycoprotein x 100 (%) after step a) And more particularly to a real-time sugar chain monitoring method using molecular weight measurement.
In addition,
(B) determining the sugar chain composition in the step (b), and determining that O-oligosaccharide is present when n (NeuAc) is greater than n (4 x fucose) .
In addition,
When the oligosaccharide composition is determined in the step b) and the content of the N-glycoside is confirmed in the step c), if the number of the HexNAc is not more than 31 (n = 4) (HexNAc) -n (NeuAc)} < n (2 x fucose) by comparing {n (HexNAc) - n (NeuAc)} with n And a method for monitoring a sugar chain protein in a real time.
The present invention also relates to a method for the preparation of HexNAc, wherein the composition of the oligosaccharide is determined in step b), and after the content of N-saccharides is confirmed in step c), when n (NeuAc) is not larger than n (4 x fucose) N (HexNAc) - n (NeuAc)} < n (2 x fucose) by comparing {n (HexNAc) - n (NeuAc)} with n Is not established, it is determined that there is no O-oligosaccharide.
The present invention also relates to a real-time sugar chain monitoring method using the measurement of the molecular weight of glycoprotein, characterized in that at least one oligosaccharide mutation among the N-oligosaccharide and the O-oligosaccharide is identified in the oligosaccharide composition determination in the step b).
The present invention also relates to a method for producing a polylactosamine or sialic acid wherein the N-glycosylation mutant is a polylactosamine in which Hex (Hexose) and HexNAc (N-Acetylhexosamine) are bonded in the vertical direction to form LacNAc (N-AcetylLactosamine) The present invention relates to a method for real-time sugar chain monitoring using a molecular weight measurement of glycoprotein.
The present invention also relates to a real-time sugar chain monitoring method using the molecular weight measurement of glycoprotein, wherein the O-sugar chain mutation is a hydroxyl group of sialic acid converted into an acetyl group.
Further, the present invention relates to a method for producing a sugar chain map using the above-described methods.
In the case of glycoprotein drugs among biopharmaceuticals, the nature and amount of the glycoprotein drugs may vary depending on the culture environment and the medium, thus affecting the quality and efficacy of the drug. Therefore, it is very important to quickly and accurately determine whether the glycosylation of glycoprotein in the production and processing of biopharmaceuticals is the same as the target standard. Since the sugar mass relative to the total mass of the glycoprotein is determined based on the mass range and the basic peak during the measurement of the total molecular weight, the composition and quantitative change of the oligosaccharide and the sugar content vary when the main oligosaccharide is differently expressed. Therefore, it is possible to quickly identify whether the sugar content is the same as the standard substance or whether the sugar content is the same between the batches by analyzing the sugar content with respect to the whole molecular weight of the glycoprotein.
According to the present invention, glycosylation of a glycoprotein having both N- and O-glycosylated glycoproteins and N- and O-glycosylated glycoproteins can be confirmed in real time at an intact glycoprotein level.
According to the present invention, the diversity of oligosaccharides and the sugar content can be confirmed by a one-step method using total molecular weight measurement of glycoprotein.
According to the present invention, since the pretreatment and analysis time are short, it is possible to check the sugar chain pattern of the large volume sample and the sugar chain of the sugar chain in real time.
In addition, according to the present invention, it is possible to rapidly confirm the major glycoprotein by measuring the total molecular weight of the glycoprotein and removing the oligosaccharide, and then using the mass value difference of the measured value of the protein molecular weight, The relative quantitative information of the main oligosaccharide can be obtained.
In addition, according to the present invention, it is possible to confirm the sugar chain binding to several sugar chains of the sugar chain to which sugar can bind, that is, the sugar chain content, and at the same time, the composition of the main sugar chain of each sugar chain can be confirmed.
Further, according to the present invention, the molecular weight content of the oligosaccharide in the glycoprotein can be confirmed.
In addition, according to the present invention, in the case of a glycoprotein having both N-glycosylation and N- and O-glycosylation, the total molecular weight of the glycoprotein, the variance of the N- and O- Can be confirmed step by step.
In addition, since the present invention performs full molecular weight measurement after only one impurity removal process using a spin column, the sample processing method is simple and the sample processing time is very short, so that diversity of the oligosaccharide can be confirmed in real time have.
Therefore, the method of the present invention can quickly confirm the shape of the sugar chain according to the production cycle such as the development stage and the culturing process of the biopharmaceutical, and can quickly and accurately determine the uniformity of sugar chain between batches in the production process of the biopharmaceutical As a quality management platform.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the method of analyzing glycoproteins of the present invention in comparison with the prior art.
FIG. 2A shows an N-oligosaccharide present in EPO (erythropoietin), and B shows an O-oligosaccharide present in EPO (erythropoietin).
3 is a flow chart of the method of analyzing the present invention for monitoring the sugar chain characteristic in real time according to the type of glycoprotein.
FIG. 4A shows the result of molecular weight analysis of the monoclonal antibody as a glycoprotein composed of N-mer gene only by the method of the present invention. A representative antibody drug, Trastuzumab, currently on the market, was administered. FIG. 4B shows the result of measuring the total molecular weight of the protein after favoring the N-oligosaccharide of Trastuzumab analyzed in A.
FIG. 5A is a result of measurement of total molecular weight of glycoprotein EPO (Erythropoietin) currently marketed by the present invention, and B is a result of favoring N- glycoprotein of glycoprotein EPO (Erythropoietin) of A and monitoring O- This is a result.
