CN114636781A - Method for evaluating sialylation level of glycoprotein oligosaccharide - Google Patents
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Abstract
The invention discloses a method for evaluating sialylation level of glycoprotein oligosaccharide. The method comprises the following steps: glycoprotein denaturation, collection and concentration, enzyme digestion of sugar chains, precipitation, sugar chain enrichment and ion chromatography analysis. The method utilizes the high-efficiency anion exchange chromatography to detect the sialylation level of the glycoprotein oligosaccharide, overcomes the problems of complex steps, low efficiency of high sialylation sugar chain labeling and the like of the traditional labeling operation, can accurately analyze the oligosaccharide map of the glycoprotein, is more favorable for accurately analyzing each sialylation component, and is suitable for evaluating the sialylation level of the glycoprotein oligosaccharide.
Description
Technical Field
The invention relates to the field of protein medicine bioengineering and technology, and relates to a method for evaluating sialylation level of glycoprotein oligosaccharide. In particular to an evaluation method of the sialylation level of oligosaccharides of a recombinant human vascular endothelial cell growth factor receptor Fc (VEGFR-Fc) fusion protein.
Background
Glycosylation is one of the most common post-translational modifications of proteins and can be divided into N-glycosylation and O-glycosylation depending on the mode of attachmentWherein N-glycosylation is-NH at an Asn residue in the sugar chain and amino acid sequence Asn-X-Ser/Thr (X is an amino acid other than Pro) of the encoded protein2And the O-glycosylation is that a sugar chain is connected with-OH on the Ser/Thr residue of the amino acid sequence of the encoded protein. At present, therapeutic protein drugs are mostly expressed by gene recombinant cells, and the glycosylation type and level of the therapeutic protein drugs have different degrees of influence on the drug effect, half-life period, stability, safety and the like of the drugs in vivo. For example, in the case of monoclonal antibodies or fusion proteins, the lower terminal sialic acid level affects the pharmacokinetics (pK) resulting in rapid clearance in vivo and a reduced half-life, and therefore, sialic acid level is an important index for assessing glycosylation level. The oligosaccharide map can display glycoform distribution of the glycoprotein with different sialic acid numbers, the glycoform proportion of the different sialic acid numbers is calculated, and the Z value is an important parameter for representing the overall sialylation degree of the glycoprotein. The detection project is mainly used for representing the sialylation degree of the recombinant therapeutic glycoprotein and investigating batch glycosylation stability.
At present, the oligosaccharide map (Z value) determination method mainly comprises high performance liquid chromatography and ion chromatography. The method for determining the Z value by the high performance liquid chromatography needs to carry out 2-AA/2-AB labeling, and has the problems of complicated operation steps (2-AB method), low labeling efficiency of high sialylation sugar chains (2-AA method) and the like. The method is favorable for deeply analyzing the sialylation degree of the complex glycoprotein, researching the difference of the sialylation degrees of different charge isomers and performing quality control on different sialylated glycoproteins in the production process.
Disclosure of Invention
The recombinant human VEGFR-Fc fusion protein is homodimeric glycoprotein, is expressed by Chinese hamster ovary cells (CHO cells), and is obtained by high purification and virus inactivation steps. The protein contains multiple glycosylation modification sites, has multiple charge isomers, and has no less than 12 bands (shown in figure 1) shown on IEF, and the difference of different bands is mainly caused by sialic acid. To ensure consistency in sialylation degree of each production batch, a stable production process and an efficient sialylation analysis method are required. In order to further carry out quality research on products, finely control the purification process and improve the product quality, the glycoprotein is denatured and reduced by guanidine hydrochloride and DTT, the denatured protein is subjected to enzyme digestion, alcohol precipitation, concentration, drying and resuspension by glycosidase F, and oligosaccharide pattern detection and analysis are carried out on the enriched sugar chains by combining a high performance anion exchange chromatography-integrated pulse amperometric detector (HPAEC-PAD) so as to guide the collection of target protein in the purification process and achieve the aim of obtaining high-quality glycoprotein drugs.