FIG. 6A is a result of measurement of the total molecular weight of the second-generation EPO of the glycoprotein drug currently marketed, B is the step of confirming the glycosyltransferase from the result of measurement of the total molecular weight of the second generation EPO of the glycoprotein drug, and C is the glycoprotein second- Of O-glycosylation after N-glycosylation.
FIG. 7 is a flow chart for identifying oligosaccharide of a glycoprotein including N- and O-glycosylation of glycoprotein EPO by site.
8 is a schematic representation of an oligosaccharide actually bound to the N- and O-positions of a second-generation EPO drug through measurement of the total molecular weight of the glycoprotein.
Figures 4, 5, 6, 8 and so on have the same meanings as those shown at the bottom of Fig. Again, the green circle means mannose, the yellow circle means galactose (Gal), the "OAc" means O-acetylation, the blue square means N-acetylglucosamine (GlcNAc), the yellow square means N-acetyl Lactosamine (GalNAc), red triangle represents fucose (Fuc), and purple rhombus represents N-acetylneuraminic acid (NeuAc).
Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited to the description of the embodiments.
1-1. Glycoprotein Total molecular weight Measure
1) The impurity removal process is performed according to the sample to be analyzed. For example, when analyzing glycoprotein drugs, impurities such as stabilizers and preservatives contained in pharmaceutical preparations were removed using a spin column (
2) DTT was added to dissociate the disulfide bond of (sugar) protein and heat treatment was performed. the sample to match the pH to 7 ~ 8 200mM NH 4 HCO 3 + 10 mM DTT and water at a ratio of 1: 1, and then boiled at 95 ° C or higher for 5 minutes. This step can be carried out if necessary for easy total molecular weight measurement.
3) Dilute the glycoprotein to an appropriate concentration for analysis (usually 0.05 μg / μl is used, but not fixed, and can be diluted to the appropriate concentration depending on the equipment used).
1-2. N- Sugar chain After glass Total molecular weight Measure
1) Dry all of the glycoproteins from which impurities have been removed.
2)
3) The buffer was exchanged (buffer: 3% acetonitrile + 0.1% aqueous formic acid solution).
4) Samples were diluted to the appropriate concentration to be analyzed (usually 0.05 μg / μl is used but this standard may vary depending on the equipment used).
2. Mass analysis
The resulting glycoprotein was mass analyzed by nanoLC chip / Q-TOF MS. More specifically, the diluted glycoprotein was analyzed using HPLC Chip / Q-TOF (Chip Quadrupole Time-of-Flight) with an autosampler (maintained at 4 ° C), a customs pump, a nanopump, an HPLC-Chip / MS interface and a 6540 Q- -Flight) MS system (Agilent Technologies, Santa Clara, Calif.). A chip packed with a C8 carbon chain was used with a nano-ESI spray tip integrated into a stationary phase of a 9 x 0.75 mm concentration column and 43 x 0.075 mm analysis column. Separation by the chromatographic method was carried out according to optimized separation conditions. Briefly, after the sample was loaded, an intact glycoprotein elution gradient solution was flowed at 0.3 μl / min. The concentration gradient solution was (A) 99.9% water and 0.1% formic acid (v / v) sap, and (B) 99.9% acetonitrile and 0.1% formic acid (v / v) 3% B, 0-1 min; 3 to 25% B, 1 to 4 min; 25 to 60% B, 4 to 8 min; 60 to 80% B, 8 to 12 min; 80 to 95% B, 12 to 15 min; 95 to 5% B, 15 to 20 min; 5 to 3%. Finally, the analytical column was re-equilibrated at 3% B for 5 minutes.
3. Data processing after mass analysis
After mass analysis, the original data of the LC / MS was analyzed using the Deconvolution algorithm (Maximum Entropy) included in Mass Hunter Qualitative Analysis software (version B.06.00 SP2, Agilent Technologies) & Bioconfirm software (version B.06.00, .
<Result>
Figure 1: Glycosylation Compare verification methods
1 is a conceptual diagram showing an assay method for confirming the oligosaccharide characteristic of a glycoprotein. Currently, three analytical methods are used collectively to identify the sugar chain characteristics of glycoproteins. First, the composition and quantification of the N-glycosylase is confirmed by enzymatic treatment of the glycoprotein to separate the N-glycoside, followed by purification and concentration analysis. Generally, the treatment of N-glycoside requires more than one day in consideration of the enzyme reaction time, the purification process of the sugar chain, and the extraction process. Secondly, O-oligosaccharide analysis proceeds separately from N-oligosaccharide analysis due to differences in oligosaccharide characteristics. The composition and quantification of O-oligosaccharide is analyzed by proceeding chemical treatment of glycoprotein, separating O-oligosaccharide, purifying and concentrating. Not only the chemical reaction time, the purification time of the sugar chain, and the extraction process require more than one day of analysis, but also the glycosylation variant may be damaged depending on the chemical reaction conditions. Third, the site-specific oligosaccharide characterization analysis proceeds through the analysis of the sugar peptide sample. Although it is possible to confirm the N-and O-oligosaccharide diversity as well as the position of the oligosaccharide, it takes considerable time for sample preprocessing and data interpretation. When all three of the above-mentioned methods are utilized, N-and O-glycosylation information, oligosaccharide diversity, and oligosaccharide position information can be obtained in a comprehensive manner. However, the above three methods must be performed separately and require significant sample preprocessing and data interpretation time for each method.