In view of the above, the present invention aims to provide a method for evaluating sialylation level of glycoprotein oligosaccharide, which overcomes the difficulties of complicated operation steps of the 2-AB method and low efficiency of labeling high sialylation sugar chains of the 2-AA method, can analyze a glycoprotein oligosaccharide map (Z value), can accurately analyze sialylation degree of glycoprotein and oligosaccharide difference of different sialylation, is helpful for collecting target protein in a purification process, and achieves the purpose of obtaining high quality glycoprotein drugs.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
the invention provides a method for evaluating sialylation level of glycoprotein oligosaccharide, which adopts high-efficiency ion chromatography for analysis and specifically comprises the following steps:
(1) performing denaturation reduction on the protein by using a denaturant and a reducing agent, and performing alkylation treatment on the denatured and reduced protein;
(2) changing the liquid of the alkylated protein, and collecting the protein into a centrifuge tube for concentration;
(3) carrying out enzyme digestion on the concentrated protein by using glycosidase F;
(4) adding an organic solvent into the protein solution after enzyme digestion, precipitating, centrifuging, and taking supernatant;
(5) concentrating, drying and resuspending the supernatant obtained in the step (4) to obtain an oligosaccharide solution to be detected;
(6) and (3) carrying out detection analysis on the oligosaccharide solution in the step (5) by using a high-efficiency anion exchange chromatography-integrated pulse amperometric detector.
Wherein the glycoprotein is a glycoprotein having a plurality of glycosylation sites.
Wherein said glycoprotein has a plurality of charge isomers.
Wherein the glycoprotein is recombinant human VEGFR-Fc fusion protein.
Wherein the denaturant used in the step 1 is guanidine hydrochloride or urea, wherein the guanidine hydrochloride is preferred.
Wherein the buffer system in the step 1 is Tris-HCl buffer solution, the reducing agent is dithiothreitol, and the alkylating reagent is iodoacetamide.
In one embodiment, denaturation can be achieved by: taking a proper amount of recombinant human VEGFR-Fc fusion protein, adding 1M Tris-HCl buffer solution, 8M guanidine hydrochloride and 1M DTT to ensure that the protein concentration in a final system is 0.5-1mg/ml, the final concentration of Tris-HCl is 50mM, the final concentration of guanidine hydrochloride is 6M and the final concentration of DTT is 10mM, mixing uniformly, and carrying out water bath at 56 ℃ for 30 min.
Wherein the step 1 is to reduce the glycoprotein with Dithiothreitol (DTT) solution; adding DTT solution with concentration of 0.5-1M, and reducing protein to obtain DTT with final concentration of 2-25 mM; further preferably the final concentration of DTT is 5-20 mM; in certain embodiments the final concentration of DTT is 2mM, 10mM or 25 mM.
Wherein step 1 is performed with Iodoacetamide (IAM) at a concentration of 5-62.5 mM, in certain embodiments 5mM, 25mM, 62.5 mM.
Wherein, in the step 2, a desalting column or an ultrafiltration centrifugal tube is used for liquid exchange, preferably the desalting column, and more preferably a Hitrap desalting column.
Wherein the final concentration of the glycosidase F used in the step 3 is 2-20U/mu l; further preferably, the final concentration of the glycosidase F is 4-10U/. mu.l; more preferably, the final concentration of glycosidase F is 5U/. mu.l.
Wherein the organic solvent used for protein precipitation in step 4 is methanol, ethanol or acetone, preferably ethanol or acetone, and in some embodiments, ethanol is further preferred.
Wherein the organic solvent is used in a volume ratio of 50% to 80%, more preferably 65% to 75%, and in certain embodiments 50%, 75%, or 80%.
In certain embodiments, 75% by volume of ethanol is used.
In a specific embodiment, the ion chromatograph uses a Thermo Scientific ICS-5000+ DC system, the column uses a Dionex CarboPac PA200(3 x 250mm), and the Guard column uses a Dionex CarboPac PA200 Guard (3 x 50 mm). Then, sample collection and data analysis processing are carried out.
The ion chromatography adopts gradient elution, and the elution conditions are as follows:
mobile phase A: 0.02M sodium hydroxide solution;
mobile phase B: 0.1M sodium hydroxide solution;
mobile phase C: 0.1M sodium hydroxide +1M sodium acetate solution;
elution setting:
a method of assessing the sialylation level of a glycoprotein oligosaccharide, comprising the steps of: (1) carrying out denaturation reduction treatment on the protein through guanidine hydrochloride and Dithiothreitol (DTT), and carrying out alkylation treatment on the protein after denaturation reduction; (2) changing the liquid of the alkylated protein by a desalting column, collecting the protein in a centrifugal tube, and concentrating the protein to a certain volume; (3) carrying out enzyme digestion on the desalted and concentrated protein by using glycosidase F; (4) collecting concentrated protein, adding 75% ethanol, precipitating, centrifuging, and collecting supernatant to obtain oligosaccharide solution containing no protein; (5) concentrating and drying the oligosaccharide solution, and suspending to a certain volume to obtain an enriched oligosaccharide solution; (6) performing oligosaccharide pattern detection analysis on the enriched sugar chains by using a high performance anion exchange chromatography-integrated pulsed amperometric detector (HPAEC-PAD), and calculating a Z value according to the following formula:
Z=（A1×1+A2×2+A3×3+A4×4）/（A0+A1+A2+A3+A4）；
wherein:
a0 is the percentage of the total peak area occupied by 0-sialylation peak area;
a1 is the percentage of 1-sialylation peak area to total peak area;
a2 is the percentage of 2-sialylation peak area to total peak area;
a3 is the percentage of the 3-sialylation peak area to the total peak area;
a4 is the percentage of 4-sialylated peak area to total peak area.