In contrast, the method of the present invention is a method capable of variously confirming the characteristics of the sugar chain within a few hours by a single analysis process. Unlike the conventional method, the total molecular weight of the glycoprotein can be measured without sample pretreatment, which separates each oligosaccharide from the protein. When considering both the glycoprotein measurement time and the data analysis time using the mass spectrometer, , Relative quantification, oligosaccharide diversity, sugar content, and sugar content versus sugar mass value. By using the assay method of the present invention, not only the analysis time is significantly shortened, but also the characteristics of the oligosaccharide can be analyzed without damage or loss of the sample.
Figure 2: Sugar chain shape
FIG. 2 shows the types and forms of oligosaccharides present in EPO (Erythropoietin). EPO is a glycoprotein that has both N- and O-oligosaccharides. The N-glycoside of EPO is a complex oligosaccharide with one fucose in the core. A maximum of four antennas can be formed, and finally one sialic acid can be bonded to each antenna. The major N-oligosaccharides of EPO are oligosaccharides with four sialic acid bound to four antennas, and specifically polylactosamine and O-acetylated oligosaccharide variants may be present. In the process of oligosaccharide synthesis by enzymes, polylactosamine in which Hex (Hexose) and HexNAc (N-Acetylhexosamine) are linked in the vertical direction and LacNAc (N-AcetylLactosamine) unit is formed may exist and the hydroxyl group of sialic acid is converted There may be variants with acetyl groups. Since up to two acetyl groups per sialic acid may be present, oligosaccharides with four sialic acids may have up to eight acetyl groups.
O-chains have eight different types of core shapes. The O-oligos of EPO is only in the form of sialic acid bound to
Figure 3: Flowchart of the method of the invention
3 is a flow chart of the method of analyzing the present invention for monitoring the oligosaccharide characteristics in real time according to the type of protein. After confirming whether or not the sugar is a protein bound through the whole molecular weight measurement, the glycoprotein having both N-and O-oligosaccharides, the glycoprotein having only N-oligosaccharides, and the glycoprotein having only O- And performs real-time sugar chain monitoring.
In the case of purified protein samples, it is possible to perform mass spectrometry without pretreatment, but in the case of impurities other than proteins, a step of removing the excipient using a spin column is optionally required (step I). For example, a glycoprotein preparation contains an excipient for protein stabilization and preservation. This component inhibits the ionization efficiency during mass spectrometry, so that only the glycoprotein should be selectively concentrated (step A).
Further, when necessary for the measurement of easy total molecular weight (when the disulfide bond and the three-dimensional structure of the protein interfere with the ionization of the (sugar) protein), a process of liberating the disulfide bond of the protein using DTT is required (Step B) .
(Step C). If the mass value shows a single peak, it is determined that the sugar is unbound (step VII-A). Ii) (D step) having a difference of one or two or more in the sum of the amino acid residues of SEQ ID NOs: 162.14 (Hexose), 203.19 (HexNAc), 146.14 (Fucose), 291.25 (NeuAc) and 307.25 After the N-glycoside is favored (Step E), total molecular weight is measured (Step F).
If only the N-oligosaccharide is selectively removed in step E and the result of measurement of the total molecular weight in step F is a single peak, it is judged to be a glycoprotein having only N-glycosylation (step G).
(Step H). In step E, only the N-glycoside is selectively removed, and the mass spectrum of the F-stage and the mass spectrum of the C-stage are identical to each other. When the glycoprotein has only the O-sugar chain, there is no difference between the result measured in the C stage and the result measured in the F stage since the O-sugar is not liberated in the E stage.
When there is a difference between the mass spectrum of the F-stage and the mass spectrum of the mass spectrum measured at Step C, the N-oligosaccharide and the O-oligosaccharide are simultaneously detected (Step H). When the glycoprotein has the N-oligosaccharide and the O-oligosaccharide at the same time, only the N-oligosaccharide is selectively liberated in the E-stage, so that the resultant value measured at the F-stage is higher than the molecular weight (Step H).
When the protein to be analyzed is a glycoprotein having only N-oligosaccharide, the result obtained in steps C and F, that is, the difference in protein molecular weight before and after treatment with the enzyme (PNGase F) But also the presence of variants of the oligosaccharide (J-I step).
If the protein to be analyzed is a glycoprotein having only an O-sugar chain, the O-oligosaccharide composition can be determined using the difference in the molecular weight of the protein determined by the amino acid sequence of the protein and the molecular weight of the protein determined through the measurement of the total molecular weight, (Step J-II).
When the protein to be analyzed is a glycoprotein having both N- and O-oligosaccharides, the composition of the N- and O-oligosaccharides can be determined by combining the methods of determining the oligosaccharides used in the J-I and J-II stages But the presence of variants in the oligosaccharide can be confirmed (Step J-III).
In addition, the carbohydrate contents can be calculated based on the difference in the molecular weight of the protein calculated using the molecular weight and the amino acid sequence of the glycoprotein determined using the whole molecular weight measurement (K-I, II, III step).
Example : N- Only sugar chain Glycoprotein analysis
FIG. 4 shows the results of molecular weight analysis of a monoclonal antibody which is a glycoprotein composed of only N-monoclonal antibody. A representative antibody drug, Trastuzumab, currently on the market, was used as a sample. Trastuzumab is composed of two identical light chains and heavy chains. There is one N-sugar site in one heavy chain, mainly neutral sugar chains.