The high-efficiency anion exchange chromatography detection method of the invention comprises the following steps:
adding 200 μ l ultrapure water into the dried oligosaccharide, dissolving completely, centrifuging at 13000rpm for 10min, and collecting the supernatant for ion chromatography detection. Ion chromatography was a Thermo Scientific ICS-5000+ DC system, and the column was run on a Dionex CarboPac PA200(3 x 250mm), after sample collection, the different sialylated peaks were integrated and the data analyzed.
The parameters of the ion chromatography detection instrument such as setting, mobile phase, gradient and the like are as follows:
flow rate: 0.5 ml/min;
column temperature: 30 ℃;
sample introduction volume: 25 mul full circle sample injection;
a mobile phase A: 0.02M sodium hydroxide solution;
mobile phase B: 0.1M sodium hydroxide solution;
and (3) mobile phase C: 0.1M sodium hydroxide +1M sodium acetate solution;
table 1: ion chromatography elution gradient
According to the invention, the enriched oligosaccharide is subjected to oligosaccharide map separation by adopting the high-efficiency anion exchange chromatographic column with high resolution, and the separated oligosaccharide is detected by using the integral pulse amperometric detector.
The invention adopts guanidine hydrochloride denaturation, desalting column liquid exchange, glycosidase F enzyme digestion, precipitation and sugar chain enrichment, avoids the complicated steps of marking sugar chains by 2-AB/2-AA, purifying the sugar chains and the like, does not need expensive consumables and equipment, does not need to mark samples, has convenient operation, and can simply and quickly finish enzyme digestion and collect the sugar chains.
The invention separates the oligosaccharide by protein denaturation, sugar chain enzyme digestion and enrichment and combining with a high-efficiency anion exchange chromatographic column, and then detects the separated oligosaccharide by an integral pulse ampere detector. Compared with the prior art, firstly, the oligosaccharide treatment process does not need to be marked, the operation is simple, the sialylation degree of the glycoprotein can be reflected more truly, secondly, the oligosaccharide atlas detection analysis is carried out on the enriched carbohydrate chains by adopting a high-efficiency anion exchange chromatography-integral pulse amperometric detector, the sensitivity is high, the sialylation degree of the complex glycoprotein can be reflected more truly, the study on the glycoform difference among different sialylation isomers is facilitated, the quality control on different sialylation isomers in the production process is facilitated, and the oligosaccharide atlas detection method has good reference significance on the glycoprotein oligosaccharide atlas analysis with various sialylation isomers.
Drawings
FIG. 1: an IEF (electrophoretic image factor) chromatogram of the recombinant human VEGFR-Fc fusion protein;
FIG. 2: recombinant human VEGFR-Fc fusion protein oligosaccharide profiles (ion chromatography);
FIG. 3: recombinant human VEGFR-Fc fusion protein oligosaccharide map (high performance liquid chromatography, 2-AB method);
FIG. 4 is a schematic view of: recombinant human VEGFR-Fc fusion protein oligosaccharide map (high performance liquid chromatography, 2-AA method);
FIG. 5: recombinant human VEGFR-Fc fusion protein oligosaccharide profiles (ion chromatography, urea denaturation);
FIG. 6: recombinant human VEGFR-Fc fusion protein oligosaccharide profiles (ion chromatography, acetone precipitation);
FIG. 7: recombinant human VEGFR-Fc fusion protein oligosaccharide map (ion chromatography, ultrafiltration exchange).
Detailed Description
In order to further illustrate the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the specific embodiments of the present invention. Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.
Example 1 analysis of oligosaccharide map (Z value) of recombinant human VEGFR-Fc fusion protein
(1) Denaturation and reduction
Adding 1M Tris-HCl, 8M guanidine hydrochloride, 1M DTT, 200 mu g protein and a proper amount of ultrapure water into a 1.5ml EP tube respectively to ensure that the final concentration of Tris-HCl in a final system is 50mM, the final concentration of guanidine hydrochloride is 6M and the final concentration of DTT is 10mM, the total reaction system is 300 mu l, simultaneously setting blank samples, sequentially adding the reagents, uniformly mixing, and carrying out 56 ℃ water bath for 30 min;
(2) alkylation
Cooling the sample to room temperature, respectively adding 7.5 μ l of 1M IAM, mixing, and reacting at room temperature in dark place for 45 min;
(3) desalting column liquid exchange
Connecting the desalting column with a high performance liquid chromatograph, balancing the chromatographic column with desalting buffer solution for 30min, and preparing for sample injection under the chromatographic conditions of flow rate: 0.8ml/min, detection wavelength: 280 nm. Sampling 275 mu l of 6M guanidine hydrochloride, prewashing a sampling needle and a pipeline, then sampling 275 mu l of the samples according to the sequence of the samples, collecting protein peaks, and collecting the volume of about 600 mu l;
(4) concentrating
Concentrating the collected sample to 300 μ l, and if the volume is less than 300 μ l, using ultrapure water to make the volume constant to 300 μ l;
(5) sugar cutter
Collecting the collected and concentrated sample, adding glycosidase F3 μ l, mixing, and performing enzyme digestion in water bath at 37 deg.C for 15-18 h;
(6) sugar chain enrichment
Adding 0.9ml of glacial ethanol stored at-30 ℃, uniformly mixing, precipitating with ethanol at-30 ℃ for 15min, and centrifuging at 13000rpm for 15min by using a refrigerated centrifuge at 4 ℃;
(7) sugar chain drying
Taking the centrifuged supernatant, putting the supernatant into a freeze concentration centrifuge for concentration until the supernatant is completely dried;
(8) ion chromatography detection
Adding 200 μ l of ultrapure water into the dried oligosaccharide, completely dissolving, centrifuging at 13000rpm for 10min, taking the supernatant, performing ion chromatography detection, wherein an ion chromatograph is a Thermo Scientific ICS-5000+ DC system, a chromatographic column adopts Dionex CarboPac PA200(3 × 250mm), and after sample collection, integrating different sialylation peaks and performing data analysis;
the parameters of the ion chromatography detection instrument such as setting, mobile phase, gradient and the like are as follows:
flow rate: 0.5 ml/min;
column temperature: 30 ℃;
sample introduction volume: injecting 25 mul full-circle sample;
mobile phase A: 0.02M sodium hydroxide solution;
mobile phase B: 0.1M sodium hydroxide solution;
and (3) mobile phase C: 0.1M sodium hydroxide +1M sodium acetate solution;
table 1: ion chromatography elution gradient
(9) Results of the experiment
Determining the peak appearance position of a solvent peak according to a blank sample, deleting the solvent peak and a gradient peak, combining component peaks in each peak cluster, wherein the first main peak cluster is a 0-sialylation peak, the second main peak cluster is a 1-sialylation peak, the third main peak cluster is a 2-sialylation peak, the fourth main peak cluster is a 3-sialylation peak, the fifth main peak cluster is a 4-sialylation peak, and the map is shown in figure 2.
The Z value was calculated according to the formula Z = (a 1 × 1+ a2 × 2+ A3 × 3+ a4 × 4)/(a 0+ a1+ a2+ A3+ a 4), where: a0 is the percentage of the total peak area occupied by 0-sialylation peak area; a1 is the percentage of 1-sialylation peak area to total peak area; a2 is the percentage of 2-sialylation peak area to total peak area; a3 is the percentage of the 3-sialylation peak area to the total peak area; a4 is the 4-sialylation peak area as a percentage of the total peak area, with the results shown in Table 2:
TABLE 2 results of Z values for recombinant human VEGFR-Fc fusion proteins
In addition, the oligosaccharide spectrum (Z value) determination methods mainly include high performance liquid chromatography and ion chromatography. The method for determining the Z value by the high performance liquid chromatography needs to carry out 2-AA/2-AB labeling, has the defects of complicated operation steps (labeling, purification, re-enrichment and re-drying are needed after sugar chains are dried), low resolution (2-AB method), and as shown in figure 3, the peak of each sialylation respectively shows 1-2 peaks and is not high in resolution, while the peak of each sialylation detected by the ion chromatography basically respectively shows 4-10 peaks and is high in resolution; the labeling efficiency of the high sialylated sugar chain was low (2-AA method), and as shown in FIG. 4, the Z value results are shown in Table 3, and the results of 2-AA method labeled high sialyl oligosaccharides, such as 3SA% oligosaccharides (2.4%) were significantly lower than 5.7% and 2SA% oligosaccharides (22.6%) were also significantly lower than 32.1% in the ion chromatography, which finally resulted in a lower Z value (2-AA method Z value of 0.95, significantly lower than 1.23 in the ion chromatography). Therefore, the advantages of detecting the oligosaccharide map by ion chromatography are further shown.