In the mass spectrometry spectrum obtained by measuring the total molecular weight of Trastuzumab (FIG. 4A and FIG. 3C), the mass value of the base peak was 148224.38 Da. After the N-glycoside was favored in the glycoprotein, (Fig. 4B, Fig. 3F), the mass value of the fundamental peak is 145172.15 Da. The composition of the oligosaccharide was confirmed by using the mass difference value (3052.23 Da) between the two basic peaks. As a result, it was confirmed that G0F (Hex3HexNAc4Fuc1NeuAc0, 1445.35 Da) and G1F (Hex4HexNAc4Fuc1NeuAc0, 1607.49 Da) were oligosaccharides. The remaining 1.96 Da is the mass value increased when asparagine (Asn), which is the N-sugar site, is converted to aspartic acid (Asp) when the N-glycoside is separated from the glycoprotein. The difference between the Asn and Asp mass values is 0.98 Da. Considering the structure of dimeric antibody, the Asn was changed to Asp and the mass value of 1.96 Da was different.
When the total molecular weight difference before and after the enzyme treatment, the molecular weight of each monosaccharide, and the possible sugar chain structure are taken into consideration, the sugar chain composition present in the glycoprotein can be confirmed. Table 1 summarizes the composition of the oligosaccharides identified as a result of total molecular weight measurement of Trastuzumab.
Example : N- and O- Sugar chains At the same time analysis of glycoprotein
FIG. 5 shows the results of measurement of the total molecular weight of the first-generation EPO of the glycoprotein drug currently marketed. The first-generation EPO consists of three N-sugar sites and one O-sugar site, with mainly acidic oligos.
The basic peak mass value of the spectrum obtained by measuring the total molecular weight of the first-generation EPO (FIG. 5A and FIG. 3C) was 30180.39 Da. The total molecular weight of the glycoprotein isolated from N- B, Fig. 3F) The basic peak mass value is 19187.14 Da.
When removing N-glycans from EPO, only the O-oligosaccharide bound to sialic acid is present in the core 1 O-oligos of
The difference in the basic peak mass values before and after removal of the N-glycans (A and B in FIG. 5) was 10995.81 Da, and the oligosaccharide composition was calculated in consideration of the three N-glycosides. As a result, it was confirmed that Hex22HexNAc19Fuc3NeuAc14 (within 15 ppm) . Also, after confirming the sugar chain composition of the base peak, the sugar chain linkage was confirmed using the difference in mass value from the peripheral peak. That is, the oligosaccharide present in consideration of the mass value difference between the peaks (365.33 Da [Hex-HexNAc], 291.25 Da [NeuAc]) and the molecular weight of the monosaccharide can be further confirmed. Based on the confirmed N & O-oligosaccharide composition, the basic composition consisting of Hex, HexNAc, and Fuc was shown in FIG. 5A in different colors and the number of bound NeuAc was specified on the peak. For example, the peak at 30180.39 Da indicates the presence of 14 sialic acids (NeuAc) in the basic composition of Hex22HexNAc19Fuc3. Also, the stated number of NeuAc is an important evidence to confirm the presence of an oligosaccharide in the theoretical position of the oligosaccharide. Considering the main oligosaccharide structure of EPO, N-oligosaccharides can contain up to four sialic acids and O-oligosaccharides can contain up to two sialic acids. That is, through the number of 14 sialic acids (NeuAc), 3 N-glycosylated sites (up to 4 NeuAc x 3 N-sites per N = 12) and 1 O- 1 O-per-seat = 2).
Table 2 lists the composition of the identified N-and O-oligosaccharide as a result of total molecular weight measurement of first-generation EPO. It is known that first-generation EPO mainly contains a complex type oligosaccharide and a part of polylactosamine having a LacNAc unit ([Hex-HexNAc]: +365.33 Da). Considering the oligosaccharide synthesis process, the complex type sugar chain may include up to four antennas. Therefore, oligos corresponding to Hex23-26 HexNAc20-23 Fuc3NeuAc12 contain up to four LacNAc units in structure.
When the N-glycans are separated from the glycoprotein, the aspartic acid (Asn), which is the N-sugar moiety, is converted to aspartic acid (Asp), thereby increasing the mass value by 0.98 Da. In the case of first-generation EPO, there is an increase of 2.94 Da (0.98 x 3) after the N-glycan separation because it has three N-valent sites.
Example : N- and O- Sugar chain , Sugar chain Mutant At the same time analysis of glycoprotein
6 shows the results of measurement of the total molecular weight of commercially available second generation EPO. The second-generation EPO is a glycoprotein drug that increases sperm count and binds sialic acid and oligosaccharide variants to improve drug efficacy and stability. Unlike the first-generation EPO, it has five N-sugar sites and one O-sugar site by increasing the number of N-sugars per sugar, and is a sugar-bound white matter drug that is recombined to increase the sugar chain and to bind sialic acid. In particular, the second-generation EPO induces O-acetylation in order to maintain the stability of sialic acid, and there are oligosaccharides with an acetyl group (42.03 Da), which is known from the paper.
As a result of measurement of the total molecular weight of the second generation EPO, mass peaks differing by 42.03 Da around the fundamental peak are continuously present (FIG. 6A). It was confirmed that a large amount of acetylated sugar chain variant was present in the second generation EPO. In Fig. 6B, it can be confirmed that the polylactosamine structure is bonded in addition to the O-acetylated mutant. The presence of variants in the oligosaccharide can be confirmed by mass accuracy in the measurement of total molecular weight without the need to perform structural analysis using the oligosaccharide structure analysis method, that is, the tandem mass spectrometry.