TABLE 3Z value results for recombinant human VEGFR-Fc fusion protein (2-AB/2-AA method)
Example 2 analysis of oligosaccharide map (Z value) of recombinant human VEGFR-Fc fusion protein (Urea denaturation)
(1) Denaturation and reduction
Taking a 1.5ml EP tube, respectively adding 1M Tris-HCl, 10M urea, 1M DTT, 200 mu g protein and a proper amount of ultrapure water to ensure that the final concentration of Tris-HCl in the final system is 50mM, the final concentration of urea is 8M and the final concentration of DTT is 10mM, the total reaction system is 300 mu l, simultaneously setting blank samples, sequentially adding the reagents, uniformly mixing, and carrying out 56 ℃ water bath for 30 min;
(2) alkylation
Cooling the sample to room temperature, respectively adding 7.5 μ l of 1M IAM, mixing, and reacting at room temperature in dark place for 45 min;
(3) desalting column liquid exchange
Connecting the desalting column with a high performance liquid chromatograph, balancing the chromatographic column with desalting buffer solution for 30min, and preparing for sample injection under the chromatographic conditions of flow rate: 0.8ml/min, detection wavelength: 280 nm. 275 mul of 8M urea sample is taken firstly, and the sample injection needle and the pipeline are prewashed. Then 275 mul of sample is injected according to the sequence of the sample, and the protein peak is collected, and the collection volume is about 600 mul;
(4) concentrating
Concentrating the collected sample to 300 μ l, and if the volume is less than 300 μ l, using ultrapure water to make the volume constant to 300 μ l;
(5) sugar cutter
Collecting the collected and concentrated sample, adding glycosidase F3 μ l, mixing, and performing enzyme digestion in water bath at 37 deg.C for 15-18 h;
(6) sugar chain enrichment
Adding 0.9ml of glacial ethanol stored at-30 ℃, uniformly mixing, precipitating with ethanol at-30 ℃ for 15min, and centrifuging at 13000rpm for 15min by using a refrigerated centrifuge at 4 ℃;
(7) sugar chain drying
Taking the centrifuged supernatant, putting the supernatant into a freeze concentration centrifuge for concentration until the supernatant is completely dried;
(8) ion chromatography detection
Adding 200 μ l ultrapure water into the dried oligosaccharide, dissolving completely, centrifuging at 13000rpm for 10min, and collecting the supernatant for ion chromatography detection. The ion chromatograph is a Thermo Scientific ICS-5000+ DC system, a Dionex CarboPac PA200(3 × 250mm) is used as a chromatographic column, and after sample collection, peaks of different sialylations are integrated and data analysis is performed;
the parameters of the ion chromatography detection instrument such as setting, mobile phase, gradient and the like are as follows:
flow rate: 0.5 ml/min;
column temperature: 30 ℃;
sample introduction volume: injecting 25 mul full-circle sample;
mobile phase A: 0.02M sodium hydroxide solution;
mobile phase B: 0.1M sodium hydroxide solution;
mobile phase C: 0.1M sodium hydroxide +1M sodium acetate solution;
table 4: ion chromatography elution gradient
(9) Results of the experiment
Determining the peak position of a solvent peak according to a blank sample, deleting the solvent peak and a gradient peak, combining component peaks in each peak cluster, wherein the first main peak cluster is a 0-sialylation peak, the second main peak cluster is a 1-sialylation peak, the third main peak cluster is a 2-sialylation peak, the fourth main peak cluster is a 3-sialylation peak, the fifth main peak cluster is a 4-sialylation peak, and the map is shown in figure 5.
The Z value was calculated according to the formula Z = (a 1 × 1+ a2 × 2+ A3 × 3+ a4 × 4)/(a 0+ a1+ a2+ A3+ a 4), where: a0 is the percentage of the total peak area occupied by 0-sialylation peak area; a1 is the percentage of 1-sialylation peak area to total peak area; a2 is the percentage of 2-sialylation peak area to total peak area; a3 is the percentage of the 3-sialylation peak area to the total peak area; a4 is the 4-sialylation peak area as a percentage of the total peak area, with the results shown in Table 5:
TABLE 5 recombinant human VEGFR-Fc fusion protein Z value results (Urea denaturation)
Example 3 analysis of oligosaccharide map (Z value) of recombinant human VEGFR-Fc fusion protein (acetone precipitation)
(1) Denaturation and reduction
Taking a 1.5ml EP tube, respectively adding 1M Tris-HCl, 8M guanidine hydrochloride, 1M DTT, 200 mu g protein and a proper amount of ultrapure water to ensure that the final concentration of Tris-HCl in the final system is 50mM, the final concentration of guanidine hydrochloride is 6M and the final concentration of DTT is 5mM, the total reaction system is 300 mu l, simultaneously setting a blank sample, sequentially adding the reagents, uniformly mixing, and carrying out water bath at 56 ℃ for 30 min;
(2) alkylation
Cooling the sample to room temperature, respectively adding 3.75 μ l of 1M IAM, mixing, and reacting at room temperature in dark place for 45 min;
(3) desalting column liquid exchange
Connecting the desalting column with a high performance liquid chromatograph, balancing the chromatographic column with desalting buffer solution for 30min, and preparing for sample injection under the chromatographic conditions of flow rate: 0.8ml/min, detection wavelength: 280 nm. 275 mul of 6M guanidine hydrochloride was injected and the injection needle and tubing were prewashed. Then 275 mul of sample is injected according to the sequence of the sample, and the protein peak is collected, and the collection volume is about 600 mul;
(4) concentrating
Concentrating the collected sample to 300 μ l, and if the volume is less than 300 μ l, using ultrapure water to make the volume constant to 300 μ l;
(5) sugar cutter
Collecting the collected and concentrated sample, adding 6 mu l of glycosidase F, uniformly mixing, and carrying out enzyme digestion in water bath at 37 ℃ for 15-18 h;
(6) sugar chain enrichment
Adding 1.2ml of acetone stored at-30 ℃ into the sample, uniformly mixing, precipitating at-30 ℃ for 15min, and centrifuging at 13000rpm for 15min by using a refrigerated centrifuge at 4 ℃;
(7) sugar chain drying
Taking the centrifuged supernatant, putting the supernatant into a freeze concentration centrifuge for concentration until the supernatant is completely dried;
(8) ion chromatography detection
Adding 200 μ l ultrapure water into the dried oligosaccharide, dissolving completely, centrifuging at 13000rpm for 10min, and collecting the supernatant for ion chromatography detection. The ion chromatograph is a Thermo Scientific ICS-5000+ DC system, a Dionex CarboPac PA200(3 x 250mm) is used as a chromatographic column, and after sample collection, peaks of different sialylations are integrated and data analysis is performed;
the parameters of the ion chromatography detection instrument such as setting, mobile phase, gradient and the like are as follows:
flow rate: 0.5 ml/min;
column temperature: 30 ℃;
sample introduction volume: injecting 25 mul full-circle sample;
mobile phase A: 0.02M sodium hydroxide solution;
mobile phase B: 0.1M sodium hydroxide solution;
mobile phase C: 0.1M sodium hydroxide +1M sodium acetate solution;
table 6: ion chromatography elution gradient
(9) Results of the experiment
Determining the peak position of a solvent peak according to a blank sample, deleting the solvent peak and a gradient peak, combining component peaks in each peak cluster, wherein the first main peak cluster is a 0-sialylation peak, the second main peak cluster is a 1-sialylation peak, the third main peak cluster is a 2-sialylation peak, the fourth main peak cluster is a 3-sialylation peak, the fifth main peak cluster is a 4-sialylation peak, and the map is shown in figure 6.