The basic peak mass value of the spectrum obtained by measuring the total molecular weight of the second generation EPO (FIG. 6A and FIG. 3C) was 37451.93 Da. The total molecular weight of the glycoprotein isolated from N- C, Fig. 3F), the basic peak mass value was 19131.93 Da. When removing the N-glycoside from EPO, only a simple structure of O-sugar is present. When O-glycans are present only in EPO, the O-oligosaccharide (Hex1HexNAc1NeuAc1) with one sialic acid and the O-oligosaccharide (Hex1HexNAc1NeuAc2) with two sialic acids through the mass value difference of 291.25 Da (NeuAc) (FIG. 6C). In addition, the presence of O-acetylated O-glycosyl variants and the number of mutants can also be confirmed at the same time through the surrounding mass peaks of 42.03 Da. When conducting O-oligosaccharide analysis using β-elimination chemistry, variants of sialic acids are degraded and there is a limit to accurate O-sugar profiling. However, when the method of the present invention is applied, since the sialic acid mutant is not damaged, the composition of the complete O-sugar chain can be confirmed.
The difference in the basic peak mass values before and after the removal of the N-glycan was found to be 18326.96 Da, and the composition of the oligosaccharide was calculated in consideration of five N-site positions. As a result, it was confirmed to be Hex36HexNAc31Fuc5NeuAc22 (within 15 ppm ). Further, after confirming the sugar chain composition of the base peak, the sugar chain linkage can be confirmed by using the difference in mass value from the peripheral peak. That is, oligos existing in consideration of the mass value difference between the peaks (365.33 Da [Hex-HexNAc], 291.25 Da [NeuAc]) and the molecular weight of the monosaccharide can be further confirmed. Based on the confirmed N-and O- oligosaccharide composition, the basic composition composed of Hex, HexNAc, and Fuc was shown in different colors in FIG. 6A, and the number of bound NeuAc was specified on the peak. For example, a peak at 37451.93 Da indicates the presence of 22 sialic acids (NeuAc) in the composition of Hex36HexNAc31Fuc5. Also, the stated number of NeuAc is an important evidence to confirm the presence of an oligosaccharide in the theoretical position of the oligosaccharide. Considering the main oligosaccharide structure of EPO, N-oligosaccharides can contain up to four sialic acids and O-oligosaccharides can contain up to two sialic acids. (Up to 4 NeuAc x 5 n-positions per N = 20) and 1 O-glycosylation site (up to 2 NeuAc x 1 O-per-seat = 2).
Table 3 and Table 4 are the composition list of the confirmed N-O-O-oligosaccharide as a result of measurement of total molecular weight of the second generation EPO. The results of summarizing the oligosaccharide composition confirmed by considering the kind of monosaccharide, oligosaccharide (O-acetylation) and oligosaccharide structure (Originally, Table 3 and Table 4 are one table, but they are divided into two tables for convenience).
When the N-glycans are separated from the glycoprotein, the aspartic acid (Asn), which is the N-sugar moiety, is converted to aspartic acid (Asp), thereby increasing the mass value by 0.98 Da. In the case of 2nd generation EPO, it has 4.9 Da (0.98 x 5) after the N-glycan separation because it has 5 N-sites. In addition, the disulfide bond is separated and reduced during the protein denaturation process, resulting in an increase in the mass value of 2.90 Da.
The method of recognizing the presence of the N-glycosyl mutation and the O-glycosyl mutation in the glycoprotein is as follows. It is primarily confirmed whether the detected peak has a mass value other than the oligosaccharide composition, ie, 42.01 Da (O-acetylation), 79.95 Da (Sulphate), and 79.96 Da (Phosphate). However, the mass value of a variant is not limited to the above three. And if the other peaks detected have a connectivity with the oligosaccharide value, the oligosaccharide is finally determined to be a glycosylated variant.
For example, in Table 3, 37493.85 Da was composed of Hex36HexNAc31Fuc5NeuAc22 and OAc (42.01Da). In the second case, the difference between Δm 291.25 (NeuAc) and 36837.17 Da was 37203.00 Da, and Δm 365.33 (162.14 (hexosu) + 203.19 (HexNAc)) was shown to show the linkage of oligosaccharide.
Glycoprotein Mass value prepare Sugar chain Mass value Content check
The content of the sugar chain mass value relative to the sugar protein mass value can be confirmed using the difference in molecular weight of the protein calculated using the total molecular weight of the glycoprotein and the amino acid sequence of the glycoprotein. The calculation formula is as shown in the following equation (1).
The content of the sugar chain mass value relative to the mass value can be calculated using the result of the whole molecular weight measurement of the antibody of Figure 4 antibody. The total molecular weight of the antibody drug was in the range of 147859.62 ~ 148709.14 Da due to the diversity of the oligosaccharide. The total molecular weight of the protein after the oligosaccharide was 145172.15 Da. As a result of calculating the ratio of the mass of sugar chains to the mass of glycoproteins using equation (1), the range of 1.81 to 2.37% was obtained.
Using the results of the measurement of the total molecular weight of the first-generation EPO drug of FIG. 5, the content of the sugar chain mass value relative to the mass value was calculated. The total molecular weight of the first generation EPO drug was measured in the range of 27627.90 ~ 31641.65 Da due to the diversity of oligosaccharides. As a result of excluding the mass value of O-oligosaccharide at the mass value measured after favoring N-glycoside, the total molecular weight of the protein was 18396.14 Da Respectively. In the first generation EPO drug, the ratio of the N-glycoside mass value to the glycoprotein mass value was measured from 33.41 to 41.86%, which was calculated using Equation (1).