The Z value was calculated according to the formula Z = (a 1 × 1+ a2 × 2+ A3 × 3+ a4 × 4)/(a 0+ a1+ a2+ A3+ a 4), where: a0 is the percentage of the total peak area occupied by 0-sialylation peak area; a1 is the percentage of 1-sialylation peak area to total peak area; a2 is the percentage of 2-sialylation peak area to total peak area; a3 is the percentage of the 3-sialylation peak area to the total peak area; a4 is the 4-sialylation peak area as a percentage of the total peak area, with the results shown in Table 7:
TABLE 7 results for Z values for recombinant human VEGFR-Fc fusion proteins (acetone precipitation)
Example 4 analysis of oligosaccharide map (Z value) of recombinant human VEGFR-Fc fusion protein (ultrafiltration exchange solution)
(1) Denaturation and reduction
Adding 1M Tris-HCl, 8M guanidine hydrochloride, 1M DTT, 200 mu g protein and a proper amount of ultrapure water into a 1.5ml EP tube respectively to ensure that the final concentration of Tris-HCl in a final system is 50mM, the final concentration of guanidine hydrochloride is 6M and the final concentration of DTT is 25mM, the total reaction system is 300 mu l, simultaneously setting blank samples, sequentially adding the reagents, uniformly mixing, and carrying out 56 ℃ water bath for 30 min;
(2) alkylation
Cooling the sample to room temperature, respectively adding 18.75 μ l of 1M IAM, mixing, and reacting at room temperature in dark for 45 min;
(3) ultrafiltration liquid exchange
Ultrafiltering the ultrafiltration buffer solution with 0.5ml and 10kD ultrafilter tube for 4 times, concentrating to 50 μ l, and diluting to 300 μ l;
(4) sugar cutter
Adding glycosidase F3 microliter into the sample after ultrafiltration liquid exchange, mixing uniformly, and performing enzyme digestion in water bath at 37 ℃ for 15-18 h;
(5) sugar chain enrichment
Adding 0.9ml of ethanol stored at-30 ℃ into the sample, mixing uniformly, precipitating with ethanol at-30 ℃ for 15min, and centrifuging at 13000rpm for 15min by using a refrigerated centrifuge at 4 ℃;
(6) sugar chain drying
Taking the centrifuged supernatant, putting the supernatant into a freeze concentration centrifuge for concentration until the supernatant is completely dried;
(7) ion chromatography detection
Adding 200 μ l ultrapure water into the dried oligosaccharide, dissolving completely, centrifuging at 13000rpm for 10min, and collecting the supernatant for ion chromatography detection. The ion chromatograph is a Thermo Scientific ICS-5000+ DC system, a Dionex CarboPac PA200(3 × 250mm) is used as a chromatographic column, and after sample collection, peaks of different sialylations are integrated and data analysis is performed;
the parameters of the ion chromatography detection instrument such as setting, mobile phase, gradient and the like are as follows:
flow rate: 0.5 ml/min;
column temperature: 30 ℃;
sample introduction volume: injecting 25 mul full-circle sample;
mobile phase A: 0.02M sodium hydroxide solution;
mobile phase B: 0.1M sodium hydroxide solution;
mobile phase C: 0.1M sodium hydroxide +1M sodium acetate solution;
table 6: ion chromatography elution gradient
(8) Results of the experiment
Determining the peak position of a solvent peak according to a blank sample, deleting the solvent peak and a gradient peak, combining component peaks in each peak cluster, wherein the first main peak cluster is a 0-sialylation peak, the second main peak cluster is a 1-sialylation peak, the third main peak cluster is a 2-sialylation peak, the fourth main peak cluster is a 3-sialylation peak, the fifth main peak cluster is a 4-sialylation peak, and the map is shown in figure 7.
The Z value was calculated according to the formula Z = (a 1 × 1+ a2 × 2+ A3 × 3+ a4 × 4)/(a 0+ a1+ a2+ A3+ a 4), where: a0 is the percentage of the total peak area of 0-sialylation peak area; a1 is the percentage of 1-sialylation peak area to total peak area; a2 is the percentage of 2-sialylation peak area to total peak area; a3 is the percentage of the 3-sialylation peak area to the total peak area; a4 is the 4-sialylation peak area as a percentage of the total peak area, with the results shown in Table 8:
TABLE 8 results for Z values for recombinant human VEGFR-Fc fusion proteins (exchange of fluid by ultrafiltration)
Claims (14)
1. A method for evaluating sialylation level of glycoprotein oligosaccharide, which adopts high performance anion exchange chromatography for analysis, and specifically comprises the following steps:
performing denaturation reduction on the protein by using a denaturant and a reducing agent, and performing alkylation treatment on the denatured and reduced protein;
changing the liquid of the alkylated protein, and collecting the protein into a centrifugal tube for concentration;
carrying out enzyme digestion on the concentrated protein by using glycosidase F;
adding an organic solvent into the protein solution after enzyme digestion, precipitating, centrifuging, and taking supernatant;
concentrating, drying and resuspending the supernatant obtained in the step (4) to obtain an oligosaccharide solution to be detected;
and (3) carrying out detection analysis on the oligosaccharide solution in the step (5) by using a high-efficiency anion exchange chromatography-integrated pulse amperometric detector.