6, the content of the sugar chain relative to the mass value was calculated using the results of the total molecular weight measurement of the second generation EPO drug. As a result of measuring the total molecular weight of the second generation EPO drug, it was measured in the range of 35847.49 ~ 38123.78 Da due to the diversity of oligosaccharide. Considering the mass value of O-oligosaccharide at the mass value measured after favoring N- The molecular weight was measured to be 18180.84 Da. In the case of the second generation EPO drug, the ratio of the sugar mass to the glycoprotein mass was 49.28 to 52.31%, which was calculated using Equation (1).
Of EPO medicines Per seat You can check your site-occupancy Hexose , HexNAc, Fucose, NeuAc and a composition list thereof
1) maximum NeuAc Number {n NeuAc ) max } And N- Per seat Calculation of Occupancy
The oligosaccharides to be identified were confined to Hex6_HexNAc5_Fuc1_NeuAc3, Hex7_HexNAc6_Fuc1_NeuAc3 , Hex7_HexNAc6_Fuc1_NeuAc4, Hex8_HexNAc7_Fuc1_NeuAc3 , and Hex8_HexNAc7_Fuc1_NeuAc4, which are major N-glycosylation components of EPO (Bioanalysis, 2013, 5: 545-559). The maximum number of NeuAc {n (NeuAc) max } that can be included according to the number of N- and O-positions of the EPO drug can be calculated using Equation (2). The maximum number of NeuAcs {n (NeuAc)} that one N-glycan can contain is four, and the maximum number of NeuAcs that an O-oligosaccharide can contain is two. For example, a second-generation EPO drug with 5 N-sites and 1 O-site can have a maximum of 22 NeuAc.
Since the N-glycans of EPO drugs are mainly composed of one fucose, the number {n (Fuc)} of fucoses in the composition of the N- (3).
Where Max (NeuAc) is the maximum number of NeuAc, and n (X) is the number of X's.
In the above equation, n (X) means the number of X's.
2) O- Per seat Check share
If the number of NeuAc {n (NeuAc)} is greater than the number of Fuc times {n (Fuc)}
If the difference between the number of HexNAc and the number of NeuAc is less than the number of Fuc multiplied by 2, it can be determined that oligosaccharide exists in the O-site.
Equation (5) can be replaced with Equation (6) using the numbers of Hex, NeuAc, and Fuc. If the difference between the number of Hex and the number of NeuAc is smaller than the number of Fuc multiplied by 3, the occupancy rate of the O-site can be determined to exist in the O-site. For example, assuming that the composition of the oligosaccharide was calculated as Hex33_HexNAc28_Fuc5_NeuAc20, the number of Fuc was 5, and the share of N-glycoside the site-occupancy can be calculated as 100% using equation (2). Also, the number of NeuAc is 20, which is equal to the number of Fuc times the number of Fuc times multiplied by 4, so that Equation (4) is not satisfied, and the number of HexNAc is 31 or less. Therefore, the occupancy rate of O-positions can be confirmed by using Equation (5) or (6). When the formula (4) is applied, since the difference between the number of HexNAc and the number of NeuAc is less than the number of Fuc multiplied by 2, it can be judged that oligosaccharide exists in the O-site.
Equations (4), (5), and (6) below are formulas for determining the presence or absence of O-oligosaccharides.
In the equations (4) to (6), n (X) denotes the number of X's.
3) Second-generation EPO Hex, HexNAc , Fuc And NeuAc Composition list
Table 4 shows the composition of oligos possible when the major N-glycans constituting the second-generation EPO are located in three or more N-valent sites. In the absence of O-oligosaccharide, polylactosamine is present if the number of Hex is greater than the number of
Equation (7) below is a formula for determining the presence or absence of polylactosamine when no O-oligosaccharide is present.
In the above equation, n (X) means the number of X's.
Table 5 shows the composition of oligos possible when there is an oligosaccharide in three or more N-per-side and one O-per-side. When the oligosaccharide is present, polylactosamine is present when the number of Hex is larger than the number of Fuc times the number multiplied by 7 times plus one (Equation 8). The Hex, HexNAc, Fuc and NeuAc compositions when polylactosamine is present are shaded in Table 5. The N-glycoprotein share shown in Table 5 was calculated using Equation (2).
For example, when the composition of the oligosaccharide is calculated as Hex23_HexNAc20_Fuc3_NeuAc13, it can be confirmed that the O-oligos exists when the relationship between the number of NeuAc {n (NeuAc)} and the number of Fuc {n (Fuc) . When the number of Hex {n (Hex)} is 23 and the formula (8) is satisfied, it can be confirmed that at least one polylactosamine exists.
Equation 8 below is a formula for determining the presence or absence of polylactosamine in the presence of an O-oligosaccharide.
In the above equation, n (X) means the number of X's.
Figure 7: N- and O- Glycosylation Of the glycoproteins involved Per seat Flow Chart for Identifying Occupancy
FIG. 7 is a flow chart for calculating the occupancy rate of N-and O-positions using a total molecular weight measurement of a glycoprotein drug product. FIG. 7 is a data processing procedure for confirming N- and O- .
First, the total molecular weight of the EPO drug is measured using a mass spectrometer, and the composition of the sugar chain is confirmed.