2. The method of claim 1, wherein the glycoprotein is a recombinant human VEGFR-Fc fusion protein.
3. The method according to claim 1, wherein the denaturant used in the step (1) is guanidine hydrochloride or urea.
4. The method according to claim 1, wherein the buffer system in step (1) is Tris-HCl buffer, the reducing agent is dithiothreitol, and the alkylating agent is iodoacetamide.
5. The method according to claim 1, wherein the denaturation in step (1) is achieved by: taking a proper amount of recombinant human VEGFR-Fc fusion protein, adding 1M Tris-HCl buffer solution, 8M guanidine hydrochloride and 1M DTT to ensure that the protein concentration in a final system is 0.5-1mg/ml, the final concentration of Tris-HCl is 50mM, the final concentration of guanidine hydrochloride is 6M and the final concentration of DTT is 10mM, uniformly mixing, and carrying out water bath at 56 ℃ for 30 min;
in the step (1), Dithiothreitol (DTT) solution is used for reducing the glycoprotein, the concentration of the added DTT solution is 0.5M-1M, and the final concentration of the DTT for reducing the protein is 2-25 mM; preferably the final concentration of DTT is 5-20 mM;
in step (1) the alkylation treatment was carried out using Iodoacetamide (IAM), wherein the IAM was used at a concentration of 5-62.5 mM.
6. The method of claim 1, wherein the step (2) comprises exchanging the solution using a desalting column or an ultrafiltration centrifuge tube.
7. The method according to claim 1, wherein the glycosidase F is used in step (3) at a final concentration of 2-20U/. mu.l, preferably at a final concentration of 4-10U/. mu.l.
8. The method according to claim 1, wherein the organic solvent used for protein precipitation in step (4) is methanol, ethanol or acetone, preferably ethanol or acetone.
9. The method according to claim 8, wherein the organic solvent used in step (4) is 50% to 80%, preferably 65% to 75% by volume.
10. The method according to claim 9, wherein the organic solvent used in the step (4) is 75% (v/v) ethanol.
11. The method of claim 1, wherein the high performance ion exchange chromatography is performed using a Dionex CarboPac PA200(3 x 250mm) column and a Dionex CarboPac PA200 Guard (3 x 50mm) column.
12. The method of claim 11, wherein the high performance ion exchange chromatography is performed using a gradient elution under the following conditions:
mobile phase A: 0.02M sodium hydroxide solution
Mobile phase B: 0.1M sodium hydroxide solution
Mobile phase C: 0.1M sodium hydroxide +1M sodium acetate solution
The elution gradient was as follows:
13. method according to any one of claims 1 to 12, characterized in that the method for evaluating the sialylation level of a glycoprotein oligosaccharide comprises the following steps: (1) carrying out denaturation reduction treatment on the protein through guanidine hydrochloride and dithiothreitol, and carrying out alkylation treatment on the protein after denaturation reduction; (2) changing the liquid of the alkylated protein by a desalting column, collecting the protein in a centrifugal tube, and concentrating the protein to a certain volume; (3) carrying out enzyme digestion on the protein subjected to desalination and concentration by using glycosidase F; (4) collecting concentrated protein, adding 75% ethanol, precipitating, centrifuging, and collecting supernatant to obtain oligosaccharide solution containing no protein; (5) concentrating and drying the oligosaccharide solution, and suspending to a certain volume to obtain an enriched oligosaccharide solution; (6) oligosaccharide mapping analysis was performed on the enriched sugar chains using high performance anion exchange chromatography-integrated pulsed amperometric detector.
14. The method of claim 13, wherein the high performance ion exchange chromatography is as follows:
adding 200 μ l of ultrapure water into the dried oligosaccharide, completely dissolving, centrifuging at 13000rpm for 10min, taking the supernatant, performing ion chromatography detection, wherein an ion chromatograph is a Thermo Scientific ICS-5000+ DC system, a chromatographic column adopts Dionex CarboPac PA200(3 × 250mm), and after sample collection, integrating different sialylation peaks and performing data analysis;
the parameters of the ion chromatography detection instrument such as setting, mobile phase, gradient and the like are as follows:
flow rate: 0.5ml/min
Column temperature: 30 deg.C
Sample introduction volume: 25 mul full loop sample injection
Mobile phase A: 0.02M sodium hydroxide solution
Mobile phase B: 0.1M sodium hydroxide solution
Mobile phase C: 0.1M sodium hydroxide +1M sodium acetate solution
The ion chromatography elution gradient was as follows:
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