Using the number of fucoses (Fuc) in the resulting oligosaccharide composition, the share of the N-oligosaccharide is confirmed (Equation 2).
The O-glucose occupancy can be determined by the following two equations (
a) The N-glycoside occupancy rate can be calculated as 100% (5/5 x 100%) since the number of Fuc {n (Fuc)} is 5.
b) The number of NeuAc {n (NeuAc)} is equal to 20 times Fuc number {n (Fuc)} multiplied by 4 and does not satisfy Eq. Further, the number of HexNAc {n (HexNAc)} is 25, which is smaller than 31, and the O-oligosaccharide occupancy is calculated using another formula.
c) The oligosaccharide having a composition of Hex33_HexNAc28_Fuc5_NeuAc20 is O (HucNAc) because the difference between the number of HexNAc {n (HexNAc)} and the number of NeuAc {n (NeuAc)} is less than 8 times the number of Fuc {n - It can be judged that the oligosaccharide exists.
8: Total molecular weight Through measurement Sugar chain Site identification and Sugar chain Mapping
8 is a schematic representation of an oligosaccharide actually bound to the N- and O-positions of a second-generation EPO drug through measurement of the total molecular weight of the glycoprotein.
The basic peak mass value in FIG. 6A is 37451,93, and it can be seen from Table 3 that the total molecular weight of the glycoprotein to which the oligosaccharide of Hex36_HexNAc31_Fuc5_NeuAc22 is bound is shown through the oligosaccharide composition list. This indicates that the oligosaccharide of Hex7_HexNAc6_Fuc1_NeuAc4 is bound to the main oligosaccharide in all five N-positions and Hex1_HexNAc1_NeuAc2 is bound to the O-site.
Therefore, the structure of the oligosaccharide bound at each site can be estimated with a pattern in which a specific mass value increases or decreases based on 37451.93 Da (Hex36_HexNAc31_Fuc5_NeuAc22_OAc16).
6C, it was confirmed that oligosaccharides having up to two sialic acids and up to four O-acetylations exist in the core 1 O- oligosaccharide in the O-per se, confirming the oligosaccharide diversity of the O-per se Respectively.
O-acetylation on sialic acid may result in up to two O-acetylations per sialic acid. For example, it was confirmed that up to 8 O-acetylated Hex7_HexNAc6_Fuc1_NeuAc4 oligosaccharides were analyzed in the oligosaccharide analysis. However, mono (1)> di (2)> tri (3) )> Hepta (7) > octa (8). Penta (5) to octa (8) O-acetylation is detectable at the oligosaccharide level using a high-resolution, high-sensitivity mass spectrometer. Mainly mono (1) or di (2) O-acetylation is detected at a high rate.
37493.77 Da (Hex36_HexNAc31_Fuc5_NeuAc22_OAc1) having an increase of 42.03 Da (OAc) relative to the reference peak suggests that O-acetylation is bound in comparison with the reference peak oligosaccharide composition (N-glycosylase: Hex7_HexNAc6_Fuc1_NeuAc4, O-oligosaccharide: Hex1_HexNAc1_NeuAc2) It is not possible to confirm that O-acetylation occurred at the site, suggesting the possibility of being bound to either the N- or O- oligosaccharide.
This indicates that there is an oligosaccharide with one NeuAc unit smaller than the reference oligosaccharide composition (N-glycoside: Hex7_HexNAc6_Fuc1_NeuAc4, O-oligosaccharide: Hex1_HexNAc1_NeuAc2) in the case of 37160.33 Da (Hex36_HexNAc31_Fuc5_NeuAc21) smaller than 291.25 Da (NeuAc) Therefore, it is suggested that Hex7_HexNAc6_Fuc1_NeuAc3 oligosaccharide exists at the N-site, or Hex1_HexNAc1_NeuAc1 oligosaccharide is bound to the O-site. In fact, it is not possible to identify which oligosaccharide is present in which position the sialic acid is reduced. Thus, a decrease in one sialic acid is considered to occur in every single place.
36795.00 Da (Hex35_HexNAc30_Fuc5_NeuAc21) is a decrease of 365.33 Da (Hex1_HexNAc1) based on 37160.33 Da. In this case, there is a possibility that the Hex6_HexNAc5_Fuc1_NeuAc3 oligosaccharide is bound to each N- position. In fact, it can not be confirmed that there is a Hex6_HexNAc5_Fuc1_NeuAc3 oligosaccharide at any position, so it is considered to be able to bind to all N-positions.
A series of processes were applied to the measurement of the total molecular weight of the glycoprotein to prepare a schematic diagram for confirming the diversity of the oligosaccharide bound to the N- and O-sites (FIG. 8).
Claims (20)
b) As a result of step a), when the mass value shows a single peak, it is judged that the sugar is not bound protein, and the difference between two peaks is 162.14 (hexose), 203.19 (HexNAc) Determining a glycoprotein when the mass value of one of 146.14 (fucose), 291.25 (NeuAc) and 307.25 (NeuGc) or the sum of two or more mass values;
c) if the protein is determined to be a glycoprotein in the step b), removing the N-glycoconjugate through an enzyme treatment and measuring the molecular weight of the N-glycosylated protein; And
d) If the mass value in the step c) shows a single peak, it is determined that the N-glycoprotein is the bound glycoprotein. If the total molecular weight measured in the step a) and the molecular weight of the N-oligosaccharide- And determining the composition of the N-glycoside by using the difference.
A) As a result of measuring the molecular weight of the protein from which the N-sugar chain was removed in the step c), two or more peaks were found and the difference between the peaks was 162.14 (Hexa), 203.19 (HexNAc), 146.14 (Fucose), 291.25 NeuAc) and 307.25 (NeuGc), or a total of two or more mass values, and determining the O-sugar chain-bound glycoprotein when the total molecular weight is the same as the measurement result of step (a). And
B) determining the composition of the O-oligosaccharide by using the difference in mass value between the peaks based on the molecular weight measurement result of the step a) or c), and performing real-time glycosylation monitoring using the glycoprotein molecular weight measurement Way.
(A) The molecular weight of the protein from which the N-linked oligosaccharide of step (c) was removed showed two or more peaks and the difference between the peaks was 162.14 (Hexa), 203.19 (HexNAc), 146.14 NeuAc) and 307.25 (NeuGc), or a sum of two or more mass values, and when there is a difference from the total molecular weight measurement result in the step a), the N- glycoprotein and the O- ;
(C) determining the N-glycosylation using the total molecular weight of the glycoprotein and the difference in mass value of the glycoprotein from which the N-sugar chain is removed in the step (c); And
C) determining the composition of the O-oligosaccharide by using the difference in mass value between peaks based on the measurement result of the molecular weight of the glycoprotein from which the N-oligosaccharide has been removed in the step c) A real - time sugar chain monitoring method using.
After the step d)
Determining the saccharide content relative to the total molecular weight using the total molecular weight of the saccharide protein (total molecular weight of glycoprotein - molecular weight measured after removing the N-glycoprotein) / total molecular weight of glycoprotein x 100 (%) Real time sugar chain monitoring method using molecular weight measurement.
After the step b)
Determining the oligosaccharide content relative to the total molecular weight using the formula (total molecular weight of glycoprotein-O-limiting molecular weight of oligosaccharide composition / total molecular weight of glycoprotein x 100 (%)) A method for real-time carbohydrate monitoring using glycoprotein molecular weight measurement.
After the step d)
The oligosaccharide content relative to the total molecular weight is determined using the formula (total molecular weight of glycoprotein-O-weight of oligosaccharide composition and mass value of N-oligosaccharide composition) / total molecular weight of glycoprotein 100 (%) Wherein the method comprises the steps of: (a) measuring the molecular weight of the glycoprotein;
In the determination of the N-glycoside composition in step d) above, the difference in mass value between the detection peaks of step a) or step c) is at least one of 42.01 Da (O-acetylation), 79.95 Da (sulfate) and 79.96 Da And determining that there is an N-glycosylation mutation when the value of the N-glycoside is represented.
When the difference in mass value between the detection peaks of step a) or step c) in the step b) of determining the O-sugar composition in step b) above represents a multiple of 1 or more of 42.01 Da (O-acetylation) Wherein the method comprises the step of measuring the molecular weight of the glycoprotein.
In the determination of the O-sugar composition in the above step (c), it is determined that there is an O-oligosaccharide mutation when the mass value difference between the detection peaks in step c) represents a multiple of 1 or more of 42.01 Da (O-acetylation) A method for real-time carbohydrate monitoring using glycoprotein molecular weight measurement.
and b) determining the sugar chain composition from the difference between the known theoretical molecular weight and the measured total molecular weight peak as a result of step a).
c) comparing the number of N-glycosides and the number of fucoses from the composition of the oligosaccharide determined in the above step b) to determine the number of the N-glycosides in the theoretically predictable N- And determining the content of the glycoprotein by measuring the molecular weight of the glycoprotein.
and d) determining the O-sugar composition from the sugar chain composition determined in step b) and the N-saccharide content determined in step c).
Wherein the saccharide content of the glycoprotein is calculated using the formula (total molecular weight of glycoprotein - theoretical protein molecular weight using protein amino acid composition) / total molecular weight of glycoprotein x 100 (%) after step a) Real time sugar chain monitoring method using molecular weight measurement.
The method according to any one of claims 1 to 5, wherein the oligosaccharide is determined to have an O-chain if n (NeuAc) is greater than n (4 x fucose) after the oligosaccharide composition is determined in the step b).
After the saccharide composition is determined in the step b) and the N-saccharide content is confirmed in the step c)
If n (NeuAc) is not greater than n (4 x fucose)
If the number of HexNAc is less than or equal to 31,
(HexNAc) -n (NeuAc)} <n (2 × fucose) by comparing n (HexNAc) -n (NeuAc) Wherein the method comprises the steps of:
After the saccharide composition is determined in the step b) and the N-saccharide content is confirmed in the step c)
If n (NeuAc) is not greater than n (4 x fucose)
If the number of HexNAc is less than or equal to 31,
(HexNAc) -n (NeuAc)} <n (2 × fucose) is not established by comparing {n (HexNAc) And measuring the molecular weight of the glycoprotein.
A method for real-time carbohydrate monitoring using the measurement of glycoprotein molecular weight, characterized in that at least one oligosaccharide mutation among the N-oligosaccharide and the O-oligosaccharide is identified in the step (b).
The N-glycosylation variant is characterized in that the hydroxyl group of polylactosamine or sialic acid in which Hex (Hexose) and HexNAc (N-Acetylhexosamine) are linked in the vertical direction and LacNAc (N-AcetylLactosamine) units are formed is converted into an acetyl group A method for real-time carbohydrate monitoring using glycoprotein molecular weight measurement.
Wherein the O-sugar chain variant is obtained by converting the hydroxyl group of sialic acid into an acetyl group.
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